Replication-associated purine asymmetry may contribute to strand-biased gene distribution

Among prokaryotic genomes, the distribution of genes on the leading and lagging strands of the replication fork is known to be biased. Several hypotheses explaining this strand-biased gene distribution (SGD) have been proposed, but none have been tested or supported by sufficient data analyses. In this work we have analyzed 211 prokaryotic genomes in terms of compositional strand asymmetries and the presence or absence of polC and have found that SGD correlates not only with polC, but also with purine asymmetry (PAS). Furthermore, SGD, PAS, and polC are all features associated with a group of low-GC, gram-positive bacteria (Firmicutes). We conclude that PAS is a characteristic of organisms with a heterodimeric DNA polymerase III alpha-subunit constituted by polC and dnaE, which may play a direct role in the maintenance of SGD.     

基因组拷贝数变异及其突变机理与人类疾病

拷贝数变异(Copy number variation,CNV)是由基因组发生重排而导致的,一般指长度为1 kb以上的基因组大片段的拷贝数增加或者减少,主要表现为亚显微水平的缺失和重复。CNV是基因组结构变异(Structural variation,SV)的重要组成部分。CNV位点的突变率远高于SNP(Single nucleotide polymorphism),是人类疾病的重要致病因素之一。目前,用来进行全基因组范围的CNV研究的方法有:基于芯片的比较基因组杂交技术(array-based comparative genomic hybridization,aCGH)、SNP分型芯片技术和新一代测序技术。CNV的形成机制有多种,并可分为DNA重组和DNA错误复制两大类。CNV可以导致呈孟德尔遗传的单基因病与罕见疾病,同时与复杂疾病也相关。其致病的可能机制有基因剂量效应、基因断裂、基因融合和位置效应等。对CNV的深入研究,可以使我们对人类基因组的构成、个体间的遗传差异、以及遗传致病因素有新的认识。     

A human genome structural variation sequencing resource reveals insights into mutational mechanisms

Understanding the prevailing mutational mechanisms responsible for human genome structural variation requires uniformity in the discovery of allelic variants and precision in terms of breakpoint delineation. We develop a resource based on capillary end sequencing of 13.8 million fosmid clones from 17 human genomes and characterize the complete sequence of 1054 large structural variants corresponding to 589 deletions, 384 insertions, and 81 inversions. We analyze the 2081 breakpoint junctions and infer potential mechanism of origin. Three mechanisms account for the bulk of germline structural variation: microhomology-mediated processes involving short (2-20 bp) stretches of sequence (28%), nonallelic homologous recombination (22%), and L1 retrotransposition (19%). The high quality and long-range continuity of the sequence reveals more complex mutational mechanisms, including repeat-mediated inversions and gene conversion, that are most often missed by other methods, such as comparative genomic hybridization, single nucleotide polymorphism microarrays, and next-generation sequencing.     

The role of mutational dynamics in genome shrinkage

Genome shrinkage occurs after whole genome duplications (WGDs) and in the evolution of parasitic or symbiotic species. The dynamics of this process, whether it occurs by single gene deletions or also by larger deletions are however unknown. In yeast, genome shrinkage has occurred after a WGD. Using a computational model of genome evolution, we show that in a random genome single gene deletions cannot explain the observed pattern of gene loss in yeast. The distribution of genes deleted per event can be very well described by a geometric distribution, with a mean of 1.1 genes per event. In terms of deletions of a stretch of base pairs, we find that a geometric distribution with an average of 500-600 base pairs per event describes the data very well. Moreover, in the model, as in the data, gene pairs that have a small intergenic distance are more likely to be both deleted. This proves that simultaneous deletion of multiple genes causes the observed pattern of gene deletions, rather than deletion of functionally clustered genes by selection. Furthermore, we found that in the bacterium Buchnera aphidicola larger deletions than in yeast are necessary to explain the clustering of deleted genes. We show that the excess clustering of deleted genes in B. aphidicola can be explained by the clustering of genes in operons. Therefore, we show that selection has little effect on the clustering of deleted genes after the WGD in yeast, while it has during genome shrinkage in B. aphidicola.     

Functional bias and spatial organization of genes in mutational hot and cold regions in the human genome

The neutral mutation rate is known to vary widely along human chromosomes, leading to mutational hot and cold regions. We provide evidence that categories of functionally related genes reside preferentially in mutationally hot or cold regions, the size of which we have measured. Genes in hot regions are biased toward extracellular communication (surface receptors, cell adhesion, immune response, etc.), while those in cold regions are biased toward essential cellular processes (gene regulation, RNA processing, protein modification, etc.). From a selective perspective, this organization of genes could minimize the mutational load on genes that need to be conserved and allow fast evolution for genes that must frequently adapt. We also analyze the effect of gene duplication and chromosomal recombination, which contribute significantly to these biases for certain categories of hot genes. Overall, our results show that genes are located nonrandomly with respect to hot and cold regions, offering the possibility that selection acts at the level of gene location in the human genome.     

Large-scale mutational analysis for the annotation of the mouse genome

After sequencing the human and mouse genomes, the annotation of these sequences with biological functions is an important challenge in genomic research. A major tool to analyse gene function on the organismal level is the analysis of mutant phenotypes. Because of its genetic and physiological similarity to man, the mouse has become the model organism of choice for the study of genetic diseases. In addition, there is at the moment no other vertebrate for which versatile techniques to manipulate the genome are as well developed. Several mouse mutagenesis projects have provided the proof-of-principle that a systematic and comprehensive mutagenesis of every gene in the mammalian genome will be feasible. An exhaustive functional annotation of the mammalian genome can only be achieved in a combination of phenotype- and gene-driven approaches in large- and small-scale academic and private projects. Major challenges will be to develop standardised phenotyping protocols for the clinical and pathological characterisation of mouse mutants, the improvement of mutation detection methods and the dissemination of resources and data. Beyond gene annotation, it will be necessary to understand how gene functions are integrated into the complex network of regulatory interactions in the cell.     

Replication-associated activities of purified human papillomavirus type 11 E1 helicase

Replication of human papillomavirus type11 (HPV11) requires both the E1 and the E2 proteins. E1 is structurally and functionally similar to SV40 large T-antigen and is a DNA helicase/NTPase that binds to the origin of replication and initiates viral DNA replication. The biochemical characterization of HPV E1 is incompletely documented in the literature in part because of difficulties in expressing and purifying the protein. Herein, we report a method for the overexpression of full-length, untagged E1 (73.5 kDa) in baculovirus-infected Trichoplusia ni insect cells and the purification to homogeneity using a two-step procedure. The purified protein is a nonspecific NTPase that hydrolyzes ATP, dATP, UTP, or GTP equally well. Point mutations were made in the putative NTPase domain to verify that the activities observed were encoded by E1. Purified mutant D523N had negligible ATPase and helicase activities but retained DNA-binding activity. Sedimentation equilibrium ultracentrifugation and glycerol gradient centrifugation demonstrated that the wild-type protein is primarily a hexamer in its purified form. Secondary structure determination by circular dichroism revealed a large percentage of alpha-helical structure consistent with secondary structure predictions. These data define a fundamental set of biochemical and kinetic parameters for HPV E1 which are a critical prerequisite to future mechanistic studies of the enzyme.     

The mutational profile of the yeast genome is shaped by replication

Despite the scrutiny that has been directed for years at the yeast genome, relatively little is known about the impact of replication on the substitution dynamics in Saccharomyces cerevisiae. Here, we show that the mutation rate increases with the replication timing by more than 30% between the earliest and the latest replicating regions. In addition, we found a mutational asymmetry associated with the polarity of replication resulting in higher rates of substitutions toward C and A than toward G and T in leading strands (reciprocally more substitutions toward G and T in lagging strands). Such mutational asymmetries applied over long evolutionary periods should generate compositional skews between the two DNA strands. Thus, we show that the leading replicating strands present an excess of C over G and of A over T in the genome of S. cerevisiae (reciprocally an excess of G + T over C + A in lagging strands). We also show that the nucleotide frequencies at mutational equilibrium predict a compositional skew at equilibrium very close to the observed skew between leading and lagging strands, suggesting that compositional equilibrium has been nearly attained in the present day genome of S. cerevisiae. Surprisingly, the direction of this skew is inverted compared with the one in the human genome.     

SNPing in the human genome

More than a million genetic markers in the form of single nucleotide polymorphisms are now available for use in genotype-phenotype studies in humans. The application of new strategies for representational cloning and sequencing from genomes combined with the mining of high-quality sequence variations in clone overlaps of genomic and/or cDNA sequences has played an important role in generating this new resource. The focus of variation analysis is now shifting from the identification of new markers to their typing in populations, and novel typing strategies are rapidly emerging. Assay readouts on oligonucleotide arrays, in microtiter plates, gels, flow cytometers and mass spectrometers have all been developed, but decreasing cost and increasing throughput of DNA typing remain key if high-density genetic maps are to be applied on a large scale.     

EMu: probabilistic inference of mutational processes and their localization in the cancer genome

The spectrum of mutations discovered in cancer genomes can be explained by the activity of a few elementary mutational processes. We present a novel probabilistic method, EMu, to infer the mutational signatures of these processes from a collection of sequenced tumors. EMu naturally incorporates the tumor-specific opportunity for different mutation types according to sequence composition. Applying EMu to breast cancer data, we derive detailed maps of the activity of each process, both genome-wide and within specific local regions of the genome. Our work provides new opportunities to study the mutational processes underlying cancer development. EMu is available at http://www.sanger.ac.uk/resources/software/emu/.     

The lack of mutational variance for fluctuating and directional asymmetry in Drosophila melanogaster.

Starting from a completely homozygous population of Drosophila melanogaster, lines were derived and independently maintained by a single brother-sister mating in each generation. Two bilateral traits--sternopleural bristle number and wing length--were individually scored on the right-(R) and left-hand (L) sides. Directional (DA) and fluctuating (FA) asymmetries were represented by the signed (R-L) and unsigned magnitude of R-L difference, respectively. Mutational variances (the mutational rate of input of genetic variation) and heritabilities (the mutational variance scaled by the environmental variance) of R, L, (R-L) and magnitude of R-L were calculated from the between-line divergence after a number of generations of mutation accumulation (bristle number: 171 lines, 122 generations; wing length: 148 lines, 170 generations). Mutational heritabilities of R and L were all significant, ranging from 0.73 x 10(-3)-2.10 x 10(-3). Those of (R-L) and magnitude of R-L were two orders of magnitude smaller and nonsignificant, ranging from -1.95 x 10(-5)-5.49 x 10(-5). These results imply that mutations affecting the DA or FA of bristle number and wing length have not been fixed in the lines or alternatively, that their effects were too small to be detected. In the population under study, the data strongly suggest that FA reflects only developmental noise due to non-genetic processes.     

Topography of mutational signatures in human cancer

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Illuminating the human genome

The identification and analysis of novel genes and their encoded protein products remains a vigorous area of research in biology today. Worldwide genomic and cDNA sequencing projects are now identifying new molecules every day and the need for methodologies to functionally characterise these proteins has never been greater. The distinct compartmental arrangement of eukaryotic cells helps define the processes which occur within or in proximity to these membranes, and as such provides one means of inferring protein function. We describe here some of the methods recently reported in the literature, which use the subcellular localisation of proteins as a first step towards their further characterisation.     

Segment duplications in the human genome

The review considers the structure, evolution, and possible mechanisms of spreading of intrachromosomal and interchromosomal segment duplications (SD), which account for more than 5% of the human genome. Most SD are mosaic and consist of multiple modules, which occur in several copies in different genome regions. SD are preferentially located in pericentric and subtelomeric regions, which are least studied on the human chromosomes. Homologous recombination between SD results in various chromosome rearrangements, contributing to the genome instability and the origin of several human hereditary disorders.     

Structural variation in the human genome

The first wave of information from the analysis of the human genome revealed SNPs to be the main source of genetic and phenotypic human variation. However, the advent of genome-scanning technologies has now uncovered an unexpectedly large extent of what we term 'structural variation' in the human genome. This comprises microscopic and, more commonly, submicroscopic variants, which include deletions, duplications and large-scale copy-number variants - collectively termed copy-number variants or copy-number polymorphisms - as well as insertions, inversions and translocations. Rapidly accumulating evidence indicates that structural variants can comprise millions of nucleotides of heterogeneity within every genome, and are likely to make an important contribution to human diversity and disease susceptibility.