Silent nucleotide substitutions and G+C content of some mitochondrial and bacterial genes

Summary The G+C content of DNA varies widely in different organisms, especially microorganisms. This variation is accompanied by changes in the nucleotide composition of silent positions in codons. (Silent positions are defined and explained in the text.) These changes are mostly neutral or near neutral, and appear to result from mutation pressure in the direction of increasing either A+T (AT pressure) or G+C(GC pressure) content. Variations in G+C content are also accompanied by substitutions at replacement positions in codons. These substituions produce changes in the amino acid content of homologous proteins. The examples studied were genes for 13 mitochondrial proteins in five species, and A and B genes for bacterial tryptophan synthase in four species.In microorganisms, varying AT and GC mutational pressures, presumably resulting from shifts in the DNA polymerase system, exert strong effects on molecular evolution by changing the G+C content of DNA. These effects may be greater than those of random drift. The effects of GC pressure on silent substitutions in the systems examined are several times as great as the effects on replacement substitutions.GC pressure is exerted on noncoding as well as coding regions in mitochondrial DNA. This is shown by the close correlation (correlation coefficient, 0.99) of the G+C content of the noncoding D loop of mitochondria with the G+C content of silent positions in the corresponding mitochondrial genes.     

Vanishing GC-rich isochores in mammalian genomes

To understand the origin and evolution of isochores-the peculiar spatial distribution of GC content within mammalian genomes-we analyzed the synonymous substitution pattern in coding sequences from closely related species in different mammalian orders. In primate and cetartiodactyls, GC-rich genes are undergoing a large excess of GC --> AT substitutions over AT --> GC substitutions: GC-rich isochores are slowly disappearing from the genome of these two mammalian orders. In rodents, our analyses suggest both a decrease in GC content of GC-rich isochores and an increase in GC-poor isochores, but more data will be necessary to assess the significance of this pattern. These observations question the conclusions of previous works that assumed that base composition was at equilibrium. Analysis of allele frequency in human polymorphism data, however, confirmed that in the GC-rich parts of the genome, GC alleles have a higher probability of fixation than AT alleles. This fixation bias appears not strong enough to overcome the large excess of GC --> AT mutations. Thus, whatever the evolutionary force (neutral or selective) at the origin of GC-rich isochores, this force is no longer effective in mammals. We propose a model based on the biased gene conversion hypothesis that accounts for the origin of GC-rich isochores in the ancestral amniote genome and for their decline in present-day mammals.     

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.      

Position-Associated GC Asymmetry of Gene Duplicates

It is well known that repositioning of a gene often exerts a strong impact on its own expression and whole development. Here we report the results of genome-wide analyses suggesting that repositioning may also radically change the evolutionary fate of gene duplicates. As an indicator of these changes, we used the GC content of gene pairs which originated by duplication. This indicator turned out to be duplicate-asymmetric, which means that genes in a pair differ significantly in GC content despite their apparent origin from a common ancestor. Such an asymmetry necessarily implies that after duplication two originally identical genes mutated in opposite directions—toward GC-rich and GC-poor content, respectively. In mammalian genomes, this trend is definitely associated with presumably methylated hypermutable CpG sites, and in a typical GC-asymmetric gene pair, its two member genes are embedded in GC-contrasting isochores. However, we unexpectedly found similar significant GC asymmetry in fish, fly, worm, and yeast. This means that neither methylation alone nor methylation in combination with isochores can be counted as a primary cause of the GC asymmetry; rather they represent specific realizations of some universal principle of genome evolution. Remarkably, genes from pairs with the greatest GC asymmetry tend to be on different chromosomes, suggesting that the mutational difference between gene duplicates is associated with translocation of a new gene to a different place in the genome, whereas GC symmetric pairs demonstrate the opposite tendency. A recently emerged extra gene copy is usually on the same chromosome as is its parent but quickly, by 0.05 substitution per synonymous site, either has perished or occupies a different chromosome. During this earliest posttranslocation period, the ratio of nonsynonymous/synonymous base substitutions is unusually high, suggesting a rapid adaptive evolution of novel functions. In a general context of evolution by gene duplication, our interpretation of this position-dependent GC asymmetry between duplicated genes is that evolution of redundant genes toward a new function has often been associated with their very early, postduplication repositioning in the genome, with a concomitant abrupt change in epigenetic control of tissue/stage-specific expression and an increase in the mutation rate. Of eight eukaryotic genomes studied, the most distinguished in this respect is the human genome.Reviewing Editor: Dr. Manyuan Long     

Can mutation or fixation biases explain the allele frequency distribution of human single nucleotide polymorphisms (SNPs)?

One of the most abiding controversies in evolutionary biology concerns the role of neutral processes in molecular evolution. A main focus of the debate has been the evolution of isochores, the strong and systematic variation of base composition in mammalian genomes. One set of hypotheses argue that regions of similar GC are owing to localised mutational biases coupled with neutral evolution. The alternatives point to either selection or biased gene conversion as mechanisms to preferentially remove A or T bases, favouring G and C instead. Using a novel method, we compare models including such fixation biases to models based on mutation bias alone, under the assumption that non-coding, non-repetitive human DNA is at compositional equilibrium. While failing to fully explain the allele frequency distributions of recent single nucleotide polymorphism data, we show that the data are best fitted if the mutation bias is assumed to be constant across the genome, while fixation bias varies with GC content. We also attempt to estimate the strength of fixation bias, which increases linearly with increasing GC. Our approximation suggests that this force exists within the necessary parameter range: it is not so weak as to be drowned by random drift, but not so strong as to lead to exclusive use of G and C alone. Together these results demonstrate that mutation bias fails to explain the evolution of isochores, and suggest that either selection or biased gene conversion are involved.     

Distinct changes of genomic biases in nucleotide substitution at the time of Mammalian radiation

Differences in the regional substitution patterns in the human genome created patterns of large-scale variation of base composition known as genomic isochores. To gain insight into the origin of the genomic isochores, we develop a maximum-likelihood approach to determine the history of substitution patterns in the human genome. This approach utilizes the vast amount of repetitive sequence deposited in the human genome over the past approximately 250 Myr. Using this approach, we estimate the frequencies of seven types of substitutions: the four transversions, two transitions, and the methyl-assisted transition of cytosine in CpG. Comparing substitutional patterns in repetitive elements of various ages, we reconstruct the history of the base-substitutional process in the different isochores for the past 250 Myr. At around 90 MYA (around the time of the mammalian radiation), we find an abrupt fourfold to eightfold increase of the cytosine transition rate in CpG pairs compared with that of the reptilian ancestor. Further analysis of nucleotide substitutions in regions with different GC content reveals concurrent changes in the substitutional patterns. Although the substitutional pattern was dependent on the regional GC content in such ways that it preserved the regional GC content before the mammalian radiation, it lost this dependence afterward. The substitutional pattern changed from an isochore-preserving to an isochore-degrading one. We conclude that isochores have been established before the radiation of the eutherian mammals and have been subject to the process of homogenization since then.     

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     

Mutational pressure in genomes of alphaherpesviruses infecting human

Genomes of the herpes simplex viruses are extremely enriched with GC. Elevated G+C level in genomes of the simplex viruses is a result of their long-term evolution under the influence of the mutational pressure. We counted the rates of nucleotide substitutions from gene coding major capsid protein (MCP) (G+C = 0.68, 3GC = 0.89) of human simplex virus 1 (HSV-1) to the MCP gene (G+C = 0.70, 3GC = 0.91) of HSV-2 (the first pair of genes) and from the same MCP gene of HSV-1 to the homologous gene (G+C = 0.73, 3GC = 0.99) from cercopithecine herpes virus 16 (the second pair of genes). The rates of transitions from A-T to G-C base pairs increases 2.17-, 3.09-, and 1.27-fold in the first, second, and third codon positions, respectively, if compared those rates between the second and first pair of genes (the growth of GC-richness is only 3%). This effect is due to an approximately 90% GC-richness of the third codon positions in all those genes. Transitions caused by the strong mutational pressure (from A-T to G-C base pairs) have a low probability to occur in the third positions, but high probability to occur in the first and second positions. For MCP gene of human herpes 3, the probability of the occurrence of transition caused by mutational pressure in the third codon position is 2.36 times higher than in MCP gene of HSV1, and 3 times higher than in MCP gene of HSV2. These data could provide an explanation of rarely occurring relapses of herpes Zoster infection and frequently occurring relapses of herpes simplex infection.     

Codon usage changes and sequence dissimilarity between human and rat

Summary This paper reports on the relationship between the number of silent differences and the codon usage changes in the lineages leading to human and rat. Examination of 102 pairs of homologous genes gives rise to four main conclusions: (1) We have previously demonstrated the existence of a codon usage change (called the minor shift) between human and rat; this was confirmed here with a larger sample. For genes with extreme C+G frequencies, the C+G level in the third codon position is less extreme in rat than in human. (2) Protein similarity and percentage of positive differences are the two main factors that discriminate homologous genes when characterized by differences between rat and human. By definition, positive differences result from silent changes between A or T and C or G with a direction implying a C+G content variation in the same direction as the overall gene variation. (3) For genes showing both codon usage change and low protein similarity, a majority of amino acid replacements contributes to C+G level variation in positions I and II in the same direction as the variation in position III. This is thus a new example of protein evolution due to constraints acting at the DNA level. (4) In heavy isochores (high C+G content) no direct correlation exists between codon usage change (measured by the dissymmetry of differences) and silent dissimilarity. In light isochores the opposite situation is observed: modification of codon usage is associated with a high synonymous dissimilarity. This result shows that, in some cases, modification of constraints acting at the DNA level could accelerate divergence between genomes.     

DNA strand biases and the mutational resilience of genes

This paper quantifies the resilience of a gene to each class of base substitution. The resilience of a gene is defined as the set of probabilities of synonymous base substitution (one for each type of base substitution on each DNA strand), and is derived from the fraction of all possible substitutions which result in no change of encoded amino acids. We discuss the resilience of the common mutational target genes, lacI and hypoxanthine-guanine phosphoribosyltransferase (hprt), and the p53 tumour suppressor gene. There are inherent strand biases to mutation in terms of the resilience differences between the non-template and template DNA strands. The ability to quantify resilience differences between the two DNA strands contributes to our understanding of strand bias to mutation.     

Evidence of distinct gene functional patterns in GC-poor and GC-rich isochores in Bos taurus

Vertebrate genomes are mosaics of megabase-size DNA segments with a fairly homogeneous base composition, called isochores. They are divided into five families characterized by different guanine-cytosine (GC) levels and linked to several functional and structural properties. The increased availability of fully sequenced genomes allows the investigation of isochores in several species, assessing their level of conservation across vertebrate genomes. In this work, we characterized the isochores in Bos taurus using the ARS-UCD1.2 genome version. The comparison of our results with the well-studied human isochores and those of other mammals revealed a large conservation in isochore families, in number, average GC levels and gene density. Exceptions to the established increase in gene density with the increase in isochores (GC%) were observed for the following gene biotypes: tRNA, small nuclear RNA, small nucleolar RNA and pseudogenes that have their maximum number in H2 and H1 isochores. Subsequently, we assessed the ontology of all gene biotypes looking for functional classes that are statistically over- or under-represented in each isochore. Receptor activity and sensory perception pathways were significantly over-represented in L1 and L2 (GC-poor) isochores. This was also validated for the horse genome. Our analysis of housekeeping genes confirmed a preferential localization in GC-rich isochores, as reported in other species. Finally, we assessed the SNP distribution of a bovine high-density SNP chip across the isochores, finding a higher density in the GC-rich families, reflecting a potential bias in the chip, widely used for genetic selection and biodiversity studies.     

Correlations between the compositional properties of human genes,codon usage,and amino acid composition of proteins

Summary We have analyzed the correlation that exists between the GC levels of third and first or second codon position for about 1400 human coding sequences. The linear relationship that was found indicates that the large differences in GC level of third codon positions of human genes are paralleled by smaller differences in GC levels of first and second codon positions. Whereas third codon position differences correspond to very large differences in codon usage within the human genome, the first and second codon position differences correspond to smaller, yet very remarkable, differences in the amino acid composition of encoded proteins. Because GC levels of codon positions are linearly correlated with the GC levels of the isochores harboring the corresponding genes, both codon usage and amino acid composition are different for proteins encoded by genes located in isochores of different GC levels. Furthermore, we have also shown that a linear relationship with a unity slope and a correlation coefficient of 0.77 exists between GC levels of introns and exons from the 238 human genes currently available for this analysis. Introns are, however, about 5% lower in GC, on average, than exons from the same genes.     

Rates of nucleotide substitution and mammalian nuclear gene evolution. Approximate and maximum-likelihood methods lead to different conclusions

Rates and patterns of synonymous and nonsynonymous substitutions have important implications for the origin and maintenance of mammalian isochores and the effectiveness of selection at synonymous sites. Previous studies of mammalian nuclear genes largely employed approximate methods to estimate rates of nonsynonymous and synonymous substitutions. Because these methods did not account for major features of DNA sequence evolution such as transition/transversion rate bias and unequal codon usage, they might not have produced reliable results. To evaluate the impact of the estimation method, we analyzed a sample of 82 nuclear genes from the mammalian orders Artiodactyla, Primates, and Rodentia using both approximate and maximum-likelihood methods. Maximum-likelihood analysis indicated that synonymous substitution rates were positively correlated with GC content at the third codon positions, but independent of nonsynonymous substitution rates. Approximate methods, however, indicated that synonymous substitution rates were independent of GC content at the third codon positions, but were positively correlated with nonsynonymous rates. Failure to properly account for transition/transversion rate bias and unequal codon usage appears to have caused substantial biases in approximate estimates of substitution rates.     

The Decline of Isochores in Mammals: An Assessment of the GC ContentVariation Along the Mammalian Phylogeny

Whether isochores, the large-scale variation of the GC content in mammalian genomes, are being maintained has recently been questioned. It has been suggested that GC-rich isochores originated in the ancestral amniote genome but that whatever force gave rise to them is no longer effective and that isochores are now disappearing from mammalian genomes. Here we investigated the evolution of the GC content of 41 coding genes in 6 to 66 species of mammals by estimating the ancestral GC content using a method which allows for different rates of substitution between sites. We found a highly significant decrease in the GC content during early mammalian evolution, as well as a weaker but still significant decrease in the GC content of GC-rich genes later in at least three groups of mammals: primates, rodents, and carnivores. These results are of interest because they confirm the recently suggested disappearance of GC-rich isochores in some mammalian genomes, and more importantly, they suggest that this disappearance started very early in mammalian evolution.This article contains online supplementary material.     

The rate, not the spectrum, of base pair substitutions changes at a GC-content transition in the human NF1 gene region: implications for the evolution of the mammalian genome structure

The human genome is composed of long stretches of DNA with distinct GC contents, called isochores or GC-content domains. A boundary between two GC-content domains in the human NF1 gene region is also a boundary between domains of early- and late-replicating sequences and of regions with high and low recombination frequencies. The perfect conservation of the GC-content distribution in this region between human and mouse demonstrates that GC-content stabilizing forces must act regionally on a fine scale at this locus. To further elucidate the nature of these forces, we report here on the spectrum of human SNPs and base pair substitutions between human and chimpanzee. The results show that the mutation rate changes exactly at the GC-content transition zone from low values in the GC-poor sequences to high values in GC-rich ones. The GC content of the GC-poor sequences can be explained by a bias in favor of GC > AT mutations, whereas the GC content of the GC-rich segment may result from a fixation bias in favor of AT > GC substitutions. This fixation bias may be explained by direct selection by the GC content or by biased gene conversion.     

SAIVGeM: spreadsheet analysis of immunoglobulin VH gene mutations

The analysis of mutations in immunoglobulin heavy chain variable (IGHV) region genes is a tedious process when performed by hand on multiple sequences. This report describes a set of linked Microsoft Excel files that perform several common analyses on large numbers of IGHV sequences. The spreadsheet analysis of immunoglobulin VH gene mutations (SAIVGeM) package determines the distribution of mutations among each nucleotide, the nature of the mutation at both the nucleotide and amino acid level, the frequency of mutation in the A/G G C/T A/T (RGYW) hotspot motifs of both strand polarity, and the distribution of replacement and silent mutations among the complementarity determining regions (CDRs) and the framework regions (FRs) of the immunoglobulin gene as defined by either the Kabat or IMGT conventions. These parameters are summarized and graphically presented where appropriate. In addition, the SAIVGeM package analyzes those mutations that occur in third positions of redundant codons. Because any nucleotide change in these positions is inherently silent, these positions can be used to study the mutational spectra without biases from the selection of protein structure.     

The elevated GC content at exonic third sites is not evidence against neutralist models of isochore evolution

The human genome is divided into isochores, large stretches (>300 kb) of genomic DNA with more or less consistent GC content. Mutational/neutralist and selectionist models have been put forward to explain their existence. A major criticism of the mutational models is that they cannot account for the higher GC content at fourfold-redundant silent sites within exons (GC4) than in flanking introns (GCi). Indeed, it has been asserted that it is hard to envisage a mutational bias explanation, as it is difficult to see how repair enzymes might act differently in exons and their flanking introns. However, this rejection, we note, ignores the effects of transposable elements (TEs), which are a major component of introns and tend to cause them to have a GC content different from (usually lower than) that dictated by point mutational processes alone. As TEs tend not to insert at the extremities of introns, this model predicts that GC content at the extremities of introns should be more like that at GC4 than are the intronic interiors. This we show to be true. The model also correctly predicts that small introns should have a composition more like that at GC4 than large introns. We conclude that the logic of the previous rejection of neutralist models is unsafe.     

Evidence of selection on silent site base composition in mammals: potential implications for the evolution of isochores and junk DNA.

It has been suggested that mutation bias is the major determinant of base composition bias at synonymous, intron, and flanking DNA sites in mammals. Here I test this hypothesis using population genetic data from the major histocompatibility genes of several mammalian species. The results of two tests are inconsistent with the mutation hypothesis in coding, noncoding, CpG-island, and non-CpG-island DNA, but are consistent with selection or biased gene conversion. It is argued that biased gene conversion is unlikely to affect silent site base composition in mammals. The results therefore suggest that selection is acting upon silent site G + C content. This may have broad implications, since silent site base composition reflects large-scale variation in G + C content along mammalian chromosomes. The results therefore suggest that selection may be acting upon the base composition of isochores and large sections of junk DNA.     

Isochore patterns and gene distributions in fish genomes

The compositional approach developed in our laboratory many years ago revealed a large-scale compositional heterogeneity in vertebrate genomes, in which GC-rich and GC-poor regions, the isochores, were found to be characterized by high and low gene densities, respectively. Here we mapped isochores on fish chromosomes and assessed gene densities in isochore families. Because of the availability of sequence data, we have concentrated our investigations on four species, zebrafish (Brachydanio rerio), medaka (Oryzias latipes), stickleback (Gasterosteus aculeatus), and pufferfish (Tetraodon nigroviridis), which belong to four distant orders and cover almost the entire GC range of fish genomes. These investigations produced isochore maps that were drastically different not only from those of mammals (in that only two major isochore families were essentially present in each genome vs five in the human genome) but also from each other (in that different isochore families were represented in different genomes). Gene density distributions for these fish genomes were also obtained and shown to follow the expected increase with increasing isochore GC. Finally, we discovered a remarkable conservation of the average size of the isochores (which match replicon clusters in the case of human chromosomes) and of the average GC levels of isochore families in both fish and human genomes. Moreover, in each genome the GC-poorest isochore families comprised a group of "long isochores" (2-20 Mb in size), which were the lowest in GC and varied in size distribution and relative amount from one genome to the other.     

Compositional heterogeneity within and among isochores in mammalian genomes. II. Some general comments

The presence of long-range correlations and/or mosaicism in DNA sequences results in GC fluctuations, even within individual isochores that are much larger than expected correlation-free 'random' sequences. Neglecting the presence of such fluctuations can lead to incorrect conclusions regarding relative homogeneity or isochore borders. In this commentary, we address these and other methodological issues raised by the variations in GC level within human isochores. We also discuss some recent misconceptions.