Directional fixation of mutations in vertebrate evolution

Summary We have made pairwise comparisons between the coding sequences of 21 genes from coldblooded vertebrates and 41 homologous sequences from warm-blooded vertebrates. In the case of 12 genes, GC levels were higher, especially in third codon positions, in warm-blooded vertebrates compared to cold-blooded vertebrates. Six genes showed no remarkable difference in GC level and three showed a lower level. In the first case, higher GC levels appear to be due to a directional fixation of mutations, presumably under the influence of body temperature (see Bernardi and Bernardi 1986b). These GC-richer genes of warm-blooded vertebrates were located, in all cases studied, in isochores higher in GC than those comprising the homologous genes of cold-blooded vertebrates. In the third case, increases appear to be due to a limited formation of GC-rich isochores which took place in some cold-blooded vertebrates after the divergence of warm-blooded vertebrates. The directional changes in the GC content of coding sequences and the evolutionary conservation of both increased and unchanged GC levels are in keeping with the existence of compositional constraints on the genome.     

The compositional patterns of the avian genomes and their evolutionary implications

The compositional distributions of large (main-band) DNA fragments from eight birds belonging to eight different orders (including both paleognathous and neognathous species) are very broad and extremely close to each other. These findings, which are paralleled by the compositional similarity of homologous coding sequences and their codon positions, support the idea that birds are a monophyletic group.The compositional distribution of third-codon positions of genes from chicken, the only avian species for which a relatively large number of coding sequences is known, is very broad and bimodal, the minor GC-richer peak reaching 100% GC. The very high compositional heterogeneity of avian genomes is accompanied (as in the case of mammalian genomes) by a very high speciation rate compared to cold-blooded vertebrates which are characterized by genomes that are much less heterogeneous. The higher GC levels attained by avian compared to mammalian genomes might be correlated with the higher body temperature (41–43°C) of birds compared to mammals (37°C).A comparison of GC levels of coding sequences and codon positions from man and chicken revealed very close average GC levels and standard deviations. Homologous coding sequences and codon positions from man and chicken showed a surprisingly high degree of compositional similarity which was, however, higher for GC-poor than for GC-rich sequences. This indicates that GC-poor isochores of warm-blooded vertebrates reflect the composition of the isochores of the genome of the common reptilian ancestor of mammals and birds, which underwent only a small compositional change at the transition from cold- to warm-blooded vertebrates. In contrast, the GC-rich isochores of birds and mammals are the result of large compositional changes at the same evolutionary transition, where were in part different in the two classes of warm-blooded vertebrates.Correspondence to: G. Bernaadi     

Thermal Adaptation of the Small Subunit Ribosomal RNA Gene: A Comparative Study

We carried out a comprehensive survey of small subunit ribosomal RNA sequences from archaeal, bacterial, and eukaryotic lineages in order to understand the general patterns of thermal adaptation in the rRNA genes. Within each lineage, we compared sequences from mesophilic, moderately thermophilic, and hyperthermophilic species. We carried out a more detailed study of the archaea, because of the wide range of growth temperatures within this group. Our results confirmed that there is a clear correlation between the GC content of the paired stem regions of the 16S rRNA genes and the optimal growth temperature, and we show that this correlation cannot be explained simply by phylogenetic relatedness among the thermophilic archaeal species. In addition, we found a significant, positive relationship between rRNA stem length and growth temperature. These correlations are found in both bacterial and archaeal rRNA genes. Finally, we compared rRNA sequences from warm-blooded and cold-blooded vertebrates. We found that, while rRNA sequences from the warm-blooded vertebrates have a higher overall GC content than those from the cold-blooded vertebrates, this difference is not concentrated in the paired regions of the molecule, suggesting that thermal adaptation is not the cause of the nucleotide differences between the vertebrate lineages. Electronic Supplementary Material Electronic Supplementary material is available for this article at and accessible for authorised users. [Reviewing Editor: Dr. Nicolas Galtier]     

Cytosine deamination plays a primary role in the evolution of mammalian isochores

DNA melting is rate-limiting for cytosine deamination, from which we infer that the rate of cytosine deamination should decline twofold for each 10% increase in GC content. Analysis of human DNA sequence data confirms that this is the case for 5-methylcytosine. Several lines of evidence further confirm that it is also the case for unmethylated cytosine and that cytosine deamination causes the majority of all C-->T and G-->A transitions in mammals. Thus, cytosine deamination and DNA base composition each affect the other, forming a positive feedback loop that facilitates divergent genetic drift to high or low GC content. Because a 10 degrees C increase in temperature in vitro increases the rate of cytosine deamination 5. 7-fold, cytosine deamination must be highly dependent on body temperature, which is consistent with the dramatic differences between the isochores of warm-blooded versus cold-blooded vertebrates. Because this process involves both DNA melting and positive feedback, it would be expected to spread progressively (in evolutionary time) down the length of the chromosome, which is consistent with the large size of isochores in modern mammals.     

Statistical analysis of vertebrate sequences reveals that long genes are scarce in GC-rich isochores

We compared the exon/intron organization of vertebrate genes belonging to different isochore classes, as predicted by their GC content at third codon position. Two main features have emerged from the analysis of sequences published in GenBank: (1) genes coding for long proteins (i.e., 500 aa) are almost two times more frequent in GC-poor than in GC-rich isochores; (2) intervening sequences (=sum of introns) are on average three times longer in GC-poor than in GC-rich isochores. These patterns are observed among human, mouse, rat, cow, and even chicken genes and are therefore likely to be common to all warm-blooded vertebrates. Analysis of Xenopus sequences suggests that the same patterns exist in cold-blooded vertebrates. It could be argued that such results do not reflect the reality because sequence databases are not representative of entire genomes. However, analysis of biases in GenBank revealed that the observed discrepancies between GC-rich and GC-poor isochores are not artifactual, and are probably largely underestimated. We investigated the distribution of microsatellites and interspersed repeats in introns of human and mouse genes from different isochores. This analysis confirmed previous studies showing that Ll repeats are almost absent from GC-rich isochores. Microsatellites and SINES (Alu, B1, B2) are found at roughly equal frequencies in introns from all isochore classes. Globally, the presence of repeated sequences does not account for the increased intron length in GC-poor isochores. The relationships between gene structure and global genome organization and evolution are discussed.     

Presence of isochore structures in reptile genomes suggested by the relationship between GC contents of intron regions and those of coding regions

Vertebrate genomes are mosaics of isochores. On the assumption that marked differences exist in the isochore structure between warm-blooded and cold-blooded animals, variations among vertebrates were previously attributed to adaptation to homeothermy. However, based on the data of coding regions from representatives of extant vertebrates, including a turtle, a crocodile (Archosauromorpha) and a few kinds of snakes (Lepidosauromorpha), it was recently hypothesized that the common ancestors of mammals, birds and extant reptiles already had the "warm-blooded" isochore structure. To test this hypothesis, the nucleotide sequences of alpha-globin genes including non-coding regions (introns) from two snakes, N. kaouthia and E. climacophora, were determined (accession number: AB104824, AB104825). The correlation between the GC contents in the introns and exons of alpha-globin genes from snakes and those from other vertebrates supports the above hypothesis. Similar analysis using data for exons and introns of other genes obtained from the GenBank (Release 131) also support the above hypothesis.     

CpG islands, genes and isochores in the genomes of vertebrates

We have shown that human genes associated with CpG islands increase in number as they increase in % of guanine + cytosine (GC) levels, and that most genes associated with CpG islands are located in the GC-richest compartment of the human genome. This is an independent confirmation of the concentration gradient of CpG islands (detected as HpaII tiny fragments, or HTF) which was demonstrated in the genome of warm-blooded vertebrates [A?ssani and Bernardi, Gene 106 (1991) 173-183]. We then reassessed the location of CpG islands using the data currently available and confirmed that CpG islands are most frequently located in the 5'-flanking sequences of genes and that they overlap genes to variable extents. We have shown that such extents increase with the increasing GC levels of genes, the GC-richest genes being completely included in CpG islands. Under such circumstances, we have investigated the properties of the 'extragenic' CpG islands located in the 5'-flanking segments of homologous genes from both warm- and cold-blooded vertebrates. We have confirmed that, in cold-blooded vertebrates, CpG islands are often absent; when present, they have lower GC and CpG levels; the latter attain, however, statistically expected values. Finally, we have shown that CpG doublets increase with the increasing GC of exons, introns and intergenic sequences (including 'extragenic' CpG islands) in the genomes from both warm- and cold-blooded vertebrates. The correlations found are the same for both classes of vertebrates, and are similar for exons, introns and intergenic sequences (including 'extragenic' CpG islands). The findings just outlined indicate that the origin and evolution of CpG islands in the vertebrate genome are associated with compositional transitions (GC increases) in genes and isochores.     

Patterns of Vertebrate Isochore Evolution Revealed by Comparison of Expressed Mammalian,Avian, and Crocodilian Genes

Vertebrate genomes are mosaics of isochores, defined as long (>100 kb) regions with relatively homogeneous within-region base composition. Birds and mammals have more GC-rich isochores than amphibians and fish, and the GC-rich isochores of birds and mammals have been suggested to be an adaptation to homeothermy. If this hypothesis is correct, all poikilothermic (cold-blooded) vertebrates, including the nonavian reptiles, are expected to lack a GC-rich isochore structure. Previous studies using various methods to examine isochore structure in crocodilians, turtles, and squamates have led to different conclusions. We collected more than 6000 expressed sequence tags (ESTs) from the American alligator to overcome sample size limitations suggested to be the fundamental problem in the previous reptilian studies. The alligator ESTs were assembled and aligned with their human, mouse, chicken, and western clawed frog orthologs, resulting in 366 alignments. Analyses of third-codon-position GC content provided conclusive evidence that the poikilothermic alligator has GC-rich isochores, like homeothermic birds and mammals. We placed these results in a theoretical framework able to unify available models of isochore evolution. The data collected for this study allowed us to reject the models that explain the evolution of GC content using changes in body temperature associated with the transition from poikilothermy to homeothermy. Falsification of these models places fundamental constraints upon the plausible pathways for the evolution of isochores. Electronic supplementary material The online version of this article (doi: ) contains supplementary material, which is available to authorized users. Reviewing Editor: Dr. Nicolas Galtier     

Changes in body temperature pattern in vertebrates do not influence the codon usages of alpha-globin genes

Codon usages are known to vary among vertebrates chiefly due to variations in isochore structure. Under the assumption that marked differences exist in isochore structure between warm-blooded and cold-blooded animals, the variations among vertebrates were previously attributed to an adaptation to homeothermy. However, based on data from a turtle species and a crocodile (Archosauromorpha), it was recently proposed that the common ancestors of mammals, birds and extent reptiles already had the "warm-blooded" isochore structure. We determined the nucleotide sequences of alpha-globin genes from two species of heterotherms, cuckoo (Cuculus canorus) and bat (Pipistrellus abramus), and three species of snakes (Lepidosauromorpha), Naja kaouthia from a tropical terrestrial habitat, Elaphe climacophora from a temperate terrestrial habitat, and Hydrophis melanocephalus from a tropical marine habitat. Our purposes were to assess the influence of differential body temperature patterns on codon usage and GC content at the third position of a codon (GC3), and to test the hypothesis concerning the phylogenetic position at which GC contents had increased in vertebrates. The results of principal component analysis (PCA) using the present data and data for other taxa from GenBank indicate that the primary difference in codon usage in globin genes among amniotes and other vertebrates lies in GC3. The codon usages (and GC3) in alpha-globin genes from two heterotherms and three snakes are similar to those in alpha-globin genes from warm-blooded vertebrates. These results refute the influence of body temperature pattern upon codon usages (and GC3) in alpha-globin genes, and support the hypothesis that the increase in GC content in the genome occurred in the common ancestor of amniotes.     

Diversity in isochore structure among cold-blooded vertebrates based on GC content of coding and non-coding sequences

To review the general consideration about the different compositional structure of warm and cold-blooded vertebrates genomes, we used of the increasing number of genetic sequences, including coding (exons) and non-coding (introns) regions, that have been deposited on the databases throughout last years. The nucleotide distributions of the third codon positions (GC3) have been analyzed in 1510 coding sequences (CDS) of fish, 1414 CDS of amphibians and 320 CDS of reptiles. Also, the relationship between GC content of 74, 56 and 25 CDS of fish, amphibians and reptiles, respectively and that of their corresponding introns (GCI) have been considerated. In accordance with recent data, sequence analysis showed the presence of very GC3-rich CDS in these poikilotherm vertebrates. However, very high diversity in compositional patterns among different orders of fish, amphibians and reptiles was found. Significant positive correlations between GC3 and GCI was also confirmed for the genes analyzed. Nevertheless, introns resulted to be poorer in GC than their corresponding CDS, this difference being larger than in human genome. Because the limited number of available sequences including exons and introns we must be cautious about the results derived from them. However, the indicious of higher GC richness of coding sequences than of their corresponding introns could aid to understand the discrepancy of sequence analysis with the ultracentrifugation studies in cold-blooded vertebrates that did not predict the existence of GC-rich isochores.     

Analysis of the phylogenetic distribution of isochores in vertebrates and a test of the thermal stability hypothesis

Warm-blooded vertebrates show large-scale variation in G + C content along their chromosomes, a pattern which appears to be largely absent from cold-blooded vertebrates. However, compositional variation in poikilotherms has generally been studied by ultracentrifugation rather than sequence analysis. In this paper, we investigate the compositional properties of coding sequences from a broad range of vertebrate poikilotherms using DNA sequence analysis. We find that on average poikilotherms have lower third-codon position GC contents (GC3) than homeotherms but that some poikilotherms have higher mean GC3 values. We find that most poikilotherms have lower variation in GC3 than homeotherms but that there is a correlation between GC12 and GC3 for some species, indicating that there is systematic variation in base composition across their genomes. We also demonstrate that the GC3 of genes in the zebrafish, Danio rerio, is correlated with that in humans, suggesting that vertebrates share a basic isochore structure. However, we find no correlation between either the mean GC3 or the standard deviation in GC3 and body temperature.     

Compositional properties of nuclear genes from cold-blooded vertebrates

Summary We have investigated the compositional properties of coding sequences from cold-blooded vertebrates and we have compared them with those from warm-blooded vertebrates. Moreover, we have studied the compositional correlations of coding sequences with the genomes in which they are contained, as well as the compositional correlations among the codon positions of the genes analyzed.The distribution of GC levels of the third codon positions of genes from cold-blooded vertebrates are distinctly different from those of warm-blooded vertebrates in that they do not reach the high values attained by the latter. Moreover, coding sequences from cold-blooded vertebrates are either equal, or, in most cases, lower in GC (not only in third, but also in first and second codon positions) than homologous coding sequences from warm-blooded vertebrates; higher values are exceptional. These results at the gene level are in agreement with the compositional differences between cold-blooded and warm-blooded vertebrates previously found at the whole genome (DNA) level (Bernardi and Bernardi 1990a,b).Two linear correlations were found: one between the GC levels of coding sequences (or of their third codon positions) and the GC levels of the genomes of cold-blooded vertebrates containing them; and another between the GC levels of third and first+ second codon positions of genes from cold-blooded vertebrates. The first correlation applies to the genomes (or genome compartments) of all vertebrates and the second to the genes of all living organisms. These correlations are tantamount to a genomic code.     

Isochores and the evolutionary genomics of vertebrates

The nuclear genomes of vertebrates are mosaics of isochores, very long stretches (>300kb) of DNA that are homogeneous in base composition and are compositionally correlated with the coding sequences that they embed. Isochores can be partitioned in a small number of families that cover a range of GC levels (GC is the molar ratio of guanine+cytosine in DNA), which is narrow in cold-blooded vertebrates, but broad in warm-blooded vertebrates. This difference is essentially due to the fact that the GC-richest 10-15% of the genomes of the ancestors of mammals and birds underwent two independent compositional transitions characterized by strong increases in GC levels. The similarity of isochore patterns across mammalian orders, on the one hand, and across avian orders, on the other, indicates that these higher GC levels were then maintained, at least since the appearance of ancestors of warm-blooded vertebrates. After a brief review of our current knowledge on the organization of the vertebrate genome, evidence will be presented here in favor of the idea that the generation and maintenance of the GC-richest isochores in the genomes of warm-blooded vertebrates were due to natural selection.     

Evolution of base composition in the insulin and insulin-like growth factor genes

The genomes of homeothermic (warm-blooded) vertebrates are mosaic interspersions of homogeneously GC-rich and GC-poor regions (isochores). Evolution of genome compartmentalization and GC-rich isochores is hypothesized to reflect either selective advantages of an elevated GC content or chromosome location and mutational pressure associated with the timing of DNA replication in germ cells. To address the present controversy regarding the origins and maintenance of isochores in homeothermic vertebrates, newly obtained as well as published nucleotide sequences of the insulin and insulin-like growth factor (IGF) genes, members of a well-characterized gene family believed to have evolved by repeated duplication and divergence, were utilized to examine the evolution of base composition in nonconstrained (flanking) and weakly constrained (introns and fourfold degenerate sites) regions. A phylogeny derived from amino acid sequences supports a common evolutionary history for the insulin/IGF family genes. In cold- blooded vertebrates, insulin and the IGFs were similar in base composition. In contrast, insulin and IGF-II demonstrate dramatic increases in GC richness in mammals, but no such trend occurred in IGF- I. Base composition of the coding portions of the insulin and IGF genes across vertebrates correlated (r = 0.90) with that of the introns and flanking regions. The GC content of homologous introns differed dramatically between insulin/IGF-II and IGF-I genes in mammals but was similar to the GC level of noncoding regions in neighboring genes. Our findings suggest that the base composition of introns and flanking regions is determined by chromosomal location and the mutational pressure of the isochore in which the sequences are embedded. An elevated GC content at codon third positions in the insulin and the IGF genes may reflect selective constraints on the usage of synonymous codons.      

LDH-A and alpha-actin as tools to assess the effects of temperature on the vertebrate genome: some problems

In a recent paper written with the purpose of shedding light on the question of whether genomic GC levels are related to temperature in vertebrates, Ream et al. [Mol. Biol. Evol. 20 (2003) 105] offered an analysis of two sets of homologous genes: those coding for alpha-actin and lactate dehydrogenase-A (LDH-A). The conclusion was that "there is no consistent relationship between adaptation temperature and the percentage of thermal stability-enhancing G+C base pairs in protein-coding genes". We argue here that the data presented neither prove nor suggest such a conclusion because of conceptual and methodological errors.     

CpG islands: features and distribution in the genomes of vertebrates

We have investigated the distribution of unmethylated CpG islands in vertebrate genomes fractionated according to their base composition. Genomes from warm-blooded vertebrates (man, mouse and chicken) are characterized by abundant CpG islands, whose frequency increases in DNA fractions of increasing % of guanine + cytosine; % G + C (GC), in parallel with the distribution of genes and CpG doublets. Small, yet significant, differences in the distribution of CpG islands were found in the three genomes. In contrast, genomes from cold-blooded vertebrates (two reptiles, one amphibian, and two fishes) were characterized by an extreme scarcity or absence of CpG islands (detected in these experiments as HpaII tiny fragments or HTF). CpG islands associated with homologous genes from cold- and warm-blooded vertebrates were then compared by analyzing CpG frequencies, GC levels, HpaII sites, rare-cutter sites and G/C boxes (GGGGCGGGGC and closely related motifs) in sequences available in gene banks. Small, yet significant, differences were again detected among the CpG islands associated with homologous genes from warm-blooded vertebrates, in that CpG islands associated with mouse or rat genes often showed low CpG and/or GC levels, as well as low numbers of HpaII sites, rare-cutter sites and G/C boxes, compared to homologous human genes; more rarely, CpG islands were just absent. As far as cold-blooded vertebrates were concerned, a number of genes showed CpG islands, which exhibited a much lower frequency of CpG doublets than that found in CpG islands of warm-blooded vertebrates, but still approached the statistically expected frequency; none of the other features of CpG islands associated with genes from warm-blooded vertebrates were present. Other genes did not show any associated CpG islands, unlike their homologues from warm-blooded vertebrates.     

Compositional properties and thermal adaptation of 18S rRNA in vertebrates

In order to investigate the influence of temperature on the GC level of the paired sequences of ribosomal 18S RNAs in vertebrates, we have studied their base composition in cold- and warm-blooded vertebrates using a stem-by-stem comparison. We observed that a number of stems of 18S ribosomal RNAs (rRNAs) are variable among species and that the majority of such stems are GC richer in warm-blooded than in cold-blooded vertebrates. We also constructed the secondary structures of the 18S rRNAs of a polar fish, a marsupial, and a monotreme to compare them with those of temperate/tropical fishes and of eutherians, respectively. In these cases, differences similar to those already mentioned were found. We conclude that there is a correlation between stem stability and body temperature even within the relatively limited temperature range of vertebrates.     

Gene conversion drives GC content evolution in mammalian histones

To examine the evolutionary influence of gene conversion on DNA base composition, I analysed an exhaustive dataset of histone paralogous genes from human and mouse. I show that those gene copies that belong to subfamilies of very similar sequences (presumably undergoing gene conversion) have a higher GC content than unique gene copies (presumably not undergoing gene conversion). Thus, it seems that gene conversion is a biased process that tends to increase the DNA GC content, a conclusion that has implications for the evolution of isochores in vertebrates.