Gene Conversion and GC-Content Evolution in Mammalian Hsp70

Grzegorz Kudla*, Aleksandra Helwak{dagger},* and Leszek Lipinski{dagger},*

* International Institute of Molecular and Cell Biology
{dagger} Institute of Biochemistry and Biophysics, Warsaw, Poland

Correspondence: E-mail: grzesiek{at}iimcb.gov.pl.

Abstract

To investigate the mechanisms regulating the nucleotide usage in mammalian genes, we analyzed the sequences of three physically linked Hsp70 paralogs in human and mouse. We report that the sequences of HSPA1A and HSPA1B genes are almost identical, whereas the HSPA1L gene contains some regions very similar to HSPA1A/B and some regions with much higher divergence. Phylogenetic analysis reveals that gene conversion has homogenized the entire coding regions of HSPA1A/B and several fragments of HSPA1L. The regions undergoing conversion are all very GC rich, contrarily to the regions not subject to conversion. The pattern of nucleotide substitution in mammalian orthologs suggests that the mechanism increasing the GC content is still functioning. To test the possibility that the high GC content facilitates the expression of Hsp70 during heat-shock, we performed in vitro translation experiments. We failed to detect any effect of GC content on the translation efficiency at high temperatures. Taken together, our data strongly support the biased gene conversion hypothesis of GC-content evolution.

Key Words: GC content • gene conversion • heat-shock

Introduction

The variability of nucleotide composition is one of the most mysterious characteristics of mammalian genomes. Bernardi et al. (1985) performed density gradient centrifugations of sheared mammalian DNA and observed several fractions with distinct average densities. The fractions were found to correspond to DNA fragments with different average GC contents, and the corresponding genome regions were called isochores. Although the question whether the isochores are truly "iso" has recently raised much controversy (Bernardi 2001; Lander et al. 2001; Li et al. 2003), there is no doubt that mammalian genomes are far from homogenous and that we do not well understand why it is so.

In the prokaryotic genomes, the main source of GC-content heterogeneity is the presence of highly expressed genes with strongly biased codon usage. Many studies have shown that the use of optimal codons is a decisive factor determining the level of gene expression in bacteria, yeast, flies, and worms and that genes requiring high translation levels are forced by selection to adopt a particular set of codons (Gouy and Gautier 1982; Grosjean and Fiers 1982; Sharp and Li 1986; Bulmer 1987; Powell and Moriyama 1997; Duret and Mouchiroud 1999).

The situation is more complicated in mammals. The GC content of large genome fragments (isochores) ranges from 30% to 60%, and the GC content at the third codon positions of genes (GC3) ranges from 25% to more than 90% (Bernardi 1995). Unlike in lower organisms, no clear correlation has been found between the codon usage of genes and their expression levels. Consequently, several other hypotheses have been put forward to explain the origin of GC-rich isochores and genes in mammals. For example, based on the observation that the genomes of homeothermic vertebrates have more GC-rich isochores than the poikilothermic ones, it has been proposed that the isochore structure of mammalian genomes is an adaptation to higher body temperatures (Bernardi et al. 1985). Although some data from genomic sequence analysis support the selective hypotheses of mammalian GC-content evolution (Zoubak et al. 1995; Hughes and Yeager 1997; Eyre-Walker 1999), most recent analyses argue against the thermal stability version of the selectionist view (Hughes, Zelus, and Mouchiroud 1999; Hamada et al. 2003; Ream, Johns, and Somero 2003).

An alternative set of hypotheses proposes that the GC content of mammalian isochores and the codon usage of genes have no selective meaning. The neutral factors that were proposed to account for the GC-content variation include mutation bias and biased gene conversion (BGC) (Sueoka 1988; Wolfe, Sharp, and Li 1989; Holmquist 1992; Eyre-Walker 1993; Eyre-Walker and Hurst 2001; Birdsell 2002). The latter theory, which has recently gained much interest (Galtier 2003; Marais 2003; Montoya-Burgos, Boursot, and Galtier 2003), states that high GC content is a consequence of a GC-biased repair of mismatches during recombination. If this theory is true, then frequently recombining genes, as well as those undergoing concerted evolution by gene conversion, should experience an increase of GC content. Indeed, it is known that GC content correlates with the local recombination rate (Eyre-Walker 1993). There is also evidence that the GC content of histone genes covaries with the number of their close paralogs in the genome, potentially indicative of gene conversion (Galtier 2003). However, the evidence for gene conversion of the histone genes is rather indirect. A model locus was needed that provided good evidence for gene conversion and in which some genes or gene fragments were known not to undergo conversion. Such a locus would enable the direct investigation of the relationship between conversion and GC content.

Here we investigate a triplet of closely linked mammalian Hsp70-family genes. Two of the genes undergo frequent conversions, and they are more GC-rich than the third gene, which is only subject to partial conversions. This supports the BGC hypothesis of GC-content evolution. Interestingly, the Hsp70-family genes are differentially expressed at high temperatures, providing an interesting framework to test the thermal hypothesis of isochore evolution. Our initial experiments suggest that high GC content is not required for the efficient translation of mammalian genes at high temperatures.

Materials and Methods

Phylogenetic Analyses
The following sequences were used in this study: human (Homo sapiens) HSPA1A (M59828), HSPA1B (M59830), HSPA1L (D85730); mouse (Mus musculus) Hspa1a (M76613), Hspa1b (M35021), Hspa1l (M32218); rat (Rattus norvegicus) Hspa1a (X77207); Hspa1b (X77208); Hspa1l (X77209); pig (Sus scrofa) HSP70 (M69100); and bovine (Bos taurus) HSP70-2 (U02892). The sequences were aligned using ClustalW at http://www.ebi.ac.uk/clustalw/ with the default parameters. Further analyses were performed on the coding regions of the genes using the MEGA2 package (Kumar et al. 2001). The synonymous and nonsynonymous substitution rates were calculated using the Nei and Gojobori method (Nei and Gojobori 1986). The phylogenetic trees were built using the neighbor-joining method, using the Tamura's three-parameter distance measure (Tamura 1992), which corrects for the GC content and transition/transversion rate biases. The bootstrap tests of phylogeny were performed using 500 replicates. To estimate the transition/transversion rate ratios between the HSPA1A orthologs, Tamura's three-parameter model was used.

Nucleotide Usage Analyses
To investigate local GC contents within and around the genes, the genomic fragments containing the human and mouse MHCIII loci were downloaded from the NCBI site (http://www.ncbi.nlm.nih.gov). To compare the local GC content and the local similarity between HSPA1A and HSPA1B, 9 kb of genomic sequence centered at the two genes was aligned using the default settings of ClustalW. The local similarity was estimated as the percentage of nucleotide matches in sliding windows of 100 bp (counting gaps as mismatches), and the local GC content was also measured in 100-bp windows. To investigate the similarity between HSPA1A and HSPA1L, only the coding regions of the genes were used.

In vitro Translation
Human/HSPA1A and HSPA8 were cloned into the pET3c plasmid (Novagen) containing a T7 polymerase promoter and a T7 terminator. After linearization, the plasmids were used as a template for the production of capped mRNA with the T7 Cap Scribe kit (Roche). Equal amounts of mRNA for HSPA1A and HSPA8 were translated for 1 h using the Reticulocyte Translation Kit Type II (Roche) and 35S-labeled Methionine (Amersham Biosciences) in a gradient thermocycler in the temperature range 26°C to 42°C.

Half of the reaction mixture was resolved on a 10% SDS-polyacrylamide gel, dried and exposed overnight to an autoradiography film (Kodak BioMax). The other half was TCA-precipitated on GFC filters (Whatman) according to the protocol enclosed in the Reticulocyte Translation Kit Type II and counted in a liquid scintillation counter.

Results and Discussion

Around 10 genes from the Hsp70 family are present in the genomes of humans and other mammals (Tavaria, Kola, and Anderson 1997). We focused our attention on the three human genes located in the MHCIII locus. Two of those genes, HSPA1A and HSPA1B, are intronless, and their expression is strongly increased in most tissues after heat-shock or other types of stress. The third gene, HSPA1L, possesses one intron, is testis specific, and is constitutively expressed even in the absence of heat-shock. An orthologous triplet of genes also exists in the MHCIII complex of the mouse (fig. 1a) and rat (Walter, Rauh, and Gunther 1994; Tavaria, Kola, and Anderson 1997; Ito et al. 1998;). It is, therefore, assumed that the duplications that led to the formation of the HSPA1A, HSPA1B, and HSPA1L genes must have taken place before the split of rodent and primate lineages.



View larger version (16K):
[in this window]
[in a new window]
 
FIG. 1. Genomic organization and phylogenetic analysis of the mammalian Hspa1 cluster. (a) Chromosomal localisation of Hspa1a, Hspa1b, and Hspa1l genes in human (top) and mouse (bottom). (b) Unrooted phylogenetic tree of the Hspa1 family genes, constructed using the entire coding regions of the genes. Numbers on the tree represent the bootstrap values supporting each node, and the scale bar represents evolutionary distance. (c) Same as (b). Constructed using gene fragments homologous to the nucleotides 583 to 858 of mouse Hspa1a. (d) Same as (b). Constructed using gene fragments homologous to the nucleotides 25 to 174

 
To identify possible gene conversion between HSPA1A, HSPA1B, and HSPA1L, we performed a phylogenetic analysis of the coding sequences of the human, mouse, and rat genes. As shown previously (Walter, Rauh, and Gunther 1994), the HSPA1A and HSPA1B paralogs in each species share more similarity with each other than with their respective orthologs in other species (fig. 1b). This suggests that gene conversion has homogenized the sequences of human HSPA1A/B genes after the primate-rodent split and the sequences of mouse and rat Hspa1a/b genes after the mouse-rat split.

To locate more precisely the regions undergoing conversion between the paralogous genes, we performed pairwise alignments of human (or mouse) genomic regions containing the genes of interest. The results of this analysis are summarized in table 1. Greater than 99% identity exists between HSPA1A and HSPA1B, as well as between mouse Hspa1a and Hspa1b. The regions of high similarity begin around 500 nucleotides upstream from the start of the open reading frames and stop near the end of the translated regions. Assuming an evolutionary rate of 2.5 silent substitutions per site per billion years (Lynch and Conery 2000), we can estimate that the last conversion event between human HSPA1A and HSPA1B took place 2 MYA. Similarly, the last conversion event in the mouse can be dated to 3 MYA. Interestingly, we also observed several regions of high local similarity between the mouse Hspa1a and Hspa1l genes (table 1). The average identity at fourfold degenerate sites between Hspa1a and Hspa1l is 57%, but there are some stretches of 50 to 90 codons with more than 90% identity at fourfold degenerate sites. A similar result was obtained for the human HSPA1A and HSPA1L genes (table 1), as well as for the rat genes (data not shown). When we repeated the phylogenetic analysis using those gene fragments, the conversion between Hspa1a/b and Hspa1l became apparent (fig. 1c and d). Interestingly, we found that similar regions undergo independent conversions in humans and mice (table 1 and fig. 1d). The extent of similarity suggests that the most similar fragments of HSPA1A/B and HSPA1L underwent conversion around 15 MYA. We conclude that frequent gene conversion homogenizes the entire coding regions of HSPA1A and HSPA1B and several fragments of HSPA1A (or HSPA1B) and HSPA1L.


View this table:
[in this window]
[in a new window]
 
Table 1 Pairwise Evolutionary Distances Between the Fragments of MHCIII-linked Hsp70-Family Genes.

 
Despite their high overall similarity, the Hsp70-family genes from the MHCIII locus show dramatic differences in GC content. The human HSPA1A and HSPA1B genes, as well as their mammalian orthologs, have GC contents of more than 90% in the third position of codons (GC3), whereas HSPA1L and its orthologs have average GC3 values around 60% (fig. 2a and b). This difference is clearly not caused by the localization of genes in different isochores, because all three genes are located in the middle of the MHCIII region, which is one of the best-characterized GC-rich isochores and is thought to have a particularly uniform base composition (Oliver et al. 2001; Li et al. 2003).



View larger version (14K):
[in this window]
[in a new window]
 
FIG. 2. GC content of MHC-linked Hsp70-family genes. The solid line represents the genomic GC content, the dashed lines above represent the GC content in the third position of codons. The arrows represent the coding regions of genes. (a) Fragment of human MHCIII. (b) Fragment of mouse MHCIII

 
To investigate the origin of the high GC content of HSPA1A and HSPA1B genes, we first compared the sequences of four mammalian HSPA1A orthologs. We wanted to know whether the mechanism that has increased the GC content of the HSPA1A genes is still functioning in mammals. As shown in table 2, in all the pairwise comparisons of HSPA1A orthologs, we detected a large excess of G-C transversional pairs as compared with other mutations. This effect is most apparent at fourfold degenerate sites, where it causes the apparent transition/transversion ratio to be as low as {kappa} = 0.17 for the human-mouse gene pair. This observation is quite surprising, because it is widely known that transitions usually occur with higher frequencies than transversions in most, if not all, known genes (Yang and Yoder 1999; Bielawski, Dunn and Yang 2000). Rather than to assume that G{leftrightarrow}C transversions occur in mammalian Hspa1a more frequently that any other type of mutation, we propose that the observed pattern is caused by a decreased fixation probability of N->A and N->T mutations. In other words, the excess of G-C transversional pairs suggests that the high GC content is actively and independently maintained in present-day mammalian HSPA1A orthologs. The recent finding that GC-rich isochores in mammals are disappearing (Duret et al. 2002) may, therefore, not be true for all GC-rich genes. A similar effect of increasing GC content was recently described for the mouse Fxy gene (Montoya-Burgos, Boursot, and Galtier 2003).


View this table:
[in this window]
[in a new window]
 
Table 2 Transition/Transversion Rate Ratios and Frequency of G-C Transversional Pairs in Pairwise Comparisons of Mammalian HSPA1A Orthologs.

 
The observed pattern of nucleotide substitutions is compatible both with selective scenarios and with the biased gene conversion hypothesis. To test whether gene conversion or selection accounts for the base composition of the Hsp70-family genes, we plotted the local similarity between the pairs of paralogs and the local GC content along the genes. We reasoned that under BGC, the GC-rich regions would correlate with the regions undergoing conversion. Translational selection, on the other hand, would produce a pattern in which the increased GC content would be limited to the coding sequence. In figure 3a and b, it can be seen that the GC-rich regions around Hspa1a and Hspa1b extend beyond the start of the coding sequence. Furthermore, the uneven pattern of GC content in Hspa1l (fig. 3cd) is not easily explained by selection for translation efficiency or translation accuracy. On the other hand, there is a very strong correlation between the nucleotide composition and the similarity between the genes (fig. 3ad). This is what one would expect if biased gene conversion increased the GC content of Hsp70-family genes. The high GC content of regions undergoing conversion between Hspa1a (or Hspa1b) and Hspa1l suggests that in most conversion events, the GC-rich gene acted as the template. Furthermore, we have estimated the average frequency of conversion between the HSPA1A and HSPA1B genes to be around one whole-gene conversion per 2 to 3 Myr. Thus, the conversions are over 100 times more frequent than mutations, so all new mutations appearing in one of the HSPA1A/B genes are soon subject to conversion. This is an important point, because it has been noted that conversion must be frequent to account for a high GC-content bias (Eyre-Walker 1999).



View larger version (32K):
[in this window]
[in a new window]
 
FIG. 3. Comparison of local GC content and local similarity between genes. (a) The local identity between mouse Hspa1a and Hspa1b genes is in black, and the average local GC content is in red. The bar represents the coding regions of genes. (b) Same as (a) using human HSPA1A and HSPA1B genes. (c) The local identity of the mouse Hspa1a and Hspa1l genes in the third codon positions is in black, and the GC content in the third codon positions of Hspa1l is in red. (d) Same as (c) using human HSPA1A and HSPA1L genes

 
Our results are, thus, compatible with the hypothesis that BGC increases the GC content in mammalian Hsp70, but they do not completely exclude the possibility that the high GC content has some selective meaning. The selective hypothesis is actually very appealing in the case of the heat-inducible Hsp70 because it nicely fits the idea that the GC-rich isochores appeared in homeothermic vertebrates as an adaptation to their higher body temperatures (Bernardi et al. 1985). The high GC content of Hsp70 could possibly play a role in the regulation of translation efficiency or fidelity at increased temperatures. However, if the extreme GC content of those genes were really caused by selection on translation efficiency, then every single mutation from an AT pair to a GC pair should increase the translation rate strongly enough to affect the fitness of the organism. As genes with low and high GC contents differ by hundreds of such nucleotide pairs, we expected the difference in their translation rates to be detectable in experimental conditions. To investigate the possibility that the high GC content of HSPA1A and HSPA1B facilitates their expression at elevated temperatures, we performed in vitro translation experiments in rabbit reticulocyte lysates. We compared the translation rates of the HSPA1A (GC3 = 92%) and HSPA8 (GC3 = 46%) genes, which share 85% identity in the amino acid sequence. At the optimal temperature for translation (28°C), the translation rates of both genes were fairly similar. Increasing the temperature to 42°C in steps of 2°C led to a gradual decrease of the amounts of both protein products, but we failed to observe any positive effect of GC content on the translation efficiencies (fig. 4). This result is perhaps not surprising, given the scarcity of published information on the effect of GC content on the translation efficiency in mammals.



View larger version (24K):
[in this window]
[in a new window]
 
FIG. 4. Influence of GC content and temperature on the in vitro translation rates of Hsp70-family genes. Human HSPA1A (GC3 = 92%) and human HSPA8 (GC3 = 46%) were in vitro translated in rabbit reticulocyte lysates for 1 hour at the indicated temperatures. The radioactive protein products were quantified by (a) SDS-PAGE followed by autoradiography or by (b) scintillation counting. In (b), the amount of each product was normalized to its yield at 26°C. White circles = HSPA8; black squares = HSPA1A. Each picture is representative of three independent experiments

 
It is conceivable that selection for DNA stability, efficiency of transcription, RNA transport, or perhaps RNA stability may increase the GC content of mammalian Hsp70. Further experiments are needed to elucidate these issues, but several arguments seem to counter these possibilities. First, the BGC mechanism is enough to explain the increased GC content of mammalian Hsp70, therefore, we do not need to invoke selective constraints. Second, although it was previously thought that thermophilic organisms are necessarily GC-rich, recent analyses show a lack of correlation between the preferred temperatures and the genomic GC content in prokaryotes (Galtier and Lobry 1997; Hurst and Merchant 2001) and vertebrates (Ream, Johns, and Somero 2003). This suggests that high GC content may not be required for the efficient expression of heat-shock genes. Indeed, the GC contents of Hsp70 homologs in lower organisms are usually similar to the average GC contents of their genomes and several mammalian heat-inducible genes, such as Hsp90, are not GC-rich (data not shown). Taken together, our data suggest that biased gene conversion is the primary mechanism that drives the high GC content in mammalian MHC-linked Hsp70. Because BGC has been recently implicated in the evolution of GC content in histones (Galtier 2003) and in the mouse Fxy gene (Montoya-Burgos, Boursot, and Galtier 2003), it is probable that biased gene conversion is the major mechanism responsible for the formation of GC-rich genes in mammals.

Acknowledgements

We thank M. Zylicz, M. Bochtler, E. Bartnik, R. I. Morimoto, J. M. Bujnicki, M. Cheetham, and the people from the M. Zylicz lab and from the Polish Children's Fund for helpful discussions. G.K. is the recipient of a scholarship from the Postgraduate School of Molecular Medicine affiliated with the Medical University of Warsaw. This work was supported by the State Committee for Scientific Research Grant number 6P04A4219 and the Foundation for Polish Science.

Footnotes

Manolo Gouy, Associate Editor Back

Literature Cited

    Bernardi, G. 1995. The human genome: organization and evolutionary history. Annu. Rev. Genet. 29:445-476.[CrossRef][ISI][Medline]

    Bernardi, G. 2001. Misunderstandings about isochores. Part 1. Gene 276:3-13.[CrossRef][ISI][Medline]

    Bernardi, G., B. Olofsson, J. Filipski, M. Zerial, J. Salinas, G. Cuny, M. Meunier-Rotival, and F. Rodier. 1985. The mosaic genome of warm-blooded vertebrates. Science 228:953-958.[ISI][Medline]

    Bielawski, J. P., K. A. Dunn, and Z. Yang. 2000. Rates of nucleotide substitution and mammalian nuclear gene evolution: approximate and maximum-likelihood methods lead to different conclusions. Genetics 156:1299-1308.[Abstract/Free Full Text]

    Birdsell, J. A. 2002. Integrating genomics, bioinformatics, and classical genetics to study the effects of recombination on genome evolution. Mol. Biol. Evol. 19:1181-1197.[Abstract/Free Full Text]

    Bulmer, M. 1987. Coevolution of codon usage and transfer RNA abundance. Nature 325:728-730.[CrossRef][ISI][Medline]

    Duret, L., and D. Mouchiroud. 1999. Expression pattern and, surprisingly, gene length shape codon usage in Caenorhabditis, Drosophila, and Arabidopsis. Proc. Natl. Acad. Sci. USA 96:4482-4487.[Abstract/Free Full Text]

    Duret, L., M. Semon, G. Piganeau, D. Mouchiroud, and N. Galtier. 2002. Vanishing GC-rich isochores in mammalian genomes. Genetics 162:1837-1847.[Abstract/Free Full Text]

    Eyre-Walker, A. 1993. Recombination and mammalian genome evolution. Proc. R. Soc. Lond. B Biol. Sci. 252:237-243.[ISI][Medline]

    Eyre-Walker, A. 1999. Evidence of selection on silent site base composition in mammals: potential implications for the evolution of isochores and junk DNA. Genetics 152:675-683.[Abstract/Free Full Text]

    Eyre-Walker, A., and L. D. Hurst. 2001. The evolution of isochores. Nat. Rev. Genet. 2:549-555.[CrossRef][ISI][Medline]

    Galtier, N. 2003. Gene conversion drives GC content evolution in mammalian histones. Trends Genet. 19:65-68.[CrossRef][ISI][Medline]

    Galtier, N., and J. R. Lobry. 1997. Relationships between genomic G+C content, RNA secondary structures, and optimal growth temperature in prokaryotes. J. Mol. Evol. 44:632-636.[ISI][Medline]

    Gouy, M., and C. Gautier. 1982. Codon usage in bacteria: correlation with gene expressivity. Nucleic Acids Res. 10:7055-7074.[Abstract]

    Grosjean, H., and W. Fiers. 1982. Preferential codon usage in prokaryotic genes: the optimal codon-anticodon interaction energy and the selective codon usage in efficiently expressed genes. Gene 18:199-209.[CrossRef][ISI][Medline]

    Hamada, K., T. Horiike, H. Ota, K. Mizuno, and T. Shinozawa. 2003. Presence of isochore structures in reptile genomes suggested by the relationship between GC contents of intron regions and those of coding regions. Genes Genet. Syst. 78:195-198.[CrossRef][ISI][Medline]

    Holmquist, G. P. 1992. Chromosome bands, their chromatin flavors, and their functional features. Am. J. Hum. Genet. 51:17-37.[ISI][Medline]

    Hughes, A. L., and M. Yeager. 1997. Comparative evolutionary rates of introns and exons in murine rodents. J. Mol. Evol. 45:125-130.[ISI][Medline]

    Hughes, S., D. Zelus, and D. Mouchiroud. 1999. Warm-blooded isochore structure in Nile crocodile and turtle. Mol. Biol. Evol. 16:1521-1527.[Abstract]

    Hurst, L. D., and A. R. Merchant. 2001. High guanine-cytosine content is not an adaptation to high temperature: a comparative analysis amongst prokaryotes. Proc. R. Soc. Lond. B Biol. Sci. 268:493-497.[CrossRef][ISI][Medline]

    International Human Genome Sequencing Consortium. 2001. Initial sequencing and analysis of the human genome. Nature 409:860-921.[CrossRef][ISI][Medline]

    Ito, Y., A. Ando, H. Ando, J. Ando, Y. Saijoh, H. Inoko, and H. Fujimoto. 1998. Genomic structure of the spermatid-specific hsp70 homolog gene located in the class III region of the major histocompatibility complex of mouse and man. J. Biochem. (Tokyo) 124:347-353.[Abstract]

    Kumar, S., K. Tamura, I. B. Jakobsen, and M. Nei. 2001. MEGA2: molecular evolutionary genetics analysis software. Bioinformatics 17:1244-1245.[Abstract/Free Full Text]

    Li, W., P. Bernaola-Galvan, P. Carpena, and J. L. Oliver. 2003. Isochores merit the prefix ‘iso’. Comput. Biol. Chem. 27:5-10.[CrossRef][ISI][Medline]

    Lynch, M., and J. S. Conery. 2000. The evolutionary fate and consequences of duplicate genes. Science 290:1151-1155.[Abstract/Free Full Text]

    Marais, G. 2003. Biased gene conversion: implications for genome and sex evolution. Trends Genet. 19:330-338.[CrossRef][ISI][Medline]

    Montoya-Burgos, J. I., P. Boursot, and N. Galtier. 2003. Recombination explains isochores in mammalian genomes. Trends Genet. 19:128-130.[CrossRef][ISI][Medline]

    Nei, M., and T. Gojobori. 1986. Simple methods for estimating the numbers of synonymous and nonsynonymous nucleotide substitutions. Mol. Biol. Evol. 3:418-426.[Abstract]

    Oliver, J. L., P. Bernaola-Galvan, P. Carpena, and R. Roman-Roldan. 2001. Isochore chromosome maps of eukaryotic genomes. Gene 276:47-56.[CrossRef][ISI][Medline]

    Powell, J. R., and E. N. Moriyama. 1997. Evolution of codon usage bias in Drosophila. Proc. Natl. Acad. Sci. USA 94:7784-7790.[Abstract/Free Full Text]

    Ream, R. A., G. C. Johns, and G. N. Somero. 2003. Base compositions of genes encoding alpha-actin and lactate dehydrogenase-A from differently adapted vertebrates show no temperature-adaptive variation in G + C content. Mol. Biol. Evol. 20:105-110.[Abstract/Free Full Text]

    Sharp, P. M., and W. H. Li. 1986. An evolutionary perspective on synonymous codon usage in unicellular organisms. J. Mol. Evol. 24:28-38.[ISI][Medline]

    Sueoka, N. 1988. Directional mutation pressure and neutral molecular evolution. Proc. Natl. Acad. Sci. USA 85:2653-2657.[Abstract]

    Tamura, K. 1992. Estimation of the number of nucleotide substitutions when there are strong transition-transversion and G+C-content biases. Mol. Biol. Evol. 9:678-687.[Abstract]

    Tavaria, M., I. Kola, and R. L. Anderson. 1997. The hsp70 genes of mice and men. Pp. 49–52 in M. J. Gething, ed. Guidebook to molecular chaperones and protein-folding catalysts. Oxford University Press, Oxford, UK.

    Walter, L., F. Rauh, and E. Gunther. 1994. Comparative analysis of the three major histocompatibility complex-linked heat shock protein 70 (Hsp70) genes of the rat. Immunogenetics 40:325-330.[ISI][Medline]

    Wolfe, K. H., P. M. Sharp, and W. H. Li. 1989. Mutation rates differ among regions of the mammalian genome. Nature 337:283-285.[CrossRef][ISI][Medline]

    Yang, Z., and A. D. Yoder. 1999. Estimation of the transition/transversion rate bias and species sampling. J. Mol. Evol. 48:274-283.[ISI][Medline]

    Zoubak, S., G. D'Onofrio, S. Caccio, and G. Bernardi. 1995. Specific compositional patterns of synonymous positions in homologous mammalian genes. J. Mol. Evol. 40:293-307.[ISI][Medline]

Accepted for publication March 26, 2004.