Widespread Adaptive Evolution in the Human Immunodeficiency Virus Type 1 Genome

We investigated variable selective pressures among amino acid sites in HIV-1 genes. Selective pressure at the amino acid level was measured by using the nonsynonymous/synonymous substitution rate ratio ( = d_N/d_S). To identify amino acid sites under positive selection with > 1, we applied maximum likelihood models that allow variable ratios among sites to analyze genomic sequences of 26 HIV-1 lineages including subtypes A, B, and C. Likelihood ratio tests detected sites under positive selection in each of the major genes in the genome: env, gag, pol, vif, and vpr. Positive selection was also detected in nef, tat, and vpu, although those genes are very small. The majority of positive selection sites is located in gp160. Positive selection was not detected if was estimated as an average across all sites, indicating the lack of power of the averaging approach. Candidate positive selection sites were mapped onto the available protein tertiary structures and immunogenic epitopes. We measured the physiochemical properties of amino acids and found that those at positive selection sites were more diverse than those at variable sites. Furthermore, amino acid residues at exposed positive selection sites were more physiochemically diverse than at buried positive selection sites. Our results demonstrate genomewide diversifying selection acting on the HIV-1.     

High-Throughput Functional Analysis of the Synechococcus elongatus PCC 7942 Genome

Synechococcus elongatus PCC 7942 was the first cyanobacterialstrain to be reliably transformed by exogenously added DNA andhas become the model organism for cyanobacterial circadian rhythms.With a small genome (2.7 Mb) and well-developed genetic tools,PCC 7942 provides an exceptional opportunity to elucidate thecircadian mechanism through genetics. We describe a projectto create mutations in every locus of the genome, both to assayeach locus for its potential contribution to the circadian clockand to archive data for the cyanobacterial community. Cosmidclones that carry inserts of PCC 7942 DNA are saturated withtransposon insertions in vitro to provide sequencing templatesand substrates for mutagenesis of the PCC 7942 genome via homologousrecombination. We have mutagenized 53% of the chromosome from50 chromosome-bearing cosmids and identified the positions ofinsertions in 31 of those cosmids and the 46 kb plasmid, pANL.PCC 7942 mutants defective for 490 different genes have beenscreened for circadian phenotypes. Mutagenesis of three apparentlyessential loci, including clpPIIclpX, resulted in circadianphenotypes. We developed an effective antisense suppressionmethod to further the analysis of essential genes. When completed,the set of comprehensive mutations will provide the communitywith a unique resource whose impact will extend beyond circadianresearch.     

A Method Enabling High-Throughput Sequencing of Human Cytomegalovirus Complete Genomes from Clinical Isolates

Human cytomegalovirus (HCMV) is a ubiquitous virus that can cause serious sequelae in immunocompromised patients and in the developing fetus. The coding capacity of the 235 kbp genome is still incompletely understood, and there is a pressing need to characterize genomic contents in clinical isolates. In this study, a procedure for the high-throughput generation of full genome consensus sequences from clinical HCMV isolates is presented. This method relies on low number passaging of clinical isolates on human fibroblasts, followed by digestion of cellular DNA and purification of viral DNA. After multiple displacement amplification, highly pure viral DNA is generated. These extracts are suitable for high-throughput next-generation sequencing and assembly of consensus sequences. Throughout a series of validation experiments, we showed that the workflow reproducibly generated consensus sequences representative for the virus population present in the original clinical material. Additionally, the performance of 454 GS FLX and/or Illumina Genome Analyzer datasets in consensus sequence deduction was evaluated. Based on assembly performance data, the Illumina Genome Analyzer was the platform of choice in the presented workflow. Analysis of the consensus sequences derived in this study confirmed the presence of gene-disrupting mutations in clinical HCMV isolates independent from in vitro passaging. These mutations were identified in genes RL5A, UL1, UL9, UL111A and UL150. In conclusion, the presented workflow provides opportunities for high-throughput characterization of complete HCMV genomes that could deliver new insights into HCMV coding capacity and genetic determinants of viral tropism and pathogenicity.     

Widespread Occurrence of Bacterial Human Virulence Determinants in Soil and Freshwater Environments

The occurrence of 22 bacterial human virulence genes (encoding toxins, adhesins, secretion systems, regulators of virulence, inflammatory mediators, and bacterial resistance) in beech wood soil, roadside soil, organic agricultural soil, and freshwater biofilm was investigated by nested PCR. The presence of clinically relevant bacterial groups known to possess virulence genes was tested by PCR of 16S and 23S rRNA genes. For each of the virulence genes detected in the environments, sequencing and NCBI BLAST analysis confirmed the identity of the PCR products. The virulence genes showed widespread environmental occurrence, as 17 different genes were observed. Sixteen genes were detected in beech wood soil, and 14 were detected in roadside and organic agricultural soils, while 11 were detected in the freshwater biofilm. All types of virulence traits were represented in all environments; however, the frequency at which they were detected was variable. A principal-component analysis suggested that several factors influenced the presence of the virulence genes; however, their distribution was most likely related to the level of contamination by polycyclic aromatic hydrocarbons and pH. The occurrence of the virulence genes in the environments generally did not appear to be the result of the presence of clinically relevant bacteria, indicating an environmental origin of the virulence genes. The widespread occurrence of the virulence traits and the high degree of sequence conservation between the environmental and clinical sequences suggest that soil and freshwater environments may constitute reservoirs of virulence determinants normally associated with human disease.     

Human Cytomegalovirus UL34 Binds to Multiple Sites within the Viral Genome

The human cytomegalovirus UL34 gene encodes a sequence-specific DNA binding protein that downregulates expression of the viral immune evasion gene US3. Analysis of the viral genome identified 14 potential UL34 binding sites. Using mobility shift experiments, UL34 bound to all predicted sites that were assayed (7 of 14). Furthermore, the UL34 binding site present within the regulatory region of the US9 gene downregulates expression in a manner similar to that seen for the US3 gene.     

Human Cytomegalovirus pUL93 Is Required for Viral Genome Cleavage and Packaging

Human cytomegalovirus (HCMV) pUL93 is essential for virus growth, but its precise function in the virus life cycle is unknown. Here, we characterize a UL93 stop mutant virus (UL93st-TB40/E-BAC) to demonstrate that the absence of this protein does not restrict viral gene expression; however, cleavage of viral DNA into unit-length genomes as well as genome packaging is abolished. Thus, pUL93 is required for viral genome cleavage and packaging.     

High-Throughput Genome Editing and Phenotyping Facilitated by High Resolution Melting Curve Analysis

With the goal to generate and characterize the phenotypes of null alleles in all genes within an organism and the recent advances in custom nucleases, genome editing limitations have moved from mutation generation to mutation detection. We previously demonstrated that High Resolution Melting (HRM) analysis is a rapid and efficient means of genotyping known zebrafish mutants. Here we establish optimized conditions for HRM based detection of novel mutant alleles. Using these conditions, we demonstrate that HRM is highly efficient at mutation detection across multiple genome editing platforms (ZFNs, TALENs, and CRISPRs); we observed nuclease generated HRM positive targeting in 1 of 6 (16%) open pool derived ZFNs, 14 of 23 (60%) TALENs, and 58 of 77 (75%) CRISPR nucleases. Successful targeting, based on HRM of G0 embryos correlates well with successful germline transmission (46 of 47 nucleases); yet, surprisingly mutations in the somatic tail DNA weakly correlate with mutations in the germline F1 progeny DNA. This suggests that analysis of G0 tail DNA is a good indicator of the efficiency of the nuclease, but not necessarily a good indicator of germline alleles that will be present in the F1s. However, we demonstrate that small amplicon HRM curve profiles of F1 progeny DNA can be used to differentiate between specific mutant alleles, facilitating rare allele identification and isolation; and that HRM is a powerful technique for screening possible off-target mutations that may be generated by the nucleases. Our data suggest that micro-homology based alternative NHEJ repair is primarily utilized in the generation of CRISPR mutant alleles and allows us to predict likelihood of generating a null allele. Lastly, we demonstrate that HRM can be used to quickly distinguish genotype-phenotype correlations within F1 embryos derived from G0 intercrosses. Together these data indicate that custom nucleases, in conjunction with the ease and speed of HRM, will facilitate future high-throughput mutation generation and analysis needed to establish mutants in all genes of an organism.     

Characterization of a Human Cytomegalovirus with Phosphorylation Site Mutations in the Immediate-Early 2 Protein

A human cytomegalovirus mutant (TNsubIE2P) was constructed with alanine substitutions of four residues (T27, S144, T233, and S234) previously shown to be phosphorylated in the immediate-early 2 (IE2) protein. This mutant grew as well as the wild type at both low and high multiplicities of infection. The mutant activated the major immediate-early, UL4, and UL44 promoters to similar levels, and with similar kinetics, as wild-type virus. However, the TNsubIE2P mutant virus transactivated an endogenous simian virus 40 early promoter 4 h earlier and to higher levels than the wild-type virus in infected human fibroblasts. The modification of the IE2 protein by SUMO-1 (i.e., its sumoylated state) was also examined.     

The Human Genome Diversity Project: past, present and future

The Human Genome Project, in accomplishing its goal of sequencing one human genome, heralded a new era of research, a component of which is the systematic study of human genetic variation. Despite delays, the Human Genome Diversity Project has started to make progress in understanding the patterns of this variation and its causes, and also promises to provide important information for biomedical studies.     

Analysis of Meiosis-Defective Mutations in Yeast by Physical Monitoring of Recombination

We have developed a method by which the extent of physical exchange of DNA molecules can be determined throughout meiosis in the yeast Saccharomyces cerevisiae. We have used this technique to analyze the effect of five meiosis-defective mutations (rad6, rad50, rad52, rad57 and spo11) on the physical exchange of DNA molecules. In the same experiments, we have also measured other meiotic parameters, such as premeiotic DNA synthesis, commitment to intragenic recombination, haploidization, ascus formation, and viability. rad50 and spo11 diploids make an undetectable amount of physically recombined DNA and less than 1% of wild-type levels of viable intragenic recombinants. In contrast, diploids homozygous for rad52, rad6 or rad57 all yield significant amounts of novel restriction fragments which arise by recombination. rad57 diploids make nearly wild-type levels of the recombined restriction fragments, although they produce less than 10% of the wild-type levels of viable intragenic recombinants. rad52 strains are also capable of a significant (33%) amount of exchange of DNA molecules, but make less than 1% of wild-type levels of viable intragenic recombinants. rad6 diploids are also capable of undergoing a high level of exchange, as measured by the appearance of the recombined restriction fragment. In addition, rad6 diploids show an unusual allele- or locus-specific variability in the level of viable intragenic recombinants produced. Although rad6 diploids produce no viable spores, they are able to complete a significant amount of haploidization upon return to vegetative growth conditions.     

Comparative Analysis of Genome Diversity in Bullmastiff Dogs

Management and preservation of genomic diversity in dog breeds is a major objective for maintaining health. The present study was undertaken to characterise genomic diversity in Bullmastiff dogs using both genealogical and molecular analysis. Genealogical analysis of diversity was conducted using a database consisting of 16,378 Bullmastiff pedigrees from year 1980 to 2013. Additionally, a total of 188 Bullmastiff dogs were genotyped using the 170,000 SNP Illumina CanineHD Beadchip. Genealogical parameters revealed a mean inbreeding coefficient of 0.047; 142 total founders (f); an effective number of founders (f_e) of 79; an effective number of ancestors (f_a) of 62; and an effective population size of the reference population of 41. Genetic diversity and the degree of genome-wide homogeneity within the breed were also investigated using molecular data. Multiple-locus heterozygosity (MLH) was equal to 0.206; runs of homozygosity (ROH) as proportion of the genome, averaged 16.44%; effective population size was 29.1, with an average inbreeding coefficient of 0.035, all estimated using SNP Data. Fine-scale population structure was analysed using NETVIEW, a population analysis pipeline. Visualisation of the high definition network captured relationships among individuals within and between subpopulations. Effects of unequal founder use, and ancestral inbreeding and selection, were evident. While current levels of Bullmastiff heterozygosity, inbreeding and homozygosity are not unusual, a relatively small effective population size indicates that a breeding strategy to reduce the inbreeding rate may be beneficial.     

Intra-protein Compensatory Mutations Analysis Highlights the tRNA Recognition Regions in Aminoacyl-tRNA Synthetases

Abstract

The aminoacyl-tRNA synthetases (aaRSs) covalently attach amino acids to their corresponding nucleic acid adapter molecules, tRNAs. The interactions in the tRNA-aaRSs complexes are mostly non-specific, and largely electrostatic. Tracing a way of aaRS-tRNA mutual adaptation throughout evolution offers a clearer view of understanding how aaRS-tRNA systems preserve patterns of tRNA recognition and binding. In this study, we used the compensatory mutations analysis to explore adaptation of aaRSs in respond to random mutations that can occur in the tRNA-recognition area. We showed that the frequency of compensatory mutations among residues that belong to the recognition region is 1.75-fold higher than that of the exposed residues. The highest frequencies of compensatory mutations are observed for pairs of charged residues, wherein one residue is located within the tRNA-recognition area, while the second is placed outside of the area, and contributes to the formation of the aaRS electrostatic landscape. Given charged residues are compensated by buried charge residues in more than 60% of the analyzed mutations. The cytoplasmatic and mitochondrial aaRSs preserve similar patterns of compensatory mutations in the tRNA recognition areas. Moreover, we found that mitochondrial aaRSs demonstrate a significant increase in the frequency of compensatory mutations in the area. Our findings shed light on the physical nature of compensatory mutations in aaRSs, thereby keeping unchanged tRNA-recognition patterns.     

Chromosomal Copy Number Variation,Selection and Uneven Rates of Recombination Reveal Cryptic Genome Diversity Linked to Pathogenicity

Pathogenic fungi constitute a growing threat to both plant and animal species on a global scale. Despite a clonal mode of reproduction dominating the population genetic structure of many fungi, putatively asexual species are known to adapt rapidly when confronted by efforts to control their growth and transmission. However, the mechanisms by which adaptive diversity is generated across a clonal background are often poorly understood. We sequenced a global panel of the emergent amphibian pathogen, Batrachochytrium dendrobatidis (Bd), to high depth and characterized rapidly changing features of its genome that we believe hold the key to the worldwide success of this organism. Our analyses show three processes that contribute to the generation of de novo diversity. Firstly, we show that the majority of wild isolates manifest chromosomal copy number variation that changes over short timescales. Secondly, we show that cryptic recombination occurs within all lineages of Bd, leading to large regions of the genome being in linkage equilibrium, and is preferentially associated with classes of genes of known importance for virulence in other pathosystems. Finally, we show that these classes of genes are under directional selection, and that this has predominantly targeted the Global Panzootic Lineage (BdGPL). Our analyses show that Bd manifests an unusually dynamic genome that may have been shaped by its association with the amphibian host. The rates of variation that we document likely explain the high levels of phenotypic variability that have been reported for Bd, and suggests that the dynamic genome of this pathogen has contributed to its success across multiple biomes and host-species.     

Genetic Analysis of Variation in Human Meiotic Recombination

The number of recombination events per meiosis varies extensively among individuals. This recombination phenotype differs between female and male, and also among individuals of each gender. In this study, we used high-density SNP genotypes of over 2,300 individuals and their offspring in two datasets to characterize recombination landscape and to map the genetic variants that contribute to variation in recombination phenotypes. We found six genetic loci that are associated with recombination phenotypes. Two of these (RNF212 and an inversion on chromosome 17q21.31) were previously reported in the Icelandic population, and this is the first replication in any other population. Of the four newly identified loci (KIAA1462, PDZK1, UGCG, NUB1), results from expression studies provide support for their roles in meiosis. Each of the variants that we identified explains only a small fraction of the individual variation in recombination. Notably, we found different sequence variants associated with female and male recombination phenotypes, suggesting that they are regulated by different genes. Characterization of genetic variants that influence natural variation in meiotic recombination will lead to a better understanding of normal meiotic events as well as of non-disjunction, the primary cause of pregnancy loss.     

Allelic Skewing of DNA Methylation Is Widespread across the Genome

DNA methylation is assumed to be complementary on both alleles across the genome, although there are exceptions, notably in regions subject to genomic imprinting. We present a genome-wide survey of the degree of allelic skewing of DNA methylation with the aim of identifying previously unreported differentially methylated regions (DMRs) associated primarily with genomic imprinting or DNA sequence variation acting in cis. We used SNP microarrays to quantitatively assess allele-specific DNA methylation (ASM) in amplicons covering 7.6% of the human genome following cleavage with a cocktail of methylation-sensitive restriction enzymes (MSREs). Selected findings were verified using bisulfite-mapping and gene-expression analyses, subsequently tested in a second tissue from the same individuals, and replicated in DNA obtained from 30 parent-child trios. Our approach detected clear examples of ASM in the vicinity of known imprinted loci, highlighting the validity of the method. In total, 2,704 (1.5%) of our 183,605 informative and stringently filtered SNPs demonstrate an average relative allele score (RAS) change ≥0.10 following MSRE digestion. In agreement with previous reports, the majority of ASM (∼90%) appears to be cis in nature, and several examples of tissue-specific ASM were identified. Our data show that ASM is a widespread phenomenon, with >35,000 such sites potentially occurring across the genome, and that a spectrum of ASM is likely, with heterogeneity between individuals and across tissues. These findings impact our understanding about the origin of individual phenotypic differences and have implications for genetic studies of complex disease.     

High-Throughput Quantitative Analysis of the Human Intestinal Microbiota with a Phylogenetic Microarray

Gut microbiota carry out key functions in health and participate in the pathogenesis of a growing number of diseases. The aim of this study was to develop a custom microarray that is able to identify hundreds of intestinal bacterial species. We used the Entrez nucleotide database to compile a data set of bacterial 16S rRNA gene sequences isolated from human intestinal and fecal samples. Identified sequences were clustered into separate phylospecies groups. Representative sequences from each phylospecies were used to develop a microbiota microarray based on the Affymetrix GeneChip platform. The designed microbiota array contains probes to 775 different bacterial phylospecies. In our validation experiments, the array correctly identified genomic DNA from all 15 bacterial species used. Microbiota array has a detection sensitivity of at least 1 pg of genomic DNA and can detect bacteria present at a 0.00025% level of overall sample. Using the developed microarray, fecal samples from two healthy children and two healthy adults were analyzed for bacterial presence. Between 227 and 232 species were detected in fecal samples from children, whereas 191 to 208 species were found in adult stools. The majority of identified phylospecies belonged to the classes Clostridia and Bacteroidetes. The microarray revealed putative differences between the gut microbiota of healthy children and adults: fecal samples from adults had more Clostridia and less Bacteroidetes and Proteobacteria than those from children. A number of other putative differences were found at the genus level.In the healthy adult, there are 10¹¹ to 10¹⁴ bacteria colonizing the intestine. This outnumbers the total tissue cells in the body by at least an order of magnitude. The composition and activity of this complex microbial system (called microbiota or microflora) have a major influence on health and disease (9). Commensal microbiota contribute to the trophic functions of the gut (producing fermentation products and vitamins that can be used by intestinal epithelial cells), stimulate the immune function of the gastrointestinal tract, transform or excrete toxic substances, protect the host against invasion by pathogenic species, and modulate gut motility (28, 36). At the same time, recent research incriminates a dysfunctional cross-talk between the host and the microbiota in the pathogenesis of a growing number of disorders, such as irritable bowel syndrome, inflammatory bowel disease, allergic diseases, and gastrointestinal cancer (28).While the intestine in a newborn contains no microbes, immediately after birth the intestine of the infant is colonized by enterobacteria and enterococci. Gradual changes in microbiota composition occur during childhood, with a general reduction in the number of aerobes and facultative anaerobes and an increase in the populations of obligate anaerobic species (27). It is considered that by 2 years of age the microbiota resembles that of an adult, which is dominated in health and disease by species from only four phyla, Firmicutes (predominantly Clostridia; 50 to 70% total bacterial numbers), Bacteroidetes (10 to 30%), Proteobacteria (up to 10%), and Actinobacteria (up to 5%), with 90% believed to be obligate anaerobes (4, 10, 11, 22).Traditionally, microorganisms were detected in intestinal samples and feces by microscopic, biochemical, or physiological methods, or by culturing on selective nutrient media. However, since most intestinal microbiota species are obligate anaerobes, their isolation and culturing are difficult (21, 38, 42). In recent years, new methods based on the use of microarray technology have been utilized for the characterization of complex microbial communities (18, 32, 41, 43, 47). Microarrays represent an excellent choice for the high-throughput analysis of bacterial populations, because many different probes can be placed on one slide or synthesized on one chip, and samples thus can be tested for the presence of many different species simultaneously. Environmental and clinical samples can be interrogated directly, circumventing any need for culturing, and thus nonculturable species can be reliably detected.Several types of microarrays have been used to date to characterize the composition of microbial communities (47). Community genome arrays are constructed using whole genomic DNA (gDNA) isolated from pure culture strains (46). Functional gene arrays contain genes encoding key enzymes that are involved in various biochemical processes, and they are useful for monitoring physiological changes in microbial communities (14, 45). Phylogenetic oligonucleotide arrays contain probes derived from rRNA sequence information and are ideally suited for the analysis of microbial community composition structure and variance. Different types of phylogenetic arrays have been designed for these purposes (26, 30, 31).A number of projects performed in the last several years focused on sampling the diversity of human microbiota by the cloning and subsequent sequencing of the 16S rRNA genes isolated from gastrointestinal and fecal samples (5, 10, 13, 23, 38). In this project, we have designed, developed, and validated a custom microbiota microarray containing 16S rRNA genes probes to 775 different microbial phylospecies of human intestinal bacteria. We also have tested the applicability of this array to profiling the microbiota populations in fecal samples isolated from two adult and two child volunteers.