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.     

Heterogeneity in regional GC content and differential usage of codons and amino acids in GC-poor and GC-rich regions of the genome of Apis mellifera

The honeybee (Apis mellifera) has a genome with a wide variation in GC content showing 2 clear modal GC values, in some ways reminiscent of an isochore-like structure. To gain insight into causes and consequences of this pattern, we used a comparative approach to study the genome-wide alignment of primarily coding sequence of A. mellifera with Drosophila melanogaster and Anopheles gambiae. The latter 2 species show a higher average GC content than A. mellifera and no indications of bimodality, suggesting that the GC-poor mode is a derived condition in honeybee. In A. mellifera, synonymous sites of genes generally adopt the GC content of the region in which they reside. A large proportion of genes in GC-poor regions have not been assigned to the honeybee assembly because of the low sequence complexity of their genome neighborhood. The synonymous substitution rate between A. mellifera and the other species is very close to saturation, but analyses of nonsynonymous substitutions as well as amino acid substitutions indicate that the GC-poor regions are not evolving faster than the GC-rich regions. We describe the codon usage and amino acid usage and show that they are remarkably heterogeneous within the honeybee genome between the 2 different GC regions. Specifically, the genes located in GC-poor regions show a much larger deviation in both codon usage bias and amino acid usage from the Dipterans than the genes located in the GC-rich regions.     

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.     

GC level and expression of human coding sequences

Several groups have addressed the issue of the influence of GC on expression levels in mammalian genes. In general, GC-rich genes appeared to be more expressed than GC-poor ones. Recently, expression levels of GC₃-rich and GC₃-poor versions of genes (GC₃ is the third codon position GC), inserted in vector plasmids, were compared in order to eliminate differences associated with their genomic context. Transfection experiments showed that GC₃-rich genes were expressed more efficiently than their GC₃-poor counterparts, indicating that GC₃ dramatically and intrinsically boosts expression efficiency. Here we show that, while the protocols used eliminated the original genomic context, they replaced it with the plasmid contexts whose compositional properties affected the results.     

Turtle isochore structure is intermediate between amphibians and other amniotes

Vertebrate genomes are comprised of isochores that are relatively long (>100 kb) regions with a relatively homogenous (either GC-rich or AT-rich) base composition and with rather sharp boundaries with neighboring isochores. Mammals and living archosaurs (birds and crocodilians) have heterogeneous genomes that include very GC-rich isochores. In sharp contrast, the genomes of amphibians and fishes are more homogeneous and they have a lower overall GC content. Because DNA with higher GC content is more thermostable, the elevated GC content of mammalian and archosaurian DNA has been hypothesized to be an adaptation to higher body temperatures. This hypothesis can be tested by examining structure of isochores across the reptilian clade, which includes the archosaurs, testudines (turtles), and lepidosaurs (lizards and snakes), because reptiles exhibit diverse body sizes, metabolic rates, and patterns of thermoregulation. This study focuses on a comparative analysis of a new set of expressed genes of the red-eared slider turtle and orthologs of the turtle genes in mammalian (human, mouse, dog, and opossum), archosaurian (chicken and alligator), and amphibian (western clawed frog) genomes. EST (expressed sequence tag) data from a turtle cDNA library enriched for genes that have specialized functions (developmental genes) revealed using the GC content of the third-codon-position to examine isochore structure requires careful consideration of the types of genes examined. The more highly expressed genes (e.g., housekeeping genes) are more likely to be GC-rich than are genes with specialized functions. However, the set of highly expressed turtle genes demonstrated that the turtle genome has a GC content that is intermediate between the GC-poor amphibians and the GC-rich mammals and archosaurs. There was a strong correlation between the GC content of all turtle genes and the GC content of other vertebrate genes, with the slope of the line describing this relationship also indicating that the isochore structure of turtles is intermediate between that of amphibians and other amniotes. These data are consistent with some thermal hypotheses of isochore evolution, but we believe that the credible set of models for isochore evolution still includes a variety of models. These data expand the amount of genomic data available from reptiles upon which future studies of reptilian genomics can build.     

Compositional compartmentalization and gene composition in the genome of vertebrates

Summary The compositional distribution of coding sequences from five vertebrates (Xenopus, chicken, mouse, rat, and human) is shifted toward higher GC values compared to that of the DNA molecules (in the 35–85-kb size range) isolated from the corresponding genomes. This shift is due to the lower GC levels of intergenic sequences compared to coding sequences. In the cold-blooded vertebrate, the two distributions are similar in that GC-poor genes and GC-poor DNA molecules are largely predominant. In contrast, in the warm-blooded vertebrates, GC-rich genes are largely predominant over GC-poor genes, whereas GC-poor DNA molecules are largely predominant over GC-rich DNA molecules. As a consequence, the genomes of warm-blooded vertebrates show a compositional gradient of gene concentration. The compositional distributions of coding sequences (as well as of DNA molecules) showed remarkable differences between chicken and mammals, and between mouse (or rat) and human. Differences were also detected in the compositional distribution of housekeeping and tissue-specific genes, the former being more abundant among GC-rich genes.     

Nature of selective constraints on synonymous codon usage of rice differs in GC-poor and GC-rich genes

Synonymous codon usage and cellular tRNA abundance are thought to be co-evolved in optimizing translational efficiencies in highly expressed genes. Here in this communication by taking the advantage of publicly available gene expression data of rice and Arabidopsis we demonstrated that tRNA gene copy number is not the only driving force favoring translational selection in all highly expressed genes of rice. We found that forces favoring translational selection differ between GC-rich and GC-poor classes of genes. Supporting our results we also showed that, in highly expressed genes of GC-poor class there is a perfect correspondence between majority of preferred codons and tRNA gene copy number that confers translational efficiencies to this group of genes. However, tRNA gene copy number is not fully consistent with models of translational selection in GC-rich group of genes, where constraints on mRNA secondary structure play a role to optimize codon usage in highly expressed genes.     

Isochore specificity of AUG initiator context of human genes

The efficiency of AUG start codon recognition in translation initiation is modulated by its sequence context. Here we investigated a non-redundant set of 5914 human genes and show that this context is different in genes located in different isochores. In particular, of the two main consensus start sequences, RCCaugR is five-fold more represented than AARaugR in genes from the GC-rich H3 isochores compared to genes from the GC-poor L isochores. Furthermore, genes located in GC-rich isochores have shorter 5' UTRs and stronger avoidance of upstream AUG than genes located in GC-poor isochores. This suggests that genes requiring highly efficient translation are located in GC-rich isochores and genes requiring fine modulation of expression are located in GC-poor isochores. This is in agreement with independent data from the literature concerning the location of housekeeping and tissue-specific genes, respectively.     

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.     

Isochores and tissue-specificity

The housekeeping (ubiquitously expressed) genes in the mammal genome were shown here to be on average slightly GC-richer than tissue-specific genes. Both housekeeping and tissue-specific genes occupy similar ranges of GC content, but the former tend to concentrate in the upper part of the range. In the human genome, tissue-specific genes show two maxima, GC-poor and GC-rich. The strictly tissue-specific human genes tend to concentrate in the GC-poor region; their distribution is left-skewed and thus reciprocal to the distribution of housekeeping genes. The intermediately tissue-specific genes show an intermediate GC content and the right-skewed distribution. Both in the human and mouse, genes specific for some tissues (e.g., parts of the central nervous system) have a higher average GC content than housekeeping genes. Since they are not transcribed in the germ line (in contrast to housekeeping genes), and therefore have a lower probability of inheritable gene conversion, this finding contradicts the biased gene conversion (BGC) explanation for elevated GC content in the heavy isochores of mammal genome. Genes specific for germ-line tissues (ovary, testes) show a low average GC content, which is also in contradiction to the BGC explanation. Both for the total data set and for the most part of tissues taken separately, a weak positive correlation was found between gene GC content and expression level. The fraction of ubiquitously expressed genes is nearly 1.5-fold higher in the mouse than in the human. This suggests that mouse tissues are comparatively less differentiated (on the molecular level), which can be related to a less pronounced isochoric structure of the mouse genome. In each separate tissue (in both species), tissue-specific genes do not form a clear-cut frequency peak (in contrast to housekeeping genes), but constitute a continuum with a gradually increasing degree of tissue-specificity, which probably reflects the path of cell differentiation and/or an independent use of the same protein in several unrelated tissues.     

The maize gene space is compositionally compartimentalized

Previous investigations by Southern hybridization of cDNA with compositional DNA fractions showed that the majority of maize genes are located in a narrow GC range of DNA fragments and that the corresponding gene space was GC-richer than the region of the genome where zein genes are found. Here, we revisited the maize gene space using new data from the maize genome sequencing initiative. We found that the maize gene space itself is formed of two compositional compartments, i.e., a GC-poor and a GC-rich, characterized by a different distribution of Opie and Huck retrotransposons. The GC-rich compartment tends to be richer in GC-rich genes than the GC-poor compartment. However, the gene space compartimentalization of maize is much simpler than that of human.     

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.     

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     

Compositional perspectives on human brain aging

Using publicly available microarray data from the frontal cortex of 30 individuals, spanning the ages of 26-106 years old, we investigate the expression patterns of compositionally distinct genes during human brain aging. Our analyses revealed that at advance ages, GC-poor genes appear to be induced while GC-rich genes are repressed. Interestingly, investigations upon two different types of genes, named pivotal (permanently expressed genes) and non-pivotal (on-off regulated genes), revealed an induction of the GC-poor pivotal genes and a repression of the GC-richer non-pivotal genes at advanced ages. Summarizing, this study shows that genes with different compositional properties have opposite age-related expression patterns, suggesting an implication of different regulation mechanisms related to their localization in different chromatin structure, which correlates with the GC level. Finally, an innovative approach on investigating human aging process is suggested, which involves the base composition of genes.     

Unusual mammalian usage of TGA stop codons reveals that sequence conservation need not imply purifying selection

The assumption that conservation of sequence implies the action of purifying selection is central to diverse methodologies to infer functional importance. GC-biased gene conversion (gBGC), a meiotic mismatch repair bias strongly favouring GC over AT, can in principle mimic the action of selection, this being thought to be especially important in mammals. As mutation is GC→AT biased, to demonstrate that gBGC does indeed cause false signals requires evidence that an AT-rich residue is selectively optimal compared to its more GC-rich allele, while showing also that the GC-rich alternative is conserved. We propose that mammalian stop codon evolution provides a robust test case. Although in most taxa TAA is the optimal stop codon, TGA is both abundant and conserved in mammalian genomes. We show that this mammalian exceptionalism is well explained by gBGC mimicking purifying selection and that TAA is the selectively optimal codon. Supportive of gBGC, we observe (i) TGA usage trends are consistent at the focal stop codon and elsewhere (in UTR sequences); (ii) that higher TGA usage and higher TAA→TGA substitution rates are predicted by a high recombination rate; and (iii) across species the difference in TAA <-> TGA substitution rates between GC-rich and GC-poor genes is largest in genomes that possess higher between-gene GC variation. TAA optimality is supported both by enrichment in highly expressed genes and trends associated with effective population size. High TGA usage and high TAA→TGA rates in mammals are thus consistent with gBGC’s predicted ability to “drive” deleterious mutations and supports the hypothesis that sequence conservation need not be indicative of purifying selection. A general trend for GC-rich trinucleotides to reside at frequencies far above their mutational equilibrium in high recombining domains supports the generality of these results.

Is sequence conservation a sign of purifying selection and hence functional importance? This analysis of why mammals use and conserve the most error-prone stop codon suggests not, consistent with GC-biased gene conversion’s predicted ability to “drive” deleterious mutations and supporting the hypothesis that sequence conservation need not be indicative of purifying selection.     

An isochore transition zone in the NF1 gene region is a conserved landmark of chromosome structure and function

The mammalian genome is organized as a mosaic of isochores, stretches of DNA with a distinct sequence composition. Isochores form the basis of the chromosomal banding pattern, which is tightly correlated with a number of structural and functional features. We have recently demonstrated that the transition from a GC-poor isochore to a GC-rich one in the NF1 gene region occurs within 5 kb and demarcates genomic regions with high and low recombination frequency. We now report that the same transition zone separates early replicating from late replicating chromatin on the molecular level. At the isochore transition the replication fork is stalled in mid-S phase and can be visualized by fiber-FISH techniques as a Y-shaped structure. The switch in GC content and in replication timing is conserved between human and mouse, emphasizing the importance of the transition zones as landmarks of chromosome organization and function.