Evidence of Selection upon Genomic GC-Content in Bacteria

The genomic GC-content of bacteria varies dramatically, from less than 20% to more than 70%. This variation is generally ascribed to differences in the pattern of mutation between bacteria. Here we test this hypothesis by examining patterns of synonymous polymorphism using datasets from 149 bacterial species. We find a large excess of synonymous GC→AT mutations over AT→GC mutations segregating in all but the most AT-rich bacteria, across a broad range of phylogenetically diverse species. We show that the excess of GC→AT mutations is inconsistent with mutation bias, since it would imply that most GC-rich bacteria are declining in GC-content; such a pattern would be unsustainable. We also show that the patterns are probably not due to translational selection or biased gene conversion, because optimal codons tend to be AT-rich, and the excess of GC→AT SNPs is observed in datasets with no evidence of recombination. We therefore conclude that there is selection to increase synonymous GC-content in many species. Since synonymous GC-content is highly correlated to genomic GC-content, we further conclude that there is selection on genomic base composition in many bacteria.     

Bendable genes of warm-blooded vertebrates.

It is shown that in the genomes of warm-blooded vertebrates the elevation of genic GC-content is associated with an increase in the bendability of the DNA helix, which is both absolute and relative as compared with random sequences. This trend takes place both in exons and introns, being more pronounced in the latter. At the same time, the free energy of melting (delta G) of exons and introns increases only absolutely with elevation of GC-content, whereas it decreases as compared with random sequences (again, this trend is stronger in the introns). In genes of cold-blooded animals, plants, and unicellular organisms, these regularities are weaker and often not consistent. Generally, there is a negative correlation between bendability and melting energy at any fixed GC-content value. This effect is stronger in the introns. These findings suggest that GC-enrichment of genes in the homeotherm vertebrates can be caused by selection for increased bendability of DNA.     

The impact of recombination on nucleotide substitutions in the human genome

Unraveling the evolutionary forces responsible for variations of neutral substitution patterns among taxa or along genomes is a major issue for detecting selection within sequences. Mammalian genomes show large-scale regional variations of GC-content (the isochores), but the substitution processes at the origin of this structure are poorly understood. We analyzed the pattern of neutral substitutions in 1 Gb of primate non-coding regions. We show that the GC-content toward which sequences are evolving is strongly negatively correlated to the distance to telomeres and positively correlated to the rate of crossovers (R²=47%). This demonstrates that recombination has a major impact on substitution patterns in human, driving the evolution of GC-content. The evolution of GC-content correlates much more strongly with male than with female crossover rate, which rules out selectionist models for the evolution of isochores. This effect of recombination is most probably a consequence of the neutral process of biased gene conversion (BGC) occurring within recombination hotspots. We show that the predictions of this model fit very well with the observed substitution patterns in the human genome. This model notably explains the positive correlation between substitution rate and recombination rate. Theoretical calculations indicate that variations in population size or density in recombination hotspots can have a very strong impact on the evolution of base composition. Furthermore, recombination hotspots can create strong substitution hotspots. This molecular drive affects both coding and non-coding regions. We therefore conclude that along with mutation, selection and drift, BGC is one of the major factors driving genome evolution. Our results also shed light on variations in the rate of crossover relative to non-crossover events, along chromosomes and according to sex, and also on the conservation of hotspot density between human and chimp.     

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.     

An in-silico study of alphaherpesviruses ICP0 genes: positive selection or strong mutational GC-pressure?

The purpose of our work was to analyze the case of the strong mutational GC-pressure influence on the ratio between nonsynonymous (DN) and synonymous (DS) distances (DN/DS ratio). We have used as the material the genes coding for ICP0 from five completely sequenced genomes of simplexviruses. DN/DS ratio, total GC-content (G + C), and GC-content in first, second, and third codon positions (1GC, 2GC, and 3GC, respectively) have been calculated separately for exon 2, nonconserved part of exon 3, and conserved part of exon 3 from ICP0 genes. Results showed that DN is more than DS only in the conserved part of exon 3 of ICP0 genes from cercopithecine herpesvirus 2 and cercopithecine herpesvirus 16. However, the cause of this result (DN/DS = 2.54) is the GC-pressure acting on the coding districts with 3GC = 99% rather than the biological process called positive selection. Only in these two viruses, because of the strong GC-pressure, 3GC has reached 99% in the conserved part of ICP0 exon 3, and so nucleotide substitutions that increase the GC-content practically cannot occur in third codon positions, where most substitutions are synonymous. In this case, GC-pressure has a substrate for nucleotide substitutions only in first and second codon positions, where most substitutions are nonsynonymous.     

Percent of highly immunogenic amino acid residues forming B-cell epitopes is higher in homologous proteins encoded by GC-rich genes

We analyzed the dependence of the percent of highly immunogenic amino acid residues included in B-cell epitopes of homologous proteins on the GC-content (G+C) of genes coding for them in twenty-seven lineages of proteins (and subsequent genes), which belong to seven Varicello and five Simplex viruses. We found out that proteins encoded by genes of a high GC-content usually contain more targets for humoral immune response than their homologs encoded by GC-poor genes. This tendency is characteristic not only to the lineages of glycoproteins, which are the main targets for humoral immune response against Simplex and Varicello viruses, but also to the lineages of capsid proteins and even "housekeeping" enzymes. The percent of amino acids included in linear B-cell epitopes has been predicted for 324 proteins by BepiPred algorithm (www.cbs.dtu.dk/services/BepiPred), the percent of highly immunogenic amino acids included in discontinuous B-cell epitopes and the percent of exposed amino acid residues have been predicted by Epitopia algorithm (http://epitopia.tau.ac.il/). Immunological consequences of the directional mutational GC-pressure are mostly due to the decrease in the total usage of highly hydrophobic amino acids and due to the increase in proline and glycine levels of usage in proteins. The weaker the negative selection on amino acid substitutions caused by symmetric mutational pressure, the higher the slope of direct dependence of the percent of highly immunogenic amino acids included in B-cell epitopes on G+C.     

Stabilization of secondary structure elements by specific combinations of hydrophilic and hydrophobic amino acid residues is more important for proteins encoded by GC-poor genes

Stabilization of secondary structure elements by specific combinations of hydrophobic and hydrophilic amino acids has been studied by the way of analysis of pentapeptide fragments from twelve partial bacterial proteomes. PDB files describing structures of proteins from species with extremely high and low genomic GC-content, as well as with average G + C were included in the study. Amino acid residues in 78,009 pentapeptides from alpha helices, beta strands and coil regions were classified into hydrophobic and hydrophilic ones. The common propensity scale for 32 possible combinations of hydrophobic and hydrophilic amino acid residues in pentapeptide has been created: specific pentapeptides for helix, sheet and coil were described. The usage of pentapeptides preferably forming alpha helices is decreasing in alpha helices of partial bacterial proteomes with the increase of the average genomic GC-content in first and second codon positions. The usage of pentapeptides preferably forming beta strands is increasing in coil regions and in helices of partial bacterial proteomes with the growth of the average genomic GC-content in first and second codon positions. Due to these circumstances the probability of coil-sheet and helix-sheet transitions should be increased in proteins encoded by GC-rich genes making them prone to form amyloid in certain conditions. Possible causes of the described fact that importance of alpha helix and coil stabilization by specific combinations of hydrophobic and hydrophilic amino acids is growing with the decrease of genomic GC-content have been discussed.     

RNA structures facilitate recombination-mediated gene swapping in HIV-1

Many viruses, including retroviruses, undergo frequent recombination, a process which can increase their rate of adaptive evolution. In the case of HIV, recombination has been responsible for the generation of numerous intersubtype recombinant variants with epidemiological importance in the AIDS pandemic. Although it is known that fragments of genetic material do not combine randomly during the generation of recombinant viruses, the mechanisms that lead to preferential recombination at specific sites are not fully understood. Here we reanalyze recent independent data defining (i) the structure of a complete HIV-1 RNA genome and (ii) favorable sites for recombination. We show that in the absence of selection acting on recombinant genomes, regions harboring RNA structures in the NL4-3 model strain are strongly predictive of recombination breakpoints in the HIV-1 env genes of primary isolates. In addition, we found that breakpoints within recombinant HIV-1 genomes sampled from human populations, which have been acted upon extensively by natural selection, also colocalize with RNA structures. Critically, junctions between genes are enriched in structured RNA elements and are also preferred sites for generating functional recombinant forms. These data suggest that RNA structure-mediated recombination allows the virus to exchange intact genes rather than arbitrary subgene fragments, which is likely to increase the overall viability and replication success of the recombinant HIV progeny.     

Detection of homologous recombination among bacteriophage P2 relatives.

Sequencing of five late genes from 18 isolates of P2-like bacteriophages showed that these are at least 96% identical to the genes of phage P2. A maximum-parsimony phylogenetic analysis of these genes showed excess homoplasy of a magnitude three to six times higher than that expected. Examination of the distribution of the number of homoplasies at parsimoniously informative sites and incompatibility matrices of such sites revealed a pattern typical for extensive recombination. It has been shown that phage P2 probably incorporated some functionally complete genes or gene modules by recombination with other phages or with different hosts, but homologous recombination within genes has previously not been shown. In this paper we demonstrate that homologous recombination between P2-like bacteriophages occurs randomly at multiple breakpoints in five late genes. The rate of recombination is high but, since some phages were sampled decades apart and in different parts of the world, this has to be viewed on an evolutionary time scale. The applicability of different methods used for detection of recombination breakpoints and estimation of rates of recombination in bacteriophages is discussed.     

GC-Content Evolution in Bacterial Genomes: The Biased Gene Conversion Hypothesis Expands

The characterization of functional elements in genomes relies on the identification of the footprints of natural selection. In this quest, taking into account neutral evolutionary processes such as mutation and genetic drift is crucial because these forces can generate patterns that may obscure or mimic signatures of selection. In mammals, and probably in many eukaryotes, another such confounding factor called GC-Biased Gene Conversion (gBGC) has been documented. This mechanism generates patterns identical to what is expected under selection for higher GC-content, specifically in highly recombining genomic regions. Recent results have suggested that a mysterious selective force favouring higher GC-content exists in Bacteria but the possibility that it could be gBGC has been excluded. Here, we show that gBGC is probably at work in most if not all bacterial species. First we find a consistent positive relationship between the GC-content of a gene and evidence of intra-genic recombination throughout a broad spectrum of bacterial clades. Second, we show that the evolutionary force responsible for this pattern is acting independently from selection on codon usage, and could potentially interfere with selection in favor of optimal AU-ending codons. A comparison with data from human populations shows that the intensity of gBGC in Bacteria is comparable to what has been reported in mammals. We propose that gBGC is not restricted to sexual Eukaryotes but also widespread among Bacteria and could therefore be an ancestral feature of cellular organisms. We argue that if gBGC occurs in bacteria, it can account for previously unexplained observations, such as the apparent non-equilibrium of base substitution patterns and the heterogeneity of gene composition within bacterial genomes. Because gBGC produces patterns similar to positive selection, it is essential to take this process into account when studying the evolutionary forces at work in bacterial genomes.     

Increased complexity of gene structure and base composition in vertebrates

How the structure and base composition of genes changed with the evolution of vertebrates remains a puzzling question. Here we analyzed 895 orthologous protein-coding genes in six multicellular animals: human, chicken, zebrafish, sea squirt, fruit fly, and worm. Our analyses reveal that many gene regions, particularly intron and 3' UTR, gradually expanded throughout the evolution of vertebrates from their invertebrate ancestors, and that the number of exons per gene increased. Studies based on all protein-coding genes in each genome provide consistent results.We also find that GC-content increased in many gene regions (especially 5' UTR) in the evolution of endotherms, except in coding-exons.Analysis of individual genomes shows that 3′ UTR demonstrated stronger length and CC-content correlation with intron than 5' UTR, and gene with large intron in all six species demonstrated relatively similar GC-content. Our data indicates a great increase in complexity in vertebrate genes and we propose that the requirement for morphological and functional changes is probably the driving force behind the evolution of structure and base composition complexity in multicellular animal genes.     

Determination and analysis of the primary structure of NM.BstSEI operone from Bacillus stearothermophilus SE-589 which produces N.BstSEI site-specific nickase

Nucleotide sequence of Bacillus stearothermophilus SE-589 DNA fragment which includes an operone for site-specific NM-system with a gene for BstSEI nickase has been determined. Analysis of the regions adjacent to nickase gene has revealed two genes encoding DNA methyltransferases, which belong to different classes. Three genes which form system operone are separated with short open reading frames (ORFs). Analysis of these ORFs has shown that they encode polypeptides which are homologous to different parts of BstSEI nickase, NatB protein and arginase. A difference in GC-content of the beginning and ending regions of the cloned DNA fragment as well as presence of short ORFs similar to genes for known proteins may indicate that NM.BstSEI system operone has evolved by horizonthal DNA transfer.     

Human recombination rates are increased around accelerated conserved regions--evidence for continued selection?

MOTIVATION: We hypothesized that recombination rates might be increased at genetic loci that are subject to more intense selection. Here, we test this hypothesis by using a recently published set of accelerated conserved regions and fine-scale recombination rate estimates provided by the HapMap project. RESULTS: We observed that fine-scale recombination rates are increased around conserved noncoding regions that show accelerated evolution in human or chimp, as compared to noncoding regions showing accelerated evolution in mouse and those being conserved between human and fugu. Recombination rates around hominid accelerated conserved regions (ACRs) are furthermore increased as compared to exonic regions. On the other hand, GC-content is reduced around ACRs, excluding a major confounding influence of GC-content on the observed variation in recombination rate. Conclusion: Our observations indicate that selection intensity could be an important determinant of local recombination rate variation and that continued positive selection might act at many ACR loci. Alternatively, a confounding factor needs to be found that causes a congruent signal in recombination rate estimates based on human polymorphism data and in the comparative genomic data. Researchers who consider the explanation involving selection as more likely may expect more common functional sequence variants at ACRs in genetic association studies.     

Genetic Control of Intrachromosomal Recombination in Saccharomyces Cerevisiae. I. Isolation and Genetic Characterization of Hyper-Recombination Mutations

Eight complementation groups have been defined for recessive mutations conferring an increased mitotic intrachromosomal recombination phenotype (hpr genes) in Saccharomyces cerevisiae. Some of the mutations preferentially increase intrachromosomal gene conversion (hpr4, hpr5 and hpr8) between repeated sequences, some increase loss of a marker between duplicated genes (hpr1 and hpr6), and some increase both types of events (hpr2, hpr3 and hpr7). New alleles of the CDC2 and CDC17 genes were recovered among these mutants. The mutants were also characterized for sensitivity to DNA damaging agents and for mutator activity. Among the more interesting mutants are hpr5, which shows a biased gene conversion in a leu2-112::URA3::leu2-k duplication; and hpr1, which has a much weaker effect on interchromosomal mitotic recombination than on intrachromosomal mitotic recombination. These analyses suggest that gene conversion and reciprocal exchange can be separated mutationally. Further studies are required to show whether different recombination pathways or different outcomes of the same recombination pathway are controlled by the genes identified in this study.     

Genome analyses and modelling the relationships between coding density, recombination rate and chromosome length

In the human genomes, recombination frequency between homologous chromosomes during meiosis is highly correlated with their physical length while it differs significantly when their coding density is considered. Furthermore, it has been observed that the recombination events are distributed unevenly along the chromosomes. We have found that many of such recombination properties can be predicted by computer simulations of population evolution based on the Monte Carlo methods. For example, these simulations have shown that the probability of acceptance of the recombination events by selection is higher at the ends of chromosomes and lower in their middle parts. The regions of high coding density are more prone to enter the strategy of haplotype complementation and to form clusters of genes, which are “recombination deserts”. The phenomenon of switching in-between the purifying selection and haplotype complementation has a phase transition character, and many relations between the effective population size, coding density, chromosome size and recombination frequency are those of the power law type.     

Gene Flow,Recombination, and Selection in Cyanobacteria: Population Structure of Geographically Related Planktothrix Freshwater Strains

Several Planktothrix strains, each producing a distinct oligopeptide profile, have been shown to coexist within Lake Steinsfjorden (Norway). Using nonribosomal peptide synthetase (NRPS) genes as markers, it has been shown that the Planktothrix community comprises distinct genetic variants displaying differences in bloom dynamics, suggesting a Planktothrix subpopulation structure. Here, we investigate the Planktothrix variants inhabiting four lakes in southeast of Norway utilizing both NRPS and non-NRPS genes. Phylogenetic analyses showed similar topologies for both NRPS and non-NRPS genes, and the lakes appear to have similar structuring of Planktothrix genetic variants. The structure of distinct variants was also supported by very low genetic diversity within variants compared to the between-variant diversity. Incongruent topologies and split decomposition revealed recombination events between Planktothrix variants. In several strains the gene variants seem to be a result of recombination. Both NRPS and non-NRPS genes are dominated by purifying selection; however, sites subjected to positive selection were also detected. The presence of similar and well-separated Planktothrix variants with low internal genetic diversity indicates gene flow within Planktothrix populations. Further, the low genetic diversity found between lakes (similar range as within lakes) indicates gene flow also between Planktothrix populations and suggests recent, or recurrent, dispersals. Our data also indicate that recombination has resulted in new genetic variants. Stability within variants and the development of new variants are likely to be influenced by selection patterns and within-variant homologous recombination.     

Recombination, GC-content and the human pseudoautosomal boundary paradox

The pseudoautosomal boundary of mammalian sex chromosomes separates a low-recombination region (X- or Y-specific) from a high-recombination region (the pseudoautosomal region), providing a good opportunity to investigate the influence of recombination on molecular evolutionary processes. The mouse and human patterns of sequence variation, however, are discordant: a striking difference of GC-content and evolutionary rate was reported between the proximal and distal sides of the pseudoautosomal boundary in the mouse genome, whereas this difference was not found in the human genome. The paradox might be explained by the mirror histories of the pseudoautosomal boundary in the two species, and by the asymmetric nature of the forces driving GC-content evolution in mammalian genomes.     

“Beijing Region” (3pter-D3S3397) of the Human Genome: Complete sequence and analysis

The Human Genome Project (HGP), undoubtedly one of the greatest endeavors in the history of natural sciences, aims at determining the DNA sequence of the 3-billion-basepair human genome that holds an extraordinary trove of information about evolution, growth, and development of humans. China officially joined HGP and became one of the six member states of the International Human Genome Sequencing Con-sortium (IHGSC), alongside France, Germany, Japan, United Kingdom, and United State…