Base Compositions of Genes Encoding -Actin and Lactate Dehydrogenase-A from Differently Adapted Vertebrates Show No Temperature-Adaptive Variation in
Hopkins Marine Station of Stanford University, Pacific Grove, Californiav'(x/1, 百拇医药
Abstractv'(x/1, 百拇医药
There is a long-standing debate in molecular evolution concerning the putative importance of GC content in adapting the thermal stabilities of DNA and RNA. Most studies of this relationship have examined broad-scale compositional patterns, for example, total GC percentages in genomes and occurrence of GC-rich isochores. Few studies have systematically examined the GC contents of individual orthologous genes from differently thermally adapted species. When this has been done, the emphasis has been on comparing large numbers of genes in only a few species. We have approached the GC-adaptation temperature hypothesis in a different manner by examining patterns of base composition of genes encoding lactate dehydrogenase-A (ldh-a) and alpha-actin ({alpha} -actin) from 51 species of vertebrates whose adaptation temperatures ranged from -1.86°C (Antarctic fishes) to approximately 45°C (desert reptile). No significant positive correlation was found between any index of GC content (GC content of the entire sequence, GC content of the third codon position [GC3], and GC content at fourfold degenerate sites [GC4]) and any index of adaptation temperature (maximal, mean, or minimal body temperature). For {alpha} -actin, slopes of regression lines for all comparisons did not differ significantly from zero. For ldh-a, negative correlations between adaptation temperature and total GC content, GC3, and GC4 were observed but were shown to be due entirely to phylogenetic influences (as revealed by independent contrast analyses). This comparison of GC content across a wide range of ectothermic ("cold-blooded") and endothermic ("warm-blooded") vertebrates revealed that frogs of the genus Xenopus, which have commonly been used as a representative cold-blooded species, in fact are outliers among ectotherms for the {alpha} -actin analyses, raising concern about the appropriateness of choosing these amphibians as representative of ectothermic vertebrates in general. Our study indicates that, whereas GC contents of isochores may show variation among different classes of vertebrates, there is no consistent relationship between adaptation temperature and the percentage of thermal stability-enhancing G + C base pairs in protein-coding genes.
Key Words: Vertebrate genome evolution • GC content • codon bias • temperature • alpha-actin • lactate dehydrogenase-A (ldh-a)a&7m, 百拇医药
Introductiona&7m, 百拇医药
Attempts to understand the large-scale structures and compositions of animal genomes have led to the discovery of isochores, long segments of compositionally homogenous DNA with characteristically different GC contents, as measured by modal buoyant densities on cesium chloride gradients . Isochore patterns and base composition differ among the classes of vertebrates. Most notably, genomes of mammals and birds contain a genome core composed of H2 and H3 isochores with substantially higher GC content than that present in the genome as a whole. These isochores represent a small fraction of the mammalian and avian genomes, but they contain an exceptionally large fraction of the genes (>50% in the case of the human genome) . In contrast to mammals and birds (endotherms, or "warm-blooded" species), fishes, amphibians, and reptiles, the vast majority of which are ectothermic ("cold-blooded") species, have genomes that are much more homogenous and lack regions of exceptionally high GC content . However, evidence from studies of hybridization of human H3 isochore DNA to genome fractions of fishes, amphibians, and reptiles, as well as comparisons of homologous genes in humans and the frog Xenopus, suggested that ectothermic vertebrates also have a genome core with a high density of genes but with a lower GC content .
To account for the differences in GC content of the isochores found in genomes of endothermic and ectothermic species, it has been hypothesized that the higher GC contents in avian and mammalian genomes reflect selection for higher thermal stability of DNA and RNA . The additional hydrogen bond found in GC pairs relative to AT pairs would give the DNA and RNA of birds and mammals a higher thermodynamic stability, thus reducing chances for disruption of structure at high body temperatures. Conversely, lower GC levels in cold-adapted ectotherms may reduce the energy required to "melt" double-stranded DNA for transcription and replication. After , we refer to this hypothesis as the thermostability hypothesis. Tests of this hypothesis have involved two different types of strategy (for review, see ). First, comparisons of whole genomes using cesium chloride gradients have generally supported the view that genomes of ectothermic vertebrates lack the asymmetry of modal buoyant densities that are indicative of the small, GC-rich isochores found in mammals and birds. Second, the GC contents of homologous genes (total %GC of the gene, third position GC content [GC3], and GC content of fourfold degenerate sites [GC4]) have been compared. These comparisons have typically focused on only a few species for which large numbers of genes have been sequenced. took a broader comparative approach by examining genes of 19 species of fish, five species of amphibians, five species of reptiles, and humans. The human genes more frequently had a higher GC3 content. However, only for one gene was there more than one ectothermic sequence to compare against the human sequence, and no explicit comparisons were made among the ectotherms with respect to their different adaptation temperatures. examined 21 genes in pairwise comparisons between ectotherms and endotherms, but only a single gene in their analysis had sequences available for more than one ectothermic species. Nonetheless, their results showed that 12 of the 21 genes showed an increase in GC3 content in endotherms, whereas six genes showed no difference and three showed higher GC3 in ectotherms. As was noted in a recent review by , these previous studies have failed to examine the precise relationship between body temperature and genomic base composition—a central tenet of the thermostability hypothesis.
The present study was designed to redress certain limitations of these earlier studies by examining the sequences of the genes encoding two widely occurring housekeeping proteins, lactate dehydrogenase-A (ldh-a) and alpha-actin ({alpha} -actin), in a large number of vertebrates with widely different adaptation temperatures. These two genes in particular were chosen for the availability of sequence data from a diverse array of vertebrate species with a broad range of physiological temperatures. We reasoned that if the thermostability hypothesis is generally correct, then the GC contents of homologous genes of vertebrates should vary in a regular manner with adaptation temperature, independently of the taxonomic grouping (endotherm vs. ectotherm) of the species. For example, ectothermic vertebrates with body temperatures equal to or higher than those of mammals and birds would be expected to have GC contents similar to, if not higher than, those of endotherms. To maximize the power of our comparisons, we selected 51 species that represent the full range of vertebrate body temperatures, from -1.86°C in cold-adapted Antarctic notothenioid fishes to approximately 45°C in a thermophilic desert reptile. Our results fail to support the thermostability hypothesis, and they call into question certain interpretations given in previous analyses of interspecific variation in GC content.
Materials and Methodsdo, 百拇医药
Analyzed Sequencesdo, 百拇医药
A total of 41 sequences of vertebrate ldh-a and 30 sequences of vertebrate -actin were analyzed. To obtain -actin from animals across a diverse range of body temperatures, it was necessary to sequence genes from five species (Trematomus bernacchii, Notothenia coriiceps, Dipsosaurus dorsalis, Lampanyctus regalis, and Sphyraena idiastes), which have been assigned the following accession numbers: AF503589, AF503590, AF503591, AF503592, and AF503593. All additional complete protein-coding sequences were obtained from GenBank in September 2000 (see online supplementary material for accession numbers).do, 百拇医药
Nucleotide Compositiondo, 百拇医药
All vertebrate sequences were aligned and then base composition was determined for each sequence using the DNAStar suite of programs (DNAStar Inc.). From this analysis the overall GC content and the GC content at each of the three codon positions (GC1, GC2, and GC3) and the fourfold degenerate sites (GC4) were calculated. For statistical correlations, mean values of GC content were taken from: (1) the four sub-Antarctic species, (2) the 13 Antarctic species, and (3) species for which there were multiple full-length coding sequences available for the same species. This was done to minimize skewing the data from extreme cases of nonindependence due to phylogenetic relatedness that, we suggest, likely represent a single adaptation event to a common environment. Additionally, independent contrasts controlling for phylogenetic nonindependence were calculated for all species for body temperature versus GC3 and GC4 for both ldh-a and {alpha} -actin using the CAIC freeware package . Phylogenies were generated from DNA sequence data in the first and second codon positions using PAUP* 4.0 software program . Phylogenies were reconstructed using a neighbor-joining method under a minimum evolution (distance) criterion using Tamura-Nei corrected divergence estimates . In general, the phylogeny based on the first two positions of the -actin gene was poorly supported for the 13 different vertebrate orders represented, whereas the ldh-a phylogeny had better bootstrap support that was in agreement with overall patterns of vertebrate evolution (with the exception of Xenopus). These phylogenies, along with bootstrap support from 500 replicates, were used in the independent contrast analyses and are available in the online supplementary materials.
Body Temperaturel, 百拇医药
Body temperatures for organisms were taken from the literature, if available, or were assigned on the basis of habitat range and behavioral adaptation in the case of ectotherms ( see online supplementary materials for complete references). To correct for the large changes in body temperature that may be experienced by several of the ectotherms in this study, we took into account the upper, mean (or preferred), and lower thermal limits of each species. Lethally high and low temperatures were not used in compiling body temperature data; rather the low and high temperatures reflect the ambient temperature ranges experienced by these species on an annual basis.l, 百拇医药
fig.ommittedl, 百拇医药
Table 1 Sequences Examined, Body Temperature Data, and Indexes of GC Contentl, 百拇医药
fig.ommittedl, 百拇医药
Table 1 Continuedl, 百拇医药
Resultsl, 百拇医药
lists the scientific and common names of the species for which sequences of genes encoding LDH-A and alpha-actin were either available in databases or were determined in our laboratory (see Materials and Methods for accession numbers). These species comprise bony fishes, cartilaginous fishes (elasmobranchs), amphibians, reptiles, birds, and mammals. Body temperatures range from -1.86°C in Antarctic notothenioid fishes, the most cold-adapted vertebrates, to approximately 45°C for a thermophilic desert reptile (Dipsosaurus dorsalis). To the degree possible, we selected ectothermic species whose body temperatures were relatively evenly distributed across the full thermal range of adaptation temperatures of vertebrates. presents three indices of adaptation temperature: maximal, mean, and lowest body temperature, as estimated from information in the literature or our own field measurements (see Materials and Methods). The sequence characteristics that were measured for each of the ldh-a and {alpha} -actin genes also are tabulated in . These data comprise the percent GC of (1) the entire gene sequence (%GC), (2) the third codon position (GC3), and (3) the fourfold degenerate sites at the third codon position (GC4).
plots GC4 of the ldh-a (panel A) and {alpha} -actin (panel C) genes as functions of mean body temperature. Independent contrasts analyses (mean body temperature versus GC4) are also shown for each gene (panel B: ldh-a; panel D: {alpha} -actin). For {alpha} -actin, the slopes of the regression lines do not differ significantly from zero (r2 = 0.111, P = 0.111; independent contrast: r2 = 0.00153, P = 0.846). For ldh-a, the slope of the regression line describing body temperature and GC4 without correction for phylogenetic effects is significantly different from zero. In contradiction to the thermostability hypothesis, the slope it is negative rather than positive (r2 = 0.381, P = 0.0022). However, when these data are corrected for phylogenetic relatedness, no significant trend is found (r2 = 0.0240, P = 0.352).m9-{, 百拇医药
fig.ommittedm9-{, 百拇医药
FIG. 1. The GC4 of ldh-a (A; r2 = 0.381, P = 0.0022) has a negative correlation with the mean body temperature, whereas the GC4 of -actin (C; r2 = 0.111, P = 0.111) shows no correlation with the mean body temperature mammals (), birds (), reptiles (), amphibians (), bony fishes (•), and elasmobranchs . Independent contrast analysis shows no correlation between the residuals of GC4 of ldh-a (B; r2 = 0.0240, P = 0.352) or -actin (D; r2 = 0.00153, P = 0.846) and mean body temperature
We also performed these analyses for total GC and GC at third codon position sites (GC3) against all three measures of body temperature (maximal, mean, and minimal), and the same trends were evident in every analysis: no significant correlation was found for {alpha} -actin, and the significant negative correlation found for ldh-a disappeared when independent contrast analysis was conducted.*, 百拇医药
Note that in the case of {alpha} -actin, the values for Xenopus (filled triangles) either lie on or just outside the lower standard deviation contour around the regression line ( and additional analyses [not shown]). The {alpha} -actin gene of Xenopus has a markedly lower total GC content compared with all of the other species we examined. The outlier quality of the {alpha} -actin data for Xenopus suggests caution when considering the suitability of Xenopus as a "representative" ectotherm (see Discussion).*, 百拇医药
Discussion*, 百拇医药
If the transition to higher GC isochores in endothermic vertebrates were a consequence of selection for increased thermostability, then one would expect to find a significant correlation between physiological temperature and GC content, irrespective of taxonomic status. That is, this relationship should hold true for all species evolving under comparable physiological temperatures, as the genomes of these various species would be experiencing the same selective agent. However, the results presented here are inconsistent with these predictions of the thermostability hypothesis: neither the ldh-a nor the {alpha} -actin gene shows a correlation between GC content and adaptation temperature when phylogenetic relatedness is controlled.
These findings represent a taxonomically diverse array of 51 vertebrate species from a wide range of physiological temperatures, including a number of ectotherms that have evolved under comparable or, in some cases, even greater physiological temperatures than those of mammals and birds . Because some ectotherms may experience a broad range of environmental temperatures, patterns associated with high, mean, and low temperatures were investigated independently, and all displayed essentially the same trends. It is reassuring to note that the conclusions are robust, regardless of whether the posited selection would be acting on maximal, mean, or minimal temperature experienced by an organism on an annual basis.yx:, 百拇医药
The observed patterns in our results are also consistent across total GC, GC3, and GC4. Each level not only represents a smaller fraction of the data set but also represents a step further away from confounding selective constraints based on limits to amino acid sequence as determined by constraints on the structure and function of the encoded proteins, alpha-actin and A4-LDH. In this regard, GC4 is the most instructive because it is free of all such selective constraints and yet does not demonstrate a significantly positive GC-temperature correlation.
The results reported for {alpha} -actin and ldh-a corroborate conclusions reached in analyses of prokaryotic genomes, where the GC content was found not to correlate with the optimal temperature of numerous prokaryote species More specifically, GC3 did not correlate with optimal temperature when phylogenetic relatedness was controlled for in the analysis .izr8+\, http://www.100md.com
The two genes that were examined here are both major housekeeping genes, essential to all vertebrates, and prime examples of the sort of "important proteins" that were suggested to be encoded by GC-rich sequences to confer a thermostability selective advantage . Thus, it is difficult to argue that these two genes are any less important or representative of a vertebrate genome than any other two such genes. Nevertheless, it is clear that two genes can no more represent vertebrate genome evolution than two species can.izr8+\, http://www.100md.com
While there are too few genes available here to refute the thermostability hypothesis or alternatives similarly based on physiological temperature, our results do suggest caution when generalizing about causative factors in vertebrate genome evolution. The lack of correlations between temperature and GC content when a more careful examination of ectothermic physiological temperatures is incorporated suggests that the dichotomy between warm-blooded and cold-blooded, while tempting in its simplicity, may not fully explain the evolution of vertebrate GC-rich isochores. The extent to which GC-rich isochores are characteristic only of birds and mammals is another issue that requires further clarification. It may be the case that GC-rich isochores evolved before the evolution of endothermy. Indeed, found evidence for GC-rich isochores in two distantly related reptiles, the Nile crocodile and the red-eared slider turtle, albeit for a limited number of sequences. A final caveat raised by our study concerns the use of Xenopus spp. as representative of cold-blooded vertebrates in general. As the analyses of {alpha} -actin consistently showed, Xenopus spp. tend to be outliers that differ from other ectothermic species.
A more complete picture of vertebrate genome evolution awaits the critical examination of GC content and codon bias in both dimensions—expanding both the numbers of genes and the diversity of species examined. Hopefully, these sorts of data will soon be tractable to collect and analyze as we emerge into a new era of comparative genomics, where complete genomes of many diverse species become available.8k(0)u0, 百拇医药
Acknowledgements8k(0)u0, 百拇医药
We thank Dr. Jonathon Stillman and the two anonymous reviewers of this paper for their constructive criticisms. These studies were supported by National Science Foundation grants IBN-0133184 to G.N.S. and OPP-9317696 to Dr. Donal Manahan, in support of the McMurdo Biology Course.8k(0)u0, 百拇医药
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Abstractv'(x/1, 百拇医药
There is a long-standing debate in molecular evolution concerning the putative importance of GC content in adapting the thermal stabilities of DNA and RNA. Most studies of this relationship have examined broad-scale compositional patterns, for example, total GC percentages in genomes and occurrence of GC-rich isochores. Few studies have systematically examined the GC contents of individual orthologous genes from differently thermally adapted species. When this has been done, the emphasis has been on comparing large numbers of genes in only a few species. We have approached the GC-adaptation temperature hypothesis in a different manner by examining patterns of base composition of genes encoding lactate dehydrogenase-A (ldh-a) and alpha-actin ({alpha} -actin) from 51 species of vertebrates whose adaptation temperatures ranged from -1.86°C (Antarctic fishes) to approximately 45°C (desert reptile). No significant positive correlation was found between any index of GC content (GC content of the entire sequence, GC content of the third codon position [GC3], and GC content at fourfold degenerate sites [GC4]) and any index of adaptation temperature (maximal, mean, or minimal body temperature). For {alpha} -actin, slopes of regression lines for all comparisons did not differ significantly from zero. For ldh-a, negative correlations between adaptation temperature and total GC content, GC3, and GC4 were observed but were shown to be due entirely to phylogenetic influences (as revealed by independent contrast analyses). This comparison of GC content across a wide range of ectothermic ("cold-blooded") and endothermic ("warm-blooded") vertebrates revealed that frogs of the genus Xenopus, which have commonly been used as a representative cold-blooded species, in fact are outliers among ectotherms for the {alpha} -actin analyses, raising concern about the appropriateness of choosing these amphibians as representative of ectothermic vertebrates in general. Our study indicates that, whereas GC contents of isochores may show variation among different classes of vertebrates, there is no consistent relationship between adaptation temperature and the percentage of thermal stability-enhancing G + C base pairs in protein-coding genes.
Key Words: Vertebrate genome evolution • GC content • codon bias • temperature • alpha-actin • lactate dehydrogenase-A (ldh-a)a&7m, 百拇医药
Introductiona&7m, 百拇医药
Attempts to understand the large-scale structures and compositions of animal genomes have led to the discovery of isochores, long segments of compositionally homogenous DNA with characteristically different GC contents, as measured by modal buoyant densities on cesium chloride gradients . Isochore patterns and base composition differ among the classes of vertebrates. Most notably, genomes of mammals and birds contain a genome core composed of H2 and H3 isochores with substantially higher GC content than that present in the genome as a whole. These isochores represent a small fraction of the mammalian and avian genomes, but they contain an exceptionally large fraction of the genes (>50% in the case of the human genome) . In contrast to mammals and birds (endotherms, or "warm-blooded" species), fishes, amphibians, and reptiles, the vast majority of which are ectothermic ("cold-blooded") species, have genomes that are much more homogenous and lack regions of exceptionally high GC content . However, evidence from studies of hybridization of human H3 isochore DNA to genome fractions of fishes, amphibians, and reptiles, as well as comparisons of homologous genes in humans and the frog Xenopus, suggested that ectothermic vertebrates also have a genome core with a high density of genes but with a lower GC content .
To account for the differences in GC content of the isochores found in genomes of endothermic and ectothermic species, it has been hypothesized that the higher GC contents in avian and mammalian genomes reflect selection for higher thermal stability of DNA and RNA . The additional hydrogen bond found in GC pairs relative to AT pairs would give the DNA and RNA of birds and mammals a higher thermodynamic stability, thus reducing chances for disruption of structure at high body temperatures. Conversely, lower GC levels in cold-adapted ectotherms may reduce the energy required to "melt" double-stranded DNA for transcription and replication. After , we refer to this hypothesis as the thermostability hypothesis. Tests of this hypothesis have involved two different types of strategy (for review, see ). First, comparisons of whole genomes using cesium chloride gradients have generally supported the view that genomes of ectothermic vertebrates lack the asymmetry of modal buoyant densities that are indicative of the small, GC-rich isochores found in mammals and birds. Second, the GC contents of homologous genes (total %GC of the gene, third position GC content [GC3], and GC content of fourfold degenerate sites [GC4]) have been compared. These comparisons have typically focused on only a few species for which large numbers of genes have been sequenced. took a broader comparative approach by examining genes of 19 species of fish, five species of amphibians, five species of reptiles, and humans. The human genes more frequently had a higher GC3 content. However, only for one gene was there more than one ectothermic sequence to compare against the human sequence, and no explicit comparisons were made among the ectotherms with respect to their different adaptation temperatures. examined 21 genes in pairwise comparisons between ectotherms and endotherms, but only a single gene in their analysis had sequences available for more than one ectothermic species. Nonetheless, their results showed that 12 of the 21 genes showed an increase in GC3 content in endotherms, whereas six genes showed no difference and three showed higher GC3 in ectotherms. As was noted in a recent review by , these previous studies have failed to examine the precise relationship between body temperature and genomic base composition—a central tenet of the thermostability hypothesis.
The present study was designed to redress certain limitations of these earlier studies by examining the sequences of the genes encoding two widely occurring housekeeping proteins, lactate dehydrogenase-A (ldh-a) and alpha-actin ({alpha} -actin), in a large number of vertebrates with widely different adaptation temperatures. These two genes in particular were chosen for the availability of sequence data from a diverse array of vertebrate species with a broad range of physiological temperatures. We reasoned that if the thermostability hypothesis is generally correct, then the GC contents of homologous genes of vertebrates should vary in a regular manner with adaptation temperature, independently of the taxonomic grouping (endotherm vs. ectotherm) of the species. For example, ectothermic vertebrates with body temperatures equal to or higher than those of mammals and birds would be expected to have GC contents similar to, if not higher than, those of endotherms. To maximize the power of our comparisons, we selected 51 species that represent the full range of vertebrate body temperatures, from -1.86°C in cold-adapted Antarctic notothenioid fishes to approximately 45°C in a thermophilic desert reptile. Our results fail to support the thermostability hypothesis, and they call into question certain interpretations given in previous analyses of interspecific variation in GC content.
Materials and Methodsdo, 百拇医药
Analyzed Sequencesdo, 百拇医药
A total of 41 sequences of vertebrate ldh-a and 30 sequences of vertebrate -actin were analyzed. To obtain -actin from animals across a diverse range of body temperatures, it was necessary to sequence genes from five species (Trematomus bernacchii, Notothenia coriiceps, Dipsosaurus dorsalis, Lampanyctus regalis, and Sphyraena idiastes), which have been assigned the following accession numbers: AF503589, AF503590, AF503591, AF503592, and AF503593. All additional complete protein-coding sequences were obtained from GenBank in September 2000 (see online supplementary material for accession numbers).do, 百拇医药
Nucleotide Compositiondo, 百拇医药
All vertebrate sequences were aligned and then base composition was determined for each sequence using the DNAStar suite of programs (DNAStar Inc.). From this analysis the overall GC content and the GC content at each of the three codon positions (GC1, GC2, and GC3) and the fourfold degenerate sites (GC4) were calculated. For statistical correlations, mean values of GC content were taken from: (1) the four sub-Antarctic species, (2) the 13 Antarctic species, and (3) species for which there were multiple full-length coding sequences available for the same species. This was done to minimize skewing the data from extreme cases of nonindependence due to phylogenetic relatedness that, we suggest, likely represent a single adaptation event to a common environment. Additionally, independent contrasts controlling for phylogenetic nonindependence were calculated for all species for body temperature versus GC3 and GC4 for both ldh-a and {alpha} -actin using the CAIC freeware package . Phylogenies were generated from DNA sequence data in the first and second codon positions using PAUP* 4.0 software program . Phylogenies were reconstructed using a neighbor-joining method under a minimum evolution (distance) criterion using Tamura-Nei corrected divergence estimates . In general, the phylogeny based on the first two positions of the -actin gene was poorly supported for the 13 different vertebrate orders represented, whereas the ldh-a phylogeny had better bootstrap support that was in agreement with overall patterns of vertebrate evolution (with the exception of Xenopus). These phylogenies, along with bootstrap support from 500 replicates, were used in the independent contrast analyses and are available in the online supplementary materials.
Body Temperaturel, 百拇医药
Body temperatures for organisms were taken from the literature, if available, or were assigned on the basis of habitat range and behavioral adaptation in the case of ectotherms ( see online supplementary materials for complete references). To correct for the large changes in body temperature that may be experienced by several of the ectotherms in this study, we took into account the upper, mean (or preferred), and lower thermal limits of each species. Lethally high and low temperatures were not used in compiling body temperature data; rather the low and high temperatures reflect the ambient temperature ranges experienced by these species on an annual basis.l, 百拇医药
fig.ommittedl, 百拇医药
Table 1 Sequences Examined, Body Temperature Data, and Indexes of GC Contentl, 百拇医药
fig.ommittedl, 百拇医药
Table 1 Continuedl, 百拇医药
Resultsl, 百拇医药
lists the scientific and common names of the species for which sequences of genes encoding LDH-A and alpha-actin were either available in databases or were determined in our laboratory (see Materials and Methods for accession numbers). These species comprise bony fishes, cartilaginous fishes (elasmobranchs), amphibians, reptiles, birds, and mammals. Body temperatures range from -1.86°C in Antarctic notothenioid fishes, the most cold-adapted vertebrates, to approximately 45°C for a thermophilic desert reptile (Dipsosaurus dorsalis). To the degree possible, we selected ectothermic species whose body temperatures were relatively evenly distributed across the full thermal range of adaptation temperatures of vertebrates. presents three indices of adaptation temperature: maximal, mean, and lowest body temperature, as estimated from information in the literature or our own field measurements (see Materials and Methods). The sequence characteristics that were measured for each of the ldh-a and {alpha} -actin genes also are tabulated in . These data comprise the percent GC of (1) the entire gene sequence (%GC), (2) the third codon position (GC3), and (3) the fourfold degenerate sites at the third codon position (GC4).
plots GC4 of the ldh-a (panel A) and {alpha} -actin (panel C) genes as functions of mean body temperature. Independent contrasts analyses (mean body temperature versus GC4) are also shown for each gene (panel B: ldh-a; panel D: {alpha} -actin). For {alpha} -actin, the slopes of the regression lines do not differ significantly from zero (r2 = 0.111, P = 0.111; independent contrast: r2 = 0.00153, P = 0.846). For ldh-a, the slope of the regression line describing body temperature and GC4 without correction for phylogenetic effects is significantly different from zero. In contradiction to the thermostability hypothesis, the slope it is negative rather than positive (r2 = 0.381, P = 0.0022). However, when these data are corrected for phylogenetic relatedness, no significant trend is found (r2 = 0.0240, P = 0.352).m9-{, 百拇医药
fig.ommittedm9-{, 百拇医药
FIG. 1. The GC4 of ldh-a (A; r2 = 0.381, P = 0.0022) has a negative correlation with the mean body temperature, whereas the GC4 of -actin (C; r2 = 0.111, P = 0.111) shows no correlation with the mean body temperature mammals (), birds (), reptiles (), amphibians (), bony fishes (•), and elasmobranchs . Independent contrast analysis shows no correlation between the residuals of GC4 of ldh-a (B; r2 = 0.0240, P = 0.352) or -actin (D; r2 = 0.00153, P = 0.846) and mean body temperature
We also performed these analyses for total GC and GC at third codon position sites (GC3) against all three measures of body temperature (maximal, mean, and minimal), and the same trends were evident in every analysis: no significant correlation was found for {alpha} -actin, and the significant negative correlation found for ldh-a disappeared when independent contrast analysis was conducted.*, 百拇医药
Note that in the case of {alpha} -actin, the values for Xenopus (filled triangles) either lie on or just outside the lower standard deviation contour around the regression line ( and additional analyses [not shown]). The {alpha} -actin gene of Xenopus has a markedly lower total GC content compared with all of the other species we examined. The outlier quality of the {alpha} -actin data for Xenopus suggests caution when considering the suitability of Xenopus as a "representative" ectotherm (see Discussion).*, 百拇医药
Discussion*, 百拇医药
If the transition to higher GC isochores in endothermic vertebrates were a consequence of selection for increased thermostability, then one would expect to find a significant correlation between physiological temperature and GC content, irrespective of taxonomic status. That is, this relationship should hold true for all species evolving under comparable physiological temperatures, as the genomes of these various species would be experiencing the same selective agent. However, the results presented here are inconsistent with these predictions of the thermostability hypothesis: neither the ldh-a nor the {alpha} -actin gene shows a correlation between GC content and adaptation temperature when phylogenetic relatedness is controlled.
These findings represent a taxonomically diverse array of 51 vertebrate species from a wide range of physiological temperatures, including a number of ectotherms that have evolved under comparable or, in some cases, even greater physiological temperatures than those of mammals and birds . Because some ectotherms may experience a broad range of environmental temperatures, patterns associated with high, mean, and low temperatures were investigated independently, and all displayed essentially the same trends. It is reassuring to note that the conclusions are robust, regardless of whether the posited selection would be acting on maximal, mean, or minimal temperature experienced by an organism on an annual basis.yx:, 百拇医药
The observed patterns in our results are also consistent across total GC, GC3, and GC4. Each level not only represents a smaller fraction of the data set but also represents a step further away from confounding selective constraints based on limits to amino acid sequence as determined by constraints on the structure and function of the encoded proteins, alpha-actin and A4-LDH. In this regard, GC4 is the most instructive because it is free of all such selective constraints and yet does not demonstrate a significantly positive GC-temperature correlation.
The results reported for {alpha} -actin and ldh-a corroborate conclusions reached in analyses of prokaryotic genomes, where the GC content was found not to correlate with the optimal temperature of numerous prokaryote species More specifically, GC3 did not correlate with optimal temperature when phylogenetic relatedness was controlled for in the analysis .izr8+\, http://www.100md.com
The two genes that were examined here are both major housekeeping genes, essential to all vertebrates, and prime examples of the sort of "important proteins" that were suggested to be encoded by GC-rich sequences to confer a thermostability selective advantage . Thus, it is difficult to argue that these two genes are any less important or representative of a vertebrate genome than any other two such genes. Nevertheless, it is clear that two genes can no more represent vertebrate genome evolution than two species can.izr8+\, http://www.100md.com
While there are too few genes available here to refute the thermostability hypothesis or alternatives similarly based on physiological temperature, our results do suggest caution when generalizing about causative factors in vertebrate genome evolution. The lack of correlations between temperature and GC content when a more careful examination of ectothermic physiological temperatures is incorporated suggests that the dichotomy between warm-blooded and cold-blooded, while tempting in its simplicity, may not fully explain the evolution of vertebrate GC-rich isochores. The extent to which GC-rich isochores are characteristic only of birds and mammals is another issue that requires further clarification. It may be the case that GC-rich isochores evolved before the evolution of endothermy. Indeed, found evidence for GC-rich isochores in two distantly related reptiles, the Nile crocodile and the red-eared slider turtle, albeit for a limited number of sequences. A final caveat raised by our study concerns the use of Xenopus spp. as representative of cold-blooded vertebrates in general. As the analyses of {alpha} -actin consistently showed, Xenopus spp. tend to be outliers that differ from other ectothermic species.
A more complete picture of vertebrate genome evolution awaits the critical examination of GC content and codon bias in both dimensions—expanding both the numbers of genes and the diversity of species examined. Hopefully, these sorts of data will soon be tractable to collect and analyze as we emerge into a new era of comparative genomics, where complete genomes of many diverse species become available.8k(0)u0, 百拇医药
Acknowledgements8k(0)u0, 百拇医药
We thank Dr. Jonathon Stillman and the two anonymous reviewers of this paper for their constructive criticisms. These studies were supported by National Science Foundation grants IBN-0133184 to G.N.S. and OPP-9317696 to Dr. Donal Manahan, in support of the McMurdo Biology Course.8k(0)u0, 百拇医药
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