Figures
Abstract
Glutamine tandem repeats are common in eukaryotic proteins. Although some studies have proposed that replication slippage plays an important role in shaping these repeats, the role of natural selection in glutamine tandem repeat evolution is somewhat unclear. In this study, we identified all of the glutamine tandem repeats containing four or more glutamines in human proteins and then estimated the nonsynonymous (dN) and synonymous (dS) substitution rates for the regions flanking the glutamine tandem repeats and the proteins containing them. The results indicated that most of the proteins containing polyglutamine (polyQ) tracts of four or more glutamines have undergone purifying selection, and that the purifying selection for the regions flanking the repeats is weaker. Additionally, we observed that the conserved repeats were under stronger selection constraints than the nonconserved repeats. Interestingly, we found that there was a higher level of purifying selection for the regions flanking the polyQ tracts encoded by pure CAG codons compared with those encoded by mixed codons. Based on our findings, we propose that selection has played a more important role than was previously speculated in constraining the expansion of polyQ tracts encoded by pure codons.
Citation: Li H, Liu J, Wu K, Chen Y (2012) Insight into Role of Selection in the Evolution of Polyglutamine Tracts in Humans. PLoS ONE 7(7): e41167. https://doi.org/10.1371/journal.pone.0041167
Editor: Reiner Albert Veitia, Institut Jacques Monod, France
Received: March 16, 2012; Accepted: June 18, 2012; Published: July 25, 2012
Copyright: © 2012 Li et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Funding: The authors have no support or funding to report.
Competing interests: The authors have declared that no competing interests exist.
Introduction
Tandem repeats of single amino acids are very common in eukaryotic proteins [1], [2]. Most of the single amino acid repeats (AARs) are embedded in intrinsically unstructured regions (IURs) that do not form compact structures under normal solvation conditions [3]–[5]. Using X-ray crystallography, Kim et al. recently reported that the secondary structure of a poly17Q region in the huntingtin protein adopts multiple conformations [6]. Numerous studies have reported that variation in the repeat size is associated with morphological evolution or changes in animals [7], [8] and certain neurological, developmental, and neuromuscular disorders in humans [9], [10].
Polyglutamine (polyQ) tracts are the most intensively studied type of AAR because several neurodegenerative disorders result from the expansion of the polyQ tracts in human proteins [9], [11], [12]. Functionally, polyQ tracts may play a role in the activation of transcription; therefore, their expansion may disrupt the transcription of the gene [13]. The most typical example is the mutant huntingtin protein, which contains expanded polyQ tracts that disrupts specific Sp1-mediated transcription [13], [14]. This expansion leads to early deleterious consequences in the brain in the form of Huntington’s disease [14]. Moreover, polyQ tracts may stabilize protein-protein interactions [15] and function as polar zippers [16].
However, the mechanism that drives the production of amino acid tandem repeats remains unclear. One hypothesis is that the selection is unrelated to the amino acid repeats and that the different rates are merely the consequence of different protein functions and CG contents [17]. Yet, studies on the correlation between the expansion of amino acid repeats and the rate of nonsynonymous substitution support a role for selection in their expansion [5], [18], [19]. Additionally, Mularoni et al. [20] found that selection has played an important role in the evolution of amino acid repeats by increasing the repeat retention rate and modulating the repeat size.
In an analysis of the role of selection in coding CAG repeats in humans, Hancock et al. [19] proposed that slippage produces polyQ tracts and that selection and point mutations modulate their expansion. Additionally, comparative genomic studies of polyQ tracts also showed that slippage plays an important role in shaping these tracts in humans [21], [22]. Nevertheless, the role of natural selection in glutamine tandem repeat evolution is still not well understood, particularly for the repeats encoded by pure CAG codons.
Knowledge of the patterns of nucleotide substitution is important for an understanding of the dynamics of molecular evolution because mutation and selection have various effects on the synonymous (dS) and nonsynonymous (dN) substitution rates [23], [24], and the estimation of these rates provides insight into the role of selection in the evolution of polyQ tracts. In this study, we identified all of the polyQ tracts of four or more glutamine residues in the human genome using phylogenetic analysis by maximum likelihood (PAML) [25] to estimate the dN, dS and dN/dS ratios for the regions flanking the polyQ tracts and the proteins containing these tracts. The results showed that selection has played a more important role than was previously speculated in constraining the expansion of the polyQ tracts encoded by pure codons in humans.
Materials and Methods
Repeat Identification
Protein sequences containing polyQ tracts of four or more glutamines were downloaded from the Ensembl database (http://www.ensembl.org/human, Version 60). Only the longest protein product of each gene was used in the analysis. We developed an in-house PERL script to locate repeats in the protein sequences and mapped the protein sequences to the DNA sequences to identify their corresponding codons. We performed an alignment using the tblastn program of BLAST to locate the codons, and manually checked for proteins that did not perfectly match their corresponding cDNA sequences.
dN/dS Calculation
Human orthologous genes in the mouse and rat genomes were downloaded from the Ensembl database, and only the longest protein/transcript of each gene was used in the analysis. The orthologous protein alignment generated by MAFFT (version 5) was transformed into a corresponding coding sequence alignment by an in-house PERL script. In our study, all of the dN/dS calculations were performed using human-mouse-rat orthologs (Ortholog group alignment in Information S1). The average dN/dS ratios were calculated using the maximum likelihood-based program yn00 in the PAML software package [25], based on the CDS alignments. This program implements the method of Yang and Nielsen [26] to estimate dN and dS in pairwise comparisons of proteins-coding DNA sequences. Unless otherwise noted, the dN and dS for the regions flanking the polyQ tracts were calculated for regions of an arbitrary length of 33 codons upstream and downstream of the repeats [19], [27]. It must be noted that other polyQ tracts were excluded from estimates of the dN and dS for a region flanking a single polyQ tract. A dN/dS ratio of less than one implies purifying (stabilizing) selection. All statistical tests were performed using SPSS (Statistical Package for the Social Sciences) Statistics 17.0.
Results
The General Organization of PolyQ Tracts in Humans
In several disease-linked proteins, the typical repeat length of the polyQ tracts is four or longer [9]. Therefore, we searched the Ensembl database for polyQ tracts. A total of 618 polyQ tracts with 4 or more residues were identified in 454 different human proteins, including nine known neurological disease-linked proteins (Information S2).
Several general organizational trends were apparent across all of the tracts. First, we observed that the number of polyglutamine tracts decreased dramatically with an increase in the size of the repeats. The observed distribution roughly fits a power law curve (Figure 1). The largest human polyQ tract contains 40 tandem glutamines, whereas the largest tracts in mice and rats are 46 and 94, respectively. Secondly, at the genomic level, only a single tract of 4 glutamines was encoded by pure CAA codons, whereas the number of polyQ tracts encoded by pure CAG codons dropped sharply when the length of the polyQ tracts increased (Figure 1). Additionally, we found that the polyQ tracts were not well conserved evolutionarily. A total of 98 polyQ tracts in human proteins were absent in the mouse and rat orthologs, and only 141 polyQ tracts, accounting for 23% of the total polyQ tracts in human proteins, were fully conserved in the orthologous proteins in mice and rats.
We identified 312, 101, 126, and 89 glutamine repeats of 4, 5, 6–9, and 10–40 residues, respectively (blue column). The numbers of glutamine repeats encoded by pure CAG codons in each size bin are 160, 41, 39, and 8, respectively (red column).
A previous study showed that conserved polyQ tracts were encoded by mixed codons in humans [28]. Conversely, we found that a portion of the glutamine tandem repeats in human proteins that were 80–100% conserved in mice and rats were encoded by pure CAG codons. Additionally, nine neurological disorder-associated polyQ tracts [9] showed extreme variability between humans and rodents.
Different Evolutionary Rates for the Regions Flanking the Repeats and the Proteins Containing the Repeats
Several authors have reported that there are weaker selection constraints for regions flanking repeats than the remainder of the protein containing them [18], [19]. Thus, we investigated whether there was a difference in the evolutionary rates between the regions flanking the tracts of four or more glutamines and the proteins containing them in humans. In this case, the dN or dS for the entire protein included the repeat, and the dN or dS for a region flanking the repeat excluded the repeat. The dN/dS ratio was taken as an indicator of the strength of purifying selection. We calculated the dN/dS ratios for 392 proteins containing polyQ tracts and 480 regions flanking the tracts (Information S3). The average dN/dS ratio of all of the proteins tested was 0.139. Most of the dN/dS ratios were less than 0.90, but one protein, C9orf144B, had a dN/dS ratio greater than 1.0 (Figure 2, pink line). These results indicated that most proteins containing polyQ tracts had undergone purifying selection.
The dN/dS ratios for more than 80% of the proteins containing polyQ tracts and more than 60% of the regions flanking the tracts are under 0.2.
As shown in Figure 2 (blue line), the dN/dS ratios for 98% of the regions flanking the tracts were less than 1.0, which indicates that most of the regions had also undergone purifying selection. However, the average dN/dS ratio for the regions flanking the tracts was significantly higher than that for the proteins containing the tracts (P<0.001, two-tailed Mann-Whitney U-test), presumably reflecting a weaker purifying selection for the regions flanking the repeats.
To understand the relationship between the dN/dS ratio and polyQ tract length, we compared the dN/dS ratios for the regions flanking the polyQ tracts of different repeat sizes. The tracts were divided into three different groups. The first group of tracts included tracts of 4 or 5 glutamines, the second group consisted of tracts of 6–9 glutamines, and the third group comprised tracts of 10 or more glutamines. The average dN/dS ratio for the regions flanking the first group of tracts was lower than that for the regions near the second group and higher than that for the regions near the third group, but the average dN/dS ratio for first and second, third groups exhibited no significant difference (Figure 3). These results indicated that there is no positive correlation between the dN/dS ratios of the regions flanking the polyQ tracts and the tract length.
The tracts were divided into three different groups. The first group of tracts included tracts of 4 or 5 glutamines, the second group consisted of tracts of 6–9 glutamines, and the third group comprised tracts of 10 or more glutamines.
Additionally, we found that the average dN/dS ratio for the C-terminal regions flanking the polyQ tracts is significantly higher than that for the N-terminal flanking regions (P = 0.001, two-tailed Mann-Whitney U-test) (Information S4), reflecting greater selection constraints on the N-terminal flanking regions.
Highly Variable Versus Highly Conserved Repeats
The analysis of the repeat size variability between humans and rodents had shown a low degree of conservation of the repeats in orthologous proteins. Therefore, we addressed whether there was any association between repeat conservation and the evolutionary rate. We investigated the difference in the evolutionary rates between two groups of proteins containing either highly conserved or highly variable polyQ tracts by estimating the dN and dS ratios for these two groups of proteins.
The highly conserved (HC) group included human proteins containing glutamine tandem repeats that were 80–100% conserved in their mouse and rat orthologs. In contrast, the highly variable (HV) group comprised human proteins containing glutamine tandem repeats that were 0–40% conserved in their orthologous mouse and rat proteins. We estimated the dN/dS ratios for the HC and HV proteins and found that the average dN/dS for the HC group was significantly lower than that for the HV group (Table 1) (P<0.001, two-tailed Mann-Whitney U-test). In the HV group, the average dN/dS ratio for the regions flanking the repeats was significantly higher than that in the HC group (Table 2) (P<0.001, two-tailed Mann-Whitney U-test). These results reflected a weaker purifying selection for the regions near the repeats in the HV group. Additionally, eight proteins encoded by genes that are associated with neurodegenerative diseases in humans were included in the HV group.
Lower Average dN/dS Ratios were Observed for the PolyQ Tracts Encoded by Pure CAG Codons than for the PolyQ Tracts Encoded by Mixed Codons
To understand the role of natural selection in the evolution of polyQ tracts, it is crucial to investigate whether a difference in selection constraints exists between the polyQ tracts encoded by pure CAG codons and those encoded by mixed codons; tandem CAG repeats are prone to slippage, whereas interrupted structures are less likely to undergo slippage [21]. By estimating the dN and dS, we found that the average dN/dS ratio for the proteins containing polyQ tracts encoded by pure CAG codons was significantly lower than that for the proteins containing polyQ tracts encoded by mixed codons (Table 1) (P<0.001, two-tailed Mann-Whitney U-test). This result indicated a stronger purifying selection for the proteins containing polyQ tracts encoded by pure CAG codons.
Surprisingly, these results showed that the average dN/dS ratio for the regions flanking polyQ tracts encoded by mixed codons was significantly higher than that for the regions flanking polyQ tracts encoded by pure CAG codons (Information S5,Table 2) (P = 0.011, two-tailed Mann-Whitney U-test). This result indicated there was a higher level of purifying selection for the regions flanking polyQ tracts encoded by pure CAG codons compared with those encoded by mixed codons.
To further investigate whether the dN/dS ratio for the regions flanking polyQ tracts is correlated with the percentage of CAG codons, the tracts were divided into three different groups based on their percentage of CAG codons. The first group of tracts consisted of pure CAG codons, the second group consisted of more than 50% CAG codons, and the third group was consisted of less than or equal to 50% CAG codons. The results indicated that the average dN/dS ratio for the regions flanking the first and second groups was significantly lower than that for the regions near the third group; however, the average dN/dS ratio for first and second groups exhibited no significant difference (Figure 4). Additionally, the conserved polyQ tracts contained some members of the first group and a large proportion of the second group but no representatives from the third group.
The labels 0–0.5, 0.5–1 and 1 indicate less than or equal to 50%, more than 50% but less than 100%, and 100% of CAG or CAA codons, respectively. Only one tract of 4 glutamines was encoded by pure CAA codons, so only two groups were encoded by more than 50% and by less than or equal to 50% CAA.
A large number of 4–5Q tracts encoded by pure codons are highly conserved, but conserved polyQ tracts of 10 or more glutamines are all encoded by mixed codons.
To exclude the possibility that CAA richness is associated with a high dN/dS ratio, we investigated the relationship between the percentage of CAA codons and the dN/dS ratio. The result showed that there was no significant difference between the average dN/dS ratio for the regions flanking the tracts encoded by more than 50% CAA codons and that for the tracts encoded by less than or equal to 50% CAA codons (Figure 4). This finding excluded the possibility that CAA richness is the factor that is associated with the high dN/dS ratio for the regions flanking the tracts in the third group.
Discussion
In this study, we found that most of the human proteins containing polyQ tracts of four or more glutamines have undergone purifying selection (Figure 2). The fact that the dN/dS ratio for the regions flanking the tracts is higher than that for the proteins containing them suggests that the regions flanking the tracts evolve more quickly than the remainder of the protein containing them, consistent with the findings of previous studies [5], [19], [27]. The weak purifying selection observed for the regions near the repeats may imply the action of slippage in the repeats.
Further analysis reveals that there is a weaker purifying selection for the regions near the nonconserved polyQ tracts than for the regions flanking the conserved tracts. In other words, the regions flanking the conserved repeats are under stronger selection constraints than the regions flanking the nonconserved repeats, which is consistent with some previous studies [18], [19]. These data suggest that the nonconserved tracts lie in regions that experience lower purifying selection.
Interestingly, we found dramatic differences between the polyQ tracts encoded by pure CAG and mixed codons. Our results showed stronger selection constraints for the proteins containing the polyQ tracts encoded by pure CAG codons. Furthermore, our analysis indicated that the average dN/dS ratio for the regions flanking the polyQ tracts encoded by mixed codons is significantly higher than that for the regions flanking the polyQ tracts encoded by pure CAG codons, suggesting that the regions near impure CAG codons evolve faster than the regions near pure CAG codons. However, a previous study showed that conserved polyglutamine tracts usually are encoded by mixed codons, whereas rapidly evolving tracts are encoded primarily by pure CAG codons [28]. A comparative analysis showed that proteins containing conserved repeats had lower evolutionary rates than did proteins with nonconserved repeats [18], [19].
The discrepancy between the previous and present studies could be due to the different data sets used. We identified all of the polyQ tracts of four or more glutamines in humans and then estimated the dN/dS ratios for the regions flanking the polyQ tracts and the proteins containing them to investigate the molecular evolution of the tracts. In previous studies, however, short tracts of 4 or 5 glutamines were excluded from the analyses [18], [19], [28]. We found that the normal repeat length of the polyQ tracts in several disease-linked proteins is four or longer [9], indicating that tracts of 4 or 5 repeats in length should be included in a comprehensive analysis. However, the polyQ tracts of 4 or 5 residues contain a large amount of the conserved tracts and tracts encoded by pure codons, which would result in overlap with regard to the relative proportion of each. In other words, a large number of the 4–5Q tracts encoded by pure codons are conserved (Figure 5), which may result in a bias between the previous and present analyses.
Additionally, a previous study found that polyQ tracts are associated with specific sequence biases [29]. To exclude the possibility that other amino acid repeats in the regions flanking the polyQ tracts introduced biases in our analysis, we identified all of the amino acid repeats of 4 residues or longer in the regions flanking the pure CAG- and mixed codon-encoded polyQ tracts and then analyzed their dN/dS ratios (Information S6). We found 23 and 41 amino acid repeats of 4 residues or longer in these regions, respectively, and determined that most of the repeats are located in the C-terminal regions flanking the polyQ tracts. Our analysis indicated that the average dN/dS ratio for the regions flanking the polyQ tracts encoded by mixed codons is also significantly higher than that for the regions flanking the polyQ tracts encoded by pure CAG codons (P = 0.009, two-tailed Mann-Whitney U-test) when the regions containing other amino acid repeats were excluded. This result indicated that the presence of other amino acid repeats in the regions flanking the polyQ tracts do not negatively affect our analysis.
Polyglutamine tracts are the most intensively studied type of amino acid tracts because expansions of these tracts in several human proteins can cause neurological disorders [9], [11], [12]. The largest class of neurological diseases results from the expansion of CAG repeats within exons. We found that there is a stronger purifying selection for the regions flanking the pure repeats and the protein containing them. We also observed that most of the longer polyQ tracts are encoded by mixed codons (Figure 1). Thus, our results suggest that selection has played a more important role than was previously speculated in constraining the expansion of the tracts encoded by pure CAG codons. This finding could help explain why only a few proteins containing polyQ tracts encoded by pure CAG codons massively expand to result in human disorders.
In conclusion, by estimating the synonymous and nonsynonymous rates of evolution for the regions flanking the tracts and the proteins containing them, our observations support the hypothesis that selection is involved in the evolution of polyQ tracts by analyzing the divergent levels of purifying selection experienced by different groups of repeats.
Supporting Information
Information S1.
Ortholog Group Alignment in Phylip format.
https://doi.org/10.1371/journal.pone.0041167.s001
(DOC)
Information S2.
A total 618 ployQ tracts of four or more glutamines in huamn proteins.
https://doi.org/10.1371/journal.pone.0041167.s002
(XLS)
Information S3.
dN and dS rates for all regions flanking ployQ tracts and for all proteins containing them.
https://doi.org/10.1371/journal.pone.0041167.s003
(XLS)
Information S4.
dN and dS rates for polyQ tracts’ N and C-terminal flanking regions.
https://doi.org/10.1371/journal.pone.0041167.s004
(XLS)
Information S5.
dN and dS rates for regions flanking polyQ tracts encoded by pure CAG and mixed codons.
https://doi.org/10.1371/journal.pone.0041167.s005
(XLS)
Information S6.
dN and dS rates for regions in which there are other amino acid repeats flanking polyQ tracts encoded by pure CAG and mixed codons.
https://doi.org/10.1371/journal.pone.0041167.s006
(XLS)
Acknowledgments
We thank Professor Changxin Wu of the College of Animal Science and Technology, China Agricultural University, for supporting the study. We thank Professor Ziding Zhang of the College of Biological Sciences, China Agricultural University, for suggestions on the study. We thank Yuan Zhou, a Ph.D. student in the College of Biological Sciences, China Agricultural University, for the estimations of dN and dS. We thank Dr. John M. Hancock of the Bioinformatics Group, MRC Harwell, Harwell, Oxfordshire, OX11 0RD, UK, for suggestions on the study.
Author Contributions
Conceived and designed the experiments: HL. Performed the experiments: HL. Analyzed the data: HL JL. Contributed reagents/materials/analysis tools: KW YC. Wrote the paper: HL.
References
- 1. Green H, Norman W (1994) Codon reiteration and the evolution of proteins. Proc. Natl .Acad .Sci. USA 91: 4298–4302.
- 2. Golding GB (1999) Simple sequence is abundant in eukaryotic proteins. Protein Science 8: 1358–1361.
- 3. Wright PE, Dyson HJ (1999) Intrinsically unstructured proteins: reassessing the protein structure-function paradigm. Journal of Molecular Biology 293: 321–331.
- 4. Brown CJ, Takayama S, Campen AM, Vise P, Marshall TW, et al. (2002) Evolutionary rate heterogeneity in proteins with long disordered regions. Journal of Molecular Evolution 55: 104–110.
- 5. Simon M, Hancock JM (2009) Tandem and cryptic amino acid repeats accumulate in disordered regions of proteins. Genome Biology 10: R59.
- 6. Kim MW, Chelliah Y, Kim SW, Otwinowski Z, Bezprozvanny I (2009) Secondary structure of Huntingtin amino-terminal region. Structure 17(9): 1205–1212.
- 7. Fondon JM, Harold R G (2004) Molecular origins of rapid and continuous morphological evolution. Proc Natl Acad Sci USA 101: 18058–18063.
- 8. Keitti A, Nobuaki Yoshida, Yuki Kataoka, Mitsuharu Sato, Hirotake Ichise, et al. (2007) Morphological change caused by loss of the taxon-specific ployalanine tract in hoxd-13. Molecular Biology and Evolution 24: 281–287.
- 9. Orr HT, Zoghbi HY (2007) Trinucleotide repeat disorders. Annu.Rev.Neurosci 30: 575–621.
- 10. Albrecht A, Mundlos S (2005) The other trinucleotide repeat: polyalanine expansion disorders. Current Opinion in Genetics & Development 15: 285–293.
- 11. Huntington’s Disease Collaborative Research Group (1993 ) A novel gene containing a trinucleotide repeat that is expanded and unstable on Huntington’s disease chromosomes. Cell 72: 971–983.
- 12. Karlin S, Burge C (1996) Trinucleotide repeats and long homopeptides in genes and proteins associated with nervous system disease and development. Proc Natl Acad Sci USA 93: 1560–1565.
- 13. Freiman RN, Tjian R (2002) A glutamine-rich trail leads to transcription factors. Science 296: 2149–2150.
- 14. Dunah AW, Jeong H, Griffin A, Kim YM, Standaert DG, et al. (2002) Sp1 and TAFII130 transcriptional activity disrupted in early Huntington’s disease. Science 296: 2238–2243.
- 15.
Schaefer MH, Wanker EE, Andrade-Navarro MA (2012) Evolution and function of CAG/polyglutamine repeats in protein–protein interaction networks. Nucleic Acids Research. January 28.[Epub ahead of print] PMID. 22287626 p.
- 16. Perutz MF, Johnson T, Suzuki M, Finch JT (1994) Glutamine repeats as polar zippers: their role in inherited neurodegenerative disease. Proc. Natl. Acad. Sci. USA 91: 5335–5358.
- 17. Cruz F, Roux J, Robinson-Rechavi M (2009) The expansion of amino-acid repeats is not associated to adaptive evolution in mammalian genes. BMC Genomics 2009 10: 619.
- 18. Mularoni L, Veitia RA, Albà MM (2007) Highly constrained proteins contain an unexpectedly large number of amino acid tandem repeats. Genomics 89: 316–325.
- 19. Hancock JM, Elizabeth A W, Santibanez-Koref MF (2001) A Role for Selection in Regulating the Evolutionary Emergence of Disease-Causing and Other Coding CAG Repeats in Humans and Mice. Molecular Biology and Evolution 18: 1014–1023.
- 20. Mularoni L, Alice L, Toll-Riera M, Albà MM (2010) Natural selection drives the accumulation of amino acid tandem repeats in human proteins. Genome Research 20: 745–754.
- 21. Albà MM, Santibanez-Koref MF, Hancock JM (2001) The comparative genomics of polyglutamine repeats: Extreme differencein the codon organization of repeat-encoding regions between mammals and Drosophila. Journal of Molecular Evolution 52: 249–259.
- 22. Albà MM, Guigo R (2004) Comparative analysis of amino acid repeats in rodents and humans. Genome Research 14: 549–554.
- 23. Ziheng Yang (1994) Estimating the pattern of nucleotide substitution. Journal of Molecular Biology 39: 105–111.
- 24. Ziheng Yang (1998) Likelihood Ratio Tests for Detecting Positive Selection and Application to Primate Lysozyme Evolution. Molecular Biology and Evolution 15: 568–573.
- 25.
Ziheng Yang (1997) Phylogenetic analysis by maximum likelihood (PAML), version 1.3. University of California, Berkeley, California, USA.
- 26. Ziheng Yang, Nielsen R (2000) Estimating nonsynonymous and synonymous substitution rates under realistic evolutionary models. Molecular Biology and Evolution 17: 32–43.
- 27. Faux NG, Gavin AH, Khalid M, Geoffrey IW, et al. (2007) RCPdb: An evolutionary classification and codon usage database for repeat-containing proteins. Genome Research 17: 1118–1127.
- 28. Albà MM, Santibanez-Koref MF, Hancock JM (1999) Conservation of polyglutamine tract size between mice and humans depends on codon interruption. Molecular Biology and Evolution 16: 1641–1644.
- 29. Matteo R, Elodie M, Choumouss K, Donatella D, Ronald M (2009) Polyglutamine Repeats Are Associated to Specific Sequence Biases That Are Conserved among Eukaryotes. PLoS ONE 7(2): e30824.