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A TALE nuclease architecture for efficient genome editing

2015-01-11T15:48:11Z

JernejMustar: /* A TALE nuclease architecture */

(Jernej Mustar)

Summarized from Miller, J. C. ''et al''. A TALE nuclease architecture for efficient genome editing. ''Nat. Biotechnol.'', vol. 29, pp. 143–148. Feb. 2011.

== Introduction to genome editing ==

Genome editing is a type of genetic engineering, where an organisms DNA is manipulated by altering; inserting, removing or replacing nucleotides, therefore changing its genome. This process is performed by the use of immensely useful tool, t. i. engineered nucleases, which are capable of cutting both strands of DNA or nicking single strand. Resulting sites are detected and repaired by endogenous repair mechanisms, which include homologous recombination (HR) or non-homologous end joining (NHEJ). The latter is error prone, therefore it is likely for mutations to occur in repair site. These repair pathways are highly conserved in diverse cell types, which is beneficial for the use of genome editing technologies. As a result, various cell types have been engineered by the use of portable cleavage-based technologies, including human stem cells [1].

Recently, there has been tremendous development in the field of synthetic biology and genome editing technology. Virtually any gene can be manipulated in diverse range of organisms. Most common approach is the use of nuclease, which can be manipulated to target specific sequences of genome. This can be achieved by exploitation of sequence-specific DNA-binding domains (ZFNs, TALENs) or by the use of complementary guide RNA (CRISPR-Cas9 system).
In this paper, the history of genome editing will first be briefly reviewed, further on genome editing will be discussed with emphasis on the TALEN’s family.

== Brief history of genome editing ==

Past studies in the field of genome editing were perusing a common goal, t. i. to obtain high specificity, which would limit the target affects [2]. The first engineered technology, based on nuclease activity, was introduced in 1991 by Pavletich and Pabo in the journal Science [2]. This technology by the name of Zinc Finger nuclease was a great breakthrough and was later on frequently applied for genome targeting. It is based on the use of cleavage domain of restriction enzyme FokI, which is linked with designated zinc finger protein (ZFP), which is responsible for targeting. By the use of different combinations of ZFP domains the selectivity for single genomic cleavage event is achieved. With the use of nuclease domains with monomeric activity there is a high probability of off-target events. This can be avoided by the use of a distinct pair of ZFNs with FokI domains that are obligate heterodimers [3]. Overtime, reports of problems and drawbacks of this technology were reported, such as targeting problem for certain triplets and reduced specificity, suffered by interactions within a zinc finger array [2].
The next technology, which emerged in 2009, is called transcription activator-like effector nuclease (TALENs). These are fusion proteins, where nuclease domain is associated with TAL effector DNA binding domain. This approach may be better in certain aspects, for example it enables a larger spectrum of targets and is more practical than ZFNs. In 2011, TALENs was proclaimed as “Method of the Year” by the journal Nature methods. The details of this system and molecular mechanisms of function are described later on.
We probably all at least heard if not read about latest breakthrough in the field of genome editing, which is CRISPR-Cas9 system. The discovery was somewhat fortuity, because researchers were studying how bacteria defend against phages and foreign plasmids. The paper was published in August 2012 in journal Science [4]. A bacterial nuclease called Cas9 is capable of altering genomes of invading viruses, consequently inactivating them. Clustered regularly interspaced short palindromic repeats (CRISPR) are DNA loci that contain short sequence repetitions, which can be transcribed, processed and then used to guide nuclease to a specific site [5]. This is in contrast with TALEN and Zinc Finger methods, where the need to prepare customized nuclease for each target remains.

== A TALE nuclease architecture ==

As mentioned before, two studies from 2009 proposed the use of TALEs effectors for targeted genome engineering. These transcriptional activators originate from Xanthomonas plant pathogens. During pathogenesis, TALEs specifically bind and regulate plant gene expression, thus aiding bacterial infection [6]. A central repeat domain, which mediates DNA recognition, is located within TALE structure [1]. Each repeat unit comprises of 33 – 35 aminoacids, these specify one target base. The units base preference is determined by two crucial adjacent aminoacids, also called the “repeat variable di-residue” (RVD). Therefore, the sequence of specified units in central repeat domain determines the target sequence on genome. TALEs generated with new repeat combinations have been shown to recognize target sequences predicted by this code.
In the light of aforementioned advances the interest in potential use of this system in combination with nuclease was initiated. Subsequently, a TALE-nuclease chimeras (TALENs) was proposed, which would allow for site-specific genome cleavage. In selected paper, the advances in the field of TALEN’s technology are discussed. TALE activity in a mammalian cell environment for targeted regulation of episomal reporters and an endogenous gene is presented, resuming with the research of the minimal TALE region required for high-affinity DNA binding. Paper also includes practical application, showing modification of endogenous human genes NTF3 and CCR5 by the use of TALEN approach.

== Retargeting a natural TALE to an endogenous mammalian sequence ==

Firstly, a suitable TAL effector for this study was chosen. Criteria for TALE selection was based on specificity and empirical evidence. The group was searching for natural TALE with high specificity for target sequence and with confirmed activity in mammalian cells. To achieve this goal, PCR amplifications for several TALEs using Xanthomonas axonopodis pathovar citri genomic DNA were performed. Native TALE-associated sequences which allow transport into plant cells were excluded. Protein products of acquired coding regions were characterized by a SELEX assay using target of interest. Outcome of this selection yielded one highly selective candidate, identified as TALE13. When combined with VP-16 activation domain, a 70-fold induction of reporter gene expression rate was observed in HEK293 cells. Based on these results, TALE13 was chosen for further work and design.
First goal was to demonstrate that chosen candidate was appropriate for mammalian gene regulation. Chosen target was NTF3 gene, which encodes a secreted nerve growth factor that has therapeutic potential for neurodegenerative diseases. Wild type TALE usually contain 18 repeats, by which they target specific sequence and bind to proximal promoter elements. TALE13 was modified to the extent of recognizing proximal promoter region of NTF3 by replacing the 18 wild-type repeat units with appropriate alternatives. This altered TALE was named NT-L (meaning NTF3 Left), which highlights its binding position on target. In order to test its activity, the truncated version of NT-L was fused with VP-16 and expressed in human HEK293 cells. By measuring the activity of endogenous NTF3 locus, a strong induction (over 20 fold) in both NTF3 transcript and protein product was detected. The subsequent SELEX analysis revealed high specificity. These results revealed, that NT-L variant is a viable candidate for endogenous mammalian gene regulation.

== Development of a TALE-nuclease architecture ==

The next step was to design TALENs chimera protein using NT-L and nuclease, which would allow implementation of targeted cleavage of its genome target. The process was realized through three stages.
First, the minimal region required for high affinity bonding was determined using gene regulation studies. This was beneficial for optimization of nuclease (FokI) cleavage domain attachment with its target. Desired outcome of this step was improved catalytic activity of chimeric tool. The truncated variant named NT-L+95 was shown to retain high specificity and at the same time contribute to improved nuclease attachment. In order to examine this, a C-terminal truncated series of TALE13 were generated and subsequently linked with FokI. The resulting candidates were screened as homodimers for cleavage of specific targets. The results have shown several interesting occurrences. For TALENs comprising of NT-L+95 no detectable nuclease activity was observed. The cause for this is unclear; it is possible that this is due to a certain effect of NT-L+95 to nuclease domain positioning. Efficient DNA cleavage was observed for certain shorter specimens, providing candidates for cellular studies.
The minimal region between TALEN binding sites for optimal activity is over 12 bp, otherwise reduce cleavage may occur. These observations are useful for design of TALENs used for in vivo studies.
In the last stage, homodimer TALEN partner for NT-L was designed and named NT-R (NTR3 Right), indicating the direction of binding. This pair was expressed in human
K562 cells as fusions with catalytic domain of FokI. Target locus was later on tested for gene modification. By using two different C-terminal subregions (+28 and +63) the designed TALENs were tested. Significant activity in in vitro cleavage study was detected. With expression at 37°C, up to 3% modification was observed by the Surveyor assay [7]. This assay reveals gene modification by the appearance of digestion products. The percentage of gene modification can be obtained by quantification of “cut” bands. Limit of detection of this assay is about 1%. The activity was higher (9% of modification) in conditions of transient hypothermia, t. i. at 30°C. Sequences from latter were analyzed by Sanger sequencing. Out of 84 analyzed alleles there were 7 mutated by the occurrence of short deletions. This observation is consistent with expectation of non-homologous end joining (NHEJ) activity. Results have also shown a NHEJ-mediated capture of an oligonucleotide duplex at the endogenous NTF3 locus. These results confirmed that NHEJ-mediated genome modification by the use of TALEN architecture is possible in mammalian environment.

== Efficient modification by NHEJ and homology directed repair (HDR) ==

To expand the practical application for this developed system, modification at an additional locus was proposed. CCR5 gene was targeted for modification. First, a set of TALENs with a range of target lengths from 13 to 17 bp and of separation distance from 5 to 27 bp were designed and tested to identify values compatible with endogenous activity. To this end, a cluster of four left and four right binding sites were chosen for targeting. This defined a matrix of 16 heterodimer targets. For each binding site, two alternative proteins were generated, with 28 or 63 residues on C-terminal (as before). Altogether, this yielded 8x8=64 combinations of left and right proteins. These were expressed by pairs in K562 cells under standard conditions. Once again, Surveyor assay was used for evaluation of allele modification. Obtained results indicate that introduction of some +63/+63 pairs yielded over 20% allele modification. TALEN treated cells seem to retain a stable modification lever after 10 day growth, suggesting that TALENs are generally well tolerated. The effect of spacer length on activity is observed once again. At least 5% modification is achieved by the use of 12 – 19 bp spacer length in the +63/+63 TALENs section. In contrast, +28/+28 section show maximal activity in narrow spacer range of 12 – 13 bp.
The next challenge was to show that it is possible to induce gene through HDR using this system. For this, a part of CCR5 region was selected for targeting, also mentioned as safe harbor previously used for transgene integration. 24 TALEN pairs were then screened for their ability to induce NHEJ. Two highly active combinations were identified, first with 18% mutation rate and second with 21%. These TALEN were introduced into K562 cells simultaneously with donor DNA fragment carrying 46 bp with restriction site BglI. The success of integration was proven by PCR and BglI digestion. About 16% of inspected alleles were modified. This is an example how TALEN architecture can be used to activate HDR.

== Conclusion ==

In presented study it is shown how TALEN architecture can be designed and generated for efficient genome editing in mammalian environment. In this case, the system consists of TALE truncation variants, linked to catalytic domain of FokI. Engineered TALENs can be used for gene modification of human cells by activating either NHEJ or HDR repair pathways. Furthermore, three distinct applications are demonstrated; NHEJ mediated gene disruption, NHEJ-mediated insertion by capture of a DNA fragment and HDR mediated gene editing. Aforementioned suggests that TALEN architecture is a compelling tool in synthetic biology.

== Discussion ==

A step to facilitate the use of this architecture would undoubtedly be the linkage of minimal central TALE repeat domain to the catalytic domain of FokI. This was previously attempted by several groups without success, whereas the lack of activity of such constructs [8]. An alternative idea is to use a full length TALE for targeting. Disadvantage of the latter is the size of linkage peptide connecting two domains, which would affect the nuclease activity. Third approach is to generate truncated variants of TALEs and link them with catalytic domain of FokI, as shown in this paper. This approach has proven affective when using truncated variants with 28 or 63 of the 278 original C-terminal residues.
During the experimental work of this study, two papers were submitted where they demonstrated the use of TALEN architecture with the use of unmodified TALE or TALE with slightly reduced C-terminus [8], [9]. These systems were used for episome cleavage in yeast, an approach that is hardly translated to more complex genomes. As expected, these proteins have extended range of cleavage compared to truncated variants.
Presented TALEN architecture is in some characteristics similar to ZFNs. One example is the high cleavage activity of TALENs, which enables high gene modification efficiency, which is in some cases superior to the one of ZFNs. A shortcoming of TALEN architecture compared to ZFNs is its relative size. TALE repeats are 3 – 4 times larger than ZFPs, which may cause difficulty in delivery methods. Also, limitations due to high levels of TALE repeat homology may complicate the assembly of constructs. Future experiments will provide a fuller picture of pros and cons of the two platforms.
Targeted endogenous gene regulation by TALE proteins is a useful tool in research and for biotechnology applications. We can use this technology in a broad sense, such as design and generation of transcriptional activators or repressors. Although TALEs were discovered as such, previous studies did not inspect this type of activity outside of their native plant context. The experiments shown in this paper suggest that TALEs can be used as transcriptional regulators.
The use of SELEX assay for identification and characterization of TALEs has previously not been reported. By using this method, it was possible to assess the DNA binding preferences of natural and designed TALE proteins. Binding sites for TALE13 and four engineered TALEs were examined. On this basis, an observation that 73 out of 76 TALE repeats facilitate base preference of RVD. An interpretation of rare exceptions is proposed, facilitating further design and use of this system. Overall data collected can be used to examine previously acquired information on RVDs and thereby gaining more insight on mechanisms of action and selectivity. Findings that common RVDs such as NI, HD, NN and NG selectively bound selectively bound adenine, cytosine, guanine and thymine, respectively, are in accordance with previous knowledge. Pattern NN has shown relaxed specificity with substantial binding to a second base, adenine. NK is therefore more suitable for specificity, due to much stronger preference for guanine. This is a potential enhancement, proposed in this paper.

== Expectations for the future ==

As already mentioned, targeting the designated loci with ZFNs or TALENs is often difficult and time consuming, due to design and generation of new custom ZF or TALE array for each locus. This is not the case in most recent approach in genome editing, that is the use of CRISPR-Cas9 system. Expression of distinct gRNAs alongside Cas9 results in Cas9–gRNA complexes with distinct cleavage specificities [10]. This approach is exploited in multiplex genome editing. Still, there are features of this system which are inferior to elderly time-demanding ZFNs or TALENs.
One of those features is size of gene constructs, which is a limitation in field of gene delivery. Cas9 is the size of about 160 kDa, whereas a typical 17,5 repeat TALEN corresponds to 105 kDa. By far the smallest are ZFNs, wherein typical four finger ZFN has about 40 kDa [10]. It is likely for this compact nature to be beneficial for certain experiments.
As for specificity, the number of bases for modification in Cas9 system is restricted to 20 and is somewhat limited by the rule of three guanine bases flanking the target site (GN19NGG) [10]. The main constriction of ZF arrays is a scarce set of fingers, resulting in inability to target certain triplets. In addition, some ZFs targeting GNN seem to be superior to other ZFs, contributing to limitations. TALE target specificity therefore seems superior, with capabilities of targeting up to 24 bases. For Cas9 system, off target studies in mammalian environment are yet to be conducted [10].
It is likely that distinct features will lead to differential use of genome editing systems for distinct applications in the fields of plant science, therapeutic sciences and biotechnology applications.

== Literature ==

[1] J. C. Miller, S. Tan, G. Qiao, K. A. Barlow, J. Wang, D. F. Xia, X. Meng, D. E. Paschon, E. Leung, S. J. Hinkley, G. P. Dulay, K. L. Hua, I. Ankoudinova, G. J. Cost, F. D. Urnov, H. S. Zhang, M. C. Holmes, L. Zhang, P. D. Gregory, and E. J. Rebar, “A TALE nuclease architecture for efficient genome editing,” Nat. Biotechnol., vol. 29, no. 2, pp. 143–148, Feb. 2011.

[2] “Genome Editing.” [Online]. Available: http://www.allelebiotech.com/genome-editing/. [Accessed: 06-Jan-2015].

[3] R. M. Gupta and K. Musunuru, “Expanding the genetic editing tool kit: ZFNs, TALENs, and CRISPR-Cas9,” J. Clin. Invest., vol. 124, no. 10, pp. 4154–4161, Oct. 2014.

[4] M. Jinek, K. Chylinski, I. Fonfara, M. Hauer, J. A. Doudna, and E. Charpentier, “A programmable dual-RNA-guided DNA endonuclease in adaptive bacterial immunity,” Science, vol. 337, no. 6096, pp. 816–821, Aug. 2012.

[5] “CRISPR,” Wikipedia, the free encyclopedia. 09-Jan-2015.

[6] “TAL effector,” Wikipedia, the free encyclopedia. 06-Jan-2015.

[7] J. C. Miller, M. C. Holmes, J. Wang, D. Y. Guschin, Y.-L. Lee, I. Rupniewski, C. M. Beausejour, A. J. Waite, N. S. Wang, K. A. Kim, P. D. Gregory, C. O. Pabo, and E. J. Rebar, “An improved zinc-finger nuclease architecture for highly specific genome editing,” Nat. Biotechnol., vol. 25, no. 7, pp. 778–785, Jul. 2007.

[8] M. Christian, T. Cermak, E. L. Doyle, C. Schmidt, F. Zhang, A. Hummel, A. J. Bogdanove, and D. F. Voytas, “Targeting DNA double-strand breaks with TAL effector nucleases,” Genetics, vol. 186, no. 2, pp. 757–761, Oct. 2010.

[9] T. Li, S. Huang, W. Z. Jiang, D. Wright, M. H. Spalding, D. P. Weeks, and B. Yang, “TAL nucleases (TALNs): hybrid proteins composed of TAL effectors and FokI DNA-cleavage domain,” Nucleic Acids Res., vol. 39, no. 1, pp. 359–372, Jan. 2011.

[10] A. Strauβ and T. Lahaye, “Zinc Fingers, TAL Effectors, or Cas9-Based DNA Binding Proteins: What’s Best for Targeting Desired Genome Loci?,” Mol. Plant, vol. 6, no. 5, pp. 1384–1387, Sep. 2013.

A TALE nuclease architecture for efficient genome editing

2015-01-11T15:46:42Z

JernejMustar: /* Retargeting a natural TALE to an endogenous mammalian sequence */

(Jernej Mustar)

Summarized from Miller, J. C. ''et al''. A TALE nuclease architecture for efficient genome editing. ''Nat. Biotechnol.'', vol. 29, pp. 143–148. Feb. 2011.

== Introduction to genome editing ==

Genome editing is a type of genetic engineering, where an organisms DNA is manipulated by altering; inserting, removing or replacing nucleotides, therefore changing its genome. This process is performed by the use of immensely useful tool, t. i. engineered nucleases, which are capable of cutting both strands of DNA or nicking single strand. Resulting sites are detected and repaired by endogenous repair mechanisms, which include homologous recombination (HR) or non-homologous end joining (NHEJ). The latter is error prone, therefore it is likely for mutations to occur in repair site. These repair pathways are highly conserved in diverse cell types, which is beneficial for the use of genome editing technologies. As a result, various cell types have been engineered by the use of portable cleavage-based technologies, including human stem cells [1].

Recently, there has been tremendous development in the field of synthetic biology and genome editing technology. Virtually any gene can be manipulated in diverse range of organisms. Most common approach is the use of nuclease, which can be manipulated to target specific sequences of genome. This can be achieved by exploitation of sequence-specific DNA-binding domains (ZFNs, TALENs) or by the use of complementary guide RNA (CRISPR-Cas9 system).
In this paper, the history of genome editing will first be briefly reviewed, further on genome editing will be discussed with emphasis on the TALEN’s family.

== Brief history of genome editing ==

Past studies in the field of genome editing were perusing a common goal, t. i. to obtain high specificity, which would limit the target affects [2]. The first engineered technology, based on nuclease activity, was introduced in 1991 by Pavletich and Pabo in the journal Science [2]. This technology by the name of Zinc Finger nuclease was a great breakthrough and was later on frequently applied for genome targeting. It is based on the use of cleavage domain of restriction enzyme FokI, which is linked with designated zinc finger protein (ZFP), which is responsible for targeting. By the use of different combinations of ZFP domains the selectivity for single genomic cleavage event is achieved. With the use of nuclease domains with monomeric activity there is a high probability of off-target events. This can be avoided by the use of a distinct pair of ZFNs with FokI domains that are obligate heterodimers [3]. Overtime, reports of problems and drawbacks of this technology were reported, such as targeting problem for certain triplets and reduced specificity, suffered by interactions within a zinc finger array [2].
The next technology, which emerged in 2009, is called transcription activator-like effector nuclease (TALENs). These are fusion proteins, where nuclease domain is associated with TAL effector DNA binding domain. This approach may be better in certain aspects, for example it enables a larger spectrum of targets and is more practical than ZFNs. In 2011, TALENs was proclaimed as “Method of the Year” by the journal Nature methods. The details of this system and molecular mechanisms of function are described later on.
We probably all at least heard if not read about latest breakthrough in the field of genome editing, which is CRISPR-Cas9 system. The discovery was somewhat fortuity, because researchers were studying how bacteria defend against phages and foreign plasmids. The paper was published in August 2012 in journal Science [4]. A bacterial nuclease called Cas9 is capable of altering genomes of invading viruses, consequently inactivating them. Clustered regularly interspaced short palindromic repeats (CRISPR) are DNA loci that contain short sequence repetitions, which can be transcribed, processed and then used to guide nuclease to a specific site [5]. This is in contrast with TALEN and Zinc Finger methods, where the need to prepare customized nuclease for each target remains.

== A TALE nuclease architecture ==

As mentioned before, two studies from 2009 proposed the use of TALEs effectors for targeted genome engineering. These transcriptional activators originate from Xanthomonas plant pathogens. During pathogenesis, TALEs specifically bind and regulate plant gene expression, thus aiding bacterial infection [6]. A central repeat domain, which mediates DNA recognition, is located within TALE structure [1]. Each repeat unit comprises of 33 – 35 amino, these specify one target base. The units base preference is determined by two crucial adjacent amino acids, also called the “repeat variable di-residue” (RVD). Therefore, the sequence of specified units in central repeat domain determines the target sequence on genome. TALEs generated with new repeat combinations have been shown to recognize target sequences predicted by this code.
In the light of aforementioned advances the interest in potential use of this system in combination with nuclease was initiated. Subsequently, a TALE-nuclease chimeras (TALENs) was proposed, which would allow for site-specific genome cleavage. In selected paper, the advances in the field of TALEN’s technology are discussed. TALE activity in a mammalian cell environment for targeted regulation of episomal reporters and an endogenous gene is presented, resuming with the research of the minimal TALE region required for high-affinity DNA binding. Paper also includes practical application, showing modification of endogenous human genes NTF3 and CCR5 by the use of TALEN approach.

== Retargeting a natural TALE to an endogenous mammalian sequence ==

Firstly, a suitable TAL effector for this study was chosen. Criteria for TALE selection was based on specificity and empirical evidence. The group was searching for natural TALE with high specificity for target sequence and with confirmed activity in mammalian cells. To achieve this goal, PCR amplifications for several TALEs using Xanthomonas axonopodis pathovar citri genomic DNA were performed. Native TALE-associated sequences which allow transport into plant cells were excluded. Protein products of acquired coding regions were characterized by a SELEX assay using target of interest. Outcome of this selection yielded one highly selective candidate, identified as TALE13. When combined with VP-16 activation domain, a 70-fold induction of reporter gene expression rate was observed in HEK293 cells. Based on these results, TALE13 was chosen for further work and design.
First goal was to demonstrate that chosen candidate was appropriate for mammalian gene regulation. Chosen target was NTF3 gene, which encodes a secreted nerve growth factor that has therapeutic potential for neurodegenerative diseases. Wild type TALE usually contain 18 repeats, by which they target specific sequence and bind to proximal promoter elements. TALE13 was modified to the extent of recognizing proximal promoter region of NTF3 by replacing the 18 wild-type repeat units with appropriate alternatives. This altered TALE was named NT-L (meaning NTF3 Left), which highlights its binding position on target. In order to test its activity, the truncated version of NT-L was fused with VP-16 and expressed in human HEK293 cells. By measuring the activity of endogenous NTF3 locus, a strong induction (over 20 fold) in both NTF3 transcript and protein product was detected. The subsequent SELEX analysis revealed high specificity. These results revealed, that NT-L variant is a viable candidate for endogenous mammalian gene regulation.

== Development of a TALE-nuclease architecture ==

The next step was to design TALENs chimera protein using NT-L and nuclease, which would allow implementation of targeted cleavage of its genome target. The process was realized through three stages.
First, the minimal region required for high affinity bonding was determined using gene regulation studies. This was beneficial for optimization of nuclease (FokI) cleavage domain attachment with its target. Desired outcome of this step was improved catalytic activity of chimeric tool. The truncated variant named NT-L+95 was shown to retain high specificity and at the same time contribute to improved nuclease attachment. In order to examine this, a C-terminal truncated series of TALE13 were generated and subsequently linked with FokI. The resulting candidates were screened as homodimers for cleavage of specific targets. The results have shown several interesting occurrences. For TALENs comprising of NT-L+95 no detectable nuclease activity was observed. The cause for this is unclear; it is possible that this is due to a certain effect of NT-L+95 to nuclease domain positioning. Efficient DNA cleavage was observed for certain shorter specimens, providing candidates for cellular studies.
The minimal region between TALEN binding sites for optimal activity is over 12 bp, otherwise reduce cleavage may occur. These observations are useful for design of TALENs used for in vivo studies.
In the last stage, homodimer TALEN partner for NT-L was designed and named NT-R (NTR3 Right), indicating the direction of binding. This pair was expressed in human
K562 cells as fusions with catalytic domain of FokI. Target locus was later on tested for gene modification. By using two different C-terminal subregions (+28 and +63) the designed TALENs were tested. Significant activity in in vitro cleavage study was detected. With expression at 37°C, up to 3% modification was observed by the Surveyor assay [7]. This assay reveals gene modification by the appearance of digestion products. The percentage of gene modification can be obtained by quantification of “cut” bands. Limit of detection of this assay is about 1%. The activity was higher (9% of modification) in conditions of transient hypothermia, t. i. at 30°C. Sequences from latter were analyzed by Sanger sequencing. Out of 84 analyzed alleles there were 7 mutated by the occurrence of short deletions. This observation is consistent with expectation of non-homologous end joining (NHEJ) activity. Results have also shown a NHEJ-mediated capture of an oligonucleotide duplex at the endogenous NTF3 locus. These results confirmed that NHEJ-mediated genome modification by the use of TALEN architecture is possible in mammalian environment.

== Efficient modification by NHEJ and homology directed repair (HDR) ==

To expand the practical application for this developed system, modification at an additional locus was proposed. CCR5 gene was targeted for modification. First, a set of TALENs with a range of target lengths from 13 to 17 bp and of separation distance from 5 to 27 bp were designed and tested to identify values compatible with endogenous activity. To this end, a cluster of four left and four right binding sites were chosen for targeting. This defined a matrix of 16 heterodimer targets. For each binding site, two alternative proteins were generated, with 28 or 63 residues on C-terminal (as before). Altogether, this yielded 8x8=64 combinations of left and right proteins. These were expressed by pairs in K562 cells under standard conditions. Once again, Surveyor assay was used for evaluation of allele modification. Obtained results indicate that introduction of some +63/+63 pairs yielded over 20% allele modification. TALEN treated cells seem to retain a stable modification lever after 10 day growth, suggesting that TALENs are generally well tolerated. The effect of spacer length on activity is observed once again. At least 5% modification is achieved by the use of 12 – 19 bp spacer length in the +63/+63 TALENs section. In contrast, +28/+28 section show maximal activity in narrow spacer range of 12 – 13 bp.
The next challenge was to show that it is possible to induce gene through HDR using this system. For this, a part of CCR5 region was selected for targeting, also mentioned as safe harbor previously used for transgene integration. 24 TALEN pairs were then screened for their ability to induce NHEJ. Two highly active combinations were identified, first with 18% mutation rate and second with 21%. These TALEN were introduced into K562 cells simultaneously with donor DNA fragment carrying 46 bp with restriction site BglI. The success of integration was proven by PCR and BglI digestion. About 16% of inspected alleles were modified. This is an example how TALEN architecture can be used to activate HDR.

== Conclusion ==

In presented study it is shown how TALEN architecture can be designed and generated for efficient genome editing in mammalian environment. In this case, the system consists of TALE truncation variants, linked to catalytic domain of FokI. Engineered TALENs can be used for gene modification of human cells by activating either NHEJ or HDR repair pathways. Furthermore, three distinct applications are demonstrated; NHEJ mediated gene disruption, NHEJ-mediated insertion by capture of a DNA fragment and HDR mediated gene editing. Aforementioned suggests that TALEN architecture is a compelling tool in synthetic biology.

== Discussion ==

A step to facilitate the use of this architecture would undoubtedly be the linkage of minimal central TALE repeat domain to the catalytic domain of FokI. This was previously attempted by several groups without success, whereas the lack of activity of such constructs [8]. An alternative idea is to use a full length TALE for targeting. Disadvantage of the latter is the size of linkage peptide connecting two domains, which would affect the nuclease activity. Third approach is to generate truncated variants of TALEs and link them with catalytic domain of FokI, as shown in this paper. This approach has proven affective when using truncated variants with 28 or 63 of the 278 original C-terminal residues.
During the experimental work of this study, two papers were submitted where they demonstrated the use of TALEN architecture with the use of unmodified TALE or TALE with slightly reduced C-terminus [8], [9]. These systems were used for episome cleavage in yeast, an approach that is hardly translated to more complex genomes. As expected, these proteins have extended range of cleavage compared to truncated variants.
Presented TALEN architecture is in some characteristics similar to ZFNs. One example is the high cleavage activity of TALENs, which enables high gene modification efficiency, which is in some cases superior to the one of ZFNs. A shortcoming of TALEN architecture compared to ZFNs is its relative size. TALE repeats are 3 – 4 times larger than ZFPs, which may cause difficulty in delivery methods. Also, limitations due to high levels of TALE repeat homology may complicate the assembly of constructs. Future experiments will provide a fuller picture of pros and cons of the two platforms.
Targeted endogenous gene regulation by TALE proteins is a useful tool in research and for biotechnology applications. We can use this technology in a broad sense, such as design and generation of transcriptional activators or repressors. Although TALEs were discovered as such, previous studies did not inspect this type of activity outside of their native plant context. The experiments shown in this paper suggest that TALEs can be used as transcriptional regulators.
The use of SELEX assay for identification and characterization of TALEs has previously not been reported. By using this method, it was possible to assess the DNA binding preferences of natural and designed TALE proteins. Binding sites for TALE13 and four engineered TALEs were examined. On this basis, an observation that 73 out of 76 TALE repeats facilitate base preference of RVD. An interpretation of rare exceptions is proposed, facilitating further design and use of this system. Overall data collected can be used to examine previously acquired information on RVDs and thereby gaining more insight on mechanisms of action and selectivity. Findings that common RVDs such as NI, HD, NN and NG selectively bound selectively bound adenine, cytosine, guanine and thymine, respectively, are in accordance with previous knowledge. Pattern NN has shown relaxed specificity with substantial binding to a second base, adenine. NK is therefore more suitable for specificity, due to much stronger preference for guanine. This is a potential enhancement, proposed in this paper.

== Expectations for the future ==

As already mentioned, targeting the designated loci with ZFNs or TALENs is often difficult and time consuming, due to design and generation of new custom ZF or TALE array for each locus. This is not the case in most recent approach in genome editing, that is the use of CRISPR-Cas9 system. Expression of distinct gRNAs alongside Cas9 results in Cas9–gRNA complexes with distinct cleavage specificities [10]. This approach is exploited in multiplex genome editing. Still, there are features of this system which are inferior to elderly time-demanding ZFNs or TALENs.
One of those features is size of gene constructs, which is a limitation in field of gene delivery. Cas9 is the size of about 160 kDa, whereas a typical 17,5 repeat TALEN corresponds to 105 kDa. By far the smallest are ZFNs, wherein typical four finger ZFN has about 40 kDa [10]. It is likely for this compact nature to be beneficial for certain experiments.
As for specificity, the number of bases for modification in Cas9 system is restricted to 20 and is somewhat limited by the rule of three guanine bases flanking the target site (GN19NGG) [10]. The main constriction of ZF arrays is a scarce set of fingers, resulting in inability to target certain triplets. In addition, some ZFs targeting GNN seem to be superior to other ZFs, contributing to limitations. TALE target specificity therefore seems superior, with capabilities of targeting up to 24 bases. For Cas9 system, off target studies in mammalian environment are yet to be conducted [10].
It is likely that distinct features will lead to differential use of genome editing systems for distinct applications in the fields of plant science, therapeutic sciences and biotechnology applications.

== Literature ==

[1] J. C. Miller, S. Tan, G. Qiao, K. A. Barlow, J. Wang, D. F. Xia, X. Meng, D. E. Paschon, E. Leung, S. J. Hinkley, G. P. Dulay, K. L. Hua, I. Ankoudinova, G. J. Cost, F. D. Urnov, H. S. Zhang, M. C. Holmes, L. Zhang, P. D. Gregory, and E. J. Rebar, “A TALE nuclease architecture for efficient genome editing,” Nat. Biotechnol., vol. 29, no. 2, pp. 143–148, Feb. 2011.

[2] “Genome Editing.” [Online]. Available: http://www.allelebiotech.com/genome-editing/. [Accessed: 06-Jan-2015].

[3] R. M. Gupta and K. Musunuru, “Expanding the genetic editing tool kit: ZFNs, TALENs, and CRISPR-Cas9,” J. Clin. Invest., vol. 124, no. 10, pp. 4154–4161, Oct. 2014.

[4] M. Jinek, K. Chylinski, I. Fonfara, M. Hauer, J. A. Doudna, and E. Charpentier, “A programmable dual-RNA-guided DNA endonuclease in adaptive bacterial immunity,” Science, vol. 337, no. 6096, pp. 816–821, Aug. 2012.

[5] “CRISPR,” Wikipedia, the free encyclopedia. 09-Jan-2015.

[6] “TAL effector,” Wikipedia, the free encyclopedia. 06-Jan-2015.

[7] J. C. Miller, M. C. Holmes, J. Wang, D. Y. Guschin, Y.-L. Lee, I. Rupniewski, C. M. Beausejour, A. J. Waite, N. S. Wang, K. A. Kim, P. D. Gregory, C. O. Pabo, and E. J. Rebar, “An improved zinc-finger nuclease architecture for highly specific genome editing,” Nat. Biotechnol., vol. 25, no. 7, pp. 778–785, Jul. 2007.

[8] M. Christian, T. Cermak, E. L. Doyle, C. Schmidt, F. Zhang, A. Hummel, A. J. Bogdanove, and D. F. Voytas, “Targeting DNA double-strand breaks with TAL effector nucleases,” Genetics, vol. 186, no. 2, pp. 757–761, Oct. 2010.

[9] T. Li, S. Huang, W. Z. Jiang, D. Wright, M. H. Spalding, D. P. Weeks, and B. Yang, “TAL nucleases (TALNs): hybrid proteins composed of TAL effectors and FokI DNA-cleavage domain,” Nucleic Acids Res., vol. 39, no. 1, pp. 359–372, Jan. 2011.

[10] A. Strauβ and T. Lahaye, “Zinc Fingers, TAL Effectors, or Cas9-Based DNA Binding Proteins: What’s Best for Targeting Desired Genome Loci?,” Mol. Plant, vol. 6, no. 5, pp. 1384–1387, Sep. 2013.

A TALE nuclease architecture for efficient genome editing

2015-01-11T15:45:12Z

JernejMustar: /* Discussion */

(Jernej Mustar)

Summarized from Miller, J. C. ''et al''. A TALE nuclease architecture for efficient genome editing. ''Nat. Biotechnol.'', vol. 29, pp. 143–148. Feb. 2011.

== Introduction to genome editing ==

Genome editing is a type of genetic engineering, where an organisms DNA is manipulated by altering; inserting, removing or replacing nucleotides, therefore changing its genome. This process is performed by the use of immensely useful tool, t. i. engineered nucleases, which are capable of cutting both strands of DNA or nicking single strand. Resulting sites are detected and repaired by endogenous repair mechanisms, which include homologous recombination (HR) or non-homologous end joining (NHEJ). The latter is error prone, therefore it is likely for mutations to occur in repair site. These repair pathways are highly conserved in diverse cell types, which is beneficial for the use of genome editing technologies. As a result, various cell types have been engineered by the use of portable cleavage-based technologies, including human stem cells [1].

Recently, there has been tremendous development in the field of synthetic biology and genome editing technology. Virtually any gene can be manipulated in diverse range of organisms. Most common approach is the use of nuclease, which can be manipulated to target specific sequences of genome. This can be achieved by exploitation of sequence-specific DNA-binding domains (ZFNs, TALENs) or by the use of complementary guide RNA (CRISPR-Cas9 system).
In this paper, the history of genome editing will first be briefly reviewed, further on genome editing will be discussed with emphasis on the TALEN’s family.

== Brief history of genome editing ==

Past studies in the field of genome editing were perusing a common goal, t. i. to obtain high specificity, which would limit the target affects [2]. The first engineered technology, based on nuclease activity, was introduced in 1991 by Pavletich and Pabo in the journal Science [2]. This technology by the name of Zinc Finger nuclease was a great breakthrough and was later on frequently applied for genome targeting. It is based on the use of cleavage domain of restriction enzyme FokI, which is linked with designated zinc finger protein (ZFP), which is responsible for targeting. By the use of different combinations of ZFP domains the selectivity for single genomic cleavage event is achieved. With the use of nuclease domains with monomeric activity there is a high probability of off-target events. This can be avoided by the use of a distinct pair of ZFNs with FokI domains that are obligate heterodimers [3]. Overtime, reports of problems and drawbacks of this technology were reported, such as targeting problem for certain triplets and reduced specificity, suffered by interactions within a zinc finger array [2].
The next technology, which emerged in 2009, is called transcription activator-like effector nuclease (TALENs). These are fusion proteins, where nuclease domain is associated with TAL effector DNA binding domain. This approach may be better in certain aspects, for example it enables a larger spectrum of targets and is more practical than ZFNs. In 2011, TALENs was proclaimed as “Method of the Year” by the journal Nature methods. The details of this system and molecular mechanisms of function are described later on.
We probably all at least heard if not read about latest breakthrough in the field of genome editing, which is CRISPR-Cas9 system. The discovery was somewhat fortuity, because researchers were studying how bacteria defend against phages and foreign plasmids. The paper was published in August 2012 in journal Science [4]. A bacterial nuclease called Cas9 is capable of altering genomes of invading viruses, consequently inactivating them. Clustered regularly interspaced short palindromic repeats (CRISPR) are DNA loci that contain short sequence repetitions, which can be transcribed, processed and then used to guide nuclease to a specific site [5]. This is in contrast with TALEN and Zinc Finger methods, where the need to prepare customized nuclease for each target remains.

== A TALE nuclease architecture ==

As mentioned before, two studies from 2009 proposed the use of TALEs effectors for targeted genome engineering. These transcriptional activators originate from Xanthomonas plant pathogens. During pathogenesis, TALEs specifically bind and regulate plant gene expression, thus aiding bacterial infection [6]. A central repeat domain, which mediates DNA recognition, is located within TALE structure [1]. Each repeat unit comprises of 33 – 35 amino, these specify one target base. The units base preference is determined by two crucial adjacent amino acids, also called the “repeat variable di-residue” (RVD). Therefore, the sequence of specified units in central repeat domain determines the target sequence on genome. TALEs generated with new repeat combinations have been shown to recognize target sequences predicted by this code.
In the light of aforementioned advances the interest in potential use of this system in combination with nuclease was initiated. Subsequently, a TALE-nuclease chimeras (TALENs) was proposed, which would allow for site-specific genome cleavage. In selected paper, the advances in the field of TALEN’s technology are discussed. TALE activity in a mammalian cell environment for targeted regulation of episomal reporters and an endogenous gene is presented, resuming with the research of the minimal TALE region required for high-affinity DNA binding. Paper also includes practical application, showing modification of endogenous human genes NTF3 and CCR5 by the use of TALEN approach.

== Retargeting a natural TALE to an endogenous mammalian sequence ==

Firstly, a suitable TAL effector for this study was chosen. Criteria for TALE selection was based on specificity and empirical evidence. The group was searching for natural TALE with high specificity for target sequence and with confirmed activity in mammalian cells. To achive this goal, PCR amplifications for several TALEs using Xanthomonas axonopodis pathovar citri genomic DNA were performed. Native TALE-associated sequences which allow transport into plant cells were excluded. Protein products of acquired coding regions were characterized by a SELEX assay using target of interest. Outcome of this selection yielded one highly selective candidate, identified as TALE13. When combined with VP-16 activation domain, a 70-fold induction of reporter gene expression rate was observed in HEK293 cells. Based on these results, TALE13 was chosen for further work and design.
First goal was to demonstrate that chosen candidate was appropriate for mammalian gene regulation. Chosen target was NTF3 gene, which encodes a secreted nerve growth factor that has therapeutic potential for neurodegenerative diseases. Wild type TALE usually contain 18 repeats, by which they target specific sequence and bind to proximal promoter elements. TALE13 was modified to the extent of recognising proximal promoter region of NTF3 by replacing the 18 wild-type repeat units with appropriate alternatives. This altered TALE was named NT-L (meaning NTF3 Left), which highlights its binding position on target. In order to test its activity, the truncated version of NT-L was fused with VP-16 and expressed in human HEK293 cells. By measuring the activity of endogenous NTF3 locus, a strong induction (over 20 fold) in both NTF3 transcript and protein product was detected. The subsequent SELEX analysis revealed high specificity. These results revealed, that NT-L variant is a viable candidate for endogenous mammalian gene regulation.

== Development of a TALE-nuclease architecture ==

The next step was to design TALENs chimera protein using NT-L and nuclease, which would allow implementation of targeted cleavage of its genome target. The process was realized through three stages.
First, the minimal region required for high affinity bonding was determined using gene regulation studies. This was beneficial for optimization of nuclease (FokI) cleavage domain attachment with its target. Desired outcome of this step was improved catalytic activity of chimeric tool. The truncated variant named NT-L+95 was shown to retain high specificity and at the same time contribute to improved nuclease attachment. In order to examine this, a C-terminal truncated series of TALE13 were generated and subsequently linked with FokI. The resulting candidates were screened as homodimers for cleavage of specific targets. The results have shown several interesting occurrences. For TALENs comprising of NT-L+95 no detectable nuclease activity was observed. The cause for this is unclear; it is possible that this is due to a certain effect of NT-L+95 to nuclease domain positioning. Efficient DNA cleavage was observed for certain shorter specimens, providing candidates for cellular studies.
The minimal region between TALEN binding sites for optimal activity is over 12 bp, otherwise reduce cleavage may occur. These observations are useful for design of TALENs used for in vivo studies.
In the last stage, homodimer TALEN partner for NT-L was designed and named NT-R (NTR3 Right), indicating the direction of binding. This pair was expressed in human
K562 cells as fusions with catalytic domain of FokI. Target locus was later on tested for gene modification. By using two different C-terminal subregions (+28 and +63) the designed TALENs were tested. Significant activity in in vitro cleavage study was detected. With expression at 37°C, up to 3% modification was observed by the Surveyor assay [7]. This assay reveals gene modification by the appearance of digestion products. The percentage of gene modification can be obtained by quantification of “cut” bands. Limit of detection of this assay is about 1%. The activity was higher (9% of modification) in conditions of transient hypothermia, t. i. at 30°C. Sequences from latter were analyzed by Sanger sequencing. Out of 84 analyzed alleles there were 7 mutated by the occurrence of short deletions. This observation is consistent with expectation of non-homologous end joining (NHEJ) activity. Results have also shown a NHEJ-mediated capture of an oligonucleotide duplex at the endogenous NTF3 locus. These results confirmed that NHEJ-mediated genome modification by the use of TALEN architecture is possible in mammalian environment.

== Efficient modification by NHEJ and homology directed repair (HDR) ==

To expand the practical application for this developed system, modification at an additional locus was proposed. CCR5 gene was targeted for modification. First, a set of TALENs with a range of target lengths from 13 to 17 bp and of separation distance from 5 to 27 bp were designed and tested to identify values compatible with endogenous activity. To this end, a cluster of four left and four right binding sites were chosen for targeting. This defined a matrix of 16 heterodimer targets. For each binding site, two alternative proteins were generated, with 28 or 63 residues on C-terminal (as before). Altogether, this yielded 8x8=64 combinations of left and right proteins. These were expressed by pairs in K562 cells under standard conditions. Once again, Surveyor assay was used for evaluation of allele modification. Obtained results indicate that introduction of some +63/+63 pairs yielded over 20% allele modification. TALEN treated cells seem to retain a stable modification lever after 10 day growth, suggesting that TALENs are generally well tolerated. The effect of spacer length on activity is observed once again. At least 5% modification is achieved by the use of 12 – 19 bp spacer length in the +63/+63 TALENs section. In contrast, +28/+28 section show maximal activity in narrow spacer range of 12 – 13 bp.
The next challenge was to show that it is possible to induce gene through HDR using this system. For this, a part of CCR5 region was selected for targeting, also mentioned as safe harbor previously used for transgene integration. 24 TALEN pairs were then screened for their ability to induce NHEJ. Two highly active combinations were identified, first with 18% mutation rate and second with 21%. These TALEN were introduced into K562 cells simultaneously with donor DNA fragment carrying 46 bp with restriction site BglI. The success of integration was proven by PCR and BglI digestion. About 16% of inspected alleles were modified. This is an example how TALEN architecture can be used to activate HDR.

== Conclusion ==

In presented study it is shown how TALEN architecture can be designed and generated for efficient genome editing in mammalian environment. In this case, the system consists of TALE truncation variants, linked to catalytic domain of FokI. Engineered TALENs can be used for gene modification of human cells by activating either NHEJ or HDR repair pathways. Furthermore, three distinct applications are demonstrated; NHEJ mediated gene disruption, NHEJ-mediated insertion by capture of a DNA fragment and HDR mediated gene editing. Aforementioned suggests that TALEN architecture is a compelling tool in synthetic biology.

== Discussion ==

A step to facilitate the use of this architecture would undoubtedly be the linkage of minimal central TALE repeat domain to the catalytic domain of FokI. This was previously attempted by several groups without success, whereas the lack of activity of such constructs [8]. An alternative idea is to use a full length TALE for targeting. Disadvantage of the latter is the size of linkage peptide connecting two domains, which would affect the nuclease activity. Third approach is to generate truncated variants of TALEs and link them with catalytic domain of FokI, as shown in this paper. This approach has proven affective when using truncated variants with 28 or 63 of the 278 original C-terminal residues.
During the experimental work of this study, two papers were submitted where they demonstrated the use of TALEN architecture with the use of unmodified TALE or TALE with slightly reduced C-terminus [8], [9]. These systems were used for episome cleavage in yeast, an approach that is hardly translated to more complex genomes. As expected, these proteins have extended range of cleavage compared to truncated variants.
Presented TALEN architecture is in some characteristics similar to ZFNs. One example is the high cleavage activity of TALENs, which enables high gene modification efficiency, which is in some cases superior to the one of ZFNs. A shortcoming of TALEN architecture compared to ZFNs is its relative size. TALE repeats are 3 – 4 times larger than ZFPs, which may cause difficulty in delivery methods. Also, limitations due to high levels of TALE repeat homology may complicate the assembly of constructs. Future experiments will provide a fuller picture of pros and cons of the two platforms.
Targeted endogenous gene regulation by TALE proteins is a useful tool in research and for biotechnology applications. We can use this technology in a broad sense, such as design and generation of transcriptional activators or repressors. Although TALEs were discovered as such, previous studies did not inspect this type of activity outside of their native plant context. The experiments shown in this paper suggest that TALEs can be used as transcriptional regulators.
The use of SELEX assay for identification and characterization of TALEs has previously not been reported. By using this method, it was possible to assess the DNA binding preferences of natural and designed TALE proteins. Binding sites for TALE13 and four engineered TALEs were examined. On this basis, an observation that 73 out of 76 TALE repeats facilitate base preference of RVD. An interpretation of rare exceptions is proposed, facilitating further design and use of this system. Overall data collected can be used to examine previously acquired information on RVDs and thereby gaining more insight on mechanisms of action and selectivity. Findings that common RVDs such as NI, HD, NN and NG selectively bound selectively bound adenine, cytosine, guanine and thymine, respectively, are in accordance with previous knowledge. Pattern NN has shown relaxed specificity with substantial binding to a second base, adenine. NK is therefore more suitable for specificity, due to much stronger preference for guanine. This is a potential enhancement, proposed in this paper.

== Expectations for the future ==

As already mentioned, targeting the designated loci with ZFNs or TALENs is often difficult and time consuming, due to design and generation of new custom ZF or TALE array for each locus. This is not the case in most recent approach in genome editing, that is the use of CRISPR-Cas9 system. Expression of distinct gRNAs alongside Cas9 results in Cas9–gRNA complexes with distinct cleavage specificities [10]. This approach is exploited in multiplex genome editing. Still, there are features of this system which are inferior to elderly time-demanding ZFNs or TALENs.
One of those features is size of gene constructs, which is a limitation in field of gene delivery. Cas9 is the size of about 160 kDa, whereas a typical 17,5 repeat TALEN corresponds to 105 kDa. By far the smallest are ZFNs, wherein typical four finger ZFN has about 40 kDa [10]. It is likely for this compact nature to be beneficial for certain experiments.
As for specificity, the number of bases for modification in Cas9 system is restricted to 20 and is somewhat limited by the rule of three guanine bases flanking the target site (GN19NGG) [10]. The main constriction of ZF arrays is a scarce set of fingers, resulting in inability to target certain triplets. In addition, some ZFs targeting GNN seem to be superior to other ZFs, contributing to limitations. TALE target specificity therefore seems superior, with capabilities of targeting up to 24 bases. For Cas9 system, off target studies in mammalian environment are yet to be conducted [10].
It is likely that distinct features will lead to differential use of genome editing systems for distinct applications in the fields of plant science, therapeutic sciences and biotechnology applications.

== Literature ==

[1] J. C. Miller, S. Tan, G. Qiao, K. A. Barlow, J. Wang, D. F. Xia, X. Meng, D. E. Paschon, E. Leung, S. J. Hinkley, G. P. Dulay, K. L. Hua, I. Ankoudinova, G. J. Cost, F. D. Urnov, H. S. Zhang, M. C. Holmes, L. Zhang, P. D. Gregory, and E. J. Rebar, “A TALE nuclease architecture for efficient genome editing,” Nat. Biotechnol., vol. 29, no. 2, pp. 143–148, Feb. 2011.

[2] “Genome Editing.” [Online]. Available: http://www.allelebiotech.com/genome-editing/. [Accessed: 06-Jan-2015].

[3] R. M. Gupta and K. Musunuru, “Expanding the genetic editing tool kit: ZFNs, TALENs, and CRISPR-Cas9,” J. Clin. Invest., vol. 124, no. 10, pp. 4154–4161, Oct. 2014.

[4] M. Jinek, K. Chylinski, I. Fonfara, M. Hauer, J. A. Doudna, and E. Charpentier, “A programmable dual-RNA-guided DNA endonuclease in adaptive bacterial immunity,” Science, vol. 337, no. 6096, pp. 816–821, Aug. 2012.

[5] “CRISPR,” Wikipedia, the free encyclopedia. 09-Jan-2015.

[6] “TAL effector,” Wikipedia, the free encyclopedia. 06-Jan-2015.

[7] J. C. Miller, M. C. Holmes, J. Wang, D. Y. Guschin, Y.-L. Lee, I. Rupniewski, C. M. Beausejour, A. J. Waite, N. S. Wang, K. A. Kim, P. D. Gregory, C. O. Pabo, and E. J. Rebar, “An improved zinc-finger nuclease architecture for highly specific genome editing,” Nat. Biotechnol., vol. 25, no. 7, pp. 778–785, Jul. 2007.

[8] M. Christian, T. Cermak, E. L. Doyle, C. Schmidt, F. Zhang, A. Hummel, A. J. Bogdanove, and D. F. Voytas, “Targeting DNA double-strand breaks with TAL effector nucleases,” Genetics, vol. 186, no. 2, pp. 757–761, Oct. 2010.

[9] T. Li, S. Huang, W. Z. Jiang, D. Wright, M. H. Spalding, D. P. Weeks, and B. Yang, “TAL nucleases (TALNs): hybrid proteins composed of TAL effectors and FokI DNA-cleavage domain,” Nucleic Acids Res., vol. 39, no. 1, pp. 359–372, Jan. 2011.

[10] A. Strauβ and T. Lahaye, “Zinc Fingers, TAL Effectors, or Cas9-Based DNA Binding Proteins: What’s Best for Targeting Desired Genome Loci?,” Mol. Plant, vol. 6, no. 5, pp. 1384–1387, Sep. 2013.

A TALE nuclease architecture for efficient genome editing

2015-01-11T15:44:28Z

JernejMustar: /* Expectations for the future */

(Jernej Mustar)

Summarized from Miller, J. C. ''et al''. A TALE nuclease architecture for efficient genome editing. ''Nat. Biotechnol.'', vol. 29, pp. 143–148. Feb. 2011.

== Introduction to genome editing ==

Genome editing is a type of genetic engineering, where an organisms DNA is manipulated by altering; inserting, removing or replacing nucleotides, therefore changing its genome. This process is performed by the use of immensely useful tool, t. i. engineered nucleases, which are capable of cutting both strands of DNA or nicking single strand. Resulting sites are detected and repaired by endogenous repair mechanisms, which include homologous recombination (HR) or non-homologous end joining (NHEJ). The latter is error prone, therefore it is likely for mutations to occur in repair site. These repair pathways are highly conserved in diverse cell types, which is beneficial for the use of genome editing technologies. As a result, various cell types have been engineered by the use of portable cleavage-based technologies, including human stem cells [1].

Recently, there has been tremendous development in the field of synthetic biology and genome editing technology. Virtually any gene can be manipulated in diverse range of organisms. Most common approach is the use of nuclease, which can be manipulated to target specific sequences of genome. This can be achieved by exploitation of sequence-specific DNA-binding domains (ZFNs, TALENs) or by the use of complementary guide RNA (CRISPR-Cas9 system).
In this paper, the history of genome editing will first be briefly reviewed, further on genome editing will be discussed with emphasis on the TALEN’s family.

== Brief history of genome editing ==

Past studies in the field of genome editing were perusing a common goal, t. i. to obtain high specificity, which would limit the target affects [2]. The first engineered technology, based on nuclease activity, was introduced in 1991 by Pavletich and Pabo in the journal Science [2]. This technology by the name of Zinc Finger nuclease was a great breakthrough and was later on frequently applied for genome targeting. It is based on the use of cleavage domain of restriction enzyme FokI, which is linked with designated zinc finger protein (ZFP), which is responsible for targeting. By the use of different combinations of ZFP domains the selectivity for single genomic cleavage event is achieved. With the use of nuclease domains with monomeric activity there is a high probability of off-target events. This can be avoided by the use of a distinct pair of ZFNs with FokI domains that are obligate heterodimers [3]. Overtime, reports of problems and drawbacks of this technology were reported, such as targeting problem for certain triplets and reduced specificity, suffered by interactions within a zinc finger array [2].
The next technology, which emerged in 2009, is called transcription activator-like effector nuclease (TALENs). These are fusion proteins, where nuclease domain is associated with TAL effector DNA binding domain. This approach may be better in certain aspects, for example it enables a larger spectrum of targets and is more practical than ZFNs. In 2011, TALENs was proclaimed as “Method of the Year” by the journal Nature methods. The details of this system and molecular mechanisms of function are described later on.
We probably all at least heard if not read about latest breakthrough in the field of genome editing, which is CRISPR-Cas9 system. The discovery was somewhat fortuity, because researchers were studying how bacteria defend against phages and foreign plasmids. The paper was published in August 2012 in journal Science [4]. A bacterial nuclease called Cas9 is capable of altering genomes of invading viruses, consequently inactivating them. Clustered regularly interspaced short palindromic repeats (CRISPR) are DNA loci that contain short sequence repetitions, which can be transcribed, processed and then used to guide nuclease to a specific site [5]. This is in contrast with TALEN and Zinc Finger methods, where the need to prepare customized nuclease for each target remains.

== A TALE nuclease architecture ==

As mentioned before, two studies from 2009 proposed the use of TALEs effectors for targeted genome engineering. These transcriptional activators originate from Xanthomonas plant pathogens. During pathogenesis, TALEs specifically bind and regulate plant gene expression, thus aiding bacterial infection [6]. A central repeat domain, which mediates DNA recognition, is located within TALE structure [1]. Each repeat unit comprises of 33 – 35 amino, these specify one target base. The units base preference is determined by two crucial adjacent amino acids, also called the “repeat variable di-residue” (RVD). Therefore, the sequence of specified units in central repeat domain determines the target sequence on genome. TALEs generated with new repeat combinations have been shown to recognize target sequences predicted by this code.
In the light of aforementioned advances the interest in potential use of this system in combination with nuclease was initiated. Subsequently, a TALE-nuclease chimeras (TALENs) was proposed, which would allow for site-specific genome cleavage. In selected paper, the advances in the field of TALEN’s technology are discussed. TALE activity in a mammalian cell environment for targeted regulation of episomal reporters and an endogenous gene is presented, resuming with the research of the minimal TALE region required for high-affinity DNA binding. Paper also includes practical application, showing modification of endogenous human genes NTF3 and CCR5 by the use of TALEN approach.

== Retargeting a natural TALE to an endogenous mammalian sequence ==

Firstly, a suitable TAL effector for this study was chosen. Criteria for TALE selection was based on specificity and empirical evidence. The group was searching for natural TALE with high specificity for target sequence and with confirmed activity in mammalian cells. To achive this goal, PCR amplifications for several TALEs using Xanthomonas axonopodis pathovar citri genomic DNA were performed. Native TALE-associated sequences which allow transport into plant cells were excluded. Protein products of acquired coding regions were characterized by a SELEX assay using target of interest. Outcome of this selection yielded one highly selective candidate, identified as TALE13. When combined with VP-16 activation domain, a 70-fold induction of reporter gene expression rate was observed in HEK293 cells. Based on these results, TALE13 was chosen for further work and design.
First goal was to demonstrate that chosen candidate was appropriate for mammalian gene regulation. Chosen target was NTF3 gene, which encodes a secreted nerve growth factor that has therapeutic potential for neurodegenerative diseases. Wild type TALE usually contain 18 repeats, by which they target specific sequence and bind to proximal promoter elements. TALE13 was modified to the extent of recognising proximal promoter region of NTF3 by replacing the 18 wild-type repeat units with appropriate alternatives. This altered TALE was named NT-L (meaning NTF3 Left), which highlights its binding position on target. In order to test its activity, the truncated version of NT-L was fused with VP-16 and expressed in human HEK293 cells. By measuring the activity of endogenous NTF3 locus, a strong induction (over 20 fold) in both NTF3 transcript and protein product was detected. The subsequent SELEX analysis revealed high specificity. These results revealed, that NT-L variant is a viable candidate for endogenous mammalian gene regulation.

== Development of a TALE-nuclease architecture ==

The next step was to design TALENs chimera protein using NT-L and nuclease, which would allow implementation of targeted cleavage of its genome target. The process was realized through three stages.
First, the minimal region required for high affinity bonding was determined using gene regulation studies. This was beneficial for optimization of nuclease (FokI) cleavage domain attachment with its target. Desired outcome of this step was improved catalytic activity of chimeric tool. The truncated variant named NT-L+95 was shown to retain high specificity and at the same time contribute to improved nuclease attachment. In order to examine this, a C-terminal truncated series of TALE13 were generated and subsequently linked with FokI. The resulting candidates were screened as homodimers for cleavage of specific targets. The results have shown several interesting occurrences. For TALENs comprising of NT-L+95 no detectable nuclease activity was observed. The cause for this is unclear; it is possible that this is due to a certain effect of NT-L+95 to nuclease domain positioning. Efficient DNA cleavage was observed for certain shorter specimens, providing candidates for cellular studies.
The minimal region between TALEN binding sites for optimal activity is over 12 bp, otherwise reduce cleavage may occur. These observations are useful for design of TALENs used for in vivo studies.
In the last stage, homodimer TALEN partner for NT-L was designed and named NT-R (NTR3 Right), indicating the direction of binding. This pair was expressed in human
K562 cells as fusions with catalytic domain of FokI. Target locus was later on tested for gene modification. By using two different C-terminal subregions (+28 and +63) the designed TALENs were tested. Significant activity in in vitro cleavage study was detected. With expression at 37°C, up to 3% modification was observed by the Surveyor assay [7]. This assay reveals gene modification by the appearance of digestion products. The percentage of gene modification can be obtained by quantification of “cut” bands. Limit of detection of this assay is about 1%. The activity was higher (9% of modification) in conditions of transient hypothermia, t. i. at 30°C. Sequences from latter were analyzed by Sanger sequencing. Out of 84 analyzed alleles there were 7 mutated by the occurrence of short deletions. This observation is consistent with expectation of non-homologous end joining (NHEJ) activity. Results have also shown a NHEJ-mediated capture of an oligonucleotide duplex at the endogenous NTF3 locus. These results confirmed that NHEJ-mediated genome modification by the use of TALEN architecture is possible in mammalian environment.

== Efficient modification by NHEJ and homology directed repair (HDR) ==

To expand the practical application for this developed system, modification at an additional locus was proposed. CCR5 gene was targeted for modification. First, a set of TALENs with a range of target lengths from 13 to 17 bp and of separation distance from 5 to 27 bp were designed and tested to identify values compatible with endogenous activity. To this end, a cluster of four left and four right binding sites were chosen for targeting. This defined a matrix of 16 heterodimer targets. For each binding site, two alternative proteins were generated, with 28 or 63 residues on C-terminal (as before). Altogether, this yielded 8x8=64 combinations of left and right proteins. These were expressed by pairs in K562 cells under standard conditions. Once again, Surveyor assay was used for evaluation of allele modification. Obtained results indicate that introduction of some +63/+63 pairs yielded over 20% allele modification. TALEN treated cells seem to retain a stable modification lever after 10 day growth, suggesting that TALENs are generally well tolerated. The effect of spacer length on activity is observed once again. At least 5% modification is achieved by the use of 12 – 19 bp spacer length in the +63/+63 TALENs section. In contrast, +28/+28 section show maximal activity in narrow spacer range of 12 – 13 bp.
The next challenge was to show that it is possible to induce gene through HDR using this system. For this, a part of CCR5 region was selected for targeting, also mentioned as safe harbor previously used for transgene integration. 24 TALEN pairs were then screened for their ability to induce NHEJ. Two highly active combinations were identified, first with 18% mutation rate and second with 21%. These TALEN were introduced into K562 cells simultaneously with donor DNA fragment carrying 46 bp with restriction site BglI. The success of integration was proven by PCR and BglI digestion. About 16% of inspected alleles were modified. This is an example how TALEN architecture can be used to activate HDR.

== Conclusion ==

In presented study it is shown how TALEN architecture can be designed and generated for efficient genome editing in mammalian environment. In this case, the system consists of TALE truncation variants, linked to catalytic domain of FokI. Engineered TALENs can be used for gene modification of human cells by activating either NHEJ or HDR repair pathways. Furthermore, three distinct applications are demonstrated; NHEJ mediated gene disruption, NHEJ-mediated insertion by capture of a DNA fragment and HDR mediated gene editing. Aforementioned suggests that TALEN architecture is a compelling tool in synthetic biology.

== Discussion ==

A step to facilitate the use of this architecture would undoubtedly be the linkage of minimal central TALE repeat domain to the catalytic domain of FokI. This was previously attempted by several groups without success, whereas the lack of activity of such constructs [8]. An alternative idea is to use a full length TALE for targeting. Disadvantage of the latter is the size of linkage peptide connecting two domains, which would affect the nuclease activity. Third approach is to generate truncated variants of TALEs and link them with catalytic domain of FokI, as shown in this paper. This approach has proven affective when using truncated variants with 28 or 63 of the 278 original C-terminal residues.
During the experimental work of this study, two papers were submitted where they demonstrated the use of TALEN architecture with the use of unmodified TALE or TALE with slightly reduced C-terminus [8], [9]. These systems were used for episome cleavage in yeast, an approach that is hardly translated to more complex genomes. As expected, these proteins have extended range of cleavage compared to truncated variants.
Presented TALEN architecture is in some characteristics similar to ZFNs. One example is the high cleavage activity of TALENs, which enables high gene modification efficiency, which is in some cases superior to the one of ZFNs. A shortcoming of TALEN architecture compared to ZFNs is its relative size. TALE repeats are 3 – 4 times larger than ZFPs, which may cause difficulty in delivery methods. Also, limitations due to high levels of TALE repeat homology may complicate the assembly of constructs. Future experiments will provide a fuller picture of pros and cons of the two platforms.
Targeted endogenous gene regulation by TALE proteins is a useful tool in research and for biotechnology applications. We can use this technology in a broad sense, such as design and generation of transcriptional activators or repressors. Although TALEs were discovered as such, previous studies did not inspect this type of activity outside of their native plant context. The experiments shown in this paper suggest that TALEs can be used as transcriptional regulators.
The use of SELEX assay for identification and characterization of TALEs has previously not been reported. By using this method, it was possible to assess the DNA binding preferences of natural and designed TALE proteins. Binding sites for TALE13 and four engineered TALEs were examined. On this basis, an observation that 73 out of 76 TALE repeats facilitate base preference of RVD. An interpretation of rare exceptions is proposed, facilitating further design and use of this system. Overall data collected can be used to examine previously acquired information on RVDs and thereby gaining more insight on mechanisms of action and selectivity. Findings that common RVDs such as NI, HD, NN and NG selectively bound selectively bound adenine, cytosine, guanine and thymine, respectively, are in accordance with previous knowledge. Pattern NN has shown relaxed specificity with substantial binding to a second base, adenine. NK is therefore more suitable for specificity, due to much stronger preference for guanine. This is a potential enhancement, proposed in this paper.

== Expectations for the future ==

As already mentioned, targeting the designated loci with ZFNs or TALENs is often difficult and time consuming, due to design and generation of new custom ZF or TALE array for each locus. This is not the case in most recent approach in genome editing, that is the use of CRISPR-Cas9 system. Expression of distinct gRNAs alongside Cas9 results in Cas9–gRNA complexes with distinct cleavage specificities [10]. This approach is exploited in multiplex genome editing. Still, there are features of this system which are inferior to elderly time-demanding ZFNs or TALENs.
One of those features is size of gene constructs, which is a limitation in field of gene delivery. Cas9 is the size of about 160 kDa, whereas a typical 17,5 repeat TALEN corresponds to 105 kDa. By far the smallest are ZFNs, wherein typical four finger ZFN has about 40 kDa [10]. It is likely for this compact nature to be beneficial for certain experiments.
As for specificity, the number of bases for modification in Cas9 system is restricted to 20 and is somewhat limited by the rule of three guanine bases flanking the target site (GN19NGG) [10]. The main constriction of ZF arrays is a scarce set of fingers, resulting in inability to target certain triplets. In addition, some ZFs targeting GNN seem to be superior to other ZFs, contributing to limitations. TALE target specificity therefore seems superior, with capabilities of targeting up to 24 bases. For Cas9 system, off target studies in mammalian environment are yet to be conducted [10].
It is likely that distinct features will lead to differential use of genome editing systems for distinct applications in the fields of plant science, therapeutic sciences and biotechnology applications.

== Literature ==

[1] J. C. Miller, S. Tan, G. Qiao, K. A. Barlow, J. Wang, D. F. Xia, X. Meng, D. E. Paschon, E. Leung, S. J. Hinkley, G. P. Dulay, K. L. Hua, I. Ankoudinova, G. J. Cost, F. D. Urnov, H. S. Zhang, M. C. Holmes, L. Zhang, P. D. Gregory, and E. J. Rebar, “A TALE nuclease architecture for efficient genome editing,” Nat. Biotechnol., vol. 29, no. 2, pp. 143–148, Feb. 2011.

[2] “Genome Editing.” [Online]. Available: http://www.allelebiotech.com/genome-editing/. [Accessed: 06-Jan-2015].

[3] R. M. Gupta and K. Musunuru, “Expanding the genetic editing tool kit: ZFNs, TALENs, and CRISPR-Cas9,” J. Clin. Invest., vol. 124, no. 10, pp. 4154–4161, Oct. 2014.

[4] M. Jinek, K. Chylinski, I. Fonfara, M. Hauer, J. A. Doudna, and E. Charpentier, “A programmable dual-RNA-guided DNA endonuclease in adaptive bacterial immunity,” Science, vol. 337, no. 6096, pp. 816–821, Aug. 2012.

[5] “CRISPR,” Wikipedia, the free encyclopedia. 09-Jan-2015.

[6] “TAL effector,” Wikipedia, the free encyclopedia. 06-Jan-2015.

[7] J. C. Miller, M. C. Holmes, J. Wang, D. Y. Guschin, Y.-L. Lee, I. Rupniewski, C. M. Beausejour, A. J. Waite, N. S. Wang, K. A. Kim, P. D. Gregory, C. O. Pabo, and E. J. Rebar, “An improved zinc-finger nuclease architecture for highly specific genome editing,” Nat. Biotechnol., vol. 25, no. 7, pp. 778–785, Jul. 2007.

[8] M. Christian, T. Cermak, E. L. Doyle, C. Schmidt, F. Zhang, A. Hummel, A. J. Bogdanove, and D. F. Voytas, “Targeting DNA double-strand breaks with TAL effector nucleases,” Genetics, vol. 186, no. 2, pp. 757–761, Oct. 2010.

[9] T. Li, S. Huang, W. Z. Jiang, D. Wright, M. H. Spalding, D. P. Weeks, and B. Yang, “TAL nucleases (TALNs): hybrid proteins composed of TAL effectors and FokI DNA-cleavage domain,” Nucleic Acids Res., vol. 39, no. 1, pp. 359–372, Jan. 2011.

[10] A. Strauβ and T. Lahaye, “Zinc Fingers, TAL Effectors, or Cas9-Based DNA Binding Proteins: What’s Best for Targeting Desired Genome Loci?,” Mol. Plant, vol. 6, no. 5, pp. 1384–1387, Sep. 2013.

A TALE nuclease architecture for efficient genome editing

2015-01-11T15:42:29Z

JernejMustar: /* Retargeting a natural TALE to an endogenous mammalian sequence */

(Jernej Mustar)

Summarized from Miller, J. C. ''et al''. A TALE nuclease architecture for efficient genome editing. ''Nat. Biotechnol.'', vol. 29, pp. 143–148. Feb. 2011.

== Introduction to genome editing ==

Genome editing is a type of genetic engineering, where an organisms DNA is manipulated by altering; inserting, removing or replacing nucleotides, therefore changing its genome. This process is performed by the use of immensely useful tool, t. i. engineered nucleases, which are capable of cutting both strands of DNA or nicking single strand. Resulting sites are detected and repaired by endogenous repair mechanisms, which include homologous recombination (HR) or non-homologous end joining (NHEJ). The latter is error prone, therefore it is likely for mutations to occur in repair site. These repair pathways are highly conserved in diverse cell types, which is beneficial for the use of genome editing technologies. As a result, various cell types have been engineered by the use of portable cleavage-based technologies, including human stem cells [1].

Recently, there has been tremendous development in the field of synthetic biology and genome editing technology. Virtually any gene can be manipulated in diverse range of organisms. Most common approach is the use of nuclease, which can be manipulated to target specific sequences of genome. This can be achieved by exploitation of sequence-specific DNA-binding domains (ZFNs, TALENs) or by the use of complementary guide RNA (CRISPR-Cas9 system).
In this paper, the history of genome editing will first be briefly reviewed, further on genome editing will be discussed with emphasis on the TALEN’s family.

== Brief history of genome editing ==

Past studies in the field of genome editing were perusing a common goal, t. i. to obtain high specificity, which would limit the target affects [2]. The first engineered technology, based on nuclease activity, was introduced in 1991 by Pavletich and Pabo in the journal Science [2]. This technology by the name of Zinc Finger nuclease was a great breakthrough and was later on frequently applied for genome targeting. It is based on the use of cleavage domain of restriction enzyme FokI, which is linked with designated zinc finger protein (ZFP), which is responsible for targeting. By the use of different combinations of ZFP domains the selectivity for single genomic cleavage event is achieved. With the use of nuclease domains with monomeric activity there is a high probability of off-target events. This can be avoided by the use of a distinct pair of ZFNs with FokI domains that are obligate heterodimers [3]. Overtime, reports of problems and drawbacks of this technology were reported, such as targeting problem for certain triplets and reduced specificity, suffered by interactions within a zinc finger array [2].
The next technology, which emerged in 2009, is called transcription activator-like effector nuclease (TALENs). These are fusion proteins, where nuclease domain is associated with TAL effector DNA binding domain. This approach may be better in certain aspects, for example it enables a larger spectrum of targets and is more practical than ZFNs. In 2011, TALENs was proclaimed as “Method of the Year” by the journal Nature methods. The details of this system and molecular mechanisms of function are described later on.
We probably all at least heard if not read about latest breakthrough in the field of genome editing, which is CRISPR-Cas9 system. The discovery was somewhat fortuity, because researchers were studying how bacteria defend against phages and foreign plasmids. The paper was published in August 2012 in journal Science [4]. A bacterial nuclease called Cas9 is capable of altering genomes of invading viruses, consequently inactivating them. Clustered regularly interspaced short palindromic repeats (CRISPR) are DNA loci that contain short sequence repetitions, which can be transcribed, processed and then used to guide nuclease to a specific site [5]. This is in contrast with TALEN and Zinc Finger methods, where the need to prepare customized nuclease for each target remains.

== A TALE nuclease architecture ==

As mentioned before, two studies from 2009 proposed the use of TALEs effectors for targeted genome engineering. These transcriptional activators originate from Xanthomonas plant pathogens. During pathogenesis, TALEs specifically bind and regulate plant gene expression, thus aiding bacterial infection [6]. A central repeat domain, which mediates DNA recognition, is located within TALE structure [1]. Each repeat unit comprises of 33 – 35 amino, these specify one target base. The units base preference is determined by two crucial adjacent amino acids, also called the “repeat variable di-residue” (RVD). Therefore, the sequence of specified units in central repeat domain determines the target sequence on genome. TALEs generated with new repeat combinations have been shown to recognize target sequences predicted by this code.
In the light of aforementioned advances the interest in potential use of this system in combination with nuclease was initiated. Subsequently, a TALE-nuclease chimeras (TALENs) was proposed, which would allow for site-specific genome cleavage. In selected paper, the advances in the field of TALEN’s technology are discussed. TALE activity in a mammalian cell environment for targeted regulation of episomal reporters and an endogenous gene is presented, resuming with the research of the minimal TALE region required for high-affinity DNA binding. Paper also includes practical application, showing modification of endogenous human genes NTF3 and CCR5 by the use of TALEN approach.

== Retargeting a natural TALE to an endogenous mammalian sequence ==

Firstly, a suitable TAL effector for this study was chosen. Criteria for TALE selection was based on specificity and empirical evidence. The group was searching for natural TALE with high specificity for target sequence and with confirmed activity in mammalian cells. To achive this goal, PCR amplifications for several TALEs using Xanthomonas axonopodis pathovar citri genomic DNA were performed. Native TALE-associated sequences which allow transport into plant cells were excluded. Protein products of acquired coding regions were characterized by a SELEX assay using target of interest. Outcome of this selection yielded one highly selective candidate, identified as TALE13. When combined with VP-16 activation domain, a 70-fold induction of reporter gene expression rate was observed in HEK293 cells. Based on these results, TALE13 was chosen for further work and design.
First goal was to demonstrate that chosen candidate was appropriate for mammalian gene regulation. Chosen target was NTF3 gene, which encodes a secreted nerve growth factor that has therapeutic potential for neurodegenerative diseases. Wild type TALE usually contain 18 repeats, by which they target specific sequence and bind to proximal promoter elements. TALE13 was modified to the extent of recognising proximal promoter region of NTF3 by replacing the 18 wild-type repeat units with appropriate alternatives. This altered TALE was named NT-L (meaning NTF3 Left), which highlights its binding position on target. In order to test its activity, the truncated version of NT-L was fused with VP-16 and expressed in human HEK293 cells. By measuring the activity of endogenous NTF3 locus, a strong induction (over 20 fold) in both NTF3 transcript and protein product was detected. The subsequent SELEX analysis revealed high specificity. These results revealed, that NT-L variant is a viable candidate for endogenous mammalian gene regulation.

== Development of a TALE-nuclease architecture ==

The next step was to design TALENs chimera protein using NT-L and nuclease, which would allow implementation of targeted cleavage of its genome target. The process was realized through three stages.
First, the minimal region required for high affinity bonding was determined using gene regulation studies. This was beneficial for optimization of nuclease (FokI) cleavage domain attachment with its target. Desired outcome of this step was improved catalytic activity of chimeric tool. The truncated variant named NT-L+95 was shown to retain high specificity and at the same time contribute to improved nuclease attachment. In order to examine this, a C-terminal truncated series of TALE13 were generated and subsequently linked with FokI. The resulting candidates were screened as homodimers for cleavage of specific targets. The results have shown several interesting occurrences. For TALENs comprising of NT-L+95 no detectable nuclease activity was observed. The cause for this is unclear; it is possible that this is due to a certain effect of NT-L+95 to nuclease domain positioning. Efficient DNA cleavage was observed for certain shorter specimens, providing candidates for cellular studies.
The minimal region between TALEN binding sites for optimal activity is over 12 bp, otherwise reduce cleavage may occur. These observations are useful for design of TALENs used for in vivo studies.
In the last stage, homodimer TALEN partner for NT-L was designed and named NT-R (NTR3 Right), indicating the direction of binding. This pair was expressed in human
K562 cells as fusions with catalytic domain of FokI. Target locus was later on tested for gene modification. By using two different C-terminal subregions (+28 and +63) the designed TALENs were tested. Significant activity in in vitro cleavage study was detected. With expression at 37°C, up to 3% modification was observed by the Surveyor assay [7]. This assay reveals gene modification by the appearance of digestion products. The percentage of gene modification can be obtained by quantification of “cut” bands. Limit of detection of this assay is about 1%. The activity was higher (9% of modification) in conditions of transient hypothermia, t. i. at 30°C. Sequences from latter were analyzed by Sanger sequencing. Out of 84 analyzed alleles there were 7 mutated by the occurrence of short deletions. This observation is consistent with expectation of non-homologous end joining (NHEJ) activity. Results have also shown a NHEJ-mediated capture of an oligonucleotide duplex at the endogenous NTF3 locus. These results confirmed that NHEJ-mediated genome modification by the use of TALEN architecture is possible in mammalian environment.

== Efficient modification by NHEJ and homology directed repair (HDR) ==

To expand the practical application for this developed system, modification at an additional locus was proposed. CCR5 gene was targeted for modification. First, a set of TALENs with a range of target lengths from 13 to 17 bp and of separation distance from 5 to 27 bp were designed and tested to identify values compatible with endogenous activity. To this end, a cluster of four left and four right binding sites were chosen for targeting. This defined a matrix of 16 heterodimer targets. For each binding site, two alternative proteins were generated, with 28 or 63 residues on C-terminal (as before). Altogether, this yielded 8x8=64 combinations of left and right proteins. These were expressed by pairs in K562 cells under standard conditions. Once again, Surveyor assay was used for evaluation of allele modification. Obtained results indicate that introduction of some +63/+63 pairs yielded over 20% allele modification. TALEN treated cells seem to retain a stable modification lever after 10 day growth, suggesting that TALENs are generally well tolerated. The effect of spacer length on activity is observed once again. At least 5% modification is achieved by the use of 12 – 19 bp spacer length in the +63/+63 TALENs section. In contrast, +28/+28 section show maximal activity in narrow spacer range of 12 – 13 bp.
The next challenge was to show that it is possible to induce gene through HDR using this system. For this, a part of CCR5 region was selected for targeting, also mentioned as safe harbor previously used for transgene integration. 24 TALEN pairs were then screened for their ability to induce NHEJ. Two highly active combinations were identified, first with 18% mutation rate and second with 21%. These TALEN were introduced into K562 cells simultaneously with donor DNA fragment carrying 46 bp with restriction site BglI. The success of integration was proven by PCR and BglI digestion. About 16% of inspected alleles were modified. This is an example how TALEN architecture can be used to activate HDR.

== Conclusion ==

In presented study it is shown how TALEN architecture can be designed and generated for efficient genome editing in mammalian environment. In this case, the system consists of TALE truncation variants, linked to catalytic domain of FokI. Engineered TALENs can be used for gene modification of human cells by activating either NHEJ or HDR repair pathways. Furthermore, three distinct applications are demonstrated; NHEJ mediated gene disruption, NHEJ-mediated insertion by capture of a DNA fragment and HDR mediated gene editing. Aforementioned suggests that TALEN architecture is a compelling tool in synthetic biology.

== Discussion ==

A step to facilitate the use of this architecture would undoubtedly be the linkage of minimal central TALE repeat domain to the catalytic domain of FokI. This was previously attempted by several groups without success, whereas the lack of activity of such constructs [8]. An alternative idea is to use a full length TALE for targeting. Disadvantage of the latter is the size of linkage peptide connecting two domains, which would affect the nuclease activity. Third approach is to generate truncated variants of TALEs and link them with catalytic domain of FokI, as shown in this paper. This approach has proven affective when using truncated variants with 28 or 63 of the 278 original C-terminal residues.
During the experimental work of this study, two papers were submitted where they demonstrated the use of TALEN architecture with the use of unmodified TALE or TALE with slightly reduced C-terminus [8], [9]. These systems were used for episome cleavage in yeast, an approach that is hardly translated to more complex genomes. As expected, these proteins have extended range of cleavage compared to truncated variants.
Presented TALEN architecture is in some characteristics similar to ZFNs. One example is the high cleavage activity of TALENs, which enables high gene modification efficiency, which is in some cases superior to the one of ZFNs. A shortcoming of TALEN architecture compared to ZFNs is its relative size. TALE repeats are 3 – 4 times larger than ZFPs, which may cause difficulty in delivery methods. Also, limitations due to high levels of TALE repeat homology may complicate the assembly of constructs. Future experiments will provide a fuller picture of pros and cons of the two platforms.
Targeted endogenous gene regulation by TALE proteins is a useful tool in research and for biotechnology applications. We can use this technology in a broad sense, such as design and generation of transcriptional activators or repressors. Although TALEs were discovered as such, previous studies did not inspect this type of activity outside of their native plant context. The experiments shown in this paper suggest that TALEs can be used as transcriptional regulators.
The use of SELEX assay for identification and characterization of TALEs has previously not been reported. By using this method, it was possible to assess the DNA binding preferences of natural and designed TALE proteins. Binding sites for TALE13 and four engineered TALEs were examined. On this basis, an observation that 73 out of 76 TALE repeats facilitate base preference of RVD. An interpretation of rare exceptions is proposed, facilitating further design and use of this system. Overall data collected can be used to examine previously acquired information on RVDs and thereby gaining more insight on mechanisms of action and selectivity. Findings that common RVDs such as NI, HD, NN and NG selectively bound selectively bound adenine, cytosine, guanine and thymine, respectively, are in accordance with previous knowledge. Pattern NN has shown relaxed specificity with substantial binding to a second base, adenine. NK is therefore more suitable for specificity, due to much stronger preference for guanine. This is a potential enhancement, proposed in this paper.

== Expectations for the future ==

As already mentioned, targeting the designated loci with ZFNs or TALENs is often difficult and time consuming, due to design and generation of new custom ZF or TALE array for each locus. This is not the case in most recent approach in genome editing, that is the use of CRISPR-Cas9 system. Expression of distinct gRNAs alongside Cas9 results in Cas9–gRNA complexes with distinct cleavage specificities [10]. This approach is exploited in multiplex genome editing. Still, there are features of this system which are inferior to elderly time-demanding ZFNs or TALENs.
One of those features is size of gene constructs, which is a limitation in field of gene delivery. Cas9 is the size of about 160 kDa, whereas a typical 17,5 repeat TALEN corresponds to 105 kDa. By far the smallest are ZFNs, wherein typical four finger ZFN has about 40 kDa [10]. It is likely for this compact nature to be beneficial for certain experiments.
As for specificity, the number of bases for modification in Cas9 system is restricted to 20 and is somewhat limited with the rule of three guanine bases flanking the target site (GN19NGG) [10]. The main constriction of ZF arrays is a scarce set of fingers, resulting in inability to target certain triplets. In addition, some ZFs targeting GNN seem to be superior to other ZFs, contributing to limitations. TALE target specificity therefore seems superior, with capabilities of targeting up to 24 bases. For Cas9 system, off target studies in mammalian environment are yet to be conducted [10].
It is likely that distinct features will lead to differential use of genome editing systems for distinct applications in the fields of plant science, therapeutic sciences and biotechnology applications.

== Literature ==

[1] J. C. Miller, S. Tan, G. Qiao, K. A. Barlow, J. Wang, D. F. Xia, X. Meng, D. E. Paschon, E. Leung, S. J. Hinkley, G. P. Dulay, K. L. Hua, I. Ankoudinova, G. J. Cost, F. D. Urnov, H. S. Zhang, M. C. Holmes, L. Zhang, P. D. Gregory, and E. J. Rebar, “A TALE nuclease architecture for efficient genome editing,” Nat. Biotechnol., vol. 29, no. 2, pp. 143–148, Feb. 2011.

[2] “Genome Editing.” [Online]. Available: http://www.allelebiotech.com/genome-editing/. [Accessed: 06-Jan-2015].

[3] R. M. Gupta and K. Musunuru, “Expanding the genetic editing tool kit: ZFNs, TALENs, and CRISPR-Cas9,” J. Clin. Invest., vol. 124, no. 10, pp. 4154–4161, Oct. 2014.

[4] M. Jinek, K. Chylinski, I. Fonfara, M. Hauer, J. A. Doudna, and E. Charpentier, “A programmable dual-RNA-guided DNA endonuclease in adaptive bacterial immunity,” Science, vol. 337, no. 6096, pp. 816–821, Aug. 2012.

[5] “CRISPR,” Wikipedia, the free encyclopedia. 09-Jan-2015.

[6] “TAL effector,” Wikipedia, the free encyclopedia. 06-Jan-2015.

[7] J. C. Miller, M. C. Holmes, J. Wang, D. Y. Guschin, Y.-L. Lee, I. Rupniewski, C. M. Beausejour, A. J. Waite, N. S. Wang, K. A. Kim, P. D. Gregory, C. O. Pabo, and E. J. Rebar, “An improved zinc-finger nuclease architecture for highly specific genome editing,” Nat. Biotechnol., vol. 25, no. 7, pp. 778–785, Jul. 2007.

[8] M. Christian, T. Cermak, E. L. Doyle, C. Schmidt, F. Zhang, A. Hummel, A. J. Bogdanove, and D. F. Voytas, “Targeting DNA double-strand breaks with TAL effector nucleases,” Genetics, vol. 186, no. 2, pp. 757–761, Oct. 2010.

[9] T. Li, S. Huang, W. Z. Jiang, D. Wright, M. H. Spalding, D. P. Weeks, and B. Yang, “TAL nucleases (TALNs): hybrid proteins composed of TAL effectors and FokI DNA-cleavage domain,” Nucleic Acids Res., vol. 39, no. 1, pp. 359–372, Jan. 2011.

[10] A. Strauβ and T. Lahaye, “Zinc Fingers, TAL Effectors, or Cas9-Based DNA Binding Proteins: What’s Best for Targeting Desired Genome Loci?,” Mol. Plant, vol. 6, no. 5, pp. 1384–1387, Sep. 2013.

A TALE nuclease architecture for efficient genome editing

2015-01-11T15:36:30Z

JernejMustar:

(Jernej Mustar)

Summarized from Miller, J. C. ''et al''. A TALE nuclease architecture for efficient genome editing. ''Nat. Biotechnol.'', vol. 29, pp. 143–148. Feb. 2011.

== Introduction to genome editing ==

Genome editing is a type of genetic engineering, where an organisms DNA is manipulated by altering; inserting, removing or replacing nucleotides, therefore changing its genome. This process is performed by the use of immensely useful tool, t. i. engineered nucleases, which are capable of cutting both strands of DNA or nicking single strand. Resulting sites are detected and repaired by endogenous repair mechanisms, which include homologous recombination (HR) or non-homologous end joining (NHEJ). The latter is error prone, therefore it is likely for mutations to occur in repair site. These repair pathways are highly conserved in diverse cell types, which is beneficial for the use of genome editing technologies. As a result, various cell types have been engineered by the use of portable cleavage-based technologies, including human stem cells [1].

Recently, there has been tremendous development in the field of synthetic biology and genome editing technology. Virtually any gene can be manipulated in diverse range of organisms. Most common approach is the use of nuclease, which can be manipulated to target specific sequences of genome. This can be achieved by exploitation of sequence-specific DNA-binding domains (ZFNs, TALENs) or by the use of complementary guide RNA (CRISPR-Cas9 system).
In this paper, the history of genome editing will first be briefly reviewed, further on genome editing will be discussed with emphasis on the TALEN’s family.

== Brief history of genome editing ==

Past studies in the field of genome editing were perusing a common goal, t. i. to obtain high specificity, which would limit the target affects [2]. The first engineered technology, based on nuclease activity, was introduced in 1991 by Pavletich and Pabo in the journal Science [2]. This technology by the name of Zinc Finger nuclease was a great breakthrough and was later on frequently applied for genome targeting. It is based on the use of cleavage domain of restriction enzyme FokI, which is linked with designated zinc finger protein (ZFP), which is responsible for targeting. By the use of different combinations of ZFP domains the selectivity for single genomic cleavage event is achieved. With the use of nuclease domains with monomeric activity there is a high probability of off-target events. This can be avoided by the use of a distinct pair of ZFNs with FokI domains that are obligate heterodimers [3]. Overtime, reports of problems and drawbacks of this technology were reported, such as targeting problem for certain triplets and reduced specificity, suffered by interactions within a zinc finger array [2].
The next technology, which emerged in 2009, is called transcription activator-like effector nuclease (TALENs). These are fusion proteins, where nuclease domain is associated with TAL effector DNA binding domain. This approach may be better in certain aspects, for example it enables a larger spectrum of targets and is more practical than ZFNs. In 2011, TALENs was proclaimed as “Method of the Year” by the journal Nature methods. The details of this system and molecular mechanisms of function are described later on.
We probably all at least heard if not read about latest breakthrough in the field of genome editing, which is CRISPR-Cas9 system. The discovery was somewhat fortuity, because researchers were studying how bacteria defend against phages and foreign plasmids. The paper was published in August 2012 in journal Science [4]. A bacterial nuclease called Cas9 is capable of altering genomes of invading viruses, consequently inactivating them. Clustered regularly interspaced short palindromic repeats (CRISPR) are DNA loci that contain short sequence repetitions, which can be transcribed, processed and then used to guide nuclease to a specific site [5]. This is in contrast with TALEN and Zinc Finger methods, where the need to prepare customized nuclease for each target remains.

== A TALE nuclease architecture ==

As mentioned before, two studies from 2009 proposed the use of TALEs effectors for targeted genome engineering. These transcriptional activators originate from Xanthomonas plant pathogens. During pathogenesis, TALEs specifically bind and regulate plant gene expression, thus aiding bacterial infection [6]. A central repeat domain, which mediates DNA recognition, is located within TALE structure [1]. Each repeat unit comprises of 33 – 35 amino, these specify one target base. The units base preference is determined by two crucial adjacent amino acids, also called the “repeat variable di-residue” (RVD). Therefore, the sequence of specified units in central repeat domain determines the target sequence on genome. TALEs generated with new repeat combinations have been shown to recognize target sequences predicted by this code.
In the light of aforementioned advances the interest in potential use of this system in combination with nuclease was initiated. Subsequently, a TALE-nuclease chimeras (TALENs) was proposed, which would allow for site-specific genome cleavage. In selected paper, the advances in the field of TALEN’s technology are discussed. TALE activity in a mammalian cell environment for targeted regulation of episomal reporters and an endogenous gene is presented, resuming with the research of the minimal TALE region required for high-affinity DNA binding. Paper also includes practical application, showing modification of endogenous human genes NTF3 and CCR5 by the use of TALEN approach.

== Retargeting a natural TALE to an endogenous mammalian sequence ==

Firstly, a suitable TAL effector for this study was chosen. Criteria for TALE selection was based on specificity and empirical evidence. The group was searching for natural TALE with high specificity for target sequence and with confirmed activity in mammalian cells. To achive this goal, PCR amplifications for several TALEs using Xanthomonas axonopodis pathovar citri genomic DNA were performed. Native TALE-associated sequences which allow transport into plant cells were excluded. Protein products of acquired coding regions were characterized by a SELEX assay using target of interest. Outcome of this selection yield one higly selective candidate, identified as TALE13. When combined with VP-16 activation domain, a 70-fold induction of reporter gene expression rate was observed in HEK293 cells. Based on these results, TALE13 was chosen for further work and design.
First goal was to demonstrate that chosen candidate was appropriate for mammalian gene regulation. Chosen target was NTF3 gene, which encodes a secreted nerve growth factor that has therapeutic potential for neurodegenerative diseases. Wild type TALE usually contain 18 repeats, by which they target specific sequence and bind to proximal promotor elements. TALE13 was modified to the extent of recognising proximal promotor region of NTF3 by replacing the 18 wild-type repeat units with appropriate alternatives. This altered TALE was named NT-L (meaning NTF3 Left), which highlits its binding position on target. In order to test its aktivity, the truncated version of NT-L was fused with VP-16 and expressed in human HEK293 cells. By measuring the activity of endogenous NTF3 locus, a strong induction (over 20 fold) in both NTF3 transcript and protein product was detected. The subsequent SELEX analysis revealed high specificity. These results revealed, that NT-L variant is a viable candidate for endogenous mammalian gene regulation.

== Development of a TALE-nuclease architecture ==

The next step was to design TALENs chimera protein using NT-L and nuclease, which would allow implementation of targeted cleavage of its genome target. The process was realized through three stages.
First, the minimal region required for high affinity bonding was determined using gene regulation studies. This was beneficial for optimization of nuclease (FokI) cleavage domain attachment with its target. Desired outcome of this step was improved catalytic activity of chimeric tool. The truncated variant named NT-L+95 was shown to retain high specificity and at the same time contribute to improved nuclease attachment. In order to examine this, a C-terminal truncated series of TALE13 were generated and subsequently linked with FokI. The resulting candidates were screened as homodimers for cleavage of specific targets. The results have shown several interesting occurrences. For TALENs comprising of NT-L+95 no detectable nuclease activity was observed. The cause for this is unclear; it is possible that this is due to a certain effect of NT-L+95 to nuclease domain positioning. Efficient DNA cleavage was observed for certain shorter specimens, providing candidates for cellular studies.
The minimal region between TALEN binding sites for optimal activity is over 12 bp, otherwise reduce cleavage may occur. These observations are useful for design of TALENs used for in vivo studies.
In the last stage, homodimer TALEN partner for NT-L was designed and named NT-R (NTR3 Right), indicating the direction of binding. This pair was expressed in human
K562 cells as fusions with catalytic domain of FokI. Target locus was later on tested for gene modification. By using two different C-terminal subregions (+28 and +63) the designed TALENs were tested. Significant activity in in vitro cleavage study was detected. With expression at 37°C, up to 3% modification was observed by the Surveyor assay [7]. This assay reveals gene modification by the appearance of digestion products. The percentage of gene modification can be obtained by quantification of “cut” bands. Limit of detection of this assay is about 1%. The activity was higher (9% of modification) in conditions of transient hypothermia, t. i. at 30°C. Sequences from latter were analyzed by Sanger sequencing. Out of 84 analyzed alleles there were 7 mutated by the occurrence of short deletions. This observation is consistent with expectation of non-homologous end joining (NHEJ) activity. Results have also shown a NHEJ-mediated capture of an oligonucleotide duplex at the endogenous NTF3 locus. These results confirmed that NHEJ-mediated genome modification by the use of TALEN architecture is possible in mammalian environment.

== Efficient modification by NHEJ and homology directed repair (HDR) ==

To expand the practical application for this developed system, modification at an additional locus was proposed. CCR5 gene was targeted for modification. First, a set of TALENs with a range of target lengths from 13 to 17 bp and of separation distance from 5 to 27 bp were designed and tested to identify values compatible with endogenous activity. To this end, a cluster of four left and four right binding sites were chosen for targeting. This defined a matrix of 16 heterodimer targets. For each binding site, two alternative proteins were generated, with 28 or 63 residues on C-terminal (as before). Altogether, this yielded 8x8=64 combinations of left and right proteins. These were expressed by pairs in K562 cells under standard conditions. Once again, Surveyor assay was used for evaluation of allele modification. Obtained results indicate that introduction of some +63/+63 pairs yielded over 20% allele modification. TALEN treated cells seem to retain a stable modification lever after 10 day growth, suggesting that TALENs are generally well tolerated. The effect of spacer length on activity is observed once again. At least 5% modification is achieved by the use of 12 – 19 bp spacer length in the +63/+63 TALENs section. In contrast, +28/+28 section show maximal activity in narrow spacer range of 12 – 13 bp.
The next challenge was to show that it is possible to induce gene through HDR using this system. For this, a part of CCR5 region was selected for targeting, also mentioned as safe harbor previously used for transgene integration. 24 TALEN pairs were then screened for their ability to induce NHEJ. Two highly active combinations were identified, first with 18% mutation rate and second with 21%. These TALEN were introduced into K562 cells simultaneously with donor DNA fragment carrying 46 bp with restriction site BglI. The success of integration was proven by PCR and BglI digestion. About 16% of inspected alleles were modified. This is an example how TALEN architecture can be used to activate HDR.

== Conclusion ==

In presented study it is shown how TALEN architecture can be designed and generated for efficient genome editing in mammalian environment. In this case, the system consists of TALE truncation variants, linked to catalytic domain of FokI. Engineered TALENs can be used for gene modification of human cells by activating either NHEJ or HDR repair pathways. Furthermore, three distinct applications are demonstrated; NHEJ mediated gene disruption, NHEJ-mediated insertion by capture of a DNA fragment and HDR mediated gene editing. Aforementioned suggests that TALEN architecture is a compelling tool in synthetic biology.

== Discussion ==

A step to facilitate the use of this architecture would undoubtedly be the linkage of minimal central TALE repeat domain to the catalytic domain of FokI. This was previously attempted by several groups without success, whereas the lack of activity of such constructs [8]. An alternative idea is to use a full length TALE for targeting. Disadvantage of the latter is the size of linkage peptide connecting two domains, which would affect the nuclease activity. Third approach is to generate truncated variants of TALEs and link them with catalytic domain of FokI, as shown in this paper. This approach has proven affective when using truncated variants with 28 or 63 of the 278 original C-terminal residues.
During the experimental work of this study, two papers were submitted where they demonstrated the use of TALEN architecture with the use of unmodified TALE or TALE with slightly reduced C-terminus [8], [9]. These systems were used for episome cleavage in yeast, an approach that is hardly translated to more complex genomes. As expected, these proteins have extended range of cleavage compared to truncated variants.
Presented TALEN architecture is in some characteristics similar to ZFNs. One example is the high cleavage activity of TALENs, which enables high gene modification efficiency, which is in some cases superior to the one of ZFNs. A shortcoming of TALEN architecture compared to ZFNs is its relative size. TALE repeats are 3 – 4 times larger than ZFPs, which may cause difficulty in delivery methods. Also, limitations due to high levels of TALE repeat homology may complicate the assembly of constructs. Future experiments will provide a fuller picture of pros and cons of the two platforms.
Targeted endogenous gene regulation by TALE proteins is a useful tool in research and for biotechnology applications. We can use this technology in a broad sense, such as design and generation of transcriptional activators or repressors. Although TALEs were discovered as such, previous studies did not inspect this type of activity outside of their native plant context. The experiments shown in this paper suggest that TALEs can be used as transcriptional regulators.
The use of SELEX assay for identification and characterization of TALEs has previously not been reported. By using this method, it was possible to assess the DNA binding preferences of natural and designed TALE proteins. Binding sites for TALE13 and four engineered TALEs were examined. On this basis, an observation that 73 out of 76 TALE repeats facilitate base preference of RVD. An interpretation of rare exceptions is proposed, facilitating further design and use of this system. Overall data collected can be used to examine previously acquired information on RVDs and thereby gaining more insight on mechanisms of action and selectivity. Findings that common RVDs such as NI, HD, NN and NG selectively bound selectively bound adenine, cytosine, guanine and thymine, respectively, are in accordance with previous knowledge. Pattern NN has shown relaxed specificity with substantial binding to a second base, adenine. NK is therefore more suitable for specificity, due to much stronger preference for guanine. This is a potential enhancement, proposed in this paper.

== Expectations for the future ==

As already mentioned, targeting the designated loci with ZFNs or TALENs is often difficult and time consuming, due to design and generation of new custom ZF or TALE array for each locus. This is not the case in most recent approach in genome editing, that is the use of CRISPR-Cas9 system. Expression of distinct gRNAs alongside Cas9 results in Cas9–gRNA complexes with distinct cleavage specificities [10]. This approach is exploited in multiplex genome editing. Still, there are features of this system which are inferior to elderly time-demanding ZFNs or TALENs.
One of those features is size of gene constructs, which is a limitation in field of gene delivery. Cas9 is the size of about 160 kDa, whereas a typical 17,5 repeat TALEN corresponds to 105 kDa. By far the smallest are ZFNs, wherein typical four finger ZFN has about 40 kDa [10]. It is likely for this compact nature to be beneficial for certain experiments.
As for specificity, the number of bases for modification in Cas9 system is restricted to 20 and is somewhat limited with the rule of three guanine bases flanking the target site (GN19NGG) [10]. The main constriction of ZF arrays is a scarce set of fingers, resulting in inability to target certain triplets. In addition, some ZFs targeting GNN seem to be superior to other ZFs, contributing to limitations. TALE target specificity therefore seems superior, with capabilities of targeting up to 24 bases. For Cas9 system, off target studies in mammalian environment are yet to be conducted [10].
It is likely that distinct features will lead to differential use of genome editing systems for distinct applications in the fields of plant science, therapeutic sciences and biotechnology applications.

== Literature ==

[1] J. C. Miller, S. Tan, G. Qiao, K. A. Barlow, J. Wang, D. F. Xia, X. Meng, D. E. Paschon, E. Leung, S. J. Hinkley, G. P. Dulay, K. L. Hua, I. Ankoudinova, G. J. Cost, F. D. Urnov, H. S. Zhang, M. C. Holmes, L. Zhang, P. D. Gregory, and E. J. Rebar, “A TALE nuclease architecture for efficient genome editing,” Nat. Biotechnol., vol. 29, no. 2, pp. 143–148, Feb. 2011.

[2] “Genome Editing.” [Online]. Available: http://www.allelebiotech.com/genome-editing/. [Accessed: 06-Jan-2015].

[3] R. M. Gupta and K. Musunuru, “Expanding the genetic editing tool kit: ZFNs, TALENs, and CRISPR-Cas9,” J. Clin. Invest., vol. 124, no. 10, pp. 4154–4161, Oct. 2014.

[4] M. Jinek, K. Chylinski, I. Fonfara, M. Hauer, J. A. Doudna, and E. Charpentier, “A programmable dual-RNA-guided DNA endonuclease in adaptive bacterial immunity,” Science, vol. 337, no. 6096, pp. 816–821, Aug. 2012.

[5] “CRISPR,” Wikipedia, the free encyclopedia. 09-Jan-2015.

[6] “TAL effector,” Wikipedia, the free encyclopedia. 06-Jan-2015.

[7] J. C. Miller, M. C. Holmes, J. Wang, D. Y. Guschin, Y.-L. Lee, I. Rupniewski, C. M. Beausejour, A. J. Waite, N. S. Wang, K. A. Kim, P. D. Gregory, C. O. Pabo, and E. J. Rebar, “An improved zinc-finger nuclease architecture for highly specific genome editing,” Nat. Biotechnol., vol. 25, no. 7, pp. 778–785, Jul. 2007.

[8] M. Christian, T. Cermak, E. L. Doyle, C. Schmidt, F. Zhang, A. Hummel, A. J. Bogdanove, and D. F. Voytas, “Targeting DNA double-strand breaks with TAL effector nucleases,” Genetics, vol. 186, no. 2, pp. 757–761, Oct. 2010.

[9] T. Li, S. Huang, W. Z. Jiang, D. Wright, M. H. Spalding, D. P. Weeks, and B. Yang, “TAL nucleases (TALNs): hybrid proteins composed of TAL effectors and FokI DNA-cleavage domain,” Nucleic Acids Res., vol. 39, no. 1, pp. 359–372, Jan. 2011.

[10] A. Strauβ and T. Lahaye, “Zinc Fingers, TAL Effectors, or Cas9-Based DNA Binding Proteins: What’s Best for Targeting Desired Genome Loci?,” Mol. Plant, vol. 6, no. 5, pp. 1384–1387, Sep. 2013.

A TALE nuclease architecture for efficient genome editing

2015-01-11T15:36:20Z

JernejMustar:

(Jernej Mustar)
Summarized from Miller, J. C. ''et al''. A TALE nuclease architecture for efficient genome editing. ''Nat. Biotechnol.'', vol. 29, pp. 143–148. Feb. 2011.

== Introduction to genome editing ==

Genome editing is a type of genetic engineering, where an organisms DNA is manipulated by altering; inserting, removing or replacing nucleotides, therefore changing its genome. This process is performed by the use of immensely useful tool, t. i. engineered nucleases, which are capable of cutting both strands of DNA or nicking single strand. Resulting sites are detected and repaired by endogenous repair mechanisms, which include homologous recombination (HR) or non-homologous end joining (NHEJ). The latter is error prone, therefore it is likely for mutations to occur in repair site. These repair pathways are highly conserved in diverse cell types, which is beneficial for the use of genome editing technologies. As a result, various cell types have been engineered by the use of portable cleavage-based technologies, including human stem cells [1].

Recently, there has been tremendous development in the field of synthetic biology and genome editing technology. Virtually any gene can be manipulated in diverse range of organisms. Most common approach is the use of nuclease, which can be manipulated to target specific sequences of genome. This can be achieved by exploitation of sequence-specific DNA-binding domains (ZFNs, TALENs) or by the use of complementary guide RNA (CRISPR-Cas9 system).
In this paper, the history of genome editing will first be briefly reviewed, further on genome editing will be discussed with emphasis on the TALEN’s family.

== Brief history of genome editing ==

Past studies in the field of genome editing were perusing a common goal, t. i. to obtain high specificity, which would limit the target affects [2]. The first engineered technology, based on nuclease activity, was introduced in 1991 by Pavletich and Pabo in the journal Science [2]. This technology by the name of Zinc Finger nuclease was a great breakthrough and was later on frequently applied for genome targeting. It is based on the use of cleavage domain of restriction enzyme FokI, which is linked with designated zinc finger protein (ZFP), which is responsible for targeting. By the use of different combinations of ZFP domains the selectivity for single genomic cleavage event is achieved. With the use of nuclease domains with monomeric activity there is a high probability of off-target events. This can be avoided by the use of a distinct pair of ZFNs with FokI domains that are obligate heterodimers [3]. Overtime, reports of problems and drawbacks of this technology were reported, such as targeting problem for certain triplets and reduced specificity, suffered by interactions within a zinc finger array [2].
The next technology, which emerged in 2009, is called transcription activator-like effector nuclease (TALENs). These are fusion proteins, where nuclease domain is associated with TAL effector DNA binding domain. This approach may be better in certain aspects, for example it enables a larger spectrum of targets and is more practical than ZFNs. In 2011, TALENs was proclaimed as “Method of the Year” by the journal Nature methods. The details of this system and molecular mechanisms of function are described later on.
We probably all at least heard if not read about latest breakthrough in the field of genome editing, which is CRISPR-Cas9 system. The discovery was somewhat fortuity, because researchers were studying how bacteria defend against phages and foreign plasmids. The paper was published in August 2012 in journal Science [4]. A bacterial nuclease called Cas9 is capable of altering genomes of invading viruses, consequently inactivating them. Clustered regularly interspaced short palindromic repeats (CRISPR) are DNA loci that contain short sequence repetitions, which can be transcribed, processed and then used to guide nuclease to a specific site [5]. This is in contrast with TALEN and Zinc Finger methods, where the need to prepare customized nuclease for each target remains.

== A TALE nuclease architecture ==

As mentioned before, two studies from 2009 proposed the use of TALEs effectors for targeted genome engineering. These transcriptional activators originate from Xanthomonas plant pathogens. During pathogenesis, TALEs specifically bind and regulate plant gene expression, thus aiding bacterial infection [6]. A central repeat domain, which mediates DNA recognition, is located within TALE structure [1]. Each repeat unit comprises of 33 – 35 amino, these specify one target base. The units base preference is determined by two crucial adjacent amino acids, also called the “repeat variable di-residue” (RVD). Therefore, the sequence of specified units in central repeat domain determines the target sequence on genome. TALEs generated with new repeat combinations have been shown to recognize target sequences predicted by this code.
In the light of aforementioned advances the interest in potential use of this system in combination with nuclease was initiated. Subsequently, a TALE-nuclease chimeras (TALENs) was proposed, which would allow for site-specific genome cleavage. In selected paper, the advances in the field of TALEN’s technology are discussed. TALE activity in a mammalian cell environment for targeted regulation of episomal reporters and an endogenous gene is presented, resuming with the research of the minimal TALE region required for high-affinity DNA binding. Paper also includes practical application, showing modification of endogenous human genes NTF3 and CCR5 by the use of TALEN approach.

== Retargeting a natural TALE to an endogenous mammalian sequence ==

Firstly, a suitable TAL effector for this study was chosen. Criteria for TALE selection was based on specificity and empirical evidence. The group was searching for natural TALE with high specificity for target sequence and with confirmed activity in mammalian cells. To achive this goal, PCR amplifications for several TALEs using Xanthomonas axonopodis pathovar citri genomic DNA were performed. Native TALE-associated sequences which allow transport into plant cells were excluded. Protein products of acquired coding regions were characterized by a SELEX assay using target of interest. Outcome of this selection yield one higly selective candidate, identified as TALE13. When combined with VP-16 activation domain, a 70-fold induction of reporter gene expression rate was observed in HEK293 cells. Based on these results, TALE13 was chosen for further work and design.
First goal was to demonstrate that chosen candidate was appropriate for mammalian gene regulation. Chosen target was NTF3 gene, which encodes a secreted nerve growth factor that has therapeutic potential for neurodegenerative diseases. Wild type TALE usually contain 18 repeats, by which they target specific sequence and bind to proximal promotor elements. TALE13 was modified to the extent of recognising proximal promotor region of NTF3 by replacing the 18 wild-type repeat units with appropriate alternatives. This altered TALE was named NT-L (meaning NTF3 Left), which highlits its binding position on target. In order to test its aktivity, the truncated version of NT-L was fused with VP-16 and expressed in human HEK293 cells. By measuring the activity of endogenous NTF3 locus, a strong induction (over 20 fold) in both NTF3 transcript and protein product was detected. The subsequent SELEX analysis revealed high specificity. These results revealed, that NT-L variant is a viable candidate for endogenous mammalian gene regulation.

== Development of a TALE-nuclease architecture ==

The next step was to design TALENs chimera protein using NT-L and nuclease, which would allow implementation of targeted cleavage of its genome target. The process was realized through three stages.
First, the minimal region required for high affinity bonding was determined using gene regulation studies. This was beneficial for optimization of nuclease (FokI) cleavage domain attachment with its target. Desired outcome of this step was improved catalytic activity of chimeric tool. The truncated variant named NT-L+95 was shown to retain high specificity and at the same time contribute to improved nuclease attachment. In order to examine this, a C-terminal truncated series of TALE13 were generated and subsequently linked with FokI. The resulting candidates were screened as homodimers for cleavage of specific targets. The results have shown several interesting occurrences. For TALENs comprising of NT-L+95 no detectable nuclease activity was observed. The cause for this is unclear; it is possible that this is due to a certain effect of NT-L+95 to nuclease domain positioning. Efficient DNA cleavage was observed for certain shorter specimens, providing candidates for cellular studies.
The minimal region between TALEN binding sites for optimal activity is over 12 bp, otherwise reduce cleavage may occur. These observations are useful for design of TALENs used for in vivo studies.
In the last stage, homodimer TALEN partner for NT-L was designed and named NT-R (NTR3 Right), indicating the direction of binding. This pair was expressed in human
K562 cells as fusions with catalytic domain of FokI. Target locus was later on tested for gene modification. By using two different C-terminal subregions (+28 and +63) the designed TALENs were tested. Significant activity in in vitro cleavage study was detected. With expression at 37°C, up to 3% modification was observed by the Surveyor assay [7]. This assay reveals gene modification by the appearance of digestion products. The percentage of gene modification can be obtained by quantification of “cut” bands. Limit of detection of this assay is about 1%. The activity was higher (9% of modification) in conditions of transient hypothermia, t. i. at 30°C. Sequences from latter were analyzed by Sanger sequencing. Out of 84 analyzed alleles there were 7 mutated by the occurrence of short deletions. This observation is consistent with expectation of non-homologous end joining (NHEJ) activity. Results have also shown a NHEJ-mediated capture of an oligonucleotide duplex at the endogenous NTF3 locus. These results confirmed that NHEJ-mediated genome modification by the use of TALEN architecture is possible in mammalian environment.

== Efficient modification by NHEJ and homology directed repair (HDR) ==

To expand the practical application for this developed system, modification at an additional locus was proposed. CCR5 gene was targeted for modification. First, a set of TALENs with a range of target lengths from 13 to 17 bp and of separation distance from 5 to 27 bp were designed and tested to identify values compatible with endogenous activity. To this end, a cluster of four left and four right binding sites were chosen for targeting. This defined a matrix of 16 heterodimer targets. For each binding site, two alternative proteins were generated, with 28 or 63 residues on C-terminal (as before). Altogether, this yielded 8x8=64 combinations of left and right proteins. These were expressed by pairs in K562 cells under standard conditions. Once again, Surveyor assay was used for evaluation of allele modification. Obtained results indicate that introduction of some +63/+63 pairs yielded over 20% allele modification. TALEN treated cells seem to retain a stable modification lever after 10 day growth, suggesting that TALENs are generally well tolerated. The effect of spacer length on activity is observed once again. At least 5% modification is achieved by the use of 12 – 19 bp spacer length in the +63/+63 TALENs section. In contrast, +28/+28 section show maximal activity in narrow spacer range of 12 – 13 bp.
The next challenge was to show that it is possible to induce gene through HDR using this system. For this, a part of CCR5 region was selected for targeting, also mentioned as safe harbor previously used for transgene integration. 24 TALEN pairs were then screened for their ability to induce NHEJ. Two highly active combinations were identified, first with 18% mutation rate and second with 21%. These TALEN were introduced into K562 cells simultaneously with donor DNA fragment carrying 46 bp with restriction site BglI. The success of integration was proven by PCR and BglI digestion. About 16% of inspected alleles were modified. This is an example how TALEN architecture can be used to activate HDR.

== Conclusion ==

In presented study it is shown how TALEN architecture can be designed and generated for efficient genome editing in mammalian environment. In this case, the system consists of TALE truncation variants, linked to catalytic domain of FokI. Engineered TALENs can be used for gene modification of human cells by activating either NHEJ or HDR repair pathways. Furthermore, three distinct applications are demonstrated; NHEJ mediated gene disruption, NHEJ-mediated insertion by capture of a DNA fragment and HDR mediated gene editing. Aforementioned suggests that TALEN architecture is a compelling tool in synthetic biology.

== Discussion ==

A step to facilitate the use of this architecture would undoubtedly be the linkage of minimal central TALE repeat domain to the catalytic domain of FokI. This was previously attempted by several groups without success, whereas the lack of activity of such constructs [8]. An alternative idea is to use a full length TALE for targeting. Disadvantage of the latter is the size of linkage peptide connecting two domains, which would affect the nuclease activity. Third approach is to generate truncated variants of TALEs and link them with catalytic domain of FokI, as shown in this paper. This approach has proven affective when using truncated variants with 28 or 63 of the 278 original C-terminal residues.
During the experimental work of this study, two papers were submitted where they demonstrated the use of TALEN architecture with the use of unmodified TALE or TALE with slightly reduced C-terminus [8], [9]. These systems were used for episome cleavage in yeast, an approach that is hardly translated to more complex genomes. As expected, these proteins have extended range of cleavage compared to truncated variants.
Presented TALEN architecture is in some characteristics similar to ZFNs. One example is the high cleavage activity of TALENs, which enables high gene modification efficiency, which is in some cases superior to the one of ZFNs. A shortcoming of TALEN architecture compared to ZFNs is its relative size. TALE repeats are 3 – 4 times larger than ZFPs, which may cause difficulty in delivery methods. Also, limitations due to high levels of TALE repeat homology may complicate the assembly of constructs. Future experiments will provide a fuller picture of pros and cons of the two platforms.
Targeted endogenous gene regulation by TALE proteins is a useful tool in research and for biotechnology applications. We can use this technology in a broad sense, such as design and generation of transcriptional activators or repressors. Although TALEs were discovered as such, previous studies did not inspect this type of activity outside of their native plant context. The experiments shown in this paper suggest that TALEs can be used as transcriptional regulators.
The use of SELEX assay for identification and characterization of TALEs has previously not been reported. By using this method, it was possible to assess the DNA binding preferences of natural and designed TALE proteins. Binding sites for TALE13 and four engineered TALEs were examined. On this basis, an observation that 73 out of 76 TALE repeats facilitate base preference of RVD. An interpretation of rare exceptions is proposed, facilitating further design and use of this system. Overall data collected can be used to examine previously acquired information on RVDs and thereby gaining more insight on mechanisms of action and selectivity. Findings that common RVDs such as NI, HD, NN and NG selectively bound selectively bound adenine, cytosine, guanine and thymine, respectively, are in accordance with previous knowledge. Pattern NN has shown relaxed specificity with substantial binding to a second base, adenine. NK is therefore more suitable for specificity, due to much stronger preference for guanine. This is a potential enhancement, proposed in this paper.

== Expectations for the future ==

As already mentioned, targeting the designated loci with ZFNs or TALENs is often difficult and time consuming, due to design and generation of new custom ZF or TALE array for each locus. This is not the case in most recent approach in genome editing, that is the use of CRISPR-Cas9 system. Expression of distinct gRNAs alongside Cas9 results in Cas9–gRNA complexes with distinct cleavage specificities [10]. This approach is exploited in multiplex genome editing. Still, there are features of this system which are inferior to elderly time-demanding ZFNs or TALENs.
One of those features is size of gene constructs, which is a limitation in field of gene delivery. Cas9 is the size of about 160 kDa, whereas a typical 17,5 repeat TALEN corresponds to 105 kDa. By far the smallest are ZFNs, wherein typical four finger ZFN has about 40 kDa [10]. It is likely for this compact nature to be beneficial for certain experiments.
As for specificity, the number of bases for modification in Cas9 system is restricted to 20 and is somewhat limited with the rule of three guanine bases flanking the target site (GN19NGG) [10]. The main constriction of ZF arrays is a scarce set of fingers, resulting in inability to target certain triplets. In addition, some ZFs targeting GNN seem to be superior to other ZFs, contributing to limitations. TALE target specificity therefore seems superior, with capabilities of targeting up to 24 bases. For Cas9 system, off target studies in mammalian environment are yet to be conducted [10].
It is likely that distinct features will lead to differential use of genome editing systems for distinct applications in the fields of plant science, therapeutic sciences and biotechnology applications.

== Literature ==

[1] J. C. Miller, S. Tan, G. Qiao, K. A. Barlow, J. Wang, D. F. Xia, X. Meng, D. E. Paschon, E. Leung, S. J. Hinkley, G. P. Dulay, K. L. Hua, I. Ankoudinova, G. J. Cost, F. D. Urnov, H. S. Zhang, M. C. Holmes, L. Zhang, P. D. Gregory, and E. J. Rebar, “A TALE nuclease architecture for efficient genome editing,” Nat. Biotechnol., vol. 29, no. 2, pp. 143–148, Feb. 2011.

[2] “Genome Editing.” [Online]. Available: http://www.allelebiotech.com/genome-editing/. [Accessed: 06-Jan-2015].

[3] R. M. Gupta and K. Musunuru, “Expanding the genetic editing tool kit: ZFNs, TALENs, and CRISPR-Cas9,” J. Clin. Invest., vol. 124, no. 10, pp. 4154–4161, Oct. 2014.

[4] M. Jinek, K. Chylinski, I. Fonfara, M. Hauer, J. A. Doudna, and E. Charpentier, “A programmable dual-RNA-guided DNA endonuclease in adaptive bacterial immunity,” Science, vol. 337, no. 6096, pp. 816–821, Aug. 2012.

[5] “CRISPR,” Wikipedia, the free encyclopedia. 09-Jan-2015.

[6] “TAL effector,” Wikipedia, the free encyclopedia. 06-Jan-2015.

[7] J. C. Miller, M. C. Holmes, J. Wang, D. Y. Guschin, Y.-L. Lee, I. Rupniewski, C. M. Beausejour, A. J. Waite, N. S. Wang, K. A. Kim, P. D. Gregory, C. O. Pabo, and E. J. Rebar, “An improved zinc-finger nuclease architecture for highly specific genome editing,” Nat. Biotechnol., vol. 25, no. 7, pp. 778–785, Jul. 2007.

[8] M. Christian, T. Cermak, E. L. Doyle, C. Schmidt, F. Zhang, A. Hummel, A. J. Bogdanove, and D. F. Voytas, “Targeting DNA double-strand breaks with TAL effector nucleases,” Genetics, vol. 186, no. 2, pp. 757–761, Oct. 2010.

[9] T. Li, S. Huang, W. Z. Jiang, D. Wright, M. H. Spalding, D. P. Weeks, and B. Yang, “TAL nucleases (TALNs): hybrid proteins composed of TAL effectors and FokI DNA-cleavage domain,” Nucleic Acids Res., vol. 39, no. 1, pp. 359–372, Jan. 2011.

[10] A. Strauβ and T. Lahaye, “Zinc Fingers, TAL Effectors, or Cas9-Based DNA Binding Proteins: What’s Best for Targeting Desired Genome Loci?,” Mol. Plant, vol. 6, no. 5, pp. 1384–1387, Sep. 2013.

A TALE nuclease architecture for efficient genome editing

2015-01-11T15:35:10Z

JernejMustar: /* Brief history of genome editing */

Summarized from Miller, J. C. ''et al''. A TALE nuclease architecture for efficient genome editing. ''Nat. Biotechnol.'', vol. 29, pp. 143–148. Feb. 2011.

== Introduction to genome editing ==

Genome editing is a type of genetic engineering, where an organisms DNA is manipulated by altering; inserting, removing or replacing nucleotides, therefore changing its genome. This process is performed by the use of immensely useful tool, t. i. engineered nucleases, which are capable of cutting both strands of DNA or nicking single strand. Resulting sites are detected and repaired by endogenous repair mechanisms, which include homologous recombination (HR) or non-homologous end joining (NHEJ). The latter is error prone, therefore it is likely for mutations to occur in repair site. These repair pathways are highly conserved in diverse cell types, which is beneficial for the use of genome editing technologies. As a result, various cell types have been engineered by the use of portable cleavage-based technologies, including human stem cells [1].

Recently, there has been tremendous development in the field of synthetic biology and genome editing technology. Virtually any gene can be manipulated in diverse range of organisms. Most common approach is the use of nuclease, which can be manipulated to target specific sequences of genome. This can be achieved by exploitation of sequence-specific DNA-binding domains (ZFNs, TALENs) or by the use of complementary guide RNA (CRISPR-Cas9 system).
In this paper, the history of genome editing will first be briefly reviewed, further on genome editing will be discussed with emphasis on the TALEN’s family.

== Brief history of genome editing ==

Past studies in the field of genome editing were perusing a common goal, t. i. to obtain high specificity, which would limit the target affects [2]. The first engineered technology, based on nuclease activity, was introduced in 1991 by Pavletich and Pabo in the journal Science [2]. This technology by the name of Zinc Finger nuclease was a great breakthrough and was later on frequently applied for genome targeting. It is based on the use of cleavage domain of restriction enzyme FokI, which is linked with designated zinc finger protein (ZFP), which is responsible for targeting. By the use of different combinations of ZFP domains the selectivity for single genomic cleavage event is achieved. With the use of nuclease domains with monomeric activity there is a high probability of off-target events. This can be avoided by the use of a distinct pair of ZFNs with FokI domains that are obligate heterodimers [3]. Overtime, reports of problems and drawbacks of this technology were reported, such as targeting problem for certain triplets and reduced specificity, suffered by interactions within a zinc finger array [2].
The next technology, which emerged in 2009, is called transcription activator-like effector nuclease (TALENs). These are fusion proteins, where nuclease domain is associated with TAL effector DNA binding domain. This approach may be better in certain aspects, for example it enables a larger spectrum of targets and is more practical than ZFNs. In 2011, TALENs was proclaimed as “Method of the Year” by the journal Nature methods. The details of this system and molecular mechanisms of function are described later on.
We probably all at least heard if not read about latest breakthrough in the field of genome editing, which is CRISPR-Cas9 system. The discovery was somewhat fortuity, because researchers were studying how bacteria defend against phages and foreign plasmids. The paper was published in August 2012 in journal Science [4]. A bacterial nuclease called Cas9 is capable of altering genomes of invading viruses, consequently inactivating them. Clustered regularly interspaced short palindromic repeats (CRISPR) are DNA loci that contain short sequence repetitions, which can be transcribed, processed and then used to guide nuclease to a specific site [5]. This is in contrast with TALEN and Zinc Finger methods, where the need to prepare customized nuclease for each target remains.

== A TALE nuclease architecture ==

As mentioned before, two studies from 2009 proposed the use of TALEs effectors for targeted genome engineering. These transcriptional activators originate from Xanthomonas plant pathogens. During pathogenesis, TALEs specifically bind and regulate plant gene expression, thus aiding bacterial infection [6]. A central repeat domain, which mediates DNA recognition, is located within TALE structure [1]. Each repeat unit comprises of 33 – 35 amino, these specify one target base. The units base preference is determined by two crucial adjacent amino acids, also called the “repeat variable di-residue” (RVD). Therefore, the sequence of specified units in central repeat domain determines the target sequence on genome. TALEs generated with new repeat combinations have been shown to recognize target sequences predicted by this code.
In the light of aforementioned advances the interest in potential use of this system in combination with nuclease was initiated. Subsequently, a TALE-nuclease chimeras (TALENs) was proposed, which would allow for site-specific genome cleavage. In selected paper, the advances in the field of TALEN’s technology are discussed. TALE activity in a mammalian cell environment for targeted regulation of episomal reporters and an endogenous gene is presented, resuming with the research of the minimal TALE region required for high-affinity DNA binding. Paper also includes practical application, showing modification of endogenous human genes NTF3 and CCR5 by the use of TALEN approach.

== Retargeting a natural TALE to an endogenous mammalian sequence ==

Firstly, a suitable TAL effector for this study was chosen. Criteria for TALE selection was based on specificity and empirical evidence. The group was searching for natural TALE with high specificity for target sequence and with confirmed activity in mammalian cells. To achive this goal, PCR amplifications for several TALEs using Xanthomonas axonopodis pathovar citri genomic DNA were performed. Native TALE-associated sequences which allow transport into plant cells were excluded. Protein products of acquired coding regions were characterized by a SELEX assay using target of interest. Outcome of this selection yield one higly selective candidate, identified as TALE13. When combined with VP-16 activation domain, a 70-fold induction of reporter gene expression rate was observed in HEK293 cells. Based on these results, TALE13 was chosen for further work and design.
First goal was to demonstrate that chosen candidate was appropriate for mammalian gene regulation. Chosen target was NTF3 gene, which encodes a secreted nerve growth factor that has therapeutic potential for neurodegenerative diseases. Wild type TALE usually contain 18 repeats, by which they target specific sequence and bind to proximal promotor elements. TALE13 was modified to the extent of recognising proximal promotor region of NTF3 by replacing the 18 wild-type repeat units with appropriate alternatives. This altered TALE was named NT-L (meaning NTF3 Left), which highlits its binding position on target. In order to test its aktivity, the truncated version of NT-L was fused with VP-16 and expressed in human HEK293 cells. By measuring the activity of endogenous NTF3 locus, a strong induction (over 20 fold) in both NTF3 transcript and protein product was detected. The subsequent SELEX analysis revealed high specificity. These results revealed, that NT-L variant is a viable candidate for endogenous mammalian gene regulation.

== Development of a TALE-nuclease architecture ==

The next step was to design TALENs chimera protein using NT-L and nuclease, which would allow implementation of targeted cleavage of its genome target. The process was realized through three stages.
First, the minimal region required for high affinity bonding was determined using gene regulation studies. This was beneficial for optimization of nuclease (FokI) cleavage domain attachment with its target. Desired outcome of this step was improved catalytic activity of chimeric tool. The truncated variant named NT-L+95 was shown to retain high specificity and at the same time contribute to improved nuclease attachment. In order to examine this, a C-terminal truncated series of TALE13 were generated and subsequently linked with FokI. The resulting candidates were screened as homodimers for cleavage of specific targets. The results have shown several interesting occurrences. For TALENs comprising of NT-L+95 no detectable nuclease activity was observed. The cause for this is unclear; it is possible that this is due to a certain effect of NT-L+95 to nuclease domain positioning. Efficient DNA cleavage was observed for certain shorter specimens, providing candidates for cellular studies.
The minimal region between TALEN binding sites for optimal activity is over 12 bp, otherwise reduce cleavage may occur. These observations are useful for design of TALENs used for in vivo studies.
In the last stage, homodimer TALEN partner for NT-L was designed and named NT-R (NTR3 Right), indicating the direction of binding. This pair was expressed in human
K562 cells as fusions with catalytic domain of FokI. Target locus was later on tested for gene modification. By using two different C-terminal subregions (+28 and +63) the designed TALENs were tested. Significant activity in in vitro cleavage study was detected. With expression at 37°C, up to 3% modification was observed by the Surveyor assay [7]. This assay reveals gene modification by the appearance of digestion products. The percentage of gene modification can be obtained by quantification of “cut” bands. Limit of detection of this assay is about 1%. The activity was higher (9% of modification) in conditions of transient hypothermia, t. i. at 30°C. Sequences from latter were analyzed by Sanger sequencing. Out of 84 analyzed alleles there were 7 mutated by the occurrence of short deletions. This observation is consistent with expectation of non-homologous end joining (NHEJ) activity. Results have also shown a NHEJ-mediated capture of an oligonucleotide duplex at the endogenous NTF3 locus. These results confirmed that NHEJ-mediated genome modification by the use of TALEN architecture is possible in mammalian environment.

== Efficient modification by NHEJ and homology directed repair (HDR) ==

To expand the practical application for this developed system, modification at an additional locus was proposed. CCR5 gene was targeted for modification. First, a set of TALENs with a range of target lengths from 13 to 17 bp and of separation distance from 5 to 27 bp were designed and tested to identify values compatible with endogenous activity. To this end, a cluster of four left and four right binding sites were chosen for targeting. This defined a matrix of 16 heterodimer targets. For each binding site, two alternative proteins were generated, with 28 or 63 residues on C-terminal (as before). Altogether, this yielded 8x8=64 combinations of left and right proteins. These were expressed by pairs in K562 cells under standard conditions. Once again, Surveyor assay was used for evaluation of allele modification. Obtained results indicate that introduction of some +63/+63 pairs yielded over 20% allele modification. TALEN treated cells seem to retain a stable modification lever after 10 day growth, suggesting that TALENs are generally well tolerated. The effect of spacer length on activity is observed once again. At least 5% modification is achieved by the use of 12 – 19 bp spacer length in the +63/+63 TALENs section. In contrast, +28/+28 section show maximal activity in narrow spacer range of 12 – 13 bp.
The next challenge was to show that it is possible to induce gene through HDR using this system. For this, a part of CCR5 region was selected for targeting, also mentioned as safe harbor previously used for transgene integration. 24 TALEN pairs were then screened for their ability to induce NHEJ. Two highly active combinations were identified, first with 18% mutation rate and second with 21%. These TALEN were introduced into K562 cells simultaneously with donor DNA fragment carrying 46 bp with restriction site BglI. The success of integration was proven by PCR and BglI digestion. About 16% of inspected alleles were modified. This is an example how TALEN architecture can be used to activate HDR.

== Conclusion ==

In presented study it is shown how TALEN architecture can be designed and generated for efficient genome editing in mammalian environment. In this case, the system consists of TALE truncation variants, linked to catalytic domain of FokI. Engineered TALENs can be used for gene modification of human cells by activating either NHEJ or HDR repair pathways. Furthermore, three distinct applications are demonstrated; NHEJ mediated gene disruption, NHEJ-mediated insertion by capture of a DNA fragment and HDR mediated gene editing. Aforementioned suggests that TALEN architecture is a compelling tool in synthetic biology.

== Discussion ==

A step to facilitate the use of this architecture would undoubtedly be the linkage of minimal central TALE repeat domain to the catalytic domain of FokI. This was previously attempted by several groups without success, whereas the lack of activity of such constructs [8]. An alternative idea is to use a full length TALE for targeting. Disadvantage of the latter is the size of linkage peptide connecting two domains, which would affect the nuclease activity. Third approach is to generate truncated variants of TALEs and link them with catalytic domain of FokI, as shown in this paper. This approach has proven affective when using truncated variants with 28 or 63 of the 278 original C-terminal residues.
During the experimental work of this study, two papers were submitted where they demonstrated the use of TALEN architecture with the use of unmodified TALE or TALE with slightly reduced C-terminus [8], [9]. These systems were used for episome cleavage in yeast, an approach that is hardly translated to more complex genomes. As expected, these proteins have extended range of cleavage compared to truncated variants.
Presented TALEN architecture is in some characteristics similar to ZFNs. One example is the high cleavage activity of TALENs, which enables high gene modification efficiency, which is in some cases superior to the one of ZFNs. A shortcoming of TALEN architecture compared to ZFNs is its relative size. TALE repeats are 3 – 4 times larger than ZFPs, which may cause difficulty in delivery methods. Also, limitations due to high levels of TALE repeat homology may complicate the assembly of constructs. Future experiments will provide a fuller picture of pros and cons of the two platforms.
Targeted endogenous gene regulation by TALE proteins is a useful tool in research and for biotechnology applications. We can use this technology in a broad sense, such as design and generation of transcriptional activators or repressors. Although TALEs were discovered as such, previous studies did not inspect this type of activity outside of their native plant context. The experiments shown in this paper suggest that TALEs can be used as transcriptional regulators.
The use of SELEX assay for identification and characterization of TALEs has previously not been reported. By using this method, it was possible to assess the DNA binding preferences of natural and designed TALE proteins. Binding sites for TALE13 and four engineered TALEs were examined. On this basis, an observation that 73 out of 76 TALE repeats facilitate base preference of RVD. An interpretation of rare exceptions is proposed, facilitating further design and use of this system. Overall data collected can be used to examine previously acquired information on RVDs and thereby gaining more insight on mechanisms of action and selectivity. Findings that common RVDs such as NI, HD, NN and NG selectively bound selectively bound adenine, cytosine, guanine and thymine, respectively, are in accordance with previous knowledge. Pattern NN has shown relaxed specificity with substantial binding to a second base, adenine. NK is therefore more suitable for specificity, due to much stronger preference for guanine. This is a potential enhancement, proposed in this paper.

== Expectations for the future ==

As already mentioned, targeting the designated loci with ZFNs or TALENs is often difficult and time consuming, due to design and generation of new custom ZF or TALE array for each locus. This is not the case in most recent approach in genome editing, that is the use of CRISPR-Cas9 system. Expression of distinct gRNAs alongside Cas9 results in Cas9–gRNA complexes with distinct cleavage specificities [10]. This approach is exploited in multiplex genome editing. Still, there are features of this system which are inferior to elderly time-demanding ZFNs or TALENs.
One of those features is size of gene constructs, which is a limitation in field of gene delivery. Cas9 is the size of about 160 kDa, whereas a typical 17,5 repeat TALEN corresponds to 105 kDa. By far the smallest are ZFNs, wherein typical four finger ZFN has about 40 kDa [10]. It is likely for this compact nature to be beneficial for certain experiments.
As for specificity, the number of bases for modification in Cas9 system is restricted to 20 and is somewhat limited with the rule of three guanine bases flanking the target site (GN19NGG) [10]. The main constriction of ZF arrays is a scarce set of fingers, resulting in inability to target certain triplets. In addition, some ZFs targeting GNN seem to be superior to other ZFs, contributing to limitations. TALE target specificity therefore seems superior, with capabilities of targeting up to 24 bases. For Cas9 system, off target studies in mammalian environment are yet to be conducted [10].
It is likely that distinct features will lead to differential use of genome editing systems for distinct applications in the fields of plant science, therapeutic sciences and biotechnology applications.

== Literature ==

[1] J. C. Miller, S. Tan, G. Qiao, K. A. Barlow, J. Wang, D. F. Xia, X. Meng, D. E. Paschon, E. Leung, S. J. Hinkley, G. P. Dulay, K. L. Hua, I. Ankoudinova, G. J. Cost, F. D. Urnov, H. S. Zhang, M. C. Holmes, L. Zhang, P. D. Gregory, and E. J. Rebar, “A TALE nuclease architecture for efficient genome editing,” Nat. Biotechnol., vol. 29, no. 2, pp. 143–148, Feb. 2011.

[2] “Genome Editing.” [Online]. Available: http://www.allelebiotech.com/genome-editing/. [Accessed: 06-Jan-2015].

[3] R. M. Gupta and K. Musunuru, “Expanding the genetic editing tool kit: ZFNs, TALENs, and CRISPR-Cas9,” J. Clin. Invest., vol. 124, no. 10, pp. 4154–4161, Oct. 2014.

[4] M. Jinek, K. Chylinski, I. Fonfara, M. Hauer, J. A. Doudna, and E. Charpentier, “A programmable dual-RNA-guided DNA endonuclease in adaptive bacterial immunity,” Science, vol. 337, no. 6096, pp. 816–821, Aug. 2012.

[5] “CRISPR,” Wikipedia, the free encyclopedia. 09-Jan-2015.

[6] “TAL effector,” Wikipedia, the free encyclopedia. 06-Jan-2015.

[7] J. C. Miller, M. C. Holmes, J. Wang, D. Y. Guschin, Y.-L. Lee, I. Rupniewski, C. M. Beausejour, A. J. Waite, N. S. Wang, K. A. Kim, P. D. Gregory, C. O. Pabo, and E. J. Rebar, “An improved zinc-finger nuclease architecture for highly specific genome editing,” Nat. Biotechnol., vol. 25, no. 7, pp. 778–785, Jul. 2007.

[8] M. Christian, T. Cermak, E. L. Doyle, C. Schmidt, F. Zhang, A. Hummel, A. J. Bogdanove, and D. F. Voytas, “Targeting DNA double-strand breaks with TAL effector nucleases,” Genetics, vol. 186, no. 2, pp. 757–761, Oct. 2010.

[9] T. Li, S. Huang, W. Z. Jiang, D. Wright, M. H. Spalding, D. P. Weeks, and B. Yang, “TAL nucleases (TALNs): hybrid proteins composed of TAL effectors and FokI DNA-cleavage domain,” Nucleic Acids Res., vol. 39, no. 1, pp. 359–372, Jan. 2011.

[10] A. Strauβ and T. Lahaye, “Zinc Fingers, TAL Effectors, or Cas9-Based DNA Binding Proteins: What’s Best for Targeting Desired Genome Loci?,” Mol. Plant, vol. 6, no. 5, pp. 1384–1387, Sep. 2013.

A TALE nuclease architecture for efficient genome editing

2015-01-11T15:30:57Z

JernejMustar: /* Introduction to genome editing */

Summarized from Miller, J. C. ''et al''. A TALE nuclease architecture for efficient genome editing. ''Nat. Biotechnol.'', vol. 29, pp. 143–148. Feb. 2011.

== Introduction to genome editing ==

Genome editing is a type of genetic engineering, where an organisms DNA is manipulated by altering; inserting, removing or replacing nucleotides, therefore changing its genome. This process is performed by the use of immensely useful tool, t. i. engineered nucleases, which are capable of cutting both strands of DNA or nicking single strand. Resulting sites are detected and repaired by endogenous repair mechanisms, which include homologous recombination (HR) or non-homologous end joining (NHEJ). The latter is error prone, therefore it is likely for mutations to occur in repair site. These repair pathways are highly conserved in diverse cell types, which is beneficial for the use of genome editing technologies. As a result, various cell types have been engineered by the use of portable cleavage-based technologies, including human stem cells [1].

Recently, there has been tremendous development in the field of synthetic biology and genome editing technology. Virtually any gene can be manipulated in diverse range of organisms. Most common approach is the use of nuclease, which can be manipulated to target specific sequences of genome. This can be achieved by exploitation of sequence-specific DNA-binding domains (ZFNs, TALENs) or by the use of complementary guide RNA (CRISPR-Cas9 system).
In this paper, the history of genome editing will first be briefly reviewed, further on genome editing will be discussed with emphasis on the TALEN’s family.

== Brief history of genome editing ==

Past studies in the field of genome editing were perusing a common goal, t. i. to obtain high specificity, which would limit the target affects [2]. The first engineered technology, based on nuclease activity, was introduced in 1991 by Pavletich and Pabo in the journal Science [2]. This technology by the name of Zinc Finger nuclease was a great breakthrough and was later on frequently applied for genome targeting. It is based on the use of cleavage domain of restriction enzyme FokI, which is linked with designated zinc finger protein (ZFP), which is responsible for targeting. By the use of different combinations of ZFP domains the selectivity for single genomic cleavage event is achieved. The use of nuclease domains with monomeric activity there is a high probability of off-target events. This can be avoided by the use of a pair of ZFNs with distinct FokI domains that are obligate heterodimers [3]. Overtime, reports of problems and drawbacks of this technology were reported, such as targeting problem for certain triplets and reduced specificity, suffered by interactions within a zinc finger array [2].
The next technology, which emerged in 2009, is called transcription activator-like effector nuclease (TALENs). These are fusion proteins, where nuclease domain is associated with TAL effector DNA binding domain. This approach may be better in certain aspects, for example it enables a larger spectrum of targets and it’s simpler and more practical than ZFNs. In 2011, TALENs was proclaimed as “Method of the Year” by the journal Nature methods. The details of this system and molecular mechanisms of function are described later on.
We probably all at least heard if not read about latest breakthrough in the field of genome editing, which is CRISPR-Cas9 system. The discovery was somewhat fortuity, because researchers were studying how bacteria defend against phages and foreign plasmids. The paper was published in August 2012 in journal Science [4]. A bacterial nuclease called Cas9 is capable of altering genomes of invading viruses, consequently inactivating them. Clustered regularly interspaced short palindromic repeats (CRISPR) are DNA loci that contain short sequence repetitions, which can be transcribed, processed and then used to guide nuclease to a specific site [5]. This is in contrast with TALEN and Zinc Finger methods, where the need to prepare customized nuclease for each target remains.

== A TALE nuclease architecture ==

As mentioned before, two studies from 2009 proposed the use of TALEs effectors for targeted genome engineering. These transcriptional activators originate from Xanthomonas plant pathogens. During pathogenesis, TALEs specifically bind and regulate plant gene expression, thus aiding bacterial infection [6]. A central repeat domain, which mediates DNA recognition, is located within TALE structure [1]. Each repeat unit comprises of 33 – 35 amino, these specify one target base. The units base preference is determined by two crucial adjacent amino acids, also called the “repeat variable di-residue” (RVD). Therefore, the sequence of specified units in central repeat domain determines the target sequence on genome. TALEs generated with new repeat combinations have been shown to recognize target sequences predicted by this code.
In the light of aforementioned advances the interest in potential use of this system in combination with nuclease was initiated. Subsequently, a TALE-nuclease chimeras (TALENs) was proposed, which would allow for site-specific genome cleavage. In selected paper, the advances in the field of TALEN’s technology are discussed. TALE activity in a mammalian cell environment for targeted regulation of episomal reporters and an endogenous gene is presented, resuming with the research of the minimal TALE region required for high-affinity DNA binding. Paper also includes practical application, showing modification of endogenous human genes NTF3 and CCR5 by the use of TALEN approach.

== Retargeting a natural TALE to an endogenous mammalian sequence ==

Firstly, a suitable TAL effector for this study was chosen. Criteria for TALE selection was based on specificity and empirical evidence. The group was searching for natural TALE with high specificity for target sequence and with confirmed activity in mammalian cells. To achive this goal, PCR amplifications for several TALEs using Xanthomonas axonopodis pathovar citri genomic DNA were performed. Native TALE-associated sequences which allow transport into plant cells were excluded. Protein products of acquired coding regions were characterized by a SELEX assay using target of interest. Outcome of this selection yield one higly selective candidate, identified as TALE13. When combined with VP-16 activation domain, a 70-fold induction of reporter gene expression rate was observed in HEK293 cells. Based on these results, TALE13 was chosen for further work and design.
First goal was to demonstrate that chosen candidate was appropriate for mammalian gene regulation. Chosen target was NTF3 gene, which encodes a secreted nerve growth factor that has therapeutic potential for neurodegenerative diseases. Wild type TALE usually contain 18 repeats, by which they target specific sequence and bind to proximal promotor elements. TALE13 was modified to the extent of recognising proximal promotor region of NTF3 by replacing the 18 wild-type repeat units with appropriate alternatives. This altered TALE was named NT-L (meaning NTF3 Left), which highlits its binding position on target. In order to test its aktivity, the truncated version of NT-L was fused with VP-16 and expressed in human HEK293 cells. By measuring the activity of endogenous NTF3 locus, a strong induction (over 20 fold) in both NTF3 transcript and protein product was detected. The subsequent SELEX analysis revealed high specificity. These results revealed, that NT-L variant is a viable candidate for endogenous mammalian gene regulation.

== Development of a TALE-nuclease architecture ==

The next step was to design TALENs chimera protein using NT-L and nuclease, which would allow implementation of targeted cleavage of its genome target. The process was realized through three stages.
First, the minimal region required for high affinity bonding was determined using gene regulation studies. This was beneficial for optimization of nuclease (FokI) cleavage domain attachment with its target. Desired outcome of this step was improved catalytic activity of chimeric tool. The truncated variant named NT-L+95 was shown to retain high specificity and at the same time contribute to improved nuclease attachment. In order to examine this, a C-terminal truncated series of TALE13 were generated and subsequently linked with FokI. The resulting candidates were screened as homodimers for cleavage of specific targets. The results have shown several interesting occurrences. For TALENs comprising of NT-L+95 no detectable nuclease activity was observed. The cause for this is unclear; it is possible that this is due to a certain effect of NT-L+95 to nuclease domain positioning. Efficient DNA cleavage was observed for certain shorter specimens, providing candidates for cellular studies.
The minimal region between TALEN binding sites for optimal activity is over 12 bp, otherwise reduce cleavage may occur. These observations are useful for design of TALENs used for in vivo studies.
In the last stage, homodimer TALEN partner for NT-L was designed and named NT-R (NTR3 Right), indicating the direction of binding. This pair was expressed in human
K562 cells as fusions with catalytic domain of FokI. Target locus was later on tested for gene modification. By using two different C-terminal subregions (+28 and +63) the designed TALENs were tested. Significant activity in in vitro cleavage study was detected. With expression at 37°C, up to 3% modification was observed by the Surveyor assay [7]. This assay reveals gene modification by the appearance of digestion products. The percentage of gene modification can be obtained by quantification of “cut” bands. Limit of detection of this assay is about 1%. The activity was higher (9% of modification) in conditions of transient hypothermia, t. i. at 30°C. Sequences from latter were analyzed by Sanger sequencing. Out of 84 analyzed alleles there were 7 mutated by the occurrence of short deletions. This observation is consistent with expectation of non-homologous end joining (NHEJ) activity. Results have also shown a NHEJ-mediated capture of an oligonucleotide duplex at the endogenous NTF3 locus. These results confirmed that NHEJ-mediated genome modification by the use of TALEN architecture is possible in mammalian environment.

== Efficient modification by NHEJ and homology directed repair (HDR) ==

To expand the practical application for this developed system, modification at an additional locus was proposed. CCR5 gene was targeted for modification. First, a set of TALENs with a range of target lengths from 13 to 17 bp and of separation distance from 5 to 27 bp were designed and tested to identify values compatible with endogenous activity. To this end, a cluster of four left and four right binding sites were chosen for targeting. This defined a matrix of 16 heterodimer targets. For each binding site, two alternative proteins were generated, with 28 or 63 residues on C-terminal (as before). Altogether, this yielded 8x8=64 combinations of left and right proteins. These were expressed by pairs in K562 cells under standard conditions. Once again, Surveyor assay was used for evaluation of allele modification. Obtained results indicate that introduction of some +63/+63 pairs yielded over 20% allele modification. TALEN treated cells seem to retain a stable modification lever after 10 day growth, suggesting that TALENs are generally well tolerated. The effect of spacer length on activity is observed once again. At least 5% modification is achieved by the use of 12 – 19 bp spacer length in the +63/+63 TALENs section. In contrast, +28/+28 section show maximal activity in narrow spacer range of 12 – 13 bp.
The next challenge was to show that it is possible to induce gene through HDR using this system. For this, a part of CCR5 region was selected for targeting, also mentioned as safe harbor previously used for transgene integration. 24 TALEN pairs were then screened for their ability to induce NHEJ. Two highly active combinations were identified, first with 18% mutation rate and second with 21%. These TALEN were introduced into K562 cells simultaneously with donor DNA fragment carrying 46 bp with restriction site BglI. The success of integration was proven by PCR and BglI digestion. About 16% of inspected alleles were modified. This is an example how TALEN architecture can be used to activate HDR.

== Conclusion ==

In presented study it is shown how TALEN architecture can be designed and generated for efficient genome editing in mammalian environment. In this case, the system consists of TALE truncation variants, linked to catalytic domain of FokI. Engineered TALENs can be used for gene modification of human cells by activating either NHEJ or HDR repair pathways. Furthermore, three distinct applications are demonstrated; NHEJ mediated gene disruption, NHEJ-mediated insertion by capture of a DNA fragment and HDR mediated gene editing. Aforementioned suggests that TALEN architecture is a compelling tool in synthetic biology.

== Discussion ==

A step to facilitate the use of this architecture would undoubtedly be the linkage of minimal central TALE repeat domain to the catalytic domain of FokI. This was previously attempted by several groups without success, whereas the lack of activity of such constructs [8]. An alternative idea is to use a full length TALE for targeting. Disadvantage of the latter is the size of linkage peptide connecting two domains, which would affect the nuclease activity. Third approach is to generate truncated variants of TALEs and link them with catalytic domain of FokI, as shown in this paper. This approach has proven affective when using truncated variants with 28 or 63 of the 278 original C-terminal residues.
During the experimental work of this study, two papers were submitted where they demonstrated the use of TALEN architecture with the use of unmodified TALE or TALE with slightly reduced C-terminus [8], [9]. These systems were used for episome cleavage in yeast, an approach that is hardly translated to more complex genomes. As expected, these proteins have extended range of cleavage compared to truncated variants.
Presented TALEN architecture is in some characteristics similar to ZFNs. One example is the high cleavage activity of TALENs, which enables high gene modification efficiency, which is in some cases superior to the one of ZFNs. A shortcoming of TALEN architecture compared to ZFNs is its relative size. TALE repeats are 3 – 4 times larger than ZFPs, which may cause difficulty in delivery methods. Also, limitations due to high levels of TALE repeat homology may complicate the assembly of constructs. Future experiments will provide a fuller picture of pros and cons of the two platforms.
Targeted endogenous gene regulation by TALE proteins is a useful tool in research and for biotechnology applications. We can use this technology in a broad sense, such as design and generation of transcriptional activators or repressors. Although TALEs were discovered as such, previous studies did not inspect this type of activity outside of their native plant context. The experiments shown in this paper suggest that TALEs can be used as transcriptional regulators.
The use of SELEX assay for identification and characterization of TALEs has previously not been reported. By using this method, it was possible to assess the DNA binding preferences of natural and designed TALE proteins. Binding sites for TALE13 and four engineered TALEs were examined. On this basis, an observation that 73 out of 76 TALE repeats facilitate base preference of RVD. An interpretation of rare exceptions is proposed, facilitating further design and use of this system. Overall data collected can be used to examine previously acquired information on RVDs and thereby gaining more insight on mechanisms of action and selectivity. Findings that common RVDs such as NI, HD, NN and NG selectively bound selectively bound adenine, cytosine, guanine and thymine, respectively, are in accordance with previous knowledge. Pattern NN has shown relaxed specificity with substantial binding to a second base, adenine. NK is therefore more suitable for specificity, due to much stronger preference for guanine. This is a potential enhancement, proposed in this paper.

== Expectations for the future ==

As already mentioned, targeting the designated loci with ZFNs or TALENs is often difficult and time consuming, due to design and generation of new custom ZF or TALE array for each locus. This is not the case in most recent approach in genome editing, that is the use of CRISPR-Cas9 system. Expression of distinct gRNAs alongside Cas9 results in Cas9–gRNA complexes with distinct cleavage specificities [10]. This approach is exploited in multiplex genome editing. Still, there are features of this system which are inferior to elderly time-demanding ZFNs or TALENs.
One of those features is size of gene constructs, which is a limitation in field of gene delivery. Cas9 is the size of about 160 kDa, whereas a typical 17,5 repeat TALEN corresponds to 105 kDa. By far the smallest are ZFNs, wherein typical four finger ZFN has about 40 kDa [10]. It is likely for this compact nature to be beneficial for certain experiments.
As for specificity, the number of bases for modification in Cas9 system is restricted to 20 and is somewhat limited with the rule of three guanine bases flanking the target site (GN19NGG) [10]. The main constriction of ZF arrays is a scarce set of fingers, resulting in inability to target certain triplets. In addition, some ZFs targeting GNN seem to be superior to other ZFs, contributing to limitations. TALE target specificity therefore seems superior, with capabilities of targeting up to 24 bases. For Cas9 system, off target studies in mammalian environment are yet to be conducted [10].
It is likely that distinct features will lead to differential use of genome editing systems for distinct applications in the fields of plant science, therapeutic sciences and biotechnology applications.

== Literature ==

[1] J. C. Miller, S. Tan, G. Qiao, K. A. Barlow, J. Wang, D. F. Xia, X. Meng, D. E. Paschon, E. Leung, S. J. Hinkley, G. P. Dulay, K. L. Hua, I. Ankoudinova, G. J. Cost, F. D. Urnov, H. S. Zhang, M. C. Holmes, L. Zhang, P. D. Gregory, and E. J. Rebar, “A TALE nuclease architecture for efficient genome editing,” Nat. Biotechnol., vol. 29, no. 2, pp. 143–148, Feb. 2011.

[2] “Genome Editing.” [Online]. Available: http://www.allelebiotech.com/genome-editing/. [Accessed: 06-Jan-2015].

[3] R. M. Gupta and K. Musunuru, “Expanding the genetic editing tool kit: ZFNs, TALENs, and CRISPR-Cas9,” J. Clin. Invest., vol. 124, no. 10, pp. 4154–4161, Oct. 2014.

[4] M. Jinek, K. Chylinski, I. Fonfara, M. Hauer, J. A. Doudna, and E. Charpentier, “A programmable dual-RNA-guided DNA endonuclease in adaptive bacterial immunity,” Science, vol. 337, no. 6096, pp. 816–821, Aug. 2012.

[5] “CRISPR,” Wikipedia, the free encyclopedia. 09-Jan-2015.

[6] “TAL effector,” Wikipedia, the free encyclopedia. 06-Jan-2015.

[7] J. C. Miller, M. C. Holmes, J. Wang, D. Y. Guschin, Y.-L. Lee, I. Rupniewski, C. M. Beausejour, A. J. Waite, N. S. Wang, K. A. Kim, P. D. Gregory, C. O. Pabo, and E. J. Rebar, “An improved zinc-finger nuclease architecture for highly specific genome editing,” Nat. Biotechnol., vol. 25, no. 7, pp. 778–785, Jul. 2007.

[8] M. Christian, T. Cermak, E. L. Doyle, C. Schmidt, F. Zhang, A. Hummel, A. J. Bogdanove, and D. F. Voytas, “Targeting DNA double-strand breaks with TAL effector nucleases,” Genetics, vol. 186, no. 2, pp. 757–761, Oct. 2010.

[9] T. Li, S. Huang, W. Z. Jiang, D. Wright, M. H. Spalding, D. P. Weeks, and B. Yang, “TAL nucleases (TALNs): hybrid proteins composed of TAL effectors and FokI DNA-cleavage domain,” Nucleic Acids Res., vol. 39, no. 1, pp. 359–372, Jan. 2011.

[10] A. Strauβ and T. Lahaye, “Zinc Fingers, TAL Effectors, or Cas9-Based DNA Binding Proteins: What’s Best for Targeting Desired Genome Loci?,” Mol. Plant, vol. 6, no. 5, pp. 1384–1387, Sep. 2013.

A TALE nuclease architecture for efficient genome editing

2015-01-11T15:28:31Z

JernejMustar:

Summarized from Miller, J. C. ''et al''. A TALE nuclease architecture for efficient genome editing. ''Nat. Biotechnol.'', vol. 29, pp. 143–148. Feb. 2011.

== Introduction to genome editing ==

Genome editing is a type of genetic engineering, where an organisms DNA is manipulated by altering; inserting, removing or replacing nucleotides, therefore changing its genome. This process is performed by the use of immensely useful tool, t. i. engineered nucleases, which are capable of cutting both strands of DNA or nicking single strand. Resulting sites are detected and repaired by endogenous repair mechanisms, which include homologous recombination (HR) or non-homologous end joining (NHEJ). The latter is error prone, therefore it is likely for mutations to occur in repair site. These repair pathways are highly conserved in diverse cell types, which is beneficial for the use of genome editing technologies. As a result, various cell types have been engineered by the use of potable cleavage-based technologies, including human stem cells [1].

Recently, there has been tremendous development in the field of synthetic biology and genome editing technology. Virtually any gene can be manipulated in diverse range of organisms. Most common approach is the use of nuclease, which can be manipulated to target specific sequences of genome. This can be achieved by exploitation of sequence-specific DNA-binding domains (ZFNs, TALENs) or by the use of complementary guide RNA (CRISPR-Cas9 system).
In this paper, the history of genome editing will first be briefly reviewed, further on genome editing will be discussed with emphasis on the TALEN’s family.

== Brief history of genome editing ==

Past studies in the field of genome editing were perusing a common goal, t. i. to obtain high specificity, which would limit the target affects [2]. The first engineered technology, based on nuclease activity, was introduced in 1991 by Pavletich and Pabo in the journal Science [2]. This technology by the name of Zinc Finger nuclease was a great breakthrough and was later on frequently applied for genome targeting. It is based on the use of cleavage domain of restriction enzyme FokI, which is linked with designated zinc finger protein (ZFP), which is responsible for targeting. By the use of different combinations of ZFP domains the selectivity for single genomic cleavage event is achieved. The use of nuclease domains with monomeric activity there is a high probability of off-target events. This can be avoided by the use of a pair of ZFNs with distinct FokI domains that are obligate heterodimers [3]. Overtime, reports of problems and drawbacks of this technology were reported, such as targeting problem for certain triplets and reduced specificity, suffered by interactions within a zinc finger array [2].
The next technology, which emerged in 2009, is called transcription activator-like effector nuclease (TALENs). These are fusion proteins, where nuclease domain is associated with TAL effector DNA binding domain. This approach may be better in certain aspects, for example it enables a larger spectrum of targets and it’s simpler and more practical than ZFNs. In 2011, TALENs was proclaimed as “Method of the Year” by the journal Nature methods. The details of this system and molecular mechanisms of function are described later on.
We probably all at least heard if not read about latest breakthrough in the field of genome editing, which is CRISPR-Cas9 system. The discovery was somewhat fortuity, because researchers were studying how bacteria defend against phages and foreign plasmids. The paper was published in August 2012 in journal Science [4]. A bacterial nuclease called Cas9 is capable of altering genomes of invading viruses, consequently inactivating them. Clustered regularly interspaced short palindromic repeats (CRISPR) are DNA loci that contain short sequence repetitions, which can be transcribed, processed and then used to guide nuclease to a specific site [5]. This is in contrast with TALEN and Zinc Finger methods, where the need to prepare customized nuclease for each target remains.

== A TALE nuclease architecture ==

As mentioned before, two studies from 2009 proposed the use of TALEs effectors for targeted genome engineering. These transcriptional activators originate from Xanthomonas plant pathogens. During pathogenesis, TALEs specifically bind and regulate plant gene expression, thus aiding bacterial infection [6]. A central repeat domain, which mediates DNA recognition, is located within TALE structure [1]. Each repeat unit comprises of 33 – 35 amino, these specify one target base. The units base preference is determined by two crucial adjacent amino acids, also called the “repeat variable di-residue” (RVD). Therefore, the sequence of specified units in central repeat domain determines the target sequence on genome. TALEs generated with new repeat combinations have been shown to recognize target sequences predicted by this code.
In the light of aforementioned advances the interest in potential use of this system in combination with nuclease was initiated. Subsequently, a TALE-nuclease chimeras (TALENs) was proposed, which would allow for site-specific genome cleavage. In selected paper, the advances in the field of TALEN’s technology are discussed. TALE activity in a mammalian cell environment for targeted regulation of episomal reporters and an endogenous gene is presented, resuming with the research of the minimal TALE region required for high-affinity DNA binding. Paper also includes practical application, showing modification of endogenous human genes NTF3 and CCR5 by the use of TALEN approach.

== Retargeting a natural TALE to an endogenous mammalian sequence ==

Firstly, a suitable TAL effector for this study was chosen. Criteria for TALE selection was based on specificity and empirical evidence. The group was searching for natural TALE with high specificity for target sequence and with confirmed activity in mammalian cells. To achive this goal, PCR amplifications for several TALEs using Xanthomonas axonopodis pathovar citri genomic DNA were performed. Native TALE-associated sequences which allow transport into plant cells were excluded. Protein products of acquired coding regions were characterized by a SELEX assay using target of interest. Outcome of this selection yield one higly selective candidate, identified as TALE13. When combined with VP-16 activation domain, a 70-fold induction of reporter gene expression rate was observed in HEK293 cells. Based on these results, TALE13 was chosen for further work and design.
First goal was to demonstrate that chosen candidate was appropriate for mammalian gene regulation. Chosen target was NTF3 gene, which encodes a secreted nerve growth factor that has therapeutic potential for neurodegenerative diseases. Wild type TALE usually contain 18 repeats, by which they target specific sequence and bind to proximal promotor elements. TALE13 was modified to the extent of recognising proximal promotor region of NTF3 by replacing the 18 wild-type repeat units with appropriate alternatives. This altered TALE was named NT-L (meaning NTF3 Left), which highlits its binding position on target. In order to test its aktivity, the truncated version of NT-L was fused with VP-16 and expressed in human HEK293 cells. By measuring the activity of endogenous NTF3 locus, a strong induction (over 20 fold) in both NTF3 transcript and protein product was detected. The subsequent SELEX analysis revealed high specificity. These results revealed, that NT-L variant is a viable candidate for endogenous mammalian gene regulation.

== Development of a TALE-nuclease architecture ==

The next step was to design TALENs chimera protein using NT-L and nuclease, which would allow implementation of targeted cleavage of its genome target. The process was realized through three stages.
First, the minimal region required for high affinity bonding was determined using gene regulation studies. This was beneficial for optimization of nuclease (FokI) cleavage domain attachment with its target. Desired outcome of this step was improved catalytic activity of chimeric tool. The truncated variant named NT-L+95 was shown to retain high specificity and at the same time contribute to improved nuclease attachment. In order to examine this, a C-terminal truncated series of TALE13 were generated and subsequently linked with FokI. The resulting candidates were screened as homodimers for cleavage of specific targets. The results have shown several interesting occurrences. For TALENs comprising of NT-L+95 no detectable nuclease activity was observed. The cause for this is unclear; it is possible that this is due to a certain effect of NT-L+95 to nuclease domain positioning. Efficient DNA cleavage was observed for certain shorter specimens, providing candidates for cellular studies.
The minimal region between TALEN binding sites for optimal activity is over 12 bp, otherwise reduce cleavage may occur. These observations are useful for design of TALENs used for in vivo studies.
In the last stage, homodimer TALEN partner for NT-L was designed and named NT-R (NTR3 Right), indicating the direction of binding. This pair was expressed in human
K562 cells as fusions with catalytic domain of FokI. Target locus was later on tested for gene modification. By using two different C-terminal subregions (+28 and +63) the designed TALENs were tested. Significant activity in in vitro cleavage study was detected. With expression at 37°C, up to 3% modification was observed by the Surveyor assay [7]. This assay reveals gene modification by the appearance of digestion products. The percentage of gene modification can be obtained by quantification of “cut” bands. Limit of detection of this assay is about 1%. The activity was higher (9% of modification) in conditions of transient hypothermia, t. i. at 30°C. Sequences from latter were analyzed by Sanger sequencing. Out of 84 analyzed alleles there were 7 mutated by the occurrence of short deletions. This observation is consistent with expectation of non-homologous end joining (NHEJ) activity. Results have also shown a NHEJ-mediated capture of an oligonucleotide duplex at the endogenous NTF3 locus. These results confirmed that NHEJ-mediated genome modification by the use of TALEN architecture is possible in mammalian environment.

== Efficient modification by NHEJ and homology directed repair (HDR) ==

To expand the practical application for this developed system, modification at an additional locus was proposed. CCR5 gene was targeted for modification. First, a set of TALENs with a range of target lengths from 13 to 17 bp and of separation distance from 5 to 27 bp were designed and tested to identify values compatible with endogenous activity. To this end, a cluster of four left and four right binding sites were chosen for targeting. This defined a matrix of 16 heterodimer targets. For each binding site, two alternative proteins were generated, with 28 or 63 residues on C-terminal (as before). Altogether, this yielded 8x8=64 combinations of left and right proteins. These were expressed by pairs in K562 cells under standard conditions. Once again, Surveyor assay was used for evaluation of allele modification. Obtained results indicate that introduction of some +63/+63 pairs yielded over 20% allele modification. TALEN treated cells seem to retain a stable modification lever after 10 day growth, suggesting that TALENs are generally well tolerated. The effect of spacer length on activity is observed once again. At least 5% modification is achieved by the use of 12 – 19 bp spacer length in the +63/+63 TALENs section. In contrast, +28/+28 section show maximal activity in narrow spacer range of 12 – 13 bp.
The next challenge was to show that it is possible to induce gene through HDR using this system. For this, a part of CCR5 region was selected for targeting, also mentioned as safe harbor previously used for transgene integration. 24 TALEN pairs were then screened for their ability to induce NHEJ. Two highly active combinations were identified, first with 18% mutation rate and second with 21%. These TALEN were introduced into K562 cells simultaneously with donor DNA fragment carrying 46 bp with restriction site BglI. The success of integration was proven by PCR and BglI digestion. About 16% of inspected alleles were modified. This is an example how TALEN architecture can be used to activate HDR.

== Conclusion ==

In presented study it is shown how TALEN architecture can be designed and generated for efficient genome editing in mammalian environment. In this case, the system consists of TALE truncation variants, linked to catalytic domain of FokI. Engineered TALENs can be used for gene modification of human cells by activating either NHEJ or HDR repair pathways. Furthermore, three distinct applications are demonstrated; NHEJ mediated gene disruption, NHEJ-mediated insertion by capture of a DNA fragment and HDR mediated gene editing. Aforementioned suggests that TALEN architecture is a compelling tool in synthetic biology.

== Discussion ==

A step to facilitate the use of this architecture would undoubtedly be the linkage of minimal central TALE repeat domain to the catalytic domain of FokI. This was previously attempted by several groups without success, whereas the lack of activity of such constructs [8]. An alternative idea is to use a full length TALE for targeting. Disadvantage of the latter is the size of linkage peptide connecting two domains, which would affect the nuclease activity. Third approach is to generate truncated variants of TALEs and link them with catalytic domain of FokI, as shown in this paper. This approach has proven affective when using truncated variants with 28 or 63 of the 278 original C-terminal residues.
During the experimental work of this study, two papers were submitted where they demonstrated the use of TALEN architecture with the use of unmodified TALE or TALE with slightly reduced C-terminus [8], [9]. These systems were used for episome cleavage in yeast, an approach that is hardly translated to more complex genomes. As expected, these proteins have extended range of cleavage compared to truncated variants.
Presented TALEN architecture is in some characteristics similar to ZFNs. One example is the high cleavage activity of TALENs, which enables high gene modification efficiency, which is in some cases superior to the one of ZFNs. A shortcoming of TALEN architecture compared to ZFNs is its relative size. TALE repeats are 3 – 4 times larger than ZFPs, which may cause difficulty in delivery methods. Also, limitations due to high levels of TALE repeat homology may complicate the assembly of constructs. Future experiments will provide a fuller picture of pros and cons of the two platforms.
Targeted endogenous gene regulation by TALE proteins is a useful tool in research and for biotechnology applications. We can use this technology in a broad sense, such as design and generation of transcriptional activators or repressors. Although TALEs were discovered as such, previous studies did not inspect this type of activity outside of their native plant context. The experiments shown in this paper suggest that TALEs can be used as transcriptional regulators.
The use of SELEX assay for identification and characterization of TALEs has previously not been reported. By using this method, it was possible to assess the DNA binding preferences of natural and designed TALE proteins. Binding sites for TALE13 and four engineered TALEs were examined. On this basis, an observation that 73 out of 76 TALE repeats facilitate base preference of RVD. An interpretation of rare exceptions is proposed, facilitating further design and use of this system. Overall data collected can be used to examine previously acquired information on RVDs and thereby gaining more insight on mechanisms of action and selectivity. Findings that common RVDs such as NI, HD, NN and NG selectively bound selectively bound adenine, cytosine, guanine and thymine, respectively, are in accordance with previous knowledge. Pattern NN has shown relaxed specificity with substantial binding to a second base, adenine. NK is therefore more suitable for specificity, due to much stronger preference for guanine. This is a potential enhancement, proposed in this paper.

== Expectations for the future ==

As already mentioned, targeting the designated loci with ZFNs or TALENs is often difficult and time consuming, due to design and generation of new custom ZF or TALE array for each locus. This is not the case in most recent approach in genome editing, that is the use of CRISPR-Cas9 system. Expression of distinct gRNAs alongside Cas9 results in Cas9–gRNA complexes with distinct cleavage specificities [10]. This approach is exploited in multiplex genome editing. Still, there are features of this system which are inferior to elderly time-demanding ZFNs or TALENs.
One of those features is size of gene constructs, which is a limitation in field of gene delivery. Cas9 is the size of about 160 kDa, whereas a typical 17,5 repeat TALEN corresponds to 105 kDa. By far the smallest are ZFNs, wherein typical four finger ZFN has about 40 kDa [10]. It is likely for this compact nature to be beneficial for certain experiments.
As for specificity, the number of bases for modification in Cas9 system is restricted to 20 and is somewhat limited with the rule of three guanine bases flanking the target site (GN19NGG) [10]. The main constriction of ZF arrays is a scarce set of fingers, resulting in inability to target certain triplets. In addition, some ZFs targeting GNN seem to be superior to other ZFs, contributing to limitations. TALE target specificity therefore seems superior, with capabilities of targeting up to 24 bases. For Cas9 system, off target studies in mammalian environment are yet to be conducted [10].
It is likely that distinct features will lead to differential use of genome editing systems for distinct applications in the fields of plant science, therapeutic sciences and biotechnology applications.

== Literature ==

[1] J. C. Miller, S. Tan, G. Qiao, K. A. Barlow, J. Wang, D. F. Xia, X. Meng, D. E. Paschon, E. Leung, S. J. Hinkley, G. P. Dulay, K. L. Hua, I. Ankoudinova, G. J. Cost, F. D. Urnov, H. S. Zhang, M. C. Holmes, L. Zhang, P. D. Gregory, and E. J. Rebar, “A TALE nuclease architecture for efficient genome editing,” Nat. Biotechnol., vol. 29, no. 2, pp. 143–148, Feb. 2011.

[2] “Genome Editing.” [Online]. Available: http://www.allelebiotech.com/genome-editing/. [Accessed: 06-Jan-2015].

[3] R. M. Gupta and K. Musunuru, “Expanding the genetic editing tool kit: ZFNs, TALENs, and CRISPR-Cas9,” J. Clin. Invest., vol. 124, no. 10, pp. 4154–4161, Oct. 2014.

[4] M. Jinek, K. Chylinski, I. Fonfara, M. Hauer, J. A. Doudna, and E. Charpentier, “A programmable dual-RNA-guided DNA endonuclease in adaptive bacterial immunity,” Science, vol. 337, no. 6096, pp. 816–821, Aug. 2012.

[5] “CRISPR,” Wikipedia, the free encyclopedia. 09-Jan-2015.

[6] “TAL effector,” Wikipedia, the free encyclopedia. 06-Jan-2015.

[7] J. C. Miller, M. C. Holmes, J. Wang, D. Y. Guschin, Y.-L. Lee, I. Rupniewski, C. M. Beausejour, A. J. Waite, N. S. Wang, K. A. Kim, P. D. Gregory, C. O. Pabo, and E. J. Rebar, “An improved zinc-finger nuclease architecture for highly specific genome editing,” Nat. Biotechnol., vol. 25, no. 7, pp. 778–785, Jul. 2007.

[8] M. Christian, T. Cermak, E. L. Doyle, C. Schmidt, F. Zhang, A. Hummel, A. J. Bogdanove, and D. F. Voytas, “Targeting DNA double-strand breaks with TAL effector nucleases,” Genetics, vol. 186, no. 2, pp. 757–761, Oct. 2010.

[9] T. Li, S. Huang, W. Z. Jiang, D. Wright, M. H. Spalding, D. P. Weeks, and B. Yang, “TAL nucleases (TALNs): hybrid proteins composed of TAL effectors and FokI DNA-cleavage domain,” Nucleic Acids Res., vol. 39, no. 1, pp. 359–372, Jan. 2011.

[10] A. Strauβ and T. Lahaye, “Zinc Fingers, TAL Effectors, or Cas9-Based DNA Binding Proteins: What’s Best for Targeting Desired Genome Loci?,” Mol. Plant, vol. 6, no. 5, pp. 1384–1387, Sep. 2013.

A TALE nuclease architecture for efficient genome editing

2015-01-11T15:26:57Z

JernejMustar:

Summarized from Miller, J. C. ''et al''. A TALE nuclease architecture for efficient genome editing. ''Nat. Biotechnol.'', vol. 29, pp. 143–148. Feb. 2011.

== Introduction to genome editing ==

Genome editing is a type of genetic engineering, where an organisms DNA is manipulated by altering; inserting, removing or replacing nucleotides, therefore changing its genome. This process is performed by the use of immensely useful tool, t. i. engineered nucleases, which are capable of cutting both strands of DNA or nicking single strand. Resulting sites are detected and repaired by endogenous repair mechanisms, which include homologous recombination (HR) or non-homologous end joining (NHEJ). The latter is error prone, therefore it is likely for mutations to occur in repair site. These repair pathways are highly conserved in diverse cell types, which is beneficial for the use of genome editing technologies. As a result, various cell types have been engineered by the use of potable cleavage-based technologies, including human stem cells [1].

Recently, there has been tremendous development in the field of synthetic biology and genome editing technology. Virtually any gene can be manipulated in diverse range of organisms. Most common approach is the use of nuclease, which can be manipulated to target specific sequences of genome. This can be achieved by exploitation of sequence-specific DNA-binding domains (ZFNs, TALENs) or by the use of complementary guide RNA (CRISPR-Cas9 system).
In this paper, the history of genome editing will first be briefly reviewed, further on genome editing will be discussed with emphasis on the TALEN’s family.

== Brief history of genome editing ==

Past studies in the field of genome editing were perusing a common goal, t. i. to obtain high specificity, which would limit the target affects [2]. The first engineered technology, based on nuclease activity, was introduced in 1991 by Pavletich and Pabo in the journal Science [2]. This technology by the name of Zinc Finger nuclease was a great breakthrough and was later on frequently applied for genome targeting. It is based on the use of cleavage domain of restriction enzyme FokI, which is linked with designated zinc finger protein (ZFP), which is responsible for targeting. By the use of different combinations of ZFP domains the selectivity for single genomic cleavage event is achieved. The use of nuclease domains with monomeric activity there is a high probability of off-target events. This can be avoided by the use of a pair of ZFNs with distinct FokI domains that are obligate heterodimers [3]. Overtime, reports of problems and drawbacks of this technology were reported, such as targeting problem for certain triplets and reduced specificity, suffered by interactions within a zinc finger array [2].
The next technology, which emerged in 2009, is called transcription activator-like effector nuclease (TALENs). These are fusion proteins, where nuclease domain is associated with TAL effector DNA binding domain. This approach may be better in certain aspects, for example it enables a larger spectrum of targets and it’s simpler and more practical than ZFNs. In 2011, TALENs was proclaimed as “Method of the Year” by the journal Nature methods. The details of this system and molecular mechanisms of function are described later on.
We probably all at least heard if not read about latest breakthrough in the field of genome editing, which is CRISPR-Cas9 system. The discovery was somewhat fortuity, because researchers were studying how bacteria defend against phages and foreign plasmids. The paper was published in August 2012 in journal Science [4]. A bacterial nuclease called Cas9 is capable of altering genomes of invading viruses, consequently inactivating them. Clustered regularly interspaced short palindromic repeats (CRISPR) are DNA loci that contain short sequence repetitions, which can be transcribed, processed and then used to guide nuclease to a specific site [5]. This is in contrast with TALEN and Zinc Finger methods, where the need to prepare customized nuclease for each target remains.

== A TALE nuclease architecture ==

As mentioned before, two studies from 2009 proposed the use of TALEs effectors for targeted genome engineering. These transcriptional activators originate from Xanthomonas plant pathogens. During pathogenesis, TALEs specifically bind and regulate plant gene expression, thus aiding bacterial infection [6]. A central repeat domain, which mediates DNA recognition, is located within TALE structure [1]. Each repeat unit comprises of 33 – 35 amino, these specify one target base. The units base preference is determined by two crucial adjacent amino acids, also called the “repeat variable di-residue” (RVD). Therefore, the sequence of specified units in central repeat domain determines the target sequence on genome. TALEs generated with new repeat combinations have been shown to recognize target sequences predicted by this code.
In the light of aforementioned advances the interest in potential use of this system in combination with nuclease was initiated. Subsequently, a TALE-nuclease chimeras (TALENs) was proposed, which would allow for site-specific genome cleavage. In selected paper, the advances in the field of TALEN’s technology are discussed. TALE activity in a mammalian cell environment for targeted regulation of episomal reporters and an endogenous gene is presented, resuming with the research of the minimal TALE region required for high-affinity DNA binding. Paper also includes practical application, showing modification of endogenous human genes NTF3 and CCR5 by the use of TALEN approach.

== Retargeting a natural TALE to an endogenous mammalian sequence ==

Firstly, a suitable TAL effector for this study was chosen. Criteria for TALE selection was based on specificity and empirical evidence. The group was searching for natural TALE with high specificity for target sequence and with confirmed activity in mammalian cells. To achive this goal, PCR amplifications for several TALEs using Xanthomonas axonopodis pathovar citri genomic DNA were performed. Native TALE-associated sequences which allow transport into plant cells were excluded. Protein products of acquired coding regions were characterized by a SELEX assay using target of interest. Outcome of this selection yield one higly selective candidate, identified as TALE13. When combined with VP-16 activation domain, a 70-fold induction of reporter gene expression rate was observed in HEK293 cells. Based on these results, TALE13 was chosen for further work and design.
First goal was to demonstrate that chosen candidate was appropriate for mammalian gene regulation. Chosen target was NTF3 gene, which encodes a secreted nerve growth factor that has therapeutic potential for neurodegenerative diseases. Wild type TALE usually contain 18 repeats, by which they target specific sequence and bind to proximal promotor elements. TALE13 was modified to the extent of recognising proximal promotor region of NTF3 by replacing the 18 wild-type repeat units with appropriate alternatives. This altered TALE was named NT-L (meaning NTF3 Left), which highlits its binding position on target. In order to test its aktivity, the truncated version of NT-L was fused with VP-16 and expressed in human HEK293 cells. By measuring the activity of endogenous NTF3 locus, a strong induction (over 20 fold) in both NTF3 transcript and protein product was detected. The subsequent SELEX analysis revealed high specificity. These resoults revealed, that NT-L variant is a viable candidate for endogenous mammalian gene regulation.

== Development of a TALE-nuclease architecture ==

The next step was to design TALENs chimera protein using NT-L and nuclease, which would allow implementation of targeted cleavage of its genome target. The process was realized through three stages.
First, the minimal region required for high affinity bonding was determined using gene regulation studies. This was beneficial for optimization of nuclease (FokI) cleavage domain attachment with its target. Desired outcome of this step was improved catalytic activity of chimeric tool. The truncated variant named NT-L+95 was shown to retain high specificity and at the same time contribute to improved nuclease attachment. In order to examine this, a C-terminal truncated series of TALE13 were generated and subsequently linked with FokI. The resulting candidates were screened as homodimers for cleavage of specific targets. The results have shown several interesting occurrences. For TALENs comprising of NT-L+95 no detectable nuclease activity was observed. The cause for this is unclear; it is possible that this is due to a certain effect of NT-L+95 to nuclease domain positioning. Efficient DNA cleavage was observed for certain shorter specimens, providing candidates for cellular studies.
The minimal region between TALEN binding sites for optimal activity is over 12 bp, otherwise reduce cleavage may occur. These observations are useful for design of TALENs used for in vivo studies.
In the last stage, homodimer TALEN partner for NT-L was designed and named NT-R (NTR3 Right), indicating the direction of binding. This pair was expressed in human
K562 cells as fusions with catalytic domain of FokI. Target locus was later on tested for gene modification. By using two different C-terminal subregions (+28 and +63) the designed TALENs were tested. Significant activity in in vitro cleavage study was detected. With expression at 37°C, up to 3% modification was observed by the Surveyor assay [7]. This assay reveals gene modification by the appearance of digestion products. The percentage of gene modification can be obtained by quantification of “cut” bands. Limit of detection of this assay is about 1%. The activity was higher (9% of modification) in conditions of transient hypothermia, t. i. at 30°C. Sequences from latter were analyzed by Sanger sequencing. Out of 84 analyzed alleles there were 7 mutated by the occurrence of short deletions. This observation is consistent with expectation of non-homologous end joining (NHEJ) activity. Results have also shown a NHEJ-mediated capture of an oligonucleotide duplex at the endogenous NTF3 locus. These results confirmed that NHEJ-mediated genome modification by the use of TALEN architecture is possible in mammalian environment.

== Efficient modification by NHEJ and homology directed repair (HDR) ==

To expand the practical application for this developed system, modification at an additional locus was proposed. CCR5 gene was targeted for modification. First, a set of TALENs with a range of target lengths from 13 to 17 bp and of separation distance from 5 to 27 bp were designed and tested to identify values compatible with endogenous activity. To this end, a cluster of four left and four right binding sites were chosen for targeting. This defined a matrix of 16 heterodimer targets. For each binding site, two alternative proteins were generated, with 28 or 63 residues on C-terminal (as before). Altogether, this yielded 8x8=64 combinations of left and right proteins. These were expressed by pairs in K562 cells under standard conditions. Once again, Surveyor assay was used for evaluation of allele modification. Obtained results indicate that introduction of some +63/+63 pairs yielded over 20% allele modification. TALEN treated cells seem to retain a stable modification lever after 10 day growth, suggesting that TALENs are generally well tolerated. The effect of spacer length on activity is observed once again. At least 5% modification is achieved by the use of 12 – 19 bp spacer length in the +63/+63 TALENs section. In contrast, +28/+28 section show maximal activity in narrow spacer range of 12 – 13 bp.
The next challenge was to show that it is possible to induce gene through HDR using this system. For this, a part of CCR5 region was selected for targeting, also mentioned as safe harbor previously used for transgene integration. 24 TALEN pairs were then screened for their ability to induce NHEJ. Two highly active combinations were identified, first with 18% mutation rate and second with 21%. These TALEN were introduced into K562 cells simultaneously with donor DNA fragment carrying 46 bp with restriction site BglI. The success of integration was proven by PCR and BglI digestion. About 16% of inspected alleles were modified. This is an example how TALEN architecture can be used to activate HDR.

== Conclusion ==

In presented study it is shown how TALEN architecture can be designed and generated for efficient genome editing in mammalian environment. In this case, the system consists of TALE truncation variants, linked to catalytic domain of FokI. Engineered TALENs can be used for gene modification of human cells by activating either NHEJ or HDR repair pathways. Furthermore, three distinct applications are demonstrated; NHEJ mediated gene disruption, NHEJ-mediated insertion by capture of a DNA fragment and HDR mediated gene editing. Aforementioned suggests that TALEN architecture is a compelling tool in synthetic biology.

== Discussion ==

A step to facilitate the use of this architecture would undoubtedly be the linkage of minimal central TALE repeat domain to the catalytic domain of FokI. This was previously attempted by several groups without success, whereas the lack of activity of such constructs [8]. An alternative idea is to use a full length TALE for targeting. Disadvantage of the latter is the size of linkage peptide connecting two domains, which would affect the nuclease activity. Third approach is to generate truncated variants of TALEs and link them with catalytic domain of FokI, as shown in this paper. This approach has proven affective when using truncated variants with 28 or 63 of the 278 original C-terminal residues.
During the experimental work of this study, two papers were submitted where they demonstrated the use of TALEN architecture with the use of unmodified TALE or TALE with slightly reduced C-terminus [8], [9]. These systems were used for episome cleavage in yeast, an approach that is hardly translated to more complex genomes. As expected, these proteins have extended range of cleavage compared to truncated variants.
Presented TALEN architecture is in some characteristics similar to ZFNs. One example is the high cleavage activity of TALENs, which enables high gene modification efficiency, which is in some cases superior to the one of ZFNs. A shortcoming of TALEN architecture compared to ZFNs is its relative size. TALE repeats are 3 – 4 times larger than ZFPs, which may cause difficulty in delivery methods. Also, limitations due to high levels of TALE repeat homology may complicate the assembly of constructs. Future experiments will provide a fuller picture of pros and cons of the two platforms.
Targeted endogenous gene regulation by TALE proteins is a useful tool in research and for biotechnology applications. We can use this technology in a broad sense, such as design and generation of transcriptional activators or repressors. Although TALEs were discovered as such, previous studies did not inspect this type of activity outside of their native plant context. The experiments shown in this paper suggest that TALEs can be used as transcriptional regulators.
The use of SELEX assay for identification and characterization of TALEs has previously not been reported. By using this method, it was possible to assess the DNA binding preferences of natural and designed TALE proteins. Binding sites for TALE13 and four engineered TALEs were examined. On this basis, an observation that 73 out of 76 TALE repeats facilitate base preference of RVD. An interpretation of rare exceptions is proposed, facilitating further design and use of this system. Overall data collected can be used to examine previously acquired information on RVDs and thereby gaining more insight on mechanisms of action and selectivity. Findings that common RVDs such as NI, HD, NN and NG selectively bound selectively bound adenine, cytosine, guanine and thymine, respectively, are in accordance with previous knowledge. Pattern NN has shown relaxed specificity with substantial binding to a second base, adenine. NK is therefore more suitable for specificity, due to much stronger preference for guanine. This is a potential enhancement, proposed in this paper.

== Expectations for the future ==

As already mentioned, targeting the designated loci with ZFNs or TALENs is often difficult and time consuming, due to design and generation of new custom ZF or TALE array for each locus. This is not the case in most recent approach in genome editing, that is the use of CRISPR-Cas9 system. Expression of distinct gRNAs alongside Cas9 results in Cas9–gRNA complexes with distinct cleavage specificities [10]. This approach is exploited in multiplex genome editing. Still, there are features of this system which are inferior to elderly time-demanding ZFNs or TALENs.
One of those features is size of gene constructs, which is a limitation in field of gene delivery. Cas9 is the size of about 160 kDa, whereas a typical 17,5 repeat TALEN corresponds to 105 kDa. By far the smallest are ZFNs, wherein typical four finger ZFN has about 40 kDa [10]. It is likely for this compact nature to be beneficial for certain experiments.
As for specificity, the number of bases for modification in Cas9 system is restricted to 20 and is somewhat limited with the rule of three guanine bases flanking the target site (GN19NGG) [10]. The main constriction of ZF arrays is a scarce set of fingers, resulting in inability to target certain triplets. In addition, some ZFs targeting GNN seem to be superior to other ZFs, contributing to limitations. TALE target specificity therefore seems superior, with capabilities of targeting up to 24 bases. For Cas9 system, off target studies in mammalian environment are yet to be conducted [10].
It is likely that distinct features will lead to differential use of genome editing systems for distinct applications in the fields of plant science, therapeutic sciences and biotechnology applications.

== Literature ==

[1] J. C. Miller, S. Tan, G. Qiao, K. A. Barlow, J. Wang, D. F. Xia, X. Meng, D. E. Paschon, E. Leung, S. J. Hinkley, G. P. Dulay, K. L. Hua, I. Ankoudinova, G. J. Cost, F. D. Urnov, H. S. Zhang, M. C. Holmes, L. Zhang, P. D. Gregory, and E. J. Rebar, “A TALE nuclease architecture for efficient genome editing,” Nat. Biotechnol., vol. 29, no. 2, pp. 143–148, Feb. 2011.

[2] “Genome Editing.” [Online]. Available: http://www.allelebiotech.com/genome-editing/. [Accessed: 06-Jan-2015].

[3] R. M. Gupta and K. Musunuru, “Expanding the genetic editing tool kit: ZFNs, TALENs, and CRISPR-Cas9,” J. Clin. Invest., vol. 124, no. 10, pp. 4154–4161, Oct. 2014.

[4] M. Jinek, K. Chylinski, I. Fonfara, M. Hauer, J. A. Doudna, and E. Charpentier, “A programmable dual-RNA-guided DNA endonuclease in adaptive bacterial immunity,” Science, vol. 337, no. 6096, pp. 816–821, Aug. 2012.

[5] “CRISPR,” Wikipedia, the free encyclopedia. 09-Jan-2015.

[6] “TAL effector,” Wikipedia, the free encyclopedia. 06-Jan-2015.

[7] J. C. Miller, M. C. Holmes, J. Wang, D. Y. Guschin, Y.-L. Lee, I. Rupniewski, C. M. Beausejour, A. J. Waite, N. S. Wang, K. A. Kim, P. D. Gregory, C. O. Pabo, and E. J. Rebar, “An improved zinc-finger nuclease architecture for highly specific genome editing,” Nat. Biotechnol., vol. 25, no. 7, pp. 778–785, Jul. 2007.

[8] M. Christian, T. Cermak, E. L. Doyle, C. Schmidt, F. Zhang, A. Hummel, A. J. Bogdanove, and D. F. Voytas, “Targeting DNA double-strand breaks with TAL effector nucleases,” Genetics, vol. 186, no. 2, pp. 757–761, Oct. 2010.

[9] T. Li, S. Huang, W. Z. Jiang, D. Wright, M. H. Spalding, D. P. Weeks, and B. Yang, “TAL nucleases (TALNs): hybrid proteins composed of TAL effectors and FokI DNA-cleavage domain,” Nucleic Acids Res., vol. 39, no. 1, pp. 359–372, Jan. 2011.

[10] A. Strauβ and T. Lahaye, “Zinc Fingers, TAL Effectors, or Cas9-Based DNA Binding Proteins: What’s Best for Targeting Desired Genome Loci?,” Mol. Plant, vol. 6, no. 5, pp. 1384–1387, Sep. 2013.

A TALE nuclease architecture for efficient genome editing

2015-01-11T15:23:44Z

JernejMustar: New page: = '''A TALE nuclease architecture for efficient genome editing.''' = Summarized from Miller, J. C. ''et al''. A TALE nuclease architecture for efficient genome editing. ''Nat. Biotechnol....

= '''A TALE nuclease architecture for efficient genome editing.''' =

Summarized from Miller, J. C. ''et al''. A TALE nuclease architecture for efficient genome editing. ''Nat. Biotechnol.'', vol. 29, 143–148. Feb. 2011.

== Introduction to genome editing ==

Genome editing is a type of genetic engineering, where an organisms DNA is manipulated by altering; inserting, removing or replacing nucleotides, therefore changing its genome. This process is performed by the use of immensely useful tool, t. i. engineered nucleases, which are capable of cutting both strands of DNA or nicking single strand. Resulting sites are detected and repaired by endogenous repair mechanisms, which include homologous recombination (HR) or non-homologous end joining (NHEJ). The latter is error prone, therefore it is likely for mutations to occur in repair site. These repair pathways are highly conserved in diverse cell types, which is beneficial for the use of genome editing technologies. As a result, various cell types have been engineered by the use of potable cleavage-based technologies, including human stem cells [1].

Recently, there has been tremendous development in the field of synthetic biology and genome editing technology. Virtually any gene can be manipulated in diverse range of organisms. Most common approach is the use of nuclease, which can be manipulated to target specific sequences of genome. This can be achieved by exploitation of sequence-specific DNA-binding domains (ZFNs, TALENs) or by the use of complementary guide RNA (CRISPR-Cas9 system).
In this paper, the history of genome editing will first be briefly reviewed, further on genome editing will be discussed with emphasis on the TALEN’s family.

== Brief history of genome editing ==

Past studies in the field of genome editing were perusing a common goal, t. i. to obtain high specificity, which would limit the target affects [2]. The first engineered technology, based on nuclease activity, was introduced in 1991 by Pavletich and Pabo in the journal Science [2]. This technology by the name of Zinc Finger nuclease was a great breakthrough and was later on frequently applied for genome targeting. It is based on the use of cleavage domain of restriction enzyme FokI, which is linked with designated zinc finger protein (ZFP), which is responsible for targeting. By the use of different combinations of ZFP domains the selectivity for single genomic cleavage event is achieved. The use of nuclease domains with monomeric activity there is a high probability of off-target events. This can be avoided by the use of a pair of ZFNs with distinct FokI domains that are obligate heterodimers [3]. Overtime, reports of problems and drawbacks of this technology were reported, such as targeting problem for certain triplets and reduced specificity, suffered by interactions within a zinc finger array [2].
The next technology, which emerged in 2009, is called transcription activator-like effector nuclease (TALENs). These are fusion proteins, where nuclease domain is associated with TAL effector DNA binding domain. This approach may be better in certain aspects, for example it enables a larger spectrum of targets and it’s simpler and more practical than ZFNs. In 2011, TALENs was proclaimed as “Method of the Year” by the journal Nature methods. The details of this system and molecular mechanisms of function are described later on.
We probably all at least heard if not read about latest breakthrough in the field of genome editing, which is CRISPR-Cas9 system. The discovery was somewhat fortuity, because researchers were studying how bacteria defend against phages and foreign plasmids. The paper was published in August 2012 in journal Science [4]. A bacterial nuclease called Cas9 is capable of altering genomes of invading viruses, consequently inactivating them. Clustered regularly interspaced short palindromic repeats (CRISPR) are DNA loci that contain short sequence repetitions, which can be transcribed, processed and then used to guide nuclease to a specific site [5]. This is in contrast with TALEN and Zinc Finger methods, where the need to prepare customized nuclease for each target remains.

== A TALE nuclease architecture ==

As mentioned before, two studies from 2009 proposed the use of TALEs effectors for targeted genome engineering. These transcriptional activators originate from Xanthomonas plant pathogens. During pathogenesis, TALEs specifically bind and regulate plant gene expression, thus aiding bacterial infection [6]. A central repeat domain, which mediates DNA recognition, is located within TALE structure [1]. Each repeat unit comprises of 33 – 35 amino, these specify one target base. The units base preference is determined by two crucial adjacent amino acids, also called the “repeat variable di-residue” (RVD). Therefore, the sequence of specified units in central repeat domain determines the target sequence on genome. TALEs generated with new repeat combinations have been shown to recognize target sequences predicted by this code.
In the light of aforementioned advances the interest in potential use of this system in combination with nuclease was initiated. Subsequently, a TALE-nuclease chimeras (TALENs) was proposed, which would allow for site-specific genome cleavage. In selected paper, the advances in the field of TALEN’s technology are discussed. TALE activity in a mammalian cell environment for targeted regulation of episomal reporters and an endogenous gene is presented, resuming with the research of the minimal TALE region required for high-affinity DNA binding. Paper also includes practical application, showing modification of endogenous human genes NTF3 and CCR5 by the use of TALEN approach.

== Retargeting a natural TALE to an endogenous mammalian sequence ==

Firstly, a suitable TAL effector for this study was chosen. Criteria for TALE selection was based on specificity and empirical evidence. The group was searching for natural TALE with high specificity for target sequence and with confirmed activity in mammalian cells. To achive this goal, PCR amplifications for several TALEs using Xanthomonas axonopodis pathovar citri genomic DNA were performed. Native TALE-associated sequences which allow transport into plant cells were excluded. Protein products of acquired coding regions were characterized by a SELEX assay using target of interest. Outcome of this selection yield one higly selective candidate, identified as TALE13. When combined with VP-16 activation domain, a 70-fold induction of reporter gene expression rate was observed in HEK293 cells. Based on these results, TALE13 was chosen for further work and design.
First goal was to demonstrate that chosen candidate was appropriate for mammalian gene regulation. Chosen target was NTF3 gene, which encodes a secreted nerve growth factor that has therapeutic potential for neurodegenerative diseases. Wild type TALE usually contain 18 repeats, by which they target specific sequence and bind to proximal promotor elements. TALE13 was modified to the extent of recognising proximal promotor region of NTF3 by replacing the 18 wild-type repeat units with appropriate alternatives. This altered TALE was named NT-L (meaning NTF3 Left), which highlits its binding position on target. In order to test its aktivity, the truncated version of NT-L was fused with VP-16 and expressed in human HEK293 cells. By measuring the activity of endogenous NTF3 locus, a strong induction (over 20 fold) in both NTF3 transcript and protein product was detected. The subsequent SELEX analysis revealed high specificity. These resoults revealed, that NT-L variant is a viable candidate for endogenous mammalian gene regulation.

== Development of a TALE-nuclease architecture ==

The next step was to design TALENs chimera protein using NT-L and nuclease, which would allow implementation of targeted cleavage of its genome target. The process was realized through three stages.
First, the minimal region required for high affinity bonding was determined using gene regulation studies. This was beneficial for optimization of nuclease (FokI) cleavage domain attachment with its target. Desired outcome of this step was improved catalytic activity of chimeric tool. The truncated variant named NT-L+95 was shown to retain high specificity and at the same time contribute to improved nuclease attachment. In order to examine this, a C-terminal truncated series of TALE13 were generated and subsequently linked with FokI. The resulting candidates were screened as homodimers for cleavage of specific targets. The results have shown several interesting occurrences. For TALENs comprising of NT-L+95 no detectable nuclease activity was observed. The cause for this is unclear; it is possible that this is due to a certain effect of NT-L+95 to nuclease domain positioning. Efficient DNA cleavage was observed for certain shorter specimens, providing candidates for cellular studies.
The minimal region between TALEN binding sites for optimal activity is over 12 bp, otherwise reduce cleavage may occur. These observations are useful for design of TALENs used for in vivo studies.
In the last stage, homodimer TALEN partner for NT-L was designed and named NT-R (NTR3 Right), indicating the direction of binding. This pair was expressed in human
K562 cells as fusions with catalytic domain of FokI. Target locus was later on tested for gene modification. By using two different C-terminal subregions (+28 and +63) the designed TALENs were tested. Significant activity in in vitro cleavage study was detected. With expression at 37°C, up to 3% modification was observed by the Surveyor assay [7]. This assay reveals gene modification by the appearance of digestion products. The percentage of gene modification can be obtained by quantification of “cut” bands. Limit of detection of this assay is about 1%. The activity was higher (9% of modification) in conditions of transient hypothermia, t. i. at 30°C. Sequences from latter were analyzed by Sanger sequencing. Out of 84 analyzed alleles there were 7 mutated by the occurrence of short deletions. This observation is consistent with expectation of non-homologous end joining (NHEJ) activity. Results have also shown a NHEJ-mediated capture of an oligonucleotide duplex at the endogenous NTF3 locus. These results confirmed that NHEJ-mediated genome modification by the use of TALEN architecture is possible in mammalian environment.

== Efficient modification by NHEJ and homology directed repair (HDR) ==

To expand the practical application for this developed system, modification at an additional locus was proposed. CCR5 gene was targeted for modification. First, a set of TALENs with a range of target lengths from 13 to 17 bp and of separation distance from 5 to 27 bp were designed and tested to identify values compatible with endogenous activity. To this end, a cluster of four left and four right binding sites were chosen for targeting. This defined a matrix of 16 heterodimer targets. For each binding site, two alternative proteins were generated, with 28 or 63 residues on C-terminal (as before). Altogether, this yielded 8x8=64 combinations of left and right proteins. These were expressed by pairs in K562 cells under standard conditions. Once again, Surveyor assay was used for evaluation of allele modification. Obtained results indicate that introduction of some +63/+63 pairs yielded over 20% allele modification. TALEN treated cells seem to retain a stable modification lever after 10 day growth, suggesting that TALENs are generally well tolerated. The effect of spacer length on activity is observed once again. At least 5% modification is achieved by the use of 12 – 19 bp spacer length in the +63/+63 TALENs section. In contrast, +28/+28 section show maximal activity in narrow spacer range of 12 – 13 bp.
The next challenge was to show that it is possible to induce gene through HDR using this system. For this, a part of CCR5 region was selected for targeting, also mentioned as safe harbor previously used for transgene integration. 24 TALEN pairs were then screened for their ability to induce NHEJ. Two highly active combinations were identified, first with 18% mutation rate and second with 21%. These TALEN were introduced into K562 cells simultaneously with donor DNA fragment carrying 46 bp with restriction site BglI. The success of integration was proven by PCR and BglI digestion. About 16% of inspected alleles were modified. This is an example how TALEN architecture can be used to activate HDR.

== Conclusion ==

In presented study it is shown how TALEN architecture can be designed and generated for efficient genome editing in mammalian environment. In this case, the system consists of TALE truncation variants, linked to catalytic domain of FokI. Engineered TALENs can be used for gene modification of human cells by activating either NHEJ or HDR repair pathways. Furthermore, three distinct applications are demonstrated; NHEJ mediated gene disruption, NHEJ-mediated insertion by capture of a DNA fragment and HDR mediated gene editing. Aforementioned suggests that TALEN architecture is a compelling tool in synthetic biology.

== Discussion ==

A step to facilitate the use of this architecture would undoubtedly be the linkage of minimal central TALE repeat domain to the catalytic domain of FokI. This was previously attempted by several groups without success, whereas the lack of activity of such constructs [8]. An alternative idea is to use a full length TALE for targeting. Disadvantage of the latter is the size of linkage peptide connecting two domains, which would affect the nuclease activity. Third approach is to generate truncated variants of TALEs and link them with catalytic domain of FokI, as shown in this paper. This approach has proven affective when using truncated variants with 28 or 63 of the 278 original C-terminal residues.
During the experimental work of this study, two papers were submitted where they demonstrated the use of TALEN architecture with the use of unmodified TALE or TALE with slightly reduced C-terminus [8], [9]. These systems were used for episome cleavage in yeast, an approach that is hardly translated to more complex genomes. As expected, these proteins have extended range of cleavage compared to truncated variants.
Presented TALEN architecture is in some characteristics similar to ZFNs. One example is the high cleavage activity of TALENs, which enables high gene modification efficiency, which is in some cases superior to the one of ZFNs. A shortcoming of TALEN architecture compared to ZFNs is its relative size. TALE repeats are 3 – 4 times larger than ZFPs, which may cause difficulty in delivery methods. Also, limitations due to high levels of TALE repeat homology may complicate the assembly of constructs. Future experiments will provide a fuller picture of pros and cons of the two platforms.
Targeted endogenous gene regulation by TALE proteins is a useful tool in research and for biotechnology applications. We can use this technology in a broad sense, such as design and generation of transcriptional activators or repressors. Although TALEs were discovered as such, previous studies did not inspect this type of activity outside of their native plant context. The experiments shown in this paper suggest that TALEs can be used as transcriptional regulators.
The use of SELEX assay for identification and characterization of TALEs has previously not been reported. By using this method, it was possible to assess the DNA binding preferences of natural and designed TALE proteins. Binding sites for TALE13 and four engineered TALEs were examined. On this basis, an observation that 73 out of 76 TALE repeats facilitate base preference of RVD. An interpretation of rare exceptions is proposed, facilitating further design and use of this system. Overall data collected can be used to examine previously acquired information on RVDs and thereby gaining more insight on mechanisms of action and selectivity. Findings that common RVDs such as NI, HD, NN and NG selectively bound selectively bound adenine, cytosine, guanine and thymine, respectively, are in accordance with previous knowledge. Pattern NN has shown relaxed specificity with substantial binding to a second base, adenine. NK is therefore more suitable for specificity, due to much stronger preference for guanine. This is a potential enhancement, proposed in this paper.

== Expectations for the future ==

As already mentioned, targeting the designated loci with ZFNs or TALENs is often difficult and time consuming, due to design and generation of new custom ZF or TALE array for each locus. This is not the case in most recent approach in genome editing, that is the use of CRISPR-Cas9 system. Expression of distinct gRNAs alongside Cas9 results in Cas9–gRNA complexes with distinct cleavage specificities [10]. This approach is exploited in multiplex genome editing. Still, there are features of this system which are inferior to elderly time-demanding ZFNs or TALENs.
One of those features is size of gene constructs, which is a limitation in field of gene delivery. Cas9 is the size of about 160 kDa, whereas a typical 17,5 repeat TALEN corresponds to 105 kDa. By far the smallest are ZFNs, wherein typical four finger ZFN has about 40 kDa [10]. It is likely for this compact nature to be beneficial for certain experiments.
As for specificity, the number of bases for modification in Cas9 system is restricted to 20 and is somewhat limited with the rule of three guanine bases flanking the target site (GN19NGG) [10]. The main constriction of ZF arrays is a scarce set of fingers, resulting in inability to target certain triplets. In addition, some ZFs targeting GNN seem to be superior to other ZFs, contributing to limitations. TALE target specificity therefore seems superior, with capabilities of targeting up to 24 bases. For Cas9 system, off target studies in mammalian environment are yet to be conducted [10].
It is likely that distinct features will lead to differential use of genome editing systems for distinct applications in the fields of plant science, therapeutic sciences and biotechnology applications.

== Literature ==

[1] J. C. Miller, S. Tan, G. Qiao, K. A. Barlow, J. Wang, D. F. Xia, X. Meng, D. E. Paschon, E. Leung, S. J. Hinkley, G. P. Dulay, K. L. Hua, I. Ankoudinova, G. J. Cost, F. D. Urnov, H. S. Zhang, M. C. Holmes, L. Zhang, P. D. Gregory, and E. J. Rebar, “A TALE nuclease architecture for efficient genome editing,” Nat. Biotechnol., vol. 29, no. 2, pp. 143–148, Feb. 2011.

[2] “Genome Editing.” [Online]. Available: http://www.allelebiotech.com/genome-editing/. [Accessed: 06-Jan-2015].

[3] R. M. Gupta and K. Musunuru, “Expanding the genetic editing tool kit: ZFNs, TALENs, and CRISPR-Cas9,” J. Clin. Invest., vol. 124, no. 10, pp. 4154–4161, Oct. 2014.

[4] M. Jinek, K. Chylinski, I. Fonfara, M. Hauer, J. A. Doudna, and E. Charpentier, “A programmable dual-RNA-guided DNA endonuclease in adaptive bacterial immunity,” Science, vol. 337, no. 6096, pp. 816–821, Aug. 2012.

[5] “CRISPR,” Wikipedia, the free encyclopedia. 09-Jan-2015.

[6] “TAL effector,” Wikipedia, the free encyclopedia. 06-Jan-2015.

[7] J. C. Miller, M. C. Holmes, J. Wang, D. Y. Guschin, Y.-L. Lee, I. Rupniewski, C. M. Beausejour, A. J. Waite, N. S. Wang, K. A. Kim, P. D. Gregory, C. O. Pabo, and E. J. Rebar, “An improved zinc-finger nuclease architecture for highly specific genome editing,” Nat. Biotechnol., vol. 25, no. 7, pp. 778–785, Jul. 2007.

[8] M. Christian, T. Cermak, E. L. Doyle, C. Schmidt, F. Zhang, A. Hummel, A. J. Bogdanove, and D. F. Voytas, “Targeting DNA double-strand breaks with TAL effector nucleases,” Genetics, vol. 186, no. 2, pp. 757–761, Oct. 2010.

[9] T. Li, S. Huang, W. Z. Jiang, D. Wright, M. H. Spalding, D. P. Weeks, and B. Yang, “TAL nucleases (TALNs): hybrid proteins composed of TAL effectors and FokI DNA-cleavage domain,” Nucleic Acids Res., vol. 39, no. 1, pp. 359–372, Jan. 2011.

[10] A. Strauβ and T. Lahaye, “Zinc Fingers, TAL Effectors, or Cas9-Based DNA Binding Proteins: What’s Best for Targeting Desired Genome Loci?,” Mol. Plant, vol. 6, no. 5, pp. 1384–1387, Sep. 2013.

SB students resources

2015-01-11T15:22:36Z

JernejMustar:

===Introduction to our students resources in Synthetic Biology===
(Marko Dolinar)

Synthetic biology made a vast progress in good 10 years since it established itself as an interdisciplinary field of research on the interface of molecular biology and engineering. University of Ljubljana Faculty of Chemistry and Chemical Technology has introduced a Synthetic Biology course as a part od Biochemistry MSc programme only in 2013/14. This is relatively late, considering a great success of Slovenian students at iGEM competitions since their first attendance in 2006. On the other hand, the field is still in its first stages if development and a complete textbook for a MSc level course is still missing. This is the reason why our students collaborated on the preparation of a Synthetic Biology textbook with the working title Synthetic Biology - A Students Textbook. It exists as a draft that is not publicly available and is actually part 1 of a (to be) 2-volumes title. Part I is subtitled Engineering Biology, while Part II (that currently doesn't exisist yet) will be subtitled Synthetic Biology Applications.

As in all highly competitive fields of science and technology, students should be following recent progress by reading articles in high quality journals. However, this is often a very difficult task, especially at the BSc level. Specificities of the scientific and technical language, push of publishers towards very short methodological chapters and limited knowledge studens might have about advanced techniques make understanding papers a very challenging task. Therefore, I decided to face MSc students with the challenge to explain selected SB articles in a manner that would make the content of these articles understandable to BSc level students and non-experts.

In 2014/15, seminars in Synthetic Biology include explanations and presentations of some of the top-cited articles from the field of Synthetic Biology. I compiled a list of 95 articles published between 2000 and 2014 having the highest number of citations according to the Web of Science database. The list ended with the paper just exceeding the 100 citations limit. Not included in the list were reviews. With 20 students enrolled in the course, the list has been further reduced to top 40 papers in the field. Students have been asked to check for content (they further eliminated 3 papers which proved to be reviews) and availabitly (they all seemed to be available as full texts with our university subscriptions). My suggestion was to avoid selecting for presentation papers with very similar content. Especially in the field of genome editing there has been a very rapid progress in the past few years resulting in a number of highly-cited articles which could appear very similar in content for a non-specialist. From the shortlist of 37 articles, students selected a topic they believed would be most interesting or easiest to explain. Presentations will be both written (in English, which is not the mother tongue of my students) and oral (in Slovenian, to establish and maintain Slovenian terminology in the field).

===List of articles for presentation===

This is the list of top-cited papers from the broader field of Synthetic Biology that students chose for explanation in 2014/15 (sorted by year of publication):

#[[A synthetic oscillatory network of transcriptional regulators]], Michael B. Elowitz & Stanislas Leibler, Letters to Nature, 2000 - Valter Bergant
#[[Construction of a genetic toggle switch in Escherichia coli]]. Gardner ''et al''., Nature, 2000 - Urban Bezeljak
#Positive feedback in eukaryotic gene networks: cell differentiation by graded to binary response conversion (2001) - Andreja Bratovš
#Chemical synthesis of poliovirus cDNA: Generation of infectious virus in the absence of natural template (2002) - Veronika Jarc
#[[Combinatorial synthesis of genetic networks]]. Guet C.C. ''et al'', Science, 2002 - Maja Remškar
#Engineering a mevalonate pathway in Escherichia coli for production of terpenoids (2003) - Ana Kapraljević
#Programmed population control by cell-cell communication and regulated killing (2004) - Alja Zottel
#Gene regulation at the single-cell level (2005) - Katarina Uršič
#[[A synthetic multicellular system for programmed pattern formation]]. (2005) - Mitja Crček
#Long-term monitoring of bacteria undergoing programmed population control in a microchemostat (2005) - Jana Verbančič
#Tuning genetic control through promoter engineering (2005) - Špela Pohleven
#Production of the antimalarial drug precursor artemisinic acid in engineered yeast (2006) - Živa Marsetič
#[[An improved zinc-finger nuclease architecture for highly specific genome editing]], Miller ''et al''., ''Nature Biotechnol''., 2007 - Eva Knapič
#Establishment of HIV-1 resistance in CD4(+) T cells by genome editing using zinc-finger nucleases (2008) - Tamara Marić
#Synthetic protein scaffolds provide modular control over metabolic flux (2009) - Ana Dolinar
#[[Creation of a bacterial cell controlled by a chemically synthesized genome]]. Gibson, D. G. ''et al.'', Science, 2010 - Eva Lucija Kozak
#[[A TALE nuclease architecture for efficient genome editing]], Miller ''et al'', ''Nature Biotechnol''., 2011 - Jernej Mustar
#Multiplex genome engineering using CRISPR/Cas systems (2013) - Uroš Stupar
#[[RNA-guided human genome engineering via Cas9]]. Mali ''et al''., Science, 2013 - Luka Smole
#[[One-step generation of mice carrying mutations in multiple genes by CRISPR/Cas-mediated genome engineering (2013)]] - Andrej Vrankar

''Please link the title of each paper with your written seminar wiki page. Expand the citation according to the following example:
''
#Emergent bistability by a growth-modulating positive feedback circuit. Tan et al., Nature Chem. Biol., 2009

User talk:JernejMustar

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JernejMustar: Removing all content from page

User talk:JernejMustar

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JernejMustar:

=A TALE nuclease architecture for efficient genome editing.=

Summarized from Miller, J. C. ''et al''. A TALE nuclease architecture for efficient genome editing. ''Nat. Biotechnol.'', vol. 29, 143–148. Feb. 2011.

== Introduction to genome editing ==

Genome editing is a type of genetic engineering, where an organisms DNA is manipulated by altering; inserting, removing or replacing nucleotides, therefore changing its genome. This process is performed by the use of immensely useful tool, t. i. engineered nucleases, which are capable of cutting both strands of DNA or nicking single strand. Resulting sites are detected and repaired by endogenous repair mechanisms, which include homologous recombination (HR) or non-homologous end joining (NHEJ). The latter is error prone, therefore it is likely for mutations to occur in repair site. These repair pathways are highly conserved in diverse cell types, which is beneficial for the use of genome editing technologies. As a result, various cell types have been engineered by the use of potable cleavage-based technologies, including human stem cells [1].

Recently, there has been tremendous development in the field of synthetic biology and genome editing technology. Virtually any gene can be manipulated in diverse range of organisms. Most common approach is the use of nuclease, which can be manipulated to target specific sequences of genome. This can be achieved by exploitation of sequence-specific DNA-binding domains (ZFNs, TALENs) or by the use of complementary guide RNA (CRISPR-Cas9 system).
In this paper, the history of genome editing will first be briefly reviewed, further on genome editing will be discussed with emphasis on the TALEN’s family.

== Brief history of genome editing ==

Past studies in the field of genome editing were perusing a common goal, t. i. to obtain high specificity, which would limit the target affects [2]. The first engineered technology, based on nuclease activity, was introduced in 1991 by Pavletich and Pabo in the journal Science [2]. This technology by the name of Zinc Finger nuclease was a great breakthrough and was later on frequently applied for genome targeting. It is based on the use of cleavage domain of restriction enzyme FokI, which is linked with designated zinc finger protein (ZFP), which is responsible for targeting. By the use of different combinations of ZFP domains the selectivity for single genomic cleavage event is achieved. The use of nuclease domains with monomeric activity there is a high probability of off-target events. This can be avoided by the use of a pair of ZFNs with distinct FokI domains that are obligate heterodimers [3]. Overtime, reports of problems and drawbacks of this technology were reported, such as targeting problem for certain triplets and reduced specificity, suffered by interactions within a zinc finger array [2].
The next technology, which emerged in 2009, is called transcription activator-like effector nuclease (TALENs). These are fusion proteins, where nuclease domain is associated with TAL effector DNA binding domain. This approach may be better in certain aspects, for example it enables a larger spectrum of targets and it’s simpler and more practical than ZFNs. In 2011, TALENs was proclaimed as “Method of the Year” by the journal Nature methods. The details of this system and molecular mechanisms of function are described later on.
We probably all at least heard if not read about latest breakthrough in the field of genome editing, which is CRISPR-Cas9 system. The discovery was somewhat fortuity, because researchers were studying how bacteria defend against phages and foreign plasmids. The paper was published in August 2012 in journal Science [4]. A bacterial nuclease called Cas9 is capable of altering genomes of invading viruses, consequently inactivating them. Clustered regularly interspaced short palindromic repeats (CRISPR) are DNA loci that contain short sequence repetitions, which can be transcribed, processed and then used to guide nuclease to a specific site [5]. This is in contrast with TALEN and Zinc Finger methods, where the need to prepare customized nuclease for each target remains.

== A TALE nuclease architecture ==

As mentioned before, two studies from 2009 proposed the use of TALEs effectors for targeted genome engineering. These transcriptional activators originate from Xanthomonas plant pathogens. During pathogenesis, TALEs specifically bind and regulate plant gene expression, thus aiding bacterial infection [6]. A central repeat domain, which mediates DNA recognition, is located within TALE structure [1]. Each repeat unit comprises of 33 – 35 amino, these specify one target base. The units base preference is determined by two crucial adjacent amino acids, also called the “repeat variable di-residue” (RVD). Therefore, the sequence of specified units in central repeat domain determines the target sequence on genome. TALEs generated with new repeat combinations have been shown to recognize target sequences predicted by this code.
In the light of aforementioned advances the interest in potential use of this system in combination with nuclease was initiated. Subsequently, a TALE-nuclease chimeras (TALENs) was proposed, which would allow for site-specific genome cleavage. In selected paper, the advances in the field of TALEN’s technology are discussed. TALE activity in a mammalian cell environment for targeted regulation of episomal reporters and an endogenous gene is presented, resuming with the research of the minimal TALE region required for high-affinity DNA binding. Paper also includes practical application, showing modification of endogenous human genes NTF3 and CCR5 by the use of TALEN approach.

== Retargeting a natural TALE to an endogenous mammalian sequence ==

Firstly, a suitable TAL effector for this study was chosen. Criteria for TALE selection was based on specificity and empirical evidence. The group was searching for natural TALE with high specificity for target sequence and with confirmed activity in mammalian cells. To achive this goal, PCR amplifications for several TALEs using Xanthomonas axonopodis pathovar citri genomic DNA were performed. Native TALE-associated sequences which allow transport into plant cells were excluded. Protein products of acquired coding regions were characterized by a SELEX assay using target of interest. Outcome of this selection yield one higly selective candidate, identified as TALE13. When combined with VP-16 activation domain, a 70-fold induction of reporter gene expression rate was observed in HEK293 cells. Based on these results, TALE13 was chosen for further work and design.
First goal was to demonstrate that chosen candidate was appropriate for mammalian gene regulation. Chosen target was NTF3 gene, which encodes a secreted nerve growth factor that has therapeutic potential for neurodegenerative diseases. Wild type TALE usually contain 18 repeats, by which they target specific sequence and bind to proximal promotor elements. TALE13 was modified to the extent of recognising proximal promotor region of NTF3 by replacing the 18 wild-type repeat units with appropriate alternatives. This altered TALE was named NT-L (meaning NTF3 Left), which highlits its binding position on target. In order to test its aktivity, the truncated version of NT-L was fused with VP-16 and expressed in human HEK293 cells. By measuring the activity of endogenous NTF3 locus, a strong induction (over 20 fold) in both NTF3 transcript and protein product was detected. The subsequent SELEX analysis revealed high specificity. These resoults revealed, that NT-L variant is a viable candidate for endogenous mammalian gene regulation.

== Development of a TALE-nuclease architecture ==

The next step was to design TALENs chimera protein using NT-L and nuclease, which would allow implementation of targeted cleavage of its genome target. The process was realized through three stages.
First, the minimal region required for high affinity bonding was determined using gene regulation studies. This was beneficial for optimization of nuclease (FokI) cleavage domain attachment with its target. Desired outcome of this step was improved catalytic activity of chimeric tool. The truncated variant named NT-L+95 was shown to retain high specificity and at the same time contribute to improved nuclease attachment. In order to examine this, a C-terminal truncated series of TALE13 were generated and subsequently linked with FokI. The resulting candidates were screened as homodimers for cleavage of specific targets. The results have shown several interesting occurrences. For TALENs comprising of NT-L+95 no detectable nuclease activity was observed. The cause for this is unclear; it is possible that this is due to a certain effect of NT-L+95 to nuclease domain positioning. Efficient DNA cleavage was observed for certain shorter specimens, providing candidates for cellular studies.
The minimal region between TALEN binding sites for optimal activity is over 12 bp, otherwise reduce cleavage may occur. These observations are useful for design of TALENs used for in vivo studies.
In the last stage, homodimer TALEN partner for NT-L was designed and named NT-R (NTR3 Right), indicating the direction of binding. This pair was expressed in human
K562 cells as fusions with catalytic domain of FokI. Target locus was later on tested for gene modification. By using two different C-terminal subregions (+28 and +63) the designed TALENs were tested. Significant activity in in vitro cleavage study was detected. With expression at 37°C, up to 3% modification was observed by the Surveyor assay [7]. This assay reveals gene modification by the appearance of digestion products. The percentage of gene modification can be obtained by quantification of “cut” bands. Limit of detection of this assay is about 1%. The activity was higher (9% of modification) in conditions of transient hypothermia, t. i. at 30°C. Sequences from latter were analyzed by Sanger sequencing. Out of 84 analyzed alleles there were 7 mutated by the occurrence of short deletions. This observation is consistent with expectation of non-homologous end joining (NHEJ) activity. Results have also shown a NHEJ-mediated capture of an oligonucleotide duplex at the endogenous NTF3 locus. These results confirmed that NHEJ-mediated genome modification by the use of TALEN architecture is possible in mammalian environment.

== Efficient modification by NHEJ and homology directed repair (HDR) ==

To expand the practical application for this developed system, modification at an additional locus was proposed. CCR5 gene was targeted for modification. First, a set of TALENs with a range of target lengths from 13 to 17 bp and of separation distance from 5 to 27 bp were designed and tested to identify values compatible with endogenous activity. To this end, a cluster of four left and four right binding sites were chosen for targeting. This defined a matrix of 16 heterodimer targets. For each binding site, two alternative proteins were generated, with 28 or 63 residues on C-terminal (as before). Altogether, this yielded 8x8=64 combinations of left and right proteins. These were expressed by pairs in K562 cells under standard conditions. Once again, Surveyor assay was used for evaluation of allele modification. Obtained results indicate that introduction of some +63/+63 pairs yielded over 20% allele modification. TALEN treated cells seem to retain a stable modification lever after 10 day growth, suggesting that TALENs are generally well tolerated. The effect of spacer length on activity is observed once again. At least 5% modification is achieved by the use of 12 – 19 bp spacer length in the +63/+63 TALENs section. In contrast, +28/+28 section show maximal activity in narrow spacer range of 12 – 13 bp.
The next challenge was to show that it is possible to induce gene through HDR using this system. For this, a part of CCR5 region was selected for targeting, also mentioned as safe harbor previously used for transgene integration. 24 TALEN pairs were then screened for their ability to induce NHEJ. Two highly active combinations were identified, first with 18% mutation rate and second with 21%. These TALEN were introduced into K562 cells simultaneously with donor DNA fragment carrying 46 bp with restriction site BglI. The success of integration was proven by PCR and BglI digestion. About 16% of inspected alleles were modified. This is an example how TALEN architecture can be used to activate HDR.

== Conclusion ==

In presented study it is shown how TALEN architecture can be designed and generated for efficient genome editing in mammalian environment. In this case, the system consists of TALE truncation variants, linked to catalytic domain of FokI. Engineered TALENs can be used for gene modification of human cells by activating either NHEJ or HDR repair pathways. Furthermore, three distinct applications are demonstrated; NHEJ mediated gene disruption, NHEJ-mediated insertion by capture of a DNA fragment and HDR mediated gene editing. Aforementioned suggests that TALEN architecture is a compelling tool in synthetic biology.

== Discussion ==

A step to facilitate the use of this architecture would undoubtedly be the linkage of minimal central TALE repeat domain to the catalytic domain of FokI. This was previously attempted by several groups without success, whereas the lack of activity of such constructs [8]. An alternative idea is to use a full length TALE for targeting. Disadvantage of the latter is the size of linkage peptide connecting two domains, which would affect the nuclease activity. Third approach is to generate truncated variants of TALEs and link them with catalytic domain of FokI, as shown in this paper. This approach has proven affective when using truncated variants with 28 or 63 of the 278 original C-terminal residues.
During the experimental work of this study, two papers were submitted where they demonstrated the use of TALEN architecture with the use of unmodified TALE or TALE with slightly reduced C-terminus [8], [9]. These systems were used for episome cleavage in yeast, an approach that is hardly translated to more complex genomes. As expected, these proteins have extended range of cleavage compared to truncated variants.
Presented TALEN architecture is in some characteristics similar to ZFNs. One example is the high cleavage activity of TALENs, which enables high gene modification efficiency, which is in some cases superior to the one of ZFNs. A shortcoming of TALEN architecture compared to ZFNs is its relative size. TALE repeats are 3 – 4 times larger than ZFPs, which may cause difficulty in delivery methods. Also, limitations due to high levels of TALE repeat homology may complicate the assembly of constructs. Future experiments will provide a fuller picture of pros and cons of the two platforms.
Targeted endogenous gene regulation by TALE proteins is a useful tool in research and for biotechnology applications. We can use this technology in a broad sense, such as design and generation of transcriptional activators or repressors. Although TALEs were discovered as such, previous studies did not inspect this type of activity outside of their native plant context. The experiments shown in this paper suggest that TALEs can be used as transcriptional regulators.
The use of SELEX assay for identification and characterization of TALEs has previously not been reported. By using this method, it was possible to assess the DNA binding preferences of natural and designed TALE proteins. Binding sites for TALE13 and four engineered TALEs were examined. On this basis, an observation that 73 out of 76 TALE repeats facilitate base preference of RVD. An interpretation of rare exceptions is proposed, facilitating further design and use of this system. Overall data collected can be used to examine previously acquired information on RVDs and thereby gaining more insight on mechanisms of action and selectivity. Findings that common RVDs such as NI, HD, NN and NG selectively bound selectively bound adenine, cytosine, guanine and thymine, respectively, are in accordance with previous knowledge. Pattern NN has shown relaxed specificity with substantial binding to a second base, adenine. NK is therefore more suitable for specificity, due to much stronger preference for guanine. This is a potential enhancement, proposed in this paper.

== Expectations for the future ==

As already mentioned, targeting the designated loci with ZFNs or TALENs is often difficult and time consuming, due to design and generation of new custom ZF or TALE array for each locus. This is not the case in most recent approach in genome editing, that is the use of CRISPR-Cas9 system. Expression of distinct gRNAs alongside Cas9 results in Cas9–gRNA complexes with distinct cleavage specificities [10]. This approach is exploited in multiplex genome editing. Still, there are features of this system which are inferior to elderly time-demanding ZFNs or TALENs.
One of those features is size of gene constructs, which is a limitation in field of gene delivery. Cas9 is the size of about 160 kDa, whereas a typical 17,5 repeat TALEN corresponds to 105 kDa. By far the smallest are ZFNs, wherein typical four finger ZFN has about 40 kDa [10]. It is likely for this compact nature to be beneficial for certain experiments.
As for specificity, the number of bases for modification in Cas9 system is restricted to 20 and is somewhat limited with the rule of three guanine bases flanking the target site (GN19NGG) [10]. The main constriction of ZF arrays is a scarce set of fingers, resulting in inability to target certain triplets. In addition, some ZFs targeting GNN seem to be superior to other ZFs, contributing to limitations. TALE target specificity therefore seems superior, with capabilities of targeting up to 24 bases. For Cas9 system, off target studies in mammalian environment are yet to be conducted [10].
It is likely that distinct features will lead to differential use of genome editing systems for distinct applications in the fields of plant science, therapeutic sciences and biotechnology applications.

== Literature ==

[1] J. C. Miller, S. Tan, G. Qiao, K. A. Barlow, J. Wang, D. F. Xia, X. Meng, D. E. Paschon, E. Leung, S. J. Hinkley, G. P. Dulay, K. L. Hua, I. Ankoudinova, G. J. Cost, F. D. Urnov, H. S. Zhang, M. C. Holmes, L. Zhang, P. D. Gregory, and E. J. Rebar, “A TALE nuclease architecture for efficient genome editing,” Nat. Biotechnol., vol. 29, no. 2, pp. 143–148, Feb. 2011.

[2] “Genome Editing.” [Online]. Available: http://www.allelebiotech.com/genome-editing/. [Accessed: 06-Jan-2015].

[3] R. M. Gupta and K. Musunuru, “Expanding the genetic editing tool kit: ZFNs, TALENs, and CRISPR-Cas9,” J. Clin. Invest., vol. 124, no. 10, pp. 4154–4161, Oct. 2014.

[4] M. Jinek, K. Chylinski, I. Fonfara, M. Hauer, J. A. Doudna, and E. Charpentier, “A programmable dual-RNA-guided DNA endonuclease in adaptive bacterial immunity,” Science, vol. 337, no. 6096, pp. 816–821, Aug. 2012.

[5] “CRISPR,” Wikipedia, the free encyclopedia. 09-Jan-2015.

[6] “TAL effector,” Wikipedia, the free encyclopedia. 06-Jan-2015.

[7] J. C. Miller, M. C. Holmes, J. Wang, D. Y. Guschin, Y.-L. Lee, I. Rupniewski, C. M. Beausejour, A. J. Waite, N. S. Wang, K. A. Kim, P. D. Gregory, C. O. Pabo, and E. J. Rebar, “An improved zinc-finger nuclease architecture for highly specific genome editing,” Nat. Biotechnol., vol. 25, no. 7, pp. 778–785, Jul. 2007.

[8] M. Christian, T. Cermak, E. L. Doyle, C. Schmidt, F. Zhang, A. Hummel, A. J. Bogdanove, and D. F. Voytas, “Targeting DNA double-strand breaks with TAL effector nucleases,” Genetics, vol. 186, no. 2, pp. 757–761, Oct. 2010.

[9] T. Li, S. Huang, W. Z. Jiang, D. Wright, M. H. Spalding, D. P. Weeks, and B. Yang, “TAL nucleases (TALNs): hybrid proteins composed of TAL effectors and FokI DNA-cleavage domain,” Nucleic Acids Res., vol. 39, no. 1, pp. 359–372, Jan. 2011.

[10] A. Strauβ and T. Lahaye, “Zinc Fingers, TAL Effectors, or Cas9-Based DNA Binding Proteins: What’s Best for Targeting Desired Genome Loci?,” Mol. Plant, vol. 6, no. 5, pp. 1384–1387, Sep. 2013.

Talk:SB students resources

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JernejMustar: Removing all content from page

Talk:SB students resources

2015-01-11T15:12:21Z

Vpliv valina in drugih aminokislin na vsebnost diacetila in 2,3-pentadiona v pivini

2014-05-16T16:12:38Z

JernejMustar:

Med fermentacijo alkoholnih pijač se iz prekurzorjev valina, levcina in izolevcina tvorijo vicinalni diketoni (VDK), kot sta diacetil in 2,3-pentadion. Te dajejo pivu maslen oz. karamelast okus, kar je pri lager stilih piva neželjeno, saj pri lagerjih težimo k čistim profilom okusa. Eden od obetavnejših pristopov za zmanjševanje produkcije diacetila je preko kontrole vsebnosti valina v pivini, saj je le ta pomemben za povratno inhibicijo encimov, ki sodelujejo v produkciji prekurzorjev diacetila.
Mejna vrednost diacetila pri lagerjih je 0,1 – 0,2 mg/L, medtem ko je ta vrednost pri ale pivih 0,1 - 0,4 mg/L. Najnižje znane vrednosti diacetila se vrtijo okoli 15 μg/L.
Diacetil in 2,3-pentadion nastajajo zunaj celic, torej v fermentacijski brozgi z spontano neencimsko oksidativno dekarboksilacijo α-acetohidroksi kislin, ki so intermediati pri biosinteznih poteh valina, izolevcina in levcina.

Znotraj celice se sinteza valina začne z pretvorbo piruvata do α-acetolaktata, ki se preko treh korakov pretvori do valina. Hitrost sinteze določa najpočasnejša reakcija, to je pretvorba α-acetolaktata do 2,3-dihidroksi-izovalerata. Med fermentacijo se zato dogaja, da α-acetolaktat uhaja čez celično membrano v pivino, kjer se neencimsko pretvori v diacetil. Kvasovke so zmožne pretvorbe/redukcije diacetila in 2,3-pentadiona v acetoin, 2,3-butanediol in 2,3-pentanediol. Slednji imajo višji prag zaznavnosti okusa, zato redko vplivajo na senzorične lastnosti piva. Ta redukcija se ponavadi začne že med primarno fermentacijo, vendar ob koncu koncentracije VDK vseeno presegajo mejne vrednosti. Zato je pri lagerjih potreben dodaten korak zorenja, kjer nivoji VDK padejo pod mejne vrednosti. Znižanje vsebnosti diacetila je torej glavni namen zorenja piva. Staranje je pogosto drag in dolgotrajen postopek, zato je pivovarnam v interesu, da se odkrije način, kako znižati vsebnost diacetila, brez da bi s tem vplivali na sestavo in kvaliteto piva.
Valin s povratno zanko inhibira encim acetohidroksiacil sintazo (AHAC), ki katalizira ireverzibilno pretvorbo piruvata do α-acetolaktata, in pretvorbo α-ketobutirata do α-acetohidroksibutirata in je ključen za tvorbo VDK. Negativno povratno zvezo lahko torej dosežemo z povečano koncentracijo valina v gojišču oz. z povečanim privzemom valina v celico.
Koncentracija ostalih aminokislin, še posebej razvejanih, lahko posredno prek vpliva na privzem valina iz okolja v celico vpliva na produkcijo diacetila.

V raziskavi so si zamislili naslednje tri poskuse:
- V prvem so testerali produkcijo diacetila in 2,3-pentadiona v gojiščih z različnimi dodatki valina (100, 200 in 300 mg/L).
- V drugem so testerali dodatek valina (300 mg/L) k (a) gojišču s standardnimi koncentracijami aminokislin in (b) gojišču s polovično vsebnostjo aminokislin.
- V tretjem so testirali dodajanje različnih skupin aminokislin v gojišče (koncentracije do 200% standardne).

== REZULTATI ==

Dodajanje različnih količin valina v pivino:

Vsi dodatki valina (100-300) so znižali maksimalno in končno koncentracijo diacetila, niso pa imeli velikega vpliva na koncentracijo 2,3-pentadiona.
Koncentracija diacetila ob koncu fermentacij nikjer ni padla pod zaznavno mejo (100 μg/L), vendar pa se je koncentracija diacetila v pivini z dodatkom 300mg/L valina tej meji približala (okoli 120 μg/L).

Dodajanje valina v pivini s standardno in reducirano vsebnostjo aminokislin:

Biomasa je bila v pivini z normalnim aminokislinskim profilom za 20% večja kot pri reduciranem, pH reduciranega gojišča je bil cel čas fermentacije nižji.
Dodatek valina je tudi v tem primeru znižal maksimalno in končno vsebnost diacetila, ni pa bistveno vplival na količino 2,3-pentadiona. Koncentracija diacetila nikjer ni padla pod zaznavno mejo, le te pa se je najbolj približala pivina s polovično vsebnostjo a.k. in dodatkom 300mg/L valina.
Privzem valina je bil prve tri dni večji v pivini z dodanim valinom kot pri kontroli, dodatek valina pa ni vplival na privzem izolevcina in levcina.
Iz vseh primerov je očitna negativna korelacija med koncentracijo diacetila in privzemom valina.

Dodajanje različnih skupin aminokislin v pivino:

Po dodatkih so izmerili majhno spremembo v pH pivine, ki je posledica narave dodanih aminokislin.
Dodatek preferenčnih (PAA) in nepreferenčnih a.k. (NPAA) je povzročil povečano koncentracijo diacetila med fermentacijo, prav tako je dodatek BCAA povzročil začetno povečanje diacetila (do 50h), v nadalnjem pa se je koncentracija diacetila zmanjšala glede na kontrolo.
Koncentracija diacetila so bile ob koncu fermentacije je bila najnižja za pivino z dodanimi razvejanimi a.k. (BCAA), medtem ko je bila koncentracija diacetila za pivini z dodanimi NPAA in PAA relativno višja.
Dodatek NPAA in BCAA je povzročil zmanjšanje relativne vsebnosti 2,3-pentadiona, dodatek PAA pa je njegovo vsebnost povišal.

== ZAKLJUČEK ==

Namen te raziskave je bil ugotoviti, kako vpliva dodatek valina v pivino in spreminjanje aminokislinskega profila na vsebnost vicinalnih diketonov v pivini. Ugotovili so, da dodatek valina zmanjša tako maksimalno kot tudi končno količino diacetila v pivini, kar bi lahko pivovarnam skrajšalo čas zorenja lager piva in jim zmanjšalo stroške. Zmanjšana koncentracija diacetila je posledica večjega privzema valina v celico, ki inhibira AHAS, posledično pa se proizvaja manj α-acetolaktata.
Podobno kot valin pa vplivata še izolevcin in levcin (skupina BCAA). Količino diacetila v pivini bi lahko torej zmanjšali z modifikacijo aminokislinskega spektra preko dodatkov ali preko spreminjanja pogojev sušenja ali slajenja.

== POVZETO PO ==
K. Krogerus, BR. Gibson. Influence of valine and other amino acids on total diacetyl and 2,3-pentanedione levels during fermentation of brewer's wort. Appl. Microbiol Biotechnol., Aug., 2013.

Vpliv valina in drugih aminokislin na vsebnost diacetila in 2,3-pentadiona v pivini

2014-05-16T16:07:56Z

JernejMustar: New page: Med fermentacijo alkoholnih pijač se iz prekurzorjev valina, levcina in izolevcina tvorijo vicinalni diketoni (VDK), kot sta diacetil in 2,3-pentadion. Te dajejo pivu maslen oz. karamela...

MBT seminarji 2014

2014-05-16T16:02:32Z

JernejMustar:

'''Seznam seminarjev iz Molekularne biotehnologije v študijskem letu 2013/14'''

V študijskem letu 13/14 izvajamo predmet Molekularna biotehnologija (in s tem tudi seminarje) prvič.
Tabela za razpored po tednih bo objavljena v spletni učilnici, vanjo pa se vpišite tudi za kratke predstavitve novic (5 min). Na tej strani bo samo seznam odobrenih člankov za seminar in povezave do člankov in do povzetkov, ki jih morate objaviti najkasneje tri dni pred predstavitvijo (petek).

Način vnosa:

# The importance of ''Arabidopsis'' glutathione peroxidase 8 for protecting ''Arabidopsis'' plant and ''E. coli'' cells against oxidative stress (A. Gaber; GM Crops & Food 5(1), 2014; http://dx.doi.org/10.4161/gmcr.26979) Pomen glutation peroksidaze 8 iz repnjakovca za zaščito rastline ''Arabidopsis thaliana'' in bakterije ''Escherichia coli'' pred oksidativnim stresom. Janez Novak, 15. marca 2014
(slovenski naslov povežete z novo stranjo, na kateri bo povzetek)

Naslovi odobrenih člankov:

# A plant factory for moth pheromone production (B-J. Ding ''et al''.; Nature Communications 5, 3353, 2014; http://www.nature.com/ncomms/2014/140225/ncomms4353/full/ncomms4353.html) [[Proizvodnja feremonov vešče v rastlinah]]. Filip Kolenc, 24. marca 2014
# Introduction of the rd29A:AtDREB2A CA gene into soybean (Glycine max L. Merril) and its molecular characterization in leaves and roots during dehydration (C. Engels ''et al''.; Genetics and Molecular Biology 36(4): 556–565, 2013; http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3873188/) [[Vstavitev gena rd29A:AtDREB2A CA v sojo in njegova molekulska karakterizacija v listih in koreninah med dehidracijo]]. Aleksander Krajnc, 24. marca 2014
# Enantioselective lactic acid production by an Enterococcus faecium strain showing potential in agro-industrial waste bioconversion: Physiological and proteomic studies (A. Pessione ''et al''.; Journal of Biotechnology 173, 31–40, 2014; http://dx.doi.org/10.1016/j.jbiotec.2014.01.014) [[Produkcija optično čiste mlečne kisline v sevu enterococcus faecium kaže potencial v biopretvorbi odpadkov kmetijske industrije: fiziološka in proteomska študija]]. Žan Železnik, 31. marca
# Isolation and characterization of formaldehyde-degrading fungi and its formaldehyde metabolism (D. Yu ''et al''.; Environmental Science and Pollution Research 2014 - v tisku; http://dx.doi.org/10.1007/s11356-014-2543-2) [[Glive, sposobne razgradnje formaldehida: izolacija, karakterizacija in njihov metabolizem formaldehida.]] Sara Sajko, 31. marca
# Generation of bispecific IgG antibodies by structure-based design of an orthogonal Fab interface (S. M. Lewis et al.; Nature Biotechnology 32, 191–198, 2014; http://www.nature.com/nbt/journal/v32/n2/full/nbt.2797.html) [[Priprava bispecifičnih IgG protiteles s pomočjo ustvarjanja strukturno baziranega ortogonalnega Fab vmesnika.]] Vito Frančič, 7. aprila
# Generation of protective immune response against anthrax by oral immunization with protective antigen plant-based vaccine (J. Gorantala, ''et al''; Journal of Biotechnology, 176, 2014, str. 1-10.; http://www.sciencedirect.com/science/article/pii/S0168165614000571) - [[Pridobitev zaščitnega imunskega odziva proti antraksu preko oralne imunizacije z zaščitnim antigenom kot cepivom, pridobljenim na osnovi rastlin]]. Sabina Kolar, 7. aprila
# Development of influenza H7N9 virus like particle (VLP) vaccine: Homologous A/Anhui/1/2013 (H7N9) protection and heterologous A/chicken/Jalisco/CPA1/2012 (H7N3) cross-protection in vaccinated mice challenged with H7N9 virus (G. E. Smith ''et al''.; Vaccine 31, 4305-4313, 2013; http://www.sciencedirect.com/science/article/pii/S0264410X13009870). [[Razvoj cepiva za virus gripe H7N9 na osnovi virusu podobnih delcev]]. Ana Dolinar, 14. aprila
# Generation of tumor-targeted human T lymphocytes from induced pluripotent stem cells for cancer therapy (M. Themeli ''et al.''; Nature Biotechnology 31, 928–933, 2013; http://www.nature.com/nbt/journal/v31/n10/full/nbt.2678.html). [[Iz induciranih pluripotentnih izvornih celic pripravljeni človeški limfociti T za terapijo raka]]. Urban Bezeljak, 14. aprila
# Engineering ''Escherichia coli'' for selective geraniol production with minimized endogenous dehydrogenation (J. Zhou; Journal of Biotechnology 169, 2014; http://www.sciencedirect.com/science/article/pii/S016816561300494X) [[Inženiring Escherichie coli za selektivno produkcijo geraniola z minimalno endogeno dehidrogenacijo]]. Maja Remškar, 5. maja
# Identifying producers of antibacterial compounds by screening for antibiotic resistance. (M. N. Thaker et al.; Nature Biotechnology 31, 922-927; 2013). [[Identifikacija proizvajalcev antibakterijskih spojin z iskanjem odpornosti proti antibiotikom]]. Špela Podjed, 5. maja
# Consolidated conversion of protein waste into biofuels and ammonia using Bacillus subtilis (K-Y. Choi ''et al''.; Metabolic Engineering 2014 - v tisku; http://dx.doi.org/10.1016/j.ymben.2014.02.007). [[Pretvorba proteinskih odpadkov v biogoriva in amonijak z bakterijo B. subtilis]] Elmina Handanović, 12. maja 2014
# Transcriptional comparison of the filamentous fungus Neurospora crassa growing on three major monosaccharides D-glucose, D-xylose and L-arabinose (J. Li ''et al''.; Biotechnology for Biofuels 7:31, 2014; http://www.biotechnologyforbiofuels.com/content/7/1/31/abstract). [[Primerjava transkriptoma filamentoznih gliv Neurospora crassa pri rasti na treh različnih vrstah monosaharidov: D-glukoze, D-ksiloze in L-arabinoze]] Luka Bevc, 12. maja
# Influence of valine and other amino acids on total diacetyl and 2,3-pentanedione levels during fermentation of brewer’s wort. (K. Krogerus, et al., Microbiol Biotechnol. 2013 Aug; http://link.springer.com/article/10.1007%2Fs00253-013-4955-1). [[Vpliv valina in drugih aminokislin na vsebnost diacetila in 2,3-pentadiona v pivini]] Jernej Mustar, 19. maja
# Xylanase and cellulase systems of Clostridium sp.: An insight on molecular approaches for strain improvement (L. Thomas ''et al''.; Bioresource Technology 2014 - v tisku; http://dx.doi.org/10.1016/j.biortech.2014.01.140) Luka Grm, 19. maja
# M Cell-Targeting Ligand and Consensus Dengue Virus Envelope Protein Domain III Fusion Protein Production in Transgenic Rice Calli (Tae-Geum K.''et al''.; Molecular Biotechnology 54, 880-887, 2013; http://link.springer.com/article/10.1007%2Fs12033-012-9637-1 ) Veronika Jarc, 26. maja
# Negative selection and stringency modulation in phage-assisted continuous evolution (Jacob C. Carlson, Ahmed H. Badran, Drago A. Guggiana-Nilo & David R. Liu; Nature chemical biology 10, 216–222, 2014; http://www.nature.com/nchembio/journal/v10/n3/full/nchembio.1453.html) Negativna selekcija in spreminjanje striktnosti pri zvezni evoluciji s pomočjo fagov. Valter Bergant, 26. maja

MBT seminarji 2014

2014-05-16T13:11:12Z

JernejMustar:

'''Seznam seminarjev iz Molekularne biotehnologije v študijskem letu 2013/14'''

V študijskem letu 13/14 izvajamo predmet Molekularna biotehnologija (in s tem tudi seminarje) prvič.
Tabela za razpored po tednih bo objavljena v spletni učilnici, vanjo pa se vpišite tudi za kratke predstavitve novic (5 min). Na tej strani bo samo seznam odobrenih člankov za seminar in povezave do člankov in do povzetkov, ki jih morate objaviti najkasneje tri dni pred predstavitvijo (petek).

Način vnosa:

# The importance of ''Arabidopsis'' glutathione peroxidase 8 for protecting ''Arabidopsis'' plant and ''E. coli'' cells against oxidative stress (A. Gaber; GM Crops & Food 5(1), 2014; http://dx.doi.org/10.4161/gmcr.26979) Pomen glutation peroksidaze 8 iz repnjakovca za zaščito rastline ''Arabidopsis thaliana'' in bakterije ''Escherichia coli'' pred oksidativnim stresom. Janez Novak, 15. marca 2014
(slovenski naslov povežete z novo stranjo, na kateri bo povzetek)

Naslovi odobrenih člankov:

# A plant factory for moth pheromone production (B-J. Ding ''et al''.; Nature Communications 5, 3353, 2014; http://www.nature.com/ncomms/2014/140225/ncomms4353/full/ncomms4353.html) [[Proizvodnja feremonov vešče v rastlinah]]. Filip Kolenc, 24. marca 2014
# Introduction of the rd29A:AtDREB2A CA gene into soybean (Glycine max L. Merril) and its molecular characterization in leaves and roots during dehydration (C. Engels ''et al''.; Genetics and Molecular Biology 36(4): 556–565, 2013; http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3873188/) [[Vstavitev gena rd29A:AtDREB2A CA v sojo in njegova molekulska karakterizacija v listih in koreninah med dehidracijo]]. Aleksander Krajnc, 24. marca 2014
# Enantioselective lactic acid production by an Enterococcus faecium strain showing potential in agro-industrial waste bioconversion: Physiological and proteomic studies (A. Pessione ''et al''.; Journal of Biotechnology 173, 31–40, 2014; http://dx.doi.org/10.1016/j.jbiotec.2014.01.014) [[Produkcija optično čiste mlečne kisline v sevu enterococcus faecium kaže potencial v biopretvorbi odpadkov kmetijske industrije: fiziološka in proteomska študija]]. Žan Železnik, 31. marca
# Isolation and characterization of formaldehyde-degrading fungi and its formaldehyde metabolism (D. Yu ''et al''.; Environmental Science and Pollution Research 2014 - v tisku; http://dx.doi.org/10.1007/s11356-014-2543-2) [[Glive, sposobne razgradnje formaldehida: izolacija, karakterizacija in njihov metabolizem formaldehida.]] Sara Sajko, 31. marca
# Generation of bispecific IgG antibodies by structure-based design of an orthogonal Fab interface (S. M. Lewis et al.; Nature Biotechnology 32, 191–198, 2014; http://www.nature.com/nbt/journal/v32/n2/full/nbt.2797.html) [[Priprava bispecifičnih IgG protiteles s pomočjo ustvarjanja strukturno baziranega ortogonalnega Fab vmesnika.]] Vito Frančič, 7. aprila
# Generation of protective immune response against anthrax by oral immunization with protective antigen plant-based vaccine (J. Gorantala, ''et al''; Journal of Biotechnology, 176, 2014, str. 1-10.; http://www.sciencedirect.com/science/article/pii/S0168165614000571) - [[Pridobitev zaščitnega imunskega odziva proti antraksu preko oralne imunizacije z zaščitnim antigenom kot cepivom, pridobljenim na osnovi rastlin]]. Sabina Kolar, 7. aprila
# Development of influenza H7N9 virus like particle (VLP) vaccine: Homologous A/Anhui/1/2013 (H7N9) protection and heterologous A/chicken/Jalisco/CPA1/2012 (H7N3) cross-protection in vaccinated mice challenged with H7N9 virus (G. E. Smith ''et al''.; Vaccine 31, 4305-4313, 2013; http://www.sciencedirect.com/science/article/pii/S0264410X13009870). [[Razvoj cepiva za virus gripe H7N9 na osnovi virusu podobnih delcev]]. Ana Dolinar, 14. aprila
# Generation of tumor-targeted human T lymphocytes from induced pluripotent stem cells for cancer therapy (M. Themeli ''et al.''; Nature Biotechnology 31, 928–933, 2013; http://www.nature.com/nbt/journal/v31/n10/full/nbt.2678.html). [[Iz induciranih pluripotentnih izvornih celic pripravljeni človeški limfociti T za terapijo raka]]. Urban Bezeljak, 14. aprila
# Engineering ''Escherichia coli'' for selective geraniol production with minimized endogenous dehydrogenation (J. Zhou; Journal of Biotechnology 169, 2014; http://www.sciencedirect.com/science/article/pii/S016816561300494X) [[Inženiring Escherichie coli za selektivno produkcijo geraniola z minimalno endogeno dehidrogenacijo]]. Maja Remškar, 5. maja
# Identifying producers of antibacterial compounds by screening for antibiotic resistance. (M. N. Thaker et al.; Nature Biotechnology 31, 922-927; 2013). [[Identifikacija proizvajalcev antibakterijskih spojin z iskanjem odpornosti proti antibiotikom]]. Špela Podjed, 5. maja
# Consolidated conversion of protein waste into biofuels and ammonia using Bacillus subtilis (K-Y. Choi ''et al''.; Metabolic Engineering 2014 - v tisku; http://dx.doi.org/10.1016/j.ymben.2014.02.007). [[Pretvorba proteinskih odpadkov v biogoriva in amonijak z bakterijo B. subtilis]] Elmina Handanović, 12. maja 2014
# Transcriptional comparison of the filamentous fungus Neurospora crassa growing on three major monosaccharides D-glucose, D-xylose and L-arabinose (J. Li ''et al''.; Biotechnology for Biofuels 7:31, 2014; http://www.biotechnologyforbiofuels.com/content/7/1/31/abstract). [[Primerjava transkriptoma filamentoznih gliv Neurospora crassa pri rasti na treh različnih vrstah monosaharidov: D-glukoze, D-ksiloze in L-arabinoze]] Luka Bevc, 12. maja
# Influence of valine and other amino acids on total diacetyl and 2,3-pentanedione levels during fermentation of brewer’s wort. (K. Krogerus, et al., Microbiol Biotechnol. 2013 Aug; http://link.springer.com/article/10.1007%2Fs00253-013-4955-1). Jernej Mustar, 19. maja
# Xylanase and cellulase systems of Clostridium sp.: An insight on molecular approaches for strain improvement (L. Thomas ''et al''.; Bioresource Technology 2014 - v tisku; http://dx.doi.org/10.1016/j.biortech.2014.01.140) Luka Grm, 19. maja
# M Cell-Targeting Ligand and Consensus Dengue Virus Envelope Protein Domain III Fusion Protein Production in Transgenic Rice Calli (Tae-Geum K.''et al''.; Molecular Biotechnology 54, 880-887, 2013; http://link.springer.com/article/10.1007%2Fs12033-012-9637-1 ) Veronika Jarc, 26. maja
# Negative selection and stringency modulation in phage-assisted continuous evolution (Jacob C. Carlson, Ahmed H. Badran, Drago A. Guggiana-Nilo & David R. Liu; Nature chemical biology 10, 216–222, 2014; http://www.nature.com/nchembio/journal/v10/n3/full/nchembio.1453.html) Negativna selekcija in spreminjanje striktnosti pri zvezni evoluciji s pomočjo fagov. Valter Bergant, 26. maja

Rizobakterijska simbioza

2013-11-08T19:32:23Z

JernejMustar:

Setten L, Soto G, Mozzicafreddo M, Fox AR, Lisi C, Cuccioloni M, Angeletti M, Pagano E, Díaz-Paleo A, Ayub ND.
Engineering Pseudomonas protegens Pf-5 for nitrogen fixation and its application to improve plant growth under nitrogen-deficient conditions. PLoS One. 2013 May 13;8(5)
________________________________________________________________________________________________________________________________________________________________________________________

UVOD:
Fiksacija oz. pretvorba atmonsferskega dušika (N2) do amoniaka (NH3) je proces, ki poteka v prostoživečih in simbiontskih talnih bakterijah. Za fiksacijo je ključen nitrogenazni kompleks, katerega aktivnost je omejena na anaerobne pogoje. Bakterije so razvile različne mehanizme, ki ščitijo nitrogenazo pred vplivom kisika. Poznamo več vrst bakterij, ki so zmožne fiksacije dušika v aerobnih in anaerobnih pogojih.
Dušik je bistvenega pomena za rast in razvoj rastlin. Glavni cilj raziskav na področju fiksacije dušika je razvoj bakterijskih vrst, ki bi s fiksacijo dušika prispevale k izboljšanju produktivnosti nestročnic. Na žalost ne poznamo bakterije, ki bi lahko hkrati fiksirala dušik in živela v simbiozi z ekonomsko pomembnimi rastlinami (koruza, riž in pšenica). V agronomiji se veliko uporabljajo rizobakterije, ki izboljšujejo rast in odpornost nestročnic. Z genskim inžiniringom jim lahko uvedemo dodatno lastnost, t.j. fiksacijo dušika.

POVZETEK REZULTATOV IN METOD:
V vseh bakterijah, ki so zmožne fiksacije dušika, najdemo gene nif, ki zapisujejo za strukturne, regulatorne in biosintezne komponente, ki ta proces omogočajo. Nekatere bakterije vsebujejo gene nif blizu skupaj na genomu v tako imenovanih otočkih, ki omogočajo fiksacijo dušika. Tak primer je bakterija Pseudomonas stutzeri A1501, ki so si ga raziskovalci izbrali kot donorski organizem genov nif. Za prejemne organizme so izbrali druge vrste bakterij iz istega rodu, saj so le te najbolj primerne za heterologno ekspresijo nitrogenaze. Izbrani prejemni organizmi so: Pseudomonas protegens Pf-5, Pseudomonas putida KT2440, Pseudomonas veronii DSM11331, Pseudomonas taetrolens IAM1653, Pseudomonas balearica SP1402 in Pseudomonas stutzeri CCUG11256. Izmed izbranih omenjajo sev P. protegens Pf-5 kot najbolj zanimiv za transformacijo in uvedbo nitrogenaznega kompleksa. Ta sev živi v rizosferi širšega spektra rastlinskih vrst, kjer tekmuje za obstoj z nativnimi mikrobi, poleg tega pa se jo uporablja komercialno kot sredstvo za biološko kontrolo, saj pomaga rastlinam pri obrambi pred patogeni.

Najprej so z mutagenezo uvedli kaseto z markerjem za odpornostjo na kanamicin na genom donorskega organizma (A1501) nif genov v bližino otočkov, ki omogočajo fiksacijo dušika. V nadaljevanju so iz modoficiranega genoma pripravili kosmidno knjižnjico in z presejalnimi testi izolirali kozmid, ki je vseboval marker in otoček za fiksacijo dušika v vključku. Slednjega so poimenovali X940 in ga uporabili za transformacijo prejemnih sevov. Največji potencial je pokazal rekombinantni sev P. protegens Pf-5 X940, ki je zmožen visoke aktivnosti v smislu fiksacije dušika in posredovanja amoniaka v okolje.
Njegov pozitivni učinek na več vrst rastlin (tudi ekonomsko pomembnih) so opazovali z dodajanjem rekombinantnega seva v gojišče rastlin in primerjavo produktivnosti le teh v odsotnosti in prisotnosti dušika. Ugotovili so, da vse analizirane rastline v kombinaciji z Pf-5 X940 kažejo povečano produktivnost glede na referenco.

DISKUSIJA IN ZAKLJUČEK:
V preteklosti so rodu Pseudomonas pripisovali splošno lastnost nezmožnosti fiksacije dušika. V zadnjem času pa odkrivajo dokaze, ki so v nasprotju z nekdanjim mišlenjem. V zadnjih študijah omenjajo, da obstaja v rodu Pseudomonas več sevov, ki so zmožni fiksacije dušika, večina ostalih pa je za to lastnost dovzetnih, kar so pokazali v opisanem članku. Do adaptacije nitrogenaznega kompleksa v tem rodu naj bi prišlo z horizontalnim prenosom genomskih otočkov.
Velja omeniti, da število esencialnih genov za sposobnost fiksacije dušika še ni znano. Teh genov je vsaj 16 in so ponavadi razpršeni po genomu, kar je raziskovalcem do danes oteževalo delo in s tem pripravo gesko spremenjenih bakterij, ki bi jim uvedli lastnost fiksacije dušika.
Na podlagi uporabljene metodologije je v prihodnosti mogoče relativno enostavno pridobiti gensko spremenjeni mikroorganizem z uvedeno sposobnostjo fiksacije dušika. V članku napovedujejo tudi študije, ki bodo razkrile število in lastnosti esencialnih genov, ki omogočajo fiksacijo dušika. Ta model pa naj bi bil uporaben tudi za študije, ki bodo razkrile dodaten informacije o nitrogenaznem kompleksu (strukturo, način sestavljanja, regulacijo,...)

Rizobakterijska simbioza

2013-11-08T19:28:34Z

JernejMustar: New page: UVOD Fiksacija oz. pretvorba atmonsferskega dušika (N2) do amoniaka (NH3) je proces, ki poteka v prostoživečih in simbiontskih talnih bakterijah. Za fiksacijo je ključen nitrogenazni k...

UVOD
Fiksacija oz. pretvorba atmonsferskega dušika (N2) do amoniaka (NH3) je proces, ki poteka v prostoživečih in simbiontskih talnih bakterijah. Za fiksacijo je ključen nitrogenazni kompleks, katerega aktivnost je omejena na anaerobne pogoje. Bakterije so razvile različne mehanizme, ki ščitijo nitrogenazo pred vplivom kisika. Poznamo več vrst bakterij, ki so zmožne fiksacije dušika v aerobnih in anaerobnih pogojih.
Dušik je bistvenega pomena za rast in razvoj rastlin. Glavni cilj raziskav na področju fiksacije dušika je razvoj bakterijskih vrst, ki bi s fiksacijo dušika prispevale k izboljšanju produktivnosti nestročnic. Na žalost ne poznamo bakterije, ki bi lahko hkrati fiksirala dušik in živela v simbiozi z ekonomsko pomembnimi rastlinami (koruza, riž in pšenica). V agronomiji se veliko uporabljajo rizobakterije, ki izboljšujejo rast in odpornost nestročnic. Z genskim inžiniringom jim lahko uvedemo dodatno lastnost, t.j. fiksacijo dušika.

POVZETEK REZULTATOV IN METOD
V vseh bakterijah, ki so zmožne fiksacije dušika, najdemo gene nif, ki zapisujejo za strukturne, regulatorne in biosintezne komponente, ki ta proces omogočajo. Nekatere bakterije vsebujejo gene nif blizu skupaj na genomu v tako imenovanih otočkih, ki omogočajo fiksacijo dušika. Tak primer je bakterija Pseudomonas stutzeri A1501, ki so si ga raziskovalci izbrali kot donorski organizem genov nif. Za prejemne organizme so izbrali druge vrste bakterij iz istega rodu, saj so le te najbolj primerne za heterologno ekspresijo nitrogenaze. Izbrani prejemni organizmi so: Pseudomonas protegens Pf-5, Pseudomonas putida KT2440, Pseudomonas veronii DSM11331, Pseudomonas taetrolens IAM1653, Pseudomonas balearica SP1402 in Pseudomonas stutzeri CCUG11256. Izmed izbranih omenjajo sev P. protegens Pf-5 kot najbolj zanimiv za transformacijo in uvedbo nitrogenaznega kompleksa. Ta sev živi v rizosferi širšega spektra rastlinskih vrst, kjer tekmuje za obstoj z nativnimi mikrobi, poleg tega pa se jo uporablja komercialno kot sredstvo za biološko kontrolo, saj pomaga rastlinam pri obrambi pred patogeni.

Najprej so z mutagenezo uvedli kaseto z markerjem za odpornostjo na kanamicin na genom donorskega organizma (A1501) nif genov v bližino otočkov, ki omogočajo fiksacijo dušika. V nadaljevanju so iz modoficiranega genoma pripravili kosmidno knjižnjico in z presejalnimi testi izolirali kozmid, ki je vseboval marker in otoček za fiksacijo dušika v vključku. Slednjega so poimenovali X940 in ga uporabili za transformacijo prejemnih sevov. Največji potencial je pokazal rekombinantni sev P. protegens Pf-5 X940, ki je zmožen visoke aktivnosti v smislu fiksacije dušika in posredovanja amoniaka v okolje.
Njegov pozitivni učinek na več vrst rastlin (tudi ekonomsko pomembnih) so opazovali z dodajanjem rekombinantnega seva v gojišče rastlin in primerjavo produktivnosti le teh v odsotnosti in prisotnosti dušika. Ugotovili so, da vse analizirane rastline v kombinaciji z Pf-5 X940 kažejo povečano produktivnost glede na referenco.

DISKUSIJA IN ZAKLJUČEK
V preteklosti so rodu Pseudomonas pripisovali splošno lastnost nezmožnosti fiksacije dušika. V zadnjem času pa odkrivajo dokaze, ki so v nasprotju z nekdanjim mišlenjem. V zadnjih študijah omenjajo, da obstaja v rodu Pseudomonas več sevov, ki so zmožni fiksacije dušika, večina ostalih pa je za to lastnost dovzetnih, kar so pokazali v opisanem članku. Do adaptacije nitrogenaznega kompleksa v tem rodu naj bi prišlo z horizontalnim prenosom genomskih otočkov.
Velja omeniti, da število esencialnih genov za sposobnost fiksacije dušika še ni znano. Teh genov je vsaj 16 in so ponavadi razpršeni po genomu, kar je raziskovalcem do danes oteževalo delo in s tem pripravo gesko spremenjenih bakterij, ki bi jim uvedli lastnost fiksacije dušika.
Na podlagi uporabljene metodologije je v prihodnosti mogoče relativno enostavno pridobiti gensko spremenjeni mikroorganizem z uvedeno sposobnostjo fiksacije dušika. V članku napovedujejo tudi študije, ki bodo razkrile število in lastnosti esencialnih genov, ki omogočajo fiksacijo dušika. Ta model pa naj bi bil uporaben tudi za študije, ki bodo razkrile dodaten informacije o nitrogenaznem kompleksu (strukturo, način sestavljanja, regulacijo,...)

Seminarji TehDNA

2013-10-08T14:14:56Z

JernejMustar:

Seminarje iz Tehnologije DNA bo v študijskem letu 2013/14 vodila asist. dr. Helena Čelešnik.

Seznam tem za seminarje:

# Mutageneza (16.10.)
# Izražanje na površini (23.10.)
# Dvohibridni sistemi (30.10.)
# Mikromrežne tehnologije (6.11.)
# GSO v agronomiji (13.11.) Niki Bursič, Petra Malavašič, Jernej Mustar
# Transgenske živali (27.11.) Andrea Grof, Eva Lucija Kozak, Špela Pohleven
# Izvorne celice (4.12.) Sara Primec, Alja Zottel, Tjaša Goričan
# DNA-diagnostika (11.12.) Tina Gregorič , Eva Knapič, Veronika Jarc
# Forenzika, arheologija, sistematika (18.12.)
# Mikromreže, genomike (8.1.)
# Gensko zdravljenje s. lat. (15.1.)

BIO2 Seminar 2011

2011-12-04T13:08:25Z

JernejMustar: /* Seznam seminarjev- datumi in seznam recenzentov še niso dokončni! */

= Biokemijski seminar =

Seminarje vodi doc. dr. Gregor Gunčar in so na urniku vsako sredo in petek po eni uri predavanj iz Biokemije.

Ocena seminarjev predstavlja 30% končne ocene in vsebuje vse točke, ki jih študent/ka lahko zbere pri seminarju in ostalih dejavnostih, ki niso del pisnega izpita.

== Seznam seminarjev- datumi in seznam recenzentov še niso dokončni! ==
Vpišite svoj izbrani naslov!!!
{| {{table}}
| align="center" style="background:#f0f0f0;"|'''Ime in priimek'''
| align="center" style="background:#f0f0f0;"|'''Naslov seminarja'''
| align="center" style="background:#f0f0f0;"|'''Rok za oddajo'''
| align="center" style="background:#f0f0f0;"|'''Rok za recenzijo'''
| align="center" style="background:#f0f0f0;"|'''Datum predstavitve'''
| align="center" style="background:#f0f0f0;"|'''Recenzent1'''
| align="center" style="background:#f0f0f0;"|'''Recenzent2'''
|-
| Ula Štok||[http://wiki.fkkt.uni-lj.si/index.php/BIO2_Povzetki_seminarjev_2011 Tipping the mind]||17.10.11||19.10.11||21.10.11||Maja Remškar||Mirjam Kmetič
|-
| Maša Mirković||[http://wiki.fkkt.uni-lj.si/index.php/BIO2_Povzetki_seminarjev_2011 The twisted way of things]||17.10.11||19.10.11||21.10.11||Eva Knapič||Marko Radojković
|-
| Sara Draščič||[http://wiki.fkkt.uni-lj.si/index.php/BIO2_Povzetki_seminarjev_2011 On the spur of a whim ]||17.10.11||19.10.11||21.10.11||Matevž Merljak||Monika Škrjanc
|-
| Katra Koman||[http://wiki.fkkt.uni-lj.si/index.php/BIO2_Povzetki_seminarjev_2011#Katra_Koman:_INZULIN Protein of the 20th century]||18.10.11||23.10.11||26.10.11||Ines Kerin||Veronika Jarc
|-
| Ana Dolinar||[http://wiki.fkkt.uni-lj.si/index.php/BIO2_Povzetki_seminarjev_2011#Ana_Dolinar:_Univerzalna_kri_.E2.80.93_prihodnost_transfuzijske_medicine.3F The juice of life]||21.10.11||25.10.11||28.10.11||Tjaša Goričan||Andreja Bratovš
|-
| Urška Rauter||[http://wiki.fkkt.uni-lj.si/index.php/BIO2_Povzetki_seminarjev_2011#Ur.C5.A1ka_Rauter:_A_Green_Glow:_zgradba_in_funkcija_encima_luciferaze A green glow]||21.10.11||25.10.11||28.10.11||Maša Mohar||Sandi Botonjić
|-
| Taja Karner||[http://wiki.fkkt.uni-lj.si/index.php/BIO2_Povzetki_seminarjev_2011#Taja_Karner:_Glavoboli_in_migrene Throb]||21.10.11||26.10.11||02.11.11||Karmen Hrovat||Tamara Marić
|-
| Rok Štemberger||[http://wiki.fkkt.uni-lj.si/index.php/BIO2_Povzetki_seminarjev_2011#Rok_.C5.A0temberger:_Protein_GABAA_.28gama_aminomaslena_kislina_A.29_-_zgradba.2C_vloga_in_zanimivosti Forbidden fruit]||21.10.11||28.10.11||04.11.11||Špela Pohleven||Maja Grdadolnik
|-
| Maša Mohar||[http://wiki.fkkt.uni-lj.si/index.php/BIO2_Povzetki_seminarjev_2011#Ma.C5.A1a_Mohar:_Mo.C5.A1ki_ali_.C5.BEenska_to_je_sedaj_vpra.C5.A1anje.3F.28SRY_-_faktor_za_dolo.C4.8Ditev_spola.29 The tenuous nature of sex]||21.10.11||28.10.11||04.11.11||Andreja Bratovš||Ines Kerin
|-
| Veronika Jarc||[http://wiki.fkkt.uni-lj.si/index.php/BIO2_Povzetki_seminarjev_2011#Veronika_Jarc:_Perforin Our hollow architecture]||21.10.11||28.10.11||04.11.11||Sabina Mavretič||Matevž Ambrožič
|-
| Mirjam Kmetič||[http://wiki.fkkt.uni-lj.si/index.php/BIO2_Povzetki_seminarjev_2011#Mirjam_Kmeti.C4.8D:_Mint_condition_.28limonen-3-hidroksilaza_in_limonen-6-hidroksilaza.29 Mint condition]||26.10.11||02.11.11||09.11.11||Sandi Botonjić||Tina Gregorič
|-
| Janez Meden||The Japanese Horseshoe Crab and Deafness||28.10.11||01.12.11||20.1.12||Veronika Jarc||Ana Dolinar
|-
| Tjaša Flis||[http://wiki.fkkt.uni-lj.si/index.php/BIO2_Povzetki_seminarjev_2011#Sandi_Botonji.C4.87:_Kokain_esteraza Life's tremors]||28.10.11||04.11.11||11.11.11||Ana Dolinar||Špela Pohleven
|-
| Sandi Botonjić||[http://wiki.fkkt.uni-lj.si/index.php/BIO2_Povzetki_seminarjev_2011#Sandi_Botonji.C4.87:_Kokain_esteraza Nature's junkie]||28.10.11||04.11.11||11.11.11||Maša Mirković||Alenka Mikuž
|-
| Kaja Javoršek||[http://wiki.fkkt.uni-lj.si/index.php/BIO2_Povzetki_seminarjev_2011#Kaja_Javor.C5.A1ek:_A_grey_matter A grey matter]||02.11.11||09.11.11||16.11.11||Dominik Kert||Tjaša Flis
|-
| Rok Vene||[http://wiki.fkkt.uni-lj.si/index.php/BIO2_Povzetki_seminarjev_2011#Rok_Vene:_A_mind_astray A mind astray]||04.11.11||11.11.11||18.11.11||Tamara Marić||Maja Remškar
|-
| Ines Šterbal||[http://wiki.fkkt.uni-lj.si/index.php/BIO2_Povzetki_seminarjev_2011 LTP1]||04.11.11||11.11.11||18.11.11||Ula Štok||Rok Vene
|-
| Matja Zalar||[http://wiki.fkkt.uni-lj.si/index.php/BIO2_Povzetki_seminarjev_2011#Matja_Zalar:_Vloga_SRK_in_SCR_proteinov_pri_prepre.C4.8Devanju_incestnega_razmno.C5.BEevanja_c Do it yourself]||04.11.11||11.11.11||18.11.11||Monika Škrjanc||Matevž Merljak
|-
| Matevž Ambrožič||[http://wiki.fkkt.uni-lj.si/index.php/BIO2_Povzetki_seminarjev_2011#Matev.C5.BE_Ambro.C5.BEi.C4.8D:_BSX_protein_in_debelost Of fidgets and food]||09.11.11||16.11.11||23.11.11||Kaja Javoršek||Petra Malavašič
|-
| Matevž Merljak||[http://wiki.fkkt.uni-lj.si/index.php/BIO2_Povzetki_seminarjev_2011#Matev.C5.BE_Merljak:_CEM15.2C_VIF_in_infektivnost_retrovirusov Protein wars]||12.12.11||19.12.11||20.1.12||Teja Banič||Urška Navodnik
|-
| Mitja Crček||[http://wiki.fkkt.uni-lj.si/index.php/BIO2_Povzetki_seminarjev_2011#Mijta_Cr.C4.8Dek:_DSIP_in_spanje When your day draws to an end]||11.11.11||18.11.11||25.11.11||Marko Radojković||Andrej Vrankar
|-
| Dominik Kert||[http://wiki.fkkt.uni-lj.si/index.php/BIO2_Povzetki_seminarjev_2011#Dominik_Kert:_FOXP2.2C_govore.C4.8Di_protein FOXP2, govoreči protein]||11.11.11||18.11.11||25.11.11||Alja Zottel||Kaja Javoršek
|-
| Petra Malavašič||[http://wiki.fkkt.uni-lj.si/index.php/BIO2_Povzetki_seminarjev_2011#Petra_Malava.C5.A1i.C4.8D:_Ureaza_bakterije_Helicobacter_pylori Going unnoticed]||16.11.11||23.11.11||30.11.11||Maja Grdadolnik||Mitja Crček
|-
| Eva Knapič||[http://wiki.fkkt.uni-lj.si/index.php/BIO2_Povzetki_seminarjev_2011#Eva_Knapi.C4.8D:_TSH3_-_Kaj_novorojen.C4.8Dkom_omogo.C4.8Da_zadihati? Life's first breath]||18.11.11||25.11.11||02.12.11||Mirjam Kmetič||Andrej Vrankar
|-
| Marko Radojković||[http://wiki.fkkt.uni-lj.si/index.php/BIO2_Povzetki_seminarjev_2011#Marko_Radojkovi.C4.87:_Fluoroscentni_proteini_in_njihova_uporaba_v_.C5.BEiv.C4.8Dnem_sistemu Paint my thoughts]||18.11.11||25.11.11||02.12.11||Sara Draščič||Urška Rode
|-
| Tjaša Goričan||Nerve regrowth: nipped by a no-go||18.11.11||25.11.11||02.12.11||Ana Remžgar||Ines Šterbal
|-
| Tina Gregorič||[http://wiki.fkkt.uni-lj.si/index.php/BIO2_Povzetki_seminarjev_2011 Grelin - hormon lakote]||23.11.11||30.11.11||07.12.11||Janez Meden||Urška Rauter
|-
| Tamara Marić||The dark side of RNA||25.11.11||02.12.11||09.12.11||Dominik Kert||Rok Štemberger
|-
| Ana Remžgar||I'll have you for supper||25.11.11||02.12.11||09.12.11||Jana Verbančič||Eva Knapič
|-
| Maja Remškar||[http://wiki.fkkt.uni-lj.si/index.php/BIO2_Povzetki_seminarjev_2011#Maja_Rem.C5.A1kar:_Okulokutani_albinizem_tipa_II_in_P_protein Questioning Colour]||25.11.11||02.12.11||09.12.11||Katra Koman||Karmen Belšak
|-
| Andreja Bratovš||[http://wiki.fkkt.uni-lj.si/index.php/BIO2_Povzetki_seminarjev_2011#Andreja_Bratov.C5.A1:_Bole.C4.8Dina_in_njen_receptor_-_TRPA1 The power behind pain]||30.11.11||07.12.11||14.12.11||Matevž Ambrožič||Teja Banič
|-
| Urška Navodnik||Darwin\'s dessert||02.12.11||09.12.11||16.12.11||Taja Karner||Karmen Hrovat
|-
| Jernej Mustar||[http://wiki.fkkt.uni-lj.si/index.php/BIO2_Povzetki_seminarjev_2011#Jernej_Mustar:_Na.2B_kanal.C4.8Dek_Nav1.7_in_bole.C4.8Dina Silent pain]||02.12.11||09.12.11||16.12.11||Petra Malavašič||Jana Verbančič
|-
| Ines Kerin||A queen\'s dinner||02.12.11||09.12.11||16.12.11||Tjaša Flis||Iza Ogris
|-
| Alja Zottel||Sleepless nights||07.12.11||14.12.11||21.12.11||Ines Šterbal||Katra Koman
|-
| Alenka Mikuž||Molecular chastity||09.12.11||16.12.11||23.12.11||Urška Rode||Janez Meden
|-
| Maja Grdadolnik||Ear of Stone||09.12.11||16.12.11||23.12.11||Tina Gregorič||Ana Potočnik
|-
| Jana Verbančič||A balanced mind||09.12.11||16.12.11||23.12.11||Alenka Mikuž||Ana Remžgar
|-
| Karmen Hrovat||The thread of life||14.12.11||21.12.11||04.01.12||Iza Ogris||Taja Karner
|-
| Andrej Vrankar||The things we forget||16.12.11||23.12.11||06.01.12||Jernej Mustar||Maša Mohar
|-
| Teja Banič||Cool news||16.12.11||23.12.11||06.01.12||Karmen Belšak||Jernej Mustar
|-
| Špela Pohleven||The making of crooked||16.12.11||23.12.11||06.01.12||Mitja Crček||Maša Mirković
|-
| Sabina Mavretič||A short story||21.12.11||04.01.12||11.01.12||Rok Vene||Sabina Mavretič
|-
| Karmen Belšak||Another dark horse||23.12.11||06.01.12||13.01.12||Urška Rauter||Sara Draščič
|-
| Iza Ogris||Love,love, love...||23.12.11||06.01.12||13.01.12||Ana Potočnik||Matja Zalar
|-
| Monika Škrjanc||The greenest of us all||23.12.11||06.01.12||13.01.12||Rok Štemberger||Tjaša Goričan
|-
| Ana Potočnik||Skin-deep||04.01.12||11.01.12||18.01.12||Matja Zalar||Ula Štok
|-
| Urška Rode||Smart sweat||06.01.12||13.01.12||20.01.12||Urška Navodnik||Alja Zottel
|-
| Ime in priimek||Naslov seminarja||06.01.12||13.01.12||20.01.12||||
|}

== Gradivo za seminarje ==
NOVO Gradivo za predavanja in seminarje najdete na http://bio.ijs.si/~zajec/bio2/
username: bio2
password: samozame

==Naloga==
'''Vaša naloga za seminar je: '''
Samostojno pripraviti seminar o enem od proteinov opisanih v [http://web.expasy.org/spotlight/back_issues/2011/ ProteinSpotlight] Poiskati morate vsaj še tri znanstvene članke, ki se nanašajo na opisano temo in jih uporabiti kot podlago za seminarsko nalogo!

V okviru seminarske naloge morate opraviti še naslednje naloge, katerih rešitve predložite na dodatni strani seminarske naloge, ki se ne šteje v kvoto obsega seminarja:

* sekvenca proteina in [http://www.uniprot.org/ UniProt] oznaka proteina
* slika strukture proteina (če je le-ta znana), ki jo naredite sami s programom Pymol. Če struktura še ni znana, vključite sliko proteina, ki je vašemu najbolj podoben po sekvenci in katerega struktura je znana
* poiskati morate, na katerem kromosomu se v človeškem genu nahaja ta protein in narisati shematsko sliko gena (eksonov in intronov) tega proteina. Če protein ni človeškega izvora, poiščite protein, ki je vašemu najbolj podoben in vse navedeno opišite za ta protein.

Za pripravo seminarja velja naslednje: 
* [[BIO2 Povzetki seminarjev 2011|Povzetek seminarja]] opišete na wikiju v približno 200 besedah - najkasneje do dne ko morate oddati seminar recenzentom.
* Povezavo do povzetka vnesete v tabelo seminarjev tekočega letnika.
* Seminar pripravite v obliki seminarske naloge na ~5-9 straneh A4 (pisava 12, enojni razmak, 2,5 cm robovi; važno je, da je obseg od 2700 do 3000 besed), vsebovati mora najmanj tri slike. Slika mora imeti legendo in v besedilu mora biti na ustreznem mestu sklic na sliko.
* Seminar oddajte do datuma oddaje, ki je naveden v tabeli vsakemu od recenzentov in docentu (docentu ga pošljite po e-pošti).
* Recenzenti do dneva določenega v tabeli določijo popravke in podajo oceno pisnega dela.
* Ustna predstavitev sledi na dan, ki je vpisan v tabeli. Za predstavitev je na voljo 20-30 minut. Recenzenti morajo biti na predstavitvi prisotni.
* Predstavitvi sledi razprava. Recenzenti podajo oceno predstavitve in postavijo najmanj dve vprašanji.
* Na dan predstavitve morate docentu oddati končno (popravljeno) in natisnjeno verzijo seminarja v enem izvodu.
* Seminarska naloga in povzetek morajo biti v slovenskem jeziku!

==Ocenjevanje seminarjev==
Recenzenti ocenijo seminar tako, da izpolnijo [https://docs.google.com/spreadsheet/viewform?formkey=dG1Pa3p2NXE2Vm1zX3FpVTZCT2dHVnc6MA recenzentsko poročilo] na spletu.

== Mnenje o predstavitvi ==
Vsak posameznik '''mora''' oceniti seminar, tako da odda svoje [https://docs.google.com/spreadsheet/viewform?formkey=dFNXUDBCRVBaVExvOFVxakpJUHRnOEE6MA mnenje] najkasneje v šestih dneh po predstavitvi. Kdor na seminarju ni bil prisoten, mnenja '''ne sme''' oddati.

==Urejanje spletnih strani na wikiju==
Wiki so razvili zato, da lahko spletne vsebine ureja vsakdo. Ukazi so preprosti, dokler si ne zamislite česa prav posebnega. Vseeno pa je Word v primerjavi z wikijem pravo čudežno orodje... Če imate težave z oblikovanjem besedila, si preberite poglavje o urejanju wiki-strani na Wikipediji ([http://en.wikipedia.org/wiki/Help:Editing tule] v angleščini in [http://sl.wikipedia.org/wiki/Wikipedija:Urejanje_strani tu] v slovenščini). Pomaga tudi, če pogledate, kako je zapisana kakšna stran, ki se vam zdi v redu: kliknite na zavihek 'Uredite stran' in si poglejte, kako so vpisane povezave, kako nov odstavek in podobno. ''Na koncu seveda pod oknom za urejanje kliknite na 'Prekliči'.''

==Citiranje virov==
Citiranje je možno po več shemah, važno je, da se v seminarju držite ene same.
Temeljno načelo je, da je treba vir navesti na tak način, da ga je mogoče nedvoumno poiskati.
Za citate v naravoslovju je najpogostejše citiranje po pravilniku ISO 690. [http://www.zveza-zotks.si/gzm/dokumenti/literatura.html Pravila], ki upoštevajo omenjeni standard, so pripravili pri ZTKS. Sicer pa ima vsaka revija lahko svoj način citiranja, ki ga je treba pri pisanju članka upoštevati. 
'''Citiranje knjig:''' 
Priimek, I. ''Naslov''. Kraj: Založba, letnica. 
Priimek, I. ''Naslov: podnaslov''. Izdaja. Kraj: Založba, letnica. Zbirka, številka. ISBN. 

Boyer, R. ''Temelji biokemije''. Ljubljana: Študentska založba, 2005. 
Glick BR in Pasternak JJ. ''Molecular biotechnology: principles and applications of recombinant DNA''. 3. izdaja. Washington: ASM Press, 2003. ISBN 1-55581-269-4. 
Če so avtorji trije, je beseda in med drugim in tretjim avtorjem. Če so avtorji več kot trije, napišemo samo prvega in dopišemo ''et al''. (in drugi, po latinsko). Vse, kar je latinsko, pišemo poševno (npr. tudi imena rastlin in živali, pojme ''in vivo'', ''in vitro'' ipd.).

'''Citiranje člankov:''' 
Priimek, I. Naslov. ''Naslov revije'', letnica, letnik, številka, strani. 
Lartigue C. ''et al''. Genome transplantation in bacteria: changing one species to another. ''Science'', 2007, letn. 317, str. 632-638.

Alternativni način citiranja (predvsem v družboslovju) je po pravilih APA, kjer članke citirajo takole: 
Priimek, I. (letnica, številka). Naslov. Naslov revije, strani. 
Lartigue C. ''et al.'' (2007, 317) Genome transplantation in bacteria: changing one species to another. ''Science'', 632-638.

Revija Science uporablja skrajšani zapis: 
C. Lartigue ''et al''. Science 317, 632 (2007) 

V diplomah na FKKT je treba navesti vire tako, da izpišete tudi naslov citiranega dela in strani od-do (ne samo začetne).

'''Citiranje spletnih virov:''' 
Priimek, I. ''Naslov dokumenta''. Izdaja. Kraj: Založnik, letnica. Datum zadnjega popravljanja. [Datum citiranja.] spletni naslov 
strangeguitars. ''On the brink of artificial life''. 6. 10. 2007. [citirano 13. 11. 2007] http://www.metafilter.com/65331/On-the-brink-of-artificial-life 
Navedemo čim več podatkov; pogosto vseh iz pravila ne boste našli.

BIO2 Povzetki seminarjev 2011

2011-12-04T13:03:54Z

JernejMustar: /* Jernej Mustar: Na+ kanalček Nav1.7 in bolečina */

== Ula Štok: Neuregulin 1 ==

Neuregulin-1 je član proteinov iz družine neuregulinov in je kodiran s strani gena NRG1. Obstaja veliko tipov Neuregulina-1, ki se razlikujejo po funkcionalnosti ter mestu v telesu na katerem delujejo. Najpogosteje delujejo v živčnem sistemu, kjer lahko z nepravilnim delovanjem med drugimi povzročajo tudi zelo razširjeno bolezen - shizofrenijo. Delujejo pa tudi na ostalih tkivih in organih (na primer: srce, pljuča, oprsje in želodec). Generalno obstajata dve poti signaliziranja Neuregulina-1, in sicer: Običajna ter neobičajna pot. Pri običajni poti je ErbB receptor aktiviran direktno, v enem koraku z vezavo Neuregulina-1. To najpogosteje povzroči dimerizacijo ali heterodimerizacijo ErbB receptorja. Dimerizacija ali heterodimerizacija sicer nista nujno potrebni, a vendar do njiju pride na skoraj vseh receptorjih ErbB. Ta združitev povzroči avto- in trans-fosforilacijo intracelularnih domen tega receptorja, kar aktivira vse nadaljnje poti signaliziranja. V končni fazi pa NRG1/ErbB signaliziranje vpliva direktno na transkripcijo. Pri neobičajni poti je postopek podoben, a vendar poteka začetna stopnja malo drugače. Na začetku namreč sodeluje JMa oblika receptorja ErbB4, ki se pod vplivom TACE cepi. Del receptorja (ErbB4-CTF) se odcepi v notranjost celice. Ta peptid je velik približno 80 kD in ima specifično izoblikovano vezavno mesto za Neuregulin-1. Nadaljnji procesi pa potekajo zelo podobno kot pri običajni signalni poti. Neuregulin-1 lahko povzroča shizofrenijo na različne načine, saj sodeluje pri zelo pomembnih procesih, kot so: tvorba sinaps, mielinizacija aksonov, razvoj oligodendrocit itd. Shizofrenija je zelo razširjena bolezen in nihče še ni odkril direktnega postopka k popolni odpravi te bolezni. A vendar, v letu 2009 se je zgodila neke vrste prelomnica v študiju shizofrenije. Odkrili so namreč, da posamezniki, ki so imeli gen za shizofrenijo niso zboleli. Še več! Napaka se jim je odrazila kot zvišanje kreativnih sposobnosti na znanstvenem ali umetniškem področju, odvisno od posameznika. Ob tem se je pojavilo mnogo vprašanj, saj bi na ta način mogoče lahko poiskali pot, da bi shizofrenija postala popolnoma ozdravljiva. A vendar, je to področje še raziskano, saj znanstveniki ne vedo po kakšnih poteh pride do tega, da te mutacije na NRG1 genu ne izrazijo v bolezenskem stanju.

== Maša Mirković: Proteinski produkti genov za disleksijo in z disleksijo povezane motnje ==

Disleksija je motnja, ki se kaže v nesposobnosti branja oziroma razumevanja prebranega, ter napakah in težavah pri izgovarjanju besed. Disleksiki,kot imenujemo posameznike, ki trpijo za disleksijo, imajo kljub normalnim intelektualnim sposobnostim, znanjem in izobrazbo, moteni veščini pisanja in branja s tendenco, da pomešajo med seboj črke ali besede med branjem ali pisanjem. V zadnjih letih, so uspeli ugotoviti mesta na kromosomih, povezana z dovzetnostjo za disleksijo. DYX1C1,KIAA0319,DCDC2 in ROBO1, so bili označeni kot kandidati, z dovzetnostjo za disleksijo. Najbolj obetaven je protein KIAA0319. Je transmembranski protein iz desetih transmembranskih vijačnic, najden v plazemski membrani nevronov. Njegov C-terminalni konec gleda v ekstracelularni matriks, manjši N-terminalni konec pa prehaja v citoplazmo nevrona. C-terminalni konec je visoko glikoziliran in nosi 5 PKD(polycystyc kidney desease) domene in eno MANEC(motif at the N terminus with eight cysteines) domeno. KIAA0319 igra vlogo pri rasti možganov in njihovi migraciji med razvojem možganov-iz tega je razvidno, da je disleksija problem v razvoju nevronov že v zgodnjih letih. Posamezniki z disleksijo nosijo izoobliko tega proteina, ki povzroči nižjo izraženost le tega. Spremembe so v 5'-regiji, ki kodira izoobliko proteina. Najopaznejše povezave z disleksijo se kažejo v 2,3 kb regiji, ki zavzema promotor, prvi nepreveden ekson in del prvega introna – odprti kromatin. Te ugotovitve vodijo, da je 5'-regija KIAA0319 gena tista lokacija alelov, ki največ prispeva k motnji branja.

== Katra Koman: INZULIN ==

Inzulin je peptidni hormon, ki sodeluje v uravnavanju ravni glukoze v krvi. Sintetizira in skladišči se v β-celicah Langerhansovih otočkov trebušne slinavke. Sinteza poteka od prekurzorske molekule preproinzulina preko proinzulina do dokončne zrele molekule inzulina, ki se shrani v skladiščnih veziklih. Ob povišanju ravni glukoze v krvi, na primer po obroku, glukoza, ki je tudi glavni stimulator sekrecije inzulina, iz krvi preide v β-celice skozi GLUT2 transporter. Tam se fosforilira v glukozo-6-fosfat, saj tako fosforilirana ne more več iz celice, lahko pa vstopi v proces glikolize, ki mu sledita še Krebsov cikel in oksidativna fosforilacija, ki povzroči pretvorbo ADP v ATP molekule. ATP molekula stimulira zaprtje kalijevih kanalčkov, kar privede do depolarizacije celične membrane, to pa sproži na odprtje kalcijevih kanalčkov in vdor Ca2+ ionov. Povišana koncentracija kalcijevih Ca2+ ionov v celici stimulira prenos in zlitje skladiščnih veziklov z inzulinom z membrano. Inzulin se tako sprosti v krvni obtok in potuje do tarčnih celic, ki imajo na površini izražene inzulinske receptorje. Ko se veže nanj, prenese signal o povišanju ravni glukoze v krvi v celico. To povzroči kaskado reakcij znotraj celice, ki pa na koncu privedejo do translokacije veziklov z GLUT4 transporterjev na površino celice. Število teh transporterjev za glukozo se na površini celične membrane poveča in glukoza lahko prehaja v celico, posledično pa pade raven glukoze v krvi. Razgradnja inzulina poteka v jetrih in ledvicah. Okvare na katerikoli stopnji poti inzulina se odražajo v diabetesu ali drugih boleznih.

== Rok Štemberger: Protein GABAA (gama aminomaslena kislina A) - zgradba, vloga in zanimivosti ==

V svoji seminarski nalogi sem raziskoval vlogo, pomen in zanimivosti proteina GABAA (gama-aminomaslena kislina A). To je receptor, ki se nahaja predvsem v centralnem živčnem sistemu in je zadolžen zato, da opravlja funkcijo inhibitorja. Lociran je na površini nevrotičnih sinaps in prekinja elektrokemični signal, tako da omogoči prehod kloridnih ionov znotraj celice. To se zgodi takrat ko se ustrezen ligand Gama veže na aktivno mesto tega receptorja. Konformacija podenot se spremeni in to omogoči aktivacijo receptorja. Znanstveniki so ugotovili, da obstaja več vrst GABAA receptorjev, kar pa je odvisno od sestave podenot. Najbolj pogoste podenote so alfa beta in gama v razmerju 2:2:1. V primeru da do prekinitve ne pride se lahko pojavijo epileptični napadi, psihiatrične motnje itd. Stres lahko v dobi odraščanja močno vpliva na GABAA receptorje in jih tudi permanentno strukturno spremeni, kar pa lahko kasneje v našem življenju vpliva predvsem na naš spanec in njegovo kvaliteto. Absint je bila v preteklosti prepovedana pijača, saj je povzročala razna obolenja zaradi substance imenovane tujon. Le ta se je vezala na GABAA receptorje in tako onemogočila njegovo delovanje, zato ker je preprečevala prehod kloridnih ionov v membrano. Sedaj potekajo raziskave teh receptorjev, saj je ključnega pomena čim boljša ozdravitev bolezni, ki nastanejo zaradi nepravilnega delovanja GABAA receptorja.

== Veronika Jarc: Perforin ==

Perforin je protein, ki nastane iz citotoksičnih limfocitov T. S pomočjo grancimov napade tarčno celico in jo uniči. Rečemo lahko, da je pomemben člen pri imunskem odzivu in sodeluje s NK celicami. Sestavljen je iz 555 aminokislin, njegova molekulska masa pa je 62-67 kD. Sestavljen je iz dveh pomembnih domen, domene MACPF in domene C2. Za domeno C2 je značilno, da ima afiniteto do Ca2+ ionov. Saj se na lipidni dvosloj veže le ob prisotnosti kalcija. Drugače obstajata dva različna tipa C2 domene, ki sta bila izolirana iz različnih organizmov. Lahko rečemo, da sta oba tipa zelo podobna v tem, da sta pri tipu 1 N-konec in C-konec obrnjena na vrh domene, kar je nasprotno kot pri tipu 2. Poznamo tri MACPF domene: Plu-MACPF, C8a MACPF in lipokalin C8g. Vse te domene primerjamo z skupino proteinov citolizinov in ugotovimo nekaj podobnosti in nekaj razlik. Na splošno, pa lahko rečemo, da je evolucija poskrbela tako, da so sta si domena MACPF in citolizini raszlični le v nekaj aminokislinah. Poznamo tri mehanizme kako perforin preide v tarčno celico in pri tem pomaga gramcimom B uničit to celico. Prvi mehanizem je prehajanje preko perforinske pore in sicer s pomočjo veziklov preide v celico. Naslednji mehanizem je endosomolitični model, pri katerem je pomemben kompleks s pomočjo katerega prehaja v celico. Kot zadnji mehanizem pa je model prehodne perforinske pore, ki pove, da perforin tvori kanalčke s pomočjo katerih grancimi B preidejo direktno v celico. Grancimi B so serinske proteaze, ki se sintetizirajo v citotoksičnih limfocitih T in NK celicah.

== Taja Karner: Glavoboli in migrene ==

Zaradi stresnega in hitrega tempa življenja, vse več ljudi trpi za občasnimi glavoboli, ki so najpogosteje posledica utrujenosti. Prav tako je vedno več ljudi, ki trpijo za močnejšimi oblikami glavobolov imenovanih migrene. V hujših oblikah migrene lahko glavobol traja do dva dni, močno migreno lahko spremljajo še drugi simptomi kot so slabost, bruhanje, občutljivost na svetlobo in močan zvok, depresija ter nespečnost. Mutacija, ki je največji krivec za nastanek bolezni se pojavlja na kromosomu 10 na genu KCNK18. Ta zapisuje protein TRESK, ki se nahaja v hrbtenjači in deluje kot kalijev kanalček. Mutacija povzroči, da ne pride do izmenjavanja ionov, kar povzroči hude glavobole. V raziskavah so odkrili zanimivo povezavo z anestetikom. Ta namreč ne glede na mutacijo ponovno aktivira kanal. To bi lahko učinkovito pozdravilo migrene, če bi ga le uspeli spraviti v primerno obliko. Ugotovili so tudi, da zdravila, ki vsebujejo citosporin in takrolimus v večini primerov povzročajo migrene v zdravstvu pa jih še vseeno pogosto uporabljajo. Odkritje te mutacije predstavlja revolucijo v zdravstvu in verjamem, da bo kmalu vodilo do odkritja učinkovitega zdravila proti migrenam.

== Ana Dolinar: Univerzalna kri – prihodnost transfuzijske medicine? ==

α-galaktozidaza (AGAL_HUMAN) je glikozil-hidrolazni encim. Spada v GH27-D (klan D, 27. družina) in ima aktivno mesto v obliki (β/α)8 sodčka. Encim zapisuje gen GLA, ki se nahaja na kromosomu X.

Ideja o univerzalni krvi, ki bi bila primerna za transfuzijo, ne glede na krvno skupino pacienta, je med znanstveniki prisotna že približno trideset let.
Razvili so tri metode za pretvorbo različnih antigenov v antigen 0 (po sistemu AB0), ki je primeren za transfuzijo v vse krvne skupine.
:#Encimska razgradnja antigenov A in B do antigena 0. Za antigene A so uporabili α-N-acetilgalaktozaminidazo, vendar so antigeni preveč kompleksni in metoda ni bila uspešna. Pri antigenih B so dosegli popolno pretvorbo v antigen 0 z uporabo α-galaktozidaze iz bakterije ''Streptomyces griseoplanus''.
:#Prekrivanje površine eritrocitov z maleimidofenil-polietilen-glikolom (Mal-Phe-PEG). Prekrije vse antigene, ne samo A ali B, vendar metoda ni uspešna, ker polietilen-glikol povzroča imunski odziv.
:#Pridobivanje univerzalnih rdečih krvnih celic iz pluripotentnih matičnih celic. Uspeli so pridobiti zrele eritrocite, ki so popolnoma funkcionalni.
Uporaba univerzalne krvi bi zmanjšala ali celo izničila imunski odziv ob transfuziji, prav tako ne bi bilo možnosti za transfuzijo napačne krvne skupne zaradi človeške napake. Metode trenutno niso dovolj izpopolnjene, da bi bilo možno pričakovati njeno uporabo v bližnji prihodnosti.

== Maša Mohar: Moški ali ženska to je sedaj vprašanje?(SRY - faktor za določitev spola) ==

SRY gen kodira Sry protein ki je član družine Sox (Sry related HMG box) transkripcijskih faktorjev. Poznamo jih okoli 20 pri človeku in miškah ter še mnogo drugih. Sox proteini imajo zelo različne vloge v embriogenezi in pri razvoju mnogih drugih organov. Tipično delujejo tako kot nekakšna stikala v diferenciaciji celic- sprožijo razvoj določenih celic. Sry je prav tako kot ostali člani te družine karakteriziran po HMG( high mobility group). HMG je drugače skupina specifičnih transkripcijskih faktorjev, ki imajo ~ 80 AK dolge strukturalno podobne domene za vezavo na DNA. Te domene oz. domena če je samo ena se veže na zaporedje (A/T)ACAA(T/A) v majhni žleb DNA. S tem ustvari zvitje DNA za približno 60- 85 stopinj. S tem ko se DNA zvije se razkrijejo mesta za izražanje drugih genov, recimo Sox9, ki kodira Sox9 protein ki pomaga pri diferenciaciji Sertoli celic in tako pri oblikovanju testisov, s tem pa determinira moški spol. Ugotovili smo tudi da obstaja veliko genskih bolezni povezanih s Sry genom in da lahko obstaja tudi ženska z XY spolnima kromosomoma, ker se pri njej zaradi mutacij Sry protein ne izrazi, prav tako pa obstajajo tudi moški z XX spolnima koromosomoma, kjer se enem od X kromosomov lahko izrazi SRY gen ob nepravilnostih pri očetovem delu zapisa. V bistvu sem prišla do zaključka da je zelo tanka meja med moškim in ženskim oblikovanjem spola, ena majhna mutacija oz. ena majhna razlika lahko privede do nastanka ženske ali moškega.

== Urška Rauter: A Green Glow: zgradba in funkcija encima luciferaze ==
Luciferaza je encim odvisen od ATP in magnezijevih ionov. Proces bioluminiscence se začne z vezavo na substrat luciferin, tvori se adenilatni intermediat in ob prisotnosti molekularnega kisika izhaja svetloba. Luciferaza je zgrajena iz dveh ločenih domen, večja se nahaja na N-koncu in manjša na C-koncu molekule, večja domena pa ima tudi svoje poddomene. Domeni sta med seboj ločeni z razpoko, kjer naj bi se po domnevanjih nahajalo tudi aktivno mesto encima. Luciferaza predstavlja tudi nov način mehanizma tvorbe adenilatnega intermediata med encimi in ponuja razlago za marsikatero metabolično pot.
Velika dilema, ki me med znanstveniki ostaja pa je razlika v barvi svetlobe, ki jo proces oksidacije luciferina emitira. Najverjetneje je za to odločilna keto tavtomerna oblika oksiluciferina in tudi resnonančna stabilizacija njegovega fenolatnega aniona, čeprav so znanstveniki odkrili tudi veliko drugih možnih vzrokov za različne barve (različne aminokisline, polarnost okolja, pH, ...).
Luciferaza se veliko uporablja v medicini, kjer služi kot marker molekul v telesu in tako pripomore k boljšem razumevanju različnih bolezni in infekcij, kot tudi sami strukturi celic in njenih organelov.

== Mirjam Kmetič: Mint condition (limonen-3-hidroksilaza in limonen-6-hidroksilaza) ==

Klasasta meta vsebuje encim limonen-6-hidroksilazo, ki sodeluje pri pridobivanju karvona. Poprova meta pa vsebuje limonen-3-hidroksilazo, ki je udeležena pri proizvodnji mentola. Obe hidroksilazi pripadata družini citokromov P450, njeni predstavniki pomembno sodelujejo pri proizvajanju različnih oksidiranih monoterpenov, ki so vir arom eteričnih olj. Karvon in mentol sta končna produkta hidroksilacije limonena. Ta encima sta si zelo podobna in njuni vezavni mesti za substrat sta zelo omejeni. Velja pravilo, da za spremembo aktivnosti v družini citokromov P450 potrebujemo določeno število mutacij, vendar je za modifikacijo vezavne aktivnosti limonenovih hidroksilaz potrebna samo ena. Ta fenilalanin v izolevcin mutacija povzroči, da se limonen-6-hidroksilaza spremeni v limonen-3-hidroksilazo! Mutiran encim je tako sposoben sinteze mentola tako kot encim v poprovi meti! Taka mutacija kaže, da sta prav ti dve aminokislini ne le nujni, temveč tudi prav zagotovo vpleteni pri orientaciji limonena v aktivnem mestu tako, da se ta hidroksilizira na ali C3 ali C6 poziciji. Posamične mutacije, ki lahko drastično spremenijo funkcijo proteina, so znanstveno zanimive. Nakazujejo ne le na zelo specifične manjše regije v sekvenici proteina, temveč so tudi ključne za razumevanje področij, kot so vezava in orientacija substrata, funkcija encima, metabolična pot in struktura vezavnega mesta.

== Sandi Botonjić: Kokain esteraza ==

Znanstveniki so v rizosferi kokinih plantaž (Erythroxylum coca) našli sev MB1, gram pozitivne bakterije Rhodococcus sp.. Tej bakteriji kokain predstavlja glavni vir ogljika in dušika in zato so znanstveniki izolirali osrednji encim njenega metabolizma tj. kokain esterazo (v nadaljevanju cocE). Encim je sestavljen iz treh domen: DOM1, ki vsebuje nabor kanoničnih α-vijačnic in β-ploskev; DOM2 - domena le z α-vijačnicami; in DOM3 je roladi podobna struktura z β-ploskvami. CocE je serinska esteraza, katere aktivno mesto se nahaja na stičišču vseh treh domen. Ta hidrolizira kokain na ekgonil metil ester in benzojsko kislino, ki nimata psihoaktivnih učinkov. CocE je pravi Ferrari v primerjavi z drugimi esterazami, saj lahko razgradi enako količino kokaina 1000 krat hitreje. Tako lahko postane neprecenljiva pri nujnih intervencijah v primeru prevelikega odmerka, saj bi intravenozni vbrizg cocE močno zmanjšal razpolovni čas kokaina. CocE je predmet številnih raziskav, v katerih znanstveniki proučujejo njeno termostabilnost in njenih mutiranih oblik, saj njen razpolovni čas pri fiziološki temperaturi traja le nekaj minut. Znanstveniki pa na podlagi ugotovitev iz raziskav cocE razvijajo tudi učinkovita protitelesa z vsaj podobnimi katalitičnimi parametri, ki bi brez imunskega odziva odlično delovala v bioloških sistemih.

==Tjaša Flis: Parkinsonizem in Parkin protein==

Parkinsonova bolezen je vse pogostejša bolezen pri starostnikih, njeni simptomi pa so tresavica, mišična otrdelost in upočasnjena motorika. Vzrok se skriva v propadu dopamnergičnih nevronskih celic. Bolezen je lahko avtosomno dominantno dedovana, kar pomeni, da pacienti podedujejo eno normalno in eno mutirano kopijo gena. Slednja prevladuje in se deduje naprej. Pri Parkinsonovi bolezni se mutacija zgodi v Park2 genu, ki kodira Parkin protein ali E3 ubikvitin ligazo. Parkin na poškodovane ali na preveč izražene proteine pripne ubikvitin (označevalni protein), ki jih nato usmeri v proteasom, to je velik razgradni kompleks v celicah.
Če mutacija poškoduje Parkin, je pot razgradnje onemogočena, to pa pomeni, da se v celici akumulirajo odvečni proteini. Tvorijo se Lewy-eva telesca polna teh proteinov, ki nadomestijo celične organele v nevronskih celicah, kar vodi do prenehanja njihovega delovanja. Ker pa ima Parkin več kot samo en substrat ki ga ubikvitinira, je točen mehanizem bolezni še dandanes uganka.
Eden izmed najbolj poznanih substratov je transmembranski protein Pael-R. Zvitje tega proteina poteka ob prisotnosti šaperonov. Prevelika koncentracija tega receptorja lahko izzove stres v endoplazmatskem retikulumu situiranem v nevronskih celicah. V primeru da je Parkin neaktiven, Pael-R povzroči celično smrt. Vendar to je le ena izmed možnih rešitev, substratov je namreč vsaj še dvajset, raziskave pa se nadaljujejo.

== Matja Zalar: Vloga SRK in SCR proteinov pri preprečevanju incestnega razmnoževanja cvetočih rastlin ==

Rastline so za zaščito pred samooplojevanjem razvile več vrst mehanizmov prepoznavanja lastnega peloda na molekularni ravni. Pri cvetočih rastlinah je najpogostejši mehanizem tipa SSI ali sporofitične lastne inkompatibilnosti. Pri družini ''Brassicaceae'' je za aktivacijo SSI ključna interakcija med transmembranskim proteinom SRK, ki predstavlja žensko determinanto odziva, in njenim ligandom - proteinom SCR, drugače imenovanim tudi moška determinanta odziva na lastno inkompatibilnost. Specifičnost vezave je zagotovljena s polimorfizmom alel obeh determinant. V posameznih vrstah je možno najti tudi do 100 različnih S-haplotipov genov za determinanti.
Vezava liganda na receptor bo uspešna le, če oba izhajata iz istega S-haplotipa. Vezava SCR na zunajcelično, N-glikolizirano domeno SRK povzroči nastanek kompleksa treh proteinov, ki s svojo aktivnostjo sproži kaksado reakcij, kar v končni fazi pripelje do preprečitve samooploditve.
Na neugodne življenske pogoje, ki so onemogočali medsebojno opraševanje, so se nekatere rastline prilagodile s favorizacijo samooplojevanja. Pri njih so mutacije S-lokusa, ki nosi zapis za SRK in SCR, povzročile nepravilno delovanje SI ali njegovo popolno odpoved. To pa seveda vodi v neprepoznavanje lastnega peloda in rastlina se samooprašuje. Najbolj znan primer take rastline je ''Arabidopsis thaliana'', ki se zaradi svojih specifičnih lastnosti uporablja kot modelni organizem v številnih študijah lastne inkompatibilnosti.

== Matevž Ambrožič: BSX protein in debelost ==

Za primeren občutek sitosti ali lakote glede na stanje energetskih zalog v telesu in odgovarjajoč vnos hrane ter porabo energije je odgovorna zapletena pot sporočanja. Začne se s tremi hormoni: inzulin, leptin in grelin. Leptin in inzulin se sprostita, ko so maščobne in hidratne zaloge v telesu polne in morata do možganov prenesti signal za prenehanje hranjenja, grelin pa ravno nasprotno. Vsi po krvi potujejo do hipotalamusa, predela možganov, ki je odgovoren za energijsko ravnovesje. V hipotalamusu sta dva tipa živčnih celic: oreksigene in anoreksigene. Prve sproščajo NPY in AgRP, nevropeptida, ki spodbujata hranjenje in zmanjšata porabo energije, druge pa α-MSH in CART, katerih učinek je nasproten. Našteti nevropeptidi se iz nevronov sprostijo po vezavi ustreznega izmed treh hormonov in prenesejo signal naprej, do končne spremembe v vnosu ali porabi energije. Glavni protein seminarja, BSX (brain specific homeobox) protein je transkripcijski faktor, ki spodbudi ekspresijo genov za AgRP in NPY, hkrati pa je odgovoren za premik organizma v iskanju hrane. Če v opisanem sistemu pride do napake, so pojavi nepotreben občutek lakote, kar je vzrok mnogih primerov debelosti. V boju z bolezensko debelostjo so ključne raziskave na BSX proteinu, saj je osrednji člen poti, ki v možgane prenese (včasih lažen) občutek lakote.

== Kaja Javoršek: A grey matter ==

Mikrocefalin je protein, ki ga kodira enakoimenski gen. Mikrocefalin naj bi kontroliral poliferacijo in diferenciacijo nevroblastov med nevrogenezo. Odkritje, da je mikrocefalin odločilen regulator velikosti možganov, je sprožilo hipotezo, da je igral vlogo v evoluciji možganov.
Razen v možganih najdemo mikrocefalin tudi v ledvicah, srcu, pljučih, vranici in skeletnih mišicah. Vendar pomen mikrocefalina v teh organih še ni znan.
Mutacije na genu mikrocefalina vodijo do nastanka mikrocefalije. To je bolezen razvoja živčnega sistema in je definirana kot resno zmanjšana velikost možganov. Pri odraslih je normalen volumen možganov od 1200 cm3 do 1600 cm3, pri odraslih s primarno mikocefalijo pa okoli 400 cm3 . Poleg mirocefalina pa povzročajo mikrocefalijo še mutacije petih genih (ASPM, MCPH2, CDK5RAP2, MCPH4, CENPJ)
Mikrocefalin ima tri BRCT domene na C – koncu. BRCT domene so prisotne v veliko ključnih proteinih, ki kontrolirajo delitev celice. Zato predvidevajo da mikrocefalija nastane, ker je ovirana normalna regulacija delitve celic v možganih.
Ugotovili so, da je protein mikrocefalin dol 835 aminokislin. Zaradi mutacije na genu mikrocefalina se ta protein skrajša na 25 aminokislin.
Znanstveniki so izvedli raziskavo ali gena mikrocefalin in ASPM vplivata na inteligenco. Na podlagi treh raziskav so zaključili, da inteligenca ni povezana z dominantnimi aleli ASPM – ja ali mikrocefalina.

== Rok Vene: A mind astray ==

Alzheimerjeva bolezen postaja vedno bolj aktualna tematika. Trenutno je na svetu več kot 26 milijonov ljudi s to obliko demence. Zaradi daljše življenjske dobe pa pričakujemo, da bo število obolelih samo še naraščalo. Alzheimerjeva bolezen prizadene centralni živčni sistem, v možganih se nalagajo snovi, ki povzročijo propad živčnih celic. Ena izmed snovi, ki se nalagajo v možganih so nefunkcionalni Tau proteini. Tau proteini sodijo v družino proteinov imenovanih microtubule-associated proteins (MAP), njihova naloga pa je je stabilizacija mikrotubulov. To dosežejo tako, da se na mikrotubule vežejo. Poleg tega predvidevajo, da imajo Tau proteini še eno nalogo. Sodelovali naj bi v kompleksu za uravnavanje vzdražnosti živčnih celic. Nefunkcionalnost Tau proteinov povezujejo z različnimi boleznimi, ki jih poznamo pod skupnim imenom tauopatije. V primeru Alzheimerjeve bolezni je Tau protein nefunkcionalen, zato ker je hiperfosforiliran, kar mu onemogoča vezavo na mikrotubule. Tau proteini zato tvorijo netopne agregate – nevrofibrilarne pentlje, ki najbrž povzročijo odmiranje živčnih celic. Pri iskanju učinkovin proti hiperfosforilaciji in agregaciji Tau proteina, so znanstveniki raziskali protein FKBP52. Ta protein ima več funkcij. Osredotočili so se predvsem na njegove šaperonske lastnosti. Ugotovili so, da se FKBP52 veže na hiperfosforiliran Tau protein, in tako prepreči agregacijo Tau proteina, ki je odgovorna za odmiranje nevronov.

== Ines Šterbal: LTP1 ==

Protein LTP1, izoliran iz ječmenovega zrna, spada v družino lipidnih prenašalnih proteinov (lipid transfer protein –LTP). Je dobro topen protein, ki se nahaja v alevronski plasti ječmenovega semena. Sestavljen je iz štirih heliksov, ki so povezani z disulfidnimi mostički. Ima dobro definiran C-terminalni konec. V razmerah in vivo je globularni protein, s stožčastim hidrofobnim jedrom, ki se razteza od enega konca molekule do drugega. Sposoben je vezati različne lipide, kot so maščobne kisline ali acetil-koencim A. LTP1 proteini so na površini aktivni proteini, so stabilni, denaturirajo šele okrog 100 °C. Vloga LTP1 proteina in vivo še ni znana. In vitro je glavni protein pri penjenju piva. Opravlja pa še številne druge funkcije, odvisno od tega, kateri ligand ima vezan. LTP1 proteini so verjetno vključeni v prenos lipidov preko membrane in celo v nastanek membrane, lahko bi imeli vlogo v transportu monomera Cutin, vlogo naj bi igrali tudi v obrambnem mehanizmu rastlin. Lipidi, ki so vezani na LTP1 bi naj imeli antibakterijsko aktivnost za bakterije in glive.
Vsi podatki kažejo, da so povezave med sladkorji in proteini, ki nastanejo kot produkt Milardove reakcije, prvi korak do nastanka pivovske pene. Kaže, da je kontrola glikacije LTP1 proteinov med slajenjem in varjenjem piva, nujna za optimalno penjenje piva.

== Mitja Crček: DSIP in spanje ==

Pred 2000 leti so ljudje verjeli, da postanemo zaspani zaradi nekakšnih želodčnih hlapov, ki gredo v možgane, se tam kondenzirajo, zamašijo pore in posledično povzročajo zaspanost. Kasneje so seveda ugotovili da temu ni tako, leta 1977 pa so odkrili majhne peptide, ki naj bi nas uspavali in jih poimenovali Delta Sleep-Inducing peptide (DSIP). DSIP je majhen peptid, sestavljen iz devetih aminokislinskih ostankov in maso 850 daltonov, prvič pa so ga odkrili pri zajcih. Sodeloval naj bi tako pri endokrini regulaciji kot pri fizioloških procesih (poveča učinkovitost oksidativne fosforilacije), pomembno vlogo pa naj bi imel tudi v medicini in pri zdravljenju bolezni. Ker naj bi podaljševal REM fazo, bi ga lahko uporabljali tudi kot dodatek pri zdravljenju alkoholizma ali ga dodajali antidepresivom in pomirjevalom, ki skrajšujejo REM fazo. Raziskave so spremljale tudi vpliv DSIP-ja na nespečnost. Ugotovili so, da DSIP rahlo povečuje kvaliteto spanja in skrajšuje latenco uspavanja, na trajanje budnosti in druge parametre pa ne vpliva, zato so si strokovnjaki enotni, da ima DSIP le rahle terapevtske učinke na nespečnost. Delovanje peptida pa še vedno ni povsem razjasnjeno in le želimo si lahko, da bodo novejše raziskave prinesle nove informacije, saj ima DSIP vsekakor velik potencial v medicini.

== Dominik Kert: FOXP2, govoreči protein ==

Ljudje in živali se razlikujejo. Za znanstvenike 19. stoletja je bilo zelo fascinantno to, da mi lahko govorimo, se sporazumevamo in pomnimo besede, medtem ko živali ne morejo. Ko se je pojavila družina KE na koncu 90. let prejšnjega stoletja, so znanstveniki ugotovili, da obstaja gen, ki kodira FOXP2. Družina KE je slovi po tem, da ima polovica njenih članov težave z govorom. Tako so ugotovili, da se mutacija prenaša avtosomno in dominantno. In verjetno na to vpliva mutacija FOXP2, FOXP2 protein pa je po vsej verjetnosti odločilen faktor pri govoru.
FOXP2 protein je sestavljen iz 715 aminokislin in spada med družino transkripcijskih faktorjev, ki se imenuje FOX (zaradi 'forkhead box' domene). Zanimivo je, da se ta gen razlikuje od gena opic (šimpanz, gorila, makaki) le za dve in od miši le za tri aminokisline. To se znanstvenikom zdi zelo zanimivo, ker je verjetno zaradi teh dve sprememb v aminokislinskem zaporedju prišlo do sprememb pri sporazumevanju. Zaradi teh dejstev so se naprej usmerili na to, ali je bil gen res pod vplivom naravne selekcije in ugotovili so, da je bil res.
FOXP2 na te spremembe vpliva v možganih, je pa prisoten tudi v pljučih, drobovju in srcu. Vendar njegova funkcija tam še ni znana.

== Petra Malavašič: Ureaza bakterije Helicobacter pylori ==

Bakterija Helicobacter pylori spada med patogene mikrobe. Znanstvenika Warren in Marshall sta leta 1987 odkrila to bakterijo ter ugotovila, da je s to bakterijo povezana razjeda na želodcu. Leta 2005 sta prejela Nobelovo nagrado. Že vsak drugi človek je okužen s to bakterijo. Naseljena je na želodčni sluznici in povzroča kronično vnetje želodčne sluznice. Bakterija se lahko naseli in se razmnožuje v prisotnosti želodčne kisline, kjer je pH okoli 2. Posebni obrambni mehanizmi omogočajo bakteriji, da lahko preživi v kislem okolju. Encim ureaza je pri tem najpomembnejši. Ureaza je encim, ki katalizira hidrolizo uree, pri čemer nastane amoniak, ki se v končni fazi veže z molekulami vode v amonijev hidroksid, ki poveča pH v neposredni okolici bakterije. Encim ureaza se nahaja v citoplazmi bakterijske celice in na njeni površini. Sam encim je zgrajen zelo kompleksno in omogoča bakteriji preživetje. Posebna kompleksna zgradba encima onemogoči, da bi kislina želodčnega soka denaturirala encim. Encim sestavljata dva kompleksa (αβ) štirih prostorsko razporejenih (αβ)3 enot.

== Matevž Merljak: CEM15, VIF in infektivnost retrovirusov ==

Ena izmed komponent obrambnega mehanizma pred retrovirusi v nekaterih človeških celicah je citidinska deaminaza CEM15 (APOBEC3G). V celicah, ki jo izražajo, se retrovirusi brez posebnega proteina (VIF, “viral infectivity factor”) ne morejo uspešno množiti, zato takim celicam pravimo “nepermisivne” celice.

CEM15 deluje tako, da med procesom reverzne transkripcije v novonastali “minus” DNA verigi številne citidinske baze pretvori v uridinske, ter s tem povzroči tako zmanjšano obstojnost z uracilom bogate DNA verige, kot tudi zamenjave gvanozinskih baz z adenozinskimi v kodirajoči (“plus”) verigi DNA. Čeprav takšna hipermutacija za nadaljno infektivnost virusa ni vedno usodna (torej lahko tako mutirana DNA v nekaterih primerih še vedno tvori funkcionalne viruse), je običajno dovolj obsežna, da onemogoči uspešno reprodukcijo virusa.

Raziskave kažejo, da CEM15 ne napade nastajajoče DNA kot lasten celični odgovor na infekcijo, pač pa se med izgradnjo novih virusov vgradi v le-te ter po infekciji nove celice povzroči omenjene spremembe v nastajajoči DNA verigi.

Že omenjen faktor VIF izhaja iz virusa HIV-1, ki primarno napada sicer nepermisivne limfocite T. Naloga VIF je preprečitev vgradnje CEM15 v nastajajoče viruse, to pa doseže tako z oteževanjem njene translacije, kot tudi z indukcijo razgradnje CEM15 v proteasomu.

== Eva Knapič: TSH3 - Kaj novorojenčkom omogoča zadihati? ==

Kaj novorojenčkom omogoča zadihati? Raziskave so pokazale, da ima eno izmed vodilnih vlog pri začetku dihanja protein teashirt homolog 3 (TSH3). To je protein, ki ga uvrščamo med transkripcijske faktorje. Po strukturi spada v družino cinkovih prstov, kjer so sekundarne strukture koordinirane s cinkovim ionom. TSH3 ima pet tako urejenih struktur in vse spadajo v Cys2His2 skupino – cinkov ion koordinira dva cisteinska in dva histidinska ostanka ßßα podenote.
Organizem brez zapisa za teashirt 3 protein se v času embrionalnega razvoja navidezno ne razlikuje od organizmov, ki ta zapis imajo. Vendar so podrobnejše raziskave pokazale, da se brez prisotnosti proteina teashirt 3 dokončno ne oblikujejo pljučni mešički, ki so funkcionalna enota pljuč, saj tam poteka izmenjava plinov. Odsotnost proteina povzroča povečano apoptozo nevronov motoričnega jedra v možganskem deblu, s tem so proteinu pripisali zmožnost inhibicije apoptoze nevronov. Prav tako so nezmožnost odziva organizma na pH spremembe okolja pripisali pomanjkanju proteina TSH3.
Iz vseh teh pomanjkljivostih, ki jih povzroča TSH3 so raziskovalci prišli do zaključka, da novorojenček brez zapisa za protein ni zmožen zadihati, ker ni sposoben odziva na spremembo okolja, predvsem pH in tako ne more vzdrževati homeostaze, ki je potreba na preživetje.

== Tjaša Goričan: Vpliv Nogo proteina na regeneracijo živčnega sistema ==

Nevroni vsebujejo mielin, ki je sestavni del mielinske ovojnice aksona in ima nalogo zagotavljanja stalnega prenosa električnih signalov. Poleg tega pa mu je dodeljena tudi nenavadna lastnost. Vsebuje namreč proteine Nogo-A, ki delujejo kot inhibirotji za rast poškodovanih aksonov. Posledično se diferencirani nevroni niso sposobni deliti. Problem se pojavi pri poškodbi živčnega sistema, saj se ni sposoben regenerirati. Bolezni, ki so povezane s poškodbami živčevja so: Poškodbe hrbtenjače, Alzheimerjeva bolezen, možganska kap, shizofrenija itd.

Nogo-A protein spada v družino proteinov retikulonov in je ena od oblik Nogo proteinov. Je transmembranski protein, ki se z domeno Nogo-66 uspešno veže na receptor in povzroči razgradnjo mikrotubulov v aksonu, kar privede do preureditve citoskeleta in posledično zaustavitve rasti aksona. Največ Nogo-A se nahaja na oligodendrocitih. Oligodendrociti so celice, ki spadajo med nevroglio in tvorijo mielinski ovoj nevronov v centralnem živčnem sistemu. Veliko več ga najdemo v centralnem živčnem sistemu v primerjavi s perifernim.

Čeprav je še veliko neznanega na področju živčnega sistema, je znanost že dosegla uspehe glede boja proti boleznimi, povezanimi z regeneracijo živčnega sistema. S protitelesi se da inhibirati protein Nogo-A in s tem preprečiti inhibicijo rasti poškodovanih nevronov.

== Marko Radojković: Fluorescentni proteini in njihova uporaba v živčnem sistemu ==

Fluorescentni proteini so členi družine homologih proteinov, ki se delijo skupno lastnost da svetlijo zaradi formiranja kromoforma znotraj lastnega polipeptidnega zaporedja. Prvi odkrit takšen protein je bil zeleni fluorescentni protein ali GFP. Od tedaj do danes so kreirani različni mutanti, ki žarijo skoraj vse barve človeškega vidnega spektra. Izkazalo se je da so zelo uporabni v mnogih bioloških disciplinah, predvsem pa so popularni v spremljanju dinamike proteinov, genske ekspresije, in tudi posledično na viši ravni, dinamike organelov ter gibanja celic znotraj tkiva.

Ne tako dolgo nazaj, je tim znanstvenikov uspel skombinirati različne barvne variante GFP-ja s sofisticiranim Cre/Lox sistemom genske rekombinacije in tako omogočil njihovo izražanje v samih možganih. Tale tehnika omogoča da se vsaki posamezni nevron obarva drugače in tako loči od sosednjih, kar omogoča detajlno analizo živčnega vezja. Brainbow strategija, kakor so jo poimenovali, daje upanje znanstvenikom da z ustvarjanem celotnega ''zemljevida'' možganov, lahko izpeljejo pomembne informacije o nevronskih povezavah in njihov nadaljni vpliv na vedenje in delovanje organizma.

== Tamara Marić: MikroRNA ==
MikroRNA je mala molekula, ki je prepisana z DNA na tak način kot mRNA. Zapis za miRNA se lahko nahaja v intronskih regijah, kodirajočih ali nekodirajočih genov. Osnovna funkcija je utišanje genov na nivoju sinteze proteinov. Da pa lahko opravi svojo nalogo mora dozoreti. Biogeneza miRNA se začne v samem jedru, kjer se 1000 nukleotidov dolg transkript s pomočjo encimskega kompleksa (Drosha-DGCR8)skrajša na 60-70 nukleotidov dolg pre-miRNA.Z eksportinom-5 se prenese iz jedra v citoplazmo do naslednjega kompleksa. Dicer veže pre-miRNA in jo skrajša na 22 nukleotidov. Nastane miRNA dupleks. Ena izmed verig prevzame vodilno funkcijo in se vmesti v kompleks istega encima v povezavi z drugimi proteini. Kompleks pripelje do komplamentarne verige mRNA in povzroči translacijsko represijo. Znanstveniki se ukvarjajo predvsem z vprašanjem,kako se miRNA izraža v številnih boleznih. Natančneje sem si pogledala proces resorpcije in obnove kosti in kako miRNA vpliva na regulacijo teh dveh procesov.

== Maja Remškar: Okulokutani albinizem tipa II in P protein ==

Melanin je pigment, ki je nujno potreben za zaščito kože pred pripekajočim soncem ter za normalno delovanje oči. Glavna sestavina za njegovo sintezo je aminokislina tirozin, ki je osnova evmelanina (črni pigment), ob dodatku cisteina pa dobimo še feomelanin (rdeče-rumen). Za običajno delovanje biosinteze melanina je potrebno kislo okolje v melanosomih, kjer se sinteza izvaja. Za vzdrževanje kislosti sta potrebna dva proteina – anionski kanalček in ATP črpalka. Anioni tu delujejo kot vaba za protone, kjučne za kisel pH. P protein naj bi deloval kot anionski transporter. Torej v njegovi odsotnosti v melanosom ne morejo dostopati anioni in posledično se v celico ne prečrpavajo protoni, kar pomeni da ni kislega pH ugodnega za sintezo melanina.
Okulokutani albinizem tipa II ali OCA2 nastane zaradi pomanjkanja količine melanina v očeh, koži in laseh. Za kožo to pomeni večjo občutljivost na UV žarke in povečano možnost za kožnega raka. Zaradi nepigmentiranih optičnih vlaken pa se pojavijo še težave z očmi, kot so škiljenje, fotofobija, nistagmus, degeneracija rumene pege, pride pa tudi do izgube biokularnega vida. OCA2 je dedna bolezen, ki se deduje recesivno. Človek le z enim okvarjenim alelom je torej prenašalec gena. Ugotovili so, da OCA2 povzroča mutacija gena P, in sicer najpogostejša je delecija 7 eksona.

== Ana Remžgar: Bacillus subtilis ==

Bacillus subtilis je grampozitivna paličasta bakterija. Ko ima v okolju dovolj hranil, se simetrično deli in vegetativno raste. Ko pa v okolju začne hranil primanjkovati, B. subtilis uvede različne mehanizme, da lahko preživi. Del populacije postane kompetenten in sprejme tujo DNA. Del populacije pa s pomočjo zapletenega sistema aktivacije proteina Spo0A vstopi v proces sporulacije. Sporulacija je počasen in energijsko potraten postopek, ki traja v idelanih razmerah vsaj 7 ur. Na koncu nastane spora, ki lahko preživi tudi več desetletji v neugodnih življenjskih razmerah. Ko celica vstopi v cikel sporulacije, začne v okolje izločati razne toksične snovi, med njimi sta najbolj učinkovita Skf in Sdp. Ko celica izloči ti dva proteina v okolje, ubije sosednje bakterijske celice Bacillis subtilisa. Zaradi njunih lasnosti, ta dva proteina pogosto zato imenujemo kanibalistična faktorja. Vendar mora celica paziti, da pri tem ne ubije še sebe. Pri tem ji pomaga medmembranski protein SdpI.
Bakterija Bacillus subtilis si tako s kanibalizmom pomaga, da celice ki vstopajo v sporulacijo dobijo dovolj hranil za dokončanje spore.

== Tina Gregorič: Grelin - hormon lakote ==

Občutek lakote je odvisen od številnih dejavnikov, med katere spadajo telesna sestava in teža, vrsta hrane, ki jo vsak dan uživamo, količina spanja in psihološki dejavniki. Večina ljudi postane lačnih, ko je čas za obrok: zajtrk, kosilo, malica, večerja. Znanstveniki so leta 1999 odkrili hormon, ki sodeluje pri nastanku lakote in poveča apetit. Imenuje se grelin, ki je poznan tudi pod imenom hormon lakote. Gen, ki kodira transkripcijo grelina, je sestavljen iz 117 aminokislin in se ob aktivaciji razcepi na 5 manjših podenot, med katerimi sta najpomembnejša grelin in obestatin. Grelin je sprva neaktiven hormon, sestavljen iz 28 aminokislin. Po esterifikaciji na serinu (Ser3) postane aktiven. Sprosti se v kri in po krvi potuje do hipofize v možganih, kjer se nahajajo grelinski receptorji, imenovani GHRS-1a receptorji. Natančna vezava grelina na receptor zaenkrat še ni znana. Grelin ni edini hormon, ki vpliva na to, kdaj nas bo zajela želja po hranjenju in kdaj nas bo minila. V telesu imamo več kot 40 snovi, ki spodbujajo in zavirajo občutek lakote. Odkritje grelina in raziskovanje njegove vloge v človeškem metabolizmu je odprlo vrata številnim raziskavam in študijam na področju debelosti in motenj, ki so povezane s prehranjevanjem. Hormon grelin je povezan z različnimi obolenji kot so anoreksija, kahesija, SW sindrom in na koncu tudi prekomerna telesna teža, vendar se njegova funkcija od bolezni do bolezni spreminja.

== Andreja Bratovš: Bolečina in njen receptor - TRPA1 ==

Ko začutimo bolečino, je to ponavadi znak, da lahko pride ali pa je že prišlo do poškodbe na ali v našem telesu – opozorilo za nas, naj ukrepamo. V zaznavanje bolečine je vpletenih veliko zapletenih mehanizmov, eno zanimivejših odkritij pa je gotovo receptor TRPA1. TRPA1 je receptorski ionski kanalček, prepusten za različne katione. Aktivirajo ga različni dražljaji: nizka temperatura, oksidativni stres in različne dražilne snovi. Med kemijskimi aktivatorji so zanimivi predvsem: alil izotiocianat (snov, ki daje pekoč okus gorčici, hrenu in wasabiju), alicin (spojina iz česna) ter akrolein (sestavina solzivca). Zanimivo je, da aktivacija TRPA1 poteka preko kovalentne vezave liganda na receptor.
TRPA1 se nahaja v nociceptorjih – to so prosti živčni končiči, ki zaznavajo bolečino – njegova funkcija pa je zaznavanje bolečine, ki jo povzročijo prej navedeni dražljaji. Udeležen je tudi pri občutenju bolečine pri vnetju tkiva, kjer deluje v povezavi z bradikininom – mediatorjem vnetja.
TRPA1 in tudi drugi TRP kanalčki so zanimive tarče za nove vrste analgetikov. Cilj novih zdravil je delovanje le na začetek poti prenosa bolečine in ne centralno na ves živčni sistem, kot je značilno za dosedanja zdravila proti bolečinam. Tako delovanje bi namreč zmanjšalo stranske učinke pri jemanju analgetikov, kot so na primer omotičnost in zaspanost.

== Jernej Mustar: Na+ kanalček Nav1.7 in bolečina ==

Prva asociacija ob besedi bolečina pri nobenem ob nas ni pozitivna. Toda če malo bolj razmislimo, kaj bi bilo brez nje, kmalu pridemo do spoznanj, ki so v nasprotju z prvo asociacijo. Bolečina ima več prednosti kot slabosti v našem življenju, je namreč izjemnega pomena za naše preživetje. Obstajajo ljudje, ki jim je pred tem globalno negativnim občutkom prizanešeno. Bolj konkretno, gre za točkovno "nonsense" mutacijo na genu SCN9A, ki povzroči nepravilno izražanje alfa podenote tipa 9 Nav1.7 proteina. Ta podenota je ključna komponenta, ki skupaj z beta podenotami sestavlja natrijev kanalček Nav1.7. Slednji je v večji količini izražen v perifernem živčevju in igra pomembno vlogo pri čutenju bolečine.
Za boljše poznavanje Nav1.7 so raziskovanje začeli na miših. Uporabili so tako imenovano "knock-out" metodo, s katero izbijejo določen gen in opazujejo posledice. Če so izbili gena SCN9A na obeh alelih (homozigoti), je to rezultiralo v poginu mišk takoj po skotitvi. Pri heterozigotih, kjer je bil odstranjen samo gen na enem alelu, do pogina ni prišlo, a je bilo opaženo zmanjšeno dojemanje bolečine. Zanimivo je dejstvo, da miške ob globalnem pomanjkanju Nav1.7 takoj poginejo, ljudje pa so popolnoma normalni, če odmislimo nesposobnost čutenja bolečine. Razlago za to najdete v moji seminarski, poleg tega pa so predstavljena tudi še druge mutacije Nav1.7 pri ljudjeh, ki rezultirajo v povečani aktivnosti le tega in posledično ojačenem čutneju bolečine.

BIO2 Povzetki seminarjev 2011

2011-12-04T13:01:59Z

JernejMustar:

== Ula Štok: Neuregulin 1 ==

Neuregulin-1 je član proteinov iz družine neuregulinov in je kodiran s strani gena NRG1. Obstaja veliko tipov Neuregulina-1, ki se razlikujejo po funkcionalnosti ter mestu v telesu na katerem delujejo. Najpogosteje delujejo v živčnem sistemu, kjer lahko z nepravilnim delovanjem med drugimi povzročajo tudi zelo razširjeno bolezen - shizofrenijo. Delujejo pa tudi na ostalih tkivih in organih (na primer: srce, pljuča, oprsje in želodec). Generalno obstajata dve poti signaliziranja Neuregulina-1, in sicer: Običajna ter neobičajna pot. Pri običajni poti je ErbB receptor aktiviran direktno, v enem koraku z vezavo Neuregulina-1. To najpogosteje povzroči dimerizacijo ali heterodimerizacijo ErbB receptorja. Dimerizacija ali heterodimerizacija sicer nista nujno potrebni, a vendar do njiju pride na skoraj vseh receptorjih ErbB. Ta združitev povzroči avto- in trans-fosforilacijo intracelularnih domen tega receptorja, kar aktivira vse nadaljnje poti signaliziranja. V končni fazi pa NRG1/ErbB signaliziranje vpliva direktno na transkripcijo. Pri neobičajni poti je postopek podoben, a vendar poteka začetna stopnja malo drugače. Na začetku namreč sodeluje JMa oblika receptorja ErbB4, ki se pod vplivom TACE cepi. Del receptorja (ErbB4-CTF) se odcepi v notranjost celice. Ta peptid je velik približno 80 kD in ima specifično izoblikovano vezavno mesto za Neuregulin-1. Nadaljnji procesi pa potekajo zelo podobno kot pri običajni signalni poti. Neuregulin-1 lahko povzroča shizofrenijo na različne načine, saj sodeluje pri zelo pomembnih procesih, kot so: tvorba sinaps, mielinizacija aksonov, razvoj oligodendrocit itd. Shizofrenija je zelo razširjena bolezen in nihče še ni odkril direktnega postopka k popolni odpravi te bolezni. A vendar, v letu 2009 se je zgodila neke vrste prelomnica v študiju shizofrenije. Odkrili so namreč, da posamezniki, ki so imeli gen za shizofrenijo niso zboleli. Še več! Napaka se jim je odrazila kot zvišanje kreativnih sposobnosti na znanstvenem ali umetniškem področju, odvisno od posameznika. Ob tem se je pojavilo mnogo vprašanj, saj bi na ta način mogoče lahko poiskali pot, da bi shizofrenija postala popolnoma ozdravljiva. A vendar, je to področje še raziskano, saj znanstveniki ne vedo po kakšnih poteh pride do tega, da te mutacije na NRG1 genu ne izrazijo v bolezenskem stanju.

== Maša Mirković: Proteinski produkti genov za disleksijo in z disleksijo povezane motnje ==

Disleksija je motnja, ki se kaže v nesposobnosti branja oziroma razumevanja prebranega, ter napakah in težavah pri izgovarjanju besed. Disleksiki,kot imenujemo posameznike, ki trpijo za disleksijo, imajo kljub normalnim intelektualnim sposobnostim, znanjem in izobrazbo, moteni veščini pisanja in branja s tendenco, da pomešajo med seboj črke ali besede med branjem ali pisanjem. V zadnjih letih, so uspeli ugotoviti mesta na kromosomih, povezana z dovzetnostjo za disleksijo. DYX1C1,KIAA0319,DCDC2 in ROBO1, so bili označeni kot kandidati, z dovzetnostjo za disleksijo. Najbolj obetaven je protein KIAA0319. Je transmembranski protein iz desetih transmembranskih vijačnic, najden v plazemski membrani nevronov. Njegov C-terminalni konec gleda v ekstracelularni matriks, manjši N-terminalni konec pa prehaja v citoplazmo nevrona. C-terminalni konec je visoko glikoziliran in nosi 5 PKD(polycystyc kidney desease) domene in eno MANEC(motif at the N terminus with eight cysteines) domeno. KIAA0319 igra vlogo pri rasti možganov in njihovi migraciji med razvojem možganov-iz tega je razvidno, da je disleksija problem v razvoju nevronov že v zgodnjih letih. Posamezniki z disleksijo nosijo izoobliko tega proteina, ki povzroči nižjo izraženost le tega. Spremembe so v 5'-regiji, ki kodira izoobliko proteina. Najopaznejše povezave z disleksijo se kažejo v 2,3 kb regiji, ki zavzema promotor, prvi nepreveden ekson in del prvega introna – odprti kromatin. Te ugotovitve vodijo, da je 5'-regija KIAA0319 gena tista lokacija alelov, ki največ prispeva k motnji branja.

== Katra Koman: INZULIN ==

Inzulin je peptidni hormon, ki sodeluje v uravnavanju ravni glukoze v krvi. Sintetizira in skladišči se v β-celicah Langerhansovih otočkov trebušne slinavke. Sinteza poteka od prekurzorske molekule preproinzulina preko proinzulina do dokončne zrele molekule inzulina, ki se shrani v skladiščnih veziklih. Ob povišanju ravni glukoze v krvi, na primer po obroku, glukoza, ki je tudi glavni stimulator sekrecije inzulina, iz krvi preide v β-celice skozi GLUT2 transporter. Tam se fosforilira v glukozo-6-fosfat, saj tako fosforilirana ne more več iz celice, lahko pa vstopi v proces glikolize, ki mu sledita še Krebsov cikel in oksidativna fosforilacija, ki povzroči pretvorbo ADP v ATP molekule. ATP molekula stimulira zaprtje kalijevih kanalčkov, kar privede do depolarizacije celične membrane, to pa sproži na odprtje kalcijevih kanalčkov in vdor Ca2+ ionov. Povišana koncentracija kalcijevih Ca2+ ionov v celici stimulira prenos in zlitje skladiščnih veziklov z inzulinom z membrano. Inzulin se tako sprosti v krvni obtok in potuje do tarčnih celic, ki imajo na površini izražene inzulinske receptorje. Ko se veže nanj, prenese signal o povišanju ravni glukoze v krvi v celico. To povzroči kaskado reakcij znotraj celice, ki pa na koncu privedejo do translokacije veziklov z GLUT4 transporterjev na površino celice. Število teh transporterjev za glukozo se na površini celične membrane poveča in glukoza lahko prehaja v celico, posledično pa pade raven glukoze v krvi. Razgradnja inzulina poteka v jetrih in ledvicah. Okvare na katerikoli stopnji poti inzulina se odražajo v diabetesu ali drugih boleznih.

== Rok Štemberger: Protein GABAA (gama aminomaslena kislina A) - zgradba, vloga in zanimivosti ==

V svoji seminarski nalogi sem raziskoval vlogo, pomen in zanimivosti proteina GABAA (gama-aminomaslena kislina A). To je receptor, ki se nahaja predvsem v centralnem živčnem sistemu in je zadolžen zato, da opravlja funkcijo inhibitorja. Lociran je na površini nevrotičnih sinaps in prekinja elektrokemični signal, tako da omogoči prehod kloridnih ionov znotraj celice. To se zgodi takrat ko se ustrezen ligand Gama veže na aktivno mesto tega receptorja. Konformacija podenot se spremeni in to omogoči aktivacijo receptorja. Znanstveniki so ugotovili, da obstaja več vrst GABAA receptorjev, kar pa je odvisno od sestave podenot. Najbolj pogoste podenote so alfa beta in gama v razmerju 2:2:1. V primeru da do prekinitve ne pride se lahko pojavijo epileptični napadi, psihiatrične motnje itd. Stres lahko v dobi odraščanja močno vpliva na GABAA receptorje in jih tudi permanentno strukturno spremeni, kar pa lahko kasneje v našem življenju vpliva predvsem na naš spanec in njegovo kvaliteto. Absint je bila v preteklosti prepovedana pijača, saj je povzročala razna obolenja zaradi substance imenovane tujon. Le ta se je vezala na GABAA receptorje in tako onemogočila njegovo delovanje, zato ker je preprečevala prehod kloridnih ionov v membrano. Sedaj potekajo raziskave teh receptorjev, saj je ključnega pomena čim boljša ozdravitev bolezni, ki nastanejo zaradi nepravilnega delovanja GABAA receptorja.

== Veronika Jarc: Perforin ==

Perforin je protein, ki nastane iz citotoksičnih limfocitov T. S pomočjo grancimov napade tarčno celico in jo uniči. Rečemo lahko, da je pomemben člen pri imunskem odzivu in sodeluje s NK celicami. Sestavljen je iz 555 aminokislin, njegova molekulska masa pa je 62-67 kD. Sestavljen je iz dveh pomembnih domen, domene MACPF in domene C2. Za domeno C2 je značilno, da ima afiniteto do Ca2+ ionov. Saj se na lipidni dvosloj veže le ob prisotnosti kalcija. Drugače obstajata dva različna tipa C2 domene, ki sta bila izolirana iz različnih organizmov. Lahko rečemo, da sta oba tipa zelo podobna v tem, da sta pri tipu 1 N-konec in C-konec obrnjena na vrh domene, kar je nasprotno kot pri tipu 2. Poznamo tri MACPF domene: Plu-MACPF, C8a MACPF in lipokalin C8g. Vse te domene primerjamo z skupino proteinov citolizinov in ugotovimo nekaj podobnosti in nekaj razlik. Na splošno, pa lahko rečemo, da je evolucija poskrbela tako, da so sta si domena MACPF in citolizini raszlični le v nekaj aminokislinah. Poznamo tri mehanizme kako perforin preide v tarčno celico in pri tem pomaga gramcimom B uničit to celico. Prvi mehanizem je prehajanje preko perforinske pore in sicer s pomočjo veziklov preide v celico. Naslednji mehanizem je endosomolitični model, pri katerem je pomemben kompleks s pomočjo katerega prehaja v celico. Kot zadnji mehanizem pa je model prehodne perforinske pore, ki pove, da perforin tvori kanalčke s pomočjo katerih grancimi B preidejo direktno v celico. Grancimi B so serinske proteaze, ki se sintetizirajo v citotoksičnih limfocitih T in NK celicah.

== Taja Karner: Glavoboli in migrene ==

Zaradi stresnega in hitrega tempa življenja, vse več ljudi trpi za občasnimi glavoboli, ki so najpogosteje posledica utrujenosti. Prav tako je vedno več ljudi, ki trpijo za močnejšimi oblikami glavobolov imenovanih migrene. V hujših oblikah migrene lahko glavobol traja do dva dni, močno migreno lahko spremljajo še drugi simptomi kot so slabost, bruhanje, občutljivost na svetlobo in močan zvok, depresija ter nespečnost. Mutacija, ki je največji krivec za nastanek bolezni se pojavlja na kromosomu 10 na genu KCNK18. Ta zapisuje protein TRESK, ki se nahaja v hrbtenjači in deluje kot kalijev kanalček. Mutacija povzroči, da ne pride do izmenjavanja ionov, kar povzroči hude glavobole. V raziskavah so odkrili zanimivo povezavo z anestetikom. Ta namreč ne glede na mutacijo ponovno aktivira kanal. To bi lahko učinkovito pozdravilo migrene, če bi ga le uspeli spraviti v primerno obliko. Ugotovili so tudi, da zdravila, ki vsebujejo citosporin in takrolimus v večini primerov povzročajo migrene v zdravstvu pa jih še vseeno pogosto uporabljajo. Odkritje te mutacije predstavlja revolucijo v zdravstvu in verjamem, da bo kmalu vodilo do odkritja učinkovitega zdravila proti migrenam.

== Ana Dolinar: Univerzalna kri – prihodnost transfuzijske medicine? ==

α-galaktozidaza (AGAL_HUMAN) je glikozil-hidrolazni encim. Spada v GH27-D (klan D, 27. družina) in ima aktivno mesto v obliki (β/α)8 sodčka. Encim zapisuje gen GLA, ki se nahaja na kromosomu X.

Ideja o univerzalni krvi, ki bi bila primerna za transfuzijo, ne glede na krvno skupino pacienta, je med znanstveniki prisotna že približno trideset let.
Razvili so tri metode za pretvorbo različnih antigenov v antigen 0 (po sistemu AB0), ki je primeren za transfuzijo v vse krvne skupine.
:#Encimska razgradnja antigenov A in B do antigena 0. Za antigene A so uporabili α-N-acetilgalaktozaminidazo, vendar so antigeni preveč kompleksni in metoda ni bila uspešna. Pri antigenih B so dosegli popolno pretvorbo v antigen 0 z uporabo α-galaktozidaze iz bakterije ''Streptomyces griseoplanus''.
:#Prekrivanje površine eritrocitov z maleimidofenil-polietilen-glikolom (Mal-Phe-PEG). Prekrije vse antigene, ne samo A ali B, vendar metoda ni uspešna, ker polietilen-glikol povzroča imunski odziv.
:#Pridobivanje univerzalnih rdečih krvnih celic iz pluripotentnih matičnih celic. Uspeli so pridobiti zrele eritrocite, ki so popolnoma funkcionalni.
Uporaba univerzalne krvi bi zmanjšala ali celo izničila imunski odziv ob transfuziji, prav tako ne bi bilo možnosti za transfuzijo napačne krvne skupne zaradi človeške napake. Metode trenutno niso dovolj izpopolnjene, da bi bilo možno pričakovati njeno uporabo v bližnji prihodnosti.

== Maša Mohar: Moški ali ženska to je sedaj vprašanje?(SRY - faktor za določitev spola) ==

SRY gen kodira Sry protein ki je član družine Sox (Sry related HMG box) transkripcijskih faktorjev. Poznamo jih okoli 20 pri človeku in miškah ter še mnogo drugih. Sox proteini imajo zelo različne vloge v embriogenezi in pri razvoju mnogih drugih organov. Tipično delujejo tako kot nekakšna stikala v diferenciaciji celic- sprožijo razvoj določenih celic. Sry je prav tako kot ostali člani te družine karakteriziran po HMG( high mobility group). HMG je drugače skupina specifičnih transkripcijskih faktorjev, ki imajo ~ 80 AK dolge strukturalno podobne domene za vezavo na DNA. Te domene oz. domena če je samo ena se veže na zaporedje (A/T)ACAA(T/A) v majhni žleb DNA. S tem ustvari zvitje DNA za približno 60- 85 stopinj. S tem ko se DNA zvije se razkrijejo mesta za izražanje drugih genov, recimo Sox9, ki kodira Sox9 protein ki pomaga pri diferenciaciji Sertoli celic in tako pri oblikovanju testisov, s tem pa determinira moški spol. Ugotovili smo tudi da obstaja veliko genskih bolezni povezanih s Sry genom in da lahko obstaja tudi ženska z XY spolnima kromosomoma, ker se pri njej zaradi mutacij Sry protein ne izrazi, prav tako pa obstajajo tudi moški z XX spolnima koromosomoma, kjer se enem od X kromosomov lahko izrazi SRY gen ob nepravilnostih pri očetovem delu zapisa. V bistvu sem prišla do zaključka da je zelo tanka meja med moškim in ženskim oblikovanjem spola, ena majhna mutacija oz. ena majhna razlika lahko privede do nastanka ženske ali moškega.

== Urška Rauter: A Green Glow: zgradba in funkcija encima luciferaze ==
Luciferaza je encim odvisen od ATP in magnezijevih ionov. Proces bioluminiscence se začne z vezavo na substrat luciferin, tvori se adenilatni intermediat in ob prisotnosti molekularnega kisika izhaja svetloba. Luciferaza je zgrajena iz dveh ločenih domen, večja se nahaja na N-koncu in manjša na C-koncu molekule, večja domena pa ima tudi svoje poddomene. Domeni sta med seboj ločeni z razpoko, kjer naj bi se po domnevanjih nahajalo tudi aktivno mesto encima. Luciferaza predstavlja tudi nov način mehanizma tvorbe adenilatnega intermediata med encimi in ponuja razlago za marsikatero metabolično pot.
Velika dilema, ki me med znanstveniki ostaja pa je razlika v barvi svetlobe, ki jo proces oksidacije luciferina emitira. Najverjetneje je za to odločilna keto tavtomerna oblika oksiluciferina in tudi resnonančna stabilizacija njegovega fenolatnega aniona, čeprav so znanstveniki odkrili tudi veliko drugih možnih vzrokov za različne barve (različne aminokisline, polarnost okolja, pH, ...).
Luciferaza se veliko uporablja v medicini, kjer služi kot marker molekul v telesu in tako pripomore k boljšem razumevanju različnih bolezni in infekcij, kot tudi sami strukturi celic in njenih organelov.

== Mirjam Kmetič: Mint condition (limonen-3-hidroksilaza in limonen-6-hidroksilaza) ==

Klasasta meta vsebuje encim limonen-6-hidroksilazo, ki sodeluje pri pridobivanju karvona. Poprova meta pa vsebuje limonen-3-hidroksilazo, ki je udeležena pri proizvodnji mentola. Obe hidroksilazi pripadata družini citokromov P450, njeni predstavniki pomembno sodelujejo pri proizvajanju različnih oksidiranih monoterpenov, ki so vir arom eteričnih olj. Karvon in mentol sta končna produkta hidroksilacije limonena. Ta encima sta si zelo podobna in njuni vezavni mesti za substrat sta zelo omejeni. Velja pravilo, da za spremembo aktivnosti v družini citokromov P450 potrebujemo določeno število mutacij, vendar je za modifikacijo vezavne aktivnosti limonenovih hidroksilaz potrebna samo ena. Ta fenilalanin v izolevcin mutacija povzroči, da se limonen-6-hidroksilaza spremeni v limonen-3-hidroksilazo! Mutiran encim je tako sposoben sinteze mentola tako kot encim v poprovi meti! Taka mutacija kaže, da sta prav ti dve aminokislini ne le nujni, temveč tudi prav zagotovo vpleteni pri orientaciji limonena v aktivnem mestu tako, da se ta hidroksilizira na ali C3 ali C6 poziciji. Posamične mutacije, ki lahko drastično spremenijo funkcijo proteina, so znanstveno zanimive. Nakazujejo ne le na zelo specifične manjše regije v sekvenici proteina, temveč so tudi ključne za razumevanje področij, kot so vezava in orientacija substrata, funkcija encima, metabolična pot in struktura vezavnega mesta.

== Sandi Botonjić: Kokain esteraza ==

Znanstveniki so v rizosferi kokinih plantaž (Erythroxylum coca) našli sev MB1, gram pozitivne bakterije Rhodococcus sp.. Tej bakteriji kokain predstavlja glavni vir ogljika in dušika in zato so znanstveniki izolirali osrednji encim njenega metabolizma tj. kokain esterazo (v nadaljevanju cocE). Encim je sestavljen iz treh domen: DOM1, ki vsebuje nabor kanoničnih α-vijačnic in β-ploskev; DOM2 - domena le z α-vijačnicami; in DOM3 je roladi podobna struktura z β-ploskvami. CocE je serinska esteraza, katere aktivno mesto se nahaja na stičišču vseh treh domen. Ta hidrolizira kokain na ekgonil metil ester in benzojsko kislino, ki nimata psihoaktivnih učinkov. CocE je pravi Ferrari v primerjavi z drugimi esterazami, saj lahko razgradi enako količino kokaina 1000 krat hitreje. Tako lahko postane neprecenljiva pri nujnih intervencijah v primeru prevelikega odmerka, saj bi intravenozni vbrizg cocE močno zmanjšal razpolovni čas kokaina. CocE je predmet številnih raziskav, v katerih znanstveniki proučujejo njeno termostabilnost in njenih mutiranih oblik, saj njen razpolovni čas pri fiziološki temperaturi traja le nekaj minut. Znanstveniki pa na podlagi ugotovitev iz raziskav cocE razvijajo tudi učinkovita protitelesa z vsaj podobnimi katalitičnimi parametri, ki bi brez imunskega odziva odlično delovala v bioloških sistemih.

==Tjaša Flis: Parkinsonizem in Parkin protein==

Parkinsonova bolezen je vse pogostejša bolezen pri starostnikih, njeni simptomi pa so tresavica, mišična otrdelost in upočasnjena motorika. Vzrok se skriva v propadu dopamnergičnih nevronskih celic. Bolezen je lahko avtosomno dominantno dedovana, kar pomeni, da pacienti podedujejo eno normalno in eno mutirano kopijo gena. Slednja prevladuje in se deduje naprej. Pri Parkinsonovi bolezni se mutacija zgodi v Park2 genu, ki kodira Parkin protein ali E3 ubikvitin ligazo. Parkin na poškodovane ali na preveč izražene proteine pripne ubikvitin (označevalni protein), ki jih nato usmeri v proteasom, to je velik razgradni kompleks v celicah.
Če mutacija poškoduje Parkin, je pot razgradnje onemogočena, to pa pomeni, da se v celici akumulirajo odvečni proteini. Tvorijo se Lewy-eva telesca polna teh proteinov, ki nadomestijo celične organele v nevronskih celicah, kar vodi do prenehanja njihovega delovanja. Ker pa ima Parkin več kot samo en substrat ki ga ubikvitinira, je točen mehanizem bolezni še dandanes uganka.
Eden izmed najbolj poznanih substratov je transmembranski protein Pael-R. Zvitje tega proteina poteka ob prisotnosti šaperonov. Prevelika koncentracija tega receptorja lahko izzove stres v endoplazmatskem retikulumu situiranem v nevronskih celicah. V primeru da je Parkin neaktiven, Pael-R povzroči celično smrt. Vendar to je le ena izmed možnih rešitev, substratov je namreč vsaj še dvajset, raziskave pa se nadaljujejo.

== Matja Zalar: Vloga SRK in SCR proteinov pri preprečevanju incestnega razmnoževanja cvetočih rastlin ==

Rastline so za zaščito pred samooplojevanjem razvile več vrst mehanizmov prepoznavanja lastnega peloda na molekularni ravni. Pri cvetočih rastlinah je najpogostejši mehanizem tipa SSI ali sporofitične lastne inkompatibilnosti. Pri družini ''Brassicaceae'' je za aktivacijo SSI ključna interakcija med transmembranskim proteinom SRK, ki predstavlja žensko determinanto odziva, in njenim ligandom - proteinom SCR, drugače imenovanim tudi moška determinanta odziva na lastno inkompatibilnost. Specifičnost vezave je zagotovljena s polimorfizmom alel obeh determinant. V posameznih vrstah je možno najti tudi do 100 različnih S-haplotipov genov za determinanti.
Vezava liganda na receptor bo uspešna le, če oba izhajata iz istega S-haplotipa. Vezava SCR na zunajcelično, N-glikolizirano domeno SRK povzroči nastanek kompleksa treh proteinov, ki s svojo aktivnostjo sproži kaksado reakcij, kar v končni fazi pripelje do preprečitve samooploditve.
Na neugodne življenske pogoje, ki so onemogočali medsebojno opraševanje, so se nekatere rastline prilagodile s favorizacijo samooplojevanja. Pri njih so mutacije S-lokusa, ki nosi zapis za SRK in SCR, povzročile nepravilno delovanje SI ali njegovo popolno odpoved. To pa seveda vodi v neprepoznavanje lastnega peloda in rastlina se samooprašuje. Najbolj znan primer take rastline je ''Arabidopsis thaliana'', ki se zaradi svojih specifičnih lastnosti uporablja kot modelni organizem v številnih študijah lastne inkompatibilnosti.

== Matevž Ambrožič: BSX protein in debelost ==

Za primeren občutek sitosti ali lakote glede na stanje energetskih zalog v telesu in odgovarjajoč vnos hrane ter porabo energije je odgovorna zapletena pot sporočanja. Začne se s tremi hormoni: inzulin, leptin in grelin. Leptin in inzulin se sprostita, ko so maščobne in hidratne zaloge v telesu polne in morata do možganov prenesti signal za prenehanje hranjenja, grelin pa ravno nasprotno. Vsi po krvi potujejo do hipotalamusa, predela možganov, ki je odgovoren za energijsko ravnovesje. V hipotalamusu sta dva tipa živčnih celic: oreksigene in anoreksigene. Prve sproščajo NPY in AgRP, nevropeptida, ki spodbujata hranjenje in zmanjšata porabo energije, druge pa α-MSH in CART, katerih učinek je nasproten. Našteti nevropeptidi se iz nevronov sprostijo po vezavi ustreznega izmed treh hormonov in prenesejo signal naprej, do končne spremembe v vnosu ali porabi energije. Glavni protein seminarja, BSX (brain specific homeobox) protein je transkripcijski faktor, ki spodbudi ekspresijo genov za AgRP in NPY, hkrati pa je odgovoren za premik organizma v iskanju hrane. Če v opisanem sistemu pride do napake, so pojavi nepotreben občutek lakote, kar je vzrok mnogih primerov debelosti. V boju z bolezensko debelostjo so ključne raziskave na BSX proteinu, saj je osrednji člen poti, ki v možgane prenese (včasih lažen) občutek lakote.

== Kaja Javoršek: A grey matter ==

Mikrocefalin je protein, ki ga kodira enakoimenski gen. Mikrocefalin naj bi kontroliral poliferacijo in diferenciacijo nevroblastov med nevrogenezo. Odkritje, da je mikrocefalin odločilen regulator velikosti možganov, je sprožilo hipotezo, da je igral vlogo v evoluciji možganov.
Razen v možganih najdemo mikrocefalin tudi v ledvicah, srcu, pljučih, vranici in skeletnih mišicah. Vendar pomen mikrocefalina v teh organih še ni znan.
Mutacije na genu mikrocefalina vodijo do nastanka mikrocefalije. To je bolezen razvoja živčnega sistema in je definirana kot resno zmanjšana velikost možganov. Pri odraslih je normalen volumen možganov od 1200 cm3 do 1600 cm3, pri odraslih s primarno mikocefalijo pa okoli 400 cm3 . Poleg mirocefalina pa povzročajo mikrocefalijo še mutacije petih genih (ASPM, MCPH2, CDK5RAP2, MCPH4, CENPJ)
Mikrocefalin ima tri BRCT domene na C – koncu. BRCT domene so prisotne v veliko ključnih proteinih, ki kontrolirajo delitev celice. Zato predvidevajo da mikrocefalija nastane, ker je ovirana normalna regulacija delitve celic v možganih.
Ugotovili so, da je protein mikrocefalin dol 835 aminokislin. Zaradi mutacije na genu mikrocefalina se ta protein skrajša na 25 aminokislin.
Znanstveniki so izvedli raziskavo ali gena mikrocefalin in ASPM vplivata na inteligenco. Na podlagi treh raziskav so zaključili, da inteligenca ni povezana z dominantnimi aleli ASPM – ja ali mikrocefalina.

== Rok Vene: A mind astray ==

Alzheimerjeva bolezen postaja vedno bolj aktualna tematika. Trenutno je na svetu več kot 26 milijonov ljudi s to obliko demence. Zaradi daljše življenjske dobe pa pričakujemo, da bo število obolelih samo še naraščalo. Alzheimerjeva bolezen prizadene centralni živčni sistem, v možganih se nalagajo snovi, ki povzročijo propad živčnih celic. Ena izmed snovi, ki se nalagajo v možganih so nefunkcionalni Tau proteini. Tau proteini sodijo v družino proteinov imenovanih microtubule-associated proteins (MAP), njihova naloga pa je je stabilizacija mikrotubulov. To dosežejo tako, da se na mikrotubule vežejo. Poleg tega predvidevajo, da imajo Tau proteini še eno nalogo. Sodelovali naj bi v kompleksu za uravnavanje vzdražnosti živčnih celic. Nefunkcionalnost Tau proteinov povezujejo z različnimi boleznimi, ki jih poznamo pod skupnim imenom tauopatije. V primeru Alzheimerjeve bolezni je Tau protein nefunkcionalen, zato ker je hiperfosforiliran, kar mu onemogoča vezavo na mikrotubule. Tau proteini zato tvorijo netopne agregate – nevrofibrilarne pentlje, ki najbrž povzročijo odmiranje živčnih celic. Pri iskanju učinkovin proti hiperfosforilaciji in agregaciji Tau proteina, so znanstveniki raziskali protein FKBP52. Ta protein ima več funkcij. Osredotočili so se predvsem na njegove šaperonske lastnosti. Ugotovili so, da se FKBP52 veže na hiperfosforiliran Tau protein, in tako prepreči agregacijo Tau proteina, ki je odgovorna za odmiranje nevronov.

== Ines Šterbal: LTP1 ==

Protein LTP1, izoliran iz ječmenovega zrna, spada v družino lipidnih prenašalnih proteinov (lipid transfer protein –LTP). Je dobro topen protein, ki se nahaja v alevronski plasti ječmenovega semena. Sestavljen je iz štirih heliksov, ki so povezani z disulfidnimi mostički. Ima dobro definiran C-terminalni konec. V razmerah in vivo je globularni protein, s stožčastim hidrofobnim jedrom, ki se razteza od enega konca molekule do drugega. Sposoben je vezati različne lipide, kot so maščobne kisline ali acetil-koencim A. LTP1 proteini so na površini aktivni proteini, so stabilni, denaturirajo šele okrog 100 °C. Vloga LTP1 proteina in vivo še ni znana. In vitro je glavni protein pri penjenju piva. Opravlja pa še številne druge funkcije, odvisno od tega, kateri ligand ima vezan. LTP1 proteini so verjetno vključeni v prenos lipidov preko membrane in celo v nastanek membrane, lahko bi imeli vlogo v transportu monomera Cutin, vlogo naj bi igrali tudi v obrambnem mehanizmu rastlin. Lipidi, ki so vezani na LTP1 bi naj imeli antibakterijsko aktivnost za bakterije in glive.
Vsi podatki kažejo, da so povezave med sladkorji in proteini, ki nastanejo kot produkt Milardove reakcije, prvi korak do nastanka pivovske pene. Kaže, da je kontrola glikacije LTP1 proteinov med slajenjem in varjenjem piva, nujna za optimalno penjenje piva.

== Mitja Crček: DSIP in spanje ==

Pred 2000 leti so ljudje verjeli, da postanemo zaspani zaradi nekakšnih želodčnih hlapov, ki gredo v možgane, se tam kondenzirajo, zamašijo pore in posledično povzročajo zaspanost. Kasneje so seveda ugotovili da temu ni tako, leta 1977 pa so odkrili majhne peptide, ki naj bi nas uspavali in jih poimenovali Delta Sleep-Inducing peptide (DSIP). DSIP je majhen peptid, sestavljen iz devetih aminokislinskih ostankov in maso 850 daltonov, prvič pa so ga odkrili pri zajcih. Sodeloval naj bi tako pri endokrini regulaciji kot pri fizioloških procesih (poveča učinkovitost oksidativne fosforilacije), pomembno vlogo pa naj bi imel tudi v medicini in pri zdravljenju bolezni. Ker naj bi podaljševal REM fazo, bi ga lahko uporabljali tudi kot dodatek pri zdravljenju alkoholizma ali ga dodajali antidepresivom in pomirjevalom, ki skrajšujejo REM fazo. Raziskave so spremljale tudi vpliv DSIP-ja na nespečnost. Ugotovili so, da DSIP rahlo povečuje kvaliteto spanja in skrajšuje latenco uspavanja, na trajanje budnosti in druge parametre pa ne vpliva, zato so si strokovnjaki enotni, da ima DSIP le rahle terapevtske učinke na nespečnost. Delovanje peptida pa še vedno ni povsem razjasnjeno in le želimo si lahko, da bodo novejše raziskave prinesle nove informacije, saj ima DSIP vsekakor velik potencial v medicini.

== Dominik Kert: FOXP2, govoreči protein ==

Ljudje in živali se razlikujejo. Za znanstvenike 19. stoletja je bilo zelo fascinantno to, da mi lahko govorimo, se sporazumevamo in pomnimo besede, medtem ko živali ne morejo. Ko se je pojavila družina KE na koncu 90. let prejšnjega stoletja, so znanstveniki ugotovili, da obstaja gen, ki kodira FOXP2. Družina KE je slovi po tem, da ima polovica njenih članov težave z govorom. Tako so ugotovili, da se mutacija prenaša avtosomno in dominantno. In verjetno na to vpliva mutacija FOXP2, FOXP2 protein pa je po vsej verjetnosti odločilen faktor pri govoru.
FOXP2 protein je sestavljen iz 715 aminokislin in spada med družino transkripcijskih faktorjev, ki se imenuje FOX (zaradi 'forkhead box' domene). Zanimivo je, da se ta gen razlikuje od gena opic (šimpanz, gorila, makaki) le za dve in od miši le za tri aminokisline. To se znanstvenikom zdi zelo zanimivo, ker je verjetno zaradi teh dve sprememb v aminokislinskem zaporedju prišlo do sprememb pri sporazumevanju. Zaradi teh dejstev so se naprej usmerili na to, ali je bil gen res pod vplivom naravne selekcije in ugotovili so, da je bil res.
FOXP2 na te spremembe vpliva v možganih, je pa prisoten tudi v pljučih, drobovju in srcu. Vendar njegova funkcija tam še ni znana.

== Petra Malavašič: Ureaza bakterije Helicobacter pylori ==

Bakterija Helicobacter pylori spada med patogene mikrobe. Znanstvenika Warren in Marshall sta leta 1987 odkrila to bakterijo ter ugotovila, da je s to bakterijo povezana razjeda na želodcu. Leta 2005 sta prejela Nobelovo nagrado. Že vsak drugi človek je okužen s to bakterijo. Naseljena je na želodčni sluznici in povzroča kronično vnetje želodčne sluznice. Bakterija se lahko naseli in se razmnožuje v prisotnosti želodčne kisline, kjer je pH okoli 2. Posebni obrambni mehanizmi omogočajo bakteriji, da lahko preživi v kislem okolju. Encim ureaza je pri tem najpomembnejši. Ureaza je encim, ki katalizira hidrolizo uree, pri čemer nastane amoniak, ki se v končni fazi veže z molekulami vode v amonijev hidroksid, ki poveča pH v neposredni okolici bakterije. Encim ureaza se nahaja v citoplazmi bakterijske celice in na njeni površini. Sam encim je zgrajen zelo kompleksno in omogoča bakteriji preživetje. Posebna kompleksna zgradba encima onemogoči, da bi kislina želodčnega soka denaturirala encim. Encim sestavljata dva kompleksa (αβ) štirih prostorsko razporejenih (αβ)3 enot.

== Matevž Merljak: CEM15, VIF in infektivnost retrovirusov ==

Ena izmed komponent obrambnega mehanizma pred retrovirusi v nekaterih človeških celicah je citidinska deaminaza CEM15 (APOBEC3G). V celicah, ki jo izražajo, se retrovirusi brez posebnega proteina (VIF, “viral infectivity factor”) ne morejo uspešno množiti, zato takim celicam pravimo “nepermisivne” celice.

CEM15 deluje tako, da med procesom reverzne transkripcije v novonastali “minus” DNA verigi številne citidinske baze pretvori v uridinske, ter s tem povzroči tako zmanjšano obstojnost z uracilom bogate DNA verige, kot tudi zamenjave gvanozinskih baz z adenozinskimi v kodirajoči (“plus”) verigi DNA. Čeprav takšna hipermutacija za nadaljno infektivnost virusa ni vedno usodna (torej lahko tako mutirana DNA v nekaterih primerih še vedno tvori funkcionalne viruse), je običajno dovolj obsežna, da onemogoči uspešno reprodukcijo virusa.

Raziskave kažejo, da CEM15 ne napade nastajajoče DNA kot lasten celični odgovor na infekcijo, pač pa se med izgradnjo novih virusov vgradi v le-te ter po infekciji nove celice povzroči omenjene spremembe v nastajajoči DNA verigi.

Že omenjen faktor VIF izhaja iz virusa HIV-1, ki primarno napada sicer nepermisivne limfocite T. Naloga VIF je preprečitev vgradnje CEM15 v nastajajoče viruse, to pa doseže tako z oteževanjem njene translacije, kot tudi z indukcijo razgradnje CEM15 v proteasomu.

== Eva Knapič: TSH3 - Kaj novorojenčkom omogoča zadihati? ==

Kaj novorojenčkom omogoča zadihati? Raziskave so pokazale, da ima eno izmed vodilnih vlog pri začetku dihanja protein teashirt homolog 3 (TSH3). To je protein, ki ga uvrščamo med transkripcijske faktorje. Po strukturi spada v družino cinkovih prstov, kjer so sekundarne strukture koordinirane s cinkovim ionom. TSH3 ima pet tako urejenih struktur in vse spadajo v Cys2His2 skupino – cinkov ion koordinira dva cisteinska in dva histidinska ostanka ßßα podenote.
Organizem brez zapisa za teashirt 3 protein se v času embrionalnega razvoja navidezno ne razlikuje od organizmov, ki ta zapis imajo. Vendar so podrobnejše raziskave pokazale, da se brez prisotnosti proteina teashirt 3 dokončno ne oblikujejo pljučni mešički, ki so funkcionalna enota pljuč, saj tam poteka izmenjava plinov. Odsotnost proteina povzroča povečano apoptozo nevronov motoričnega jedra v možganskem deblu, s tem so proteinu pripisali zmožnost inhibicije apoptoze nevronov. Prav tako so nezmožnost odziva organizma na pH spremembe okolja pripisali pomanjkanju proteina TSH3.
Iz vseh teh pomanjkljivostih, ki jih povzroča TSH3 so raziskovalci prišli do zaključka, da novorojenček brez zapisa za protein ni zmožen zadihati, ker ni sposoben odziva na spremembo okolja, predvsem pH in tako ne more vzdrževati homeostaze, ki je potreba na preživetje.

== Tjaša Goričan: Vpliv Nogo proteina na regeneracijo živčnega sistema ==

Nevroni vsebujejo mielin, ki je sestavni del mielinske ovojnice aksona in ima nalogo zagotavljanja stalnega prenosa električnih signalov. Poleg tega pa mu je dodeljena tudi nenavadna lastnost. Vsebuje namreč proteine Nogo-A, ki delujejo kot inhibirotji za rast poškodovanih aksonov. Posledično se diferencirani nevroni niso sposobni deliti. Problem se pojavi pri poškodbi živčnega sistema, saj se ni sposoben regenerirati. Bolezni, ki so povezane s poškodbami živčevja so: Poškodbe hrbtenjače, Alzheimerjeva bolezen, možganska kap, shizofrenija itd.

Nogo-A protein spada v družino proteinov retikulonov in je ena od oblik Nogo proteinov. Je transmembranski protein, ki se z domeno Nogo-66 uspešno veže na receptor in povzroči razgradnjo mikrotubulov v aksonu, kar privede do preureditve citoskeleta in posledično zaustavitve rasti aksona. Največ Nogo-A se nahaja na oligodendrocitih. Oligodendrociti so celice, ki spadajo med nevroglio in tvorijo mielinski ovoj nevronov v centralnem živčnem sistemu. Veliko več ga najdemo v centralnem živčnem sistemu v primerjavi s perifernim.

Čeprav je še veliko neznanega na področju živčnega sistema, je znanost že dosegla uspehe glede boja proti boleznimi, povezanimi z regeneracijo živčnega sistema. S protitelesi se da inhibirati protein Nogo-A in s tem preprečiti inhibicijo rasti poškodovanih nevronov.

== Marko Radojković: Fluorescentni proteini in njihova uporaba v živčnem sistemu ==

Fluorescentni proteini so členi družine homologih proteinov, ki se delijo skupno lastnost da svetlijo zaradi formiranja kromoforma znotraj lastnega polipeptidnega zaporedja. Prvi odkrit takšen protein je bil zeleni fluorescentni protein ali GFP. Od tedaj do danes so kreirani različni mutanti, ki žarijo skoraj vse barve človeškega vidnega spektra. Izkazalo se je da so zelo uporabni v mnogih bioloških disciplinah, predvsem pa so popularni v spremljanju dinamike proteinov, genske ekspresije, in tudi posledično na viši ravni, dinamike organelov ter gibanja celic znotraj tkiva.

Ne tako dolgo nazaj, je tim znanstvenikov uspel skombinirati različne barvne variante GFP-ja s sofisticiranim Cre/Lox sistemom genske rekombinacije in tako omogočil njihovo izražanje v samih možganih. Tale tehnika omogoča da se vsaki posamezni nevron obarva drugače in tako loči od sosednjih, kar omogoča detajlno analizo živčnega vezja. Brainbow strategija, kakor so jo poimenovali, daje upanje znanstvenikom da z ustvarjanem celotnega ''zemljevida'' možganov, lahko izpeljejo pomembne informacije o nevronskih povezavah in njihov nadaljni vpliv na vedenje in delovanje organizma.

== Tamara Marić: MikroRNA ==
MikroRNA je mala molekula, ki je prepisana z DNA na tak način kot mRNA. Zapis za miRNA se lahko nahaja v intronskih regijah, kodirajočih ali nekodirajočih genov. Osnovna funkcija je utišanje genov na nivoju sinteze proteinov. Da pa lahko opravi svojo nalogo mora dozoreti. Biogeneza miRNA se začne v samem jedru, kjer se 1000 nukleotidov dolg transkript s pomočjo encimskega kompleksa (Drosha-DGCR8)skrajša na 60-70 nukleotidov dolg pre-miRNA.Z eksportinom-5 se prenese iz jedra v citoplazmo do naslednjega kompleksa. Dicer veže pre-miRNA in jo skrajša na 22 nukleotidov. Nastane miRNA dupleks. Ena izmed verig prevzame vodilno funkcijo in se vmesti v kompleks istega encima v povezavi z drugimi proteini. Kompleks pripelje do komplamentarne verige mRNA in povzroči translacijsko represijo. Znanstveniki se ukvarjajo predvsem z vprašanjem,kako se miRNA izraža v številnih boleznih. Natančneje sem si pogledala proces resorpcije in obnove kosti in kako miRNA vpliva na regulacijo teh dveh procesov.

== Maja Remškar: Okulokutani albinizem tipa II in P protein ==

Melanin je pigment, ki je nujno potreben za zaščito kože pred pripekajočim soncem ter za normalno delovanje oči. Glavna sestavina za njegovo sintezo je aminokislina tirozin, ki je osnova evmelanina (črni pigment), ob dodatku cisteina pa dobimo še feomelanin (rdeče-rumen). Za običajno delovanje biosinteze melanina je potrebno kislo okolje v melanosomih, kjer se sinteza izvaja. Za vzdrževanje kislosti sta potrebna dva proteina – anionski kanalček in ATP črpalka. Anioni tu delujejo kot vaba za protone, kjučne za kisel pH. P protein naj bi deloval kot anionski transporter. Torej v njegovi odsotnosti v melanosom ne morejo dostopati anioni in posledično se v celico ne prečrpavajo protoni, kar pomeni da ni kislega pH ugodnega za sintezo melanina.
Okulokutani albinizem tipa II ali OCA2 nastane zaradi pomanjkanja količine melanina v očeh, koži in laseh. Za kožo to pomeni večjo občutljivost na UV žarke in povečano možnost za kožnega raka. Zaradi nepigmentiranih optičnih vlaken pa se pojavijo še težave z očmi, kot so škiljenje, fotofobija, nistagmus, degeneracija rumene pege, pride pa tudi do izgube biokularnega vida. OCA2 je dedna bolezen, ki se deduje recesivno. Človek le z enim okvarjenim alelom je torej prenašalec gena. Ugotovili so, da OCA2 povzroča mutacija gena P, in sicer najpogostejša je delecija 7 eksona.

== Ana Remžgar: Bacillus subtilis ==

Bacillus subtilis je grampozitivna paličasta bakterija. Ko ima v okolju dovolj hranil, se simetrično deli in vegetativno raste. Ko pa v okolju začne hranil primanjkovati, B. subtilis uvede različne mehanizme, da lahko preživi. Del populacije postane kompetenten in sprejme tujo DNA. Del populacije pa s pomočjo zapletenega sistema aktivacije proteina Spo0A vstopi v proces sporulacije. Sporulacija je počasen in energijsko potraten postopek, ki traja v idelanih razmerah vsaj 7 ur. Na koncu nastane spora, ki lahko preživi tudi več desetletji v neugodnih življenjskih razmerah. Ko celica vstopi v cikel sporulacije, začne v okolje izločati razne toksične snovi, med njimi sta najbolj učinkovita Skf in Sdp. Ko celica izloči ti dva proteina v okolje, ubije sosednje bakterijske celice Bacillis subtilisa. Zaradi njunih lasnosti, ta dva proteina pogosto zato imenujemo kanibalistična faktorja. Vendar mora celica paziti, da pri tem ne ubije še sebe. Pri tem ji pomaga medmembranski protein SdpI.
Bakterija Bacillus subtilis si tako s kanibalizmom pomaga, da celice ki vstopajo v sporulacijo dobijo dovolj hranil za dokončanje spore.

== Tina Gregorič: Grelin - hormon lakote ==

Občutek lakote je odvisen od številnih dejavnikov, med katere spadajo telesna sestava in teža, vrsta hrane, ki jo vsak dan uživamo, količina spanja in psihološki dejavniki. Večina ljudi postane lačnih, ko je čas za obrok: zajtrk, kosilo, malica, večerja. Znanstveniki so leta 1999 odkrili hormon, ki sodeluje pri nastanku lakote in poveča apetit. Imenuje se grelin, ki je poznan tudi pod imenom hormon lakote. Gen, ki kodira transkripcijo grelina, je sestavljen iz 117 aminokislin in se ob aktivaciji razcepi na 5 manjših podenot, med katerimi sta najpomembnejša grelin in obestatin. Grelin je sprva neaktiven hormon, sestavljen iz 28 aminokislin. Po esterifikaciji na serinu (Ser3) postane aktiven. Sprosti se v kri in po krvi potuje do hipofize v možganih, kjer se nahajajo grelinski receptorji, imenovani GHRS-1a receptorji. Natančna vezava grelina na receptor zaenkrat še ni znana. Grelin ni edini hormon, ki vpliva na to, kdaj nas bo zajela želja po hranjenju in kdaj nas bo minila. V telesu imamo več kot 40 snovi, ki spodbujajo in zavirajo občutek lakote. Odkritje grelina in raziskovanje njegove vloge v človeškem metabolizmu je odprlo vrata številnim raziskavam in študijam na področju debelosti in motenj, ki so povezane s prehranjevanjem. Hormon grelin je povezan z različnimi obolenji kot so anoreksija, kahesija, SW sindrom in na koncu tudi prekomerna telesna teža, vendar se njegova funkcija od bolezni do bolezni spreminja.

== Andreja Bratovš: Bolečina in njen receptor - TRPA1 ==

Ko začutimo bolečino, je to ponavadi znak, da lahko pride ali pa je že prišlo do poškodbe na ali v našem telesu – opozorilo za nas, naj ukrepamo. V zaznavanje bolečine je vpletenih veliko zapletenih mehanizmov, eno zanimivejših odkritij pa je gotovo receptor TRPA1. TRPA1 je receptorski ionski kanalček, prepusten za različne katione. Aktivirajo ga različni dražljaji: nizka temperatura, oksidativni stres in različne dražilne snovi. Med kemijskimi aktivatorji so zanimivi predvsem: alil izotiocianat (snov, ki daje pekoč okus gorčici, hrenu in wasabiju), alicin (spojina iz česna) ter akrolein (sestavina solzivca). Zanimivo je, da aktivacija TRPA1 poteka preko kovalentne vezave liganda na receptor.
TRPA1 se nahaja v nociceptorjih – to so prosti živčni končiči, ki zaznavajo bolečino – njegova funkcija pa je zaznavanje bolečine, ki jo povzročijo prej navedeni dražljaji. Udeležen je tudi pri občutenju bolečine pri vnetju tkiva, kjer deluje v povezavi z bradikininom – mediatorjem vnetja.
TRPA1 in tudi drugi TRP kanalčki so zanimive tarče za nove vrste analgetikov. Cilj novih zdravil je delovanje le na začetek poti prenosa bolečine in ne centralno na ves živčni sistem, kot je značilno za dosedanja zdravila proti bolečinam. Tako delovanje bi namreč zmanjšalo stranske učinke pri jemanju analgetikov, kot so na primer omotičnost in zaspanost.

== Jernej Mustar: Na+ kanalček Nav1.7 in bolečina ==

Prva asociacija ob besedi bolečina pri nobenem ob nas ni pozitivna. Toda če malo bolj razmislimo, kaj bi bilo brez nje, kmalu pridemo do spoznanj, ki so v nasprotju z prvo asociacijo. Bolečina ima več prednosti kot slabosti v našem življenju, je namreč izjemnega pomena za naše preživetje. Obstajajo ljudje, ki jim je pred tem globalno negativnim občutkom prizanešeno. Bolj konkretno, gre za točkovno "nonsense" mutacijo na genu SCN9A, ki povzroči nepravilno izražanje alfa podenote tipa 9 Nav1.7 proteina. Ta podenota je ključna komponenta, ki skupaj z beta podenotami sestavlja natrijev kanalček Nav1.7. Slednji je v večji količini izražen v perifernem živčevju in igra pomembno vlogo pri čutenju bolečine.
Za boljše poznavanje Nav1.7 so raziskovanje začeli na miših. Uporabili so tako imenovano "knock-out" metodo, s katero izbijejo določen gen in opazujejo posledice. Če so izbili gena SCN9A na obeh alelih (homozigoti), je to rezultiralo v poginu mišk takoj po skotitvi. Pri heterozigotih, kjer je bil odstranjen samo gen na enem alelu, do pogina ni prišlo, a je bilo opaženo zmanjšeno dojemanje bolečine. Zanimivo je dejstvo, da miške ob globalnem pomanjkanju Nav1.7 takoj poginejo, ljudje pa so bili popolnoma normalni, če odmislimo nesposobnost čutenja bolečine. Razlago za to najdete v moji seminarski, poleg tega pa so predstavljena tudi še druge mutacije Nav1.7 pri ljudjeh, ki rezultirajo v povečani aktivnosti le tega in posledično ojačenem čutneju bolečine.

BIO2 Seminar 2011

2011-10-09T10:23:27Z

JernejMustar: /* Seznam seminarjev - datumi še niso dokončni, listka na katerem imam napisano kdaj kdo ne more nimam doma in bom to popravil v ponedeljek */

= Biokemijski seminar =

Seminarje vodi doc. dr. Gregor Gunčar in so na urniku vsako sredo in petek po eni uri predavanj iz Biokemije.

Ocena seminarjev predstavlja 30% končne ocene in vsebuje vse točke, ki jih študent/ka lahko zbere pri seminarju in ostalih dejavnostih, ki niso del pisnega izpita.

== Seznam seminarjev - datumi še niso dokončni, listka na katerem imam napisano kdaj kdo ne more nimam doma in bom to popravil v ponedeljek==
Vpišite svoj izbrani naslov!!!
{| {{table}}
| align="center" style="background:#f0f0f0;"|'''Ime in priimek'''
| align="center" style="background:#f0f0f0;"|'''Naslov seminarja'''
| align="center" style="background:#f0f0f0;"|'''Rok za oddajo'''
| align="center" style="background:#f0f0f0;"|'''Rok za recenzijo'''
| align="center" style="background:#f0f0f0;"|'''Datum predstavitve'''
| align="center" style="background:#f0f0f0;"|'''Recenzent1'''
| align="center" style="background:#f0f0f0;"|'''Recenzent2'''
|-
| Ula Štok||Tipping the mind||17.10.11||19.10.11||21.10.11||||
|-
| Maša Mirković||Naslov seminarja||17.10.11||19.10.11||21.10.11||||
|-
| Sara Draščič||Naslov seminarja||17.10.11||19.10.11||21.10.11||||
|-
| Katra Koman||Naslov seminarja||18.10.11||23.10.11||26.10.11||||
|-
| Iza Ogris||Naslov seminarja||21.10.11||25.10.11||28.10.11||||
|-
| Ana Remžgar||Naslov seminarja||21.10.11||25.10.11||28.10.11||||
|-
| Urška Rauter||Naslov seminarja||21.10.11||25.10.11||28.10.11||||
|-
| Taja Karner||Naslov seminarja||21.10.11||26.10.11||02.11.11||||
|-
| Rok Štemberger||Naslov seminarja||21.10.11||28.10.11||04.11.11||||
|-
| Maša Mohar||Naslov seminarja||21.10.11||28.10.11||04.11.11||||
|-
| Veronika Jarc||Naslov seminarja||21.10.11||28.10.11||04.11.11||||
|-
| Mirjam Kmetič||Naslov seminarja||26.10.11||02.11.11||09.11.11||||
|-
| Janez Meden||Naslov seminarja||28.10.11||04.11.11||11.11.11||||
|-
| Tjaša Flis||Naslov seminarja||28.10.11||04.11.11||11.11.11||||
|-
| Sandi Botonjić||Naslov seminarja||28.10.11||04.11.11||11.11.11||||
|-
| Kaja Javoršek||Naslov seminarja||02.11.11||09.11.11||16.11.11||||
|-
| Rok Vene||Naslov seminarja||04.11.11||11.11.11||18.11.11||||
|-
| Ines Šterbal||Naslov seminarja||04.11.11||11.11.11||18.11.11||||
|-
| Andreja Bratovš||Naslov seminarja||04.11.11||11.11.11||18.11.11||||
|-
| Matevž Ambrožič||Naslov seminarja||09.11.11||16.11.11||23.11.11||||
|-
| Matevž Merljak||Naslov seminarja||11.11.11||18.11.11||25.11.11||||
|-
| Mitja Crček||Naslov seminarja||11.11.11||18.11.11||25.11.11||||
|-
| Dominik Kert||Naslov seminarja||11.11.11||18.11.11||25.11.11||||
|-
| Petra Malavašič||Naslov seminarja||16.11.11||23.11.11||30.11.11||||
|-
| Eva Knapič||Naslov seminarja||18.11.11||25.11.11||02.12.11||||
|-
| Marko Radojković||Naslov seminarja||18.11.11||25.11.11||02.12.11||||
|-
| Tjaša Goričan||Naslov seminarja||18.11.11||25.11.11||02.12.11||||
|-
| Tina Gregorič||Naslov seminarja||23.11.11||30.11.11||07.12.11||||
|-
| Tamara Marić||Naslov seminarja||25.11.11||02.12.11||09.12.11||||
|-
| Ana Dolinar||Naslov seminarja||25.11.11||02.12.11||09.12.11||||
|-
| Maja Remškar||Naslov seminarja||25.11.11||02.12.11||09.12.11||||
|-
| Matja Zalar||Naslov seminarja||30.11.11||07.12.11||14.12.11||||
|-
| Urška Navodnik||Naslov seminarja||02.12.11||09.12.11||16.12.11||||
|-
| Jernej Mustar||Silent pain||02.12.11||09.12.11||16.12.11||||
|-
| Ines Kerin||Naslov seminarja||02.12.11||09.12.11||16.12.11||||
|-
| Alja Zottel||Naslov seminarja||07.12.11||14.12.11||21.12.11||||
|-
| Alenka Mikuž||Naslov seminarja||09.12.11||16.12.11||23.12.11||||
|-
| Maja Grdadolnik||Ear of Stone||09.12.11||16.12.11||23.12.11||||
|-
| Jana Verbančič||Naslov seminarja||09.12.11||16.12.11||23.12.11||||
|-
| Petra Gorečan||Naslov seminarja||14.12.11||21.12.11||04.01.12||||
|-
| Karmen Hrovat||Naslov seminarja||16.12.11||23.12.11||06.01.12||||
|-
| Andrej Vrankar||The things we forget||16.12.11||23.12.11||06.01.12||||
|-
| Teja Banič||Naslov seminarja||16.12.11||23.12.11||06.01.12||||
|-
| Špela Pohleven||Naslov seminarja||21.12.11||04.01.12||11.01.12||||
|-
| Ime in priimek ||Naslov seminarja||23.12.11||06.01.12||13.01.12||||
|-
| Ime in priimek ||Naslov seminarja||23.12.11||06.01.12||13.01.12||||
|-
| Ime in priimek ||Naslov seminarja||23.12.11||06.01.12||13.01.12||||
|-
| Ime in priimek ||Naslov seminarja||04.01.12||11.01.12||18.01.12||||
|-
| Ime in priimek ||Naslov seminarja||06.01.12||13.01.12||20.01.12||||
|-
| Ime in priimek ||Naslov seminarja||06.01.12||13.01.12||20.01.12||||
|-
| Ime in priimek ||Naslov seminarja||06.01.12||13.01.12||20.01.12||||
|}

== Gradivo za seminarje ==
Gradivo za predavanja in seminarje najdete na http://bio.ijs.si/~zajec/bio2/
username: bio2
password: samozame

==Naloga==
'''Vaša naloga za seminar je: '''
Samostojno pripraviti seminar o enem od proteinov opisanih v [http://web.expasy.org/spotlight/back_issues/2011/ ProteinSpotlight] Poiskati morate vsaj še tri znanstvene članke, ki se nanašajo na opisano temo in jih uporabiti kot podlago za seminarsko nalogo!
V seminarsko nalogo mora biti vključeno:
* sekvenca proteina in SwissProt oznaka proteina
* slika strukture proteina (če je le-ta znana), ki jo naredite sami s programom Pymol. Če struktura še ni znana, vključite sliko proteina, ki je vašemu najbolj podoben po sekvenci in katerega struktura je znana
* poiskati morate, na katerem kromosomu se v človeškem genu nahaja ta protein in narisati shematsko sliko gena (eksonov in intronov) tega proteina. Če protein ni človeškega izvora, poiščite protein, ki je vašemu najbolj podoben in vse navedeno opišite za ta protein.

Za pripravo seminarja velja naslednje: 
* [[BIO2 Povzetki seminarjev 2011|Povzetek seminarja]] opišete na wikiju v približno 200 besedah, besedilo naj vsebuje sliko strukture proteina, ki jo sami narišete s programom PyMol - najkasneje do dne ko morate oddati seminar recenzentom.
* Povezavo do povzetka vnesete v tabelo seminarjev tekočega letnika.
* Seminar pripravite v obliki seminarske naloge na ~5-9 straneh A4 (pisava 12, enojni razmak, 2,5 cm robovi; važno je, da je obseg od 2700 do 3000 besed), vsebovati mora najmanj tri slike. Slika mora imeti legendo in v besedilu mora biti na ustreznem mestu sklic na sliko.
* Natisnjen seminar oddajte dva tedna pred predstavitvijo vsakemu od recenzentov (docentu ga pošljite po e-pošti v formatu .doc ali .docx).
* Recenzenti do dneva določenega v tabeli določijo popravke in podajo oceno pisnega dela.
* Ustna predstavitev sledi na dan, ki je vpisan v tabeli. Za predstavitev je na voljo 20-30 minut. Recenzenti morajo biti na predstavitvi prisotni.
* Predstavitvi sledi razprava. Recenzenti podajo oceno predstavitve in postavijo najmanj dve vprašanji.
* Na dan predstavitve morate docentu oddati končno (popravljeno) in natisnjeno verzijo seminarja v enem izvodu.
* Seminarska naloga in povzetek morajo biti v slovenskem jeziku!

==Ocenjevanje seminarjev==
Recenzenti ocenijo seminar tako, da izpolnijo [[https://spreadsheets.google.com/viewform?hl=en&formkey=dE1aOFU1aE1iMlBrNEJzLTRGeTdWZXc6MQ#gid=0 recenzentsko poročilo]] na spletu.

== Mnenje o predstavitvi ==
Vsak posameznik '''mora''' oceniti seminar, tako da odda svoje [https://spreadsheets.google.com/viewform?hl=en&formkey=dDlsbDlnclNrc3dIS2otRFdxUEFTNnc6MQ#gid=0 mnenje] najkasneje v treh dneh po predstavitvi. Kdor na seminarju ni bil prisoten, mnenja '''ne sme''' oddati.

==Urejanje spletnih strani na wikiju==
Wiki so razvili zato, da lahko spletne vsebine ureja vsakdo. Ukazi so preprosti, dokler si ne zamislite česa prav posebnega. Vseeno pa je Word v primerjavi z wikijem pravo čudežno orodje... Če imate težave z oblikovanjem besedila, si preberite poglavje o urejanju wiki-strani na Wikipediji ([http://en.wikipedia.org/wiki/Help:Editing tule] v angleščini in [http://sl.wikipedia.org/wiki/Wikipedija:Urejanje_strani tu] v slovenščini). Pomaga tudi, če pogledate, kako je zapisana kakšna stran, ki se vam zdi v redu: kliknite na zavihek 'Uredite stran' in si poglejte, kako so vpisane povezave, kako nov odstavek in podobno. ''Na koncu seveda pod oknom za urejanje kliknite na 'Prekliči'.''

==Citiranje virov==
Citiranje je možno po več shemah, važno je, da se v seminarju držite ene same.
Temeljno načelo je, da je treba vir navesti na tak način, da ga je mogoče nedvoumno poiskati.
Za citate v naravoslovju je najpogostejše citiranje po pravilniku ISO 690. [http://www.zveza-zotks.si/gzm/dokumenti/literatura.html Pravila], ki upoštevajo omenjeni standard, so pripravili pri ZTKS. Sicer pa ima vsaka revija lahko svoj način citiranja, ki ga je treba pri pisanju članka upoštevati. 
'''Citiranje knjig:''' 
Priimek, I. ''Naslov''. Kraj: Založba, letnica. 
Priimek, I. ''Naslov: podnaslov''. Izdaja. Kraj: Založba, letnica. Zbirka, številka. ISBN. 

Boyer, R. ''Temelji biokemije''. Ljubljana: Študentska založba, 2005. 
Glick BR in Pasternak JJ. ''Molecular biotechnology: principles and applications of recombinant DNA''. 3. izdaja. Washington: ASM Press, 2003. ISBN 1-55581-269-4. 
Če so avtorji trije, je beseda in med drugim in tretjim avtorjem. Če so avtorji več kot trije, napišemo samo prvega in dopišemo ''et al''. (in drugi, po latinsko). Vse, kar je latinsko, pišemo poševno (npr. tudi imena rastlin in živali, pojme ''in vivo'', ''in vitro'' ipd.).

'''Citiranje člankov:''' 
Priimek, I. Naslov. ''Naslov revije'', letnica, letnik, številka, strani. 
Lartigue C. ''et al''. Genome transplantation in bacteria: changing one species to another. ''Science'', 2007, letn. 317, str. 632-638.

Alternativni način citiranja (predvsem v družboslovju) je po pravilih APA, kjer članke citirajo takole: 
Priimek, I. (letnica, številka). Naslov. Naslov revije, strani. 
Lartigue C. ''et al.'' (2007, 317) Genome transplantation in bacteria: changing one species to another. ''Science'', 632-638.

Revija Science uporablja skrajšani zapis: 
C. Lartigue ''et al''. Science 317, 632 (2007) 

V diplomah na FKKT je treba navesti vire tako, da izpišete tudi naslov citiranega dela in strani od-do (ne samo začetne).

'''Citiranje spletnih virov:''' 
Priimek, I. ''Naslov dokumenta''. Izdaja. Kraj: Založnik, letnica. Datum zadnjega popravljanja. [Datum citiranja.] spletni naslov 
strangeguitars. ''On the brink of artificial life''. 6. 10. 2007. [citirano 13. 11. 2007] http://www.metafilter.com/65331/On-the-brink-of-artificial-life 
Navedemo čim več podatkov; pogosto vseh iz pravila ne boste našli.

BIO1-seminar 2011

2011-05-19T16:21:23Z

JernejMustar: /* Seznam seminarjev */

= Temelji biokemije- seminar =

Seminarje vodi doc. dr. Gregor Gunčar in so na urniku vsak ponedeljek od 10:00 do 11:30.

Ocena seminarjev predstavlja ??% končne ocene in vsebuje vse točke, ki jih študent/ka lahko zbere pri seminarju in ostalih dejavnostih, ki niso del pisnega izpita.

== Seznam seminarjev ==
{| border="1" cellpadding="4" cellspacing="0" style="border:#c9c9c9 1px solid; margin: 1em 1em 1em 0; border-collapse: collapse;"
| align="center" style="background:#f0f0f0;"|'''Ime in priimek'''
| align="center" style="background:#f0f0f0;"|'''Slovenski naslov članka'''
| align="center" style="background:#f0f0f0;"|'''Faktor vpliva revije'''
| align="center" style="background:#f0f0f0;"|'''Rok za oddajo'''
| align="center" style="background:#f0f0f0;"|'''Rok za recenzijo'''
| align="center" style="background:#f0f0f0;"|'''Datum predstavitve'''
| align="center" style="background:#f0f0f0;"|'''Recenzent 1'''
| align="center" style="background:#f0f0f0;"|'''Recenzent 2'''
| align="center" style="background:#f0f0f0;"|'''Recenzent 3'''
|-
| BOTONJIĆ SANDI||[http://wiki.fkkt.uni-lj.si/index.php/BIO1_Povzetki_seminarjev#Sandi_Botonji.C4.87:_Tioredoksinu_podoben_protein_.28TXNL2.29_.C5.A1.C4.8Diti_kancerogene_celice_pred_oksidativnim_stresom Tioredoksinu podoben protein (TXNL2) ščiti kancerogene celice pred oksidativnim stresom]
||15.387||28.02.||03.03.||07.03.||RODE URŠKA||KERIN INES||OGRIS IZA
|-
| VRANKAR ANDREJ||Število lasno-mešičnih matičnih celic se v plešastem lasišču moških z androgeno alopecijo ohranja za razliko od števila CD200-rich in CD34-positive lasno-mešičnih predniških celic||||28.02.||03.03.||07.03.||HROVAT KARMEN||BOHNEC IVO||JAVORŠEK KAJA
|-
| ZALAR MATJA||Protein p53||||28.02.||03.03.||07.03.||OGRIS IZA||CRČEK MITJA||ZOTTEL ALJA
|-
| ZOTTEL ALJA||Vloga imunskega sistema pri aterosklerozi||31.434||07.03.||10.03.||14.03.||RADOJKOVIĆ MARKO||KERT DOMINIK||HROVAT KARMEN
|-
| DOLINAR ANA||[http://wiki.fkkt.uni-lj.si/index.php/BIO1_Povzetki_seminarjev#Ana_Dolinar:_Prilagojena_ali_prilagodljiva_imunost.3F_Primer_naravnih_celic_ubijalk Prirojena ali prilagodljiva imunost? Primer naravnih celic ubijalk]||28||07.03.||10.03.||14.03.||RAUTER URŠKA||MOHAR MAŠA||VERBANČIČ JANA
|-
| RAUTER URŠKA||Razvojna vloga Srf, kortikalnega citoskeleta in celične oblike v orientaciji epidermalnega vretena||19.527||07.03.||10.03.||14.03.||MUSTAR JERNEJ||JAVORŠEK KAJA||MOHAR MAŠA
|-
| MOHAR MAŠA||Sladkorna bolezen tipa 2 kot bolezen imunskega sistema||30,006||14.03.||17.03.||21.03.||VENE ROK||RAUTER URŠKA||GORIČAN TJAŠA
|-
| POHLEVEN ŠPELA||Prioni||34||14.03.||17.03.||21.03.||KEPIC LEA||RADOJKOVIĆ MARKO||DOLINAR ANA
|-
| KEPIC LEA||Agonisti adrenoreceptorjev β2||34.48||14.03.||17.03.||21.03.||VRANKAR ANDREJ||BRATOVŠ ANDREJA||MUSTAR JERNEJ
|-
| KMETIČ MIRJAM||Celična regulacija metabolizma železa||5,371||14.03.||17.03.||21.03.||MARIĆ TAMARA||REMŠKAR MAJA||KOMAN KATRA
|-
| JARC VERONIKA||Eksperimentalni modeli za študijo imunobiologije hepatitisa C||3.26||14.03.||21.03.||28.03.||REMŠKAR MAJA||MUSTAR JERNEJ||KEPIC LEA
|-
| KOMAN KATRA||[http://wiki.fkkt.uni-lj.si/index.php/BIO1_Povzetki_seminarjev#Katra_Koman:_.09Pomen_dendritskih_celic_.28DCs.29_in_celic_ubijalk_.28NK.29_v_imunskem_odzivu_na_oku.C5.BEbo_z_virusom_HIV-1 Pomen dendritskih celic (DCs) in celic ubijalk (NK) v imunskem odzivu na okužbo z virusom HIV-1]||32.245||21.03.||25.03.||28.03.||ČUPOVIĆ VANA||KARNER TAJA||KMETIČ MIRJAM
|-
| OGRIS IZA||Zakaj imajo možgani glikogen?||5,125||14.03.||21.03.||28.03.||KNAPIČ EVA||BRGLEZ ŽIVA||VRANKAR ANDREJ
|-
| KERIN INES||Kanabinoidi za zdravljenje shizofrenije? Uravnotežena nevrokemična sestava za škodljive in terapevtske učinke uživanja konoplje||4.458||14.03.||21.03.||28.03.||ŠTOK ULA||ŠTEMBERGER ROK||KERT DOMINIK
|-
| VERBANČIČ JANA||Apoptozi podobna smrt v bakterijah, ki jo povzroča HAMLET, človeški mlečni lipidno-proteinski kompleks||4.351||21.03.||28.03.||04.04.||KARNER TAJA||ZOTTEL ALJA||KNAPIČ EVA
|-
| KNAPIČ EVA||Kako virusi vodijo delovanje celice.||14.101||21.03.||28.03.||04.04.||ZALAR MATJA||POHLEVEN ŠPELA||LORBEK SARA
|-
| REMŽGAR ANA||Črevesna absorpcija vitamina D ne poteka le s pasivno difuzijo: dokazi za vpletenost enakih transporterjev kot pri holesterolu||4.356||21.03.||28.03.||04.04.||BOTONJIĆ SANDI||LORBEK SARA||ČUPOVIĆ VANA
|-
| GRDADOLNIK MAJA||Jedrni in nejedrni receptorji za estrogene||5.328||21.03.||28.03.||04.04.||MOHAR MAŠA||REMŽGAR ANA||FRANKO NIK
|-
| JAVORŠEK KAJA||Potencial matičnih celic pri Parkinsonovi bolezni in molekularni faktorji za tvorbo dopaminskih nevronov||4.139||28.03.||04.04.||11.04.||GEC KARMEN||MARIĆ TAMARA||RADOJKOVIĆ MARKO
|-
| BRATOVŠ ANDREJA||Vloga GPCR v patologiji Alzheimerjeve bolezni||26||28.03.||04.04.||11.04.||ZOTTEL ALJA||ČUPOVIĆ VANA||GRDADOLNIK MAJA
|-
| CRČEK MITJA||Matične celice in njihova vloga pri zdravljenju bolezni in poškodb||7.365||28.03.||04.04.||11.04.||BOHNEC IVO||KMETIČ MIRJAM||BRATOVŠ ANDREJA
|-
| MARIĆ TAMARA||Organizacija jedra||9.58||28.03.||04.04.||11.04.||NAVODNIK URŠKA||GEC KARMEN||REMŠKAR MAJA
|-
| ŠTEMBERGER ROK||povečano izražanje in imunogenosti HIV proteinov po inaktivaciji encimske aktivnosti||3.616||04.04.||11.04.||18.04.||JAVORŠEK KAJA||VRANKAR ANDREJ||BOTONJIĆ SANDI
|-
| LORBEK SARA||Sovplivanje maščobnih kislin ter genov na adipokine in debelost||3.072||04.04.||11.04.||18.04.||POHLEVEN ŠPELA||KNAPIČ EVA||VENE ROK
|-
| REMŠKAR MAJA||Evolucijska dinamika transponibilnih elementov (TE) v majhnem RNA svetu||8.689||04.04.||11.04.||18.04.||KERIN INES||POVŠE KATJA||CRČEK MITJA
|-
| ČUPOVIĆ VANA||naslov||||04.04.||11.04.||18.04.||REMŽGAR ANA||VERBANČIČ JANA||RODE URŠKA
|-
| RODE URŠKA||vpliv c-reaktivnega proteina na patogenezo simptomov metaboličnega sindroma||6.614||03.05.||06.05.||09.05.||GRDADOLNIK MAJA||ŠTEMBERGER ROK||MARIĆ TAMARA
|-
| RADOJKOVIĆ MARKO||[http://wiki.fkkt.uni-lj.si/index.php/BIO1_Povzetki_seminarjev#Marko_Radojkovi.C4.87:_Vpliv_rakastih_celic_in_sepse_na_izra.C5.BEenost_krvnega_proteina_trombina Vpliv rakastih celic in sepse na izraženost krvnega proteina trombina]||14.608||03.05.||06.05.||09.05.||FRANKO NIK||VENE ROK||POVŠE KATJA
|-
| VENE ROK||[http://wiki.fkkt.uni-lj.si/index.php/BIO1_Povzetki_seminarjev#Rok_Vene:_Spremembe_nivoja_metilacije_DNA_so_sorazmerne_s_starostjo_.C4.8Dlove.C5.A1kih_mo.C5.BEganov Spremembe nivoja metilacije DNA so sorazmerne s starostjo človeških možganov]||7.386||03.05.||06.05.||09.05.||VERBANČIČ JANA||NAVODNIK URŠKA||ZALAR MATJA
|-
| FRANKO NIK||naslov||||03.05.||06.05.||09.05.||xx||HROVAT KARMEN||BOHNEC IVO
|-
| HROVAT KARMEN||Ciljanje kemokinih receptorjev v alergijskih boleznih||5,155||04.05.||09.05.||16.05.||KERT DOMINIK||JARC VERONIKA||KARNER TAJA
|-
| AMBROŽIČ MATEVŽ||[http://wiki.fkkt.uni-lj.si/index.php/BIO1_Povzetki_seminarjev#Matev.C5.BE_Ambro.C5.BEi.C4.8D:_Termogene_snovi_in_regulacija_telesne_te.C5.BEe Termogene snovi in regulacija telesne teže]||4,434||04.05.||09.05.||16.05.||LORBEK SARA||KEPIC LEA||REMŽGAR ANA
|-
| NAVODNIK URŠKA||Stabilnost RNA/DNA in DNA/DNA dupleksa vpliva na mRNA transkripcijo||||04.05.||09.05.||16.05.||AMBROŽIČ MATEVŽ||ŠTOK ULA||ŠTEMBERGER ROK
|-
| BRGLEZ ŽIVA||[http://wiki.fkkt.uni-lj.si/index.php/BIO1_Povzetki_seminarjev#.C5.BDiva_Brglez:_Kompleks_Mre11 Kompleks Mre11]||42.198||09.05.||16.05.||23.05.||DOLINAR ANA||BOTONJIĆ SANDI||JARC VERONIKA
|-
| KARNER TAJA||Alkohol omogoča lažje nalaganje CD1d molekul, s tem aktivira NKT celice in zmanjša pojavljanje sladkorne bolezni pri NOD miškah
||||12.05.||17.05.||23.05.||KOMAN KATRA||OGRIS IZA||NAVODNIK URŠKA
|-
| KERT DOMINIK||[http://wiki.fkkt.uni-lj.si/index.php/BIO1_Povzetki_seminarjev#Dominik_Kert:_Kako_osteokalcin_vpliva_na_reprodukcijo_organizmov Kako osteokalcin vpliva na reprodukcijo organizmov]||||09.05.||16.05.||23.05.||GORIČAN TJAŠA||GRDADOLNIK MAJA||RAUTER URŠKA
|-
| MUSTAR JERNEJ||[http://wiki.fkkt.uni-lj.si/index.php/BIO1_Povzetki_seminarjev#Jernej_Mustar:_Odpornost_srpastih_celic_na_oku.C5.BEbo_z_plazmodijem Odpornost srpastih celic na okužbo z plazmodijem]||31||16.05.||23.05.||30.05.||JARC VERONIKA||AMBROŽIČ MATEVŽ||BRGLEZ ŽIVA
|-
| GEC KARMEN||Učinki vadbe in/ali dodajanja antioksidantov na izražanje genov endotelnih celic||3.469||16.05.||23.05.||30.05.||POVŠE KATJA||ZALAR MATJA||AMBROŽIČ MATEVŽ
|-
| GORIČAN TJAŠA||Molekulske tarče oksidativnega stresa||17,902||16.05.||23.05.||30.05.||KMETIČ MIRJAM||RODE URŠKA||POHLEVEN ŠPELA
|-
| BOHNEC IVO||naslov||||23.05.||30.05.||06.06.||CRČEK MITJA||GORIČAN TJAŠA||ŠTOK ULA
|-
| ŠTOK ULA||Mutacija mitohondrijske DNA v povezavi z rakom debelega črevesa kot posledica abnormalnega delovanja citokroma c oksidaze||||23.05.||30.05.||06.06.||BRGLEZ ŽIVA||DOLINAR ANA||KERIN INES
|-
| nihce ||naslov||||23.05.||30.05.||06.06.||BRATOVŠ ANDREJA||KOMAN KATRA||GEC KARMEN
|}

==Naloga==
* samostojno pripraviti seminar, katerega osnova je znanstveni članek s področja biokemije, ki ga po želji izberete v reviji s področja biokemije, ki ima faktor vpliva večji kot 3 in je bil objavljen v letu 2011. Poleg tega članka morate za seminar uporabiti še najmanj pet drugih virov! http://www.cobiss.si/scripts/cobiss?command=CONNECT&base=JCR
* osnovni članek in naslov pošljete meni, najkasneje pet dni pred rokom za oddajo (rok-5), da ocenim, če je primeren za predstavitev. Naslov vpišete v tabelo, takoj ko ste si ga izbrali!
* [[BIO1 Povzetki seminarjev|Povzetek seminarja]] opišete na wikiju v približno 200 besedah - najkasneje do dne ko morate oddati seminar recenzentom. Povezave do slik so dobrodošle, niso pa nujne.
* Povezavo do povzetka vnesete v tabelo seminarjev tekočega letnika.
* Seminar pripravite v obliki seminarske naloge (pisava 12, enojni razmak, 2,5 cm robovi; važno je, da je obseg od 1800 do 2000 besed), vsebovati mora najmanj eno sliko. Slika mora imeti legendo in v besedilu mora biti na ustreznem mestu sklic na sliko.
* Natisnjen seminar oddajte do roka vsakemu od recenzentov (docentu ga pošljite po e-pošti v formatu .doc ali .docx).
* Recenzenti do dneva določenega v tabeli določijo popravke in podajo oceno pisnega dela, v predpisanem formatu elektronskega obrazca na internetu.
* Ustna predstavitev sledi na dan, ki je vpisan v tabeli. Za predstavitev je na voljo 15 minut. Recenzenti morajo biti na predstavitvi prisotni.
* Predstavitvi sledi razprava- 5 minut. Recenzenti podajo oceno predstavitve in postavijo vsak vsaj dve vprašanji.
* Na dan predstavitve morate docentu oddati končno (popravljeno) in natisnjeno verzijo seminarja v enem izvodu.
* Seminarska naloga in povzetek na wikiju morajo biti v slovenskem jeziku!

==Ocenjevanje seminarjev==
Recenzenti ocenijo seminar tako, da izpolnijo [[https://spreadsheets.google.com/viewform?formkey=dFM2SktfM3Q4VU1wNUQzdU45OTlWVXc6MA recenzentsko poročilo]] na spletu.

== Mnenje o predstavitvi ==
Vsak posameznik '''mora''' oceniti seminar, tako da odda svoje [https://spreadsheets.google.com/viewform?formkey=dFd3TGhLV3ZSa2xsLVlmMVVUaEFURWc6MA mnenje] najkasneje v treh dneh po predstavitvi. Kdor na seminarju ni bil prisoten, mnenja '''ne sme''' oddati.

==Urejanje spletnih strani na wikiju==
Wiki so razvili zato, da lahko spletne vsebine ureja vsakdo. Ukazi so preprosti, dokler si ne zamislite česa prav posebnega. Vseeno pa je Word v primerjavi z wikijem pravo čudežno orodje... Če imate težave z oblikovanjem besedila, si preberite poglavje o urejanju wiki-strani na Wikipediji ([http://en.wikipedia.org/wiki/Help:Editing tule] v angleščini in [http://sl.wikipedia.org/wiki/Wikipedija:Urejanje_strani tu] v slovenščini). Pomaga tudi, če pogledate, kako je zapisana kakšna stran, ki se vam zdi v redu: kliknite na zavihek 'Uredite stran' in si poglejte, kako so vpisane povezave, kako nov odstavek in podobno. ''Na koncu seveda pod oknom za urejanje kliknite na 'Prekliči'.''

==Faktor vpliva==
Faktor vpliva (angl. impact factor) neke revije pove, kolikokrat so bili v poprečju citirani članki v tej reviji v dveh letih skupaj pred objavo tega faktorja. Faktorje vpliva za posamezno revijo lahko najdete v [http://www.cobiss.si/scripts/cobiss?command=CONNECT&base=JCR COBISS-u]. V polje "Naslov revije" vnesite ime revije za katero želite izvedeti faktor vpliva in pritisnite na gumb POIŠČI. V skrajnem desnem stolpcu se bodo izpisali faktorji vpliva za revije, ki ustrezajo vašim iskalnim kriterijem. Zadetkov za posamezno revijo je več zato, ker so navedeni faktorji vpliva za posamezno leto. Za leto 2011 faktorji vpliva še niso objavljeni, zato se orientirajte po faktorjih vpliva zadnjih par let. Če faktorja vpliva za vašo izbrano revijo ne najdete v bazi COBISS, potem izberite članek iz kakšne druge revije.

==Citiranje virov==
Citiranje je možno po več shemah, važno je, da se v seminarju držite ene same.
Temeljno načelo je, da je treba vir navesti na tak način, da ga je mogoče nedvoumno poiskati.
Za citate v naravoslovju je najpogostejše citiranje po pravilniku ISO 690. [http://www.google.com/url?sa=t&source=web&cd=6&sqi=2&ved=0CEUQFjAF&url=http%3A%2F%2Fwww.tre.sik.si%2Fmain%2Fpomoc%2Ffiles%2Fcitiranje_in_navajanje_virov.pdf&rct=j&q=citiranje%20po%20pravilniku%20ISO%20690&ei=jPBqTe6FC9DKswaWk-TmDA&usg=AFQjCNF8r6X9Y781sanDObaXNdCew4suUg&sig2=cTqKObSJsTicekWGRGa72g&cad=rja Pravila], ki upoštevajo omenjeni standard, so pripravili pri ZTKS. Sicer pa ima vsaka revija lahko svoj način citiranja, ki ga je treba pri pisanju članka upoštevati. 
'''Citiranje knjig:''' 
Priimek, I. ''Naslov''. Kraj: Založba, letnica. 
Priimek, I. ''Naslov: podnaslov''. Izdaja. Kraj: Založba, letnica. Zbirka, številka. ISBN. 

Boyer, R. ''Temelji biokemije''. Ljubljana: Študentska založba, 2005. 
Glick BR in Pasternak JJ. ''Molecular biotechnology: principles and applications of recombinant DNA''. 3. izdaja. Washington: ASM Press, 2003. ISBN 1-55581-269-4. 
Če so avtorji trije, je beseda in med drugim in tretjim avtorjem. Če so avtorji več kot trije, napišemo samo prvega in dopišemo ''et al''. (in drugi, po latinsko). Vse, kar je latinsko, pišemo poševno (npr. tudi imena rastlin in živali, pojme ''in vivo'', ''in vitro'' ipd.).

'''Citiranje člankov:''' 
Priimek, I. Naslov. ''Naslov revije'', letnica, letnik, številka, strani. 
Lartigue C. ''et al''. Genome transplantation in bacteria: changing one species to another. ''Science'', 2007, letn. 317, str. 632-638.

Alternativni način citiranja (predvsem v družboslovju) je po pravilih APA, kjer članke citirajo takole: 
Priimek, I. (letnica, številka). Naslov. Naslov revije, strani. 
Lartigue C. ''et al.'' (2007, 317) Genome transplantation in bacteria: changing one species to another. ''Science'', 632-638.

Revija Science uporablja skrajšani zapis: 
C. Lartigue ''et al''. Science 317, 632 (2007) 

V diplomah na FKKT je treba navesti vire tako, da izpišete tudi naslov citiranega dela in strani od-do (ne samo začetne).

'''Citiranje spletnih virov:''' 
Priimek, I. ''Naslov dokumenta''. Izdaja. Kraj: Založnik, letnica. Datum zadnjega popravljanja. [Datum citiranja.] spletni naslov 
strangeguitars. ''On the brink of artificial life''. 6. 10. 2007. [citirano 13. 11. 2007] http://www.metafilter.com/65331/On-the-brink-of-artificial-life 
Navedemo čim več podatkov; pogosto vseh iz pravila ne boste našli.

User talk:JernejMustar

2011-05-19T16:19:31Z

JernejMustar: Removing all content from page

BIO1 Povzetki seminarjev 2011

2011-05-19T16:19:02Z

JernejMustar:

== Alja Zottel: Vloga imunskega sistema pri nastanku ateroskleroze ==
Glavni vzrok nastanka ateroskleroze je imunski odgovor na lipoproteine majhne gostote oz LDL, ki se kopiči pod endotelom arterijskih žil. Apolipoprotein B100, ki je komponenta LDL, se veže na proteoglikane zunajceličnega matriksa in se pod vplivom različnih radikalov oksidira. OxLDL nato aktivira endotelijske celice, da začnejo proizvajati adhezijske beljakovine, kot sta E-selektin in VCAM-1. Te beljakovine skupaj s kemokini povlečejo monocite, T limfocite in in dendritske celice v endotelijsko plast žile. Monociti se nato pod vplivom M-CSF citokina diferencirajo v makrofage. Makrofagi nato začnejo proizvajati odstranjevalne receptorje. Ti tako lahko prepoznajo oxLDL in ga z endocitozo vsrkajo. Makrofagi se zato napihnejo in spremenijo v »foam cell«. Te celice so najštevilčnejše celice v aterosklerotskih plakih. Dejavniki, ki pospešujejo nastanej ateroskleroze so signalni proteini PRR, T levkociti in proteini CRP. T celice pomagalke izločajo interferon gama, ki privlači monocite. Protein CRP se veže na navadni LDL in tako ga lahko makrofagi, ki imajo receptorje za CRP vsrkajo. Dejavniki, ki preprečujejo nastanek ateroskleroze so B limfociti in protein PPAR. PPAR je receptorski protein oz. transkripcijski faktor, ki preprečuje nastanek »foam cell« celic in vsrkavanje LDL v makrofage. Preprečuje tudi razvoj T celic in povečuje količino HDL v krvi.

== Veronika Jarc: Hepatitis C ==
Hepatitis C(HCV) je nalezljiva bolezen, ki napade ljudi, šimpanze ter nekatere majhne modelne živali. HCV spada med RNA viruse z ovojnico.Razvrščen pa je v rod hepacivirus ter družino flaviviridae. Sestavljen je iz 6 genotipov (1-6), ki se razlikujejo v nukleotidni sekvenci od 30-35%, sedmega pa so odkrili leta 2008 (Gottwein et al., 2008). HCV vsebuje pozitiven trak gena (9,6 kb), ki je sestavljen iz 5´-NCR( non-coding region), 3´- NCR in IRES( internal ribosome entry side). IRES vsebuje odprto bralno ogrodje, ki šifrira strukturne in ne strukturne proteine. Med strukturne proteine spadajo proteinsko jedro, virusna RNA ter dva glikoproteina E1 in E2. Sestavni deli ne strukturnih proteinov pa so hidrofoben protein p7, NS2-3 proteaza, NS3 serin proteaza, NS4A polipeptid, NS4B protein, NS5A protein in NS5B RNA odvisna RNA polimeraza (RdRp).
S pomočjo različnih odkritij, kot so HCVpp(sestavljen iz lipidne ovojnice z E1-E2 proteini, na retrovirusni nukleokapsidi), izoliranje kloniranega gena 2a ter s pomočjo tega gena HCVcc( cell-culture produced HCV), so znanstveniki začeli preučevati življenski cikel in celično strukturo hepatitisa C. To so dosegli z preučevanjem različnih eksperimentalnih modelov kot so imunski odzivi, NK celice in dendritske celice.
Poznamo tudi proteine, ki jih HCv sreča v hepatocitski celici in ti so in tegrin RGE/RGD, LDL receptor, HDL receptor, klaudin okludin in tetraspanin CD81.

== Matja Zalar: Protein p53 ==
Protein p53, včasih imenovan tudi varuh genoma, kodira gen TP53 na sedemnajstem kromosomu. Je eden izmed tako imenovanih tumor-supresorskih proteinov, ki, kot to sporoča že samo ime, zavirajo nastanek in rast tumorjev. Na področju razumevanja delovanja, vloge in strukture proteina p53 in njegovih mutantov se izvaja veliko raziskav. Trenutno je p53 najbolj raziskan tumor-supresorski protein, še zdaleč pa ni edini. Gre za protein, ki se kopiči v jedru in z vezavo na DNA v obliki teramera nadzoruje in regulira procese kot so apoptoza, zaustavitev celičnega cikla in popravljanje poškodovane DNA. Za raziskovalce je še posebno zanimiv zaradi dejstva, da v nemutirani obliki zavira nastanek in rast tumojev, njegove GOF mutirane oblike pa pripomorejo k nenadzorovani delitvi celic in nastanku rakastih tkiv. Veliko raziskav se ukvarjaja z iskanjem snovi, ki bi obnovile osnovno obliko p53, oziroma uničile mutantske oblike p53 v rakastih celicah ter s tem uničile tumor. To pa bi lahko bistveno izboljšalo tehnike zdravljenja rakavih obolenj in odziv človeškega organizma na ta zdravljenja. Odkrili so že kar nekaj takšnih snovi (RITA, PRIMA, nutlin3), ki pa jih še vedno testirajo in še niso v redni uporabi pri zdravljenju rakavih obolenj.

== Andrej Vrankar: Androgena alopecija ==
Na podlagi raziskav, ki so jih znanstveniki izvedli na celičnih vzorcih posameznikov z androgeno alopecijo, so ugotovili, da je bila domneva, da je za nastanek AGA kriv propad matičnih celic v lasnem mešičku oziroma, propad samega lasnega mešička napačna. Raziskave so pokazale ravno nasprotno in sicer, da se matične celice tudi v plešastem lasišču posameznika z AGA ohranjajo in da lasni mešički ne propadejo, vendar se le zelo skrčijo. So pa ugotovili, da se število celic imenovanih predniške celice v plešastem lasišču močno zmanjša, kar je eden od glavnih vzrokov za nastanek AGA, saj so prav predniške celice tiste, ki so zaslužene za rast las. Čeprav se dednost smatra kot glavni vzrok za nastanek AGA, pa tudi hormoni igrajo pomembno vlogo. Pri moških je to moški hormon testosteron, ki se s pomočjo encima 5-α-reduktaze v lasno mešičnih celicah pretvarja v svojo bolj aktivno obliko dihidrotestosteron (DHT). Ta se se nato s posebno vezjo veže na androgene receptorje v lasnih mešičkih, kar sproži posebne procese, ki skrajšajo anageno fazo celičnega cikla. Zaradi skrajšanja te faze las prej prestopi v telogeno fazo in izpade. Kako občutljivi so lasni mešički na androgene pa je seveda gensko pogojeno.

== Sandi Botonjić: Tioredoksinu podoben protein (TXNL2) ščiti kancerogene celice pred oksidativnim stresom ==
Kisikovi radikali, ki povzročajo oksidativni stres lahko v skrajnem primeru poškodujejo DNA in tako povzročijo nenadzorovano delitev celic, kar pomeni nastanek raka v organizmu. Hkrati pa je raven kisikovih radikalov v rakastih celicah višja, kot v zdravih, in sicer zaradi onkogenih stimulacij, povečane presnovne aktivnosti ter okvare mitohondrijev. Toda rakave celice imajo, kot protiutež tudi močan antioksidantni mehanizem s katerim zavirajo programirano celično smrt.

Raziskovalci so tekom analiziranja večih tkiv, ki so obolela z različnimi vrstami raka ugotovili, da je pri vseh povečana raven [http://www.thesgc.org/structures/structure_images/2WZ9_400x400.png tioredoksinu podobnega proteina - TXNL2]. Zatem so izvajali poskuse na miših tako, da so jim vbrizgali kancerogene eritrocite in ko so se pojavili simptomi tumorja – so jim vbrizgali še protein TXNL2. Ugotovili so, da protein TXNL2 zavira rast rakavih celic. Proučevali so tudi vpliv proteina TXNL2 v mišjih zarodkih. Prišli so do zaključka, da protein TXNL2 regulira raven kisikovih radikalov tako pri živečih organizmih, kot med embriogenezo. Znanstveniki so prepričani, da je protein TXNL2 potencialna tarča bioloških zdravil v prihodnosti.

== Ana Dolinar: Prilagojena ali prilagodljiva imunost? Primer naravnih celic ubijalk ==
Naravne celice ubijalke (NK celice) so vrsta levkocitov. V človeškem telesu so zadolžene za uničevanje patogenih organizmov s pomočjo za celice strupenih snovi. Na površini imajo pet skupin receptorjev: aktivacijske, inhibitorne, kemotaksične in citokine ter adhezijske receptorje.

Njihova aktivacija je odvisna od vezave ligandov na površinske receptorje NK celice. Če je vezanih več inhibitornih ligandov kot aktivacijskih, potem se NK celica ne aktivira, ker inhibitorni ligandi zavrejo delovanje NK celice. V primeru, da se veže več aktivacijskih kot inhibitornih ligandov ali pa se slednji sploh ne vežejo, se NK celica aktivira ([http://www.georg-speyer-haus.de/agkoch/research/subframe_en.htm aktivirana NK celica-rumeno, tarčna celica-rdeče]). Vezava kemotaksičnih ligandov vpliva na gibanje molekule zaradi kemičnih signalov, vezava citokinov spodbuja rast celic ali sintezo snovi, ki jih potrebuje imunski sistem, vezava adhezijskih ligandov pa omogoča pritrjanje NK celice na tarčno celico.

Raziskovalci se trudijo, da bi našli optimalno imunoterapijo, pri kateri bi sodelovale NK celice. Te terapije bi bile uporabne predvsem pri rakavih obolenjih, vendar so možnosti tudi pri obolenjih z virusom HIV ali z virusom hepatitisa C. Ta način imunoterapije je mogoč, ker večina tumorskih celic in virusov ne izraža MHC tipa 1, pomembnega inhibitorskega liganda za NK celice. [http://media.wiley.com/CurrentProtocols/IM/ima01n/ima01n-fig-0004-1-full.gif Zgradba MHC-1 molekule, prikazana z Ribbonovim diagramom in vezanim peptidom (A) ter površinska struktura molekule z vezanim peptidom (C). Slika B prikazuje molekulo MHC-2 z vezanim peptidom.]

== Urška Rauter: Razvojne vloge Srf, kortikalnega citoskeleta in celične oblike pri orientaciji epidermalnega vretena ==
Mehanizem nastajanja polariziranega epidermalnega sloja, ki s procesoma stratifikacije in diferenciacije tvori kožo, regulira več različnih med seboj v komplekse povezanih bioloških molekul. Trije najbolj osnovni procesi so delovanje proteinov aktina, orientacija vretena in sistem celične signalizacije. Znanstveniki pa so v obširni raziskavi potrdili tudi pomembno vlogo t. i. Srf proteina (serum response factor protein), transkripcijskega dejavnika, katerega pomembna vloga je regulacija celične diferenciacije.

Srf je transkripcijski dejavnik, ki se veže na določen, njemu ustrezen receptorski element; Sre (serum response element), to so predvsem geni v zgodnjem razvoju, geni za razvoj nevronov in mišična gena (proteina) aktin in miozin. Ker je njegova primarna funkcija regulacija ekspresije naštetih genov, odločilno vpliva na celično rast in diferenciacijo, prenos med nevroni in razvoj mišic.

Namen raziskave je obširen. Rezultati obetajoči. Dokazali so pomembno vlogo Srf proteina pri marsikaterem mehanizmu/procesu v embrionalnem razvoju. Tako recimo Srf odločilno vpliva na diferenciacijo celic, saj izguba le-tega povzroči kaotično deljenje in diferenciacijo celic med več plastmi epidermisa. Nadalje vpliva tudi na pravilno vzpostavitev polarnosti bazalne lamine in še najbolj ključno na tvorbo aktinsko-miozinskega skeleta, ki je nujen za pravilno mitozo, posledično za obliko in trdnost celice. Orientacija vretena in asimetrično dedovanje sta po zadnjih raziskavah osrednja mehanizma, ki omogočata matičnim celicam samostojno obnovi in diferenciacijo v pravilni smeri. Rezultati kažejo, da lahko takšne signale pošiljamo preko Srf proteina in aktinsko-miozinskega skeleta, za pravilno tvorbo in nadzirano regulacijo orientacije vretena, asimetrične celične delitve in nasploh usodo posamezne celice. Rezultati razkrivajo nove pojasnitve bioloških procesov, ki sodelujejo pri tvorbi morfologije epidermisa.

== Špela Pohleven: Prioni ==

Prioni so patogeni proteini, ki se od svojih nepatogenih, normalnih, v zaporedju aminokislin enakih dvojnikov, razlikujejo v 3D strukturi – imajo večji del β ploskev. Poznamo več vrst prionov, toda običajno govorimo le o proteinu PrP, ki je prisoten pri ljudeh in živalih. Ostali so namreč značilni za glive, ki so tako primerne za razne raziskave.
Za prione je značilno povezovanje v nitaste polimere, ki jih imenjujemo amiloidi. Znanstveniki domnevajo, da je prav njihova urejena struktura tista, zaradi katere so slabo topni v detergentih in odporni na proteaze.
Najbolj nenavadna lastnost prionov pa je njihova zmožnost širjenja brez potrebe po DNA in RNA. V zvezi s tem potekajo številne raziskave, saj prioni povzročajo številne smrtne bolezni, kot so Creutzfeldt-Jakobova bolezen, smrtonosna družinska nespečnost in druge. Z informacijami, ki jih tako pridobivajo, je možnost za odkritje zdravila večja.
Pri eni od nedavnih raziskav so tako ugotovili, da obstajata dve prionski obliki proteina PrP – infektivna in toksična. Za raziskave so uporabili miši z različnim izražanjem gena PRNP za PrP protein. Vse so okužili s prioni praskavca (ena od prionskih bolezni). Vse so dosegle enak prag infektivnosti, toda smrt ni nastopila istočasno. Iz meritev so znanstveniki prišli do zaključka, da morata obstajati dve različni obliki. To pa je le izhodišče za nove raziskave.

== Maša Mohar: Sladkorna bolezen, kot bolezen imunskega sistema ==

Diabetes mellitus je kronična motnja metabolizma beljakovin, lipidov in ogljikovih hidratov. Nastane zaradi zmanjšane funkcije proizvajanja insulina v telesu. Njen vzrok pa je lahko studi zmanjšana sposobnost telesnih celic za pravilno izkoriščanje insulina. Tip 2 je od insulina neodvisen diabetes (NIDDM). Ta tip ima 80-90% vseh pacientov in se pojavi v odraslem obdobju življenja, spodbudijo ga lahko različni mehanizmi, in za nekatere se še ne ve točno kako pride do tega, je pa res da k temu veliko pripomore nezdrav način življenja in seveda dednost. Prav tako se diabetes tipa 2 deli v dve skupini in sicer na debeli tip, ki ga ima približno 80% vse populacije in na ne debeli tip.
Da je T2D bolezen imunskega sistema pa ugotovimo s tem ko vidimo kako se telo odzovena določene mehanizme, ki sprožijo to bolezen. To so oksidativni stres, stres ER( endoplazemski retikel), lipotoksičnost in glukotoksičnost. Prav tako je potrebno poudariti, da ima diabetes tipa 2 svoje metabolne karakteristike in skupaj s temi patogenimi mehanizmi tvori formulo za nastanek bolezni. Seveda lahko pri T2D pride tudi do dolgoročnih komplikacij, kot so makro in mikro- vaskularne bolezni, problemi z ledvicami, očmi in živci. Te pa so glavni dejavniki za povzročitev hujšega bolezenskega stanja in ne nazadnje tudi smrti zaradi diabetesa.

== Mirjam Kmetič: Regulacija celičnega metabolizma železa ==

Železo je pomemben mikroelement, ki ga vezanega na proteine, vsebujejo skoraj vsa živa bitja. Celice sesalcev potrebujejo zadostno količino železa, da zadovoljijo metabolne potrebe ali dosežejo specializirane funkcije. Vsekakor pa je železo potencialno strupeno, še posebej v obliki Fe2+ ionov, ki katalizirajo pretvorbo vodikovega peroksida v proste radikale, ti pa poškodujejo veliko celičnih struktur (DNA, proteine, lipide...) in posledično celica lahko celo odmre. Vse oblike življenja se temu izognejo tako, da vežejo železove ione na proteine in tako hkrati izkoristijo njegove ugodnosti. Železo se prenaša v tkivo ob pomoči kroženja transferina, prenašalca, ki veže železo v plazmi, katerega predvsem sproščajo črevesne resice in retikuloendotelni makrofagi. Z železom bogat transferin se veže na membranski transferin receptor 1, kar se odraža z endocitozo in sprejemom te kovine. Sprejeto železo se prenese do mitohondrija za sintezo hema ali železo-žveplovih proteinov, ki so bistveni deli mnogih metaloproteinov. Presežno železo se skladišči in detoksificira v feritinu, ki je v citosolu. Metabolizem železa je nadzorovan na različnih nivojih in z raznovrstnimi mehanizmi. Pri uravnavanju je zelo pomemben sistem IRE (iron-responsive element)/IRP (iron-regulatory protein), dobro poznano post-transkripcijsko regulatorno vezje, ki ne le vzdržuje homeostazo v različnih tipih celic, ampak tudi prispeva k sistemskemu ravnovesju železa.

== Lea Kepic: Agonisti adrenoreceptorjev β2 ==

Vloga receptorjev v organizmih je zelo pomembna saj prenaša vse potrebne informacije za delovanje. Delimo jih na ionotropne in metabotropne. Največja skupina metabotropnih receptorjev pripada receptorjem, ki so sklopljeni s proteinom G. Mednje spadajo tudi adrenergični receptorji ali adrenoreceptorji. Adrenoreceptorji so tarčni za katekolamine (fight or flight hormoni) med katere spadajo adrenalin, noradrenalin in dopamin. V svojem seminarju sem se posvetila predvsem podskupini β2 (β2-AR) in njihovim agonistom. Agonisti so spojine, ki se selektivno vežejo na specifične receptorje, ki sprožijo nadaljnji odziv. Njegova naloga je posnemanje naravno obstoječih (endogenih) molekul, kot so na primer hormoni. Najbolj pogost in učinkovit agonist za β2-AR je izoprenalin, med hormoni pa je najboljši adrenalin. S pomočjo eksperimentov znanstveniki raziskujejo posebnosti v zgradbi predvsem kristalnih struktur, tvorbo vezi z različnimi spojinami, konformacijske spremembe, vpliv inhibitorjev, ravnotežna stanja ter energijska pretvarjanja. Rezultati teh raziskav so izhodišče za praktično uporabnost agonistov. Zaradi njihovih lastnosti jih vedno več uporabljamo v medicini za zdravnjenje plujčnih bolezni; predvsem astme in bronhitisa. To področje za enkrat še ni do dobra raziskano zato jih navadno uporabljamo le kot dodatke drugim zdravilom. Raziskani pa so že tudi nekateri negativni učinki na telo.

== Iza Ogris: Zakaj imajo možgani glikogen? ==

Glikogen se v možganih nahaja v precej manjših koncentracijah kot v jetrih in mišicah.Pojavi se vprašanje o njegovi vlogi v možganih in kje se nahaja. Glikogen vsebujejo astrocite- glia celice, ki obdajajo nevrone in skbijo za koncentracijo ionov v izvenceličnem prostoru ter dovajanje določenih snovi nevronom. Ko se med aktivnostjo nevronov v izvenceličnem prostoru kopičijo kalijevi ioni, jih astrocite začnejo privzemati z K/Na ATPazo. Posledično se v astrocitah zviša nivo AMP, kar stimulira delovanje encima glikogen fosforilaze (razgradnja glikogena). Astrocite med nevronsko aktivnostjo privzemajo tudi živnčni prenašalec glutamat iz sinaps, ki tudi posredno povzroča padec energije v astrocitah. Ko se nivo glukoze v dejavnih nevronih znižuje, se medtem v astrocitih povečuje. Koncentracija glukoze je nato v astrocitih večja kot v izvencelični tekočini in nevronih, zato se ustvari koncentracijski gradient kar omogoči pot glukoze iz astrocitov v nevrone. Pri vzdrževanju glukoze se tako razgradnja glikogena izkaže za bolj učinkovito kot le privzem glukoze iz krvi. Razkriva se izvor in usoda glukozne rezerve.

== Ines Kerin: Kanabinoidi za zdravljenje shizofrenije? Uravnotežena nevrokemična sestava za škodljive in terapevtske učinke uživanja konoplje ==

Že desetletja velja prepričanje, da je uživanje konoplje eden pomembnih dejavnikov za nastanek in razvoj shizofrenije. Vendar so v novejših raziskavah odkrili, da naj bi kanabinoidi, psihoaktivne substance v konoplji, izboljšali nevropsihološke učinke in negativne simptome, ter imeli antipsihotične lastnosti pri ljudeh s shizofrenijo. Shizofrenija je huda duševna bolezen iz skupine psihoz. Simptome shizofrenije povzroča spremenjena količina določenih snovi v možganih, in sicer živčnih prenašalcev, ki omogočajo medsebojno komunikacijo možganskih celic. Motnje v komunikaciji pa povzročajo spremembo v delovanju možganov. Pomembno vlogo ima pri bolezni dopamin, ki lahko s prevelikim sproščanjem izzove nekatere simptome.
Shizofrenijo zdravijo s pomočjo antipsihotikov, ki imajo podobne lastnosti kot kanabinoidi v konoplji. Vendar se učinki konoplje od učinkov antipsihotikov nekoliko razlikujejo. Pri negativnih simptomih konoplja, tako kot antipsihotiki, spodbuja sproščanje in delovanje dopamina. Manj znano pa je, ali zavira ali spodbuja delovanje ostalih petih nevrotransmiterjev (serotonina, acetilholina, noradrenalina, glutamina in GABA). Na pozitivne simptome ima konoplja, kot je vidno v tabeli lahko tako koristne kot nekosristne učinke. Simptome lahko izboljša z zaviranjem sproščanja serotonina, acetilholina in noradrenalina. V primeru dopamina, glutamata in GABA ima konoplja negative učinke, saj v nasprotju z antipsihotiki, poveča sproščanje dopamina in zavira delovanje glutamata in GABA. Obstajajo dokazi, da imajo kanabinoidi zdravilne učinke na pozitivne in negativne simptome pri shizofreniji. Vendar to poglavje še ni zaključeno in se izvajajo še nadalnje raziskave v tej smeri.

== Eva Knapič: Kako virusi vodijo delovanje celice. ==
Virusi so geni obdani z zaščitno proteinsko ovojnico. Za izražanje teh genov, da lahko naredijo proteine in podvojijo kromosome, je potrebno, da vstopijo v celico in uporabijo celične mehanizme, saj sami tega niso zmožni. Poznamo več vrst virusov. Posebnost evkariontskih virusov je sposobnost posnemanja kratkih linearnih motivov proteinov poznanih pod kratico SLiMs. To so deli proteinov, ki so odgovorni za posredovanje med nekaterimi celičnimi funkcijami. So zelo kratki, večinoma nekje od 3 do 10 aminokislin. Motivi sodelujejo pri vezavi proteinih, pri prepoznavanju post-translacijske modifikacije encimov, pri usmerjanju proteinov v celične razdelke in pa so prisotni na cepitvenih mestih proteina. S posnemanjem različnih motivov lahko virusi prevzamejo nadzor nad celico. Najpogostejši mehanizmi prevzema nadzora so uporaba celičnega transporta, manipuliranje signalnega transporta, nadzor proteinov v celici, regulacija prepisovanja, sprememba modifikacije gostiteljevega proteina in usmerjanje modifikacije proteinov.
Uporaba proteinskih motivov v celici in lahko posnemanje le teh predstavlja šibkost v celični organiziranosti, saj virusi s pridom izkoriščajo to v svojo korist. Posnemanje motivov virusom omogoča, da sami vodijo delovanje celice in se sami s pomočjo celičnih mehanizmov enostavno razmnožujejo in tako hitro okužijo celoten organizem.
V nadalje bodo potekale raziskave za izkoriščanje posnemanja motivov v namene zdravljenja virusnih okužb.
== Katra Koman: Pomen dendritskih celic (DCs) in celic ubijalk (NK) v imunskem odzivu na okužbo z virusom HIV-1 ==
Dendritske celice (DCs - dendritic cells) in celice ubijalke (NK – natural killer cells) sta dva tipa celic prirojenega imunskega sistema, ki imata zelo pomembno vlogo pri protivirusni odpornosti. Tako dendritske celice, kot tudi celice ubijalke so sicer pomemben del (nespecifičnega) prirojenega imunskega sistema, a hkrati vplivajo tudi na učinkovit razvoj (specifičnega) prilagojenega imunskega odziva. DC so ključnega pomena za aktiviranje za virus specifičnih T celic, kar pa je močno odvisno od prejšnjega, prirojenega imunskega odziva. NK celice pa ovirajo zgodnje širjenje virusov, tako da proizvajajo citokine in s fagocitozo neposredno uničujejo okužene celice. Razumevanje delovanja in funkcije teh celic pa ima pomemben vpliv na razvijanje nove strategije cepiva proti virusu HIV-1, katere uspeh bo odvisen od primernega razumevanja delovanja teh celic.

== Jana Verbančič: Apoptozi podobna smrt v bakterijah, ki jo povzroča HAMLET, lipidno-proteinski kompleks v človeškem mleku ==
Apoptoza oz. programirana celična smrt je eden najpomembnejših procesov v evkariontskih celicah. Organizem je z apoptozo sposoben sam uravnavati število živih celic. Uniči jih lahko, ker so poškodovane, stare ali ker ne opravljajo več svoje naloge, lahko pa uniči tudi popolnoma zdrave celice, ki jih ne potrebuje več (npr. pri embrionalnem razvoju). Pomemben dejavnik pri apoptozi so encimi kaspaze, ki cepijo in aktivirajo druge proteine, vse skupaj pa lahko poteka po dveh poteh. Prva je notranja in vključuje mitohondrije in citokrom c, ki deluje kot signalna molekula v apoptotskem ciklu ter tako sproži delovanje kaspaz in posledično apoptozo. Druga pot je zunanja in vključuje aktivacijo proteinskih receptorjev (t. i. receptorjev smrti) na zunanji strani membrane. Oblikuje se kompleks iz receptorja, adaptorskega proteina in vezane kaspaze (DISC), ki povzroča cepljenje in aktivacijo nadaljnjih kaspaz; to pa spet vodi v apoptozo. V mehanizme so lahko vključeni mnogi drugi proteini ali neproteinski signali.
Programirane celične smrti pa nimajo samo evkarionti, ampak so dokazali, da so tudi prokarionti sposobni procesov, ki so zelo podobni apoptozi. Raziskave so delali na streptokokih in tumorskih celicah, ki so jim dodali kompleks HAMLET (human alpha-lactalbumin made lethal to tumor cells), ki ga lahko najdemo v človeškem mleku. Kompleks je deloval kot signalna molekula za začetek apoptoze v tumorskih celicah oz. za začetek apoptozi podobnega procesa v bakterijah.

== Ana Remžgar: Črevesna absorpcija vitamina D ne poteka le s pasivno difuzijo: dokazi za vpletenost enakih transporterjev kot pri holesterolu ==
Vitamin D je hormon, ki ga telo lahko proizvede samo s pomočjo obsevanja kože z ultravijolično svetlobo, vendar je hipovitaminoza D razširjena v mnogih državah in je pomemben svetovni zdravstveni problem. Vitamin D je nujno potreben za uravnavanje ravnovesja med kalcijem in fosfati v telesu ter za normalno rast kosti.

Dolgo časa je veljalo, da se v črevesju vitamin D absorbira le s pomočjo pasivne difuzije. Znanstveniki so kulturi človeških embrionalnih ledvičnih (HEK) celic dodali vsaj enega od teh membranskih proteinov (SR-BI, CD36, NPC1L1). Ti trije proteini so pomembni pri absorpciji holesterola. Zaradi podobne zgradbe holesterola in vitamina D, so znanstveniki sklepali, da so lahko ti trije proteini pomembni tudi pri absorpciji vitamina D. Ko so HEK celicam dodali te proteine, se je absorpcija vitamina D opazno povečala. Ko pa so HEK celicam dodali poleg proteinov še njihove inhibitorje, se je absorpcija močno zmanjšala.
Vpliv SR-BI so opazovali tudi in vivo. Uporabili so wild type miši ter miši z mnogo bolj izraženim Scavenger receptorjem razreda B tipa I (SR-BI). Tudi tu se je pri SR-BI miših povečala absorpcija vitamina D.

Ti rezultati nam prvič pokažejo, da se vitamin D v črevesju ne absorbira le preko pasivne difuzije vendar je v ta proces vključenih kar nekaj transporterjev.

== Maja Grdadolnik: Jedrni in nejedrni receptorji za estrogene. ==
Receptorji za estrogene oz. estrogenski receptorji so proteinske molekule z vlogo specifičnega mesta vezave ustreznega liganda. Nahajajo se v vseh celicah tkiv, ki so tarčne celice estrogena. Lahko se nahajajo v jedru celice, v neposredni bližini DNA, lahko pa so vezani na posebna mesta na membrani celice, t.i. caveole.
Estrogeni (estron (E1), estradiol (E2), estriol (E3)) so lipidopolarni in brez večjih težav prehajajo skozi lipidni dvosloj. Nato se vežejo na lipoproteine v krvi, ki jih prenesejo do jedra tarčne celice. Tarčne celice so po navadi celice jajčnikov, testisov, nadledvičnih žlez, jeter in prsi. V jedru se nato vežejo na estrogenski receptor, s katerim tvorijo kompleks. Ta ligand-receptor kompleks se nato s posebnim mestom (domeno E) veže na specifično mesto na DNA, imenovano estrogen response element (ESE). S tem sodeluje pri procesu transkripcije in uravnava sintezo ustreznih proteinov.
Nejedrni estrogenski receptorji so vezani na posebna mesta na membrani celice, t.i. caveole. Na ta mesta so vezani z integralnim proteinom, za vezavo pa potrebujejo aminokislinski substrat. Receptorje na membrani lahko povezujemo z interakcijo z različnimi ligandi, imajo pa tudi pomembno vlogo posredne aktivacije endotelijske NO sintaze, ki pozitivno vpliva na srce in ožilje. Nejedrne estrogenske receptorje že povezujejo s procesi, ki blagodejno vplivajo na kardiovaskularne bolezni in tkivo endotelija.

== Andreja Bratovš: Vloga GPCR v patologiji Alzheimerjeve bolezni. ==
Alzheimerjeva bolezen je najpogostejša oblika demence. Zaradi odmiranja nevronov pride do zmanjšanja obsega možganov in pešanja razumskih funkcij. Eden glavnih razlogov za nastanek bolezni so amiloidni plaki. Ti nastajajo s kopičenjem amiloidnih peptidov beta. APP (amyloid precursor protein) je membranski protein, ki ga pri zdravem človeku cepi najprej α-sekretaza (nastane sAPPα), nato pa še γ-sekretaza – nastane topen delec p3. Kadar pa APP cepi β-sekretaza, nastane najprej sAPPβ, po cepitvi z γ-sekretazo pa nastane amiloidni peptid beta.
Pri iskanju zdravila za Alzheimerjevo bolezen se trenutno osredotočajo prav na amiloidne plake oz. na preprečevanje njihovega nastajanja ter njihovo razgradnjo. Alternativen pristop imunoterapiji je regulacija receptorjev, sklopljenih z G-proteini, saj so ti udeleženi v več fazah nastajanja plakov. Možnih je več poti, in sicer: zaviranje nastajanja amiloidnih peptidov beta z regulacijo α-, β- ali γ-sekretaze ter sproščanje encimov za razgradnjo plakov. Pri regulaciji α-sekretaze gre za promoviranje njenega delovanja, saj se tako poleg tega, da ne nastajajo amiloidni peptidi beta, tudi sprošča sAPPα, ki ima vlogo pri zaščiti nevronov. Za β-sekretazo je sicer znanih veliko inhibitorjev, vendar jih iz možganov eksportira P-glikoprotein. Problem pri γ-sekretaze je, da ta sekretaza cepi tudi del proteina Notch, zato bi z njeno inhibicijo vplivali tudi na Notch signalno pot.

== Kaja Javoršek: Potencial matičnih celic pri Parkinsonovi bolezni in molekularni faktorji za tvorbo dopaminskih nevronov. ==
Parkinsonova bolezen je nevrodegenerativna bolezen bazalnih ganglijev. ta bolezen prizadane predvsem telesno gibanje, nastane pa ker se zmanjša koncentracija dopamina v striatumu. Kot posledica tega, začnejo propadati dopaminski nevroni v substanti nigri. Prav propadanje dopaminskih nevronov pa je vrzok za Parkinsonovo bolezen. Vzrok za propadanje teh nevronov pa še vedno ni znan. Znano je da dopaminski nevroni s starostjo pospešeno propadajo. To je tudi razlog, zakaj se ta bolezen pojavlja šele pri starejših ljudeh.
Danes se v medicini uporablja veliko terapij, ki pa le lajšajo simptome in bolezni ne pozdravijo. Prav to je razlog za tako veliko število raziskav povezanih s Parkinsonovo boleznijo. Čeprav mehanizmi razvoja dopaminskih nevronov še niso povsem znani, so raziskovalci odkrili kar nekaj molekularnih faktorjev, ki vplivajo na njihovo tvorbo, na primer Fox proteini in receptor sirota Nurr1. Fox proteini so transkripcijski faktorji, ki vežejo DNA. Med temi proteini igrata najpomembnejšo vlogo v nastanku dopaminskih nevronov FoxA1 in FoxA2 proteina. Receptor sirota Nurr1 pa je pomemben pri nastanku L-DOPE, ki je vmesen produkt pri nastanku dopamina iz L-tirozina. Za nastanek L-DOPE mora biti prisoten encim tirozin hidroksilaza. Za izražanje tega encima pa je pomemben receptor sirota Nurr1 in mutacije tega receptorja so povezane s Parkinsonovo boleznijo in shizofrenijo.
Poleg vseh načinov zdravljenja, pa poskušajo Parkinsonovo bolezen pozdraviti tudi s pomočjo matičnih celic, saj so se zmožne diferencirati v katero koli vrsto celic, vključno z dopaminskimi nevroni.

== Tamara Marić: Organizacija jedra. ==
Organizacija genoma v jedru je zelo kompleksna in dinamična in prav to je znanstvenike privedlo do mišljenja, da ima jedro neko globjo strukturo, kjer mora vladati red. S pomočjo novih tehnoloških metod (3C, FISH, 4C) so odkrili kar nekaj zanimivh stvari o sestavi samega jedra. Jedro si moramo predstavljati kot 3D strukturo, v kateri se neprestano nekaj dogaja. Sestavljen je iz dveh glavnih domen, obrobja in centra. Na obrobju sta še dva pododdelka. Ob jedrnih porah se nahajajo aktivni geni, ki so povezani s številnimi proteini, speči geni pa se nahajajo ob lamini. V centru se pododdelki medseboj razlikujejo po funkcijah. V jedrcu se nahajajo geni za rRNA, v transkripcijskih tovarnah se nahajajo vse »sestavine«, ki jih geni potrebujejo za prepis, polycombska telesca imajo bistveno vlogo pri ohranjevanju represije in perinuklearni prostor je specializiran za replikacijo heterokromatina. Ker pa to ne miruje je logično da kromatinske zanke med seboj interagirajo. Poznamo homologne (kjer gre za podobno zgrajene/iste kromosome) in nehomologne(se med seboj razlikujejo) interkromosomske interakcije. Pri prvi je pomembno, da se podobna kromosoma »zmenita«, kateri bo aktiven, pri drugi pa je diferenciacija celice odvisna od aktivnosti nekega gena.

== Mitja Crček: Matične celice in njihova vloga pri zdravljenju bolezni in poškodb. ==
Regeneracija je proces, pri katerem nadomestimo poškodovane telesne dele. V človeškem telesu imajo to nalogo matične celice (MC), ki skribijo za delno regeneracijo in celjenje poškodb. Matične celice so nediferencirane celice odraslege človeka ali zarodka, ki imajo izjemen potencial, da se defirencirajo v mnogo različnih tipov celic v telesu. V tri do pet dni starih zarodkih iz matičnih celic nastane celotno telo organizma, pri odraslih ljudeh pa nas matične celice ohranjajo pri življenju. Glede na potentnost jih razdelimo v štiri razrede: totipotentne in pluripotentne MC so celice, ki se lahko diferencirajo v praktično vse celice telesa, medtem ko so multipotentne in unipotentne bolj omejene. Drugo delitev lahko opravimo glede na njihov izvor: embrionalne MC izvirajo iz zarodka, medtem ko MC odraslih tkiv in organov najdemo med že diferenciranimi celicami. Zaradi vseh njihovih lastnosti imajo velik potencial pri zdravljenju bolezni, že vrsto let jih uporabljajo za zdravljenje levkemije in limfoma. Z diferenciacijo MC v nevrone bi lahko pozdravili poškodbe hrbtenjače in možganov, ob sproščanju kemičnih signalov iz MC proti lasnim mešičkom bi lahko pozdravili plešavost. V teoriji bi lahko nadomestili tudi izgubljen zob, zdravili slabovidnost in gluhost, pa tudi sladkorno bolezen in neplodnost. Velik potencial imajo tudi pri zdravljenju poškodb kosti in mišic. Pri zlomih ter poškodbah hrustanca in vezi služijo predvsem za hitrejšo regeneracijo, omogočajo pa tudi zdravljenje mišične distrofije ali pa povečanje mišične mase in moči, kar bi lahko s pridom izkoriščali športniki in starejši ljudje.

== Sara Lorbek: Sovplivanje maščobnih kislin ter genov na adipokine in debelost. ==
Belo maščobno tkivo ni namenjeno zgolj shranjevanju zalog maščobe, temveč ima velik vpliv na prisotnost in stopnjo vnetja v telesu, saj na le-to vpliva s sekrecijo adipokinov. Adipokini so proteini, ki se izločajo iz celic maščobnega tkiva, do danes pa je poznanih že več kot 100 različnih. Njihov vpliv je zelo različen, v nalogi pa sem se osredotočila na vpliv aipokinov na vnetje, za katerega velja, da ga sproža debelost. Adipokina, ki odločilno prispevata k vnetnemu stanju sta TNF in interlevkin-6 (IL-6), njuna količina pa je močno odvisna tudi od telesne teže: večja kot je telesna teža posameznika, več je teh dveh adipokinov, ki promovirata vnetje, zato je tudi stopnja vnetja večja pri debelejših osebkih. Maščobne kisline veljajo za snovi, ki so sposobne regulirati proizvodnjo adipokinov in s tem vplivati na stopnjo vnetja, toda natančni molekulski mehanizmi tovrstne aktivnosti maščobnih kislin še niso pojasnjeni. Kljub temu imamo že dovolj dokazov, da lahko z gotovostjo trdimo, da različni tipi maščobnih kislin različno ugodno/neugodno vplivajo na promocijo vnetja v organizmu, tako npr. uživanje večkrat nenasičenih in omega-3 m.k. znižuje količino IL-6 in TNF- torej zavira vnetje, uživanje nasičenih m.k. pa vnetje promovira, saj zvišuje količino IL-6 in TNF v organizmu. Odziv številnih adipokinov na različne m.k. do danes še ni bil raziskan, kar predstavlja nov izziv za področje nutrigenomike.

== Maja Remškar: Evolucijska dinamika transponibilnih elementov v majhnem RNA svetu ==
Genom si pogosto predstavljamo kot nekaj statičnega, a ni tako. V zadnjem času so odkrili transponibilne elemente, samostojne dele DNA, ki se lahko premeščajo po genomu in sprožajo mutacije. Če se vgradijo v v stukturne gene, običajno uničijo njihovo informacijo, če pa se vgradijo v regulacijske regije vplivajo na izražanje genov, navadno jih naredijo neaktivne. Vsebujejo gene za podvajanje in premeščanje. Sestojijo iz obrnjenih ponovitev na vsakem koncu in iz vsaj še gena za transpozazo, ki mu omogoča premeščanje. Vmes imajo lahko poljubno število genov. Transpozoni naj bi bili odvečna in sebična DNA in dolgo je bila naravna selekcija edini poznan pojav, ki je nadzoroval njihovo pretirano razmnoževanje. Dandanes vemo, da je mehanizmov njihovega zaviranja več, in sicer, lahko delujejo samorepresorsko (za vrste, ki se razmnožujejo nespolno), pri zaviranju lahko pomaga represorski alel, ki je navadno s škodljivimi TE v stiku, lahko pa jih nadzorujeta mehanizma siRNA in piRNA, ki vztrajno popravljata napake povzročene s strani transpozonov. Na dinamiko represorskih alelov vpliva genetski zdrs – zaradi zmanjšanja populacije, se poruši naravno ravnovesje in lahko pride do fiksacije škodljivejših alelov – in rekombinacija, ki prekine povezave med represorskimi aleli in njihovim tarčnim mestom vezave ter prepreči njihovo fiksacijo. Transpozone so preučevali na koruzi, v bakterijah, vinski mušici in tudi pri človeku, saj povzročajo dedne bolezni kot sta hemofiliji A in B ter Duchennova mišična distrofija.

== Rok Štemberger: virus HIv in povečana ekspresija ter imunogenost HIV-1 proteaze po deaktivacija encimske aktivnosti ==
Virus HIV spada v skupino retrovirusov in je povzročitelj ene najhujših svetovnih pandemij, ki vsako leto terja skoraj 3 milijone žrtev. Virus HIV potrebuje za svoje razmnoževanje gostiteljsko celico, ker nima svojih lastnih mehanizmov, s katerimi bi se lahko razmnoževal. Njegov razmnoževalni cikel obsega veliko procesov, ki se morajo izvršiti, da se virus HIV lahko ustrezno replicira. V moji raziskavi je bil pod drobnogled vzet eden izmed treh encimov, ki sodelujejo pri razmnoževanju HIV-a, in sicer HIV-1 proteaza. HIV-1 proteaza je encim, ki dolge verige proteinov cepi na manjše dele. Če se HIV-1 proteazo z inhibitorji blokira, bi to pomenilo da virus HIV ne bi imel potrebnih encimov za svoje razmnoževanje, saj je dolga veriga proteinov popolnoma neuporabna, če niso razrezani na manjše dele. V raziskavi so ugotovili, da če uporabimo mutirano HIV-1 proteazo, se ji aktivnost drastično zmanjša po drugi strani pa so opazili veliko ekspresijo. Ta ekspresija se je pokazal v tem, da je inducirala imunski odziv in HIV-1 proteaza je bila trača predvsem CD8+ T celic pomagalk. Kasneje so ugotovili, da lahko prav te T celice popolnoma uničijo mutirano HIV-1 proteazo iz telesa in jo s tem odstranijo iz našega sistema. Poskusi so bili narejeni tudi na transgenih miših, ki dajejo bolj verodostojne rezultate kot ostale miši. Ta raziskava bo osnova vsem nadaljnjim raziskavam, ki se bodo ukvarjali predvsem z HIV-1 proteazo, saj ta do sedaj ni bila deležna velike pozornosti. Različni Inhibitorji HIV-1 proteaze pa bodo v prihodnosti bili še bolj pogosto uporabljeni v mešanici zdravil proti bolezni HIV.

== Rok Vene: Spremembe nivoja metilacije DNA so sorazmerne s starostjo človeških možganov ==
V zaporedju DNA se nahajajo posebna zaporedja nukleotidov, ki so edina, na katerih lahko poteče metilacija. Ta mesta sestavlja dinukleotid CpG – cytosine-phosphat-guanine (Od 5' konca DNA verige proti 3' koncu sta citozin in gvanin zaporedno vezana s fosfodiestersko vezjo – 5'-CG-3'). Metilacija DNA je proces v katerem se na ta posebna t.i. CpG mesta veže metilna skupina (-CH3). Taka mesta so v DNA redkejša, kot bi statistično gledano smela biti. Večina CpG mest je metiliranih. CpG mesta se lokacijsko na DNA lahko nahajajo v skupkih imenovanih CpG otočki (CpG islands), ali posamič. Direktna posledica metiliranih CpG mest je utišanje genov, na katerih se ta metilirana mesta nahajajo. Skupaj z ostalimi epigenetskimi faktorji pa indirektno vplivajo še na diferenciacijo celic. Destabilizacija epigenetskih faktorjev je lahko vzrok za številne bolezni (rak, sindromi Rett, ICF, Prader-Willi,...).

V raziskavi iz članka so znanstveniki primerjali količino in lokacijo metilirane DNA v različnih možganskih tkivih. Precejšen del rezultatov je specifičen glede na vrsto tkiva (preko petsto lokusov), vendar obstajajo določene povezave, ki so značilne za vsa raziskovana tkiva možganov. Odkrili so deset specifičnih lokusov, ki vsi vsebujejo metilirana CpG mesta. Odkrili so tudi, da nivo metilirane DNA s starostjo tkiva narašča. Starost tkiva je bila v kar 32-75% primerov glavni razlog za spremembe v količini metilirane DNA. Nekatere izmed destih lokusov so že pred to raziskavo povezovali z starostnimi spremembami v metilaciji DNA, vendar so šest izmed desetih najpomembnejših lokusov odkrili na novo.

== Karmen Hrovat: Ciljanje kemokinih receptorjev v alergijskih boleznih ==
Kemokini so družina majhnih proteinov, velikosti 8-10 kDa. Sodelujejo v procesu agiogeneze, embriogeneze, za nas pa je bistveno, da spodbujajo premikanje levkocitov, bazofilcev, monocitov, jih usmerjajo in nadzorujejo njihov prehod iz krvi v tkiva. Dandanes jih uvrščamo med mnoge raziskave, povezujemo jih tako z aterosklerozo, prenosljivimi boleznimi kot sta virus HIV in malarija, rakom, luskavico in alergijskimi boleznimi med katere sodijo astma, alergijski rinitis in atopijski dermatitis. Poznamo štiri vrste kemokinov: CC,CCX, C in CX3C. Nekateri kemokini se vežejo na več različnih receptorjev in obratno.V članku je opisanih več poskusov na kemokinih receptorjih v miših obolelih za alergijskimi boleznimi.

Farmacevti si prizadevajo odkriti kemokine receptorje antagoniste, saj se je aktivacija GPCR kompleksa izkazala uporabna za zdravilo. V miših obolelih za alergijskimi boleznimi je bilo do sedaj tako in vitro kot tudi in vivo dokazanih že veliko antagonistov kemokinih receptorjev. Kljub temu številni zaradi vprašanja varnosti in farmacevtskih dogm niso dočakali kliničnega sojenja.Lahko rečemo, da napredek ovira tudi pomanjkljivo razumevanje funkcije kemokinov in njihovih receptorjev v alergijskih boleznih. Kljub temu pa se bodo v prihodnost namenili k iskanju antagonistov potencialnega kandidata kemokinega receptorja CCR3.

== Matevž Ambrožič: Termogene snovi in regulacija telesne teže ==
Eden izmed glavnih problemov modernega človeka je prekomerna teža in z njo povezane zdravstvene težave. Strokovnjaki so si edini, da je ključ do uspeha pozitivno razmerje med porabljeno in vnešeno energijo. Veliko pomoč pri porabi energije nudijo snovi, ki spodbujajo termogenezo, po možnosti prek oksidacije maščob. Katehini in kofein so termogene snovi, ki jih najdemo v mnogih naravnih virih, vsi skupaj pa se pojavljajo v čaju. Najboljša vira sta zeleni in beli čaj, saj sta manj obdelana. Pri izgubi telesne teže je vedno cilj večja poraba energije in uničevanje maščobnih zalog. Maščobe, ki jih zaužijemo, so v obliki triacilglicerolov in se shranjujejo v belem in rjavem maščobnem tkivu. Služijo nam kot rezervna zaloga energije, mehanska zaščita in pomoč pri vzdrževanju temperature. V določenih pogojih simpatični živčni sistem z izločanjem hormona epinefrina sproži pretvarjanje shranjenih triacilglicerolov v maščobne kisline (proces lipolize), te pa se lahko v procesu oksidacije porabijo za sintezo ATP. Da pospešimo porabo maščob, se s termogenimi snovmi skušamo vmešavati v metabolizem maščob na raznih stopnjah. Katehini in kofein katalizirajo lipolizo na različne načine, večinoma z inhibicijo zaviralcev lipolize. Rezultati raziskav sicer rahlo variirajo, vendar v splošnem velja, da katehini in kofein pomagajo pri regulaciji telesne teže.

== Marko Radojković: Vpliv rakastih celic in sepse na izraženost krvnega proteina trombina ==
Nedavne študije so pokazale kako anti-koagulanti pomagajo pri zdravljenju in preprečevanju raka, vendar natančen mehanizem ki opisuje kako sta strjevanje krvi in napredovanje raka povezana, ni bil znan do sedaj. Znastveniki so odkrili kako celice pod stresom povečajo proizvodnjo enega izmed ključnih faktorjev strjevanja krvi – trombina. Količina trombina ki ga naše celice proizvajajo je kontrolirana z dvema vrstami proteinov : proteini ki zavirajo produkcijo (FBP2 in FBP3) , ter proteini ki jo pospešujejo (hnRNPI, U2AF65 in U2AF35). Obe vrsti proteinov delujeta tako da se vežeta na celične ''stroje'' na protrombinski mRNA , in v normalnih pogojih, proteini inhibitorji vzdržujejo nizko koncentracijo trombina. Ko naše celice pridejo v stanje stresa, v primeru ko je povzročitelj vnetje, en drugi protein ki se imenuje p38 MAPK, reagira tako da pripne fosfatne skupine ihibitornim proteinom. To povzroči da se le ti težje vežejo na celične ''stroje'' za produkcijo trombina, in omogoča stimulatornim proteinom da prevzamejo glavno vlogo v mehanizmu. Torej, vnetje zaradi raka bi lahko pripeljalo do povečane ravni trombina in, kot je trombin glavni agent strjevanja krvi, bi to lahko pojasnilo, zakaj se pri bolnikih z rakom pogosteje pojavljajo krvni strdki.
Znastveniki so ugotovili da p38 MAPK protein tudi vpliva na proizvodnjo trombina v sepsi. Znana tudi kot zastrupitev krvi, sepsa se pojavi, ko bakterije ali drugi povzročitelji bolezni pridejo v kri, ki vodi k razširjenosti okužbe in nastanku težav pri strjevanu krvi. Ko so analizirali vzorce jeter odvzetih iz miši s sepso in iz bolnikov z rakom, so znanstveniki odkrili da je povečana produkcija trombina odgovor, tako kot na široko vnetje v primeru sepse, kot na lokalizirano vnetje v invaziji tumorja , kje se rakave celice širijo v bližnje tkivo.

== Urška Rode: Vpliv C- reaktivnega proteina na patogenezo simptomov metaboličnega sindroma ==
C-reaktivni protein je protein, akutne faze, ki nastaja v jetrih, njegova koncentracija v krvi se poviša ob razih vnetjih in okužbah, kot del imunskega odziva. Njegova vloga in pomen še nista povsem jasna. Najbolj znana njegova vloga je pri vezavi na fosfoholin na membrano bakterije ali poškovodovane celice. s tem ko se veže z eno strenjo na fosfoholin se na njegovo drugo stran veže prva komponenta klasične proti imunskega odziva.
njegova vloga na cel organizem še ni popolnoma pojasnjena. V zadnjem času poteka veliko raziskav, ki poučujejo njegov vpliv na sindrome metaboličnega sindroma, to so povečan krvni tlak, diabetes,... Dokazali so namreč. de ima CRP vpliv na povišan krvni tlak, saj so to bolezen odkrili tudi pri ljudjeh, ki so imeli povečan CRP in niso imeli povečan LDL-holesterol, ki je glavni povzročitekj te bolezni. V članku so znanstveniki preučevali zvišane koncentracije človeškega C-reaktivnega proteina v transgenih miših, ki so imele ižražen človeški CRP. raziskovali so kako povečan protein CRP vpliva na razvoj simptomov metaboličnega sindroma. pri miših, kiso imele povečan CRP so ugotovili povečan krvni tlak, vendar je pri vplivu CRP na cel organizem še veliko nejasnosti. Zato bo potrebno še veliko raziskav, da bo znano ali ina človeški C-reaktivni protein neposredno vlogo, pri patogenezi simptomov metaboličnega sindroma

== Urška Navodnik: Stabilnnost DNA/DNA in RNA/DNA dupleksa vpliva na mRNA transkripcijo ==
Nukleinske kisline nam s svojo specifično kemijsko in fizikalno zgradbo zagotavljajo shranjevanje genetskih informacij. Zaradi teh lastnosti imata vijačnici DNA in RNA sposobnost tvoriti medmolekulske interakcije in vodikove vezi, kateri sta glavni razlog, da lahko tvorita dvojno vijačnico – dupleks. Pojavi se vprašanje, če lahko nukleinske kisline posedujejo še druge lastnosti, ki prispevajo k biološkim funkcijam. Eno izmed zanimivih vprašanj se pojavi, ko poskušamo razložiti pojav intron – ekson. Nekateri izmed intronov naj bi imeli vlogo uravnavanja izražanja genov, pa vendar se pri zorenju mRNA izrežejo iz zapisa. V tej raziskavi je predstavljeno, kako lahko termodinamična stabilnost DNA/DNA in RNA/DNA dupleksa vpliva na prepis mRNA. Raziskave so izvajali predvsem na vrsti Saccharomyces z metodo najbližjega soseda. Rezultati so pokazali, da so kodirane regije termodinamsko bolj stabilne od nekodiranih zaporedij – intronov, 3´ - neprevedenih regij in medgenskih področij. Povrh, odprti bralni predelni imajo bolj stabilno smerno RNA/DNA dvojno vijačnico, kot potencialno ujemajoč protismeren dupleks. Raziskava temelji na izračunih, koliko proste energije je potrebno, da se dvojna vijačnica razvije. Več energije, kot je potrebno stabilnejša je struktura. Rezultati torej prikazujejo, da so geni stabilnejši od medgenskih področij. Torej lahko povzamemo, da stabilnost DNA/DNA in RNA/DNA dupleksa vpliva na mRNA prepis.

== Dominik Kert: Kako osteokalcin vpliva na reprodukcijo organizmov ==
Nove raziskave kažejo povezave med reprodukcijo in skeletnim sistemom. Kosti so sestavljene iz osteoblastov. Produkt le teh je hormon osteokalcin. Ta hormon pa zelo vpliva na produkcijo testosterona, ampak ne neposredno. Deluje namreč na leydigove celice, ki se nahajajo med tubuli v testisih. Raziskovalci so odkrivali te rezultate tako, da bo samcem miši vbrizgali osteokalcin-takrat se je raven testosterona dvignila. Po drugi strani pa so miškam odstranili gen, ki kodira ta hormon. Prišlo je do osupljivih rezultatov. Mišim se je zmanjšala plodnost, in sicer tako, da se je zmanjšala količina sperme, zmanjšala število zdravih spermijev in veliko jih je tudi pomrlo že v modih. Dokazali so tudi veliko signalnih poti med reproduciranjem in okostjem. In sicer če organizem dobro funkcionira in ima dovolj zalog hrane se je tudi sposoben razmnoževati. In pa tudi povezavo med debelostjo in neplodnostjo. Ampak to še ni vse. Mišim se je tudi spremenilo partnersko vedenje: manjkrat so postavljali gnezda. To je bila posledica nabiranja luteinizirajočega hormona. Poleg slednjega hormona se je začela pojavljati tudi večja količina estrogena. Te dve posledici pa lahko povežemo s staranjem moškega, ko testosteron začne vpadati, estrogen in luteinizirajoči hormon pa narasteta. Zaradi teh ugotovitev bi bilo lahko v prihodnje možno zdravljenje neplodnosti pri ljudeh.

== Živa Brglez: Kompleks Mre11 ==
Med podvojevanjem dvojne vijačnice DNA včasih pride do napak, ki jih je nujno potrebno popraviti, oziroma obnoviti v njeno izvirno obliko, če je prišlo do poškodbe zaradi mutagenih dejavnikov iz okolja, da se ne prenesejo naprej v hčerinske celice in prihodnje rodove. Za to skrbijo raznovrstni popravljalni mehanizmi. V primeru dvojnega preloma verige DNA (Double-Strand Breaks – DBS) je kompleks Mre11, sestavljen iz treh različnih proteinov: dimera mejotske rekombinacije 11 (Mre11), dimera Rad50 in Nbs1, ključnega pomena za celični odziv na poškodbe DNA. Mre11 in Nbs1 skupaj z delom Rad50 tvorita glavno domeno kompleksa, iz katere izraščajo obvite vijačnice (coiled-coil) Rad50. Poznana sta dva načina poprave preloma dvojne verige DNA, homologna rekombinacija (HR) in sklepanje nehomolognih koncev verig DNA (NHEJ). Kompleks Mre11 sodeluje pri obeh, strukturno in encimatsko. Strukturno tako, da glavno domeno veže poškodovan del DNA, medtem ko se obviti vijačnici povežeta z obvitima vijačnicama drugega kompleksa Mre11 in držita skupaj prelomljene verige. Encimatsko pa s spodbujanjem odstranitve koncev in posredovanjem informacije ATM (ataxia-telengastia-mutated), katerega del se fosforilira na glavni domeni kompleksa Mre11 in tako vpokliče še ostale molekule odgovorne za popravo. Kompleks Mre11 skrbi tudi za homeostazo telomerov z regulira njihove dolžine, in posredno za razvoj imunskega sistema. V primeru mutacije v katerem izmed genov, ki kodirajo gradbene proteine kompleksa Mre11, pride do različnih dednih bolezni, ki se kažejo fenotipsko podobno – značilni sta hiperobčutljivost na radiacijo in mikrocefalija.

== Taja Karner: Alkohol omogoča lažje nalaganje CD1d molekul, s tem aktivira NKT celice in zmanjša pojavljanje sladkorne bolezni pri NOD miškah ==
V raziskavi, ki sem si jo izbrala raziskujejo pozitivne učinke alkohola. Vse več raziskav kaže, da zmerno uživanje alkoholnih pijač pripomore k boljšemu delovanju našega mehanizma. V tej raziskavi pa jih je zanimalo predvsem kakšen je vpliv alkohola na prirojen imunski sistem. Raziskave so potekale in vitro z uporabo α-GalCer molekul in in vivo na NOD miših. NKT celice, ki jih omenjam v svoji seminarski, pa so heterogene skupine, sestavljene iz NK celic in T celic. Te celice prepoznavajo antigene CD1d, ki se izražajo na površini antigen-predstavitvenih celic. To so celice, ki imajo sposobnost preoblikovanja antigenov, tako da jih T-celice prepoznajo in ustvarijo ustrezen odgovor. Ugotovili so, da alkohol izboljša nalaganje CD1d molekul, s tem aktivacijo NKT celic in tako zmanjša možnost za razvoj diabetesa. Pri NOD miših, ki so jim dajali 5 % alkohola se je pokazalo zmanjšano število diabetesa in manjša koncentracija glukoze ob testiranju. Zanimivo je, da te miši niso kazale nikakršnih znakov alkoholizma. Pri ljudeh bi takšna koncentracija, preračunana glede na maso človeka, povzročila visoko stopnjo opojnost. Kljub temu pa je bila v mišji krvi razmeroma mala količina alkohola, kar kaže na bistveno večjo zmožnost razstrupljanja alkohola pri NOD miših, kot jo imamo ljudje.

== Karmen Gec: Učinki vadbe in/ali dodajanja antioksidantov na gene endotelnih celic ==
Teoretično je raziskano, da so antioksidanti v sadju in zelenjavi pomembni pri zaviraju oksidativnih mehanizmov, ki vodijo do različnih degenerativnih bolezni, tudi srčno-žilnih bolezni. V obravnavani raziskavi avtorji na podganah ugotavljajo različno izražanje genov endotelnih celic glede na dodatek antioksidantov, glede na vadbo ali kombinacijo obojega. Navajajo, da je pri ednoteliju zelo pomembno, da razlikujemo med fiziološkimi in patološkimi t.i. Reaktivnimi kisikovimi zvrstmi (v nadaljevanju RKZ). Izražanje genov endotelnih celic so ugotavljali v področju srčnega endotelija (levi ventrikel) in žilnega endotelija (koronarna arterija). V raziskavi so ugotovili, da je gen RhoA, ki je pomemben pri srčno-žilnih boleznih kazal znižan učinek pri vadbi ter povišan pri dodatku antioksidantov v področju levega ventrikla. Poleg tega pa je še IL-6, pomemben gen pri vnetju, znižal učinek pri vseh treh dodanih tretmajih. Tako izražanje obeh genov z dodajanjem vadbe in/ali antioksidantov poda vpogled v molekulske mehanizme srčnožilne bolezni.

== Tjaša Goričan: Molekulske tarče oksidativnega stresa ==
Aerobni organizmi so življensko odvisni od procesov celičnega dihanja, pri čemer pa vedno nastajajo za biološke makromolekule (lipide, DNA, proteine) škodljivi kisikovi radikali. Organizmi so razvili obrambne mehanizme, ki preprečujejo potencialno škodo. Pri tem se ohranja neko ravnovesje med kisikovimi radikali in antioksidanti, katere mora organizem pridobiti s hrano. Antioksidanti (vitamini C, E, koencim Q10, karotenoidi itd.) so snovi, ki delujejo kot katalizatorji in celice varujejo pred oksidacijo. Porušeno ravnotežje povzroči oksidativni stres, posledice katerega so lahko različne bolezni (Alzheimerjeva bolezen, Parkinsonova bolezen, rak, itd.)in staranje. Zmerna oksidacija sproži apoptozo (programirano celično smrt), hujši in intenzivni oksidativni stres pa lahko povzroči celično smrt in celo nekrozo (odmrtje celic/ tkiva). V mojem članku so poskuse izvajali na bakterijah in kvasovkah ter ugotovili, da so pri obeh prvotne tarče ROS (reaktivne spojine, ki vsebujejo atom kisika) različne. Rezultati: pri prokariontih je DNA prvotna tarča, pri evkariontih pa ne. Vzrok za to je različen prag občutljivosti molekul pri različnih organizmih. Identifikacija primarnih tarč oksidativnega stresa bi odprla nove možnosti za terapije bolezni povezane z njim.

== Jernej Mustar: Odpornost srpastih celic na okužbo z plazmodijem ==
V mojem članku so raziskovali fenomen, ki se pojavlja pri obolelih za anemijo srpastih celic(HbS homozigotni) in HbS heterozigotnih, in sicer toleranca do okužbe z Plasmodijem. V raziskavi so uporabili Plasmodium berghei, ki je modelni organizem za razumevanje človeške malarije. Ta povzroča t.i. možgansko malarijo (experimental cerebral malaria-ECM). Znanstveniki so prišli do zelo zanimivih odkritij. Ugotovili so namreč, da se glavni vzrok imunosti skriva v nalaganju nizkih količin prostega hema v krvi in povečane ekspresije stresno-odgovornega encima HemOxigenaze1(ki razgrajuje prosti hem). Pri katalizi hema nastaja CO, ki se veže na hemoglobin in prepreči odcepitev prostetične skupine (hema), saj je le ta glavni vzrok patogeneze ECM. Ta spoznanja so, po mojem mnenju, človeštvo privedla korak bližje k odkritju zdravila za malarijo.

User talk:JernejMustar

2011-05-19T16:08:48Z

JernejMustar: /* Jernej Mustar: Odpornost srpastih celic na okužbo z plazmodijem */

V mojem članku so raziskovali fenomen, ki se pojavlja pri obolelih za anemijo srpastih celic(HbS homozigotni) in HbS heterozigotnih, in sicer toleranca do okužbe z Plasmodijem. V raziskavi so uporabili Plasmodium berghei, ki je modelni organizem za razumevanje človeške malarije. Ta povzroča t.i. možgansko malarijo (experimental cerebral malaria-ECM). Znanstveniki so prišli do zelo zanimivih odkritij. Ugotovili so namreč, da se glavni vzrok imunosti skriva v nalaganju nizkih količin prostega hema v krvi in povečane ekspresije stresno-odgovornega encima HemOxigenaze1(ki razgrajuje prosti hem). Pri katalizi hema nastaja CO, ki se veže na hemoglobin in prepreči odcepitev prostetične skupine (hema), saj je le ta glavni vzrok patogeneze ECM. Ta spoznanja so, po mojem mnenju ,človeštvo privedla korak bližje k odkritju zdravila za malarijo.

User talk:JernejMustar

2011-05-19T15:53:53Z

JernejMustar: /* Jernej Mustar: Odpornost srpastih celic na okužbo z plazmodijem */

Raziskovali so fenomen, ki se pojavlja pri obolelih za SCA in HbS heterozigotnih, in sicer toleranca do okužbe z Plasmodijem. V raziskavi so uporabili Plasmodium berghei, ki je modelni organizem za razumevanje človeške malarije. Povzroča t.i. možgansko malarijo (experimental cerebral malaria-ECM). Poskuse so opravili na C57BL/6 laboratorijskih miškah, imenovanih tudi "black 6" miške.

User talk:JernejMustar

2011-05-19T15:48:00Z

JernejMustar: Removing all content from page

User talk:JernejMustar

2011-05-19T15:47:35Z

JernejMustar: Odpornost srpastih celic na okužbo z plazmodijem

BIO1-seminar 2011

2011-05-13T20:19:10Z

JernejMustar: /* Seznam seminarjev */

= Temelji biokemije- seminar =

Seminarje vodi doc. dr. Gregor Gunčar in so na urniku vsak ponedeljek od 10:00 do 11:30.

Ocena seminarjev predstavlja ??% končne ocene in vsebuje vse točke, ki jih študent/ka lahko zbere pri seminarju in ostalih dejavnostih, ki niso del pisnega izpita.

== Seznam seminarjev ==
{| border="1" cellpadding="4" cellspacing="0" style="border:#c9c9c9 1px solid; margin: 1em 1em 1em 0; border-collapse: collapse;"
| align="center" style="background:#f0f0f0;"|'''Ime in priimek'''
| align="center" style="background:#f0f0f0;"|'''Slovenski naslov članka'''
| align="center" style="background:#f0f0f0;"|'''Faktor vpliva revije'''
| align="center" style="background:#f0f0f0;"|'''Rok za oddajo'''
| align="center" style="background:#f0f0f0;"|'''Rok za recenzijo'''
| align="center" style="background:#f0f0f0;"|'''Datum predstavitve'''
| align="center" style="background:#f0f0f0;"|'''Recenzent 1'''
| align="center" style="background:#f0f0f0;"|'''Recenzent 2'''
| align="center" style="background:#f0f0f0;"|'''Recenzent 3'''
|-
| BOTONJIĆ SANDI||[http://wiki.fkkt.uni-lj.si/index.php/BIO1_Povzetki_seminarjev#Sandi_Botonji.C4.87:_Tioredoksinu_podoben_protein_.28TXNL2.29_.C5.A1.C4.8Diti_kancerogene_celice_pred_oksidativnim_stresom Tioredoksinu podoben protein (TXNL2) ščiti kancerogene celice pred oksidativnim stresom]
||15.387||28.02.||03.03.||07.03.||RODE URŠKA||KERIN INES||OGRIS IZA
|-
| VRANKAR ANDREJ||Število lasno-mešičnih matičnih celic se v plešastem lasišču moških z androgeno alopecijo ohranja za razliko od števila CD200-rich in CD34-positive lasno-mešičnih predniških celic||||28.02.||03.03.||07.03.||HROVAT KARMEN||BOHNEC IVO||JAVORŠEK KAJA
|-
| ZALAR MATJA||Protein p53||||28.02.||03.03.||07.03.||OGRIS IZA||CRČEK MITJA||ZOTTEL ALJA
|-
| ZOTTEL ALJA||Vloga imunskega sistema pri aterosklerozi||31.434||07.03.||10.03.||14.03.||RADOJKOVIĆ MARKO||KERT DOMINIK||HROVAT KARMEN
|-
| DOLINAR ANA||[http://wiki.fkkt.uni-lj.si/index.php/BIO1_Povzetki_seminarjev#Ana_Dolinar:_Prilagojena_ali_prilagodljiva_imunost.3F_Primer_naravnih_celic_ubijalk Prirojena ali prilagodljiva imunost? Primer naravnih celic ubijalk]||28||07.03.||10.03.||14.03.||RAUTER URŠKA||MOHAR MAŠA||VERBANČIČ JANA
|-
| RAUTER URŠKA||Razvojna vloga Srf, kortikalnega citoskeleta in celične oblike v orientaciji epidermalnega vretena||19.527||07.03.||10.03.||14.03.||MUSTAR JERNEJ||JAVORŠEK KAJA||MOHAR MAŠA
|-
| MOHAR MAŠA||Sladkorna bolezen tipa 2 kot bolezen imunskega sistema||30,006||14.03.||17.03.||21.03.||VENE ROK||RAUTER URŠKA||GORIČAN TJAŠA
|-
| POHLEVEN ŠPELA||Prioni||34||14.03.||17.03.||21.03.||KEPIC LEA||RADOJKOVIĆ MARKO||DOLINAR ANA
|-
| KEPIC LEA||Agonisti adrenoreceptorjev β2||34.48||14.03.||17.03.||21.03.||VRANKAR ANDREJ||BRATOVŠ ANDREJA||MUSTAR JERNEJ
|-
| KMETIČ MIRJAM||Celična regulacija metabolizma železa||5,371||14.03.||17.03.||21.03.||MARIĆ TAMARA||REMŠKAR MAJA||KOMAN KATRA
|-
| JARC VERONIKA||Eksperimentalni modeli za študijo imunobiologije hepatitisa C||3.26||14.03.||21.03.||28.03.||REMŠKAR MAJA||MUSTAR JERNEJ||KEPIC LEA
|-
| KOMAN KATRA||[http://wiki.fkkt.uni-lj.si/index.php/BIO1_Povzetki_seminarjev#Katra_Koman:_.09Pomen_dendritskih_celic_.28DCs.29_in_celic_ubijalk_.28NK.29_v_imunskem_odzivu_na_oku.C5.BEbo_z_virusom_HIV-1 Pomen dendritskih celic (DCs) in celic ubijalk (NK) v imunskem odzivu na okužbo z virusom HIV-1]||32.245||21.03.||25.03.||28.03.||ČUPOVIĆ VANA||KARNER TAJA||KMETIČ MIRJAM
|-
| OGRIS IZA||Zakaj imajo možgani glikogen?||5,125||14.03.||21.03.||28.03.||KNAPIČ EVA||BRGLEZ ŽIVA||VRANKAR ANDREJ
|-
| KERIN INES||Kanabinoidi za zdravljenje shizofrenije? Uravnotežena nevrokemična sestava za škodljive in terapevtske učinke uživanja konoplje||4.458||14.03.||21.03.||28.03.||ŠTOK ULA||ŠTEMBERGER ROK||KERT DOMINIK
|-
| VERBANČIČ JANA||Apoptozi podobna smrt v bakterijah, ki jo povzroča HAMLET, človeški mlečni lipidno-proteinski kompleks||4.351||21.03.||28.03.||04.04.||KARNER TAJA||ZOTTEL ALJA||KNAPIČ EVA
|-
| KNAPIČ EVA||Kako virusi vodijo delovanje celice.||14.101||21.03.||28.03.||04.04.||ZALAR MATJA||POHLEVEN ŠPELA||LORBEK SARA
|-
| REMŽGAR ANA||Črevesna absorpcija vitamina D ne poteka le s pasivno difuzijo: dokazi za vpletenost enakih transporterjev kot pri holesterolu||4.356||21.03.||28.03.||04.04.||BOTONJIĆ SANDI||LORBEK SARA||ČUPOVIĆ VANA
|-
| GRDADOLNIK MAJA||Jedrni in nejedrni receptorji za estrogene||5.328||21.03.||28.03.||04.04.||MOHAR MAŠA||REMŽGAR ANA||FRANKO NIK
|-
| JAVORŠEK KAJA||Potencial matičnih celic pri Parkinsonovi bolezni in molekularni faktorji za tvorbo dopaminskih nevronov||4.139||28.03.||04.04.||11.04.||GEC KARMEN||MARIĆ TAMARA||RADOJKOVIĆ MARKO
|-
| BRATOVŠ ANDREJA||Vloga GPCR v patologiji Alzheimerjeve bolezni||26||28.03.||04.04.||11.04.||ZOTTEL ALJA||ČUPOVIĆ VANA||GRDADOLNIK MAJA
|-
| CRČEK MITJA||Matične celice in njihova vloga pri zdravljenju bolezni in poškodb||7.365||28.03.||04.04.||11.04.||BOHNEC IVO||KMETIČ MIRJAM||BRATOVŠ ANDREJA
|-
| MARIĆ TAMARA||Organizacija jedra||9.58||28.03.||04.04.||11.04.||NAVODNIK URŠKA||GEC KARMEN||REMŠKAR MAJA
|-
| ŠTEMBERGER ROK||povečano izražanje in imunogenosti HIV proteinov po inaktivaciji encimske aktivnosti||3.616||04.04.||11.04.||18.04.||JAVORŠEK KAJA||VRANKAR ANDREJ||BOTONJIĆ SANDI
|-
| LORBEK SARA||Sovplivanje maščobnih kislin ter genov na adipokine in debelost||3.072||04.04.||11.04.||18.04.||POHLEVEN ŠPELA||KNAPIČ EVA||VENE ROK
|-
| REMŠKAR MAJA||Evolucijska dinamika transponibilnih elementov (TE) v majhnem RNA svetu||8.689||04.04.||11.04.||18.04.||KERIN INES||POVŠE KATJA||CRČEK MITJA
|-
| ČUPOVIĆ VANA||naslov||||04.04.||11.04.||18.04.||REMŽGAR ANA||VERBANČIČ JANA||RODE URŠKA
|-
| RODE URŠKA||vpliv c-reaktivnega proteina na patogenezo simptomov metaboličnega sindroma||6.614||03.05.||06.05.||09.05.||GRDADOLNIK MAJA||ŠTEMBERGER ROK||MARIĆ TAMARA
|-
| RADOJKOVIĆ MARKO||Vpliv rakastih celic in sepse na izraženost krvnega proteina trombina||14.608||03.05.||06.05.||09.05.||FRANKO NIK||VENE ROK||POVŠE KATJA
|-
| VENE ROK||[http://wiki.fkkt.uni-lj.si/index.php/BIO1_Povzetki_seminarjev#Rok_Vene:_Spremembe_nivoja_metilacije_DNA_so_sorazmerne_s_starostjo_.C4.8Dlove.C5.A1kih_mo.C5.BEganov Spremembe nivoja metilacije DNA so sorazmerne s starostjo človeških možganov]||7.386||03.05.||06.05.||09.05.||VERBANČIČ JANA||NAVODNIK URŠKA||ZALAR MATJA
|-
| FRANKO NIK||naslov||||03.05.||06.05.||09.05.||xx||HROVAT KARMEN||BOHNEC IVO
|-
| HROVAT KARMEN||Ciljanje kemokinih receptorjev v alergijskih boleznih||5,155||04.05.||09.05.||16.05.||KERT DOMINIK||JARC VERONIKA||KARNER TAJA
|-
| AMBROŽIČ MATEVŽ||[http://wiki.fkkt.uni-lj.si/index.php/BIO1_Povzetki_seminarjev#Matev.C5.BE_Ambro.C5.BEi.C4.8D:_Termogene_snovi_in_regulacija_telesne_te.C5.BEe Termogene snovi in regulacija telesne teže]||4,434||04.05.||09.05.||16.05.||LORBEK SARA||KEPIC LEA||REMŽGAR ANA
|-
| NAVODNIK URŠKA||Stabilnost RNA/DNA in DNA/DNA dupleksa vpliva na mRNA transkripcijo||||04.05.||09.05.||16.05.||AMBROŽIČ MATEVŽ||ŠTOK ULA||ŠTEMBERGER ROK
|-
| BRGLEZ ŽIVA||[http://wiki.fkkt.uni-lj.si/index.php/BIO1_Povzetki_seminarjev#.C5.BDiva_Brglez:_Kompleks_Mre11 Kompleks Mre11]||42.198||09.05.||16.05.||23.05.||DOLINAR ANA||BOTONJIĆ SANDI||JARC VERONIKA
|-
| KARNER TAJA||Alkohol omogoča lažje nalaganje CD1d molekul, s tem aktivira NKT celice in zmanjša pojavljanje sladkorne bolezni pri NOD miškah
||||12.05.||17.05.||23.05.||KOMAN KATRA||OGRIS IZA||NAVODNIK URŠKA
|-
| KERT DOMINIK||[http://wiki.fkkt.uni-lj.si/index.php/BIO1_Povzetki_seminarjev#Dominik_Kert:_Kako_osteokalcin_vpliva_na_reprodukcijo_organizmov Kako osteokalcin vpliva na reprodukcijo organizmov]||||09.05.||16.05.||23.05.||GORIČAN TJAŠA||GRDADOLNIK MAJA||RAUTER URŠKA
|-
| MUSTAR JERNEJ||Odpornost srpastih celic na okužbo z plazmodijem||31||16.05.||23.05.||30.05.||JARC VERONIKA||AMBROŽIČ MATEVŽ||BRGLEZ ŽIVA
|-
| GEC KARMEN||Učinki vadbe in uporabe antioksidantov na izražanje endotelnih genov||||16.05.||23.05.||30.05.||POVŠE KATJA||ZALAR MATJA||AMBROŽIČ MATEVŽ
|-
| GORIČAN TJAŠA||Molekulske tarče oksidativnega stresa||||16.05.||23.05.||30.05.||KMETIČ MIRJAM||RODE URŠKA||POHLEVEN ŠPELA
|-
| BOHNEC IVO||naslov||||23.05.||30.05.||06.06.||CRČEK MITJA||GORIČAN TJAŠA||ŠTOK ULA
|-
| ŠTOK ULA||Mutacija mitohondrijske DNA v povezavi z rakom debelega črevesa kot posledica abnormalnega delovanja citokroma c oksidaze||||23.05.||30.05.||06.06.||BRGLEZ ŽIVA||DOLINAR ANA||KERIN INES
|-
| nihce ||naslov||||23.05.||30.05.||06.06.||BRATOVŠ ANDREJA||KOMAN KATRA||GEC KARMEN
|}

==Naloga==
* samostojno pripraviti seminar, katerega osnova je znanstveni članek s področja biokemije, ki ga po želji izberete v reviji s področja biokemije, ki ima faktor vpliva večji kot 3 in je bil objavljen v letu 2011. Poleg tega članka morate za seminar uporabiti še najmanj pet drugih virov! http://www.cobiss.si/scripts/cobiss?command=CONNECT&base=JCR
* osnovni članek in naslov pošljete meni, najkasneje pet dni pred rokom za oddajo (rok-5), da ocenim, če je primeren za predstavitev. Naslov vpišete v tabelo, takoj ko ste si ga izbrali!
* [[BIO1 Povzetki seminarjev|Povzetek seminarja]] opišete na wikiju v približno 200 besedah - najkasneje do dne ko morate oddati seminar recenzentom. Povezave do slik so dobrodošle, niso pa nujne.
* Povezavo do povzetka vnesete v tabelo seminarjev tekočega letnika.
* Seminar pripravite v obliki seminarske naloge (pisava 12, enojni razmak, 2,5 cm robovi; važno je, da je obseg od 1800 do 2000 besed), vsebovati mora najmanj eno sliko. Slika mora imeti legendo in v besedilu mora biti na ustreznem mestu sklic na sliko.
* Natisnjen seminar oddajte do roka vsakemu od recenzentov (docentu ga pošljite po e-pošti v formatu .doc ali .docx).
* Recenzenti do dneva določenega v tabeli določijo popravke in podajo oceno pisnega dela, v predpisanem formatu elektronskega obrazca na internetu.
* Ustna predstavitev sledi na dan, ki je vpisan v tabeli. Za predstavitev je na voljo 15 minut. Recenzenti morajo biti na predstavitvi prisotni.
* Predstavitvi sledi razprava- 5 minut. Recenzenti podajo oceno predstavitve in postavijo vsak vsaj dve vprašanji.
* Na dan predstavitve morate docentu oddati končno (popravljeno) in natisnjeno verzijo seminarja v enem izvodu.
* Seminarska naloga in povzetek na wikiju morajo biti v slovenskem jeziku!

==Ocenjevanje seminarjev==
Recenzenti ocenijo seminar tako, da izpolnijo [[https://spreadsheets.google.com/viewform?formkey=dFM2SktfM3Q4VU1wNUQzdU45OTlWVXc6MA recenzentsko poročilo]] na spletu.

== Mnenje o predstavitvi ==
Vsak posameznik '''mora''' oceniti seminar, tako da odda svoje [https://spreadsheets.google.com/viewform?formkey=dFd3TGhLV3ZSa2xsLVlmMVVUaEFURWc6MA mnenje] najkasneje v treh dneh po predstavitvi. Kdor na seminarju ni bil prisoten, mnenja '''ne sme''' oddati.

==Urejanje spletnih strani na wikiju==
Wiki so razvili zato, da lahko spletne vsebine ureja vsakdo. Ukazi so preprosti, dokler si ne zamislite česa prav posebnega. Vseeno pa je Word v primerjavi z wikijem pravo čudežno orodje... Če imate težave z oblikovanjem besedila, si preberite poglavje o urejanju wiki-strani na Wikipediji ([http://en.wikipedia.org/wiki/Help:Editing tule] v angleščini in [http://sl.wikipedia.org/wiki/Wikipedija:Urejanje_strani tu] v slovenščini). Pomaga tudi, če pogledate, kako je zapisana kakšna stran, ki se vam zdi v redu: kliknite na zavihek 'Uredite stran' in si poglejte, kako so vpisane povezave, kako nov odstavek in podobno. ''Na koncu seveda pod oknom za urejanje kliknite na 'Prekliči'.''

==Faktor vpliva==
Faktor vpliva (angl. impact factor) neke revije pove, kolikokrat so bili v poprečju citirani članki v tej reviji v dveh letih skupaj pred objavo tega faktorja. Faktorje vpliva za posamezno revijo lahko najdete v [http://www.cobiss.si/scripts/cobiss?command=CONNECT&base=JCR COBISS-u]. V polje "Naslov revije" vnesite ime revije za katero želite izvedeti faktor vpliva in pritisnite na gumb POIŠČI. V skrajnem desnem stolpcu se bodo izpisali faktorji vpliva za revije, ki ustrezajo vašim iskalnim kriterijem. Zadetkov za posamezno revijo je več zato, ker so navedeni faktorji vpliva za posamezno leto. Za leto 2011 faktorji vpliva še niso objavljeni, zato se orientirajte po faktorjih vpliva zadnjih par let. Če faktorja vpliva za vašo izbrano revijo ne najdete v bazi COBISS, potem izberite članek iz kakšne druge revije.

==Citiranje virov==
Citiranje je možno po več shemah, važno je, da se v seminarju držite ene same.
Temeljno načelo je, da je treba vir navesti na tak način, da ga je mogoče nedvoumno poiskati.
Za citate v naravoslovju je najpogostejše citiranje po pravilniku ISO 690. [http://www.google.com/url?sa=t&source=web&cd=6&sqi=2&ved=0CEUQFjAF&url=http%3A%2F%2Fwww.tre.sik.si%2Fmain%2Fpomoc%2Ffiles%2Fcitiranje_in_navajanje_virov.pdf&rct=j&q=citiranje%20po%20pravilniku%20ISO%20690&ei=jPBqTe6FC9DKswaWk-TmDA&usg=AFQjCNF8r6X9Y781sanDObaXNdCew4suUg&sig2=cTqKObSJsTicekWGRGa72g&cad=rja Pravila], ki upoštevajo omenjeni standard, so pripravili pri ZTKS. Sicer pa ima vsaka revija lahko svoj način citiranja, ki ga je treba pri pisanju članka upoštevati. 
'''Citiranje knjig:''' 
Priimek, I. ''Naslov''. Kraj: Založba, letnica. 
Priimek, I. ''Naslov: podnaslov''. Izdaja. Kraj: Založba, letnica. Zbirka, številka. ISBN. 

Boyer, R. ''Temelji biokemije''. Ljubljana: Študentska založba, 2005. 
Glick BR in Pasternak JJ. ''Molecular biotechnology: principles and applications of recombinant DNA''. 3. izdaja. Washington: ASM Press, 2003. ISBN 1-55581-269-4. 
Če so avtorji trije, je beseda in med drugim in tretjim avtorjem. Če so avtorji več kot trije, napišemo samo prvega in dopišemo ''et al''. (in drugi, po latinsko). Vse, kar je latinsko, pišemo poševno (npr. tudi imena rastlin in živali, pojme ''in vivo'', ''in vitro'' ipd.).

'''Citiranje člankov:''' 
Priimek, I. Naslov. ''Naslov revije'', letnica, letnik, številka, strani. 
Lartigue C. ''et al''. Genome transplantation in bacteria: changing one species to another. ''Science'', 2007, letn. 317, str. 632-638.

Alternativni način citiranja (predvsem v družboslovju) je po pravilih APA, kjer članke citirajo takole: 
Priimek, I. (letnica, številka). Naslov. Naslov revije, strani. 
Lartigue C. ''et al.'' (2007, 317) Genome transplantation in bacteria: changing one species to another. ''Science'', 632-638.

Revija Science uporablja skrajšani zapis: 
C. Lartigue ''et al''. Science 317, 632 (2007) 

V diplomah na FKKT je treba navesti vire tako, da izpišete tudi naslov citiranega dela in strani od-do (ne samo začetne).

'''Citiranje spletnih virov:''' 
Priimek, I. ''Naslov dokumenta''. Izdaja. Kraj: Založnik, letnica. Datum zadnjega popravljanja. [Datum citiranja.] spletni naslov 
strangeguitars. ''On the brink of artificial life''. 6. 10. 2007. [citirano 13. 11. 2007] http://www.metafilter.com/65331/On-the-brink-of-artificial-life 
Navedemo čim več podatkov; pogosto vseh iz pravila ne boste našli.