Patterns of nucleotide asymmetries in plant and animal genomes

Symmetry in biology provides many intriguing puzzles to the scientist's mind. Chargaff's second parity rule states a symmetric distribution of oligonucleotides within a single strand of double-stranded DNA. While this rule has been verified in a wide range of microbial genomes, it still awaits explanation. In our study, we inquired into patterns of mono- and trinucleotide intra-strand parity in complex plant genomic sequences that became available during the last few years, and compared these to equally complex animal genomes. The degree and patterns of deviation from Chargaff's second rule were different between plant and animal species. We observed a universal inter-chromosomal homogeneity of mononucleotide skews in coding sequences of plant chromosomes, while the base composition of animal coding sequences differed between chromosomes even within a single species. We also found differences in the base composition of dicot introns in comparison to those of monocots. These genome-wide patterns were limited to genic regions and were not encountered in inter-genic sequences. We discuss the implications of our findings in relation to hypotheses about functional correlations of intra-strand parity which have hitherto been put forward. Furthermore, we propose more recent polyploidization and subsequent homogenization of homoeologues as a possible reason for more homogeneous skew patterns in plants.     

Relative roles of primary sequence and (G + C)% in determining the hierarchy of frequencies of complementary trinucleotide pairs in DNAs of different species

To an approximation Chargaff's rule (%A = %T; %G = %C) applies to single-stranded DNA. In long sequences, not only complementary bases but also complementary oligonucleotides are present in approximately equal frequencies. This applies to all species studied. However, species usually differ in base composition. With the goal of understanding the evolutionary forces involved, I have compared the frequencies of trinucleotides in long sequences and their shuffled counterparts. Among the 32 complementary trinucleotide pairs there is a hierarchy of frequencies which is influenced both by base composition (not affected by shuffling the order of the bases) and by base order (affected by shuffling). The influence of base order is greatest in DNA of 50% G + C and seems to reflects a more fundamental hierarchy of dinucleotide frequencies. Thus if TpA is at low frequency, all eight TpA-containing trinucleotides are at low frequency. Mammals and their viruses share similar hierarchies, with intra- and intergenomic differences being mainly associated with differences in base composition (percentage G + C). E. coli and, to a lesser extent, Drosophila melanogaster hierarchies differ from mammalian hierarchies; this is associated with differences both in base composition and in base order. It is proposed that Chargaff's rule applies to single-stranded DNA because there has been an evolutionary selection pressure favoring mutations that generate complementary oligonucleotides in close proximity, thus creating a potential to form stem-loops. These are dispersed throughout genomes and are rate-limiting in recombination. Differences in (G + C)% between species would impair interspecies recombination by interfering with stem-loop interactions.     

Genomic Conflict Settled in Favour of the Species Rather Than the Gene at Extreme GC Percentage Values

Wada and colleagues have shown that, whether prokaryotic or eukaryotic, each gene has a "homostabilising propensity" to adopt a relatively uniform GC percentage (GC%). Accordingly, each gene can be viewed as a "microisochore" occupying a discrete GC% niche of relatively uniform base composition amongst its fellow genes. Although first, second and third codon positions usually differ in GC%, each position tends to maintain a uniform, gene-specific GC% value. Thus, within a genome, genic GC% values can cover a wide range. This is most evident at third codon positions, which are least constrained by amino acid encoding needs. In 1991, Wada and colleagues further noted that, within a phylogenetic group, genomic GC% values can also cover a wide range. This is again most evident at third codon positions. Thus, the dispersion of GC% values among genes within a genome matches the dispersion of GC% values among genomes within a phylogenetic group. Wada described the context-independence of plots of different codon position GC% values against total GC% as a "universal" characteristic. Several studies relate this to recombination. We have confirmed that third codon positions usually relate more to the genes that contain them than to the species. However, in genomes with extreme GC% values (low or high), third codon positions tend to maintain a constant GC%, thus relating more to the species than to the genes that contain them. Genes in an extreme-GC% genome collectively span a smaller GC% range, and mainly rely on first and second codon positions for differentiation as "microisochores". Our results are consistent with the view that differences in GC% serve to recombinationally isolate both genome sectors (facilitating gene duplication) and genomes (facilitating genome duplication, e.g. speciation). In intermediate-GC% genomes, conflict between the needs of the species and the needs of individual genes within that species is minimal. However, in extreme-GC% genomes there is a conflict, which is settled in favour of the species (i.e. group selection) rather than in favour of the gene (genic selection).     

Estimating Effective Population Size or Mutation Rate Using the Frequencies of Mutations of Various Classes in a Sample of DNA Sequences

Mutations resulting in segregating sites of a sample of DNA sequences can be classified by size and type and the frequencies of mutations of different sizes and types can be inferred from the sample. A framework for estimating the essential parameter θ = 4Nu utilizing the frequencies of mutations of various sizes and types is developed in this paper, where N is the effective size of a population and μ is mutation rate per sequence per generation. The framework is a combination of coalescent theory, general linear model and Monte-Carlo integration, which leads to two new estimators θ(ξ) and θ(η) as well as a general Watterson''s estimator θ(K) and a general Tajima''s estimator θ(π). The greatest strength of the framework is that it can be used under a variety of population models. The properties of the framework and the four estimators θ(K), θ(π), θ(ξ) and θ(η) are investigated under three important population models: the neutral Wright-Fisher model, the neutral model with recombination and the neutral Wright''s finite-islands model. Under all these models, it is shown that θ(ξ) is the best estimator among the four even when recombination rate or migration rate has to be estimated. Under the neutral Wright-Fisher model, it is shown that the new estimator θ(ξ) has a variance close to a lower bound of variances of all unbiased estimators of θ which suggests that θ(ξ) is a very efficient estimator.     

The correlation coefficient of GC content of the genome-wide genes is positively correlated with animal evolutionary relationships

In this study, we present a new method for evaluating animal evolutionary relationships. We used the GC% levels of genome-wide genes to determine the correlation between the GC% content and evolutionary relationship. The correlation coefficients of the GC% content of the orthologous genes of the paired animal species were calculated for a total of 21 species, and the evolutionary branching dates of these 21 species were derived from fossil records. The correlation coefficient of the GC% content of the orthologous genes of the species pair under study served as an indicator of their evolutionary relationship. Moreover, there was a decreasing linear relationship between the correlation coefficient and evolutionary branching date (R² = 0.930).     

Fixations of the HIV-1 env gene refute neutralism: New evidence for pan-selective evolution

We examined 103 nucleotide sequences of the HIV-1 env gene, sampled from 35 countries and tested: I) the random (neutral) distribution of the number of nucleotide changes; II) the proportion of bases at molecular equilibrium; III) the neutral expected homogeneity of the distribution of new fixated bases; IV) the hypothesis of the neighbor influence on the mutation rates in a site. The expected random number of fixations per site was estimated by Bose-Einstein statistics, and the expected frequencies of bases by matrices of mutation-fixation rates. The homogeneity of new fixations was analyzed using χ2 and trinomial tests for homogeneity. Fixations of the central base in trinucleotides were used to test the neighbor influence on base substitutions. Neither the number of fixations nor the frequencies of bases fitted the expected neutral distribution. There was a highly significant heterogeneity in the distribution of new fixations, and several sites showed more transversions than transitions, showing that each nucleotide site has its own pattern of change. These three independent results make the neutral theory, the nearly neutral and the neighbor influence hypotheses untenable and indicate that evolution of env is rather highly selective.     

Neutral effect of recombination on base composition in Drosophila

Recombination is thought to have various evolutionary effects on genome evolution. In this study, we investigated the relationship between the base composition and recombination rate in the Drosophila melanogaster genome. Because of a current debate about the accuracy of the estimates of recombination rate in Drosophila, we used eight different measures of recombination rate from recent work. We confirmed that the G + C content of large introns and flanking regions is positively correlated with recombination rate, suggesting that recombination has a neutral effect on base composition in Drosophila. We also confirmed that this neutral effect of recombination is the main determinant of the correlation between synonymous codon usage bias and recombination rate in Drosophila.     

Genome-wide analysis of DNA replication and DNA double-strand breaks using TrAEL-seq

Faithful replication of the entire genome requires replication forks to copy large contiguous tracts of DNA, and sites of persistent replication fork stalling present a major threat to genome stability. Understanding the distribution of sites at which replication forks stall, and the ensuing fork processing events, requires genome-wide methods that profile replication fork position and the formation of recombinogenic DNA ends. Here, we describe Transferase-Activated End Ligation sequencing (TrAEL-seq), a method that captures single-stranded DNA 3′ ends genome-wide and with base pair resolution. TrAEL-seq labels both DNA breaks and replication forks, providing genome-wide maps of replication fork progression and fork stalling sites in yeast and mammalian cells. Replication maps are similar to those obtained by Okazaki fragment sequencing; however, TrAEL-seq is performed on asynchronous populations of wild-type cells without incorporation of labels, cell sorting, or biochemical purification of replication intermediates, rendering TrAEL-seq far simpler and more widely applicable than existing replication fork direction profiling methods. The specificity of TrAEL-seq for DNA 3′ ends also allows accurate detection of double-strand break sites after the initiation of DNA end resection, which we demonstrate by genome-wide mapping of meiotic double-strand break hotspots in a dmc1Δ mutant that is competent for end resection but not strand invasion. Overall, TrAEL-seq provides a flexible and robust methodology with high sensitivity and resolution for studying DNA replication and repair, which will be of significant use in determining mechanisms of genome instability.

TrAEL-seq provides genome-wide base pair resolution maps of exposed DNA 3’ ends; this reveals replication fork stalling and normal replication profiles in asynchronous, unlabelled wildtype cell populations, along with the sites of resected DNA breaks.     

Haplotypic Divergence Coupled with Lack of Diversity at the Arabidopsis Thaliana Alcohol Dehydrogenase Locus: Roles for Both Balancing and Directional Selection?

We designate a region of the alcohol dehydrogenase locus (Adh) of the weedy crucifer, Arabidopsis thaliana, as ``hypervariable'''' on the basis of a comparison of sequences from ecotypes Columbia and Landsberg. We found eight synonymous and two replacement mutations in the first 262 nucleotides of exon 4, and an additional two mutations in the contiguous region of intron 3. The rest of the sequence (2611 bp) has just three mutations, all of them confined to noncoding regions. Our survey of the hypervariable region among 37 ecotypes of A. thaliana revealed two predominant haplotypes, corresponding to the Columbia and Landsberg sequences. We identified five additional haplotypes and 4 additional segregating sites. The lack of haplotype diversity is presumably in part a function of low rates of recombination between haplotypes conferred by A. thaliana''s tendency to self-fertilize. However, an analysis in 32 ecotypes of 12 genome-wide polymorphic markers distinguishing Columbia and Landsberg ecotypes indicated levels of outcrossing sufficient at least to erode linkage disequilibrium between dispersed markers. We discuss possible evolutionary explanations for the coupled observation of marked divergence within the hypervariable region and a lack of haplotype diversity among ecotypes. The sequence of the region for closely related species argues against the possibility that one allele is the product of introgression. We note (1) that several loss of function mutations (both naturally and chemically induced) map to the hypervariable region, and (2) the presence of two amino acid replacement polymorphisms, one of which causes the mobility difference between the two major classes of A. thaliana Adh electrophoretic alleles. We argue that protein polymorphism in such a functionally significant part of the molecule may be subject to balancing selection. The observed pattern of extensive divergence between the alleles is consistent with this explanation because balancing selection on a particular site maintains linked neutral polymorphisms at intermediate frequencies.     

A genome-wide departure from the standard neutral model in natural populations of Drosophila

We analyze nucleotide polymorphism data for a large number of loci in areas of normal to high recombination in Drosophila melanogaster and D. simulans (24 and 16 loci, respectively). We find a genome-wide, systematic departure from the neutral expectation for a panmictic population at equilibrium in natural populations of both species. The distribution of sequence-based estimates of 2Nc across loci is inconsistent with the assumptions of the standard neutral theory, given the observed levels of nucleotide diversity and accepted values for recombination and mutation rates. Under these assumptions, most estimates of 2Nc are severalfold too low; in other words, both species exhibit greater intralocus linkage disequilibrium than expected. Variation in recombination or mutation rates is not sufficient to account for the excess of linkage disequilibrium. While an equilibrium island model does not seem to account for the data, more complicated forms of population structure may. A proper test of alternative demographic models will require loci to be sampled in a more consistent fashion.     

The relation between genetic recombination and base sequence complementarity

A non-sliding and sliding model of illegitimate recombination utilizing recent experimentally determined free energies are presented. In each model two random sequences of nucleotide bases were brought into contact and the free energy of stabilization was computed.Combinatorial and Monte-Carlo versions were developed to investigate the role of base sequence complementarity in regions of non-homology over varying lengths. It was found that short sequences of complementarity enhanced the frequency of recombination. When the energy threshold wasequivalent to ten contiguous base pairs, the results conformed to experimental recombination frequencies. Recombination is slightly enhanced by increasing the size of exposed contact regions. Skewing the G-C composition does not significantly effect recombination. There was a significant difference in frequencies between sliding and non-sliding models. Application of the models to other genetic events such as nonhomologous sticking of circular DNA and hair-pin formation indicate that these events are essentially non-random processes.     

Antimutator role of the DNA glycosylase mutY gene in Helicobacter pylori

Helicobacter pylori has a highly variable genome with ongoing diversification via inter- and intragenomic recombination and spontaneous mutation. DNA repair genes modulating mutation and recombination rates that influence diversification have not been well characterized for H. pylori. To examine the role of putative base excision repair ung and mutY glycosylase and xthA apurinic/apyrimidinic endonuclease genes in H. pylori, mutants of each were constructed in strain JP26 by allelic exchange. Spontaneous mutation frequencies of JP26 mutY mutants, assessed by rifampin resistance, were consistently higher (26-fold) than that of the wild type, whereas the ung and xthA mutants showed smaller increases. In trans complementation of the JP26 mutY mutant restored spontaneous mutation frequencies to wild-type levels. In cross-species studies, H. pylori mutY complemented an Escherichia coli mutY mutant and vice versa. In contrast, the ung and mutY mutants did not show higher frequencies of intergenomic recombination or greater sensitivity to UV-induced DNA damage than the wild type. The H. pylori mutY open reading frame contains an eight-adenine homonucleotide tract; we provide evidence that this is subject to slipped-strand mispairing, leading to frameshifts that eliminate gene function. Our findings indicate that H. pylori possesses phase-variable base excision repair, consistent with a tension between repair and mutation.     

TransTerm: a database of translational signals.

The TransTerm database of sequence contexts of stop and start codons has been expanded to include approximately 50% more species than last year's release. It now contains 148 organisms and >39 500 coding sequences; it is now available on the World Wide Web. The database includes: (i) initiation and termination sequence contexts organized by species; (ii) summary parameters about the individual sequences (sequence length, GC%, GC3, Nc, CAI) in addition to tables of base frequencies for each species' stop and start codon sequence context; (iii) species codon usage tables; and (iv) summary tables of stop signal frequency.     

Over-representation of Chi sequences caused by di-codon increase in Escherichia coli K-12

Chi sequences (5'-GCTGGTGG-3') are cis-acting 8 bp sequence elements that enhance homologous recombination promoted by the RecBCD pathway in Escherichia coli. The genome of E. coli K-12 MG1655 contains 1009 Chi sequences and this frequency far exceeds the expected value for occurrence of an 8 bp sequence in a genome of this size. It is generally thought that the over-representation of Chi sequences indicates that they have been selected for during evolution because of their function in recombination. The genes from three E. coli strains (K-12, O157 and CFT) were classified into three categories (island, match to other E. coli, and backbone). Island genes have a different base composition and codon usage in comparison with those in the backbone genes, therefore they were relatively new and not yet adapted to the base composition patterns and codon usage typical of the recipient genome. The over-representation of Chi sequences was examined by comparing Chi frequencies and codon frequencies between island and backbone genes. The difference in the CTGGTG di-codon frequency between the backbone and island genes was correlated with the frequency of Chi sequences which were translated in the Leu-Val (-G/CTG/GTG/G-) reading frame in the K-12 strain. These results suggest that the main reading frame of Chi sequences increased as a result of the di-codon CTG-GTG increasing under a genome-wide pressure for adapting to the codon usage and base composition of the E. coli K-12 strain, and that the RecBCD recombinase might adjust its recognition sequence to a frequently occurring oligomer such as G-CTG-GTG-G.     

War and world of Erwin Chargaff (Dedicated to 110th anniversary of birth)

The article shortly describes the life path of Erwin Chargaff, one of the most famous figures in the history of molecular biology and genetics. Chargaff was born in Chernivtsi (Austria-Hungary, now Ukraine) but during the First World War his family was forced to move to Vienna. After graduating from the University of Vienna, Chargaff worked in Berlin, where he studied bacterial lipids. Due to Nazis coming to power in Germany, Chargaff moved to Paris and later (1935) emigrated to the USA and obtained a position at the Columbia University, where he initially invastigated the role of phospholipids in blood clotting. In year 1944, applying novel methods Chargaff initiated intensive investigation of the chemical composition of nucleic acids from taxonomically distant species and established two rules, which were later named after him. The first Chargaff's rule provided a significant support to Watson and Crick in construction of their double helical DNA model. The explosion of atomic bombs over Hiroshima and Nagasaki forced Chargaff to think about the moral responsibility of researchers and science to mankind. He began to raise these issues in the press and manifested himself as a talented journalist, who criticized the bureaucratization of science and its transformation into a way of earning money. Despite decades of life in America, spiritually Erwin Chargaff always remained a European, who never forgot his roots and always remembered his native land.     

A circular dichroism study of the primary structures of three crab satellite DNA's rich in A:T base pairs

We have measured the circular dichroism spectra and have obtained melting curves for samples of purified satellite DNA's from three species of crab, Cancer borealis, Gecarcinus lateralis, and Callinectes sapidus. Although all three satellite DNA's have the same density in neutral CsCl, their circular dichroism spectra and melting and reassociation characteristics are different, as are other properties of these DNA's. The circular dichroism spectra are analyzed to give estimates of nearest-neighbor frequencies. We estimate that about 90% of the Cancer borealis satellite DNA and over 80% of the Gecarcinus lateralis satellite DNA consist of d(AT)_n:d(AT)_n sequences, while the serially repeating unit of the Callinectes sapidus satellite DNA may not contain any extended d(AT)_n:d(AT)_n sequences. The results from the circular dichroism spectra for the Cancer borealis, Gecarcinus lateralis, and the Callinectes sapidus satellite DNA's are in agreement with base composition analyses, which give 2.4, 8, and 25% G+C, respectively, for these DNA's.     

Natural Selection and Recombination Rate Variation Shape Nucleotide Polymorphism Across the Genomes of Three Related Populus Species

A central aim of evolutionary genomics is to identify the relative roles that various evolutionary forces have played in generating and shaping genetic variation within and among species. Here we use whole-genome resequencing data to characterize and compare genome-wide patterns of nucleotide polymorphism, site frequency spectrum, and population-scaled recombination rates in three species of Populus: Populus tremula, P. tremuloides, and P. trichocarpa. We find that P. tremuloides has the highest level of genome-wide variation, skewed allele frequencies, and population-scaled recombination rates, whereas P. trichocarpa harbors the lowest. Our findings highlight multiple lines of evidence suggesting that natural selection, due to both purifying and positive selection, has widely shaped patterns of nucleotide polymorphism at linked neutral sites in all three species. Differences in effective population sizes and rates of recombination largely explain the disparate magnitudes and signatures of linked selection that we observe among species. The present work provides the first phylogenetic comparative study on a genome-wide scale in forest trees. This information will also improve our ability to understand how various evolutionary forces have interacted to influence genome evolution among related species.     

Transduction of the cellular src gene and 3'' adjacent sequences in avian sarcoma virus PR2257.

When injected into chickens, a transformation-defective mutant of the Prague C strain of Rous sarcoma virus induced tumors at low incidence and after a long latency. One such tumor released a replication-defective virus designated PR2257. We molecularly cloned and sequenced the proviral DNA from quail fibroblasts transformed by PR2257. Comparison of PR2257 sequence with that of Prague C, cellular src, and 3' adjacent cellular DNA showed that the spliced version of the c-src gene and about 950 base pairs (bp) of 3'-flanking cellular DNA were transduced into PR2257. This transduction eliminated nearly all replicative genes, since the gag gene splice donor site was linked to the splice acceptor site of the src gene and, on the 3' side, recombination occurred in the end of env gene. Insertion of two extra cytosines 23 bp before and 19 bp after the c-src stop codon resulted in an extension of the coding portion up to 587 amino acids, divergence of sequences after Pro-525 and replacement of Tyr-527 by a valine residue. In addition, it appears that the 5' and 3' untranslated regions of PR2257 result from multiple recombinations between exogenous and endogenous virus genomes. Limited digestion of p66src encoded by PR2257 with Staphylococcus aureus V8 protease yielded a V2 peptide (C-terminal moiety) with an apparent molecular mass of 31 kilodaltons, consistent with the 5.7-kilodalton increase expected from the DNA sequence. The structure of PR2257 suggests that the first step in the capture of c-src gene by avian lymphomatosis viruses is the trans splicing of the viral leader mRNA to exon 1 of c-src.