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PubMed2004

Detection and nucleotide sequencing of a DNA-packaging protein gene of equine gammaherpesviruses.

In previous studies, novel putative viral pathogens designated that asinine herpesvirus 4 (AsHV4) and asinine herpesvirus 5 (AsHV5) were associated with fatal interstitial pneumonia in donkeys (Equus asinus). Nucleotide sequence analysis of a portion of the DNA polymerase gene identified these putative pathogens as herpesviruses and possibly as members of the Gammaherpesvirinae subfamily. Although similar to equine herpesvirus 2 (EHV2) and equine herpesvirus 5 (EHV5), sequence diversity was observed among the detected viruses. In this study, novel sequence is reported for a DNA-packaging protein gene of EHV5 plus AsHV4, AsHV5, and a newly described putative pathogen herein designated asinine herpesvirus 6 (AsHV6). Phylogenetic analysis of these sequences suggested that the equine gammaherpesviruses may form a separate clade within the Gammaherpesvirinae subfamily. Based on the sequence of EHV2 and the novel sequences reported in this study, a PCR assay was developed to detect equine gammaherpesviruses. Products of the predicted size were produced after amplification of DNA from EHV2, EHV5, AsHV4, AsHV5, and AsHV6. This nonnested assay was shown to consistently amplify approximately 10 genomic copies of EHV2. Amplification products were not produced from DNA template of other alpha- and gammaherpesviruses. Because the role of gammaherpesviruses has not been well defined in equine disease, it is envisioned that a single, sensitive PCR assay to detect these potential pathogens will facilitate further assessment of their role in disease.

Amino Acid Sequence↗
PubMed1985

Diversity of circumsporozoite antigen genes from two strains of the malarial parasite Plasmodium knowlesi.

The complete nucleotide sequence of the coding region of the circumsporozoite antigen gene (CS gene) of the Nuri strain of the malarial parasite Plasmodium knowlesi is presented. The gene from the Nuri strain exhibits a novel form of sequence diversity when compared to the CS gene from the H strain. Instead of the 12 tandem repeating 36-base pair units of the H strain, the Nuri strain contains 16 tandem repeating 27-base pair units of a different nucleotide sequence that encodes a different repeating peptide. In contrast, the 5' and 3' coding and noncoding sequences flanking the repeats are 98 percent conserved in both strains.

Amino Acid Sequence↗
PubMed2001

The interaction of protein structure, selection, and recombination on the evolution of the type-1 fimbrial major subunit (fimA) from Escherichia coli.

Fimbrial adhesins allow bacteria to interact with and attach to their environment. The bacteria possibly benefit from these interactions, but all external structures including adhesins also allow bacteria to be identified by other organisms. Thus adhesion molecules might be under multiple forms of selection including selection to constrain functional interactions or evolve novel epitopes to avoid recognition. We address these issues by studying genetic diversity in the Escherichia coli type-1 fimbrial major subunit, fimA. Overall, sequence diversity in fimA is high (pi = 0.07) relative to that in other E. coli genes. High diversity is a function of positive diversifying selection, as detected by d(N)/d(S) ratios higher than 1.0, and amino acid residuces subject to diversifying selection are nonrandomly clustered on the exterior surface of the peptide. In addition, McDonald and Kreitman tests suggest that there has been historical but not current directional selection at fimA between E. coli and Salmonella. Finally, some regions of the fimA peptide appear to be under strong structural constraint within E. coli, particularly the interior regions of the molecule that is involved in subunit to subunit interaction. Recombination also plays a major role contributing to E. coli fimA allelic variation and estimates of recombination (2N(e)c) and mutation (2N(e)mu) are about the same. Recombination may act to separate the diverse evolutionary forces in different regions of the fimA peptide.

Adhesins, Escherichia coli↗
PubMed2005

The evolution of immune-related genes from disease carrying mosquitoes: diversity in a peptidoglycan- and a thioester-recognizing protein.

Adaptive polymorphism may be common in immune system genes as co-evolutionary interactions foster diversity; either through ongoing positive selection (arms races), or balancing selection. DNA sequence diversity in two putative immune system genes was examined in species of the genus Anopheles and from Aedes aegypti. For one gene, encoding the peptidoglycan recognizing protein PGRPLB, there was evidence of purifying selection, suggesting that selection acts to eliminate sequence variation. For another gene, encoding the thioester-containing protein TEP3, higher levels of amino acid replacement were found than would be expected under neutral models of evolution - an indication that this gene has been subject to repeated bouts of positive selection.

Aedes↗
PubMed1981

Complete amino acid sequence of light chain variable regions derived from five monoclonal anti-p-azophenylarsonate antibodies differing with respect to a crossreactive idiotype.

The induced antibody response to the hapten p-azophenylarsonate in the A/J mouse has provided a model system for the detailed examination of a heritable crossreactive idiotype and its fine structural and serologic analysis. While earlier studies used to apparent homogeneity in the serum response for structural studies, a more complete understanding of the arsonate idiotypic system became possible with the development of monoclonal antibodies differing with respect to these determinants. Five monoclonal antibodies, four crossreactive idiotype positive and one crossreactive idiotype negative, were selected for complete amino acid sequence analysis. The sequences of the light chain variable regions of these molecules are presented here. The data indicate considerable sequence divergence of the monoclonal light chains from the serum light chains. However, there is a striking degree of homology among the monoclonal light chains regardless of the idiotype character of the parent molecule. Although minor variations are apparent throughout the variable regions, the joining regions are identical among light chains in all of these anti-arsonate antibodies. A particularly notable focus of variation is found at positions 92 and 93 in the third hypervariable region. The possible role of this region in the contribution of the light chain to the arsonate crossreactive idiotype is discussed. These data are consistent with the concept that the anti-arsonate monoclonal light chains originate from the joining of a specific J kappa gene segment to a single germ-line V kappa gene segment. These coding segments are likely further subject to a variety of somatic alterations that generate the modest sequence diversity found among the final protein products.

Amino Acid Sequence↗
PubMed2000

Dichotomy of single-nucleotide polymorphism haplotypes in olfactory receptor genes and pseudogenes.

Substantial efforts are focused on identifying single-nucleotide polymorphisms (SNPs) throughout the human genome, particularly in coding regions (cSNPs), for both linkage disequilibrium and association studies. Less attention, however, has been directed to the clarification of evolutionary processes that are responsible for the variability in nucleotide diversity among different regions of the genome. We report here the population sequence diversity of genomic segments within a 450-kb cluster of olfactory receptor (OR) genes on human chromosome 17. We found a dichotomy in the pattern of nucleotide diversity between OR pseudogenes and introns on the one hand and the closely interspersed intact genes on the other. We suggest that weak positive selection is responsible for the observed patterns of genetic variation. This is inferred from a lower ratio of polymorphism to divergence in genes compared with pseudogenes or introns, high non-synonymous substitution rates in OR genes, and a small but significant overall reduction in variability in the entire OR gene cluster compared with other genomic regions. The dichotomy among functionally different segments within a short genomic distance requires high recombination rates within this OR cluster. Our work demonstrates the impact of weak positive selection on human nucleotide diversity, and has implications for the evolution of the olfactory repertoire.

Alleles↗
PubMed2002

Extensive CDR3H length heterogeneity exists in bovine foetal VDJ rearrangements.

Analysis of seven variable-diversity-joining (VDJ) gene rearrangements in B splenocytes from a 125-day-old bovine foetus revealed an extensive heavy-chain complementarity-determining region 3 (CDR3H) length variation (9-56 codons). Indeed, the global CDR3H size spectratyping of foetal VDJ rearrangements substantiated such an extensive heterogeneity and was comparable with that noted in peripheral B lymphocytes of adult cattle. These observations are in contrast to species such as humans with extensive germline combinatorial capability where shorter CDR3H length is noted early during B-cell development. Exceptionally long CDR3H (as in adult cattle) was noted in two foetal VDJ rearrangements encoded by a single germline VH gene. Further, two VH genes (gl.110.20 and BF2B5) were preferentially expressed in the foetal VDJ rearrangements. The DH gene-encoded CDR3H region of foetal VDJ rearrangements is remarkable for repetitive GGT (glycine) and TAT (tyrosine) codons that favour the recruitment of somatic hypermutations. It appears that closely related germline DH genes, preferentially used in the hydrophilic reading frame, encode varying CDR3H lengths early during B-cell ontogeny in cattle. A comparison of germline and expressed VH genes, especially in the CDR1 and CDR2, confirms that somatic hypermutations contribute to immunoglobulin (Ig)M antibody diversification in cattle. The biased nucleotide base use and high occurrence of 'hot-spot' triplet (AGPy; AG pyrimidine base) in the CDRs predisposes to somatic hypermutations. Overall, these observations suggest that extensive CDR3H length heterogeneity, including the generation of exceptionally long CDR3H (up to 56 amino acids), and somatic hypermutations contribute to IgM antibody diversification in cattle. The extensive CDR3H length heterogeneity early during the B-cell development may compensate for constraints imposed on antibody diversification owing to the limited germline sequence diversity of genetic elements in cattle.

Amino Acid Sequence↗
PubMed2004

Molecular characterization of echovirus 11 isolates from India.

BACKGROUND & OBJECTIVES: Echovirus 11 (ECV11) is one of the most frequent non-polio enteroviruses isolated from stool samples of children with acute flaccid paralysis in north India. The present work was undertaken to study the sequence variability in the 440 bp of 5'-non-translated region of ECV11 genome using heteroduplex mobility assay (HMA). METHODS: Twelve ECV11 isolates were studied for sequence variability in the 5'-non-translated region (5'NTR) using the HMA followed by nucleotide sequencing. HMA was used to determine sequence diversity between Indian ECV11 isolates and prototype Gregory strain. HMA results were confirmed by 5'NTR nucleotide sequencing of five Indian ECV11 isolates. RESULTS: HMA results showed high genomic diversity between the prototype Gregory strain and Indian ECV11 isolates. All isolates were grouped into five different types of heteroduplex mobility patterns with respect to Gregory strain. A 440 bp 5'NTR fragment of five ECV11 isolates representing different heteroduplex patterns, was sequenced. The sequence alignment showed that 5'NTR of Indian isolates was different from prototype Gregory strain and identical to the ECV11 isolates of Finland and Hungary. Phylogenetic analysis including ECV11 isolate sequences from different parts of the world showed that Indian ECV11 isolates represented a different subgroup. INTERPRETATION & CONCLUSION: The results of the present study suggested that the HMA could be successfully used as a preliminary screening method for sequence variability determination of enterovirus field isolates. The sequence data generated on ECV11 isolates from India will be useful for future studies of endemic genotypes of echovirus.

5' Untranslated Regions↗
PubMed2003

Quantitative estimates of sequence divergence for comparative analyses of mammalian genomes.

Comparative sequence analyses on a collection of carefully chosen mammalian genomes could facilitate identification of functional elements within the human genome and allow quantification of evolutionary constraint at the single nucleotide level. High-resolution quantification would be informative for determining the distribution of important positions within functional elements and for evaluating the relative importance of nucleotide sites that carry single nucleotide polymorphisms (SNPs). Because the level of resolution in comparative sequence analyses is a direct function of sequence diversity, we propose that the information content of a candidate mammalian genome be defined as the sequence divergence it would add relative to already-sequenced genomes. We show that reliable estimates of genomic sequence divergence can be obtained from small genomic regions. On the basis of a multiple sequence alignment of approximately 1.4 megabases each from eight mammals, we generate such estimates for five unsequenced mammals. Estimates of the neutral divergence in these data suggest that a small number of diverse mammalian genomes in addition to human, mouse, and rat would allow single nucleotide resolution in comparative sequence analyses.

Animals↗
PubMed1993

Reconstitution of wild type viral DNA in simian cells transfected with early and late SV40 defective genomes.

The DNAs of polyomaviruses ordinarily exist as a single circular molecule of approximately 5000 base pairs. Variants of SV40, BKV and JCV have been described which contain two complementing defective DNA molecules. These defectives, which form a bipartite genome structure, contain either the viral early region or the late region. The defectives have the unique property of being able to tolerate variable sized reiterations of regulatory and terminus region sequences, and portions of the coding region. They can also exchange coding region sequences with other polyomaviruses. It has been suggested that the bipartite genome structure might be a stage in the evolution of polyomaviruses which can uniquely sustain genome and sequence diversity. However, it is not known if the regulatory and terminus region sequences are highly mutable. Also, it is not known if the bipartite genome structure is reversible and what the conditions might be which would favor restoration of the monomolecular genome structure. We addressed the first question by sequencing the reiterated regulatory and terminus regions of E- and L-SV40 DNAs. This revealed a large number of mutations in the regulatory regions of the defective genomes, including deletions, insertions, rearrangements and base substitutions. We also detected insertions and base substitutions in the T-antigen gene. We addressed the second question by introducing into permissive simian cells, E- and L-SV40 genomes which had been engineered to contain only a single regulatory region. Analysis of viral DNA from transfected cells demonstrated recombined genomes containing a wild type monomolecular DNA structure. However, the complete defectives, containing reiterated regulatory regions, could often compete away the wild type genomes. The recombinant monomolecular genomes were isolated, cloned and found to be infectious. All of the DNA alterations identified in one of the regulatory regions of E-SV40 DNA were present in the recombinant monomolecular genomes. These and other findings indicate that the bipartite genome state can sustain many mutations which wtSV40 cannot directly sustain. However, the mutations can later be introduced into the wild type genomes when the E- and L-SV40 DNAs recombine to generate a new monomolecular genome structure.

Animals↗
PubMed2003

Imitating the humoral immune response.

The immune system makes use of two distinct mechanisms to mount an efficient response against almost every foreign macromolecular substance. First, antibodies with their robust immunoglobulin domain architecture provide a rigid scaffold to support six hypervariable loops, capable of forming highly diverse binding sites. Second, an efficient genetic mechanism has evolved to create sequence diversity at the somatic level in a step-wise process, whereby random recombination of an inherited set of gene segments is followed by hypermutation events. Insight into the corresponding molecular mechanisms is developing rapidly and enables adaptation of the emerging principles to the creation of artificial binding proteins in vitro, using the techniques of combinatorial biotechnology. Thus, novel types of receptor molecules have been constructed from alternative scaffolds, including alpha-helical bundle and beta-barrel proteins. These may provide superior tools for the recognition, targeting or separation of a wide range of biomolecular structures or substances in biological research, technology, and even medicine.

Antibodies↗
PubMed2005

Post-amplification Klenow fragment treatment alleviates PCR bias caused by partially single-stranded amplicons.

Partially single-stranded amplicons, formed during PCR amplification of single and mixed templates, are a potential source of bias in genetic diversity studies. The analysis of 16S rRNA gene diversity in mixed template samples by the fingerprinting technique terminal restriction fragment length polymorphism (T-RFLP) analysis can be biased by the occurrence of pseudo-T-RFs, i.e., restriction fragments occurring in addition to the expected terminal restriction fragments of single amplicons. This bias originates from PCR products, which are single-stranded at their terminal restriction site. Here we show that treatment of PCR amplicons with Klenow fragment prior to restriction digest and T-RFLP analysis minimized effectively the occurrence of pseudo-T-RFs. Klenow fragment activity filled in bases into the partially single-stranded amplicons and thereby restored the affected amplicons to complete double strands. Our method allowed to improve the assessment of genetic diversity and gene ratios from T-RFLP analysis of an original environmental sample. Since partially single-stranded amplicons might influence many PCR-based techniques, post-amplification treatment with Klenow fragment may be useful for a wide range of applications, which assess the composition of amplicon pools, e.g., the analysis of marker gene diversity in mixed template samples by fingerprinting techniques or the analysis of sequence diversity by cloning.

Animals↗
PubMed2002

Extensive profiling of a complex microbial community by high-throughput sequencing.

Complex microbial communities remain poorly characterized despite their ubiquity and importance to human and animal health, agriculture, and industry. Attempts to describe microbial communities by either traditional microbiological methods or molecular methods have been limited in both scale and precision. The availability of genomics technologies offers an unprecedented opportunity to conduct more comprehensive characterizations of microbial communities. Here we describe the application of an established molecular diagnostic method based on the chaperonin-60 sequence, in combination with high-throughput sequencing, to the profiling of a microbial community: the pig intestinal microbial community. Four libraries of cloned cpn60 sequences were generated by two genomic DNA extraction procedures in combination with two PCR protocols. A total of 1,125 cloned cpn60 sequences from the four libraries were sequenced. Among the 1,125 cloned cpn60 sequences, we identified 398 different nucleotide sequences encoding 280 unique peptide sequences. Pairwise comparisons of the 398 unique nucleotide sequences revealed a high degree of sequence diversity within the library. Identification of the likely taxonomic origins of cloned sequences ranged from imprecise, with clones assigned to a taxonomic subclass, to precise, for cloned sequences with 100% DNA sequence identity with a species in our reference database. The compositions of the four libraries were compared and differences related to library construction parameters were observed. Our results indicate that this method is an alternative to 16S rRNA sequence-based studies which can be scaled up for the purpose of performing a potentially comprehensive assessment of a given microbial community or for comparative studies.

Animals↗
PubMed1996

Genetic and phylogenetic divergence of feline immunodeficiency virus in the puma (Puma concolor).

Feline immunodeficiency virus (FIV) is a lentivirus which causes an AIDS-like disease in domestic cats (Felis catus). A number of other felid species, including the puma (Puma concolor), carry a virus closely related to domestic cat FIV. Serological testing revealed the presence of antibodies to FIV in 22% of 434 samples from throughout the geographic range of the puma. FIV-Pco pol gene sequences isolated from pumas revealed extensive sequence diversity, greater than has been documented in the domestic cat. The puma sequences formed two highly divergent groups, analogous to the clades which have been defined for domestic cat and lion (Panthera leo) FIV. The puma clade A was made up of samples from Florida and California, whereas clade B consisted of samples from other parts of North America, Central America, and Brazil. The difference between these two groups was as great as that reported among three lion FIV clades. Within puma clades, sequence variation is large, comparable to between-clade differences seen for domestic cat clades, allowing recognition of 15 phylogenetic lineages (subclades) among puma FIV-Pco. Large sequence divergence among isolates, nearly complete species monophyly, and widespread geographic distribution suggest that FIV-Pco has evolved within the puma species for a long period. The sequence data provided evidence for vertical transmission of FIV-Pco from mothers to their kittens, for coinfection of individuals by two different viral strains, and for cross-species transmission of FIV from a domestic cat to a puma. These factors may all be important for understanding the epidemiology and natural history of FIV in the puma.

Amino Acid Sequence↗
PubMed2005

Gain and loss of multiple genes during the evolution of Helicobacter pylori.

Sequence diversity and gene content distinguish most isolates of Helicobacter pylori. Even greater sequence differences differentiate distinct populations of H. pylori from different continents, but it was not clear whether these populations also differ in gene content. To address this question, we tested 56 globally representative strains of H. pylori and four strains of Helicobacter acinonychis with whole genome microarrays. Of the weighted average of 1,531 genes present in the two sequenced genomes, 25% are absent in at least one strain of H. pylori and 21% were absent or variable in H. acinonychis. We extrapolate that the core genome present in all isolates of H. pylori contains 1,111 genes. Variable genes tend to be small and possess unusual GC content; many of them have probably been imported by horizontal gene transfer. Phylogenetic trees based on the microarray data differ from those based on sequences of seven genes from the core genome. These discrepancies are due to homoplasies resulting from independent gene loss by deletion or recombination in multiple strains, which distort phylogenetic patterns. The patterns of these discrepancies versus population structure allow a reconstruction of the timing of the acquisition of variable genes within this species. Variable genes that are located within the cag pathogenicity island were apparently first acquired en bloc after speciation. In contrast, most other variable genes are of unknown function or encode restriction/modification enzymes, transposases, or outer membrane proteins. These seem to have been acquired prior to speciation of H. pylori and were subsequently lost by convergent evolution within individual strains. Thus, the use of microarrays can reveal patterns of gene gain or loss when examined within a phylogenetic context that is based on sequences of core genes.

Evolution, Molecular↗
PubMed2006

Identification of Tn5397-like and Tn916-like transposons and diversity of the tetracycline resistance gene tet(M) in enterococci from humans, pigs and poultry.

OBJECTIVES: To analyse the sequence diversity of the tetracycline resistance gene tet(M) and its location on mobile elements in Enterococcus faecium and Enterococcus faecalis from humans, pigs and poultry in Denmark. METHODS: A total of 76 isolates were screened for Tn916/Tn1545-like and Tn5397-like transposons using PCR. tet(M) was sequenced in 15 of the isolates and compared with tet(M) sequences submitted to GenBank (phylogenetic analysis and signs of recombination). Plasmids were extracted, filter-mating experiments were performed and Tn5397-like transposons were further characterized in selected isolates. RESULTS: In 8 of 13 isolates of E. faecium from broilers, tet(M) was present on Tn5397-like transposons, whereas tet(M) was predominantly associated with Tn916/Tn1545-like transposons in E. faecium from pigs and humans, as well as in E. faecalis from humans, pigs and broilers (50 of 63 isolates). The tet(M) genes were divided into three major subgroups according to the phylogenetic analysis. Subgroup I consisted of tet(M) from Clostridium difficile and E. faecium associated with Tn5397-like elements, subgroup II consisted of tet(M) located on Tn916/Tn1545 family transposons and subgroup III consisted of tet(M) associated with composite elements containing several resistance genes. We found evidence of recombination both within and between these groups. Moreover, we identified an E. faecium isolate with both Tn916/Tn1545-like and Tn5397-like elements. CONCLUSIONS: This study showed that enterococci contain diverse tet(M) genes present on different mobile elements, which may suggest that enterococci play an important role in the evolution and horizontal spread of mobile elements carrying tet(M). This is the first report of Tn5397-like elements in enterococci.

Animals↗
PubMedDate not supplied

Elephant seal genetic variation and the use of simulation models to investigate historical population bottlenecks.

Because the northern elephant seal (Mirounga angustirostrus) was heavily exploited during the 19th century, it experienced an extreme population bottleneck. Since then, under legislative protection in the United States and Mexico, northern elephant seals have recovered dramatically in number, although their genomic diversity was greatly reduced, apparently as a consequence of the bottleneck. In this study we investigated DNA sequence diversity in two mtDNA regions (the control region and 16S RNA) and found low genetic variation in the northern elephant seal: there were only two control region haplotypes (sequence difference = 1%), which was consistent with an extreme founder event in the recent history of the northern species. We also reaffirmed the lack of allozyme diversity in this species. In contrast, the southern elephant seal (M. leonina), which though similarly exploited never fell below 1,000 animals, had 23 control region mtDNA haplotypes (average sequence difference = 2.3%). To investigate the extent of the founder event in the northern elephant seal we devised a simulation model based on extensive demographic data. This allowed a statistical analysis of the likely outcome of bottlenecks of different size and duration. Given these historical data, our results indicate (within 95% confidence) a bottleneck of less than 30 seals and 20-year duration, or, if hunting was the primary pressure on the population, a single-year bottleneck of less than 20 seals.

Animals↗
PubMed2006

Evolutionary history of a mosquito endosymbiont revealed through mitochondrial hitchhiking.

Due to cytoplasmic inheritance, spread of maternally inherited Wolbachia symbionts can result in reduction of mitochondrial variation in populations. We examined sequence diversity of the mitochondrial NADH dehydrogenase subunit 4 (ND4) gene in Wolbachia-infected (South Africa (SA), California and Thailand) and uninfected (SA) Culex pipiens complex populations. In total, we identified 12 haplotypes (A-L). In infected populations, 99% of individuals had haplotype K. In the uninfected SA population, 11 haplotypes were present, including K. Nuclear allozyme diversity was similar between infected and uninfected SA populations. Analysis of nuclear DNA sequences suggested that haplotype K presence in uninfected SA Cx. pipiens was probably due to a shared ancestral polymorphism rather than hybrid introgression. These data indicate that Wolbachia spread has resulted in drastic reduction of mitochondrial variability in widely separated Cx. pipiens complex populations. In contrast, the uninfected SA population is probably a cryptic species where Wolbachia introgression has been prevented by reproductive isolation, maintaining ancestral levels of mitochondrial diversity. Molecular clock analyses suggest that the Wolbachia sweep occurred within the last 47000 years. The effect of Wolbachia on mitochondrial dynamics can provide insight on the potential for Wolbachia to spread transgenes into mosquito populations to control vector-borne diseases.

Animals↗