Diversity in the 18S SSU rRNA V4 hyper-variable region of Theileria spp. in Cape buffalo (Syncerus caffer) and cattle from southern Africa.

PubMed

Mans, Ben J; Pienaar, Ronel; Latif, Abdalla A; Potgieter, Fred T

2011-05-01

Sequence variation within the 18S SSU rRNA V4 hyper-variable region can affect the accuracy of real-time hybridization probe-based diagnostics for the detection of Theileria spp. infections. This is relevant for assays that use non-specific primers, such as the real-time hybridization assay for T. parva (Sibeko et al. 2008). To assess the effect of sequence variation on this test, the Theileria 18S gene from 62 buffalo and 49 cattle samples was cloned and ∼1000 clones sequenced. Twenty-six genotypes were detected which included known and novel genotypes for the T. buffeli, T. mutans, T. taurotragi and T. velifera clades. A novel genotype related to T. sp. (sable) was also detected in 1 bovine sample. Theileria genotypic diversity was higher in buffalo compared to cattle. Polymorphism within the T. parva hyper-variable region was confirmed by aberrant real-time melting peaks and supported by sequencing of the S5 ribosomal gene. Analysis of the S5 gene suggests that this gene can be a marker for species differentiation. T. parva, T. sp. (buffalo) and T. sp. (bougasvlei) remain the only genotypes amplified by the primer set of the hybridization assay. Therefore, the 18S sequence diversity observed does not seem to affect the current real-time hybridization assay for T. parva.

Spatiotemporal Phylogenetic Analysis and Molecular Characterisation of Infectious Bursal Disease Viruses Based on the VP2 Hyper-Variable Region

PubMed Central

Dolz, Roser; Valle, Rosa; Perera, Carmen L.; Bertran, Kateri; Frías, Maria T.; Majó, Natàlia; Ganges, Llilianne; Pérez, Lester J.

2013-01-01

Background Infectious bursal disease is a highly contagious and acute viral disease caused by the infectious bursal disease virus (IBDV); it affects all major poultry producing areas of the world. The current study was designed to rigorously measure the global phylogeographic dynamics of IBDV strains to gain insight into viral population expansion as well as the emergence, spread and pattern of the geographical structure of very virulent IBDV (vvIBDV) strains. Methodology/Principal Findings Sequences of the hyper-variable region of the VP2 (HVR-VP2) gene from IBDV strains isolated from diverse geographic locations were obtained from the GenBank database; Cuban sequences were obtained in the current work. All sequences were analysed by Bayesian phylogeographic analysis, implemented in the Bayesian Evolutionary Analysis Sampling Trees (BEAST), Bayesian Tip-association Significance testing (BaTS) and Spatial Phylogenetic Reconstruction of Evolutionary Dynamics (SPREAD) software packages. Selection pressure on the HVR-VP2 was also assessed. The phylogeographic association-trait analysis showed that viruses sampled from individual countries tend to cluster together, suggesting a geographic pattern for IBDV strains. Spatial analysis from this study revealed that strains carrying sequences that were linked to increased virulence of IBDV appeared in Iran in 1981 and spread to Western Europe (Belgium) in 1987, Africa (Egypt) around 1990, East Asia (China and Japan) in 1993, the Caribbean Region (Cuba) by 1995 and South America (Brazil) around 2000. Selection pressure analysis showed that several codons in the HVR-VP2 region were under purifying selection. Conclusions/Significance To our knowledge, this work is the first study applying the Bayesian phylogeographic reconstruction approach to analyse the emergence and spread of vvIBDV strains worldwide. PMID:23805195

Spatiotemporal Phylogenetic Analysis and Molecular Characterisation of Infectious Bursal Disease Viruses Based on the VP2 Hyper-Variable Region.

PubMed

Alfonso-Morales, Abdulahi; Martínez-Pérez, Orlando; Dolz, Roser; Valle, Rosa; Perera, Carmen L; Bertran, Kateri; Frías, Maria T; Majó, Natàlia; Ganges, Llilianne; Pérez, Lester J

2013-01-01

Infectious bursal disease is a highly contagious and acute viral disease caused by the infectious bursal disease virus (IBDV); it affects all major poultry producing areas of the world. The current study was designed to rigorously measure the global phylogeographic dynamics of IBDV strains to gain insight into viral population expansion as well as the emergence, spread and pattern of the geographical structure of very virulent IBDV (vvIBDV) strains. Sequences of the hyper-variable region of the VP2 (HVR-VP2) gene from IBDV strains isolated from diverse geographic locations were obtained from the GenBank database; Cuban sequences were obtained in the current work. All sequences were analysed by Bayesian phylogeographic analysis, implemented in the Bayesian Evolutionary Analysis Sampling Trees (BEAST), Bayesian Tip-association Significance testing (BaTS) and Spatial Phylogenetic Reconstruction of Evolutionary Dynamics (SPREAD) software packages. Selection pressure on the HVR-VP2 was also assessed. The phylogeographic association-trait analysis showed that viruses sampled from individual countries tend to cluster together, suggesting a geographic pattern for IBDV strains. Spatial analysis from this study revealed that strains carrying sequences that were linked to increased virulence of IBDV appeared in Iran in 1981 and spread to Western Europe (Belgium) in 1987, Africa (Egypt) around 1990, East Asia (China and Japan) in 1993, the Caribbean Region (Cuba) by 1995 and South America (Brazil) around 2000. Selection pressure analysis showed that several codons in the HVR-VP2 region were under purifying selection. To our knowledge, this work is the first study applying the Bayesian phylogeographic reconstruction approach to analyse the emergence and spread of vvIBDV strains worldwide.

Phylogeography of Barbary macaques (Macaca sylvanus) and the origin of the Gibraltar colony.

PubMed

Modolo, Lara; Salzburger, Walter; Martin, Robert D

2005-05-17

The Barbary macaque (Macaca sylvanus) is the earliest offshoot of the genus Macaca and the only extant African representative, all other species being Asiatic. Once distributed throughout North Africa, M. sylvanus is now restricted to isolated forest fragments in Algeria and Morocco. The species is threatened; the maximum total wild population size is estimated at 10,000 individuals. Relationships among surviving wild subpopulations in Algeria (96 samples) and Morocco (116 samples) were examined by using 468-bp sequences from hypervariable region I of the mitochondrial DNA control region. Twenty-four different haplotypes were identified, differing by 1-26 mutational steps (0.2-5.6%) and 1 insertion. With one exception (attributable to secondary introduction in coastal Morocco), Algerian and Moroccan haplotypes are clearly distinct. However, whereas Moroccan subpopulations show little divergence in hypervariable region I sequences and little correspondence with geographical distribution, there is a deep division between two main subpopulations in Algeria and one marked secondary division, with haplotypes generally matching geographical distribution. Accepting an origin of the genus Macaca of 5.5 million years ago, the Moroccan population and the two main Algerian subpopulations diverged approximately 1.6 million years ago. Distinction between Moroccan and Algerian haplotypes permitted analysis of the origin of the Gibraltar colony of Barbary macaques (68 samples; 30% of the population). It is generally held that the present Gibraltar population descended from a dozen individuals imported during World War II. However, the Gibraltar sample was found to include Algerian and Moroccan haplotypes separated by at least 16 mutational steps, revealing a dual origin of the founding females.

Phylogeography of Barbary macaques (Macaca sylvanus) and the origin of the Gibraltar colony

PubMed Central

Modolo, Lara; Salzburger, Walter; Martin, Robert D.

2005-01-01

The Barbary macaque (Macaca sylvanus) is the earliest offshoot of the genus Macaca and the only extant African representative, all other species being Asiatic. Once distributed throughout North Africa, M. sylvanus is now restricted to isolated forest fragments in Algeria and Morocco. The species is threatened; the maximum total wild population size is estimated at 10,000 individuals. Relationships among surviving wild subpopulations in Algeria (96 samples) and Morocco (116 samples) were examined by using 468-bp sequences from hypervariable region I of the mitochondrial DNA control region. Twenty-four different haplotypes were identified, differing by 1-26 mutational steps (0.2-5.6%) and 1 insertion. With one exception (attributable to secondary introduction in coastal Morocco), Algerian and Moroccan haplotypes are clearly distinct. However, whereas Moroccan subpopulations show little divergence in hypervariable region I sequences and little correspondence with geographical distribution, there is a deep division between two main subpopulations in Algeria and one marked secondary division, with haplotypes generally matching geographical distribution. Accepting an origin of the genus Macaca of 5.5 million years ago, the Moroccan population and the two main Algerian subpopulations diverged ≈1.6 million years ago. Distinction between Moroccan and Algerian haplotypes permitted analysis of the origin of the Gibraltar colony of Barbary macaques (68 samples; 30% of the population). It is generally held that the present Gibraltar population descended from a dozen individuals imported during World War II. However, the Gibraltar sample was found to include Algerian and Moroccan haplotypes separated by at least 16 mutational steps, revealing a dual origin of the founding females. PMID:15870193

High-Resolution Melting (HRM) of Hypervariable Mitochondrial DNA Regions for Forensic Science.

PubMed

Dos Santos Rocha, Alípio; de Amorim, Isis Salviano Soares; Simão, Tatiana de Almeida; da Fonseca, Adenilson de Souza; Garrido, Rodrigo Grazinoli; Mencalha, Andre Luiz

2018-03-01

Forensic strategies commonly are proceeding by analysis of short tandem repeats (STRs); however, new additional strategies have been proposed for forensic science. Thus, this article standardized the high-resolution melting (HRM) of DNA for forensic analyzes. For HRM, mitochondrial DNA (mtDNA) from eight individuals were extracted from mucosa swabs by DNAzol reagent, samples were amplified by PCR and submitted to HRM analysis to identify differences in hypervariable (HV) regions I and II. To confirm HRM, all PCR products were DNA sequencing. The data suggest that is possible discriminate DNA from different samples by HRM curves. Also, uncommon dual-dissociation was identified in a single PCR product, increasing HRM analyzes by evaluation of melting peaks. Thus, HRM is accurate and useful to screening small differences in HVI and HVII regions from mtDNA and increase the efficiency of laboratory routines based on forensic genetics. © 2017 American Academy of Forensic Sciences.

Characterization of a prototype strain of hepatitis E virus.

PubMed

Tsarev, S A; Emerson, S U; Reyes, G R; Tsareva, T S; Legters, L J; Malik, I A; Iqbal, M; Purcell, R H

1992-01-15

A strain of hepatitis E virus (SAR-55) implicated in an epidemic of enterically transmitted non-A, non-B hepatitis, now called hepatitis E, was characterized extensively. Six cynomolgus monkeys (Macaca fascicularis) were infected with a strain of hepatitis E virus from Pakistan. Reverse transcription-polymerase chain reaction was used to determine the pattern of virus shedding in feces, bile, and serum relative to hepatitis and induction of specific antibodies. Virtually the entire genome of SAR-55 (7195 nucleotides) was sequenced. Comparison of the sequence of SAR-55 with that of a Burmese strain revealed a high level of homology except for one region encoding 100 amino acids of a putative nonstructural polyprotein. Identification of this region as hypervariable was obtained by partial sequencing of a third isolate of hepatitis E virus from Kirgizia.

Analyzing Population Genetics Using the Mitochondrial Control Region and Bioinformatics

ERIC Educational Resources Information Center

Sato, Takumi; Phillips, Bonnie; Latourelle, Sandra M.; Elwess, Nancy L.

2010-01-01

The 14-base pair hypervariable region in mitochondrial DNA (mtDNA) of Asian populations, specifically Japanese and Chinese students at Plattsburgh State University, was examined. Previous research on this 14-base pair region showed it to be susceptible to mutations and as a result indicated direct correlation with specific ethnic populations.…

Utility of combining morphological characters, nuclear and mitochondrial genes: An attempt to resolve the conflicts of species identification for ciliated protists.

PubMed

Zhao, Yan; Yi, Zhenzhen; Gentekaki, Eleni; Zhan, Aibin; Al-Farraj, Saleh A; Song, Weibo

2016-01-01

Ciliates comprise a highly diverse protozoan lineage inhabiting all biotopes and playing crucial roles in regulating microbial food webs. Nevertheless, subtle morphological differences and tiny sizes hinder proper species identification for many ciliates. Here, we use the species-rich taxon Frontonia and employ both nuclear and mitochondrial loci. We attempt to assess the level of genetic diversity and evaluate the potential of each marker in delineating species of Frontonia. Morphological features and ecological characteristics are also integrated into genetic results, in an attempt to resolve conflicts of species identification based on morphological and molecular methods. Our studies reveal: (1) the mitochondrial cox1 gene, nuclear ITS1 and ITS2 as well as the hypervariable D2 region of LSU rDNA are promising candidates for species delineation; (2) the cox1 gene provides the best resolution for analyses below the species level; (3) the V2 and V4 hypervariable regions of SSU rDNA, and D1 of LSU rDNA as well as the 5.8S rDNA gene do not show distinct barcoding gap due to overlap between intra- and inter-specific genetic divergences; (4) morphological character-based analysis shows promise for delimitation of Frontonia species; and (5) all gene markers and character-based analyses demonstrate that the genus Frontonia consists of three groups and monophyly of the genus Frontonia is questionable. Copyright © 2015 Elsevier Inc. All rights reserved.

A molecular epidemiology study based on VP2 gene sequences reveals that a new genotype of infectious bursal disease virus is dominantly prevalent in Italy.

PubMed

Lupini, Caterina; Giovanardi, Davide; Pesente, Patrizia; Bonci, Michela; Felice, Viviana; Rossi, Giulia; Morandini, Emilio; Cecchinato, Mattia; Catelli, Elena

2016-08-01

A distinctive infectious bursal disease (IBD) virus genotype (ITA) was detected in IBD-live vaccinated broilers in Italy without clinical signs of IBD. It was isolated in specific-pathogen-free eggs and molecularly characterized in the hypervariable region of the virus protein (VP) 2. Phylogenetic analysis showed that ITA strains clustered separately from other homologous reference sequences of IBDVs, either classical or very virulent, retrieved from GenBank or previously reported in Italy, and from vaccine strains. The new genotype shows peculiar molecular characteristics in key positions of the VP2 hypervariable region, which affect charged or potentially glycosylated amino acids virtually associated with important changes in virus properties. Characterization of 41 IBDV strains detected in Italy between 2013 and 2014 showed that ITA is emergent in densely populated poultry areas of Italy, being 68% of the IBDV detections made during routine diagnostic activity over a two-year period, in spite of the immunity induced by large-scale vaccination. Four very virulent strains (DV86) and one classical strain (HPR2), together with eight vaccine strains, were also detected. The currently available epidemiological and clinical data do not allow the degree of pathogenicity of the ITA genotype to be defined. Only in vivo experimental pathogenicity studies conducted in secure isolation conditions, through the evaluation of clinical signs and macro/microscopic lesions, will clarify conclusively the virulence of the new Italian genotype.

Proposal of a Consensus Set of Hypervariable Mycobacterial Interspersed Repetitive-Unit–Variable-Number Tandem-Repeat Loci for Subtyping of Mycobacterium tuberculosis Beijing Isolates

PubMed Central

Allix-Béguec, Caroline; Wahl, Céline; Hanekom, Madeleine; Nikolayevskyy, Vladyslav; Drobniewski, Francis; Maeda, Shinji; Campos-Herrero, Isolina; Mokrousov, Igor; Niemann, Stefan; Kontsevaya, Irina; Rastogi, Nalin; Samper, Sofia; Sng, Li-Hwei; Warren, Robin M.

2014-01-01

Mycobacterium tuberculosis Beijing strains represent targets of special importance for molecular surveillance of tuberculosis (TB), especially because they are associated with spread of multidrug resistance in some world regions. Standard 24-locus mycobacterial interspersed repetitive-unit–variable-number tandem-repeat (MIRU-VNTR) typing lacks resolution power for accurately discriminating closely related clones that often compose Beijing strain populations. Therefore, we evaluated a set of 7 additional, hypervariable MIRU-VNTR loci for better resolution and tracing of such strains, using a collection of 535 Beijing isolates from six world regions where these strains are known to be prevalent. The typeability and interlaboratory reproducibility of these hypervariable loci were lower than those of the 24 standard loci. Three loci (2163a, 3155, and 3336) were excluded because of their redundant variability and/or more frequent noninterpretable results compared to the 4 other markers. The use of the remaining 4-locus set (1982, 3232, 3820, and 4120) increased the number of types by 52% (from 223 to 340) and reduced the clustering rate from 58.3 to 36.6%, when combined with the use of the standard 24-locus set. Known major clonal complexes/24-locus-based clusters were all subdivided, although the degree of subdivision varied depending on the complex. Only five single-locus variations were detected among the hypervariable loci of an additional panel of 92 isolates, representing 15 years of clonal spread of a single Beijing strain in a geographically restricted setting. On this calibrated basis, we propose this 4-locus set as a consensus for subtyping Beijing clonal complexes and clusters, after standard typing. PMID:24172154

Proposal of a consensus set of hypervariable mycobacterial interspersed repetitive-unit-variable-number tandem-repeat loci for subtyping of Mycobacterium tuberculosis Beijing isolates.

PubMed

Allix-Béguec, Caroline; Wahl, Céline; Hanekom, Madeleine; Nikolayevskyy, Vladyslav; Drobniewski, Francis; Maeda, Shinji; Campos-Herrero, Isolina; Mokrousov, Igor; Niemann, Stefan; Kontsevaya, Irina; Rastogi, Nalin; Samper, Sofia; Sng, Li-Hwei; Warren, Robin M; Supply, Philip

2014-01-01

Mycobacterium tuberculosis Beijing strains represent targets of special importance for molecular surveillance of tuberculosis (TB), especially because they are associated with spread of multidrug resistance in some world regions. Standard 24-locus mycobacterial interspersed repetitive-unit-variable-number tandem-repeat (MIRU-VNTR) typing lacks resolution power for accurately discriminating closely related clones that often compose Beijing strain populations. Therefore, we evaluated a set of 7 additional, hypervariable MIRU-VNTR loci for better resolution and tracing of such strains, using a collection of 535 Beijing isolates from six world regions where these strains are known to be prevalent. The typeability and interlaboratory reproducibility of these hypervariable loci were lower than those of the 24 standard loci. Three loci (2163a, 3155, and 3336) were excluded because of their redundant variability and/or more frequent noninterpretable results compared to the 4 other markers. The use of the remaining 4-locus set (1982, 3232, 3820, and 4120) increased the number of types by 52% (from 223 to 340) and reduced the clustering rate from 58.3 to 36.6%, when combined with the use of the standard 24-locus set. Known major clonal complexes/24-locus-based clusters were all subdivided, although the degree of subdivision varied depending on the complex. Only five single-locus variations were detected among the hypervariable loci of an additional panel of 92 isolates, representing 15 years of clonal spread of a single Beijing strain in a geographically restricted setting. On this calibrated basis, we propose this 4-locus set as a consensus for subtyping Beijing clonal complexes and clusters, after standard typing.

The skin microbiome in psoriatic arthritis: methodology development and pilot data.

PubMed

Castelino, Madhura; Eyre, Stephen; Moat, John; Fox, Graeme; Martin, Paul; Ijaz, Umer; Quince, Christopher; Ho, Pauline; Upton, Mathew; Barton, Anne

2015-02-26

Skin microbiota are likely to be important in the development of conditions such as psoriatic arthritis. Profiling the bacterial community in the psosriatic plaques will contribute to our understanding of the role of the skin microbiome in these conditions. The aim of this work was to determine the optimum study design for work on the skin microbiome with use of the MiSeq platform. The objectives were to compare data generated from two platforms for two primer pairs in a low density mock bacterial community. DNA was obtained from two low density mock communities of 11 diverse bacterial strains (with and without human DNA supplementation) and from swabs taken from the skin of four healthy volunteers. The DNA was amplified with primer pairs covering hypervariable regions of the 16S rRNA gene: primers 63F and 519R (V1-V3), and 347F and 803R (V3-V4). The resultant libraries were indexed for the MiSeq and Roche454 platforms and sequenced. Both datasets were de-noised, cleaned of chimeras, and analysed by use of QIIME software (version 1.8.0). No significant difference in the diversity indices at the phylum and the genus level between the platforms was seen. Comparison of the diversity indices for the mock community data for the two primer pairs demonstrated that the V3-V4 hypervariable region had significantly better capture of bacterial diversity than did the V1-V3 region. Amplification with the same primer pairs showed strong concordance within each platform (98·9-99·8%), with negligible effect of spiked human DNA contamination. Comparison at the family level classification between samples processed on the MiSeq and Roche454 platforms using the V3-V4 hypervariable region also showed a high level of concordance (87%), although less so for the V1-V3 primers (10%). The pilot data from healthy volunteers were similar. Results obtained from the V3-V4 16S rRNA hypervariable region, sequencing on the MiSeq and Roche454 platforms, were concordant between replicates, and between each other. These findings suggest that the MiSeq platform, and these primers, is a comparable method for determining skin microbiota to the widely used Roche454 methodology. NIHR Manchester Musculoskeletal Biomedical Research Unit. Copyright © 2015 Elsevier Ltd. All rights reserved.

Molecular characterization of infectious bursal disease virus isolates from Nepal based on hypervariable region of VP2 gene.

PubMed

Sharma, K; Hair-Bejo, M; Omar, A R; Aini, I

2005-01-01

Two Infectious bursal disease virus (IBDV) isolates, NP1SSH and NP2K were obtained from a severe infectious bursal disease (IBD) outbreak in Nepal in 2002. The hypervariable (HV) region of VP2 gene (1326 bp) of the isolates was generated by RT-PCR and sequenced. The obtained nucleotide sequences were compared with those of twenty other IBDV isolates/strains. Phylogenetic analysis based on this comparison revealed that NP1SSH and NP2K clustered with very virulent (vv) IBDV strains of serotype 1. In contrast, classical, Australian classical and attenuated strains of serotype 1 and avirulent IBDV strains of serotype 2 formed a different cluster. The deduced amino acid sequences of the two isolates showed a 98.3% identity with each other and 97.1% and 98.3% identities, respectively with very virulent IBDV (vvIBDV) isolates/strains. Three amino acids substitutions at positions 300 (E-->A), 308 (I-->F) and 334 (A-->P) within the HV region were common for both the isolates. The amino acids substitutions at positions 27 (S-->T), 28 (I-->T), 31 (D-->A), 36 (H-->Y), 135 (E-->G), 223 (G-->S), 225 (V-->I), 351 (L-->I), 352 (V-->E) and 399 (I-->S) for NP1SSH and at position 438 (I-->S) for NP2K were unique and differed from other IBDV isolates/strains. NP1SSH and NP2K showed highest similarity (97.8%) with the BD399 strain from Bangladesh as compared with other vvIBDV isolates/strains. We conclude that the NP1SSH and NP2K isolates of IBDV from Nepal represent vvIBDV of serotype 1.

Revisiting the phylogeny of Zoanthidea (Cnidaria: Anthozoa): Staggered alignment of hypervariable sequences improves species tree inference.

PubMed

Swain, Timothy D

2018-01-01

The recent rapid proliferation of novel taxon identification in the Zoanthidea has been accompanied by a parallel propagation of gene trees as a tool of species discovery, but not a corresponding increase in our understanding of phylogeny. This disparity is caused by the trade-off between the capabilities of automated DNA sequence alignment and data content of genes applied to phylogenetic inference in this group. Conserved genes or segments are easily aligned across the order, but produce poorly resolved trees; hypervariable genes or segments contain the evolutionary signal necessary for resolution and robust support, but sequence alignment is daunting. Staggered alignments are a form of phylogeny-informed sequence alignment composed of a mosaic of local and universal regions that allow phylogenetic inference to be applied to all nucleotides from both hypervariable and conserved gene segments. Comparisons between species tree phylogenies inferred from all data (staggered alignment) and hypervariable-excluded data (standard alignment) demonstrate improved confidence and greater topological agreement with other sources of data for the complete-data tree. This novel phylogeny is the most comprehensive to date (in terms of taxa and data) and can serve as an expandable tool for evolutionary hypothesis testing in the Zoanthidea. Spanish language abstract available in Text S1. Translation by L. O. Swain, DePaul University, Chicago, Illinois, 60604, USA. Copyright © 2017 Elsevier Inc. All rights reserved.

Characterization of a prototype strain of hepatitis E virus.

PubMed Central

Tsarev, S A; Emerson, S U; Reyes, G R; Tsareva, T S; Legters, L J; Malik, I A; Iqbal, M; Purcell, R H

1992-01-01

A strain of hepatitis E virus (SAR-55) implicated in an epidemic of enterically transmitted non-A, non-B hepatitis, now called hepatitis E, was characterized extensively. Six cynomolgus monkeys (Macaca fascicularis) were infected with a strain of hepatitis E virus from Pakistan. Reverse transcription-polymerase chain reaction was used to determine the pattern of virus shedding in feces, bile, and serum relative to hepatitis and induction of specific antibodies. Virtually the entire genome of SAR-55 (7195 nucleotides) was sequenced. Comparison of the sequence of SAR-55 with that of a Burmese strain revealed a high level of homology except for one region encoding 100 amino acids of a putative nonstructural polyprotein. Identification of this region as hypervariable was obtained by partial sequencing of a third isolate of hepatitis E virus from Kirgizia. Images PMID:1731327

Multiplexed Microsphere Suspension-Array Assay for Urine Mitochondrial DNA Typing by C-Stretch Length in Hypervariable Regions.

PubMed

Aoki, Kimiko; Tanaka, Hiroyuki; Kawahara, Takashi

2018-07-01

The standard method for personal identification and verification of urine samples in doping control is short tandem repeat (STR) analysis using nuclear DNA (nDNA). The DNA concentration of urine is very low and decreases under most conditions used for sample storage; therefore, the amount of DNA from cryopreserved urine samples may be insufficient for STR analysis. We aimed to establish a multiplexed assay for urine mitochondrial DNA typing containing only trace amounts of DNA, particularly for Japanese populations. A multiplexed suspension-array assay using oligo-tagged microspheres (Luminex MagPlex-TAG) was developed to measure C-stretch length in hypervariable region 1 (HV1) and 2 (HV2), five single nucleotide polymorphisms (SNPs), and one polymorphic indel. Based on these SNPs and the indel, the Japanese population can be classified into five major haplogroups (D4, B, M7a, A, D5). The assay was applied to DNA samples from urine cryopreserved for 1 - 1.5 years (n = 63) and fresh blood (n = 150). The assay with blood DNA enabled Japanese subjects to be categorized into 62 types, exhibiting a discriminatory power of 0.960. The detection limit for cryopreserved urine was 0.005 ng of nDNA. Profiling of blood and urine pairs revealed that 5 of 63 pairs showed different C-stretch patterns in HV1 or HV2. The assay described here yields valuable information in terms of the verification of urine sample sources employing only trace amounts of recovered DNA. However, blood cannot be used as a reference sample.

Genome-wide analysis of differential transcriptional and epigenetic variability across human immune cell types.

PubMed

Ecker, Simone; Chen, Lu; Pancaldi, Vera; Bagger, Frederik O; Fernández, José María; Carrillo de Santa Pau, Enrique; Juan, David; Mann, Alice L; Watt, Stephen; Casale, Francesco Paolo; Sidiropoulos, Nikos; Rapin, Nicolas; Merkel, Angelika; Stunnenberg, Hendrik G; Stegle, Oliver; Frontini, Mattia; Downes, Kate; Pastinen, Tomi; Kuijpers, Taco W; Rico, Daniel; Valencia, Alfonso; Beck, Stephan; Soranzo, Nicole; Paul, Dirk S

2017-01-26

A healthy immune system requires immune cells that adapt rapidly to environmental challenges. This phenotypic plasticity can be mediated by transcriptional and epigenetic variability. We apply a novel analytical approach to measure and compare transcriptional and epigenetic variability genome-wide across CD14 + CD16 - monocytes, CD66b + CD16 + neutrophils, and CD4 + CD45RA + naïve T cells from the same 125 healthy individuals. We discover substantially increased variability in neutrophils compared to monocytes and T cells. In neutrophils, genes with hypervariable expression are found to be implicated in key immune pathways and are associated with cellular properties and environmental exposure. We also observe increased sex-specific gene expression differences in neutrophils. Neutrophil-specific DNA methylation hypervariable sites are enriched at dynamic chromatin regions and active enhancers. Our data highlight the importance of transcriptional and epigenetic variability for the key role of neutrophils as the first responders to inflammatory stimuli. We provide a resource to enable further functional studies into the plasticity of immune cells, which can be accessed from: http://blueprint-dev.bioinfo.cnio.es/WP10/hypervariability .

Diversity and Plasticity of the Intracellular Plant Pathogen and Insect Symbiont “Candidatus Liberibacter asiaticus” as Revealed by Hypervariable Prophage Genes with Intragenic Tandem Repeats ▿ †

PubMed Central

Zhou, Lijuan; Powell, Charles A.; Hoffman, Michele T.; Li, Wenbin; Fan, Guocheng; Liu, Bo; Lin, Hong; Duan, Yongping

2011-01-01

“Candidatus Liberibacter asiaticus” is a psyllid-transmitted, phloem-limited alphaproteobacterium and the most prevalent species of “Ca. Liberibacter” associated with a devastating worldwide citrus disease known as huanglongbing (HLB). Two related and hypervariable genes (hyvI and hyvII) were identified in the prophage regions of the Psy62 “Ca. Liberibacter asiaticus” genome. Sequence analyses of the hyvI and hyvII genes in 35 “Ca. Liberibacter asiaticus” DNA isolates collected globally revealed that the hyvI gene contains up to 12 nearly identical tandem repeats (NITRs, 132 bp) and 4 partial repeats, while hyvII contains up to 2 NITRs and 4 partial repeats and shares homology with hyvI. Frequent deletions or insertions of these repeats within the hyvI and hyvII genes were observed, none of which disrupted the open reading frames. Sequence conservation within the individual repeats but an extensive variation in repeat numbers, rearrangement, and the sequences flanking the repeat region indicate the diversity and plasticity of “Ca. Liberibacter asiaticus” bacterial populations in the world. These differences were found not only in samples of distinct geographical origins but also in samples from a single origin and even from a single “Ca. Liberibacter asiaticus”-infected sample. This is the first evidence of different “Ca. Liberibacter asiaticus” populations coexisting in a single HLB-affected sample. The Florida “Ca. Liberibacter asiaticus” isolates contain both hyvI and hyvII, while all other global “Ca. Liberibacter asiaticus” isolates contain either one or the other. Interclade assignments of the putative HyvI and HyvII proteins from Florida isolates with other global isolates in phylogenetic trees imply multiple “Ca. Liberibacter asiaticus” populations in the world and a multisource introduction of the “Ca. Liberibacter asiaticus” bacterium into Florida. PMID:21784907

Analysis of new isolates reveals new genome organization and a hypervariable region in infectious myonecrosis virus (IMNV).

PubMed

Dantas, Márcia Danielle A; Chavante, Suely F; Teixeira, Dárlio Inácio A; Lima, João Paulo M S; Lanza, Daniel C F

2015-05-04

Infectious myonecrosis virus (IMNV) has been the cause of many losses in shrimp farming since 2002, when the first myonecrosis outbreak was reported at Brazilian's northeast coast. Two additional genomes of Brazilian IMNV isolates collected in 2009 and 2013 were sequenced and analyzed in the present study. The sequencing revealed extra 643 bp and 22 bp, at 5' and 3' ends of IMNV genome respectively, confirming that its actual size is at least 8226 bp long. Considering these additional sequences in genome extremities, ORF1 can starts at nt 470, encoding a 1708 aa polyprotein. Computational predictions reveal two stem loops and two pseudoknots in the 5' end and a putative stem loop and a slippery motif located at 3' end, indicating that these regions can be involved in the start and termination of translation. Through a careful phylogenetic analysis, a higher genetic variability among Brazilian isolates could be observed, comparing with Indonesian IMNV isolates. It was also observed that the most variable region of IMNV genome is located in the first half of ORF1, coinciding with a region which probably encodes the capsid protrusions. The results presented here are a starting point to elucidate the viral's translational regulation and the mechanisms involved in virulence. Copyright © 2015 Elsevier B.V. All rights reserved.

Full-Length Characterization of Hepatitis C Virus Subtype 3a Reveals Novel Hypervariable Regions under Positive Selection during Acute Infection▿

PubMed Central

Humphreys, Isla; Fleming, Vicki; Fabris, Paolo; Parker, Joe; Schulenberg, Bodo; Brown, Anthony; Demetriou, Charis; Gaudieri, Silvana; Pfafferott, Katja; Lucas, Michaela; Collier, Jane; Huang, Kuan-Hsiang Gary; Pybus, Oliver G.; Klenerman, Paul; Barnes, Eleanor

2009-01-01

Hepatitis C virus subtype 3a is a highly prevalent and globally distributed strain that is often associated with infection via injection drug use. This subtype exhibits particular phenotypic characteristics. In spite of this, detailed genetic analysis of this subtype has rarely been performed. We performed full-length viral sequence analysis in 18 patients with chronic HCV subtype 3a infection and assessed genomic viral variability in comparison to other HCV subtypes. Two novel regions of intragenotypic hypervariability within the envelope protein E2, of HCV genotype 3a, were identified. We named these regions HVR495 and HVR575. They consisted of flanking conserved hydrophobic amino acids and central variable residues. A 5-amino-acid insertion found only in genotype 3a and a putative glycosylation site is contained within HVR575. Evolutionary analysis of E2 showed that positively selected sites within genotype 3a infection were largely restricted to HVR1, HVR495, and HVR575. Further analysis of clonal viral populations within single hosts showed that viral variation within HVR495 and HVR575 were subject to intrahost positive selecting forces. Longitudinal analysis of four patients with acute HCV subtype 3a infection sampled at multiple time points showed that positively selected mutations within HVR495 and HVR575 arose early during primary infection. HVR495 and HVR575 were not present in HCV subtypes 1a, 1b, 2a, or 6a. Some variability that was not subject to positive selection was present in subtype 4a HVR575. Further defining the functional significance of these regions may have important implications for genotype 3a E2 virus-receptor interactions and for vaccine studies that aim to induce cross-reactive anti-E2 antibodies. PMID:19740991

Chloroplast Genome Differences between Asian and American Equisetum arvense (Equisetaceae) and the Origin of the Hypervariable trnY-trnE Intergenic Spacer

PubMed Central

Kim, Hyoung Tae; Kim, Ki-Joong

2014-01-01

Comparative analyses of complete chloroplast (cp) DNA sequences within a species may provide clues to understand the population dynamics and colonization histories of plant species. Equisetum arvense (Equisetaceae) is a widely distributed fern species in northeastern Asia, Europe, and North America. The complete cp DNA sequences from Asian and American E. arvense individuals were compared in this study. The Asian E. arvense cp genome was 583 bp shorter than that of the American E. arvense. In total, 159 indels were observed between two individuals, most of which were concentrated on the hypervariable trnY-trnE intergenic spacer (IGS) in the large single-copy (LSC) region of the cp genome. This IGS region held a series of 19 bp repeating units. The numbers of the 19 bp repeat unit were responsible for 78% of the total length difference between the two cp genomes. Furthermore, only other closely related species of Equisetum also show the hypervariable nature of the trnY-trnE IGS. By contrast, only a single indel was observed in the gene coding regions: the ycf1 gene showed 24 bp differences between the two continental individuals due to a single tandem-repeat indel. A total of 165 single-nucleotide polymorphisms (SNPs) were recorded between the two cp genomes. Of these, 52 SNPs (31.5%) were distributed in coding regions, 13 SNPs (7.9%) were in introns, and 100 SNPs (60.6%) were in intergenic spacers (IGS). The overall difference between the Asian and American E. arvense cp genomes was 0.12%. Despite the relatively high genetic diversity between Asian and American E. arvense, the two populations are recognized as a single species based on their high morphological similarity. This indicated that the two regional populations have been in morphological stasis. PMID:25157804

Single nucleotide polymorphisms in the Mycobacterium bovis genome resolve phylogenetic relationships

USDA-ARS?s Scientific Manuscript database

Mycobacterium bovis isolates carry restricted allelic variation yet exhibit a range of disease phenotypes and host preferences. Conventional genotyping methods target small hyper-variable regions of their genome and provide anonymous biallelic information insufficient to develop phylogeny. To resolv...

Evolution of ribonuclease in relation to polypeptide folding mechanisms.

NASA Technical Reports Server (NTRS)

Barnard, E. A.; Cohen, M. S.; Gold, M. H.; Kim, J.-K.

1972-01-01

Comparisons of the N-terminal region of pancreatic RNAase in seven species are presented, taking into account cow, bison, deer, rat, pig, kangaroo, and turtle. The available limited evidence on hypervariable regions indicates that there is still an evolutionary constraint on them. It is proposed that there is a selection pressure acting on all regions of a protein sequence in evolution. Mutations that tend to obstruct the folding process can lead to various intensities of selection pressure.

Analysis of base and codon usage by rubella virus.

PubMed

Zhou, Yumei; Chen, Xianfeng; Ushijima, Hiroshi; Frey, Teryl K

2012-05-01

Rubella virus (RUBV), a small, plus-strand RNA virus that is an important human pathogen, has the unique feature that the GC content of its genome (70%) is the highest (by 20%) among RNA viruses. To determine the effect of this GC content on genomic evolution, base and codon usage were analyzed across viruses from eight diverse genotypes of RUBV. Despite differences in frequency of codon use, the favored codons in the RUBV genome matched those in the human genome for 18 of the 20 amino acids, indicating adaptation to the host. Although usage patterns were conserved in corresponding genes in the diverse genotypes, within-genome comparison revealed that both base and codon usages varied regionally, particularly in the hypervariable region (HVR) of the P150 replicase gene. While directional mutation pressure was predominant in determining base and codon usage within most of the genome (with the strongest tendency being towards C's at third codon positions), natural selection was predominant in the HVR region. The GC content of this region was the highest in the genome (>80%), and it was not clear if selection at the nucleotide level accompanied selection at the amino acid level. Dinucleotide frequency analysis of the RUBV genome revealed that TpA usage was lower than expected, similar to mammalian genes; however, CpG usage was not suppressed, and TpG usage was not enhanced, as is the case in mammalian genes.

Automated antibody structure prediction using Accelrys tools: Results and best practices

PubMed Central

Fasnacht, Marc; Butenhof, Ken; Goupil-Lamy, Anne; Hernandez-Guzman, Francisco; Huang, Hongwei; Yan, Lisa

2014-01-01

We describe the methodology and results from our participation in the second Antibody Modeling Assessment experiment. During the experiment we predicted the structure of eleven unpublished antibody Fv fragments. Our prediction methods centered on template-based modeling; potential templates were selected from an antibody database based on their sequence similarity to the target in the framework regions. Depending on the quality of the templates, we constructed models of the antibody framework regions either using a single, chimeric or multiple template approach. The hypervariable loop regions in the initial models were rebuilt by grafting the corresponding regions from suitable templates onto the model. For the H3 loop region, we further refined models using ab initio methods. The final models were subjected to constrained energy minimization to resolve severe local structural problems. The analysis of the models submitted show that Accelrys tools allow for the construction of quite accurate models for the framework and the canonical CDR regions, with RMSDs to the X-ray structure on average below 1 Å for most of these regions. The results show that accurate prediction of the H3 hypervariable loops remains a challenge. Furthermore, model quality assessment of the submitted models show that the models are of quite high quality, with local geometry assessment scores similar to that of the target X-ray structures. Proteins 2014; 82:1583–1598. © 2014 The Authors. Proteins published by Wiley Periodicals, Inc. PMID:24833271

Forensic Analysis of Canine DNA Samples in the Undergraduate Biochemistry Laboratory

ERIC Educational Resources Information Center

Carson, Tobin M.; Bradley, Sharonda Q.; Fekete, Brenda L.; Millard, Julie T.; LaRiviere, Frederick J.

2009-01-01

Recent advances in canine genomics have allowed the development of highly distinguishing methods of analysis for both nuclear and mitochondrial DNA. We describe a laboratory exercise suitable for an undergraduate biochemistry course in which the polymerase chain reaction is used to amplify hypervariable regions of DNA from dog hair and saliva…

A polymorphic and hypervariable locus in the pseudoautosomal region of the CBA/H mouse sex chromosomes

DOE Office of Scientific and Technical Information (OSTI.GOV)

Fennelly, J.; Laval, S.; Wright, E.

1996-04-01

We have identified a genomic locus (DXYH1) that is polymorphic and hypervariable within the CBA/H colony. Using a panel of C57BL/6 x Mus spretus backcross offspring, it was mapped to the distal end of the X chromosome. Pseudoautosomal inheritance was demonstrated through three generations of CBA/H x CBA/H and CBA/H x C57BL/6 crosses and confirmed through linkage to the Sxr locus in X/Y Sxr x 3H1 crosses. Meiotic recombination frequencies place DXYH1 {approximately}28% into the pseudoautosomal region from the boundary. The de novo generation of CBA/H variant DXYH1 restriction fragment length polymorphisms during spermatogenesis is suggestive of the germline instabilitymore » associated with hypermutable human minisatellites. The absence of DXY1-related sequences in Mus spretus provides DNA sequence evidence to support the observed failure of X-Y pairing during meiosis and consequent hybrid infertility in C57BL/6 x Mus spretus male F1 offspring. 19 refs., 4 figs.« less

Evaluation of different PCR primers for denaturing gradient gel electrophoresis (DGGE) analysis of fungal community structure in traditional fermentation starters used for Hong Qu glutinous rice wine.

PubMed

Lv, Xu-Cong; Jiang, Ya-Jun; Liu, Jie; Guo, Wei-Ling; Liu, Zhi-Bin; Zhang, Wen; Rao, Ping-Fan; Ni, Li

2017-08-16

Denaturing gradient gel electrophoresis (DGGE) has become a widely used tool to examine microbial community structure. However, when DGGE is applied to evaluate the fungal community of traditional fermentation starters, the choice of hypervariable ribosomal RNA gene regions is still controversial. In the current study, several previously published fungal PCR primer sets were compared and evaluated using PCR-DGGE, with the purpose of screening a suitable primer set to study the fungal community of traditional fermentation starters for Hong Qu glutinous rice wine. Firstly, different primer sets were used to amplify different hypervariable regions from pure fungal cultures. Except NS1/FR1+ and ITS1fGC/ITS4, other primer sets (NL1+/LS2R, NL3A/NL4GC, FF390/FR1+, NS1/GCFung, NS3+/YM951r and ITS1fGC/ITS2r) amplified the target DNA sequences successfully. Secondly, the selected primer sets were further evaluated based on their resolution to distinguish different fungal cultures through DGGE fingerprints. Three primer sets (NL1+/LS2R, NS1/GCFung and ITS1fGC/ITS2r) were finally selected for investigating the fungal community structure of different traditional fermentation starters for Hong Qu glutinous rice wine. The internal transcribed spacer (ITS) region amplified by ITS1fGC/ITS2r, which is more hypervariable than the 18S rRNA gene and 26S rRNA gene, provides an excellent tool to separate amplification products of different fungal species. Results indicated that PCR-DGGE profile using ITS1fGC/ITS2r showed more abundant fungal species than that using NL1+/LS2R and NS1/GCFung. Therefore, ITS1fGC/ITS2r is the most suitable primer set for PCR-DGGE analysis of fungal community structure in traditional fermentation starters for Hong Qu glutinous rice wine. DGGE profiles based on ITS1fGC/ITS2r revealed the presence of twenty-four fungal species in traditional fermentation starter. A significant difference of fungal community can be observed directly from DGGE fingerprints and principal component analysis. The statistical analysis results based on the band intensities of fungal DGGE profile showed that Saccharomyces cerevisiae, Saccharomycopsis fibuligera, Rhizopus oryzae, Monascus purpureus and Aspergillus niger were the dominant fungal species. In conclusion, the comparison of several primer sets for fungal PCR-DGGE would be useful to enrich our knowledge of the fungal community structures associated with traditional fermentation starters, which may facilitate the development of better starter cultures for manufacturing Chinese Hong Qu glutinous rice wine. Copyright © 2017 Elsevier B.V. All rights reserved.

Repeat-Associated Plasticity in the Helicobacter pylori RD Gene Family▿ †

PubMed Central

Shak, Joshua R.; Dick, Jonathan J.; Meinersmann, Richard J.; Perez-Perez, Guillermo I.; Blaser, Martin J.

2009-01-01

The bacterium Helicobacter pylori is remarkable for its ability to persist in the human stomach for decades without provoking sterilizing immunity. Since repetitive DNA can facilitate adaptive genomic flexibility via increased recombination, insertion, and deletion, we searched the genomes of two H. pylori strains for nucleotide repeats. We discovered a family of genes with extensive repetitive DNA that we have termed the H. pylori RD gene family. Each gene of this family is composed of a conserved 3′ region, a variable mid-region encoding 7 and 11 amino acid repeats, and a 5′ region containing one of two possible alleles. Analysis of five complete genome sequences and PCR genotyping of 42 H. pylori strains revealed extensive variation between strains in the number, location, and arrangement of RD genes. Furthermore, examination of multiple strains isolated from a single subject's stomach revealed intrahost variation in repeat number and composition. Despite prior evidence that the protein products of this gene family are expressed at the bacterial cell surface, enzyme-linked immunosorbent assay and immunoblot studies revealed no consistent seroreactivity to a recombinant RD protein by H. pylori-positive hosts. The pattern of repeats uncovered in the RD gene family appears to reflect slipped-strand mispairing or domain duplication, allowing for redundancy and subsequent diversity in genotype and phenotype. This novel family of hypervariable genes with conserved, repetitive, and allelic domains may represent an important locus for understanding H. pylori persistence in its natural host. PMID:19749042

Repeat-associated plasticity in the Helicobacter pylori RD gene family.

PubMed

Shak, Joshua R; Dick, Jonathan J; Meinersmann, Richard J; Perez-Perez, Guillermo I; Blaser, Martin J

2009-11-01

The bacterium Helicobacter pylori is remarkable for its ability to persist in the human stomach for decades without provoking sterilizing immunity. Since repetitive DNA can facilitate adaptive genomic flexibility via increased recombination, insertion, and deletion, we searched the genomes of two H. pylori strains for nucleotide repeats. We discovered a family of genes with extensive repetitive DNA that we have termed the H. pylori RD gene family. Each gene of this family is composed of a conserved 3' region, a variable mid-region encoding 7 and 11 amino acid repeats, and a 5' region containing one of two possible alleles. Analysis of five complete genome sequences and PCR genotyping of 42 H. pylori strains revealed extensive variation between strains in the number, location, and arrangement of RD genes. Furthermore, examination of multiple strains isolated from a single subject's stomach revealed intrahost variation in repeat number and composition. Despite prior evidence that the protein products of this gene family are expressed at the bacterial cell surface, enzyme-linked immunosorbent assay and immunoblot studies revealed no consistent seroreactivity to a recombinant RD protein by H. pylori-positive hosts. The pattern of repeats uncovered in the RD gene family appears to reflect slipped-strand mispairing or domain duplication, allowing for redundancy and subsequent diversity in genotype and phenotype. This novel family of hypervariable genes with conserved, repetitive, and allelic domains may represent an important locus for understanding H. pylori persistence in its natural host.

Long-Term Evolution of the Hypervariable Region of Hepatitis C Virus in a Common-Source-Infected Cohort

PubMed Central

McAllister, Jane; Casino, Carmela; Davidson, Fiona; Power, Joan; Lawlor, Emer; Yap, Peng Lee; Simmonds, Peter; Smith, Donald B.

1998-01-01

The long-term evolution of the hepatitis C virus hypervariable region (HVR) and flanking regions of the E1 and E2 envelope proteins have been studied in a cohort of women infected from a common source of anti-D immunoglobulin. Whereas virus sequences in the infectious source were relatively homogeneous, distinct HVR variants were observed in each anti-D recipient, indicating that this region can evolve in multiple directions from the same point. Where HVR variants with dissimilar sequences were present in a single individual, the frequency of synonymous substitution in the flanking regions suggested that the lineages diverged more than a decade previously. Even where a single major HVR variant was present in an infected individual, this lineage was usually several years old. Multiple lineages can therefore coexist during long periods of chronic infection without replacement. The characteristics of amino acid substitution in the HVR were not consistent with the random accumulation of mutations and imply that amino acid replacement in the HVR was strongly constrained. Another variable region of E2 centered on codon 60 shows similar constraints, while HVR2 was relatively unconstrained. Several of these features are difficult to explain if a neutralizing immune response against the HVR is the only selective force operating on E2. The impact of PCR artifacts such as nucleotide misincorporation and the shuffling of dissimilar templates is discussed. PMID:9573256

Sequences from the hypervariable V3-V5 regions of the 16S rRNA gene amplified from the porcine proximal colon

USDA-ARS?s Scientific Manuscript database

Helminths, including GI nematodes, colonize > 1/3 of the world’s population and have evolved with humans and their microbiome. Parasites inherently regulate the host immune response to ensure their survival through mechanisms that dampen host inflammation. These unique properties of nematodes have b...

Skin Microbiome Surveys Are Strongly Influenced by Experimental Design.

PubMed

Meisel, Jacquelyn S; Hannigan, Geoffrey D; Tyldsley, Amanda S; SanMiguel, Adam J; Hodkinson, Brendan P; Zheng, Qi; Grice, Elizabeth A

2016-05-01

Culture-independent studies to characterize skin microbiota are increasingly common, due in part to affordable and accessible sequencing and analysis platforms. Compared to culture-based techniques, DNA sequencing of the bacterial 16S ribosomal RNA (rRNA) gene or whole metagenome shotgun (WMS) sequencing provides more precise microbial community characterizations. Most widely used protocols were developed to characterize microbiota of other habitats (i.e., gastrointestinal) and have not been systematically compared for their utility in skin microbiome surveys. Here we establish a resource for the cutaneous research community to guide experimental design in characterizing skin microbiota. We compare two widely sequenced regions of the 16S rRNA gene to WMS sequencing for recapitulating skin microbiome community composition, diversity, and genetic functional enrichment. We show that WMS sequencing most accurately recapitulates microbial communities, but sequencing of hypervariable regions 1-3 of the 16S rRNA gene provides highly similar results. Sequencing of hypervariable region 4 poorly captures skin commensal microbiota, especially Propionibacterium. WMS sequencing, which is resource and cost intensive, provides evidence of a community's functional potential; however, metagenome predictions based on 16S rRNA sequence tags closely approximate WMS genetic functional profiles. This study highlights the importance of experimental design for downstream results in skin microbiome surveys. Copyright © 2016 The Authors. Published by Elsevier Inc. All rights reserved.

Skin microbiome surveys are strongly influenced by experimental design

PubMed Central

Meisel, Jacquelyn S.; Hannigan, Geoffrey D.; Tyldsley, Amanda S.; SanMiguel, Adam J.; Hodkinson, Brendan P.; Zheng, Qi; Grice, Elizabeth A.

2016-01-01

Culture-independent studies to characterize skin microbiota are increasingly common, due in part to affordable and accessible sequencing and analysis platforms. Compared to culture-based techniques, DNA sequencing of the bacterial 16S ribosomal RNA (rRNA) gene or whole metagenome shotgun (WMS) sequencing provide more precise microbial community characterizations. Most widely used protocols were developed to characterize microbiota of other habitats (i.e. gastrointestinal), and have not been systematically compared for their utility in skin microbiome surveys. Here we establish a resource for the cutaneous research community to guide experimental design in characterizing skin microbiota. We compare two widely sequenced regions of the 16S rRNA gene to WMS sequencing for recapitulating skin microbiome community composition, diversity, and genetic functional enrichment. We show that WMS sequencing most accurately recapitulates microbial communities, but sequencing of hypervariable regions 1-3 of the 16S rRNA gene provides highly similar results. Sequencing of hypervariable region 4 poorly captures skin commensal microbiota, especially Propionibacterium. WMS sequencing, which is resource- and cost-intensive, provides evidence of a community’s functional potential; however, metagenome predictions based on 16S rRNA sequence tags closely approximate WMS genetic functional profiles. This work highlights the importance of experimental design for downstream results in skin microbiome surveys. PMID:26829039

Receptor-like genes in the major resistance locus of lettuce are subject to divergent selection.

PubMed Central

Meyers, B C; Shen, K A; Rohani, P; Gaut, B S; Michelmore, R W

1998-01-01

Disease resistance genes in plants are often found in complex multigene families. The largest known cluster of disease resistance specificities in lettuce contains the RGC2 family of genes. We compared the sequences of nine full-length genomic copies of RGC2 representing the diversity in the cluster to determine the structure of genes within this family and to examine the evolution of its members. The transcribed regions range from at least 7.0 to 13.1 kb, and the cDNAs contain deduced open reading frames of approximately 5. 5 kb. The predicted RGC2 proteins contain a nucleotide binding site and irregular leucine-rich repeats (LRRs) that are characteristic of resistance genes cloned from other species. Unique features of the RGC2 gene products include a bipartite LRR region with >40 repeats. At least eight members of this family are transcribed. The level of sequence diversity between family members varied in different regions of the gene. The ratio of nonsynonymous (Ka) to synonymous (Ks) nucleotide substitutions was lowest in the region encoding the nucleotide binding site, which is the presumed effector domain of the protein. The LRR-encoding region showed an alternating pattern of conservation and hypervariability. This alternating pattern of variation was also found in all comparisons within families of resistance genes cloned from other species. The Ka /Ks ratios indicate that diversifying selection has resulted in increased variation at these codons. The patterns of variation support the predicted structure of LRR regions with solvent-exposed hypervariable residues that are potentially involved in binding pathogen-derived ligands. PMID:9811792

Effect of migration patterns on maternal genetic structure: a case of Tai-Kadai migration from China to Thailand.

PubMed

Kampuansai, Jatupol; Kutanan, Wibhu; Tassi, Francesca; Kaewgahya, Massupa; Ghirotto, Silvia; Kangwanpong, Daoroong

2017-02-01

The migration of the Tai-Kadai speaking people from southern China to northern Thailand over the past hundreds of years has revealed numerous patterns that have likely been influenced by routes, purposes and periods of time. To study the effects of different migration patterns on Tai-Kadai maternal genetic structure, mitochondrial DNA hypervariable region I sequences from the Yong and the Lue people having well-documented histories in northern Thailand were analyzed. Although the Yong and Lue people were historically close relatives who shared Xishuangbanna Dai ancestors, significant genetic differences have been observed among them. The Yong people who have been known to practice mass migration have exhibited a closer genetic affinity to their Dai ancestors than have the Lue people. Genetic heterogeneity and a sudden reduced effective population size within the Lue group is likely a direct result of the circumstances of the founder effect.

Historically low mitochondrial DNA diversity in koalas (Phascolarctos cinereus)

PubMed Central

2012-01-01

Background The koala (Phascolarctos cinereus) is an arboreal marsupial that was historically widespread across eastern Australia until the end of the 19th century when it suffered a steep population decline. Hunting for the fur trade, habitat conversion, and disease contributed to a precipitous reduction in koala population size during the late 1800s and early 1900s. To examine the effects of these reductions in population size on koala genetic diversity, we sequenced part of the hypervariable region of mitochondrial DNA (mtDNA) in koala museum specimens collected in the 19th and 20th centuries, hypothesizing that the historical samples would exhibit greater genetic diversity. Results The mtDNA haplotypes present in historical museum samples were identical to haplotypes found in modern koala populations, and no novel haplotypes were detected. Rarefaction analyses suggested that the mtDNA genetic diversity present in the museum samples was similar to that of modern koalas. Conclusions Low mtDNA diversity may have been present in koala populations prior to recent population declines. When considering management strategies, low genetic diversity of the mtDNA hypervariable region may not indicate recent inbreeding or founder events but may reflect an older historical pattern for koalas. PMID:23095716

Compression-based distance (CBD): a simple, rapid, and accurate method for microbiota composition comparison

PubMed Central

2013-01-01

Background Perturbations in intestinal microbiota composition have been associated with a variety of gastrointestinal tract-related diseases. The alleviation of symptoms has been achieved using treatments that alter the gastrointestinal tract microbiota toward that of healthy individuals. Identifying differences in microbiota composition through the use of 16S rRNA gene hypervariable tag sequencing has profound health implications. Current computational methods for comparing microbial communities are usually based on multiple alignments and phylogenetic inference, making them time consuming and requiring exceptional expertise and computational resources. As sequencing data rapidly grows in size, simpler analysis methods are needed to meet the growing computational burdens of microbiota comparisons. Thus, we have developed a simple, rapid, and accurate method, independent of multiple alignments and phylogenetic inference, to support microbiota comparisons. Results We create a metric, called compression-based distance (CBD) for quantifying the degree of similarity between microbial communities. CBD uses the repetitive nature of hypervariable tag datasets and well-established compression algorithms to approximate the total information shared between two datasets. Three published microbiota datasets were used as test cases for CBD as an applicable tool. Our study revealed that CBD recaptured 100% of the statistically significant conclusions reported in the previous studies, while achieving a decrease in computational time required when compared to similar tools without expert user intervention. Conclusion CBD provides a simple, rapid, and accurate method for assessing distances between gastrointestinal tract microbiota 16S hypervariable tag datasets. PMID:23617892

Compression-based distance (CBD): a simple, rapid, and accurate method for microbiota composition comparison.

PubMed

Yang, Fang; Chia, Nicholas; White, Bryan A; Schook, Lawrence B

2013-04-23

Perturbations in intestinal microbiota composition have been associated with a variety of gastrointestinal tract-related diseases. The alleviation of symptoms has been achieved using treatments that alter the gastrointestinal tract microbiota toward that of healthy individuals. Identifying differences in microbiota composition through the use of 16S rRNA gene hypervariable tag sequencing has profound health implications. Current computational methods for comparing microbial communities are usually based on multiple alignments and phylogenetic inference, making them time consuming and requiring exceptional expertise and computational resources. As sequencing data rapidly grows in size, simpler analysis methods are needed to meet the growing computational burdens of microbiota comparisons. Thus, we have developed a simple, rapid, and accurate method, independent of multiple alignments and phylogenetic inference, to support microbiota comparisons. We create a metric, called compression-based distance (CBD) for quantifying the degree of similarity between microbial communities. CBD uses the repetitive nature of hypervariable tag datasets and well-established compression algorithms to approximate the total information shared between two datasets. Three published microbiota datasets were used as test cases for CBD as an applicable tool. Our study revealed that CBD recaptured 100% of the statistically significant conclusions reported in the previous studies, while achieving a decrease in computational time required when compared to similar tools without expert user intervention. CBD provides a simple, rapid, and accurate method for assessing distances between gastrointestinal tract microbiota 16S hypervariable tag datasets.

Genetic Assessment of African Swine Fever Isolates Involved in Outbreaks in the Democratic Republic of Congo between 2005 and 2012 Reveals Co-Circulation of p72 Genotypes I, IX and XIV, Including 19 Variants

PubMed Central

Mulumba–Mfumu, Leopold K.; Achenbach, Jenna E.; Mauldin, Matthew R.; Dixon, Linda K.; Tshilenge, Curé Georges; Thiry, Etienne; Moreno, Noelia; Blanco, Esther; Saegerman, Claude; Lamien, Charles E.; Diallo, Adama

2017-01-01

African swine fever (ASF) is a devastating disease of domestic pigs. It is a socioeconomically important disease, initially described from Kenya, but subsequently reported in most Sub-Saharan countries. ASF spread to Europe, South America and the Caribbean through multiple introductions which were initially eradicated—except for Sardinia—followed by re‑introduction into Europe in 2007. In this study of ASF within the Democratic Republic of the Congo, 62 domestic pig samples, collected between 2005–2012, were examined for viral DNA and sequencing at multiple loci: C-terminus of the B646L gene (p72 protein), central hypervariable region (CVR) of the B602L gene, and the E183L gene (p54 protein). Phylogenetic analyses identified three circulating genotypes: I (64.5% of samples), IX (32.3%), and XIV (3.2%). This is the first evidence of genotypes IX and XIV within this country. Examination of the CVR revealed high levels of intra-genotypic variation, with 19 identified variants. PMID:28218698

Molecular epidemiological investigation of velogenic Newcastle disease viruses from village chickens in Cambodia.

PubMed

Choi, Kang-Seuk; Kye, Soo-Jeong; Kim, Ji-Ye; Damasco, Vanessa R; Sorn, San; Lee, Youn-Jeong; Choi, Jun-Gu; Kang, Hyun-Mi; Kim, Kwang-Il; Song, Byung-Min; Lee, Hee-Soo

2013-10-01

Three isolates of Newcastle disease virus (NDV) were isolated from tracheal samples of dead village chickens in two provinces (Phnom Penh and Kampong Cham) in Cambodia during 2011-2012. All of these Cambodian NDV isolates were categorized as velogenic pathotype, based on in vivo pathogenicity tests and F cleavage site motif sequence ((112)RRRKRF(117)). The phylogenetic analysis and the evolutionary distances based on the sequences of the F gene revealed that all the three field isolates of NDV from Cambodia form a distinct cluster (VIIh) together with three Indonesian strains and were assigned to the genotype VII within the class II. Further phylogenetic analysis based on the hyper-variable region of the F gene revealed that some of NDV strains from Malaysia since the mid-2000s were also classified into the VIIh virus. This indicates that the VIIh NDVs are spreading through Southeast Asia. The present investigation, therefore, emphasizes the importance of further surveillance of NDV in neighboring countries as well as throughout Southeast Asia to contain further spreading of these VIIh viruses.

Genetic Assessment of African Swine Fever Isolates Involved in Outbreaks in the Democratic Republic of Congo between 2005 and 2012 Reveals Co-Circulation of p72 Genotypes I, IX and XIV, Including 19 Variants.

PubMed

Mulumba-Mfumu, Leopold K; Achenbach, Jenna E; Mauldin, Matthew R; Dixon, Linda K; Tshilenge, Curé Georges; Thiry, Etienne; Moreno, Noelia; Blanco, Esther; Saegerman, Claude; Lamien, Charles E; Diallo, Adama

2017-02-18

African swine fever (ASF) is a devastating disease of domestic pigs. It is a socioeconomically important disease, initially described from Kenya, but subsequently reported in most Sub-Saharan countries. ASF spread to Europe, South America and the Caribbean through multiple introductions which were initially eradicated-except for Sardinia-followed by re‑introduction into Europe in 2007. In this study of ASF within the Democratic Republic of the Congo, 62 domestic pig samples, collected between 2005-2012, were examined for viral DNA and sequencing at multiple loci: C-terminus of the B646L gene (p72 protein), central hypervariable region (CVR) of the B602L gene, and the E183L gene (p54 protein). Phylogenetic analyses identified three circulating genotypes: I (64.5% of samples), IX (32.3%), and XIV (3.2%). This is the first evidence of genotypes IX and XIV within this country. Examination of the CVR revealed high levels of intra-genotypic variation, with 19 identified variants.

DGR mutagenic transposition occurs via hypermutagenic reverse transcription primed by nicked template RNA

PubMed Central

Naorem, Santa S.; Han, Jin; Wang, Shufang; Lee, William R.; Heng, Xiao; Miller, Jeff F.

2017-01-01

Diversity-generating retroelements (DGRs) are molecular evolution machines that facilitate microbial adaptation to environmental changes. Hypervariation occurs via a mutagenic retrotransposition process from a template repeat (TR) to a variable repeat (VR) that results in adenine-to-random nucleotide conversions. Here we show that reverse transcription of the Bordetella phage DGR is primed by an adenine residue in TR RNA and is dependent on the DGR-encoded reverse transcriptase (bRT) and accessory variability determinant (Avd ), but is VR-independent. We also find that the catalytic center of bRT plays an essential role in site-specific cleavage of TR RNA for cDNA priming. Adenine-specific mutagenesis occurs during reverse transcription and does not involve dUTP incorporation, indicating it results from bRT-catalyzed misincorporation of standard deoxyribonucleotides. In vivo assays show that this hybrid RNA-cDNA molecule is required for mutagenic transposition, revealing a unique mechanism of DNA hypervariation for microbial adaptation. PMID:29109248

Structure-Directed and Tailored Diversity Synthetic Antibody Libraries Yield Novel Anti-EGFR Antagonists.

PubMed

Miersch, Shane; Maruthachalam, Bharathikumar Vellalore; Geyer, C Ronald; Sidhu, Sachdev S

2017-05-19

We tested whether grafting an interaction domain into the hypervariable loop of a combinatorial antibody library could promote targeting to a specific epitope. Formation of the epidermal growth factor receptor (EGFR) signaling heterodimer involves extensive contacts mediated by a "dimerization loop." We grafted the dimerization loop into the third hypervariable loop of a synthetic antigen-binding fragment (Fab) library and diversified other loops using a tailored diversity strategy. This structure-directed Fab library and a naı̈ve synthetic Fab library were used to select Fabs against EGFR. Both libraries yielded high affinity Fabs that bound to overlapping epitopes on cell-surface EGFR, inhibited receptor activation, and targeted epitopes distinct from those of cetuximab and panitumumab. Epitope mapping experiments revealed complex sites of interaction, comprised of domains I and II but not exclusively localized to the receptor dimerization loop. These results validate the grafting approach for designing Fab libraries and also underscore the versatility of naı̈ve synthetic libraries.

The ancient Yakuts: a population genetic enigma

PubMed Central

Keyser, Christine; Hollard, Clémence; Gonzalez, Angela; Fausser, Jean-Luc; Rivals, Eric; Alexeev, Anatoly Nikolayevich; Riberon, Alexandre; Crubézy, Eric; Ludes, Bertrand

2015-01-01

This study is part of an ongoing project aiming at determining the ethnogenesis of an eastern Siberian ethnic group, the Yakuts, on the basis of archaeological excavations carried out over a period of 10 years in three regions of Yakutia: Central Yakutia, the Vilyuy River basin and the Verkhoyansk area. In this study, genetic analyses were carried out on skeletal remains from 130 individuals of unknown ancestry dated mainly from the fifteenth to the nineteenth century AD. Kinship studies were conducted using sets of commercially available autosomal and Y-chromosomal short tandem repeats (STRs) along with hypervariable region I sequences of the mitochondrial DNA. An unexpected and intriguing finding of this work was that the uniparental marker systems did not always corroborate results from autosomal DNA analyses; in some cases, false-positive relationships were observed. These discrepancies revealed that 15 autosomal STR loci are not sufficient to discriminate between first degree relatives and more distantly related individuals in our ancient Yakut sample. The Y-STR analyses led to similar conclusions, because the current Y-STR panels provided the limited resolution of the paternal lineages. PMID:25487336

Detection and molecular characterization of infectious bronchitis virus isolated from recent outbreaks in broiler flocks in Thailand.

PubMed

Pohuang, Tawatchai; Chansiripornchai, Niwat; Tawatsin, Achara; Sasipreeyajan, Jiroj

2009-09-01

Thirteen field isolates of infectious bronchitis virus (IBV) were isolated from broiler flocks in Thailand between January and June 2008. The 878-bp of the S1 gene covering a hypervariable region was amplified and sequenced. Phylogenetic analysis based on that region revealed that these viruses were separated into two groups (I and II). IBV isolates in group I were not related to other IBV strains published in the GenBank database. Group 1 nucleotide sequence identities were less than 85% and amino acid sequence identities less than 84% in common with IBVs published in the GenBank database. This group likely represents the strains indigenous to Thailand. The isolates in group II showed a close relationship with Chinese IBVs. They had nucleotide sequence identities of 97-98% and amino acid sequence identities 96-98% in common with Chinese IBVs (strain A2, SH and QXIBV). This finding indicated that the recent Thai IBVs evolved separately and at least two groups of viruses are circulating in Thailand.

Phylogenetic Diversity of Koala Retrovirus within a Wild Koala Population.

PubMed

Chappell, K J; Brealey, J C; Amarilla, A A; Watterson, D; Hulse, L; Palmieri, C; Johnston, S D; Holmes, E C; Meers, J; Young, P R

2017-02-01

Koala populations are in serious decline across many areas of mainland Australia, with infectious disease a contributing factor. Koala retrovirus (KoRV) is a gammaretrovirus present in most wild koala populations and captive colonies. Five subtypes of KoRV (A to E) have been identified based on amino acid sequence divergence in a hypervariable region of the receptor binding domain of the envelope protein. However, analysis of viral genetic diversity has been conducted primarily on KoRV in captive koalas housed in zoos in Japan, the United States, and Germany. Wild koalas within Australia have not been comparably assessed. Here we report a detailed analysis of KoRV genetic diversity in samples collected from 18 wild koalas from southeast Queensland. By employing deep sequencing we identified 108 novel KoRV envelope sequences and determined their phylogenetic diversity. Genetic diversity in KoRV was abundant and fell into three major groups; two comprised the previously identified subtypes A and B, while the third contained the remaining hypervariable region subtypes (C, D, and E) as well as four hypervariable region subtypes that we newly define here (F, G, H, and I). In addition to the ubiquitous presence of KoRV-A, which may represent an exclusively endogenous variant, subtypes B, D, and F were found to be at high prevalence, while subtypes G, H, and I were present in a smaller number of animals. Koala retrovirus (KoRV) is thought to be a significant contributor to koala disease and population decline across mainland Australia. This study is the first to determine KoRV subtype prevalence among a wild koala population, and it significantly expands the total number of KoRV sequences available, providing a more precise picture of genetic diversity. This understanding of KoRV subtype prevalence and genetic diversity will be important for conservation efforts attempting to limit the spread of KoRV. Furthermore, KoRV is one of the only retroviruses shown to exist in both endogenous (transmitted vertically to offspring in the germ line DNA) and exogenous (horizontally transmitted between infected individuals) forms, a division of fundamental evolutionary importance. Copyright © 2017 American Society for Microbiology.

Molecular evolution of respiratory syncytial virus subgroup A genotype NA1 and ON1 attachment glycoprotein (G) gene in central Vietnam.

PubMed

Yoshihara, Keisuke; Le, Minh Nhat; Nagasawa, Koo; Tsukagoshi, Hiroyuki; Nguyen, Hien Anh; Toizumi, Michiko; Moriuchi, Hiroyuki; Hashizume, Masahiro; Ariyoshi, Koya; Dang, Duc Anh; Kimura, Hirokazu; Yoshida, Lay-Myint

2016-11-01

We performed molecular evolutionary analyses of the G gene C-terminal 3rd hypervariable region of RSV-A genotypes NA1 and ON1 strains from the paediatric acute respiratory infection patients in central Vietnam during the 2010-2012 study period. Time-scaled phylogenetic analyses were performed using Bayesian Markov Chain Monte Carlo (MCMC) method, and pairwise distances (p-distances) were calculated. Bayesian Skyline Plot (BSP) was constructed to analyze the time-trend relative genetic diversity of central Vietnam RSV-A strains. We also estimated the N-glycosylation sites within G gene hypervariable region. Amino acid substitutions under positive and negative selection pressure were examined using Conservative Single Likelihood Ancestor Counting (SLAC), Fixed Effects Likelihood (FEL), Internal Fixed Effects Likelihood (IFEL) and Mixed Effects Model for Episodic Diversifying Selection (MEME) models. The majority of central Vietnam ON1 strains detected in 2012 were classified into lineage 1 with few positively selected substitutions. As for the Vietnamese NA1 strains, four lineages were circulating during the study period with a few positive selection sites. Shifting patterns of the predominantly circulating NA1 lineage were observed in each year during the investigation period. Median p-distance of central Vietnam NA1 strains was wider (p-distance=0.028) than that of ON1 (p-distance=0.012). The molecular evolutionary rate of central Vietnam ON1 strains was estimated to be 2.55×10 -2 (substitutions/site/year) and was faster than NA1 (7.12×10 -3 (substitutions/site/year)). Interestingly, the evolutionary rates of both genotypes ON1 and NA1 strains from central Vietnam were faster than the global strains respectively. Furthermore, the shifts of N-glycosylation pattern within the G gene 3rd hypervariable region of Vietnamese NA1 strains were observed in each year. BSP analysis indicated the rapid growth of RSV-A effective population size in early 2012. These results suggested that the molecular evolution of RSV-A G gene detected in central Vietnam was fast with unique evolutionary dynamics. Copyright © 2016 Elsevier B.V. All rights reserved.

A genetic investigation of Korean mummies from the Joseon Dynasty.

PubMed

Kim, Na Young; Lee, Hwan Young; Park, Myung Jin; Yang, Woo Ick; Shin, Kyoung-Jin

2011-01-01

Two Korean mummies (Danwoong-mirra and Yoon-mirra) found in medieval tombs in the central region of the Korean peninsula were genetically investigated by analysis of mitochondrial DNA (mtDNA), Y-chromosomal short tandem repeat (Y-STR) and the ABO gene. Danwoong-mirra is a male child mummy and Yoon-mirra is a pregnant female mummy, dating back about 550 and 450 years, respectively. DNA was extracted from soft tissues or bones. mtDNA, Y-STR and the ABO gene were amplified using a small size amplicon strategy and were analyzed according to the criteria of ancient DNA analysis to ensure that authentic DNA typing results were obtained from these ancient samples. Analysis of mtDNA hypervariable region sequence and coding region single nucleotide polymorphism (SNP) information revealed that Danwoong-mirra and Yoon-mirra belong to the East Asian mtDNA haplogroups D4 and M7c, respectively. The Y-STRs were analyzed in the male child mummy (Danwoong-mirra) using the AmpFlSTR® Yfiler PCR Amplification Kit and an in-house Y-miniplex plus system, and could be characterized in 4 loci with small amplicon size. The analysis of ABO gene SNPs using multiplex single base extension methods revealed that the ABO blood types of Danwoong-mirra and Yoon-mirra are AO01 and AB, respectively. The small size amplicon strategy and the authentication process in the present study will be effectively applicable to future genetic analyses of various forensic and ancient samples.

Genetic diversity of Streptococcus equi subsp. zooepidemicus and doxycycline resistance in kennelled dogs.

PubMed

Chalker, Victoria J; Waller, Andrew; Webb, Katy; Spearing, Emma; Crosse, Patricia; Brownlie, Joe; Erles, Kerstin

2012-06-01

The genetic diversity and antibiotic resistance profiles of 38 Streptococcus equi subsp. zooepidemicus isolates were determined from a kennelled canine population during two outbreaks of hemorrhagic pneumonia (1999 to 2002 and 2007 to 2010). Analysis of the szp gene hypervariable region and the 16S-23S rRNA intergenic spacer region and multilocus sequence typing (MLST) indicated a predominant tetO-positive, doxycycline-resistant ST-10 strain during 1999 to 2002 and a predominant tetM-positive doxycycline-resistant ST-62 strain during 2007 to 2010.

Genetic Diversity of Streptococcus equi subsp. zooepidemicus and Doxycycline Resistance in Kennelled Dogs

PubMed Central

Chalker, Victoria J.; Waller, Andrew; Webb, Katy; Spearing, Emma; Crosse, Patricia; Brownlie, Joe

2012-01-01

The genetic diversity and antibiotic resistance profiles of 38 Streptococcus equi subsp. zooepidemicus isolates were determined from a kennelled canine population during two outbreaks of hemorrhagic pneumonia (1999 to 2002 and 2007 to 2010). Analysis of the szp gene hypervariable region and the 16S-23S rRNA intergenic spacer region and multilocus sequence typing (MLST) indicated a predominant tetO-positive, doxycycline-resistant ST-10 strain during 1999 to 2002 and a predominant tetM-positive doxycycline-resistant ST-62 strain during 2007 to 2010. PMID:22495558

Thermal Unfolding Simulations of Bacterial Flagellin: Insight into its Refolding Before Assembly

PubMed Central

Chng, Choon-Peng; Kitao, Akio

2008-01-01

Flagellin is the subunit of the bacterial filament, the micrometer-long propeller of a bacterial flagellum. The protein is believed to undergo unfolding for transport through the channel of the filament and to refold in a chamber at the end of the channel before being assembled into the growing filament. We report a thermal unfolding simulation study of S. typhimurium flagellin in aqueous solution as an attempt to gain atomic-level insight into the refolding process. Each molecule comprises two filament-core domains {D0, D1} and two hypervariable-region domains {D2, D3}. D2 can be separated into subdomains D2a and D2b. We observed a similar unfolding order of the domains as reported in experimental thermal denaturation. D2a and D3 exhibited high thermal stability and contained persistent three-stranded β-sheets in the denatured state which could serve as folding cores to guide refolding. A recent mutagenesis study on flagellin stability seems to suggest the importance of the folding cores. Using crude size estimates, our data suggests that the chamber might be large enough for either denatured hypervariable-region domains or filament-core domains, but not whole flagellin; this implicates a two-staged refolding process. PMID:18263660

Characterisation of caecum and crop microbiota of Indian indigenous chicken targeting multiple hypervariable regions within 16S rRNA gene.

PubMed

Saxena, S; Saxena, V K; Tomar, S; Sapcota, D; Gonmei, G

2016-06-01

A comparative analysis of caecum and crop microbiota of chick, grower and adult stages of Indian indigenous chickens was conducted to investigate the role of the microbiota of the gastrointestinal tract, which play an important role in host performance, health and immunity. High-throughput Illumina sequencing was performed for V3, V4 and V4-V6 hypervariable regions of the 16S rRNA gene. M5RNA and M5NR databases under MG-RAST were used for metagenomic datasets annotation. In the crop, Firmicutes (~78%) and Proteobacteria (~16%) were the predominant phyla whereas in the caecum, Firmicutes (~50%), Bacteroidetes (~29%) and Actinobacteria (~10%) were predominant. The Shannon-Wiener diversity index suggested that sample richness and diversity increased as the chicken aged. For the first time, the presence of Lactobacillus species such as L. frumenti, L. antri, L. mucosae in the chicken crop along with Kineococcus radiotolerans, Desulfohalobium retbaense and L. jensenii in the caecum are reported. Many of these bacterial species have been found to be involved in immune response modulation and disease prevention in pigs and humans. The gut microbiome of the indigenous chicken was enriched with microbes having probiotic potential which might be essential for their adaptability.

Genetic examination of the putative skull of Jan Kochanowski reveals its female sex

PubMed Central

Kupiec, Tomasz; Branicki, Wojciech

2011-01-01

We report the results of genetic examination of the putative skull of Jan Kochanowski (1530-1584), a great Polish renaissance poet. The skull was retrieved in 1791 by historian Tadeusz Czacki from the Kochanowski family tomb and became the property of the Czartoryskis Museum in Krakow. An anthropological study in 1926 questioned its male origin, which raised doubts about its authenticity. Our report presents genetic evidence that resolves this dispute. From the sole tooth we obtained a sufficient amount of DNA to perform the analysis of nuclear markers. The analysis of the sex-informative part of intron 1 in amelogenin, genotyped using AmpFiSTR® NGM PCR Amplification Kit and Powerplex® ESI17 Kit human identification systems, revealed the female origin of the tooth. The female origin was further confirmed by the analysis of a portion of amelogenin intron 2, a microsatellite marker located on the X chromosome, as well as by a lack of signal from Y chromosomal microsatellite markers and the sex-determining region Y marker. Data obtained for two hypervariable regions, HVI and HVII, in mitochondrial DNA showed that mtDNA haplotype was relatively frequent among contemporary Europeans. The analysis of a set of single nucleotide polymorphisms relevant for prediction of the iris color indicated an 87% probability that the woman had hazel or brown eye color. PMID:21674838

Genetic examination of the putative skull of Jan Kochanowski reveals its female sex.

PubMed

Kupiec, Tomasz; Branicki, Wojciech

2011-06-01

We report the results of genetic examination of the putative skull of Jan Kochanowski (1530-1584), a great Polish renaissance poet. The skull was retrieved in 1791 by historian Tadeusz Czacki from the Kochanowski family tomb and became the property of the Czartoryskis Museum in Krakow. An anthropological study in 1926 questioned its male origin, which raised doubts about its authenticity. Our report presents genetic evidence that resolves this dispute. From the sole tooth we obtained a sufficient amount of DNA to perform the analysis of nuclear markers. The analysis of the sex-informative part of intron 1 in amelogenin, genotyped using AmpFiSTR® NGM PCR Amplification Kit and Powerplex® ESI17 Kit human identification systems, revealed the female origin of the tooth. The female origin was further confirmed by the analysis of a portion of amelogenin intron 2, a microsatellite marker located on the X chromosome, as well as by a lack of signal from Y chromosomal microsatellite markers and the sex-determining region Y marker. Data obtained for two hypervariable regions, HVI and HVII, in mitochondrial DNA showed that mtDNA haplotype was relatively frequent among contemporary Europeans. The analysis of a set of single nucleotide polymorphisms relevant for prediction of the iris color indicated an 87% probability that the woman had hazel or brown eye color.

Positive selection of digestive Cys proteases in herbivorous Coleoptera.

PubMed

Vorster, Juan; Rasoolizadeh, Asieh; Goulet, Marie-Claire; Cloutier, Conrad; Sainsbury, Frank; Michaud, Dominique

2015-10-01

Positive selection is thought to contribute to the functional diversification of insect-inducible protease inhibitors in plants in response to selective pressures exerted by the digestive proteases of their herbivorous enemies. Here we assessed whether a reciprocal evolutionary process takes place on the insect side, and whether ingestion of a positively selected plant inhibitor may translate into a measurable rebalancing of midgut proteases in vivo. Midgut Cys proteases of herbivorous Coleoptera, including the major pest Colorado potato beetle (Leptinotarsa decemlineata), were first compared using a codon-based evolutionary model to look for the occurrence of hypervariable, positively selected amino acid sites among the tested sequences. Hypervariable sites were found, distributed within -or close to- amino acid regions interacting with Cys-type inhibitors of the plant cystatin protein family. A close examination of L. decemlineata sequences indicated a link between their assignment to protease functional families and amino acid identity at positively selected sites. A function-diversifying role for positive selection was further suggested empirically by in vitro protease assays and a shotgun proteomic analysis of L. decemlineata Cys proteases showing a differential rebalancing of protease functional family complements in larvae fed single variants of a model cystatin mutated at positively selected amino acid sites. These data confirm overall the occurrence of hypervariable, positively selected amino acid sites in herbivorous Coleoptera digestive Cys proteases. They also support the idea of an adaptive role for positive selection, useful to generate functionally diverse proteases in insect herbivores ingesting functionally diverse, rapidly evolving dietary cystatins. Copyright © 2015 Elsevier Ltd. All rights reserved.

Distinct composition signatures of archaeal and bacterial phylotypes in the Wanda Glacier forefield, Antarctic Peninsula.

PubMed

Pessi, Igor S; Osorio-Forero, César; Gálvez, Eric J C; Simões, Felipe L; Simões, Jefferson C; Junca, Howard; Macedo, Alexandre J

2015-01-01

Several studies have shown that microbial communities in Antarctic environments are highly diverse. However, considering that the Antarctic Peninsula is among the regions with the fastest warming rates, and that regional climate change has been linked to an increase in the mean rate of glacier retreat, the microbial diversity in Antarctic soil is still poorly understood. In this study, we analysed more than 40 000 sequences of the V5-V6 hypervariable region of the 16S rRNA gene obtained by 454 pyrosequencing from four soil samples from the Wanda Glacier forefield, King George Island, Antarctic Peninsula. Phylotype diversity and richness were surprisingly high, and taxonomic assignment of sequences revealed that communities are dominated by Proteobacteria, Bacteroidetes and Euryarchaeota, with a high frequency of archaeal and bacterial phylotypes unclassified at the genus level and without cultured representative strains, representing a distinct microbial community signature. Several phylotypes were related to marine microorganisms, indicating the importance of the marine environment as a source of colonizers for this recently deglaciated environment. Finally, dominant phylotypes were related to different microorganisms possessing a large array of metabolic strategies, indicating that early successional communities in Antarctic glacier forefield can be also functionally diverse. © FEMS 2014. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.

Pyrosequencing analysis of microbial communities reveals dominant cosmopolitan phylotypes in deep-sea sediments of the eastern Mediterranean Sea.

PubMed

Polymenakou, Paraskevi N; Christakis, Christos A; Mandalakis, Manolis; Oulas, Anastasis

2015-06-01

The deep eastern basin of the Mediterranean Sea is considered to be one of the world's most oligotrophic areas in the world. Here we performed pyrosequenicng analysis of bacterial and archaeal communities in oxic nutrient-poor sediments collected from the eastern Mediterranean at 1025-4393 m depth. Microbial communities were surveyed by targeting the hypervariable V5-V6 regions of the 16S ribosomal RNA gene using bar-coded pyrosequencing. With a total of 13,194 operational taxonomic units (OTUs) or phylotypes at 97% sequence similarities, the phylogenetic affiliation of microbes was assigned to 23 bacterial and 2 archaeal known phyla, 23 candidate divisions at the phylum level and distributed into 186 families. It was further revealed that the microbial consortia inhabiting all sampling sites were highly diverse, but dominated by phylotypes closely related to members of the genus Pseudomonas and Marine Group I archaea. Such pronounced and widespread enrichment probably manifests the cosmopolitan character of these species and raises questions about their metabolic adaptation to the physical stressors and low nutrient availability of the deep eastern Mediterranean Sea. Copyright © 2015 Institut Pasteur. Published by Elsevier Masson SAS. All rights reserved.

Egypt's Red Sea coast: phylogenetic analysis of cultured microbial consortia in industrialized sites.

PubMed

Mustafa, Ghada A; Abd-Elgawad, Amr; Abdel-Haleem, Alyaa M; Siam, Rania

2014-01-01

The Red Sea possesses a unique geography, and its shores are rich in mangrove, macro-algal and coral reef ecosystems. Various sources of pollution affect Red Sea biota, including microbial life. We assessed the effects of industrialization on microbes along the Egyptian Red Sea coast at eight coastal sites and two lakes. The bacterial communities of sediment samples were analyzed using bacterial 16S rDNA pyrosequencing of V6-V4 hypervariable regions. The taxonomic assignment of 131,402 significant reads to major bacterial taxa revealed five main bacterial phyla dominating the sampled sites: Proteobacteria (68%), Firmicutes (13%), Fusobacteria (12%), Bacteriodetes (6%), and Spirochetes (0.03%). Further analysis revealed distinct bacterial consortia that primarily included (1) marine Vibrio spp.-suggesting a "marine Vibrio phenomenon"; (2) potential human pathogens; and (3) oil-degrading bacteria. We discuss two divergent microbial consortia that were sampled from Solar Lake West near Taba/Eilat and Saline Lake in Ras Muhammad; these consortia contained the highest abundance of human pathogens and no pathogens, respectively. Our results draw attention to the effects of industrialization on the Red Sea and suggest the need for further analysis to overcome the hazardous effects observed at the impacted sites.

Community Shifts in the Surface Microbiomes of the Coral Porites astreoides with Unusual Lesions

PubMed Central

Meyer, Julie L.; Paul, Valerie J.; Teplitski, Max

2014-01-01

Apical lesions on Porites astreoides were characterized by the appearance of a thin yellow band, which was preceded by bleaching of the coral tissues and followed by a completely denuded coral skeleton, which often harbored secondary macroalgal colonizers. These characteristics have not been previously described in Porites and do not match common Caribbean coral diseases. The lesions were observed only in warmer months and at shallow depths on the fore reef in Belize. Analysis of the microbial community composition based on the V4 hypervariable region of 16S ribosomal RNA genes revealed that the surface microbiomes associated with nonsymptomatic corals were dominated by the members of the genus Endozoicomonas, consistent with other studies. Comparison of the microbiomes of nonsymptomatic and lesioned coral colonies sampled in July and September revealed two distinct groups, inconsistently related to the disease state of the coral, but showing some temporal signal. The loss of Endozoicomonas was characteristic of lesioned corals, which also harbored potential opportunistic pathogens such as Alternaria, Stenotrophomonas, and Achromobacter. The presence of lesions in P. astreoides coincided with a decrease in the relative abundance of Endozoicomonas, rather than the appearance of specific pathogenic taxa. PMID:24937478

A Study of the 5S Ribosomal RNAs of the Vibrionaceae

DTIC Science & Technology

1984-01-01

codon (UAA, UAG, or UGA) TBE Tris-borate-EDTA buffer ug microgram, i.e., 10-’ gram 6 ul microliter. iJe., 10- 6 liter UPG unweighted pair-group UPGMA ...Psy~ww~w .......................... .. 4.------------------ 0 IC 5b. The UPGMA , or UPS average linkage, dendrogram resulting from the...cluster, and the V. damsela - Q. anguillarus doublet are identical to that predicted by UPGMA analysis. C. CONSERVED AND HYPERVARIABLE REGIONS As

Microbial Diversity of Acidic Hot Spring (Kawah Hujan B) in Geothermal Field of Kamojang Area, West Java-Indonesia

PubMed Central

Aditiawati, Pingkan; Yohandini, Heni; Madayanti, Fida; Akhmaloka

2009-01-01

Microbial communities in an acidic hot spring, namely Kawah Hujan B, at Kamojang geothermal field, West Java-Indonesia was examined using culture dependent and culture independent strategies. Chemical analysis of the hot spring water showed a characteristic of acidic-sulfate geothermal activity that contained high sulfate concentrations and low pH values (pH 1.8 to 1.9). Microbial community present in the spring was characterized by 16S rRNA gene combined with denaturing gradient gel electrophoresis (DGGE) analysis. The majority of the sequences recovered from culture-independent method were closely related to Crenarchaeota and Proteobacteria phyla. However, detail comparison among the member of Crenarchaeota showing some sequences variation compared to that the published data especially on the hypervariable and variable regions. In addition, the sequences did not belong to certain genus. Meanwhile, the 16S Rdna sequences from culture-dependent samples revealed mostly close to Firmicute and gamma Proteobacteria. PMID:19440252

The unusually large Plasmodium telomerase reverse-transcriptase localizes in a discrete compartment associated with the nucleolus

PubMed Central

Figueiredo, Luisa M.; Rocha, Eduardo P. C.; Mancio-Silva, Liliana; Prevost, Christine; Hernandez-Verdun, Danièle; Scherf, Artur

2005-01-01

Telomerase replicates chromosome ends, a function necessary for maintaining genome integrity. We have identified the gene that encodes the catalytic reverse transcriptase (RT) component of this enzyme in the malaria parasite Plasmodium falciparum (PfTERT) as well as the orthologous genes from two rodent and one simian malaria species. PfTERT is predicted to encode a basic protein that contains the major sequence motifs previously identified in known telomerase RTs (TERTs). At ∼2500 amino acids, PfTERT is three times larger than other characterized TERTs. We observed remarkable sequence diversity between TERT proteins of different Plasmodial species, with conserved domains alternating with hypervariable regions. Immunofluorescence analysis revealed that PfTERT is expressed in asexual blood stage parasites that have begun DNA synthesis. Surprisingly, rather than at telomere clusters, PfTERT typically localizes into a discrete nuclear compartment. We further demonstrate that this compartment is associated with the nucleolus, hereby defined for the first time in P.falciparum. PMID:15722485

Identification of novel Theileria genotypes from Grant's gazelle

PubMed Central

Hooge, Janis; Howe, Laryssa; Ezenwa, Vanessa O.

2015-01-01

Blood samples collected from Grant's gazelles (Nanger granti) in Kenya were screened for hemoparasites using a combination of microscopic and molecular techniques. All 69 blood smears examined by microscopy were positive for hemoparasites. In addition, Theileria/Babesia DNA was detected in all 65 samples screened by PCR for a ~450-base pair fragment of the V4 hypervariable region of the 18S rRNA gene. Sequencing and BLAST analysis of a subset of PCR amplicons revealed widespread co-infection (25/39) and the existence of two distinct Grant's gazelle Theileria subgroups. One group of 11 isolates clustered as a subgroup with previously identified Theileria ovis isolates from small ruminants from Europe, Asia and Africa; another group of 3 isolates clustered with previously identified Theileria spp. isolates from other African antelope. Based on extensive levels of sequence divergence (1.2–2%) from previously reported Theileria species within Kenya and worldwide, the Theileria isolates detected in Grant's gazelles appear to represent at least two novel Theileria genotypes. PMID:25973394

Identification of novel Theileria genotypes from Grant's gazelle.

PubMed

Hooge, Janis; Howe, Laryssa; Ezenwa, Vanessa O

2015-08-01

Blood samples collected from Grant's gazelles (Nanger granti) in Kenya were screened for hemoparasites using a combination of microscopic and molecular techniques. All 69 blood smears examined by microscopy were positive for hemoparasites. In addition, Theileria/Babesia DNA was detected in all 65 samples screened by PCR for a ~450-base pair fragment of the V4 hypervariable region of the 18S rRNA gene. Sequencing and BLAST analysis of a subset of PCR amplicons revealed widespread co-infection (25/39) and the existence of two distinct Grant's gazelle Theileria subgroups. One group of 11 isolates clustered as a subgroup with previously identified Theileria ovis isolates from small ruminants from Europe, Asia and Africa; another group of 3 isolates clustered with previously identified Theileria spp. isolates from other African antelope. Based on extensive levels of sequence divergence (1.2-2%) from previously reported Theileria species within Kenya and worldwide, the Theileria isolates detected in Grant's gazelles appear to represent at least two novel Theileria genotypes.

Population differentiation in the red-legged kittiwake (Rissa brevirostris) as revealed by mitochondrial DNA

USGS Publications Warehouse

Patirana, A.; Hatcher, S.A.; Friesen, Vicki L.

2002-01-01

Population decline in red-legged kittiwakes (Rissa brevirostris) over recent decades has necessitated the collection of information on the distribution of genetic variation within and among colonies for implementation of suitable management policies. Here we present a preliminary study of the extent of genetic structuring and gene flow among the three principal breeding locations of red-legged kittiwakes using the hypervariable Domain I of the mitochondrial control region. Genetic variation was high relative to other species of seabirds, and was similar among locations. Analysis of molecular variance indicated that population genetic structure was statistically significant, and nested clade analysis suggested that kittiwakes breeding on Bering Island maybe genetically isolated from those elsewhere. However, phylogeographic structure was weak. Although this analysis involved only a single locus and a small number of samples, it suggests that red-legged kittiwakes probably constitute a single evolutionary significant unit; the possibility that they constitute two management units requires further investigation.

Microbial diversity of acidic hot spring (kawah hujan B) in geothermal field of kamojang area, west java-indonesia.

PubMed

Aditiawati, Pingkan; Yohandini, Heni; Madayanti, Fida; Akhmaloka

2009-01-01

Microbial communities in an acidic hot spring, namely Kawah Hujan B, at Kamojang geothermal field, West Java-Indonesia was examined using culture dependent and culture independent strategies. Chemical analysis of the hot spring water showed a characteristic of acidic-sulfate geothermal activity that contained high sulfate concentrations and low pH values (pH 1.8 to 1.9). Microbial community present in the spring was characterized by 16S rRNA gene combined with denaturing gradient gel electrophoresis (DGGE) analysis. The majority of the sequences recovered from culture-independent method were closely related to Crenarchaeota and Proteobacteria phyla. However, detail comparison among the member of Crenarchaeota showing some sequences variation compared to that the published data especially on the hypervariable and variable regions. In addition, the sequences did not belong to certain genus. Meanwhile, the 16S Rdna sequences from culture-dependent samples revealed mostly close to Firmicute and gamma Proteobacteria.

Genetic Characterization of Circulating African Swine Fever Viruses in Nigeria (2007-2015).

PubMed

Luka, P D; Achenbach, J E; Mwiine, F N; Lamien, C E; Shamaki, D; Unger, H; Erume, J

2017-10-01

Sequencing and analysis of three discrete genome regions of African swine fever viruses (ASFV) from archival samples collected in 2007-2011 and active and passive surveillance between 2012 and 2015 in Nigeria were carried out. Analysis was conducted by genotyping of three single-copy African swine fever (ASF) genes. The E183L and B646L genes that encode structural proteins p54 and p72, respectively, were utilized to delineate genotypes before intragenotypic resolution by characterization of the tetrameric amino acid repeat region within the hypervariable central variable region of the B602L gene. The results showed no variation in the p72 and p54 gene regions sequenced. Phylogeny of p72 sequences revealed that all the Nigerian isolates belonged to genotype I, while that of the p54 recovered the Ia genotype. Analysis of B602L gene revealed the differences in the number of tetrameric repeats. Four new variants (Tet-15, Tet-17a, Tet-17b and Tet-48) were recovered, while a fifth variant (Tet-20) was the most widely distributed in the country displacing Tet-36 reported previously in 2003-2006. The viruses responsible for ASF outbreaks in Nigeria are from very closely related but mutated variants of the virus that have been circulating since 1997. A practical implication of the genetic variability of the Nigerian viral isolates in this study is the need for continuous sampling and analysis of circulating viruses, which will provide epidemiological information on the evolution of ASFV in the field versus new incursion for informed strategic control of the disease in the country. © 2016 Blackwell Verlag GmbH.

Complete mtDNA genomes of Filipino ethnolinguistic groups: a melting pot of recent and ancient lineages in the Asia-Pacific region

PubMed Central

Delfin, Frederick; Min-Shan Ko, Albert; Li, Mingkun; Gunnarsdóttir, Ellen D; Tabbada, Kristina A; Salvador, Jazelyn M; Calacal, Gayvelline C; Sagum, Minerva S; Datar, Francisco A; Padilla, Sabino G; De Ungria, Maria Corazon A; Stoneking, Mark

2014-01-01

The Philippines is a strategic point in the Asia-Pacific region for the study of human diversity, history and origins, as it is a cross-road for human migrations and consequently exhibits enormous ethnolinguistic diversity. Following on a previous in-depth study of Y-chromosome variation, here we provide new insights into the maternal genetic history of Filipino ethnolinguistic groups by surveying complete mitochondrial DNA (mtDNA) genomes from a total of 14 groups (11 groups in this study and 3 groups previously published) including previously published mtDNA hypervariable segment (HVS) data from Filipino regional center groups. Comparison of HVS data indicate genetic differences between ethnolinguistic and regional center groups. The complete mtDNA genomes of 14 ethnolinguistic groups reveal genetic aspects consistent with the Y-chromosome, namely: diversity and heterogeneity of groups, no support for a simple dichotomy between Negrito and non-Negrito groups, and different genetic affinities with Asia-Pacific groups that are both ancient and recent. Although some mtDNA haplogroups can be associated with the Austronesian expansion, there are others that associate with South Asia, Near Oceania and Australia that are consistent with a southern migration route for ethnolinguistic group ancestors into the Asia-Pacific, with a timeline that overlaps with the initial colonization of the Asia-Pacific region, the initial colonization of the Philippines and a possible separate post-colonization migration into the Philippine archipelago. PMID:23756438

A novel multi-variant epitope ensemble vaccine against avian leukosis virus subgroup J.

PubMed

Wang, Xiaoyu; Zhou, Defang; Wang, Guihua; Huang, Libo; Zheng, Qiankun; Li, Chengui; Cheng, Ziqiang

2017-12-04

The hypervariable antigenicity and immunosuppressive features of avian leukosis virus subgroup J (ALV-J) has led to great challenges to develop effective vaccines. Epitope vaccine will be a perspective trend. Previously, we identified a variant antigenic neutralizing epitope in hypervariable region 1 (hr1) of ALV-J, N-LRDFIA/E/TKWKS/GDDL/HLIRPYVNQS-C. BLAST analysis showed that the mutation of A, E, T and H in this epitope cover 79% of all ALV-J strains. Base on this data, we designed a multi-variant epitope ensemble vaccine comprising the four mutation variants linked with glycine and serine. The recombinant multi-variant epitope gene was expressed in Escherichia coli BL21. The expressed protein of the variant multi-variant epitope gene can react with positive sera and monoclonal antibodies of ALV-J, while cannot react with ALV-J negative sera. The multi-variant epitope vaccine that conjugated Freund's adjuvant complete/incomplete showed high immunogenicity that reached the titer of 1:64,000 at 42 days post immunization and maintained the immune period for at least 126 days in SPF chickens. Further, we demonstrated that the antibody induced by the variant multi-variant ensemble epitope vaccine recognized and neutralized different ALV-J strains (NX0101, TA1, WS1, BZ1224 and BZ4). Protection experiment that was evaluated by clinical symptom, viral shedding, weight gain, gross and histopathology showed 100% chickens that inoculated the multi-epitope vaccine were well protected against ALV-J challenge. The result shows a promising multi-variant epitope ensemble vaccine against hypervariable viruses in animals. Copyright © 2017 Elsevier Ltd. All rights reserved.

Variable sequences outside the SAM-binding core critically influence the conformational dynamics of the SAM-III/SMK box riboswitch

PubMed Central

Lu, Changrui; Smith, Angela M; Ding, Fang; Chowdhury, Anirban; Henkin, Tina M; Ke, Ailong

2012-01-01

The SMK box (SAM-III) translational riboswitches were identified in S-adenosyl-L-methionine (SAM) synthetase metK genes in members of the Lactobacillales. This riboswitch switches between two alternative conformations in response to the intracellular SAM concentration and controls metK expression at the level of translation initiation. We previously reported the crystal structure of the SAM-bound SMK box riboswitch. In this study we combined SHAPE chemical probing with mutagenesis to probe the ligand-induced conformational switching mechanism. We revealed that while the majority of the apo SMK box RNA molecules exist in an alternatively base paired (ON) conformation, a subset of them pre-organize into a SAM-bound-like (READY) conformation, which upon SAM exposure is selectively stabilized into the SAM-bound (OFF) conformation through an induced-fit mechanism. Mutagenesis showed that the ON state is only slightly more stable than the READY state, as several single-nucleotide substitutions in a hypervariable region outside the SAM-binding core can alter the folding landscape to favor the READY state. Such SMK variants display a “constitutively-OFF” behavior both in vitro and in vivo. Time-resolved and temperature-dependent SHAPE analyses revealed adaptation of the SMK box RNA to its mesothermal working environment. The latter analysis revealed that the SAM-bound SMK box RNA follows a two-step folding/unfolding process. PMID:21549712

Hypervariability of ribosomal DNA at multiple chromosomal sites in lake trout (Salvelinus namaycush).

PubMed

Zhuo, L; Reed, K M; Phillips, R B

1995-06-01

Variation in the intergenic spacer (IGS) of the ribosomal DNA (rDNA) of lake trout (Salvelinus namaycush) was examined. Digestion of genomic DNA with restriction enzymes showed that almost every individual had a unique combination of length variants with most of this variation occurring within rather than between populations. Sequence analysis of a 2.3 kilobase (kb) EcoRI-DraI fragment spanning the 3' end of the 28S coding region and approximately 1.8 kb of the IGS revealed two blocks of repetitive DNA. Putative transcriptional termination sites were found approximately 220 bases (b) downstream from the end of the 28S coding region. Comparison of the 2.3-kb fragments with two longer (3.1 kb) fragments showed that the major difference in length resulted from variation in the number of short (89 b) repeats located 3' to the putative terminator. Repeat units within a single nucleolus organizer region (NOR) appeared relatively homogeneous and genetic analysis found variants to be stably inherited. A comparison of the number of spacer-length variants with the number of NORs found that the number of length variants per individual was always less than the number of NORs. Examination of spacer variants in five populations showed that populations with more NORs had more spacer variants, indicating that variants are present at different rDNA sites on nonhomologous chromosomes.

Systematics of Cladophora spp. (Chlorophyta) from North Carolina, USA, based upon morphology and DNA sequence data with a description of Cladophora subtilissima sp. nov.

PubMed

Taylor, Robin L; Bailey, Jeffrey Craig; Freshwater, David Wilson

2017-06-01

Identification of Cladophora species is challenging due to conservation of gross morphology, few discrete autapomorphies, and environmental influences on morphology. Twelve species of marine Cladophora were reported from North Carolina waters. Cladophora specimens were collected from inshore and offshore marine waters for DNA sequence and morphological analyses. The nuclear-encoded rRNA internal transcribed spacer regions (ITS) were sequenced for 105 specimens and used in molecular assisted identification. The ITS1 and ITS2 region was highly variable, and sequences were sorted into ITS Sets of Alignable Sequences (SASs). Sequencing of short hyper-variable ITS1 sections from Cladophora type specimens was used to positively identify species represented by SASs when the types were made available. Secondary structures for the ITS1 locus were also predicted for each specimen and compared to predicted structures from Cladophora sequences available in GenBank. Nine ITS SASs were identified and representative specimens chosen for phylogenetic analyses of 18S and 28S rRNA gene sequences to reveal relationships with other Cladophora species. Phylogenetic analyses indicated that marine Cladophorales were polyphyletic and separated into two clades, the Cladophora clade and the "Siphonocladales" clade. Morphological analyses were performed to assess the consistency of character states within species, and complement the DNA sequence analyses. These analyses revealed intra- and interspecific character state variation, and that combined molecular and morphological analyses were required for the identification of species. One new report, Cladophora dotyana, and one new species Cladophora subtilissima sp. nov., were revealed, and increased the biodiversity of North Carolina marine Cladophora to 14 species. © 2017 Phycological Society of America.

Genetic divergence among invasive and native populations of the yellow peacock cichlid Cichla kelberi.

PubMed

Marques, A C P B; Franco, A C S; Salgueiro, F; García-Berthou, E; Santos, L N

2016-12-01

This study used the hypervariable domain of the mitochondrial DNA (mtDNA) control region (CR) to assess the genetic divergence among native and invasive populations of Cichla kelberi, which is considered the first peacock cichlid introduced and established throughout Brazil and is among the most invasive populations of this genus worldwide. The maximum likelihood tree based on 53 CR sequences with strong bootstrap support revealed that C. kelberi forms a monophyletic clade, confirming that all 30 C. kelberi studied belong to this morphotype. Additionally, the haplotype analysis of the C. kelberi sequences from 11 sampling sites revealed that invasive populations are much less diverse than native ones and largely dominated by a single haplotype that prevailed in reservoirs at the Paraíba do Sul River basin. Two haplotypes were recorded exclusively in an invasive population at Porto Rico, southern Brazil, and one private haplotype was detected in two reservoirs from Paraíba do Sul (Pereira Passos and Paracambi), suggesting more than one introduction event and that native populations should be better evaluated to encompass the entire genetic diversity of native C. kelberi. The possible route and pathways of C. kelberi introduction are also briefly discussed. © 2016 The Fisheries Society of the British Isles.

Egypt's Red Sea coast: phylogenetic analysis of cultured microbial consortia in industrialized sites

PubMed Central

Mustafa, Ghada A.; Abd-Elgawad, Amr; Abdel-Haleem, Alyaa M.; Siam, Rania

2014-01-01

The Red Sea possesses a unique geography, and its shores are rich in mangrove, macro-algal and coral reef ecosystems. Various sources of pollution affect Red Sea biota, including microbial life. We assessed the effects of industrialization on microbes along the Egyptian Red Sea coast at eight coastal sites and two lakes. The bacterial communities of sediment samples were analyzed using bacterial 16S rDNA pyrosequencing of V6-V4 hypervariable regions. The taxonomic assignment of 131,402 significant reads to major bacterial taxa revealed five main bacterial phyla dominating the sampled sites: Proteobacteria (68%), Firmicutes (13%), Fusobacteria (12%), Bacteriodetes (6%), and Spirochetes (0.03%). Further analysis revealed distinct bacterial consortia that primarily included (1) marine Vibrio spp.—suggesting a “marine Vibrio phenomenon”; (2) potential human pathogens; and (3) oil-degrading bacteria. We discuss two divergent microbial consortia that were sampled from Solar Lake West near Taba/Eilat and Saline Lake in Ras Muhammad; these consortia contained the highest abundance of human pathogens and no pathogens, respectively. Our results draw attention to the effects of industrialization on the Red Sea and suggest the need for further analysis to overcome the hazardous effects observed at the impacted sites. PMID:25157243

Genetic origin of goat populations in Oman revealed by mitochondrial DNA analysis.

PubMed

Al-Araimi, Nasser Ali; Gaafar, Osman Mahgoub; Costa, Vânia; Neira, Agusto Luzuriaga; Al-Atiyat, Raed Mahmoud; Beja-Pereira, Albano

2017-01-01

The Sultanate of Oman has a complex mosaic of livestock species and production systems, but the genetic diversity, demographic history or origins of these Omani animals has not been expensively studied. Goats might constitute one of the most abundant and important domestic livestock species since the Neolithic transition. Here, we examined the genetic diversity, origin, population structure and demographic history of Omani goats. Specifically, we analyzed a 525-bp fragment of the first hypervariable region of the mitochondrial DNA (mtDNA) control region from 69 Omani individuals and compared this fragment with 17 mtDNA sequences from Somalia and Yemen as well as 18 wild goat species and 1,198 previously published goat sequences from neighboring countries. The studied goat breeds show substantial diversity. The haplotype and nucleotide diversities of Omani goats were found equal to 0.983 ± 0.006 and 0.0284 ± 0.014, respectively. The phylogenetic analyses allowed us to classify Omani goats into three mtDNA haplogroups (A, B and G): haplogroup A was found to be predominant and widely distributed and accounted for 80% of all samples, and haplogroups B and G exhibited low frequencies. Phylogenetic comparisons with wild goats revealed that five of the native Omani goat populations originate from Capra aegagrus. Furthermore, most comparisons of pairwise population FST values within and between these five Omani goat breeds as well as between Omani goats and nine populations from nearby countries were not significant. These results suggest strong gene flow among goat populations caused by the extensive transport of goats and the frequent movements of human populations in ancient Arabia. The findings improve our understanding of the migration routes of modern goats from their region of domestication into southeastern Arabia and thereby shed light on human migratory and commercial networks during historical times.

Distribution of Barley yellow dwarf virus-PAV in the Sub-Antarctic Kerguelen Islands and Characterization of Two New Luteovirus Species

PubMed Central

Svanella-Dumas, Laurence; Candresse, Thierry; Hullé, Maurice; Marais, Armelle

2013-01-01

A systematic search for viral infection was performed in the isolated Kerguelen Islands, using a range of polyvalent genus-specific PCR assays. Barley yellow dwarf virus (BYDV) was detected in both introduced and native grasses such as Poa cookii. The geographical distribution of BYDV and its prevalence in P. cookii were analyzed using samples collected from various sites of the archipelago. We estimate the average prevalence of BYDV to be 24.9% in P. cookii, with significant variability between sites. BYDV genetic diversity was assessed using sequence information from two genomic regions: the P3 open reading frame (ORF) (encoding the coat protein) and the hypervariable P6 ORF region. The phylogenetic analysis in the P3 region showed that BYDV sequences segregate into three major lineages, the most frequent of which (Ker-I cluster) showed close homology with BYDV-PAV-I isolates and had very low intra-lineage diversity (0.6%). A similarly low diversity was also recorded in the hypervariable P6 region, suggesting that Ker-I isolates derive from the recent introduction of BYDV-PAV-I. Divergence time estimation suggests that BYDV-PAV-I was likely introduced in the Kerguelen environment at the same time frame as its aphid vector, Rhopalosiphum padi, whose distribution shows good overlap with that of BYDV-Ker-I. The two other lineages show more than 22% amino acid divergence in the P3 region with other known species in the BYDV species complex, indicating that they represent distinct BYDV species. Using species-specific amplification primers, the distribution of these novel species was analyzed. The high prevalence of BYDV on native Poaceae and the presence of the vector R. padi, raises the question of its impact on the vulnerable plant communities of this remote ecosystem. PMID:23825645

Molecular epidemiology of human respiratory syncytial virus among children in Japan during three seasons and hospitalization risk of genotype ON1

PubMed Central

Hibino, Akinobu; Taniguchi, Kiyosu; Zaraket, Hassan; Shobugawa, Yugo; Matsui, Tamano; Suzuki, Hiroshi

2018-01-01

We investigated the genetic diversity, the circulation patterns, and risk for hospital admission of human respiratory syncytial virus (HRSV) strains in Japan between 2012 through 2015. During the study period, 744 HRSV-positive cases were identified by rapid diagnostic test. Of these, 572 samples were positive by real-time PCR; 400 (69.9%) were HRSV-A, and 172 (30.1%) were HRSV-B. HRSV-A and -B alternated as the dominant strain in the subsequent seasons. Phylogenetic tree analysis of the second hyper-variable region of the G protein classified the HRSV-A specimens into NA1 (n = 242) and ON1 (n = 114) genotypes and the HRSV-B specimens into BA9 (n = 60), and BA10 (n = 27). The ON1 genotype, containing a 72-nucleotide duplication in the G protein’s second hyper-variable region, was first detected in the 2012–2013 season but it predominated and replaced the older NA1 HRSV-A in the 2014–2015 season, which also coincided with a record number of HRSV cases reported to the National Infectious Disease Surveillance in Japan. The risk of hospitalization was 6.9 times higher for the ON1 genotype compared to NA1. In conclusion, our data showed that the emergence and predominance of the relatively new ON1 genotype in Japan was associated with a record high number of cases and increased risk for hospitalization. PMID:29377949

KDNA Genetic Signatures Obtained by LSSP-PCR Analysis of Leishmania (Leishmania) infantum Isolated from the New and the Old World

PubMed Central

Alvarenga, Janaína Sousa Campos; Ligeiro, Carla Maia; Gontijo, Célia Maria Ferreira; Cortes, Sofia; Campino, Lenea; Vago, Annamaria Ravara; Melo, Maria Norma

2012-01-01

Background Visceral Leishmaniasis (VL) caused by species from the Leishmania donovani complex is the most severe form of the disease, lethal if untreated. VL caused by Leishmania infantum is a zoonosis with an increasing number of human cases and millions of dogs infected in the Old and the New World. In this study, L. infantum (syn. L.chagasi) strains were isolated from human and canine VL cases. The strains were obtained from endemic areas from Brazil and Portugal and their genetic polymorphism was ascertained using the LSSP-PCR (Low-Stringency Single Specific Primer PCR) technique for analyzing the kinetoplastid DNA (kDNA) minicircles hypervariable region. Principal Findings KDNA genetic signatures obtained by minicircle LSSP-PCR analysis of forty L. infantum strains allowed the grouping of strains in several clades. Furthermore, LSSP-PCR profiles of L. infantum subpopulations were closely related to the host origin (human or canine). To our knowledge this is the first study which used this technique to compare genetic polymorphisms among strains of L. infantum originated from both the Old and the New World. Conclusions LSSP-PCR profiles obtained by analysis of L. infantum kDNA hypervariable region of parasites isolated from human cases and infected dogs from Brazil and Portugal exhibited a genetic correlation among isolates originated from the same reservoir, human or canine. However, no association has been detected among the kDNA signatures and the geographical origin of L. infantum strains. PMID:22912862

A 454 Survey Reveals the Community Composition and Core Microbiome of the Common Bed Bug (Cimex lectularius) across an Urban Landscape

PubMed Central

Meriweather, Matthew; Matthews, Sara; Rio, Rita; Baucom, Regina S.

2013-01-01

Elucidating the spatial dynamic and core constituents of the microbial communities found in association with arthropod hosts is of crucial importance for insects that may vector human or agricultural pathogens. The hematophagous Cimex lectularius (Hemiptera: Cimicidae), known as the human bed bug, has made a recent resurgence in North America, as well as worldwide, potentially owing to increased travel, climate change and resistance to insecticides. A comprehensive survey of the bed bug microbiome has not been performed to date, nor has an assessment of the spatial dynamics of its microbiome. Here we present a survey of internal and external bed bug microbial communities by amplifying the V4–V6 hypervariable region of the 16S rDNA gene region followed by 454 Titanium sequencing using 31 individuals from eight distinct collection locations obtained from residences in Cincinnati, OH. Across all samples, 97% of the microbial community is made up of two dominant OTUs, previously identified as the α-proteobacterium Wolbachia and an unnamed γ-proteobacterium from the Enterobacteriaceae. Microbial communities varied among host locations for measures of community diversity and exhibited structure according to collection location. This broad survey represents the most in-depth assessment, to date, of the microbes that associate with bed bugs. PMID:23585900

A 454 survey reveals the community composition and core microbiome of the common bed bug (Cimex lectularius) across an Urban Landscape.

PubMed

Meriweather, Matthew; Matthews, Sara; Rio, Rita; Baucom, Regina S

2013-01-01

Elucidating the spatial dynamic and core constituents of the microbial communities found in association with arthropod hosts is of crucial importance for insects that may vector human or agricultural pathogens. The hematophagous Cimex lectularius (Hemiptera: Cimicidae), known as the human bed bug, has made a recent resurgence in North America, as well as worldwide, potentially owing to increased travel, climate change and resistance to insecticides. A comprehensive survey of the bed bug microbiome has not been performed to date, nor has an assessment of the spatial dynamics of its microbiome. Here we present a survey of internal and external bed bug microbial communities by amplifying the V4-V6 hypervariable region of the 16S rDNA gene region followed by 454 Titanium sequencing using 31 individuals from eight distinct collection locations obtained from residences in Cincinnati, OH. Across all samples, 97% of the microbial community is made up of two dominant OTUs, previously identified as the α-proteobacterium Wolbachia and an unnamed γ-proteobacterium from the Enterobacteriaceae. Microbial communities varied among host locations for measures of community diversity and exhibited structure according to collection location. This broad survey represents the most in-depth assessment, to date, of the microbes that associate with bed bugs.

Origins and genetic features of the Okhotsk people, revealed by ancient mitochondrial DNA analysis.

PubMed

Sato, Takehiro; Amano, Tetsuya; Ono, Hiroko; Ishida, Hajime; Kodera, Haruto; Matsumura, Hirofumi; Yoneda, Minoru; Masuda, Ryuichi

2007-01-01

In order to investigate the phylogenetic status of the Okhotsk people that were distributed in northern and eastern Hokkaido as well as southern Sakhalin during the fifth to the thirteenth centuries, DNA was carefully extracted from human bone and tooth remains excavated from archaeological sites. The hypervariable region 1 sequences of the mitochondrial DNA (mtDNA) control region were successfully amplified and 16 mtDNA haplotypes were identified from 37 individuals of the Okhotsk people. Of the 16 haplotypes found, 6 were unique to the Okhotsk people, whereas the other 10 were shared by northeastern Asian people that are currently distributed around Sakhalin and downstream of the Amur River. The phylogenetic relationships inferred from mtDNA sequences showed that the Okhotsk people were more closely related to the Nivkhi and Ulchi people among populations of northeastern Asia. In addition, the Okhotsk people had a relatively closer genetic affinity with the Ainu people of Hokkaido, and were likely intermediates of gene flow from the northeastern Asian people to the Ainu people. These findings support the hypothesis that the Okhotsk culture joined the Satsumon culture (direct descendants of the Jomon people) resulting in the Ainu culture, as suggested by previous archaeological and anthropological studies.

Factor H binds to the hypervariable region of many Streptococcus pyogenes M proteins but does not promote phagocytosis resistance or acute virulence.

PubMed

Gustafsson, Mattias C U; Lannergård, Jonas; Nilsson, O Rickard; Kristensen, Bodil M; Olsen, John E; Harris, Claire L; Ufret-Vincenty, Rafael L; Stålhammar-Carlemalm, Margaretha; Lindahl, Gunnar

2013-01-01

Many pathogens express a surface protein that binds the human complement regulator factor H (FH), as first described for Streptococcus pyogenes and the antiphagocytic M6 protein. It is commonly assumed that FH recruited to an M protein enhances virulence by protecting the bacteria against complement deposition and phagocytosis, but the role of FH-binding in S. pyogenes pathogenesis has remained unclear and controversial. Here, we studied seven purified M proteins for ability to bind FH and found that FH binds to the M5, M6 and M18 proteins but not the M1, M3, M4 and M22 proteins. Extensive immunochemical analysis indicated that FH binds solely to the hypervariable region (HVR) of an M protein, suggesting that selection has favored the ability of certain HVRs to bind FH. These FH-binding HVRs could be studied as isolated polypeptides that retain ability to bind FH, implying that an FH-binding HVR represents a distinct ligand-binding domain. The isolated HVRs specifically interacted with FH among all human serum proteins, interacted with the same region in FH and showed species specificity, but exhibited little or no antigenic cross-reactivity. Although these findings suggested that FH recruited to an M protein promotes virulence, studies in transgenic mice did not demonstrate a role for bound FH during acute infection. Moreover, phagocytosis tests indicated that ability to bind FH is neither sufficient nor necessary for S. pyogenes to resist killing in whole human blood. While these data shed new light on the HVR of M proteins, they suggest that FH-binding may affect S. pyogenes virulence by mechanisms not assessed in currently used model systems.

Factor H Binds to the Hypervariable Region of Many Streptococcus pyogenes M Proteins but Does Not Promote Phagocytosis Resistance or Acute Virulence

PubMed Central

Kristensen, Bodil M.; Olsen, John E.; Harris, Claire L.; Ufret-Vincenty, Rafael L.; Stålhammar-Carlemalm, Margaretha; Lindahl, Gunnar

2013-01-01

Many pathogens express a surface protein that binds the human complement regulator factor H (FH), as first described for Streptococcus pyogenes and the antiphagocytic M6 protein. It is commonly assumed that FH recruited to an M protein enhances virulence by protecting the bacteria against complement deposition and phagocytosis, but the role of FH-binding in S. pyogenes pathogenesis has remained unclear and controversial. Here, we studied seven purified M proteins for ability to bind FH and found that FH binds to the M5, M6 and M18 proteins but not the M1, M3, M4 and M22 proteins. Extensive immunochemical analysis indicated that FH binds solely to the hypervariable region (HVR) of an M protein, suggesting that selection has favored the ability of certain HVRs to bind FH. These FH-binding HVRs could be studied as isolated polypeptides that retain ability to bind FH, implying that an FH-binding HVR represents a distinct ligand-binding domain. The isolated HVRs specifically interacted with FH among all human serum proteins, interacted with the same region in FH and showed species specificity, but exhibited little or no antigenic cross-reactivity. Although these findings suggested that FH recruited to an M protein promotes virulence, studies in transgenic mice did not demonstrate a role for bound FH during acute infection. Moreover, phagocytosis tests indicated that ability to bind FH is neither sufficient nor necessary for S. pyogenes to resist killing in whole human blood. While these data shed new light on the HVR of M proteins, they suggest that FH-binding may affect S. pyogenes virulence by mechanisms not assessed in currently used model systems. PMID:23637608

GTP Binding and Oncogenic Mutations May Attenuate Hypervariable Region (HVR)-Catalytic Domain Interactions in Small GTPase K-Ras4B, Exposing the Effector Binding Site*

PubMed Central

Lu, Shaoyong; Banerjee, Avik; Jang, Hyunbum; Zhang, Jian; Gaponenko, Vadim; Nussinov, Ruth

2015-01-01

K-Ras4B, a frequently mutated oncogene in cancer, plays an essential role in cell growth, differentiation, and survival. Its C-terminal membrane-associated hypervariable region (HVR) is required for full biological activity. In the active GTP-bound state, the HVR interacts with acidic plasma membrane (PM) headgroups, whereas the farnesyl anchors in the membrane; in the inactive GDP-bound state, the HVR may interact with both the PM and the catalytic domain at the effector binding region, obstructing signaling and nucleotide exchange. Here, using molecular dynamics simulations and NMR, we aim to figure out the effects of nucleotides (GTP and GDP) and frequent (G12C, G12D, G12V, G13D, and Q61H) and infrequent (E37K and R164Q) oncogenic mutations on full-length K-Ras4B. The mutations are away from or directly at the HVR switch I/effector binding site. Our results suggest that full-length wild-type GDP-bound K-Ras4B (K-Ras4BWT-GDP) is in an intrinsically autoinhibited state via tight HVR-catalytic domain interactions. The looser association in K-Ras4BWT-GTP may release the HVR. Some of the oncogenic mutations weaken the HVR-catalytic domain association in the K-Ras4B-GDP/-GTP bound states, which may facilitate the HVR disassociation in a nucleotide-independent manner, thereby up-regulating oncogenic Ras signaling. Thus, our results suggest that mutations can exert their effects in more than one way, abolishing GTP hydrolysis and facilitating effector binding. PMID:26453300

Role of Sequencing the Measles Virus Hemagglutinin Gene and Hypervariable Region in the Measles Outbreak Investigations in Sweden During 2013-2014.

PubMed

Harvala, Heli; Wiman, Åsa; Wallensten, Anders; Zakikhany, Katherina; Englund, Hélène; Brytting, Maria

2016-02-15

It is increasingly difficult to differentiate measles viruses (MeVs) relating to certain outbreaks on the basis of the nucleoprotein (N) gene sequence only, as the diversity of circulating MeV strains has decreased. We studied genomic regions that could provide better molecular discrimination between epidemiologically linked and unlinked MeV variants identified in Sweden during 2013-2014. The hemagglutinin (H) gene and hypervariable region between the fusion and matrix genes (MF-HVR) from 53 MeV-positive samples were amplified and sequenced. Data on phylogenetic clustering of MeVs on the basis of N, H, and MF-HVR sequences were compared to epidemiological data. MeVs were genotyped: 27 were B3, and 26 were D8. One genotype B3 cluster based on the N gene sequence contained epidemiologically unrelated viruses from 4 outbreaks, whereas analysis of H and MF-HVR sequences separated them into phylogenetic clusters consistent with the epidemiological data. Similarly, the single cluster of viruses with a genotype D8 N gene could be divided into the 5 outbreak groups on the basis of the phylogeny of MF-HVR sequences. A detailed picture of MeV circulation with more-defined links between outbreaks was obtained by sequencing the H gene and MF-HVR. Further identification and better genetic characterization of MeVs internationally is essential in identifying sources and routes of MeV spread within and beyond Europe in the elimination end game. © The Author 2015. Published by Oxford University Press for the Infectious Diseases Society of America. All rights reserved. For permissions, e-mail journals.permissions@oup.com.

Unexpected sequences and structures of mtDNA required for efficient transcription from the first heavy-strand promoter

PubMed Central

Uchida, Akira; Murugesapillai, Divakaran; Kastner, Markus; Wang, Yao; Lodeiro, Maria F; Prabhakar, Shaan; Oliver, Guinevere V; Arnold, Jamie J; Maher, L James; Williams, Mark C; Cameron, Craig E

2017-01-01

Human mtDNA contains three promoters, suggesting a need for differential expression of the mitochondrial genome. Studies of mitochondrial transcription have used a reductionist approach, perhaps masking differential regulation. Here we evaluate transcription from light-strand (LSP) and heavy-strand (HSP1) promoters using templates that mimic their natural context. These studies reveal sequences upstream, hypervariable in the human population (HVR3), and downstream of the HSP1 transcription start site required for maximal yield. The carboxy-terminal tail of TFAM is essential for activation of HSP1 but not LSP. Images of the template obtained by atomic force microscopy show that TFAM creates loops in a discrete region, the formation of which correlates with activation of HSP1; looping is lost in tail-deleted TFAM. Identification of HVR3 as a transcriptional regulatory element may contribute to between-individual variability in mitochondrial gene expression. The unique requirement of HSP1 for the TFAM tail may enable its regulation by post-translational modifications. DOI: http://dx.doi.org/10.7554/eLife.27283.001 PMID:28745586

Tilting the balance between canonical and noncanonical conformations for the H1 hypervariable loop of a llama VHH through point mutations.

PubMed

Mahajan, Sai Pooja; Velez-Vega, Camilo; Escobedo, Fernando A

2013-01-10

Nanobodies are single-domain antibodies found in camelids. These are the smallest naturally occurring binding domains and derive functionality via three hypervariable loops (H1-H3) that form the binding surface. They are excellent candidates for antibody engineering because of their favorable characteristics like small size, high solubility, and stability. To rationally engineer antibodies with affinity for a specific target, the hypervariable loops can be tailored to obtain the desired binding surface. As a first step toward such a goal, we consider the design of loops with a desired conformation. In this study, we focus on the H1 loop of the anti-hCG llama nanobody that exhibits a noncanonical conformation. We aim to "tilt" the stability of the H1 loop structure from a noncanonical conformation to a (humanized) type 1 canonical conformation by studying the effect of selected mutations to the amino acid sequence of the H1, H2, and proximal residues. We use all-atomistic, explicit-solvent, biased molecular dynamic simulations to simulate the wild-type and mutant loops in a prefolded framework. We thus find mutants with increasing propensity to form a stable type 1 canonical conformation of the H1 loop. Free energy landscapes reveal the existence of conformational isomers of the canonical conformation that may play a role in binding different antigenic surfaces. We also elucidate the approximate mechanism and kinetics of transitions between such conformational isomers by using a Markovian model. We find that a particular three-point mutant has the strongest thermodynamic propensity to form the H1 type 1 canonical structure but also to exhibit transitions between conformational isomers, while a different, more rigid three-point mutant has the strongest propensity to be kinetically trapped in such a canonical structure.

Structural insights into the evolution of a sexy protein: novel topology and restricted backbone flexibility in a hypervariable pheromone from the red-legged salamander, Plethodon shermani.

PubMed

Wilburn, Damien B; Bowen, Kathleen E; Doty, Kari A; Arumugam, Sengodagounder; Lane, Andrew N; Feldhoff, Pamela W; Feldhoff, Richard C

2014-01-01

In response to pervasive sexual selection, protein sex pheromones often display rapid mutation and accelerated evolution of corresponding gene sequences. For proteins, the general dogma is that structure is maintained even as sequence or function may rapidly change. This phenomenon is well exemplified by the three-finger protein (TFP) superfamily: a diverse class of vertebrate proteins co-opted for many biological functions - such as components of snake venoms, regulators of the complement system, and coordinators of amphibian limb regeneration. All of the >200 structurally characterized TFPs adopt the namesake "three-finger" topology. In male red-legged salamanders, the TFP pheromone Plethodontid Modulating Factor (PMF) is a hypervariable protein such that, through extensive gene duplication and pervasive sexual selection, individual male salamanders express more than 30 unique isoforms. However, it remained unclear how this accelerated evolution affected the protein structure of PMF. Using LC/MS-MS and multidimensional NMR, we report the 3D structure of the most abundant PMF isoform, PMF-G. The high resolution structural ensemble revealed a highly modified TFP structure, including a unique disulfide bonding pattern and loss of secondary structure, that define a novel protein topology with greater backbone flexibility in the third peptide finger. Sequence comparison, models of molecular evolution, and homology modeling together support that this flexible third finger is the most rapidly evolving segment of PMF. Combined with PMF sequence hypervariability, this structural flexibility may enhance the plasticity of PMF as a chemical signal by permitting potentially thousands of structural conformers. We propose that the flexible third finger plays a critical role in PMF:receptor interactions. As female receptors co-evolve, this flexibility may allow PMF to still bind its receptor(s) without the immediate need for complementary mutations. Consequently, this unique adaptation may establish new paradigms for how receptor:ligand pairs co-evolve, in particular with respect to sexual conflict.

Use of synthetic oligonucleotide DNA probes for the identification of Bacteroides gingivalis.

PubMed Central

Moncla, B J; Braham, P; Dix, K; Watanabe, S; Schwartz, D

1990-01-01

Six different oligonucleotide probes complementary to the hypervariable regions of 16S rRNA of Bacteroides gingivalis were tested for specificity and sensitivity against 77 field strains of B. gingivalis and 105 strains of 12 other Bacteroides species. The data demonstrated that these probes were very specific (range, 0.85 to 1.00) and sensitive (1.00). Some limited cross-reactions with other Bacteroides species were observed. Four of these probes should be useful for rapid detection and identification of B. gingivalis. Images PMID:1690217

Cultivation Versus Molecular Analysis of Banana (Musa sp.) Shoot-Tip Tissue Reveals Enormous Diversity of Normally Uncultivable Endophytic Bacteria.

PubMed

Thomas, Pious; Sekhar, Aparna Chandra

2017-05-01

The interior of plants constitutes a unique environment for microorganisms with various organisms inhabiting as endophytes. Unlike subterranean plant parts, aboveground parts are relatively less explored for endophytic microbial diversity. We employed a combination of cultivation and molecular approaches to study the endophytic bacterial diversity in banana shoot-tips. Cultivable bacteria from 20 sucker shoot-tips of cv. Grand Naine included 37 strains under 16 genera and three phyla (Proteobacteria, Actinobacteria, Firmicutes). 16S rRNA gene-ribotyping approach on 799f and 1492r PCR-amplicons to avoid plant organelle sequences was ineffective showing limited bacterial diversity. 16S rRNA metagene profiling targeting the V3-V4 hypervariable region after filtering out the chloroplast (74.2 %), mitochondrial (22.9 %), and unknown sequences (1.1 %) revealed enormous bacterial diversity. Proteobacteria formed the predominant phylum (64 %) succeeded by Firmicutes (12.1 %), Actinobacteria (9.5 %), Bacteroidetes (6.4 %), Planctomycetes, Cyanobacteria, and minor shares (<1 %) of 14 phyla including several candidate phyla besides the domain Euryarchaeota (0.2 %). Microbiome analysis of single shoot-tips through 16S rRNA V3 region profiling showed similar taxonomic richness and diversity and was less affected by plant sequence interferences. DNA extraction kit ominously influenced the phylogenetic diversity. The study has revealed vast diversity of normally uncultivable endophytic bacteria prevailing in banana shoot-tips (20 phyla, 46 classes) with about 2.6 % of the deciphered 269 genera and 1.5 % of the 656 observed species from the same source of shoot-tips attained through cultivation. The predominant genera included several agriculturally important bacteria. The study reveals an immense ecosystem of endophytic bacteria in banana shoot tissues endorsing the earlier documentation of intracellular "Cytobacts" and "Peribacts" with possible roles in plant holobiome and hologenome.

Characterization of bacterial community associated with phytoplankton bloom in a eutrophic lake in South Norway using 16S rRNA gene amplicon sequence analysis.

PubMed

Parulekar, Niranjan Nitin; Kolekar, Pandurang; Jenkins, Andrew; Kleiven, Synne; Utkilen, Hans; Johansen, Anette; Sawant, Sangeeta; Kulkarni-Kale, Urmila; Kale, Mohan; Sæbø, Mona

2017-01-01

Interactions between different phytoplankton taxa and heterotrophic bacterial communities within aquatic environments can differentially support growth of various heterotrophic bacterial species. In this study, phytoplankton diversity was studied using traditional microscopic techniques and the bacterial communities associated with phytoplankton bloom were studied using High Throughput Sequencing (HTS) analysis of 16S rRNA gene amplicons from the V1-V3 and V3-V4 hypervariable regions. Samples were collected from Lake Akersvannet, a eutrophic lake in South Norway, during the growth season from June to August 2013. Microscopic examination revealed that the phytoplankton community was mostly represented by Cyanobacteria and the dinoflagellate Ceratium hirundinella. The HTS results revealed that Proteobacteria (Alpha, Beta, and Gamma), Bacteriodetes, Cyanobacteria, Actinobacteria and Verrucomicrobia dominated the bacterial community, with varying relative abundances throughout the sampling season. Species level identification of Cyanobacteria showed a mixed population of Aphanizomenon flos-aquae, Microcystis aeruginosa and Woronichinia naegeliana. A significant proportion of the microbial community was composed of unclassified taxa which might represent locally adapted freshwater bacterial groups. Comparison of cyanobacterial species composition from HTS and microscopy revealed quantitative discrepancies, indicating a need for cross validation of results. To our knowledge, this is the first study that uses HTS methods for studying the bacterial community associated with phytoplankton blooms in a Norwegian lake. The study demonstrates the value of considering results from multiple methods when studying bacterial communities.

Phylogeography and origin of Chinese domestic chicken.

PubMed

Wu, Y P; Huo, J H; Xie, J F; Liu, L X; Wei, Q P; Xie, M G; Kang, Z F; Ji, H Y; Ma, Y H

2014-04-01

The loss of local chicken breeds as result of replacement with cosmopolitan breeds indicates the need for conservation measures to protect the future of local genetic stocks. The aim of this study is to describe the patterns of polymorphism of the hypervariable control region of mitochondrial DNA (HVR1) in domestic chicken in China's Jiangxi province to investigate genetic diversity, genetic structure and phylo-dynamics. To this end, we sequenced the mtDNA HVR1 in 231 chickens including 22 individuals which belonged to previously published sequences. A neighbor-joining tree revealed that these samples clustered into five lineages (Lineages A, B, C, E and G). The highest haplotype diversity and nucleotide diversity were both found in Anyi tile-liked gray breed. We estimated that the most recent common ancestor of the local chicken existed approximately 16 million years ago. The mismatch distribution analysis showed two major peaks at positions 4 and 9, while the neutrality test (Tajima's D = -2.19, p < 0.05) and Fu's F-statistics (-8.59, p < 0.05) revealed a significant departure from the neutrality assumption. These results support the idea that domestication of chickens facilitated population increases. Results of a global AMOVA indicated that there was no obvious geographic structure among the local chicken breeds analyzed in this study. The data obtained in this study will assist future conservation management of local breeds and also reveals intriguing implications for the history of human population movements and commerce.

Multilocus Microsatellite Typing (MLMT) of Strains from Turkey and Cyprus Reveals a Novel Monophyletic L. donovani Sensu Lato Group

PubMed Central

Amro, Ahmad; Mentis, Andreas; Pratlong, Francine; Dedet, Jean-Pierre; Votypka, Jan; Volf, Petr; Ozensoy Toz, Seray; Kuhls, Katrin; Schönian, Gabriele; Soteriadou, Ketty

2012-01-01

Background New foci of human CL caused by strains of the Leishmania donovani (L. donovani) complex have been recently described in Cyprus and the Çukurova region in Turkey (L. infantum) situated 150 km north of Cyprus. Cypriot strains were typed by Multilocus Enzyme Electrophoresis (MLEE) using the Montpellier (MON) system as L. donovani zymodeme MON-37. However, multilocus microsatellite typing (MLMT) has shown that this zymodeme is paraphyletic; composed of distantly related genetic subgroups of different geographical origin. Consequently the origin of the Cypriot strains remained enigmatic. Methodology/Principal Findings The Cypriot strains were compared with a set of Turkish isolates obtained from a CL patient and sand fly vectors in south-east Turkey (Çukurova region; CUK strains) and from a VL patient in the south-west (Kuşadasi; EP59 strain). These Turkish strains were initially analyzed using the K26-PCR assay that discriminates MON-1 strains by their amplicon size. In line with previous DNA-based data, the strains were inferred to the L. donovani complex and characterized as non MON-1. For these strains MLEE typing revealed two novel zymodemes; L. donovani MON-309 (CUK strains) and MON-308 (EP59). A population genetic analysis of the Turkish isolates was performed using 14 hyper-variable microsatellite loci. The genotypic profiles of 68 previously analyzed L. donovani complex strains from major endemic regions were included for comparison. Population structures were inferred by combination of Bayesian model-based and distance-based approaches. MLMT placed the Turkish and Cypriot strains in a subclade of a newly discovered, genetically distinct L. infantum monophyletic group, suggesting that the Cypriot strains may originate from Turkey. Conclusion The discovery of a genetically distinct L. infantum monophyletic group in the south-eastern Mediterranean stresses the importance of species genetic characterization towards better understanding, monitoring and controlling the spread of leishmaniasis in this region. PMID:22348162

Population characteristics of DNA fingerprints in humpback whales (Megaptera novaeangliae).

PubMed

Baker, C S; Gilbert, D A; Weinrich, M T; Lambertsen, R; Calambokidis, J; McArdle, B; Chambers, G K; O'Brien, S J

1993-01-01

Humpback whales exhibit a remarkable social organization that is characterized by seasonal long-distance migration (> 10,000 km/year) between summer feeding grounds in high latitudes and winter calving and breeding grounds in tropical or near-tropical waters. All populations are currently considered endangered as a result of intensive commercial exploitation during the last 200 years. Using three hypervariable minisatellite DNA probes (33.15, 3'HVR, and M13) originally developed for studies of human genetic variation, we examined genetic variation within and among three regional subpopulations of humpback whales from the North Pacific and one from the North Atlantic oceans. Analysis of DNA extracted from skin tissues collected by biopsy darting from free-ranging whales revealed considerable variation in each subpopulation. The extent of this variation argues against a recent history of inbreeding among humpback whales as a result of nineteenth- and twentieth-century hunting. A canonical variate analysis suggested a relationship between scaled genetic distance, based on similarities of DNA fingerprints, and geographic distance (i.e., longitude of regional subpopulation). Significant categorical differences were found between the two oceanic populations using a multivariate analysis of variance (MANOVA) with a modification of the Mantel nonparametric permutation test. The relationship between DNA fingerprint similarities and geographic distance suggests that nuclear gene flow between regional subpopulations within the North Pacific is restricted by relatively low rates of migratory interchange between breeding grounds or assortative mating on common wintering grounds.

Humanized Antibodies for Antiviral Therapy

NASA Astrophysics Data System (ADS)

Co, Man Sung; Deschamps, Marguerite; Whitley, Richard J.; Queen, Cary

1991-04-01

Antibody therapy holds great promise for the treatment of cancer, autoimmune disorders, and viral infections. Murine monoclonal antibodies are relatively easy to produce but are severely restricted for therapeutic use by their immunogenicity in humans. Production of human monoclonal antibodies has been problematic. Humanized antibodies can be generated by introducing the six hypervariable regions from the heavy and light chains of a murine antibody into a human framework sequence and combining it with human constant regions. We humanized, with the aid of computer modeling, two murine monoclonal antibodies against herpes simplex virus gB and gD glycoproteins. The binding, virus neutralization, and cell protection results all indicate that both humanized antibodies have retained the binding activities and the biological properties of the murine monoclonal antibodies.

Genetic Diversity and Structure among Isolated Populations of the Endangered Gees Golden Langur in Assam, India

PubMed Central

Biswas, Jihosuo; Nag, Sudipta; Shil, Joydeep; Umapathy, Govindhaswamy

2016-01-01

Gee’s golden langur (Trachypithecus geei) is an endangered colobine primate, endemic to the semi-evergreen and mixed-deciduous forests of Indo-Bhutan border. During the last few decades, extensive fragmentation has caused severe population decline and local extinction of golden langur from several fragments. However, no studies are available on the impact of habitat fragmentation and the genetic diversity of golden langur in the fragmented habitats. The present study aimed to estimate the genetic diversity in the Indian population of golden langur. We sequenced and analyzed around 500 bases of the mitochondrial DNA (mtDNA) hypervariable region-I from 59 fecal samples of wild langur collected from nine forest fragments. Overall, genetic diversity was high (h = 0.934, π = 0.0244) and comparable with other colobines. Populations in smaller fragments showed lower nucleotide diversity compared to the larger forest fragments. The median-joining network of haplotypes revealed a genetic structure that corresponded with the geographical distribution. The Aie and Champabati Rivers were found to be a barrier to gene flow between golden langur populations. In addition, it also established that T. geei is monophyletic but revealed possible hybridization with capped langur, T. pileatus, in the wild. It is hoped that these findings would result in a more scientific approach towards managing the fragmented populations of this enigmatic species. PMID:27564405

Microbial Communities on Seafloor Basalts at Dorado Outcrop Reflect Level of Alteration and Highlight Global Lithic Clades

PubMed Central

Lee, Michael D.; Walworth, Nathan G.; Sylvan, Jason B.; Edwards, Katrina J.; Orcutt, Beth N.

2015-01-01

Areas of exposed basalt along mid-ocean ridges and at seafloor outcrops serve as conduits of fluid flux into and out of a subsurface ocean, and microbe–mineral interactions can influence alteration reactions at the rock–water interface. Located on the eastern flank of the East Pacific Rise, Dorado Outcrop is a site of low-temperature (<20°C) hydrothermal venting and represents a new end-member in the current survey of seafloor basalt biomes. Consistent with prior studies, a survey of 16S rRNA gene sequence diversity using universal primers targeting the V4 hypervariable region revealed much greater richness and diversity on the seafloor rocks than in surrounding seawater. Overall, Gamma-, Alpha-, and Deltaproteobacteria, and Thaumarchaeota dominated the sequenced communities, together making up over half of the observed diversity, though bacterial sequences were more abundant than archaeal in all samples. The most abundant bacterial reads were closely related to the obligate chemolithoautotrophic, sulfur-oxidizing Thioprofundum lithotrophicum, suggesting carbon and sulfur cycling as dominant metabolic pathways in this system. Representatives of Thaumarchaeota were detected in relatively high abundance on the basalts in comparison to bottom water, possibly indicating ammonia oxidation. In comparison to other sequence datasets from globally distributed seafloor basalts, this study reveals many overlapping and cosmopolitan phylogenetic groups and also suggests that substrate age correlates with community structure. PMID:26779122

Mitochondrial DNA history of Sri Lankan ethnic people: their relations within the island and with the Indian subcontinental populations.

PubMed

Ranaweera, Lanka; Kaewsutthi, Supannee; Win Tun, Aung; Boonyarit, Hathaichanoke; Poolsuwan, Samerchai; Lertrit, Patcharee

2014-01-01

Located only a short distance off the southernmost shore of the Greater Indian subcontinent, the island of Sri Lanka has long been inhabited by various ethnic populations. Mainly comprising the Vedda, Sinhalese (Up- and Low-country) and Tamil (Sri Lankan and Indian); their history of settlements on the island and the biological relationships among them have remained obscure. It has been hypothesized that the Vedda was probably the earliest inhabitants of the area, followed by Sinhalese and Tamil from the Indian mainland. This study, in which 271 individuals, representing the Sri Lankan ethnic populations mentioned, were typed for their mitochondrial DNA (mtDNA) hypervariable segment 1 (HVS-1) and part of hypervariable segment 2 (HVS-2), provides implications for their settlement history on the island. From the phylogenetic, principal coordinate and analysis of molecular variance results, the Vedda occupied a position separated from all other ethnic people of the island, who formed relatively close affiliations among themselves, suggesting a separate origin of the former. The haplotypes and analysis of molecular variance revealed that Vedda people's mitochondrial sequences are more related to the Sinhalese and Sri Lankan Tamils' than the Indian Tamils' sequences. MtDNA haplogroup analysis revealed that several West Eurasian haplogroups as well as Indian-specific mtDNA clades were found amongst the Sri Lankan populations. Through a comparison with the mtDNA HVS-1 and part of HVS-2 of Indian database, both Tamils and Sinhalese clusters were affiliated with Indian subcontinent populations than Vedda people who are believed to be the native population of the island of Sri Lanka.

Comparison of morphological and genetic analyses reveals cryptic divergence and morphological plasticity in Stylophora (Cnidaria, Scleractinia)

NASA Astrophysics Data System (ADS)

Stefani, Fabrizio; Benzoni, F.; Yang, S.-Y.; Pichon, M.; Galli, P.; Chen, C. A.

2011-12-01

A combined morphological and genetic study of the coral genus Stylophora investigated species boundaries in the Gulf of Aden, Yemen. Two mitochondrial regions, including the hypervariable IGS9 spacer and the control region, and a fragment of rDNA were used for phylogenetic analysis. Results were compared by multivariate analysis on the basis of branch morphology and corallite morphometry. Two species were clearly discriminated by both approaches. The first species was characterised by small corallites and a low morphological variability and was ascribed to a new geographical record of Stylophora madagascarensis on the basis of its phylogenetic distinction and its morphological similarity to the type material. The second species was characterised by larger corallite size and greater morphological variability and was ascribed to Stylophora pistillata. The analysis was extended to the intrageneric level for other S. pistillata populations from the Red Sea and the Pacific Ocean. Strong internal divergence was evident in the genus Sty lophora. S. pistillata populations were split into two highly divergent Red Sea/Gulf of Aden and western Pacific lineages with significant morphological overlap, which suggests they represent two distinct cryptic species. The combined use of morphological and molecular approaches, so far proved to be a powerful tool for the re-delineation of species boundaries in corals, provided novel evidence of cryptic divergence in this group of marine metazoans.

Comparison of Species Richness Estimates Obtained Using Nearly Complete Fragments and Simulated Pyrosequencing-Generated Fragments in 16S rRNA Gene-Based Environmental Surveys▿ †

PubMed Central

Youssef, Noha; Sheik, Cody S.; Krumholz, Lee R.; Najar, Fares Z.; Roe, Bruce A.; Elshahed, Mostafa S.

2009-01-01

Pyrosequencing-based 16S rRNA gene surveys are increasingly utilized to study highly diverse bacterial communities, with special emphasis on utilizing the large number of sequences obtained (tens to hundreds of thousands) for species richness estimation. However, it is not yet clear how the number of operational taxonomic units (OTUs) and, hence, species richness estimates determined using shorter fragments at different taxonomic cutoffs correlates with the number of OTUs assigned using longer, nearly complete 16S rRNA gene fragments. We constructed a 16S rRNA clone library from an undisturbed tallgrass prairie soil (1,132 clones) and used it to compare species richness estimates obtained using eight pyrosequencing candidate fragments (99 to 361 bp in length) and the nearly full-length fragment. Fragments encompassing the V1 and V2 (V1+V2) region and the V6 region (generated using primer pairs 8F-338R and 967F-1046R) overestimated species richness; fragments encompassing the V3, V7, and V7+V8 hypervariable regions (generated using primer pairs 338F-530R, 1046F-1220R, and 1046F-1392R) underestimated species richness; and fragments encompassing the V4, V5+V6, and V6+V7 regions (generated using primer pairs 530F-805R, 805F-1046R, and 967F-1220R) provided estimates comparable to those obtained with the nearly full-length fragment. These patterns were observed regardless of the alignment method utilized or the parameter used to gauge comparative levels of species richness (number of OTUs observed, slope of scatter plots of pairwise distance values for short and nearly complete fragments, and nonparametric and parametric species richness estimates). Similar results were obtained when analyzing three other datasets derived from soil, adult Zebrafish gut, and basaltic formations in the East Pacific Rise. Regression analysis indicated that these observed discrepancies in species richness estimates within various regions could readily be explained by the proportions of hypervariable, variable, and conserved base pairs within an examined fragment. PMID:19561178

Identification of Forensic Samples via Mitochondrial DNA in the Undergraduate Biochemistry Laboratory

NASA Astrophysics Data System (ADS)

Millard, Julie T.; Pilon, André M.

2003-04-01

A recent forensic approach for identification of unknown biological samples is mitochondrial DNA (mtDNA) sequencing. We describe a laboratory exercise suitable for an undergraduate biochemistry course in which the polymerase chain reaction is used to amplify a 440 base pair hypervariable region of human mtDNA from a variety of "crime scene" samples (e.g., teeth, hair, nails, cigarettes, envelope flaps, toothbrushes, and chewing gum). Amplification is verified via agarose gel electrophoresis and then samples are subjected to cycle sequencing. Sequence alignments are made via the program CLUSTAL W, allowing students to compare samples and solve the "crime."

Some maternal lineages of domestic horses may have origins in East Asia revealed with further evidence of mitochondrial genomes and HVR-1 sequences.

PubMed

Ma, Hongying; Wu, Yajiang; Xiang, Hai; Yang, Yunzhou; Wang, Min; Zhao, Chunjiang; Wu, Changxin

2018-01-01

There are large populations of indigenous horse ( Equus caballus ) in China and some other parts of East Asia. However, their matrilineal genetic diversity and origin remained poorly understood. Using a combination of mitochondrial DNA (mtDNA) and hypervariable region (HVR-1) sequences, we aim to investigate the origin of matrilineal inheritance in these domestic horses. To investigate patterns of matrilineal inheritance in domestic horses, we conducted a phylogenetic study using 31 de novo mtDNA genomes together with 317 others from the GenBank. In terms of the updated phylogeny, a total of 5,180 horse mitochondrial HVR-1 sequences were analyzed. Eightteen haplogroups (Aw-Rw) were uncovered from the analysis of the whole mitochondrial genomes. Most of which have a divergence time before the earliest domestication of wild horses (about 5,800 years ago) and during the Upper Paleolithic (35-10 KYA). The distribution of some haplogroups shows geographic patterns. The Lw haplogroup contained a significantly higher proportion of European horses than the horses from other regions, while haplogroups Jw, Rw, and some maternal lineages of Cw, have a higher frequency in the horses from East Asia. The 5,180 sequences of horse mitochondrial HVR-1 form nine major haplogroups (A-I). We revealed a corresponding relationship between the haplotypes of HVR-1 and those of whole mitochondrial DNA sequences. The data of the HVR-1 sequences also suggests that Jw, Rw, and some haplotypes of Cw may have originated in East Asia while Lw probably formed in Europe. Our study supports the hypothesis of the multiple origins of the maternal lineage of domestic horses and some maternal lineages of domestic horses may have originated from East Asia.

Two ways of legumin-precursor processing in conifers. Characterization and evolutionary relationships of Metasequoia cDNAs representing two divergent legumin gene subfamilies.

PubMed

Häger, K P; Wind, C

1997-06-15

Subunit monomers and oligomers of crystalloid-type legumins are major components of SDS-soluble fractions from Metasequoia glyptostroboides (Dawn redwood, Taxodiaceae) seed proteins. The subunits are made up of disulfide linked alpha-polypeptides and beta-polypeptides with molecular masses of 33 kDa and 23-25 kDa, respectively. Unusually for legumins, those from Metasequoia are glycosylated and the carbohydrate moieties are residing in the C-terminal region of the respective beta-polypeptides. A Metasequoia endosperm cDNA library has been constructed and legumin-encoding transcripts representing two divergent gene subfamilies have been characterized. Intersubfamily comparisons reveal 75% identity at the amino acid level and the values range from 53-35% when the legumin precursors deduced were compared with those from angiosperms. The predicted sequences together with data from amino acid sequencing prove that post-translational processing of Metasequoia prolegumins is directed to two different processing sites, each of them specific for one of the legumin subfamilies. The sites involved differ in their relative position and in the junction to be cleaved: Metasequoia legumin precursors MgLeg18 and MgLeg26 contain the conventional post-translational Asn-Gly processing site, which is generally regarded as highly conserved. In contrast, the MgLeg4 precursor is lacking this site and post-translational cleavage is directed to an unusual Asn-Thr processing site located in its hypervariable region, causing N-terminal extension of the beta-polypeptide relative to those hitherto known. Evidence is given that the unusual variant of processing also occurs in other conifers. Phylogenetic analysis reveals the precursors concerned as representatives of a distinct legumin subfamily, originating from duplication of an ancestral gene prior to or at the beginning of Taxodiaceae diversification.

A hypervariable STR polymorphism in the CFI gene: southern origin of East Asian-specific group H alleles.

PubMed

Yuasa, Isao; Jin, Feng; Harihara, Shinji; Matsusue, Aya; Fujihara, Junko; Takeshita, Haruo; Akane, Atsushi; Umetsu, Kazuo; Saitou, Naruya; Chattopadhyay, Prasanta K

2013-09-01

Previous studies of four populations revealed that a hypervariable short tandem repeat (iSTR) in intron 7 of the human complement factor I (CFI) gene on chromosome 4q was unique, with 17 possible East Asian-specific group H alleles observed at relatively high frequencies. To develop a deeper anthropological and forensic understanding of iSTR, 1161 additional individuals from 11 Asian populations were investigated. Group H alleles of iSTR and c.1217A allele of a SNP in exon 11 of the CFI gene were associated with each other and were almost entirely confined to East Asian populations. Han Chinese in Changsha, southern China, showed the highest frequency for East Asian-specific group H alleles (0.201) among 15 populations. Group H alleles were observed to decrease gradually from south to north in 11 East Asian populations. This expansion of group H alleles provides evidence that southern China and Southeast Asia are a hotspot of Asian diversity and a genetic reservoir of Asians after they entered East Asia. The expected heterozygosity values of iSTR ranged from 0.927 in Thais to 0.874 in Oroqens, higher than those of an STR in the fibrinogen alpha chain (FGA) gene on chromosome 4q. Thus, iSTR is a useful marker for anthropological and forensic genetics. Copyright © 2013 Elsevier Ireland Ltd. All rights reserved.

Pre-Historic and Recent Vicariance Events Shape Genetic Structure and Diversity in Endangered Lion-Tailed Macaque in the Western Ghats: Implications for Conservation

PubMed Central

Ram, Muthuvarmadam S.; Marne, Minal; Gaur, Ajay; Kumara, Honnavalli N.; Singh, Mewa; Kumar, Ajith; Umapathy, Govindhaswamy

2015-01-01

Genetic isolation of populations is a potent force that helps shape the course of evolution. However, small populations in isolation, especially in fragmented landscapes, are known to lose genetic variability, suffer from inbreeding depression and become genetically differentiated among themselves. In this study, we assessed the genetic diversity of lion-tailed macaques (Macaca silenus) inhabiting the fragmented landscape of Anamalai hills and examined the genetic structure of the species across its distributional range in the Western Ghats. We sequenced around 900 bases of DNA covering two mitochondrial regions–hypervariable region-I and partial mitochondrial cytochrome b–from individuals sampled both from wild and captivity, constructed and dated phylogenetic trees. We found that the lion-tailed macaque troops in the isolated forest patches in Anamalai hills have depleted mitochondrial DNA diversity compared to troops in larger and continuous forests. Our results also revealed an ancient divergence in the lion-tailed macaque into two distinct populations across the Palghat gap, dating to 2.11 million years ago. In light of our findings, we make a few suggestions on the management of wild and captive populations. PMID:26561307

Composition of soil microbiome along elevation gradients in southwestern highlands of Saudi Arabia.

PubMed

Yasir, Muhammad; Azhar, Esam I; Khan, Imran; Bibi, Fehmida; Baabdullah, Rnda; Al-Zahrani, Ibrahim A; Al-Ghamdi, Ahmed K

2015-03-14

Saudi Arabia is mostly barren except the southwestern highlands that are susceptible to environmental changes, a hotspot for biodiversity, but poorly studied for microbial diversity and composition. In this study, 454-pyrosequencing of 16S rRNA gene hypervariable region V6 was used to analyze soil bacterial community along elevation gradients of the southwestern highlands. In general, lower percentage of total soil organic matter (SOM) and nitrogen were detected in the analyzed soil samples. Total 33 different phyla were identified across the samples, including dominant phyla Proteobacteria, Actinobacteria and Acidobacteria. Representative OTUs were grouped into 329 and 508 different taxa at family and genus level taxonomic classification, respectively. The identified OTUs unique to each sample were very low irrespective of the altitude. Jackknifed principal coordinates analysis (PCoA) revealed, overall differences in the bacterial community were more related to the quantity of specific OTUs than to their diversity among the studied samples. Bacterial diversity and soil physicochemical properties did not show consistent changes along the elevation gradients. The large number of OTUs shared between the studied samples suggest the presence of a core soil bacterial community in the southwestern highlands of Saudi Arabia.

Shape-shifting corals: Molecular markers show morphology is evolutionarily plastic in Porites

PubMed Central

Forsman, Zac H; Barshis, Daniel J; Hunter, Cynthia L; Toonen, Robert J

2009-01-01

Background Corals are notoriously difficult to identify at the species-level due to few diagnostic characters and variable skeletal morphology. This 'coral species problem' is an impediment to understanding the evolution and biodiversity of this important and threatened group of organisms. We examined the evolution of the nuclear ribosomal internal transcribed spacer (ITS) and mitochondrial markers (COI, putative control region) in Porites, one of the most taxonomically challenging and ecologically important genera of reef-building corals. Results Nuclear and mitochondrial markers were congruent, clearly resolving many traditionally recognized species; however, branching and mounding varieties were genetically indistinguishable within at least two clades, and specimens matching the description of 'Porites lutea' sorted into three genetically divergent groups. Corallite-level features were generally concordant with genetic groups, although hyper-variability in one group (Clade I) overlapped and obscured several others, and Synarea (previously thought to be a separate subgenus) was closely related to congeners despite its unique morphology. Scanning electron microscopy revealed subtle differences between genetic groups that may have been overlooked previously as taxonomic characters. Conclusion This study demonstrates that the coral skeleton can be remarkably evolutionarily plastic, which may explain some taxonomic difficulties, and obscure underlying patterns of endemism and diversity. PMID:19239678

Characterization of the gut microbiota in the red panda (Ailurus fulgens).

PubMed

Kong, Fanli; Zhao, Jiangchao; Han, Shushu; Zeng, Bo; Yang, Jiandong; Si, Xiaohui; Yang, Benqing; Yang, Mingyao; Xu, Huailiang; Li, Ying

2014-01-01

The red panda is the only living species of the genus Ailurus. Like giant pandas, red pandas are also highly specialized to feed mainly on highly fibrous bamboo. Although several studies have focused on the gut microbiota in the giant panda, little is known about the gut microbiota of the red panda. In this study, we characterized the fecal microbiota from both wild (n = 16) and captive (n = 6) red pandas using a pyrosequecing based approach targeting the V1-V3 hypervariable regions of the 16S rRNA gene. Distinct bacterial communities were observed between the two groups based on both membership and structure. Wild red pandas maintained significantly higher community diversity, richness and evenness than captive red pandas, the communities of which were skewed and dominated by taxa associated with Firmicutes. Phylogenetic analysis of the top 50 OTUs revealed that 10 of them were related to known cellulose degraders. To the best of our knowledge, this is the first study of the gut microbiota of the red panda. Our data suggest that, similar to the giant panda, the gut microbiota in the red panda might also play important roles in the digestion of bamboo.

Characterization of the Gut Microbiota in the Red Panda (Ailurus fulgens)

PubMed Central

Han, Shushu; Zeng, Bo; Yang, Jiandong; Si, Xiaohui; Yang, Benqing; Yang, Mingyao; Xu, Huailiang; Li, Ying

2014-01-01

The red panda is the only living species of the genus Ailurus. Like giant pandas, red pandas are also highly specialized to feed mainly on highly fibrous bamboo. Although several studies have focused on the gut microbiota in the giant panda, little is known about the gut microbiota of the red panda. In this study, we characterized the fecal microbiota from both wild (n = 16) and captive (n = 6) red pandas using a pyrosequecing based approach targeting the V1-V3 hypervariable regions of the 16S rRNA gene. Distinct bacterial communities were observed between the two groups based on both membership and structure. Wild red pandas maintained significantly higher community diversity, richness and evenness than captive red pandas, the communities of which were skewed and dominated by taxa associated with Firmicutes. Phylogenetic analysis of the top 50 OTUs revealed that 10 of them were related to known cellulose degraders. To the best of our knowledge, this is the first study of the gut microbiota of the red panda. Our data suggest that, similar to the giant panda, the gut microbiota in the red panda might also play important roles in the digestion of bamboo. PMID:24498390

Changes in community structure of active protistan assemblages from the lower Pearl River to coastal Waters of the South China Sea.

PubMed

Li, Ran; Jiao, Nianzhi; Warren, Alan; Xu, Dapeng

2018-04-01

Protists make up an important component of aquatic ecosystems, playing crucial roles in biogeochemical processes on local and global scales. To reveal the changes of diversity and community structure of protists along the salinity gradients, community compositions of active protistan assemblages were characterized along a transect from the lower Pearl River estuary to the open waters of the South China Sea (SCS), using high-throughput sequencing of the hyper-variable V9 regions of 18S rRNA. This study showed that the alpha diversity of protists, both in the freshwater and in the coastal SCS stations was higher than that in the estuary. The protist community structure also changed along the salinity gradient. The relative sequence abundance of Stramenopiles was highest at stations with lower salinity and decreased with the increasing of salinity. By contrast, the contributions of Alveolata, Hacrobia and Rhizaria to the protistan communities generally increased with the increasing of salinity. The composition of the active protistan community was strongly correlated with salinity, indicating that salinity was the dominant factor among measured environmental parameters affecting protistan community composition and structure. Copyright © 2018 Elsevier GmbH. All rights reserved.

The Genetic Legacy of the Pre-Colonial Period in Contemporary Bolivians

PubMed Central

Vidal-Bralo, Laura; Gómez-Carballa, Alberto; Catelli, Laura; Pardo-Seco, Jacobo; Pastoriza, Ana; Carracedo, Ángel; Torres-Balanza, Antonio; Rocabado, Omar; Vullo, Carlos

2013-01-01

Only a few genetic studies have been carried out to date in Bolivia. However, some of the most important (pre)historical enclaves of South America were located in these territories. Thus, the (sub)-Andean region of Bolivia was part of the Inca Empire, the largest state in Pre-Columbian America. We have genotyped the first hypervariable region (HVS-I) of 720 samples representing the main regions in Bolivia, and these data have been analyzed in the context of other pan-American samples (>19,000 HVS-I mtDNAs). Entire mtDNA genome sequencing was also undertaken on selected Native American lineages. Additionally, a panel of 46 Ancestry Informative Markers (AIMs) was genotyped in a sub-set of samples. The vast majority of the Bolivian mtDNAs (98.4%) were found to belong to the main Native American haplogroups (A: 14.3%, B: 52.6%, C: 21.9%, D: 9.6%), with little indication of sub-Saharan and/or European lineages; however, marked patterns of haplogroup frequencies between main regions exist (e.g. haplogroup B: Andean [71%], Sub-Andean [61%], Llanos [32%]). Analysis of entire genomes unraveled the phylogenetic characteristics of three Native haplogroups: the pan-American haplogroup B2b (originated ∼21.4 thousand years ago [kya]), A2ah (∼5.2 kya), and B2o (∼2.6 kya). The data suggest that B2b could have arisen in North California (an origin even in the north most region of the American continent cannot be disregarded), moved southward following the Pacific coastline and crossed Meso-America. Then, it most likely spread into South America following two routes: the Pacific path towards Peru and Bolivia (arriving here at about ∼15.2 kya), and the Amazonian route of Venezuela and Brazil southwards. In contrast to the mtDNA, Ancestry Informative Markers (AIMs) reveal a higher (although geographically variable) European introgression in Bolivians (25%). Bolivia shows a decreasing autosomal molecular diversity pattern along the longitudinal axis, from the Altiplano to the lowlands. Both autosomes and mtDNA revealed a low impact (1–2%) of a sub-Saharan component in Bolivians. PMID:23527064

The genetic legacy of the pre-colonial period in contemporary Bolivians.

PubMed

Taboada-Echalar, Patricia; Alvarez-Iglesias, Vanesa; Heinz, Tanja; Vidal-Bralo, Laura; Gómez-Carballa, Alberto; Catelli, Laura; Pardo-Seco, Jacobo; Pastoriza, Ana; Carracedo, Angel; Torres-Balanza, Antonio; Rocabado, Omar; Vullo, Carlos; Salas, Antonio

2013-01-01

Only a few genetic studies have been carried out to date in Bolivia. However, some of the most important (pre)historical enclaves of South America were located in these territories. Thus, the (sub)-Andean region of Bolivia was part of the Inca Empire, the largest state in Pre-Columbian America. We have genotyped the first hypervariable region (HVS-I) of 720 samples representing the main regions in Bolivia, and these data have been analyzed in the context of other pan-American samples (>19,000 HVS-I mtDNAs). Entire mtDNA genome sequencing was also undertaken on selected Native American lineages. Additionally, a panel of 46 Ancestry Informative Markers (AIMs) was genotyped in a sub-set of samples. The vast majority of the Bolivian mtDNAs (98.4%) were found to belong to the main Native American haplogroups (A: 14.3%, B: 52.6%, C: 21.9%, D: 9.6%), with little indication of sub-Saharan and/or European lineages; however, marked patterns of haplogroup frequencies between main regions exist (e.g. haplogroup B: Andean [71%], Sub-Andean [61%], Llanos [32%]). Analysis of entire genomes unraveled the phylogenetic characteristics of three Native haplogroups: the pan-American haplogroup B2b (originated ∼21.4 thousand years ago [kya]), A2ah (∼5.2 kya), and B2o (∼2.6 kya). The data suggest that B2b could have arisen in North California (an origin even in the north most region of the American continent cannot be disregarded), moved southward following the Pacific coastline and crossed Meso-America. Then, it most likely spread into South America following two routes: the Pacific path towards Peru and Bolivia (arriving here at about ∼15.2 kya), and the Amazonian route of Venezuela and Brazil southwards. In contrast to the mtDNA, Ancestry Informative Markers (AIMs) reveal a higher (although geographically variable) European introgression in Bolivians (25%). Bolivia shows a decreasing autosomal molecular diversity pattern along the longitudinal axis, from the Altiplano to the lowlands. Both autosomes and mtDNA revealed a low impact (1-2%) of a sub-Saharan component in Bolivians.

Sequence analysis of the canine mitochondrial DNA control region from shed hair samples in criminal investigations.

PubMed

Berger, C; Berger, B; Parson, W

2012-01-01

In recent years, evidence from domestic dogs has increasingly been analyzed by forensic DNA testing. Especially, canine hairs have proved most suitable and practical due to the high rate of hair transfer occurring between dogs and humans. Starting with the description of a contamination-free sample handling procedure, we give a detailed workflow for sequencing hypervariable segments (HVS) of the mtDNA control region from canine evidence. After the hair material is lysed and the DNA extracted by Phenol/Chloroform, the amplification and sequencing strategy comprises the HVS I and II of the canine control region and is optimized for DNA of medium-to-low quality and quantity. The sequencing procedure is based on the Sanger Big-dye deoxy-terminator method and the separation of the sequencing reaction products is performed on a conventional multicolor fluorescence detection capillary electrophoresis platform. Finally, software-aided base calling and sequence interpretation are addressed exemplarily.

The assembly of the plant urease activation complex and the essential role of the urease accessory protein G (UreG) in delivery of nickel to urease.

PubMed

Myrach, Till; Zhu, Anting; Witte, Claus-Peter

2017-09-01

Urease is a ubiquitous nickel metalloenzyme. In plants, its activation requires three urease accessory proteins (UAPs), UreD, UreF, and UreG. In bacteria, the UAPs interact with urease and facilitate activation, which involves the channeling of two nickel ions into the active site. So far this process has not been investigated in eukaryotes. Using affinity pulldowns of Strep-tagged UAPs from Arabidopsis and rice transiently expressed in planta , we demonstrate that a urease-UreD-UreF-UreG complex exists in plants and show its stepwise assembly. UreG is crucial for nickel delivery because UreG-dependent urease activation in vitro was observed only with UreG obtained from nickel-sufficient plants. This activation competence could not be generated in vitro by incubation of UreG with nickel, bicarbonate, and GTP. Compared with their bacterial orthologs, plant UreGs possess an N-terminal extension containing a His- and Asp/Glu-rich hypervariable region followed by a highly conserved sequence comprising two potential H X H metal-binding sites. Complementing the ureG-1 mutant of Arabidopsis with N-terminal deletion variants of UreG demonstrated that the hypervariable region has a minor impact on activation efficiency, whereas the conserved region up to the first H X H motif is highly beneficial and up to the second H X H motif strictly required for activation. We also show that urease reaches its full activity several days after nickel becomes available in the leaves, indicating that urease activation is limited by nickel accessibility in vivo Our data uncover the crucial role of UreG for nickel delivery during eukaryotic urease activation, inciting further investigations of the details of this process. © 2017 by The American Society for Biochemistry and Molecular Biology, Inc.

The hypervariable region 1 protein of hepatitis C virus broadly reactive with sera of patients with chronic hepatitis C has a similar amino acid sequence with the consensus sequence.

PubMed

Watanabe, K; Yoshioka, K; Ito, H; Ishigami, M; Takagi, K; Utsunomiya, S; Kobayashi, M; Kishimoto, H; Yano, M; Kakumu, S

1999-11-10

Hypervariable region 1 (HVR1) proteins of hepatitis C virus (HCV) have been reported to react broadly with sera of patients with HCV infection. However, the variability of the broad reactivity of individual HVR1 proteins has not been elucidated. We assessed the reactivity of 25 different HVR1 proteins (genotype 1b) with sera of 81 patients with HCV infection (genotype 1b) by Western blot. HVR1 proteins reacted with 2-60 sera. The number of sera reactive with each HVR1 protein significantly correlated with the number of amino acid residues identical to the consensus sequence defined by Puntoriero et al. (G. Puntoriero, A. Lahm, S. Zucchelli, B. B. Ercole, R. Tafi, M. Penzzanera, M. U. Mondelli, R. Cortese, A. Tramontano, G. Galfre', and A. Nicosia. 1998. EMBO J. 17, 3521-3533. ) (r = 0.561, P < 0.005). The most widely reactive HVR1 protein, 12-22, had a sequence similar to the consensus sequence. The peptide with C-terminal 13-amino-acids sequence of HVR1 protein 12-22 (NH2-CSFTSLFTPGPSQK) was injected into rabbits as an immunogen. The rabbit immune sera reacted with 9 of 25 HVR1 proteins of genotype 1b including HVR1 protein 12-22 and with 3 of 12 proteins of genotype 2a. These results indicate that the HVR1 protein broadly reactive with patients' sera has a sequence similar to the consensus sequence, can induce broadly reactive sera, and could be one of the candidate immunogens in a prophylactic vaccine against HCV. Copyright 1999 Academic Press.

[Correlation between polymorphisms in the coagulation factor VII gene hypervariable region 4 site and the risk of coronary heart disease in population with different ethnic backgrounds: a Meta-analysis].

PubMed

Wang, Li-li; Ma, Bin; Qian, Dun; Pang, Jun; Yao, Ya-li

2013-12-01

To assess the correlation between polymorphisms in the coagulation factor VII (F VII)gene hypervariable region 4 (HVR4)site and risk related to coronary heart disease (CHD)in different ethnic populations, especially the Asian populations. Publications up to April 2013, from CBM, CNKI, Wanfang Database,VIP, PubMed, Cochrane Library and Embase were searched to collect data from case-control studies related to F VII gene HVR4 site and CHD in populations from different ethnicities. Quality of studies was evaluated, available data extracted and both RevMan 5.1 and Stata 11.0 softwares were used for Meta-analysis. Fifteen case-control studies were included, involving 3167 cases with CHD group and 3168 cases in the control group. on this Meta-analysis showed that:a)polymorphism of the F VII gene HVR4 site H7/H6+H5 and CHD, b)H7H7/H6H6 + H7H6 and CHD were both slightly correlated between people with different ethnic backgrounds. However, the H6 allele versus H7+H5 allele and CHD showed different results-a high correlation seen in different ethnic groups. H5 allele versus H6+H7 allele and CHD did not appear significant difference(OR = 1.20, 95%CI:0.76-1.90, P = 0.43). Both F VII gene HVR4 polymorphisms H7 allele and the H7H7 genotype might have served as protective factors for CHD in different ethnic groups, H6 allele might serve as a risk factor for CHD, but H5 allele was likely not to be associated with CHD in different ethnic groups.

Bacterial communities in soil samples from the Mingyong Glacier of southwestern China.

PubMed

Li, Haoyu; Taj, Muhammad Kamran; Ji, Xiuling; Zhang, Qi; Lin, Liangbing; Zhou, Zhimei; Wei, Yunlin

2017-05-01

The present study was an effort to determine the bacterial diversity of soils in Mingyong Glacier located at the Meili Snow Mountains of southwestern China. Mingyong Glacier has different climatic zones within a very narrow area, and bacterial community diversity in this low temperature area remains largely unknown. In this study, soil samples were collected from four different climatic zones: M11A (dry warm valley), M14 (forest), M15 (grass land), and M16 (glacier zones). Phylogenetic analysis based on 16S rRNA gene V6 hypervariable region showed high bacterial abundance in the glacier. The number of Operational Taxonomic Units ranged from 2.24×10 3 to 5.56×10 3 in soil samples. Statistical analysis of 16S rRNA gene clone libraries results showed that bacterial diversity in zones M11A，M14 and M16 are higher than in zone M15. The bacterial community structures are clearly distinguishable, and phylogenetic analysis showed that the predominant phyla were Proteobacteria, Deinococcus-Thermus, Firmicutes, Actinobacteria, and Nitrospirae in Mingyong Glacier. Seventy-nine different orders from four zones have been isolated. Bacterial diversity and distribution of bacterial communities related to the anthropogenic perturbations in zone (M15) were confirmed by diversity index analysis, and the diversity index of other three zones was satisfactory through this analysis software. The results suggest that bacterial diversity and distribution analyses using bacterial 16S rRNA gene V6 hypervariable region were successful, and bacterial communities in this area not only had the same bacterial phyla compared to other glaciers but also had their own rare species.

Pyrosequencing as a tool for the identification of common isolates of Mycobacterium sp.

PubMed

Tuohy, Marion J; Hall, Gerri S; Sholtis, Mary; Procop, Gary W

2005-04-01

Pyrosequencing technology, sequencing by addition, was evaluated for categorization of mycobacterial isolates. One hundred and eighty-nine isolates, including 18 ATCC and Trudeau Mycobacterial Culture Collection (TMC) strains, were studied. There were 38 Mycobacterium tuberculosis complex, 27 M. kansasii, 27 MAI complex, 21 M. marinum, 14 M. gordonae, 20 M. chelonae-abscessus group, 10 M. fortuitum, 5 M. xenopi, 3 M. celatum, 2 M. terrae complex, 20 M. mucogenicum, and 2 M. scrofulaceum. Nucleic acid extracts were prepared from solid media or MGIT broth. Traditional PCR was performed with one of the primers biotinylated; the assay targeted a portion of the 16S rRNA gene that contains a hypervariable region, which has been previously shown to be useful for the identification of mycobacteria. The PSQ Sample Preparation Kit was used, and the biotinylated PCR product was processed to a single-stranded DNA template. The sequencing primer was hybridized to the DNA template in a PSQ96 plate. Incorporation of the complementary nucleotides resulted in light generation peaks, forming a pyrogram, which was evaluated by the instrument software. Thirty basepairs were used for isolate categorization. Manual interpretation of the sequences was performed if the quality of the 30-bp sequence was in doubt or if more than 4 bp homopolymers were recognized. Sequences with more than 5 bp of bad quality were deemed unacceptable. When blasted against GenBank, 179 of 189 sequences (94.7%) assigned isolates to the correct molecular genus or group. Ten M. gordonae isolates had more than 5 bp of bad quality sequence and were not accepted. Pyrosequencing of this hypervariable region afforded rapid and acceptable characterization of common, routinely isolated clinical Mycobacterium sp. Algorithms are recommended for further differentiation with an additional sequencing primer or additional biochemicals.

Use of a Molecular Decoy to Segregate Transport from Antigenicity in the FrpB Iron Transporter from Neisseria meningitidis

PubMed Central

Saleem, Muhammad; Prince, Stephen M.; Rigby, Stephen E. J.; Imran, Muhammad; Patel, Hema; Chan, Hannah; Sanders, Holly; Maiden, Martin C. J.; Feavers, Ian M.; Derrick, Jeremy P.

2013-01-01

FrpB is an outer membrane transporter from Neisseria meningitidis, the causative agent of meningococcal meningitis. It is a member of the TonB-dependent transporter (TBDT) family and is responsible for iron uptake into the periplasm. FrpB is subject to a high degree of antigenic variation, principally through a region of hypervariable sequence exposed at the cell surface. From the crystal structures of two FrpB antigenic variants, we identify a bound ferric ion within the structure which induces structural changes on binding which are consistent with it being the transported substrate. Binding experiments, followed by elemental analysis, verified that FrpB binds Fe3+ with high affinity. EPR spectra of the bound Fe3+ ion confirmed that its chemical environment was consistent with that observed in the crystal structure. Fe3+ binding was reduced or abolished on mutation of the Fe3+-chelating residues. FrpB orthologs were identified in other Gram-negative bacteria which showed absolute conservation of the coordinating residues, suggesting the existence of a specific TBDT sub-family dedicated to the transport of Fe3+. The region of antigenic hypervariability lies in a separate, external sub-domain, whose structure is conserved in both the F3-3 and F5-1 variants, despite their sequence divergence. We conclude that the antigenic sub-domain has arisen separately as a result of immune selection pressure to distract the immune response from the primary transport function. This would enable FrpB to function as a transporter independently of antibody binding, by using the antigenic sub-domain as a ‘molecular decoy’ to distract immune surveillance. PMID:23457610

Use of a molecular decoy to segregate transport from antigenicity in the FrpB iron transporter from Neisseria meningitidis.

PubMed

Saleem, Muhammad; Prince, Stephen M; Rigby, Stephen E J; Imran, Muhammad; Patel, Hema; Chan, Hannah; Sanders, Holly; Maiden, Martin C J; Feavers, Ian M; Derrick, Jeremy P

2013-01-01

FrpB is an outer membrane transporter from Neisseria meningitidis, the causative agent of meningococcal meningitis. It is a member of the TonB-dependent transporter (TBDT) family and is responsible for iron uptake into the periplasm. FrpB is subject to a high degree of antigenic variation, principally through a region of hypervariable sequence exposed at the cell surface. From the crystal structures of two FrpB antigenic variants, we identify a bound ferric ion within the structure which induces structural changes on binding which are consistent with it being the transported substrate. Binding experiments, followed by elemental analysis, verified that FrpB binds Fe(3+) with high affinity. EPR spectra of the bound Fe(3+) ion confirmed that its chemical environment was consistent with that observed in the crystal structure. Fe(3+) binding was reduced or abolished on mutation of the Fe(3+)-chelating residues. FrpB orthologs were identified in other Gram-negative bacteria which showed absolute conservation of the coordinating residues, suggesting the existence of a specific TBDT sub-family dedicated to the transport of Fe(3+). The region of antigenic hypervariability lies in a separate, external sub-domain, whose structure is conserved in both the F3-3 and F5-1 variants, despite their sequence divergence. We conclude that the antigenic sub-domain has arisen separately as a result of immune selection pressure to distract the immune response from the primary transport function. This would enable FrpB to function as a transporter independently of antibody binding, by using the antigenic sub-domain as a 'molecular decoy' to distract immune surveillance.

Prophage-Mediated Dynamics of ‘Candidatus Liberibacter asiaticus’ Populations, the Destructive Bacterial Pathogens of Citrus Huanglongbing

PubMed Central

Zhou, Lijuan; Powell, Charles A.; Li, Wenbin; Irey, Mike; Duan, Yongping

2013-01-01

Prophages are highly dynamic components in the bacterial genome and play an important role in intraspecies variations. There are at least two prophages in the chromosomes of Candidatus Liberibacter asiaticus’ (Las) Floridian isolates. Las is both unculturable and the most prevalent species of Liberibacter pathogens that cause huanglongbing (HLB), a worldwide destructive disease of citrus. In this study, seven new prophage variants resulting from two hyper-variable regions were identified by screening clone libraries of infected citrus, periwinkle and psyllids. Among them, Types A and B share highly conserved sequences and localize within the two prophages, FP1 and FP2, respectively. Although Types B and C were abundant in all three libraries, Type A was much more abundant in the libraries from the Las-infected psyllids than from the Las-infected plants, and Type D was only identified in libraries from the infected host plants but not from the infected psyllids. Sequence analysis of these variants revealed that the variations may result from recombination and rearrangement events. Conventional PCR results using type-specific molecular markers indicated that A, B, C and D are the four most abundant types in Las-infected citrus and periwinkle. However, only three types, A, B and C are abundant in Las-infected psyllids. Typing results for Las-infected citrus field samples indicated that mixed populations of Las bacteria present in Floridian isolates, but only the Type D population was correlated with the blotchy mottle symptom. Extended cloning and sequencing of the Type D region revealed a third prophage/phage in the Las genome, which may derive from the recombination of FP1 and FP2. Dramatic variations in these prophage regions were also found among the global Las isolates. These results are the first to demonstrate the prophage/phage-mediated dynamics of Las populations in plant and insect hosts, and their correlation with insect transmission and disease development. PMID:24349235

Genetic origin of goat populations in Oman revealed by mitochondrial DNA analysis

PubMed Central

Gaafar, Osman Mahgoub; Costa, Vânia; Neira, Agusto Luzuriaga; Al-Atiyat, Raed Mahmoud; Beja-Pereira, Albano

2017-01-01

The Sultanate of Oman has a complex mosaic of livestock species and production systems, but the genetic diversity, demographic history or origins of these Omani animals has not been expensively studied. Goats might constitute one of the most abundant and important domestic livestock species since the Neolithic transition. Here, we examined the genetic diversity, origin, population structure and demographic history of Omani goats. Specifically, we analyzed a 525-bp fragment of the first hypervariable region of the mitochondrial DNA (mtDNA) control region from 69 Omani individuals and compared this fragment with 17 mtDNA sequences from Somalia and Yemen as well as 18 wild goat species and 1,198 previously published goat sequences from neighboring countries. The studied goat breeds show substantial diversity. The haplotype and nucleotide diversities of Omani goats were found equal to 0.983 ± 0.006 and 0.0284 ± 0.014, respectively. The phylogenetic analyses allowed us to classify Omani goats into three mtDNA haplogroups (A, B and G): haplogroup A was found to be predominant and widely distributed and accounted for 80% of all samples, and haplogroups B and G exhibited low frequencies. Phylogenetic comparisons with wild goats revealed that five of the native Omani goat populations originate from Capra aegagrus. Furthermore, most comparisons of pairwise population FST values within and between these five Omani goat breeds as well as between Omani goats and nine populations from nearby countries were not significant. These results suggest strong gene flow among goat populations caused by the extensive transport of goats and the frequent movements of human populations in ancient Arabia. The findings improve our understanding of the migration routes of modern goats from their region of domestication into southeastern Arabia and thereby shed light on human migratory and commercial networks during historical times. PMID:29281717

Prophage-mediated dynamics of 'Candidatus Liberibacter asiaticus' populations, the destructive bacterial pathogens of citrus huanglongbing.

PubMed

Zhou, Lijuan; Powell, Charles A; Li, Wenbin; Irey, Mike; Duan, Yongping

2013-01-01

Prophages are highly dynamic components in the bacterial genome and play an important role in intraspecies variations. There are at least two prophages in the chromosomes of Candidatus Liberibacter asiaticus' (Las) Floridian isolates. Las is both unculturable and the most prevalent species of Liberibacter pathogens that cause huanglongbing (HLB), a worldwide destructive disease of citrus. In this study, seven new prophage variants resulting from two hyper-variable regions were identified by screening clone libraries of infected citrus, periwinkle and psyllids. Among them, Types A and B share highly conserved sequences and localize within the two prophages, FP1 and FP2, respectively. Although Types B and C were abundant in all three libraries, Type A was much more abundant in the libraries from the Las-infected psyllids than from the Las-infected plants, and Type D was only identified in libraries from the infected host plants but not from the infected psyllids. Sequence analysis of these variants revealed that the variations may result from recombination and rearrangement events. Conventional PCR results using type-specific molecular markers indicated that A, B, C and D are the four most abundant types in Las-infected citrus and periwinkle. However, only three types, A, B and C are abundant in Las-infected psyllids. Typing results for Las-infected citrus field samples indicated that mixed populations of Las bacteria present in Floridian isolates, but only the Type D population was correlated with the blotchy mottle symptom. Extended cloning and sequencing of the Type D region revealed a third prophage/phage in the Las genome, which may derive from the recombination of FP1 and FP2. Dramatic variations in these prophage regions were also found among the global Las isolates. These results are the first to demonstrate the prophage/phage-mediated dynamics of Las populations in plant and insect hosts, and their correlation with insect transmission and disease development.

Evolution of the complementary sex-determination gene of honey bees: balancing selection and trans-species polymorphisms.

PubMed

Cho, Soochin; Huang, Zachary Y; Green, Daniel R; Smith, Deborah R; Zhang, Jianzhi

2006-11-01

The mechanism of sex determination varies substantively among evolutionary lineages. One important mode of genetic sex determination is haplodiploidy, which is used by approximately 20% of all animal species, including >200,000 species of the entire insect order Hymenoptera. In the honey bee Apis mellifera, a hymenopteran model organism, females are heterozygous at the csd (complementary sex determination) locus, whereas males are hemizygous (from unfertilized eggs). Fertilized homozygotes develop into sterile males that are eaten before maturity. Because homozygotes have zero fitness and because common alleles are more likely than rare ones to form homozygotes, csd should be subject to strong overdominant selection and negative frequency-dependent selection. Under these selective forces, together known as balancing selection, csd is expected to exhibit a high degree of intraspecific polymorphism, with long-lived alleles that may be even older than the species. Here we sequence the csd genes as well as randomly selected neutral genomic regions from individuals of three closely related species, A. mellifera, Apis cerana, and Apis dorsata. The polymorphic level is approximately seven times higher in csd than in the neutral regions. Gene genealogies reveal trans-species polymorphisms at csd but not at any neutral regions. Consistent with the prediction of rare-allele advantage, nonsynonymous mutations are found to be positively selected in csd only in early stages after their appearances. Surprisingly, three different hypervariable repetitive regions in csd are present in the three species, suggesting variable mechanisms underlying allelic specificities. Our results provide a definitive demonstration of balancing selection acting at the honey bee csd gene, offer insights into the molecular determinants of csd allelic specificities, and help avoid homozygosity in bee breeding.

Sequence variation in the env gene of simian immunodeficiency virus recovered from immunized macaques is predominantly in the V1 region.

PubMed

Almond, N; Jenkins, A; Heath, A B; Kitchin, P

1993-05-01

Three cynomolgus macaques were immunized with recombinant envelope protein preparations derived from simian immunodeficiency virus (SIV). Although humoral and cellular responses were elicited by the immunization regime, all macaques became infected upon challenge with 10 MID50 of the 11/88 virus challenge stock of SIVmac251-32H. The polymerase chain reaction was used to amplify proviral SIV gp120 sequences present in the blood of both immunized and control macaques at 2 months post-infection. A comparison of the predominant sequences found in the region from V2 to V5 of gp120 failed to differentiate provirus recovered from either immunized or control animals. A detailed investigation of sequences obtained from the hypervariable V1 region identified a mixture of sequences in both immunized and control macaques. Some sequences were identical to those previously detected in the virus challenge stock, whereas others had not been detected previously. Phenogram analysis of the new V1 sequences found in immunized animals revealed that they were quite distinct from those from the virus challenge stock and that they included alterations to potential N-linked glycosylation sites. In contrast, new sequence variants recovered from the control animals were closely related to sequences from the virus challenge stock. The difference in diversity of new V1 sequences recovered from immunized and control macaques was highly significant (P < 0.001). Thus, the presence of pre-existing immune responses to SIV envelope protein is associated with greater genetic change in the V1 region of gp120. These data are discussed in relation to the epitopes of SIV gp120 that may confer protection from in vivo challenge.

Recognition and stabilization of geranylgeranylated human Rab5 by the GDP Dissociation Inhibitor (GDI).

PubMed

Edler, Eileen; Stein, Matthias

2017-10-25

The small GTPase Rab5 is the key regulator of early endosomal fusion. It is post-translationally modified by covalent attachment of two geranylgeranyl (GG) chains to adjacent cysteine residues of the C-terminal hypervariable region (HVR). The GDP dissociation inhibitor (GDI) recognizes membrane-associated Rab5(GDP) and serves to release it into the cytoplasm where it is kept in a soluble state. A detailed new structural and dynamic model for human Rab5(GDP) recognition and binding with human GDI at the early endosome membrane and in its dissociated state is presented. In the cytoplasm, the GDI protein accommodates the GG chains in a transient hydrophobic binding pocket. In solution, two different binding modes of the isoprenoid chains inserted into the hydrophobic pocket of the Rab5(GDP):GDI complex can be identified. This equilibrium between the two states helps to stabilize the protein-protein complex in solution. Interprotein contacts between the Rab5 switch regions and characteristic patches of GDI residues from the Rab binding platform (RBP) and the C-terminus coordinating region (CCR) reveal insight on the formation of such a stable complex. GDI binding to membrane-anchored Rab5(GDP) is initially mediated by the solvent accessible switch regions of the Rab-specific RBP. Formation of the membrane-associated Rab5(GDP):GDI complex induces a GDI reorientation to establish additional interactions with the Rab5 HVR. These results allow to devise a detailed structural model for the process of extraction of GG-Rab5(GDP) by GDI from the membrane and the dissociation from targeting factors and effector proteins prior to GDI binding.

Forensic strategy to ensure the quality of sequencing data of mitochondrial DNA in highly degraded samples.

PubMed

Adachi, Noboru; Umetsu, Kazuo; Shojo, Hideki

2014-01-01

Mitochondrial DNA (mtDNA) is widely used for DNA analysis of highly degraded samples because of its polymorphic nature and high number of copies in a cell. However, as endogenous mtDNA in deteriorated samples is scarce and highly fragmented, it is not easy to obtain reliable data. In the current study, we report the risks of direct sequencing mtDNA in highly degraded material, and suggest a strategy to ensure the quality of sequencing data. It was observed that direct sequencing data of the hypervariable segment (HVS) 1 by using primer sets that generate an amplicon of 407 bp (long-primer sets) was different from results obtained by using newly designed primer sets that produce an amplicon of 120-139 bp (mini-primer sets). The data aligned with the results of mini-primer sets analysis in an amplicon length-dependent manner; the shorter the amplicon, the more evident the endogenous sequence became. Coding region analysis using multiplex amplified product-length polymorphisms revealed the incongruence of single nucleotide polymorphisms between the coding region and HVS 1 caused by contamination with exogenous mtDNA. Although the sequencing data obtained using long-primer sets turned out to be erroneous, it was unambiguous and reproducible. These findings suggest that PCR primers that produce amplicons shorter than those currently recognized should be used for mtDNA analysis in highly degraded samples. Haplogroup motif analysis of the coding region and HVS should also be performed to improve the reliability of forensic mtDNA data. Copyright © 2013 Elsevier Ireland Ltd. All rights reserved.

Regional spread of HIV-1 M subtype B in middle-aged patients by random env-C2V4 region sequencing

PubMed Central

Stürmer, Martin; Zimmermann, Katrin; Fritzsche, Carlos; Reisinger, Emil; Doelken, Gottfried; Berger, Annemarie; Doerr, Hans W.; Eberle, Josef

2010-01-01

A transmission cluster of HIV-1 M:B was identified in 11 patients with a median age of 52 (range 26–65) in North-East Germany by C2V4 region sequencing of the env gene of HIV-1, who—except of one—were not aware of any risky behaviour. The 10 male and 1 female patients deteriorated immunologically, according to their information made available, within 4 years after a putative HIV acquisition. Nucleic acid sequence analysis showed a R5 virus in all patients and in 7 of 11 a crown motif of the V3 loop, GPGSALFTT, which is found rarely. Analysis of formation of this cluster showed that there is still a huge discrepancy between awareness and behaviour regarding HIV transmission in middle-aged patients, and that a local outbreak can be detected by nucleic acid analysis of the hypervariable env region. PMID:20217125

'Mitominis': multiplex PCR analysis of reduced size amplicons for compound sequence analysis of the entire mtDNA control region in highly degraded samples.

PubMed

Eichmann, Cordula; Parson, Walther

2008-09-01

The traditional protocol for forensic mitochondrial DNA (mtDNA) analyses involves the amplification and sequencing of the two hypervariable segments HVS-I and HVS-II of the mtDNA control region. The primers usually span fragment sizes of 300-400 bp each region, which may result in weak or failed amplification in highly degraded samples. Here we introduce an improved and more stable approach using shortened amplicons in the fragment range between 144 and 237 bp. Ten such amplicons were required to produce overlapping fragments that cover the entire human mtDNA control region. These were co-amplified in two multiplex polymerase chain reactions and sequenced with the individual amplification primers. The primers were carefully selected to minimize binding on homoplasic and haplogroup-specific sites that would otherwise result in loss of amplification due to mis-priming. The multiplexes have successfully been applied to ancient and forensic samples such as bones and teeth that showed a high degree of degradation.

The evolution of Dscam genes across the arthropods.

PubMed

Armitage, Sophie A O; Freiburg, Rebecca Y; Kurtz, Joachim; Bravo, Ignacio G

2012-04-13

One way of creating phenotypic diversity is through alternative splicing of precursor mRNAs. A gene that has evolved a hypervariable form is Down syndrome cell adhesion molecule (Dscam-hv), which in Drosophila melanogaster can produce thousands of isoforms via mutually exclusive alternative splicing. The extracellular region of this protein is encoded by three variable exon clusters, each containing multiple exon variants. The protein is vital for neuronal wiring where the extreme variability at the somatic level is required for axonal guidance, and it plays a role in immunity where the variability has been hypothesised to relate to recognition of different antigens. Dscam-hv has been found across the Pancrustacea. Additionally, three paralogous non-hypervariable Dscam-like genes have also been described for D. melanogaster. Here we took a bioinformatics approach, building profile Hidden Markov Models to search across species for putative orthologs to the Dscam genes and for hypervariable alternatively spliced exons, and inferring the phylogenetic relationships among them. Our aims were to examine whether Dscam orthologs exist outside the Bilateria, whether the origin of Dscam-hv could lie outside the Pancrustacea, when the Dscam-like orthologs arose, how many alternatively spliced exons of each exon cluster were present in the most common recent ancestor, and how these clusters evolved. Our results suggest that the origin of Dscam genes may lie after the split between the Cnidaria and the Bilateria and supports the hypothesis that Dscam-hv originated in the common ancestor of the Pancrustacea. Our phylogeny of Dscam gene family members shows six well-supported clades: five containing Dscam-like genes and one containing all the Dscam-hv genes, a seventh clade contains arachnid putative Dscam genes. Furthermore, the exon clusters appear to have experienced different evolutionary histories. Dscam genes have undergone independent duplication events in the insects and in an arachnid genome, which adds to the more well-known tandem duplications that have taken place within Dscam-hv genes. Therefore, two forms of gene expansion seem to be active within this gene family. The evolutionary history of this dynamic gene family will be further unfolded as genomes of species from more disparate groups become available.

The evolution of Dscam genes across the arthropods

PubMed Central

2012-01-01

Background One way of creating phenotypic diversity is through alternative splicing of precursor mRNAs. A gene that has evolved a hypervariable form is Down syndrome cell adhesion molecule (Dscam-hv), which in Drosophila melanogaster can produce thousands of isoforms via mutually exclusive alternative splicing. The extracellular region of this protein is encoded by three variable exon clusters, each containing multiple exon variants. The protein is vital for neuronal wiring where the extreme variability at the somatic level is required for axonal guidance, and it plays a role in immunity where the variability has been hypothesised to relate to recognition of different antigens. Dscam-hv has been found across the Pancrustacea. Additionally, three paralogous non-hypervariable Dscam-like genes have also been described for D. melanogaster. Here we took a bioinformatics approach, building profile Hidden Markov Models to search across species for putative orthologs to the Dscam genes and for hypervariable alternatively spliced exons, and inferring the phylogenetic relationships among them. Our aims were to examine whether Dscam orthologs exist outside the Bilateria, whether the origin of Dscam-hv could lie outside the Pancrustacea, when the Dscam-like orthologs arose, how many alternatively spliced exons of each exon cluster were present in the most common recent ancestor, and how these clusters evolved. Results Our results suggest that the origin of Dscam genes may lie after the split between the Cnidaria and the Bilateria and supports the hypothesis that Dscam-hv originated in the common ancestor of the Pancrustacea. Our phylogeny of Dscam gene family members shows six well-supported clades: five containing Dscam-like genes and one containing all the Dscam-hv genes, a seventh clade contains arachnid putative Dscam genes. Furthermore, the exon clusters appear to have experienced different evolutionary histories. Conclusions Dscam genes have undergone independent duplication events in the insects and in an arachnid genome, which adds to the more well-known tandem duplications that have taken place within Dscam-hv genes. Therefore, two forms of gene expansion seem to be active within this gene family. The evolutionary history of this dynamic gene family will be further unfolded as genomes of species from more disparate groups become available. PMID:22500922

Genetic mosaic in a marine species flock.

PubMed

McCartney, Michael A; Acevedo, Jenny; Heredia, Christine; Rico, Ciro; Quenoville, Brice; Bermingham, Eldredge; McMillan, W Owen

2003-11-01

We used molecular approaches to study the status of speciation in coral reef fishes known as hamlets (Serranidae: Hypoplectrus). Several hamlet morphospecies coexist on Caribbean reefs, and mate assortatively with respect to their strikingly distinct colour patterns. We provide evidence that, genetically, the hamlets display characteristics common in species flocks on land and in freshwaters. Substitutions within two mitochondrial DNA (mtDNA) protein-coding genes place hamlets within a monophyletic group relative to members of two related genera (Serranus and Diplectrum), and establish that the hamlet radiation must have been very recent. mtDNA distances separating hamlet morphospecies were slight (0.6 +/- 0.04%), yielding a coalescent estimate for the age of the hamlet flock of approximately 430 000 years. Morphospecies did not sort into distinct mtDNA haplotype phylogroups, and alleles at five hypervariable microsatellite loci were shared broadly across species boundaries. None the less, molecular variation was not distributed at random. Analyses of mtDNA haplotype frequencies and nested clades in haplotype networks revealed significant genetic differences between geographical regions and among colour morphospecies. We also observed significant microsatellite differentiation between geographical regions and in Puerto Rico, among colour morphospecies; the latter providing evidence for reproductive isolation between colour morphospecies at this locale. In our Panama collection, however, colour morphospecies were mostly genetically indistinguishable. This mosaic pattern of DNA differentiation implies a complex interaction between population history, mating behaviour and geography and suggests that porous boundaries separate species in this flock of brilliantly coloured coral reef fishes.

Microbial communities associated with ferromanganese nodules and the surrounding sediments

PubMed Central

Tully, Benjamin J.; Heidelberg, John F.

2013-01-01

The formation and maintenance of deep-sea ferromanganese/polymetallic nodules still remains a mystery 140 years after their discovery. The wealth of rare metals concentrated in these nodules has spurred global interest in exploring the mining potential of these resources. The prevailing theory of abiotic formation has been called into question and the role of microbial metabolisms in nodule development is now an area of active research. To understand the community structure of microbes associated with nodules and their surrounding sediment, we performed targeted sequencing of the V4 hypervariable region of the 16S rRNA gene from three nodules collected from the central South Pacific. Results have shown that the microbial communities of the nodules are significantly distinct from the communities in the surrounding sediments, and that the interiors of the nodules harbor communities different from the exterior. This suggests not only differences in potential metabolisms between the nodule and sediment communities, but also differences in the dominant metabolisms of interior and exterior communities. We identified several operational taxonomic units (OTUs) unique to both the nodule and sediment environments. The identified OTUs were assigned putative taxonomic identifications, including two OTUs only found associated with the nodules, which were assigned to the α-Proteobacteria. Finally, we explored the diversity of the most assigned taxonomic group, the Thaumarchaea MG-1, which revealed novel OTUs compared to previous research from the region and suggests a potential role as a source of fixed carbon for ammonia oxidizing archaea in the environment. PMID:23805131

Subspecies composition and founder contribution of the captive U.S. chimpanzee (Pan troglodytes) population.

PubMed

Ely, John J; Dye, Brent; Frels, William I; Fritz, Jo; Gagneux, Pascal; Khun, Henry H; Switzer, William M; Lee, D Rick

2005-10-01

Chimpanzees are presently classified into three subspecies: Pan troglodytes verus from west Africa, P.t. troglodytes from central Africa, and P.t. schweinfurthii from east Africa. A fourth subspecies (P.t. vellerosus), from Cameroon and northern Nigeria, has been proposed. These taxonomic designations are based on geographical origins and are reflected in sequence variation in the first hypervariable region (HVR-I) of the mtDNA D-loop. Although advances have been made in our understanding of chimpanzee phylogenetics, little has been known regarding the subspecies composition of captive chimpanzees. We sequenced part of the mtDNA HVR-I region in 218 African-born population founders and performed a phylogenetic analysis with previously characterized African sequences of known provenance to infer subspecies affiliations. Most founders were P.t. verus (95.0%), distantly followed by the troglodytes schweinfurthii clade (4.6%), and a single P.t. vellerosus (0.4%). Pedigree-based estimates of genomic representation in the descendant population revealed that troglodytes schweinfurthii founder representation was reduced in captivity, vellerosus representation increased due to prolific breeding by a single male, and reproductive variance resulted in uneven representation among male P.t.verus founders. No increase in mortality was evident from between-subspecies interbreeding, indicating a lack of outbreeding depression. Knowledge of subspecies and their genomic representation can form the basis for phylogenetically informed genetic management of extant chimpanzees to preserve rare genetic variation for research, conservation, or possible future breeding. Copyright 2005 Wiley-Liss, Inc.

Comparison of base composition analysis and Sanger sequencing of mitochondrial DNA for four U.S. population groups.

PubMed

Kiesler, Kevin M; Coble, Michael D; Hall, Thomas A; Vallone, Peter M

2014-01-01

A set of 711 samples from four U.S. population groups was analyzed using a novel mass spectrometry based method for mitochondrial DNA (mtDNA) base composition profiling. Comparison of the mass spectrometry results with Sanger sequencing derived data yielded a concordance rate of 99.97%. Length heteroplasmy was identified in 46% of samples and point heteroplasmy was observed in 6.6% of samples in the combined mass spectral and Sanger data set. Using discrimination capacity as a metric, Sanger sequencing of the full control region had the highest discriminatory power, followed by the mass spectrometry base composition method, which was more discriminating than Sanger sequencing of just the hypervariable regions. This trend is in agreement with the number of nucleotides covered by each of the three assays. Published by Elsevier Ireland Ltd.

Genetic analysis and ethnic affinities from two Scytho-Siberian skeletons.

PubMed

Ricaut, François-Xavier; Keyser-Tracqui, Christine; Cammaert, Laurence; Crubézy, Eric; Ludes, Bertrand

2004-04-01

We extracted DNA from two skeletons belonging to the Sytho-Siberian population, which were excavated from the Sebÿstei site (dating back 2,500 years) in the Altai Republic (Central Asia). Ancient DNA was analyzed by autosomal short tandem repeats (STRs) and by the sequencing of the hypervariable region 1 (HV1) of the mitochondrial DNA (mtDNA) control region. The results showed that these two skeletons were not close relatives. Moreover, their haplogroups were characteristic of Asian populations. Comparison with the haplogroup of 3,523 Asian and American individuals linked one skeleton with a putative ancestral paleo-Asiatic population and the other with Chinese populations. It appears that the genetic study of ancient populations of Central Asia brings important elements to the understanding of human population movements in Asia. Copyright 2003 Wiley-Liss, Inc.

Molecular cloning and characterization of an acetylcholinesterase cDNA in the brown planthopper, Nilaparvata lugens.

PubMed

Yang, Zhifan; Chen, Jun; Chen, Yongqin; Jiang, Sijing

2010-01-01

A full cDNA encoding an acetylcholinesterase (AChE, EC 3.1.1.7) was cloned and characterized from the brown planthopper, Nilaparvata lugens Stål (Hemiptera: Delphacidae). The complete cDNA (2467 bp) contains a 1938-bp open reading frame encoding 646 amino acid residues. The amino acid sequence of the AChE deduced from the cDNA consists of 30 residues for a putative signal peptide and 616 residues for the mature protein with a predicted molecular weight of 69,418. The three residues (Ser242, Glu371, and His485) that putatively form the catalytic triad and the six Cys that form intra-subunit disulfide bonds are completely conserved, and 10 out of the 14 aromatic residues lining the active site gorge of the AChE are also conserved. Northern blot analysis of poly(A)+ RNA showed an approximately 2.6-kb transcript, and Southern blot analysis revealed there likely was just a single copy of this gene in N. lugens. The deduced protein sequence is most similar to AChE of Nephotettix cincticeps with 83% amino acid identity. Phylogenetic analysis constructed with 45 AChEs from 30 species showed that the deduced N. lugens AChE formed a cluster with the other 8 insect AChE2s. Additionally, the hypervariable region and amino acids specific to insect AChE2 also existed in the AChE of N. lugens. The results revealed that the AChE cDNA cloned in this work belongs to insect AChE2 subgroup, which is orthologous to Drosophila AChE. Comparison of the AChEs between the susceptible and resistant strains revealed a point mutation, Gly185Ser, is likely responsible for the insensitivity of the AChE to methamidopho in the resistant strain.

Multiple maternal origins and weak phylogeographic structure in domestic goats

PubMed Central

Luikart, Gordon; Gielly, Ludovic; Excoffier, Laurent; Vigne, Jean-Denis; Bouvet, Jean; Taberlet, Pierre

2001-01-01

Domestic animals have played a key role in human history. Despite their importance, however, the origins of most domestic species remain poorly understood. We assessed the phylogenetic history and population structure of domestic goats by sequencing a hypervariable segment (481 bp) of the mtDNA control region from 406 goats representing 88 breeds distributed across the Old World. Phylogeographic analysis revealed three highly divergent goat lineages (estimated divergence >200,000 years ago), with one lineage occurring only in eastern and southern Asia. A remarkably similar pattern exists in cattle, sheep, and pigs. These results, combined with recent archaeological findings, suggest that goats and other farm animals have multiple maternal origins with a possible center of origin in Asia, as well as in the Fertile Crescent. The pattern of goat mtDNA diversity suggests that all three lineages have undergone population expansions, but that the expansion was relatively recent for two of the lineages (including the Asian lineage). Goat populations are surprisingly less genetically structured than cattle populations. In goats only ≈10% of the mtDNA variation is partitioned among continents. In cattle the amount is ≥50%. This weak structuring suggests extensive intercontinental transportation of goats and has intriguing implications about the importance of goats in historical human migrations and commerce. PMID:11344314

New insights into mitogenomic phylogeny and copy number in eight indigenous sheep populations based on the ATP synthase and cytochrome c oxidase genes.

PubMed

Xiao, P; Niu, L L; Zhao, Q J; Chen, X Y; Wang, L J; Li, L; Zhang, H P; Guo, J Z; Xu, H Y; Zhong, T

2017-11-16

The origins and phylogeny of different sheep breeds has been widely studied using polymorphisms within the mitochondrial hypervariable region. However, little is known about the mitochondrial DNA (mtDNA) content and phylogeny based on mtDNA protein-coding genes. In this study, we assessed the phylogeny and copy number of the mtDNA in eight indigenous (population size, n=184) and three introduced (n=66) sheep breeds in China based on five mitochondrial coding genes (COX1, COX2, ATP8, ATP6 and COX3). The mean haplotype and nucleotide diversities were 0.944 and 0.00322, respectively. We identified a correlation between the lineages distribution and the genetic distance, whereby Valley-type Tibetan sheep had a closer genetic relationship with introduced breeds (Dorper, Poll Dorset and Suffolk) than with other indigenous breeds. Similarly, the Median-joining profile of haplotypes revealed the distribution of clusters according to genetic differences. Moreover, copy number analysis based on the five mitochondrial coding genes was affected by the genetic distance combining with genetic phylogeny; we also identified obvious non-synonymous mutations in ATP6 between the different levels of copy number expressions. These results imply that differences in mitogenomic compositions resulting from geographical separation lead to differences in mitochondrial function.

Genetic characterization and phylogeny of pigeon paramyxovirus isolate (PPMV-1) from Pakistan.

PubMed

Akhtar, Sameera; Muneer, Muhammad Akram; Muhammad, Khushi; Tipu, Muhammad Yasin; Rabbani, Masood; Ul-Rahman, Aziz; Shabbir, Muhammad Zubair

2016-01-01

Knowing the genome characteristics of circulating Newcastle disease viruses [avian paramyxoviruses (APMV-1) and pigeon paramyxoviruses (PPMV-1)] is important to devise appropriate diagnostics and control strategies. APMVs originating from chicken and wildlife in Pakistan are well-elucidated; nevertheless, molecular characterization for the circulating PPMV-1 is largely unknown. Here, we have performed fusion (F) and hemagglutinin (HN) gene based characterization of PPMV-1 isolated from an outbreak in a pigeon flock. With F0 proteolytic cleavage site (112RRQKR↓F117), characteristic of velogenic/mesogenic serotype, the complete F and HN gene based sequence analysis of the isolate revealed evolutionary relationship to genotype VI. Further analysis of hyper-variable region of F-gene demonstrated clustering of the study isolate with genotype VIb. The deduced residue analysis for both F and HN protein showed a number of substitution mutations in the functional domains distinct from representative strains of each genotype including the vaccine strains; some of them were found exclusive to the study isolate. Though limited and preliminary data, the findings enhance our knowledge towards circulating strains of PPMVs in Pakistan. Further studies are needed to ascertain its potential for transmission in the wild birds, commercial and backyard poultry and its subsequent shedding into the environment.

Modular community structure suggests metabolic plasticity during the transition to polar night in ice-covered Antarctic lakes.

PubMed

Vick-Majors, Trista J; Priscu, John C; Amaral-Zettler, Linda A

2014-04-01

High-latitude environments, such as the Antarctic McMurdo Dry Valley lakes, are subject to seasonally segregated light-dark cycles, which have important consequences for microbial diversity and function on an annual basis. Owing largely to the logistical difficulties of sampling polar environments during the darkness of winter, little is known about planktonic microbial community responses to the cessation of photosynthetic primary production during the austral sunset, which lingers from approximately February to April. Here, we hypothesized that changes in bacterial, archaeal and eukaryotic community structure, particularly shifts in favor of chemolithotrophs and mixotrophs, would manifest during the transition to polar night. Our work represents the first concurrent molecular characterization, using 454 pyrosequencing of hypervariable regions of the small-subunit ribosomal RNA gene, of bacterial, archaeal and eukaryotic communities in permanently ice-covered lakes Fryxell and Bonney, before and during the polar night transition. We found vertically stratified populations that varied at the community and/or operational taxonomic unit-level between lakes and seasons. Network analysis based on operational taxonomic unit level interactions revealed nonrandomly structured microbial communities organized into modules (groups of taxa) containing key metabolic potential capacities, including photoheterotrophy, mixotrophy and chemolithotrophy, which are likely to be differentially favored during the transition to polar night.

Functional bottlenecks for generation of HIV-1 intersubtype Env recombinants.

PubMed

Bagaya, Bernard S; Vega, José F; Tian, Meijuan; Nickel, Gabrielle C; Li, Yuejin; Krebs, Kendall C; Arts, Eric J; Gao, Yong

2015-05-23

Intersubtype recombination is a powerful driving force for HIV evolution, impacting both HIV-1 diversity within an infected individual and within the global epidemic. This study examines if viral protein function/fitness is the major constraint shaping selection of recombination hotspots in replication-competent HIV-1 progeny. A better understanding of the interplay between viral protein structure-function and recombination may provide insights into both vaccine design and drug development. In vitro HIV-1 dual infections were used to recombine subtypes A and D isolates and examine breakpoints in the Env glycoproteins. The entire env genes of 21 A/D recombinants with breakpoints in gp120 were non-functional when cloned into the laboratory strain, NL4-3. Likewise, cloning of A/D gp120 coding regions also produced dead viruses with non-functional Envs. 4/9 replication-competent viruses with functional Env's were obtained when just the V1-V5 regions of these same A/D recombinants (i.e. same A/D breakpoints as above) were cloned into NL4-3. These findings on functional A/D Env recombinants combined with structural models of Env suggest a conserved interplay between the C1 domain with C5 domain of gp120 and extracellular domain of gp41. Models also reveal a co-evolution within C1, C5, and ecto-gp41 domains which might explain the paucity of intersubtype recombination in the gp120 V1-V5 regions, despite their hypervariability. At least HIV-1 A/D intersubtype recombination in gp120 may result in a C1 from one subtype incompatible with a C5/gp41 from another subtype.

Land, language, and loci: mtDNA in Native Americans and the genetic history of Peru.

PubMed

Lewis, Cecil M; Tito, Raúl Y; Lizárraga, Beatriz; Stone, Anne C

2005-07-01

Despite a long history of complex societies and despite extensive present-day linguistic and ethnic diversity, relatively few populations in Peru have been sampled for population genetic investigations. In order to address questions about the relationships between South American populations and about the extent of correlation between genetic distance, language, and geography in the region, mitochondrial DNA (mtDNA) hypervariable region I sequences and mtDNA haplogroup markers were examined in 33 individuals from the state of Ancash, Peru. These sequences were compared to those from 19 American Indian populations using diversity estimates, AMOVA tests, mismatch distributions, a multidimensional scaling plot, and regressions. The results show correlations between genetics, linguistics, and geographical affinities, with stronger correlations between genetics and language. Additionally, the results suggest a pattern of differential gene flow and drift in western vs. eastern South America, supporting previous mtDNA and Y chromosome investigations. (c) 2004 Wiley-Liss, Inc

Cloning, expression and phylogenetic analysis of Hemolin, from the Chinese oak silkmoth, Antheraea pernyi.

PubMed

Li, Wenli; Terenius, Olle; Hirai, Makoto; Nilsson, Anders S; Faye, Ingrid

2005-01-01

The Chinese oak silk moth Antheraea pernyi is an important silk producer. To understand microbial resistance of this moth, we cloned Hemolin, encoding a multifunctional immune protein belonging to the immunoglobulin superfamily, and examined the expression in gonads and fat body. The ApHemolin amino acid sequence was compared to other Hemolin sequences in order to predict functional sites. Several sites were conserved; among them a phosphate binding site, which according to 3D structure modelling does not appear in neuroglian, the phylogenetically closest related protein. In addition, two conserved KDG sequences in the C-C' loop of immunoglobulin domains 1 and 3, give rise to gamma-turns, which is a common motif in the C'-C'' loop of the hypervariable region L2 in vertebrate immunoglobulins. The comparisons also show variable regions of specific interest for future studies of hemolin and its interaction with microbial entities.

Association between mitochondrial DNA variations and schizophrenia in the northern Chinese Han population.

PubMed

Xu, Feng-Ling; Ding, Mei; Yao, Jun; Shi, Zhang-Sen; Wu, Xue; Zhang, Jing-Jing; Pang, Hao; Xing, Jia-Xin; Xuan, Jin-Feng; Wang, Bao-Jie

2017-01-01

To determine whether mitochondrial DNA (mtDNA) variations are associated with schizophrenia, 313 patients with schizophrenia and 326 unaffected participants of the northern Chinese Han population were included in a prospective study. Single-nucleotide polymorphisms (SNPs) including C5178A, A10398G, G13708A, and C13928G were analyzed by polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP). Hypervariable regions I and II (HVSI and HVSII) were analyzed by sequencing. The results showed that the 4 SNPs and 11 haplotypes, composed of the 4 SNPs, did not differ significantly between patient and control groups. No significant association between haplogroups and the risk of schizophrenia was ascertained after Bonferroni correction. Drawing a conclusion, there was no evidence of an association between mtDNA (the 4 SNPs and the control region) and schizophrenia in the northern Chinese Han population.

DNA analyses of the remains of the Prince Branciforte Barresi family.

PubMed

Rickards, O; Martínez-Labarga, C; Favaro, M; Frezza, D; Mallegni, F

2001-01-01

The five skeletons found buried in the church of Militello di Catania, Sicily, were tentatively identified by morphological analysis and historical reports as the remains of Prince Branciforte Barresi, two of his children, his brother and another juvenile member of the family (sixteenth and seventeenth centuries). In order to attempt to clarify the degree of relationships of the five skeletons, sex testing and mitochondrial DNA (mtDNA) sequence analysis of the hypervariable segments I and II (HV1 and HV2) of control region were performed. Moreover, the 9 bp-deletion marker of region V (COII/tRNAlys) was examined. Molecular genetic analyses were consistent with historical expectations, although they did not directly demonstrate that these are in fact the remains of the Prince and his relatives, due to the impossibility of obtaining DNA from living maternal relatives of the Prince.

Protistan Biogeography: A Snapshot Across a Major Shipping Corridor Spanning Two Oceans.

PubMed

Pagenkopp Lohan, Katrina M; Fleischer, Robert C; Torchin, Mark E; Ruiz, Gregory M

2017-04-01

Deciphering patterns of protistan taxa is a crucial step for understanding anthropogenic and environmental impacts on biogeography. We characterized and compared protistan communities from environmental samples collected along a major shipping corridor, the Panama Canal, and the Bocas del Toro archipelago. We used metabarcoding with high throughput sequencing (HTS) with the V4 hypervariable region of the ribosomal gene complex (rDNA). We detected many protistan taxa, including a variety of parasitic and toxic taxa. There were 1,296 OTUs shared across all three regions, with an additional 342-1,526 OTUs occurring across two or more regions, suggesting some mixing within the Caribbean and across the Isthmus. In general, this mixing did not impact community similarity, which was primarily distinct across regions. When OTUs identified as gregarines were analyzed separately, most samples grouped by region and communities were distinct across the Canal. Shipping traffic through the Panama Canal could move some taxa across regions; however, different environmental conditions in the two oceans may limit their establishment. Overall our results suggest that contemporary protistan biogeographic patterns are likely caused by a complex combination of factors, including anthropogenic dispersal and environmental tolerance. Published by Elsevier GmbH.

A new mitochondrial C1 lineage from the prehistory of Uruguay: population genocide, ethnocide, and continuity.

PubMed

Sans, Monica; Figueiro, Gonzalo; Hidalgo, Pedro C

2012-06-01

Uruguayan population has been considered as of European descent, as its Native populations victims of genocide apparently disappeared in the 19th century. Contradicting this national belief, genetic studies have shown a substantial Native contribution. However, the continuity between prehistoric, historic, and present populations remains unproved. With the aim of adding elements to prove a possible population continuity, we studied a mitochondrial lineage, part of haplogroup C1, analyzing the complete genome of a modern Uruguayan individual and the hypervariable region I (HVRI) in prehistoric, historic, and contemporary individuals. Several individuals carried the mutations that characterize this lineage: two from an archaeological mound located in the east of the country, the Charrúa Indian chief Vaimaca Perú and five individuals from the present population. The lineage was initially characterized by its HVRI sequence, having the four typical C1 mutations and adding 16051G and 16288C; other mutations were also found: 16140C was found in all but the oldest individual, dated 1,610 years BP, while 16209C, 16422C, and 16519C were found only in some individuals. Hypervariable region II showed the typical C1 mutations and 194T. The coding region, analyzed in modern individuals, was characterized by 12378T, while other mutations found were not common to all of them. In summary, we have found and described a new lineage that shows continuity from prehistoric mound builders to the present population, through a representative of the extinct Charrúa Indians. The lineage appeared at least 1,600 years ago and is carried by approximately 0.7% of the modern Uruguayan population. The continuity of the lineage supports alternative perspectives about Uruguayan national identity and the meaning of the genocide, best labeled as ethnocide because of its consequences. It also contributes to the discussion about who the prehistoric mound builders were, and to the origin, at least in the maternal line, of a Charrúa Indian. From a more general perspective, we can conclude that the characteristics, evolution, and expansion of founder haplogroup C in America have not yet been elucidated.

Design and Evaluation of Illumina MiSeq-Compatible, 18S rRNA Gene-Specific Primers for Improved Characterization of Mixed Phototrophic Communities.

PubMed

Bradley, Ian M; Pinto, Ameet J; Guest, Jeremy S

2016-10-01

The use of high-throughput sequencing technologies with the 16S rRNA gene for characterization of bacterial and archaeal communities has become routine. However, the adoption of sequencing methods for eukaryotes has been slow, despite their significance to natural and engineered systems. There are large variations among the target genes used for amplicon sequencing, and for the 18S rRNA gene, there is no consensus on which hypervariable region provides the most suitable representation of diversity. Additionally, it is unclear how much PCR/sequencing bias affects the depiction of community structure using current primers. The present study amplified the V4 and V8-V9 regions from seven microalgal mock communities as well as eukaryotic communities from freshwater, coastal, and wastewater samples to examine the effect of PCR/sequencing bias on community structure and membership. We found that degeneracies on the 3' end of the current V4-specific primers impact read length and mean relative abundance. Furthermore, the PCR/sequencing error is markedly higher for GC-rich members than for communities with balanced GC content. Importantly, the V4 region failed to reliably capture 2 of the 12 mock community members, and the V8-V9 hypervariable region more accurately represents mean relative abundance and alpha and beta diversity. Overall, the V4 and V8-V9 regions show similar community representations over freshwater, coastal, and wastewater environments, but specific samples show markedly different communities. These results indicate that multiple primer sets may be advantageous for gaining a more complete understanding of community structure and highlight the importance of including mock communities composed of species of interest. The quantification of error associated with community representation by amplicon sequencing is a critical challenge that is often ignored. When target genes are amplified using currently available primers, differential amplification efficiencies result in inaccurate estimates of community structure. The extent to which amplification bias affects community representation and the accuracy with which different gene targets represent community structure are not known. As a result, there is no consensus on which region provides the most suitable representation of diversity for eukaryotes. This study determined the accuracy with which commonly used 18S rRNA gene primer sets represent community structure and identified particular biases related to PCR amplification and Illumina MiSeq sequencing in order to more accurately study eukaryotic microbial communities. Copyright © 2016, American Society for Microbiology. All Rights Reserved.

An accurate and efficient experimental approach for characterization of the complex oral microbiota.

PubMed

Zheng, Wei; Tsompana, Maria; Ruscitto, Angela; Sharma, Ashu; Genco, Robert; Sun, Yijun; Buck, Michael J

2015-10-05

Currently, taxonomic interrogation of microbiota is based on amplification of 16S rRNA gene sequences in clinical and scientific settings. Accurate evaluation of the microbiota depends heavily on the primers used, and genus/species resolution bias can arise with amplification of non-representative genomic regions. The latest Illumina MiSeq sequencing chemistry has extended the read length to 300 bp, enabling deep profiling of large number of samples in a single paired-end reaction at a fraction of the cost. An increasingly large number of researchers have adopted this technology for various microbiome studies targeting the 16S rRNA V3-V4 hypervariable region. To expand the applicability of this powerful platform for further descriptive and functional microbiome studies, we standardized and tested an efficient, reliable, and straightforward workflow for the amplification, library construction, and sequencing of the 16S V1-V3 hypervariable region using the new 2 × 300 MiSeq platform. Our analysis involved 11 subgingival plaque samples from diabetic and non-diabetic human subjects suffering from periodontitis. The efficiency and reliability of our experimental protocol was compared to 16S V3-V4 sequencing data from the same samples. Comparisons were based on measures of observed taxonomic richness and species evenness, along with Procrustes analyses using beta(β)-diversity distance metrics. As an experimental control, we also analyzed a total of eight technical replicates for the V1-V3 and V3-V4 regions from a synthetic community with known bacterial species operon counts. We show that our experimental protocol accurately measures true bacterial community composition. Procrustes analyses based on unweighted UniFrac β-diversity metrics depicted significant correlation between oral bacterial composition for the V1-V3 and V3-V4 regions. However, measures of phylotype richness were higher for the V1-V3 region, suggesting that V1-V3 offers a deeper assessment of population diversity and community ecology for the complex oral microbiota. This study provides researchers with valuable experimental evidence for the selection of appropriate 16S amplicons for future human oral microbiome studies. We expect that the tested 16S V1-V3 framework will be widely applicable to other types of microbiota, allowing robust, time-efficient, and inexpensive examination of thousands of samples for population, phylogenetic, and functional crossectional and longitutidal studies.

Taming the late Quaternary phylogeography of the Eurasiatic wild ass through ancient and modern DNA

PubMed Central

Peters, Joris; Arbuckle, Benjamin S.; Guimaraes, Silvia; Pruvost, Mélanie; Bar-David, Shirli; Davis, Simon J. M.; Gautier, Mathieu; Kaczensky, Petra; Kuehn, Ralph; Mashkour, Marjan; Morales-Muñiz, Arturo; Pucher, Erich; Tournepiche, Jean-François; Uerpmann, Hans-Peter; Bălăşescu, Adrian; Germonpré, Mietje; Gündem, Can Y.; Hemami, Mahmoud-Reza; Moullé, Pierre-Elie; Ötzan, Aliye; Uerpmann, Margarete; Walzer, Chris

2017-01-01

Taxonomic over-splitting of extinct or endangered taxa, due to an incomplete knowledge of both skeletal morphological variability and the geographical ranges of past populations, continues to confuse the link between isolated extant populations and their ancestors. This is particularly problematic with the genus Equus. To more reliably determine the evolution and phylogeographic history of the endangered Asiatic wild ass, we studied the genetic diversity and inter-relationships of both extinct and extant populations over the last 100,000 years, including samples throughout its previous range from Western Europe to Southwest and East Asia. Using 229 bp of the mitochondrial hypervariable region, an approach which allowed the inclusion of information from extremely poorly preserved ancient samples, we classify all non-African wild asses into eleven clades that show a clear phylogeographic structure revealing their phylogenetic history. This study places the extinct European wild ass, E. hydruntinus, the phylogeny of which has been debated since the end of the 19th century, into its phylogenetic context within the Asiatic wild asses and reveals recent mitochondrial introgression between populations currently regarded as separate species. The phylogeographic organization of clades resulting from these efforts can be used not only to improve future taxonomic determination of a poorly characterized group of equids, but also to identify historic ranges, interbreeding events between various populations, and the impact of ancient climatic changes. In addition, appropriately placing extant relict populations into a broader phylogeographic and genetic context can better inform ongoing conservation strategies for this highly-endangered species. PMID:28422966

Taming the late Quaternary phylogeography of the Eurasiatic wild ass through ancient and modern DNA.

PubMed

Bennett, E Andrew; Champlot, Sophie; Peters, Joris; Arbuckle, Benjamin S; Guimaraes, Silvia; Pruvost, Mélanie; Bar-David, Shirli; Davis, Simon J M; Gautier, Mathieu; Kaczensky, Petra; Kuehn, Ralph; Mashkour, Marjan; Morales-Muñiz, Arturo; Pucher, Erich; Tournepiche, Jean-François; Uerpmann, Hans-Peter; Bălăşescu, Adrian; Germonpré, Mietje; Gündem, Can Y; Hemami, Mahmoud-Reza; Moullé, Pierre-Elie; Ötzan, Aliye; Uerpmann, Margarete; Walzer, Chris; Grange, Thierry; Geigl, Eva-Maria

2017-01-01

Taxonomic over-splitting of extinct or endangered taxa, due to an incomplete knowledge of both skeletal morphological variability and the geographical ranges of past populations, continues to confuse the link between isolated extant populations and their ancestors. This is particularly problematic with the genus Equus. To more reliably determine the evolution and phylogeographic history of the endangered Asiatic wild ass, we studied the genetic diversity and inter-relationships of both extinct and extant populations over the last 100,000 years, including samples throughout its previous range from Western Europe to Southwest and East Asia. Using 229 bp of the mitochondrial hypervariable region, an approach which allowed the inclusion of information from extremely poorly preserved ancient samples, we classify all non-African wild asses into eleven clades that show a clear phylogeographic structure revealing their phylogenetic history. This study places the extinct European wild ass, E. hydruntinus, the phylogeny of which has been debated since the end of the 19th century, into its phylogenetic context within the Asiatic wild asses and reveals recent mitochondrial introgression between populations currently regarded as separate species. The phylogeographic organization of clades resulting from these efforts can be used not only to improve future taxonomic determination of a poorly characterized group of equids, but also to identify historic ranges, interbreeding events between various populations, and the impact of ancient climatic changes. In addition, appropriately placing extant relict populations into a broader phylogeographic and genetic context can better inform ongoing conservation strategies for this highly-endangered species.

Characteristics of polymorphism at a VNTR locus 3[prime] to the apolipoprotein B gene in five human populations

DOE Office of Scientific and Technical Information (OSTI.GOV)

Deka, R.; DeCroo, S.; Ferrell, R.E.

1992-12-01

The authors have analyzed the allele frequency distribution at the hypervariable locus 3[prime] to the apolipoprotein B gene (ApoB 3[prime] VNTR) in five well-defined human populations (Kacharis of northeast India, New Guinea Highlanders of Papua New Guinea, Dogrib Indians of Canada, Pehuenche Indians of Chile, and a relatively homogeneous Caucasian population of northern German extraction) by using the PCR technique. A total of 12 segregating alleles were detected in the pooled sample of 319 individuals. A fairly consistent bimodal pattern of allele frequency distribution, apparent in most of these geographically and genetically diverse populations, suggests that the ApoB 3[prime] VNTRmore » polymorphism predates the geographic dispersal of ancestral human populations. In spite of the observed high degree of polymorphism at this locus (expected heterozygosity levels 55%-78%), the genotype distributions in all populations (irrespective of their tribal or cosmopolitan nature) conform to their respective Hardy-Weinberg predictions. Furthermore, analysis of the congruence between expected heterozygosity and the observed number of alleles reveals that, in general, the allele frequency distributions at this locus are in agreement with the predictions of the classical mutation-drift models. The data also show that alleles that are shared by all populations have the highest average frequency within populations. These findings demonstrate the potential utility of highly informative hypervariable loci such as the ApoB 3[prime] VNTR locus in population genetic research, as well as in forensic medicine and determination of biological relatedness of individuals. 38 refs., 2 figs., 3 tabs.« less

Characterization of the HLA-DRβ1 third hypervariable region amino acid sequence according to charge and parental inheritance in systemic sclerosis.

PubMed

Gentil, Coline A; Gammill, Hilary S; Luu, Christine T; Mayes, Maureen D; Furst, Dan E; Nelson, J Lee

2017-03-07

Specific HLA class II alleles are associated with systemic sclerosis (SSc) risk, clinical characteristics, and autoantibodies. HLA nomenclature initially developed with antibodies as typing reagents defining DRB1 allele groups. However, alleles from different DRB1 allele groups encode the same third hypervariable region (3rd HVR) sequence, the primary T-cell recognition site, and 3rd HVR charge differences can affect interactions with T cells. We considered 3rd HVR sequences (amino acids 67-74) irrespective of the allele group and analyzed parental inheritance considered according to the 3rd HVR charge, comparing SSc patients with controls. In total, 306 families (121 SSc and 185 controls) were HLA genotyped and parental HLA-haplotype origin was determined. Analysis was conducted according to DRβ1 3rd HVR sequence, charge, and parental inheritance. The distribution of 3rd HVR sequences differed in SSc patients versus controls (p = 0.007), primarily due to an increase of specific DRB1*11 alleles, in accord with previous observations. The 3rd HVR sequences were next analyzed according to charge and parental inheritance. Paternal transmission of DRB1 alleles encoding a +2 charge 3rd HVR was significantly reduced in SSc patients compared with maternal transmission (p = 0.0003, corrected for analysis of four charge categories p = 0.001). To a lesser extent, paternal transmission was increased when charge was 0 (p = 0.021, corrected for multiple comparisons p = 0.084). In contrast, paternal versus maternal inheritance was similar in controls. SSc patients differed from controls when DRB1 alleles were categorized according to 3rd HVR sequences. Skewed parental inheritance was observed in SSc patients but not in controls when the DRβ1 3rd HVR was considered according to charge. These observations suggest that epigenetic modulation of HLA merits investigation in SSc.

Investigating the diversity of the 18S SSU rRNA hyper-variable region of Theileria in cattle and Cape buffalo (Syncerus caffer) from southern Africa using a next generation sequencing approach.

PubMed

Mans, Ben J; Pienaar, Ronel; Ratabane, John; Pule, Boitumelo; Latif, Abdalla A

2016-07-01

Molecular classification and systematics of the Theileria is based on the analysis of the 18S rRNA gene. Reverse line blot or conventional sequencing approaches have disadvantages in the study of 18S rRNA diversity and a next-generation 454 sequencing approach was investigated. The 18S rRNA gene was amplified using RLB primers coupled to 96 unique sequence identifiers (MIDs). Theileria positive samples from African buffalo (672) and cattle (480) from southern Africa were combined in batches of 96 and sequenced using the GS Junior 454 sequencer to produce 825711 informative sequences. Sequences were extracted based on MIDs and analysed to identify Theileria genotypes. Genotypes observed in buffalo and cattle were confirmed in the current study, while no new genotypes were discovered. Genotypes showed specific geographic distributions, most probably linked with vector distributions. Host specificity of buffalo and cattle specific genotypes were confirmed and prevalence data as well as relative parasitemia trends indicate preference for different hosts. Mixed infections are common with African buffalo carrying more genotypes compared to cattle. Associative or exclusion co-infection profiles were observed between genotypes that may have implications for speciation and systematics: specifically that more Theileria species may exist in cattle and buffalo than currently recognized. Analysis of primers used for Theileria parva diagnostics indicate that no new genotypes will be amplified by the current primer sets confirming their specificity. T. parva SNP variants that occur in the 18S rRNA hypervariable region were confirmed. A next generation sequencing approach is useful in obtaining comprehensive knowledge regarding 18S rRNA diversity and prevalence for the Theileria, allowing for the assessment of systematics and diagnostic assays based on the 18S gene. Copyright © 2016 Elsevier GmbH. All rights reserved.

Metagenomic Analysis of Hot Springs in Central India Reveals Hydrocarbon Degrading Thermophiles and Pathways Essential for Survival in Extreme Environments.

PubMed

Saxena, Rituja; Dhakan, Darshan B; Mittal, Parul; Waiker, Prashant; Chowdhury, Anirban; Ghatak, Arundhuti; Sharma, Vineet K

2016-01-01

Extreme ecosystems such as hot springs are of great interest as a source of novel extremophilic species, enzymes, metabolic functions for survival and biotechnological products. India harbors hundreds of hot springs, the majority of which are not yet explored and require comprehensive studies to unravel their unknown and untapped phylogenetic and functional diversity. The aim of this study was to perform a large-scale metagenomic analysis of three major hot springs located in central India namely, Badi Anhoni, Chhoti Anhoni, and Tattapani at two geographically distinct regions (Anhoni and Tattapani), to uncover the resident microbial community and their metabolic traits. Samples were collected from seven distinct sites of the three hot spring locations with temperature ranging from 43.5 to 98°C. The 16S rRNA gene amplicon sequencing of V3 hypervariable region and shotgun metagenome sequencing uncovered a unique taxonomic and metabolic diversity of the resident thermophilic microbial community in these hot springs. Genes associated with hydrocarbon degradation pathways, such as benzoate, xylene, toluene, and benzene were observed to be abundant in the Anhoni hot springs (43.5-55°C), dominated by Pseudomonas stutzeri and Acidovorax sp., suggesting the presence of chemoorganotrophic thermophilic community with the ability to utilize complex hydrocarbons as a source of energy. A high abundance of genes belonging to methane metabolism pathway was observed at Chhoti Anhoni hot spring, where methane is reported to constitute >80% of all the emitted gases, which was marked by the high abundance of Methylococcus capsulatus . The Tattapani hot spring, with a high-temperature range (61.5-98°C), displayed a lower microbial diversity and was primarily dominated by a nitrate-reducing archaeal species Pyrobaculum aerophilum . A higher abundance of cell metabolism pathways essential for the microbial survival in extreme conditions was observed at Tattapani. Taken together, the results of this study reveal a novel consortium of microbes, genes, and pathways associated with the hot spring environment.

Geography has more influence than language on maternal genetic structure of various northeastern Thai ethnicities.

PubMed

Kutanan, Wibhu; Ghirotto, Silvia; Bertorelle, Giorgio; Srithawong, Suparat; Srithongdaeng, Kanokpohn; Pontham, Nattapon; Kangwanpong, Daoroong

2014-09-01

Several literatures have shown the influence of geographic and linguistic factors in shaping genetic variation patterns, but their relative impact, if any, in the very heterogeneous northeastern region of Thailand has not yet been studied. This area, called Isan, is geographically structured in two wide basins, the Sakon Nakorn Basin and the Korat Basin, serving today as home to diverse ethnicities encompassing two different linguistic families, that is, the Austro-Asiatic; Suay (Kui), Mon, Chaobon (Nyahkur), So and Khmer, and the Tai-Kadai; Saek, Nyaw, Phu Tai, Kaleung and Lao Isan. In this study, we evaluated the relative role of geographic distance and barriers as well as linguistic differences as possible causes affecting the maternal genetic distances among northeastern Thai ethnicities. A 596-bp segment of the hypervariable region I mitochondrial DNA was utilized to elucidate the genetic structure and biological affinity from 433 individuals. Different statistical analyses agreed in suggesting that most ethnic groups in the Sakon Nakorn Basin are closely related. Mantel test revealed that genetic distances were highly associated to geographic (r = 0.445, P<0.01) but not to linguistic (r = 0.001, P>0.01) distances. Three evolutionary models were compared by Approximate Bayesian Computation. The posterior probability of the scenario, which assumed an initial population divergence possibly related to reduced gene flow among basins, was equal or higher than 0.87. All analyses exhibited concordant results supporting that geography was the most relevant factor in determining the maternal genetic structure of northeastern Thai populations.

The mtDNA haplogroup P of modern Asian cattle: A genetic legacy of Asian aurochs?

PubMed

Noda, Aoi; Yonesaka, Riku; Sasazaki, Shinji; Mannen, Hideyuki

2018-01-01

Aurochs (Bos primigenius) were distributed throughout large parts of Eurasia and Northern Africa during the late Pleistocene and the early Holocene, and all modern cattle are derived from the aurochs. Although the mtDNA haplogroups of most modern cattle belong to haplogroups T and I, several additional haplogroups (P, Q, R, C and E) have been identified in modern cattle and aurochs. Haplogroup P was the most common haplogroup in European aurochs, but so far, it has been identified in only three of >3,000 submitted haplotypes of modern Asian cattle. We sequenced the complete mtDNA D-loop region of 181 Japanese Shorthorn cattle and analyzed these together with representative bovine mtDNA sequences. The haplotype P of Japanese Shorthorn cattle was analyzed along with that of 36 previously published European aurochs and three modern Asian cattle sequences using the hypervariable 410 bp of the D-loop region. We detected the mtDNA haplogroup P in Japanese Shorthorn cattle with an extremely high frequency (83/181). Phylogenetic networks revealed two main clusters, designated as Pa for haplogroup P in European aurochs and Pc in modern Asian cattle. We also report the genetic diversity of haplogroup P compared with the sequences of extinct aurochs. No shared haplotypes are observed between the European aurochs and the modern Asian cattle. This finding suggests the possibility of local and secondary introgression events of haplogroup P in northeast Asian cattle, and will contribute to a better understanding of its origin and genetic diversity.

The mtDNA haplogroup P of modern Asian cattle: A genetic legacy of Asian aurochs?

PubMed Central

Noda, Aoi; Yonesaka, Riku; Sasazaki, Shinji

2018-01-01

Background Aurochs (Bos primigenius) were distributed throughout large parts of Eurasia and Northern Africa during the late Pleistocene and the early Holocene, and all modern cattle are derived from the aurochs. Although the mtDNA haplogroups of most modern cattle belong to haplogroups T and I, several additional haplogroups (P, Q, R, C and E) have been identified in modern cattle and aurochs. Haplogroup P was the most common haplogroup in European aurochs, but so far, it has been identified in only three of >3,000 submitted haplotypes of modern Asian cattle. Methodology We sequenced the complete mtDNA D-loop region of 181 Japanese Shorthorn cattle and analyzed these together with representative bovine mtDNA sequences. The haplotype P of Japanese Shorthorn cattle was analyzed along with that of 36 previously published European aurochs and three modern Asian cattle sequences using the hypervariable 410 bp of the D-loop region. Conclusions We detected the mtDNA haplogroup P in Japanese Shorthorn cattle with an extremely high frequency (83/181). Phylogenetic networks revealed two main clusters, designated as Pa for haplogroup P in European aurochs and Pc in modern Asian cattle. We also report the genetic diversity of haplogroup P compared with the sequences of extinct aurochs. No shared haplotypes are observed between the European aurochs and the modern Asian cattle. This finding suggests the possibility of local and secondary introgression events of haplogroup P in northeast Asian cattle, and will contribute to a better understanding of its origin and genetic diversity. PMID:29304129

Next-Generation Sequencing Reveals Frequent Opportunities for Exposure to Hepatitis C Virus in Ghana

PubMed Central

Phillips, Richard O.; Mora, Nallely; Xia, Guo-liang; Campo, David S.; Purdy, Michael A.; Dimitrova, Zoya E.; Owusu, Dorcas O.; Punkova, Lili T.; Skums, Pavel; Owusu-Ofori, Shirley; Sarfo, Fred Stephen; Vaughan, Gilberto; Roh, Hajung; Opare-Sem, Ohene K.; Cooper, Richard S.; Khudyakov, Yury E.

2015-01-01

Globally, hepatitis C Virus (HCV) infection is responsible for a large proportion of persons with liver disease, including cancer. The infection is highly prevalent in sub-Saharan Africa. West Africa was identified as a geographic origin of two HCV genotypes. However, little is known about the genetic composition of HCV populations in many countries of the region. Using conventional and next-generation sequencing (NGS), we identified and genetically characterized 65 HCV strains circulating among HCV-positive blood donors in Kumasi, Ghana. Phylogenetic analysis using consensus sequences derived from 3 genomic regions of the HCV genome, 5'-untranslated region, hypervariable region 1 (HVR1) and NS5B gene, consistently classified the HCV variants (n = 65) into genotypes 1 (HCV-1, 15%) and genotype 2 (HCV-2, 85%). The Ghanaian and West African HCV-2 NS5B sequences were found completely intermixed in the phylogenetic tree, indicating a substantial genetic heterogeneity of HCV-2 in Ghana. Analysis of HVR1 sequences from intra-host HCV variants obtained by NGS showed that three donors were infected with >1 HCV strain, including infections with 2 genotypes. Two other donors share an HCV strain, indicating HCV transmission between them. The HCV-2 strain sampled from one donor was replaced with another HCV-2 strain after only 2 months of observation, indicating rapid strain switching. Bayesian analysis estimated that the HCV-2 strains in Ghana were expanding since the 16th century. The blood donors in Kumasi, Ghana, are infected with a very heterogeneous HCV population of HCV-1 and HCV-2, with HCV-2 being prevalent. The detection of three cases of co- or super-infections and transmission linkage between 2 cases suggests frequent opportunities for HCV exposure among the blood donors and is consistent with the reported high HCV prevalence. The conditions for effective HCV-2 transmission existed for ~ 3–4 centuries, indicating a long epidemic history of HCV-2 in Ghana. PMID:26683463

Distinctive archaebacterial species associated with anaerobic rumen protozoan Entodinium caudatum.

PubMed

Tóthová, T; Piknová, M; Kisidayová, S; Javorský, P; Pristas, P

2008-01-01

The diversity of archaebacteria associated with anaerobic rumen protozoan Entodinium caudatum in long term in vitro culture was investigated by denaturing gradient gel electrophoresis (DGGE) analysis of hypervariable V3 region of archaebacterial 16S rRNA gene. PCR was accomplished directly from DNA extracted from a single protozoal cell and from total community genomic DNA and the obtained fingerprints were compared. The analysis indicated the presence of a solitary intensive band present in Entodinium caudatum single cell DNA, which had no counterparts in the profile from total DNA. The identity of archaebacterium represented by this band was determined by sequence analysis which showed that the sequence fell to the cluster of ciliate symbiotic methanogens identified recently by 16S gene library approach.

The complementarity-determining region sequences in IgY antivenom hypervariable regions.

PubMed

da Rocha, David Gitirana; Fernandez, Jorge Hernandez; de Almeida, Claudia Maria Costa; da Silva, Claudia Letícia; Magnoli, Fabio Carlos; da Silva, Osmair Élder; da Silva, Wilmar Dias

2017-08-01

The data presented in this article are related to the research article entitled "Development of IgY antibodies against anti-snake toxins endowed with highly lethal neutralizing activity" (da Rocha et al., 2017) [1]. Complementarity-determining region (CDR) sequences are variable antibody (Ab) sequences that respond with specificity, duration and strength to identify and bind to antigen (Ag) epitopes. B lymphocytes isolated from hens immunized with Bitis arietans (Ba) and anti- Crotalus durissus terrificus (Cdt) venoms and expressing high specificity, affinity and toxicity neutralizing antibody titers were used as DNA sources. The VLF1, CDR1, CDR2, VLR1 and CDR3 sequences were validated by BLASTp, and values corresponding to IgY V L and V H anti-Ba or anti-Cdt venoms were identified, registered [ Gallus gallus IgY Fv Light chain (GU815099)/ Gallus gallus IgY Fv Heavy chain (GU815098)] and used for molecular modeling of IgY scFv anti-Ba. The resulting CDR1, CDR2 and CDR3 sequences were combined to construct the three - dimensional structure of the Ab paratope.

Population structure and historical demography of South American sea lions provide insights into the catastrophic decline of a marine mammal population.

PubMed

Hoffman, J I; Kowalski, G J; Klimova, A; Eberhart-Phillips, L J; Staniland, I J; Baylis, A M M

2016-07-01

Understanding the causes of population decline is crucial for conservation management. We therefore used genetic analysis both to provide baseline data on population structure and to evaluate hypotheses for the catastrophic decline of the South American sea lion (Otaria flavescens) at the Falkland Islands (Malvinas) in the South Atlantic. We genotyped 259 animals from 23 colonies across the Falklands at 281 bp of the mitochondrial hypervariable region and 22 microsatellites. A weak signature of population structure was detected, genetic diversity was moderately high in comparison with other pinniped species, and no evidence was found for the decline being associated with a strong demographic bottleneck. By combining our mitochondrial data with published sequences from Argentina, Brazil, Chile and Peru, we also uncovered strong maternally directed population structure across the geographical range of the species. In particular, very few shared haplotypes were found between the Falklands and South America, and this was reflected in correspondingly low migration rate estimates. These findings do not support the prominent hypothesis that the decline was caused by migration to Argentina, where large-scale commercial harvesting operations claimed over half a million animals. Thus, our study not only provides baseline data for conservation management but also reveals the potential for genetic studies to shed light upon long-standing questions pertaining to the history and fate of natural populations.

Bathymetric and geographic population structure in the pan-Atlantic deep-sea bivalve Deminucula atacellana (Schenck, 1939).

PubMed

Zardus, John D; Etter, Ron J; Chase, Michael R; Rex, Michael A; Boyle, Elizabeth E

2006-03-01

The deep-sea soft-sediment environment hosts a diverse and highly endemic fauna of uncertain origin. We know little about how this fauna evolved because geographic patterns of genetic variation, the essential information for inferring patterns of population differentiation and speciation are poorly understood. Using formalin-fixed specimens from archival collections, we quantify patterns of genetic variation in the protobranch bivalve Deminucula atacellana, a species widespread throughout the Atlantic Ocean at bathyal and abyssal depths. Samples were taken from 18 localities in the North American, West European and Argentine basins. A hypervariable region of mitochondrial 16S rDNA was amplified by polymerase chain reaction (PCR) and sequenced from 130 individuals revealing 21 haplotypes. Except for several important exceptions, haplotypes are unique to each basin. Overall gene diversity is high (h = 0.73) with pronounced population structure (Phi(ST) = 0.877) and highly significant geographic associations (P < 0.0001). Sequences cluster into four major clades corresponding to differences in geography and depth. Genetic divergence was much greater among populations at different depths within the same basin, than among those at similar depths but separated by thousands of kilometres. Isolation by distance probably explains much of the interbasin variation. Depth-related divergence may reflect historical patterns of colonization or strong environmental selective gradients. Broadly distributed deep-sea organisms can possess highly genetically divergent populations, despite the lack of any morphological divergence.

Effect of Bifidobacterium breve on the Intestinal Microbiota of Coeliac Children on a Gluten Free Diet: A Pilot Study.

PubMed

Quagliariello, Andrea; Aloisio, Irene; Bozzi Cionci, Nicole; Luiselli, Donata; D'Auria, Giuseppe; Martinez-Priego, Llúcia; Pérez-Villarroya, David; Langerholc, Tomaž; Primec, Maša; Mičetić-Turk, Dušanka; Di Gioia, Diana

2016-10-22

Coeliac disease (CD) is associated with alterations of the intestinal microbiota. Although several Bifidobacterium strains showed anti-inflammatory activity and prevention of toxic gliadin peptides generation in vitro, few data are available on their efficacy when administered to CD subjects. This study evaluated the effect of administration for three months of a food supplement based on two Bifidobacterium breve strains (B632 and BR03) to restore the gut microbial balance in coeliac children on a gluten free diet (GFD). Microbial DNA was extracted from faeces of 40 coeliac children before and after probiotic or placebo administration and 16 healthy children (Control group). Sequencing of the amplified V3-V4 hypervariable region of 16S rRNA gene as well as qPCR of Bidobacterium spp., Lactobacillus spp., Bacteroides fragilis group Clostridium sensu stricto and enterobacteria were performed. The comparison between CD subjects and Control group revealed an alteration in the intestinal microbial composition of coeliacs mainly characterized by a reduction of the Firmicutes/Bacteroidetes ratio, of Actinobacteria and Euryarchaeota . Regarding the effects of the probiotic, an increase of Actinobacteria was found as well as a re-establishment of the physiological Firmicutes/Bacteroidetes ratio. Therefore, a three-month administration of B. breve strains helps in restoring the healthy percentage of main microbial components.

A comparative analysis of microbial profile of Guinea fowl and chicken using metagenomic approach

PubMed Central

Bhogoju, Sarayu; Wang, Xiaofei; Darris, Carl; Kilonzo-Nthenge, Agnes

2018-01-01

Probiotics are live microbial feed supplements that promote growth and health to the host by minimizing non-essential and pathogenic microorganisms in the host’s gastrointestinal tract (GIT). The campaign to minimize excessive use of antibiotics in poultry production has necessitated development of probiotics with broad application in multiple poultry species. Design of such probiotics requires understanding of the diversity or similarity in microbial profiles among avian species of economic importance. Therefore, the objective of this research was to establish and compare the microbial profiles of the GIT of Guinea fowl and chicken and to establish the microbial diversity or similarity between the two avian species. A metagenomic approach consisting of the amplification and sequence analysis of the hypervariable regions V1-V9 of the 16S rRNA gene was used to identify the GIT microbes. Collectively, we detected more than 150 microbial families. The total number of microbial species detected in the chicken GIT was higher than that found in the Guinea Fowl GIT. Our studies also revealed phylogenetic diversity among the microbial species found in chicken and guinea fowl. The phylum Firmicutes was most abundant in both avian species whereas Phylum Actinobacteria was most abundant in chickens than Guinea fowls. The diversity of the microbial profiles found in broiler chickens and Guinea fowls suggest that the design of effective avian probiotics would require species specificity. PMID:29494648

Genotyping of single spore isolates of a Pasteuria penetrans population occurring in Florida using SNP-based markers.

PubMed

Joseph, S; Schmidt, L M; Danquah, W B; Timper, P; Mekete, T

2017-02-01

To generate single spore lines of a population of bacterial parasite of root-knot nematode (RKN), Pasteuria penetrans, isolated from Florida and examine genotypic variation and virulence characteristics exist within the population. Six single spore lines (SSP), 16SSP, 17SSP, 18SSP, 25SSP, 26SSP and 30SSP were generated. Genetic variability was evaluated by comparing single-nucleotide polymorphisms (SNPs) in six protein-coding genes and the 16S rRNA gene. An average of one SNP was observed for every 69 bp in the 16S rRNA, whereas no SNPs were observed in the protein-coding sequences. Hierarchical cluster analysis of 16S rRNA sequences placed the clones into three distinct clades. Bio-efficacy analysis revealed significant heterogeneity in the level virulence and host specificity between the individual clones. The SNP markers developed to the 5' hypervariable region of the 16S rRNA gene may be useful in biotype differentiation within a population of P. penetrans. This study demonstrates an efficient method for generating single spore lines of P. penetrans and gives a deep insight into genetic heterogeneity and varying level of virulence exists within a population parasitizing a specific Meloidogyne sp. host. The results also suggest that the application of generalist spore lines in nematode management may achieve broad RKN control. © 2016 The Society for Applied Microbiology.

Population structure and historical demography of South American sea lions provide insights into the catastrophic decline of a marine mammal population

PubMed Central

Hoffman, J. I.; Kowalski, G. J.; Klimova, A.; Staniland, I. J.; Baylis, A. M. M.

2016-01-01

Understanding the causes of population decline is crucial for conservation management. We therefore used genetic analysis both to provide baseline data on population structure and to evaluate hypotheses for the catastrophic decline of the South American sea lion (Otaria flavescens) at the Falkland Islands (Malvinas) in the South Atlantic. We genotyped 259 animals from 23 colonies across the Falklands at 281 bp of the mitochondrial hypervariable region and 22 microsatellites. A weak signature of population structure was detected, genetic diversity was moderately high in comparison with other pinniped species, and no evidence was found for the decline being associated with a strong demographic bottleneck. By combining our mitochondrial data with published sequences from Argentina, Brazil, Chile and Peru, we also uncovered strong maternally directed population structure across the geographical range of the species. In particular, very few shared haplotypes were found between the Falklands and South America, and this was reflected in correspondingly low migration rate estimates. These findings do not support the prominent hypothesis that the decline was caused by migration to Argentina, where large-scale commercial harvesting operations claimed over half a million animals. Thus, our study not only provides baseline data for conservation management but also reveals the potential for genetic studies to shed light upon long-standing questions pertaining to the history and fate of natural populations. PMID:27493782

Oligotrophic lagoons of the South Pacific Ocean are home to a surprising number of novel eukaryotic microorganisms.

PubMed

Kim, Eunsoo; Sprung, Ben; Duhamel, Solange; Filardi, Christopher; Kyoon Shin, Mann

2016-12-01

The diversity of microbial eukaryotes was surveyed by environmental sequencing from tropical lagoon sites of the South Pacific, collected through the American Museum of Natural History (AMNH)'s Explore21 expedition to the Solomon Islands in September 2013. The sampled lagoons presented low nutrient concentrations typical of oligotrophic waters, but contained levels of chlorophyll a, a proxy for phytoplankton biomass, characteristic of meso- to eutrophic waters. Two 18S rDNA hypervariable sites, the V4 and V8-V9 regions, were amplified from the total of eight lagoon samples and sequenced on the MiSeq system. After assembly, clustering at 97% similarity, and removal of singletons and chimeras, a total of 2741 (V4) and 2606 (V8-V9) operational taxonomic units (OTUs) were identified. Taxonomic annotation of these reads, including phylogeny, was based on a combination of automated pipeline and manual inspection. About 18.4% (V4) and 13.8% (V8-V9) of the OTUs could not be assigned to any of the known eukaryotic groups. Of these, we focused on OTUs that were not divergent and possessed multiple sources of evidence for their existence. Phylogenetic analyses of these sequences revealed more than ten branches that might represent new deeply-branching lineages of microbial eukaryotes, currently without any cultured representatives or morphological information. © 2016 Society for Applied Microbiology and John Wiley & Sons Ltd.

Faecal microbiota of healthy adults in south India: Comparison of a tribal & a rural population.

PubMed

Ramadass, Balamurugan; Rani, B Sandya; Pugazhendhi, Srinivasan; John, K R; Ramakrishna, Balakrishnan S

2017-02-01

The relevance of the gut microbiota to human health is increasingly appreciated. The objective of this study was to compare the gut microbiota of a group of adult tribals with that of healthy adult villagers in Tamil Nadu, India. Faeces were collected from 10 healthy tribal adults (TAs) in the Jawadhi hills and from 10 healthy villagers [rural adults (RAs)] in Vellore district, Tamil Nadu. DNA was extracted, and 456 bp segments comprising hypervariable regions 3 and 4 of the 16S rRNA gene were amplified, barcoded and 454 sequenced. Totally 227,710 good-quality reads were analyzed. TAs consumed a millets-based diet, ate pork every day, and did not consume milk or milk products. RAs consumed a rice-based diet with meat intake once a week. In both groups, Firmicutes was the most abundant phylum, followed by Proteobacteria, Bacteroidetes and Actinobacteria. The median Firmicutes-to-Bacteroidetes ratio was 34.0 in TA and 92.9 in RA groups. Actinobacteria were significantly low in TA, possibly due to non-consumption of milk. Clostridium constituted the most abundant genus in both groups, but was significantly more abundant in TAs than RAs, while Streptococcus was significantly more abundant in RA (P<0.05). Analyses of genetic distance revealed that the microbiota were distinctly different between TA and RA, and principal component analysis using 550 distinct taxonomically identifiable sequences revealed a clear separation of microbiota composition in the two groups. Phylogenetic analysis of major microbiota indicated clustering of microbial groups at different major branch points for TAs and RAs. Phylum Firmicutes and genus Clostridium constituted the bulk of the faecal microbiota, while significant differences in composition between the groups were probably due to differences in diet and lifestyle.

Comprehensive analysis of mutations in the hepatitis delta virus genome based on full-length sequencing in a nationwide cohort study and evolutionary pattern during disease progression.

PubMed

Shirvani-Dastgerdi, E; Amini-Bavil-Olyaee, S; Alavian, S Moayed; Trautwein, C; Tacke, F

2015-05-01

Delta hepatitis, caused by co-infection or super-infection of hepatitis D virus (HDV) in hepatitis B virus (HBV) -infected patients, is the most severe form of chronic hepatitis, often progressing to liver cirrhosis and liver failure. Although 15 million individuals are affected worldwide, molecular data on the HDV genome and its proteins, small and large delta antigen (S-/L-HDAg), are limited. We therefore conducted a nationwide study in HBV-HDV-infected patients from Iran and successfully amplified 38 HDV full genomes and 44 L-HDAg sequences from 34 individuals. Phylogenetic analyses of full-length HDV and L-HDAg isolates revealed that all strains clustered with genotype 1 and showed high genotypic distances to HDV genotypes 2 to 8, with a maximal distance to genotype 3. Longitudinal analyses in individual patients indicated a reverse evolutionary trend, especially in L-HDAg amino acid composition, over time. Besides multiple sequence variations in the hypervariable region of HDV, nucleotide substitutions preferentially occurred in the stabilizing P4 domain of the HDV ribozyme. A high rate of single amino acid changes was detected in structural parts of L-HDAg, whereas its post-translational modification sites were highly conserved. Interestingly, several non-synonymous mutations were positively selected that affected immunogenic epitopes of L-HDAg towards CD8 T-cell- and B-cell-driven immune responses. Hence, our comprehensive molecular analysis comprising a nationwide cohort revealed phylogenetic relationships and provided insight into viral evolution within individual hosts. Moreover, preferential areas of frequent mutations in the HDV ribozyme and antigen protein were determined in this study. Copyright © 2014 European Society of Clinical Microbiology and Infectious Diseases. Published by Elsevier Ltd. All rights reserved.

Highly diverse microbiota in dental root canals in cases of apical periodontitis (data of illumina sequencing).

PubMed

Vengerfeldt, Veiko; Špilka, Katerina; Saag, Mare; Preem, Jens-Konrad; Oopkaup, Kristjan; Truu, Jaak; Mändar, Reet

2014-11-01

Chronic apical periodontitis (CAP) is a frequent condition that has a considerable effect on a patient's quality of life. We aimed to reveal root canal microbial communities in antibiotic-naive patients by applying Illumina sequencing (Illumina Inc, San Diego, CA). Samples were collected under strict aseptic conditions from 12 teeth (5 with primary CAP, 3 with secondary CAP, and 4 with a periapical abscess [PA]) and characterized by profiling the microbial community on the basis of the V6 hypervariable region of the 16S ribosomal RNA gene by using Illumina HiSeq2000 sequencing combinatorial sequence-tagged polymerase chain reaction products. Root canal specimens displayed highly polymicrobial communities in all 3 patient groups. One sample contained 5-8 (mean = 6.5) phyla of bacteria. The most numerous were Firmicutes and Bacteroidetes, but Actinobacteria, Fusobacteria, Proteobacteria, Spirochaetes, Tenericutes, and Synergistetes were also present in most of the patients. One sample contained 30-70 different operational taxonomic units; the mean (± standard deviation) was lower in the primary CAP group (36 ± 4) than in the PA (45 ± 4) and secondary CAP (43 ± 13) groups (P < .05). The communities were individually different, but anaerobic bacteria predominated as the rule. Enterococcus faecalis was found only in patients with secondary CAP. One PA sample displayed a significantly high proportion (47%) of Proteobacteria, mainly at the expense of Janthinobacterium lividum. This study provided an in-depth characterization of the microbiota of periapical tissues, revealing highly polymicrobial communities and minor differences between the study groups. A full understanding of the etiology of periodontal disease will only be possible through further in-depth systems-level analyses of the host-microbiome interaction. Copyright © 2014 American Association of Endodontists. Published by Elsevier Inc. All rights reserved.

Faecal microbiota of healthy adults in south India: Comparison of a tribal & a rural population

PubMed Central

Ramadass, Balamurugan; Rani, B. Sandya; Pugazhendhi, Srinivasan; John, K.R.; Ramakrishna, Balakrishnan S.

2017-01-01

Background & objectives: The relevance of the gut microbiota to human health is increasingly appreciated. The objective of this study was to compare the gut microbiota of a group of adult tribals with that of healthy adult villagers in Tamil Nadu, India. Methods: Faeces were collected from 10 healthy tribal adults (TAs) in the Jawadhi hills and from 10 healthy villagers [rural adults (RAs)] in Vellore district, Tamil Nadu. DNA was extracted, and 456 bp segments comprising hypervariable regions 3 and 4 of the 16S rRNA gene were amplified, barcoded and 454 sequenced. Results: Totally 227,710 good-quality reads were analyzed. TAs consumed a millets-based diet, ate pork every day, and did not consume milk or milk products. RAs consumed a rice-based diet with meat intake once a week. In both groups, Firmicutes was the most abundant phylum, followed by Proteobacteria, Bacteroidetes and Actinobacteria. The median Firmicutes-to-Bacteroidetes ratio was 34.0 in TA and 92.9 in RA groups. Actinobacteria were significantly low in TA, possibly due to non-consumption of milk. Clostridium constituted the most abundant genus in both groups, but was significantly more abundant in TAs than RAs, while Streptococcus was significantly more abundant in RA (P<0.05). Analyses of genetic distance revealed that the microbiota were distinctly different between TA and RA, and principal component analysis using 550 distinct taxonomically identifiable sequences revealed a clear separation of microbiota composition in the two groups. Phylogenetic analysis of major microbiota indicated clustering of microbial groups at different major branch points for TAs and RAs. Interpretation & conclusions: Phylum Firmicutes and genus Clostridium constituted the bulk of the faecal microbiota, while significant differences in composition between the groups were probably due to differences in diet and lifestyle. PMID:28639601

Babesia lengau sp. nov., a novel Babesia species in cheetah (Acinonyx jubatus, Schreber, 1775) populations in South Africa.

PubMed

Bosman, Anna-Mari; Oosthuizen, Marinda C; Peirce, Michael A; Venter, Estelle H; Penzhorn, Barend L

2010-08-01

In a previous paper, we reported on a large number of cheetah blood specimens that gave positive signals only for Babesia and/or Theileria genus-specific probes on the reverse line blot (RLB) assay, indicating the presence of a novel species or variant of an existing species. Some of these specimens were investigated further by microscopic, serological, sequencing, and phylogenetic analyses. The near-full-length 18S rRNA genes of 13 samples, as well as the second internal transcribed spacer (ITS2) region, were amplified, cloned, and sequenced. A species-specific RLB probe, designed to target the hypervariable V4 region of the 18S rRNA gene for detection of the novel Babesia sp., was used to screen an additional 137 cheetah blood specimens for the presence of the species. The prevalence of infection was 28.5%. Here we describe the morphology and phylogenetic relationships of the novel species, which we have named Babesia lengau sp. nov.

Babesia lengau sp. nov., a Novel Babesia Species in Cheetah (Acinonyx jubatus, Schreber, 1775) Populations in South Africa ▿

PubMed Central

Bosman, Anna-Mari; Oosthuizen, Marinda C.; Peirce, Michael A.; Venter, Estelle H.; Penzhorn, Barend L.

2010-01-01

In a previous paper, we reported on a large number of cheetah blood specimens that gave positive signals only for Babesia and/or Theileria genus-specific probes on the reverse line blot (RLB) assay, indicating the presence of a novel species or variant of an existing species. Some of these specimens were investigated further by microscopic, serological, sequencing, and phylogenetic analyses. The near-full-length 18S rRNA genes of 13 samples, as well as the second internal transcribed spacer (ITS2) region, were amplified, cloned, and sequenced. A species-specific RLB probe, designed to target the hypervariable V4 region of the 18S rRNA gene for detection of the novel Babesia sp., was used to screen an additional 137 cheetah blood specimens for the presence of the species. The prevalence of infection was 28.5%. Here we describe the morphology and phylogenetic relationships of the novel species, which we have named Babesia lengau sp. nov. PMID:20519464

Rheumatoid arthritis and Swine influenza vaccine: a case report.

PubMed

Basra, Gurjot; Jajoria, Praveen; Gonzalez, Emilio

2012-01-01

Rheumatoid arthritis (RA) is the most common chronic inflammatory joint disease. Multiple scientific articles have documented that vaccinations for influenza, MMR, and HBV, to name a few, could be triggers of RA in genetically predisposed individuals. However, there is limited data regarding the association of swine flu vaccine (H1N1) and RA. We report the case of a Mexican American female who developed RA right after vaccination with H1N1 vaccine. Genetically, RA has consistently been associated with an epitope in the third hypervariable region of the HLA-DR β chains, known as the "shared epitope", which is found primarily in DR4 and DR1 regions. The presence of HLA-DRB1 alleles is associated with susceptibility to RA in Mexican Americans. Hence, certain individuals with the presence of the "shared epitope" may develop RA following specific vaccinations. To our knowledge, this is the first reported case of RA following vaccination with the swine flu vaccine.

Vaccination with dendritic cells pulsed with hepatitis C pseudo particles induces specific immune responses in mice

PubMed Central

Weigand, Kilian; Voigt, Franziska; Encke, Jens; Hoyler, Birgit; Stremmel, Wolfgang; Eisenbach, Christoph

2012-01-01

AIM: To explore dendritic cells (DCs) multiple functions in immune modulation. METHODS: We used bone-marrow derived dendritic cells from BALB/c mice pulsed with pseudo particles from the hepatitis C virus to vaccinate naive BALB/c mice. Hepatitis C virus (HCV) pseudo particles consist of the genotype 1b derived envelope proteins E1 and E2, covering a non-HCV core structure. Thus, not a single epitope, but the whole “viral surface” induces immunogenicity. For vaccination, mature and activated DC were injected subcutaneously twice. RESULTS: Humoral and cellular immune responses measured by enzyme-linked immunosorbent assay and interferon-gamma enzyme-linked immunosorbent spot test showed antibody production as well as T-cells directed against HCV. Furthermore, T-cell responses confirmed two highly immunogenic regions in E1 and E2 outside the hypervariable region 1. CONCLUSION: Our results indicate dendritic cells as a promising vaccination model for HCV infection that should be evaluated further. PMID:22371638

Differential diagnosis between complete mole and hydropic abortus by deoxyribonucleic acid fingerprints.

PubMed

Nobunaga, T; Azuma, C; Kimura, T; Tokugawa, Y; Takemura, M; Kamiura, S; Saji, F; Tanizawa, O

1990-08-01

We used a new method of deoxyribonucleic acid fingerprint analysis to obtain the differential diagnosis between complete mole and hydropic abortus. This method with a deoxyribonucleic acid minisatellite probe requires only a small amount of tissue sample and peripheral blood, and presents individual specific restriction fragment length polymorphisms (deoxyribonucleic acid "fingerprints") by simultaneous detection of many hypervariable regions (minisatellite regions) widely dispersed in the human genome. Southern blot hybridization showed that in cases of complete mole, all polymorphic fragments were exclusively inherited from the father. Some of the polymorphic bands of paternal deoxyribonucleic acid were not observed in molar deoxyribonucleic acid. However, in the hydropic abortus, the polymorphic fragments could be traced back to its parent. These results indicate that deoxyribonucleic acid fingerprints could distinguish the abnormal fertilization of complete mole (androgenesis) from the normal fertilization of hydropic abortus by identifying the difference in genetic variations between complete mole and hydropic abortus at the deoxyribonucleic acid level.

Mutation mechanisms that underlie turnover of a human telomere-adjacent segmental duplication containing an unstable minisatellite.

PubMed

Hills, Mark; Jeyapalan, Jennie N; Foxon, Jennifer L; Royle, Nicola J

2007-04-01

Subterminal regions, juxtaposed to telomeres on human chromosomes, contain a high density of segmental duplications, but relatively little is known about the evolutionary processes that underlie sequence turnover in these regions. We have characterized a segmental duplication adjacent to the Xp/Yp telomere, each copy containing a hypervariable array of the DXYS14 minisatellite. Both DXYS14 repeat arrays mutate at a high rate (0.3 and 0.2% per gamete) but linkage disequilibrium analysis across 27 SNPs and a direct crossover assay show that recombination during meiosis is suppressed. Therefore instability at DXYS14a and b is dominated by intra-allelic processes or possibly conversion limited to the repeat arrays. Furthermore some chromosomes (14%) carry only one copy of the duplicon, including one DXYS14 repeat array that is also highly mutable (1.2% per gamete). To explain these and other observations, we propose there is another low-rate mutation process that causes copy number change in part or all of the duplicon.

First report of bacterial community from a Bat Guano using Illumina next-generation sequencing.

PubMed

De Mandal, Surajit; Zothansanga; Panda, Amritha Kumari; Bisht, Satpal Singh; Senthil Kumar, Nachimuthu

2015-06-01

V4 hypervariable region of 16S rDNA was analyzed for identifying the bacterial communities present in Bat Guano from the unexplored cave - Pnahkyndeng, Meghalaya, Northeast India. Metagenome comprised of 585,434 raw Illumina sequences with a 59.59% G+C content. A total of 416,490 preprocessed reads were clustered into 1282 OTUs (operational taxonomical units) comprising of 18 bacterial phyla. The taxonomic profile showed that the guano bacterial community is dominated by Chloroflexi, Actinobacteria and Crenarchaeota which account for 70.73% of all sequence reads and 43.83% of all OTUs. Metagenome sequence data are available at NCBI under the accession no. SRP051094. This study is the first to characterize Bat Guano bacterial community using next-generation sequencing approach.

First report of bacterial community from a Bat Guano using Illumina next-generation sequencing

PubMed Central

De Mandal, Surajit; Zothansanga; Panda, Amritha Kumari; Bisht, Satpal Singh; Senthil Kumar, Nachimuthu

2015-01-01

V4 hypervariable region of 16S rDNA was analyzed for identifying the bacterial communities present in Bat Guano from the unexplored cave — Pnahkyndeng, Meghalaya, Northeast India. Metagenome comprised of 585,434 raw Illumina sequences with a 59.59% G+C content. A total of 416,490 preprocessed reads were clustered into 1282 OTUs (operational taxonomical units) comprising of 18 bacterial phyla. The taxonomic profile showed that the guano bacterial community is dominated by Chloroflexi, Actinobacteria and Crenarchaeota which account for 70.73% of all sequence reads and 43.83% of all OTUs. Metagenome sequence data are available at NCBI under the accession no. SRP051094. This study is the first to characterize Bat Guano bacterial community using next-generation sequencing approach. PMID:26484190

Paternity after bone marrow transplantation following conditioning with total body irradiation.

PubMed

Pakkala, S; Lukka, M; Helminen, P; Koskimies, S; Ruutu, T

1994-04-01

A 28-year-old man with chronic myeloid leukaemia received an allogeneic bone marrow transplant after conditioning with daunorubicin, cyclophosphamide and fractionated total body irradiation (TBI). Four years later his wife gave birth to a healthy child. Although the patient was azospermic serologic HLA testing suggested that the patient was the father of the child. DNA fingerprinting as well as analysis of three variable number of tandem repeats (VNTR) loci D1S80 (MCT118), D17S30 (YNZ22) and the apolipoprotein B hypervariable region (apo B 3') gave unequivocal results showing that the patient was the father. Fathering a child after TBI-containing regimen has been very rare and this is the first case where the paternity has been proven with DNA methodology.

Molecular Cloning and Characterization of an Acetylcholinesterase cDNA in the Brown Planthopper, Nilaparvata lugens

PubMed Central

Yang, Zhifan; Chen, Jun; Chen, Yongqin; Jiang, Sijing

2010-01-01

A full cDNA encoding an acetylcholinesterase (AChE, EC 3.1.1.7) was cloned and characterized from the brown planthopper, Nilaparvata lugens Stål (Hemiptera: Delphacidae). The complete cDNA (2467 bp) contains a 1938-bp open reading frame encoding 646 amino acid residues. The amino acid sequence of the AChE deduced from the cDNA consists of 30 residues for a putative signal peptide and 616 residues for the mature protein with a predicted molecular weight of 69,418. The three residues (Ser242, Glu371, and His485) that putatively form the catalytic triad and the six Cys that form intra-subunit disulfide bonds are completely conserved, and 10 out of the 14 aromatic residues lining the active site gorge of the AChE are also conserved. Northern blot analysis of poly(A)+ RNA showed an approximately 2.6-kb transcript, and Southern blot analysis revealed there likely was just a single copy of this gene in N. lugens. The deduced protein sequence is most similar to AChE of Nephotettix cincticeps with 83% amino acid identity. Phylogenetic analysis constructed with 45 AChEs from 30 species showed that the deduced N. lugens AChE formed a cluster with the other 8 insect AChE2s. Additionally, the hypervariable region and amino acids specific to insect AChE2 also existed in the AChE of N. lugens. The results revealed that the AChE cDNA cloned in this work belongs to insect AChE2 subgroup, which is orthologous to Drosophila AChE. Comparison of the AChEs between the susceptible and resistant strains revealed a point mutation, Gly185Ser, is likely responsible for the insensitivity of the AChE to methamidopho in the resistant strain. PMID:20874389

Evolutionary dynamics of an expressed MHC class IIβ locus in the Ranidae (Anura) uncovered by genome walking and high-throughput amplicon sequencing

USGS Publications Warehouse

Mulder, Kevin P.; Cortazar-Chinarro, Maria; Harris, D. James; Crottini, Angelica; Grant, Evan H. Campbell; Fleischer, Robert C.; Savage, Anna E.

2017-01-01

The Major Histocompatibility Complex (MHC) is a genomic region encoding immune loci that are important and frequently used markers in studies of adaptive genetic variation and disease resistance. Given the primary role of infectious diseases in contributing to global amphibian declines, we characterized the hypervariable exon 2 and flanking introns of the MHC Class IIβ chain for 17 species of frogs in the Ranidae, a speciose and cosmopolitan family facing widespread pathogen infections and declines. We find high levels of genetic variation concentrated in the Peptide Binding Region (PBR) of the exon. Ten codons are under positive selection, nine of which are located in the mammal-defined PBR. We hypothesize that the tenth codon (residue 21) is an amphibian-specific PBR site that may be important in disease resistance. Trans-species and trans-generic polymorphisms are evident from exon-based genealogies, and co-phylogenetic analyses between intron, exon and mitochondrial based reconstructions reveal incongruent topologies, likely due to different locus histories. We developed two sets of barcoded adapters that reliably amplify a single and likely functional locus in all screened species using both 454 and Illumina based sequencing methods. These primers provide a resource for multiplexing and directly sequencing hundreds of samples in a single sequencing run, avoiding the labour and chimeric sequences associated with cloning, and enabling MHC population genetic analyses. Although the primers are currently limited to the 17 species we tested, these sequences and protocols provide a useful genetic resource and can serve as a starting point for future disease, adaptation and conservation studies across a range of anuran taxa.

Pyrosequencing-Derived Bacterial, Archaeal, and Fungal Diversity of Spacecraft Hardware Destined for Mars

PubMed Central

Vaishampayan, Parag; Nilsson, Henrik R.; Torok, Tamas; Venkateswaran, Kasthuri

2012-01-01

Spacecraft hardware and assembly cleanroom surfaces (233 m2 in total) were sampled, total genomic DNA was extracted, hypervariable regions of the 16S rRNA gene (bacteria and archaea) and ribosomal internal transcribed spacer (ITS) region (fungi) were subjected to 454 tag-encoded pyrosequencing PCR amplification, and 203,852 resulting high-quality sequences were analyzed. Bioinformatic analyses revealed correlations between operational taxonomic unit (OTU) abundance and certain sample characteristics, such as source (cleanroom floor, ground support equipment [GSE], or spacecraft hardware), cleaning regimen applied, and location about the facility or spacecraft. National Aeronautics and Space Administration (NASA) cleanroom floor and GSE surfaces gave rise to a larger number of diverse bacterial communities (619 OTU; 20 m2) than colocated spacecraft hardware (187 OTU; 162 m2). In contrast to the results of bacterial pyrosequencing, where at least some sequences were generated from each of the 31 sample sets examined, only 13 and 18 of these sample sets gave rise to archaeal and fungal sequences, respectively. As was the case for bacteria, the abundance of fungal OTU in the GSE surface samples dramatically diminished (9× less) once cleaning protocols had been applied. The presence of OTU representative of actinobacteria, deinococci, acidobacteria, firmicutes, and proteobacteria on spacecraft surfaces suggests that certain bacterial lineages persist even following rigorous quality control and cleaning practices. The majority of bacterial OTU observed as being recurrent belonged to actinobacteria and alphaproteobacteria, supporting the hypothesis that the measures of cleanliness exerted in spacecraft assembly cleanrooms (SAC) inadvertently select for the organisms which are the most fit to survive long journeys in space. PMID:22729532

Microbial eukaryote plankton communities of high-mountain lakes from three continents exhibit strong biogeographic patterns.

PubMed

Filker, Sabine; Sommaruga, Ruben; Vila, Irma; Stoeck, Thorsten

2016-05-01

Microbial eukaryotes hold a key role in aquatic ecosystem functioning. Yet, their diversity in freshwater lakes, particularly in high-mountain lakes, is relatively unknown compared with the marine environment. Low nutrient availability, low water temperature and high ultraviolet radiation make most high-mountain lakes extremely challenging habitats for life and require specific molecular and physiological adaptations. We therefore expected that these ecosystems support a plankton diversity that differs notably from other freshwater lakes. In addition, we hypothesized that the communities under study exhibit geographic structuring. Our rationale was that geographic dispersal of small-sized eukaryotes in high-mountain lakes over continental distances seems difficult. We analysed hypervariable V4 fragments of the SSU rRNA gene to compare the genetic microbial eukaryote diversity in high-mountain lakes located in the European Alps, the Chilean Altiplano and the Ethiopian Bale Mountains. Microbial eukaryotes were not globally distributed corroborating patterns found for bacteria, multicellular animals and plants. Instead, the plankton community composition emerged as a highly specific fingerprint of a geographic region even on higher taxonomic levels. The intraregional heterogeneity of the investigated lakes was mirrored in shifts in microbial eukaryote community structure, which, however, was much less pronounced compared with interregional beta-diversity. Statistical analyses revealed that on a regional scale, environmental factors are strong predictors for plankton community structures in high-mountain lakes. While on long-distance scales (>10 000 km), isolation by distance is the most plausible scenario, on intermediate scales (up to 6000 km), both contemporary environmental factors and historical contingencies interact to shift plankton community structures. © 2016 John Wiley & Sons Ltd.

Evolutionary dynamics of an expressed MHC class IIβ locus in the Ranidae (Anura) uncovered by genome walking and high-throughput amplicon sequencing.

PubMed

Mulder, Kevin P; Cortazar-Chinarro, Maria; Harris, D James; Crottini, Angelica; Campbell Grant, Evan H; Fleischer, Robert C; Savage, Anna E

2017-11-01

The Major Histocompatibility Complex (MHC) is a genomic region encoding immune loci that are important and frequently used markers in studies of adaptive genetic variation and disease resistance. Given the primary role of infectious diseases in contributing to global amphibian declines, we characterized the hypervariable exon 2 and flanking introns of the MHC Class IIβ chain for 17 species of frogs in the Ranidae, a speciose and cosmopolitan family facing widespread pathogen infections and declines. We find high levels of genetic variation concentrated in the Peptide Binding Region (PBR) of the exon. Ten codons are under positive selection, nine of which are located in the mammal-defined PBR. We hypothesize that the tenth codon (residue 21) is an amphibian-specific PBR site that may be important in disease resistance. Trans-species and trans-generic polymorphisms are evident from exon-based genealogies, and co-phylogenetic analyses between intron, exon and mitochondrial based reconstructions reveal incongruent topologies, likely due to different locus histories. We developed two sets of barcoded adapters that reliably amplify a single and likely functional locus in all screened species using both 454 and Illumina based sequencing methods. These primers provide a resource for multiplexing and directly sequencing hundreds of samples in a single sequencing run, avoiding the labour and chimeric sequences associated with cloning, and enabling MHC population genetic analyses. Although the primers are currently limited to the 17 species we tested, these sequences and protocols provide a useful genetic resource and can serve as a starting point for future disease, adaptation and conservation studies across a range of anuran taxa. Copyright © 2017 Elsevier Ltd. All rights reserved.

Mitochondrial DNA and two Y-chromosome genes of common long-tailed macaques (Macaca fascicularis fascicularis) throughout Thailand and vicinity.

PubMed

Bunlungsup, Srichan; Imai, Hiroo; Hamada, Yuzuru; Matsudaira, Kazunari; Malaivijitnond, Suchinda

2017-02-01

Macaca fascicularis fascicularis is distributed over a wide area of Southeast Asia. Thailand is located at the center of their distribution range and is the bridge connecting the two biogeographic regions of Indochina and Sunda. However, only a few genetic studies have explored the macaques in this region. To shed some light on the evolutionary history of M. f. fascicularis, including hybridization with M. mulatta, M. f. fascicularis and M. mulatta samples of known origins throughout Thailand and the vicinity were analyzed by molecular phylogenetics using mitochondrial DNA (mtDNA), including the hypervariable region 1, and Y-chromosomal DNA, including SRY and TSPY genes. The mtDNA phylogenetic analysis divided M. f. fascicularis into five subclades (Insular Indonesia, Sundaic Thai Gulf, Vietnam, Sundaic Andaman sea coast, and Indochina) and revealed genetic differentiation between the two sides of the Thai peninsula, which had previously been reported as a single group of Malay peninsular macaques. From the estimated divergence time of the Sundaic Andaman sea coast subclade, it is proposed that after M. f. fascicularis dispersed throughout Southeast Asia, some populations on the south-easternmost Indochina (eastern Thailand, southern Cambodia and southern Vietnam at the present time) migrated south-westwards across the land bridge, which was exposed during the glacial period of the late Pleistocene epoch, to the southernmost Thailand/northern peninsular Malaysia. Then, some of them migrated north and south to colonize the Thai Andaman sea coast and northern Sumatra, respectively. The SRY-TSPY phylogenetic analysis suggested that male-mediated gene flow from M. mulatta southward to M. f. fascicularis was restricted south of, but close to, the Isthmus of Kra. There was a strong impact of the geographical factors in Thailand, such as the Isthmus of Kra, Nakhon Si Thammarat, and Phuket ranges and Sundaland, on M. f. fascicularis biogeography and their hybridization with M. mulatta. © 2016 Wiley Periodicals, Inc.

New Insights into the Lake Chad Basin Population Structure Revealed by High-Throughput Genotyping of Mitochondrial DNA Coding SNPs

PubMed Central

Černý, Viktor; Carracedo, Ángel

2011-01-01

Background Located in the Sudan belt, the Chad Basin forms a remarkable ecosystem, where several unique agricultural and pastoral techniques have been developed. Both from an archaeological and a genetic point of view, this region has been interpreted to be the center of a bidirectional corridor connecting West and East Africa, as well as a meeting point for populations coming from North Africa through the Saharan desert. Methodology/Principal Findings Samples from twelve ethnic groups from the Chad Basin (n = 542) have been high-throughput genotyped for 230 coding region mitochondrial DNA (mtDNA) Single Nucleotide Polymorphisms (mtSNPs) using Matrix-Assisted Laser Desorption/Ionization Time-Of-Flight (MALDI-TOF) mass spectrometry. This set of mtSNPs allowed for much better phylogenetic resolution than previous studies of this geographic region, enabling new insights into its population history. Notable haplogroup (hg) heterogeneity has been observed in the Chad Basin mirroring the different demographic histories of these ethnic groups. As estimated using a Bayesian framework, nomadic populations showed negative growth which was not always correlated to their estimated effective population sizes. Nomads also showed lower diversity values than sedentary groups. Conclusions/Significance Compared to sedentary population, nomads showed signals of stronger genetic drift occurring in their ancestral populations. These populations, however, retained more haplotype diversity in their hypervariable segments I (HVS-I), but not their mtSNPs, suggesting a more ancestral ethnogenesis. Whereas the nomadic population showed a higher Mediterranean influence signaled mainly by sub-lineages of M1, R0, U6, and U5, the other populations showed a more consistent sub-Saharan pattern. Although lifestyle may have an influence on diversity patterns and hg composition, analysis of molecular variance has not identified these differences. The present study indicates that analysis of mtSNPs at high resolution could be a fast and extensive approach for screening variation in population studies where labor-intensive techniques such as entire genome sequencing remain unfeasible. PMID:21533064

Genotypic variations in field isolates of Theileria species infecting giraffes (Giraffa camelopardalis tippelskirchi and Giraffa camelopardalis reticulata) in Kenya.

PubMed

Githaka, Naftaly; Konnai, Satoru; Skilton, Robert; Kariuki, Edward; Kanduma, Esther; Murata, Shiro; Ohashi, Kazuhiko

2013-10-01

Recently, mortalities among giraffes, attributed to infection with unique species of piroplasms were reported in South Africa. Although haemoparasites are known to occur in giraffes of Kenya, the prevalence, genetic diversity and pathogenicity of these parasites have not been investigated. In this study, blood samples from 13 giraffes in Kenya were investigated microscopically and genomic DNA extracted. PCR amplicons of the hyper-variable region 4 (V4) of Theileria spp. small subunit ribosomal RNA (18S rRNA) gene were hybridized to a panel of genus- and species-specific oligonucleotide probes by reverse line blot (RLB). Two newly designed oligonucleotide probes specific for previously identified Theileria spp. of giraffes found single infections in eight of the specimens and mixed infections in the remaining five samples. Partial 18S rRNA genes were successfully amplified from 9 samples and the PCR amplicons were cloned. A total of 28 plasmid clones representing the Kenyan isolates were analyzed in the present study and compared with those of closely-related organisms retrieved from GenBank. In agreement with RLB results, the nucleotide sequence alignment indicated the presence of mixed infections in the giraffes. In addition, sequence alignment with the obtained 18S rRNA gene sequences revealed extensive microheterogeneities within and between isolates, characterized by indels in the V4 regions and point mutations outside this region. Phylogeny with 18S rRNA gene sequences from the detected parasites and those of related organisms places Theileria of giraffes into two major groups, within which are numerous clades that include the isolates reported in South Africa. Collectively, these data suggest the existence of at least two distinct Theileria species among giraffes, and extensive genetic diversity within the two parasite groups. Copyright © 2013 Elsevier Ireland Ltd. All rights reserved.

Systematic cloning of human minisatellites from ordered array charomid libraries.

PubMed

Armour, J A; Povey, S; Jeremiah, S; Jeffreys, A J

1990-11-01

We present a rapid and efficient method for the isolation of minisatellite loci from human DNA. The method combines cloning a size-selected fraction of human MboI DNA fragments in a charomid vector with hybridization screening of the library in ordered array. Size-selection of large MboI fragments enriches for the longer, more variable minisatellites and reduces the size of the library required. The library was screened with a series of multi-locus probes known to detect a large number of hypervariable loci in human DNA. The gridded library allowed both the rapid processing of positive clones and the comparative evaluation of the different multi-locus probes used, in terms of both the relative success in detecting hypervariable loci and the degree of overlap between the sets of loci detected. We report 23 new human minisatellite loci isolated by this method, which map to 14 autosomes and the sex chromosomes.

Serotype-Specific Neutralizing Antibody Epitopes of Human Adenovirus Type 3 (HAdV-3) and HAdV-7 Reside in Multiple Hexon Hypervariable Regions

PubMed Central

Qiu, Hongling; Li, Xiao; Tian, Xingui; Zhou, Zhichao; Xing, Ke; Li, Haitao; Tang, Ni; Liu, Wenkuan; Bai, Peisheng

2012-01-01

Human adenovirus types 3 and 7 (HAdV-3 and HAdV-7) occur epidemically and contribute greatly to respiratory diseases, but there is no currently available licensed recombinant HAdV-3/HAdV-7 bivalent vaccine. Identification of serotype-specific neutralizing antibody (NAb) epitopes for HAdV-3 and HAdV-7 will be beneficial for development of recombinant HAdV-3/HAdV-7 bivalent vaccines. In this study, four NAb epitopes within hexon hypervariable regions (HVRs) were predicted for HAdV-3 and HAdV-7, respectively, by using bioinformatics. Eight hexon chimeric adenovirus vectors with the alternation of only one predicted neutralizing epitope were constructed. Further in vitro and in vivo neutralization assays indicated that E2 (residing in HVR2) and E3 (residing in HVR5) are NAb epitopes for HAdV-7, and E3 plays a more important role in generating NAb responses. Cross-neutralization assays indicated that all four predicted epitopes, R1 to R4, are NAb epitopes for HAdV-3, and R1 (residing in HVR1) plays the most important role in generating NAb responses. Humoral immune responses elicited by the recombinant rAdH7R1 (containing the R1 epitope) were significantly and durably suppressed by HAdV-3-specific NAbs. Surprisingly, the rAdΔE3GFP-specific neutralizing epitope responses induced by rAdMHE3 (R3 replaced by E3) and rAdMHE4 (R4 replaced by E4) were weaker than those of rAdMHE1 (R1 replaced by E1) or rAdMHE2 (R2 relaced by E2) in vitro and in vivo. Furthermore, rAdMHE4 replicated more slowly in HEp-2 cells, and the final yield was about 10-fold lower than that of rAdΔE3GFP. The current findings contribute not only to the development of new adenovirus vaccine candidates, but also to the construction of new gene delivery vectors. PMID:22623776

Mycobacterium tuberculosis Population in Northwestern Russia: An Update from Russian-EU/Latvian Border Region

PubMed Central

Mokrousov, Igor; Vyazovaya, Anna; Otten, Tatiana; Zhuravlev, Viacheslav; Pavlova, Elena; Tarashkevich, Larisa; Krishevich, Valery; Vishnevsky, Boris; Narvskaya, Olga

2012-01-01

This study aimed to characterize the population structure of Mycobacterium tuberculosis in Pskov oblast in northwestern Russia, to view it in the geographical context, to compare drug resistance properties across major genetic families. Ninety M. tuberculosis strains from tuberculosis (TB) patients, permanent residents in Pskov oblast were subjected to LAM-specific IS6110-PCR and spoligotyping, followed by comparison with SITVITWEB and MIRU-VNTRplus databases. The Beijing genotype (n = 40) was found the most prevalent followed by LAM (n = 18), T (n = 13), Haarlem (n = 10), Ural (n = 5), and Manu2 (n = 1); the family status remained unknown for 3 isolates. The high rate of Beijing genotype and prevalence of LAM family are similar to those in the other Russian settings. A feature specific for M. tuberculosis population in Pskov is a relatively higher rate of Haarlem and T types. Beijing strains were further typed with 12-MIRU (followed by comparison with proprietary global database) and 3 hypervariable loci QUB-3232, VNTR-3820, VNTR-4120. The 12-MIRU typing differentiated 40 Beijing strains into 14 types (HGI = 0.82) while two largest types were M2 (223325153533) prevalent throughout former USSR and M11 (223325173533) prevalent in Russia and East Asia. The use of 3 hypervariable loci increased a discrimination of the Beijing strains (18 profiles, HGI = 0.89). Both major families Beijing and LAM had similar rate of MDR strains (62.5 and 55.6%, respectively) that was significantly higher than in other strains (21.9%; P = 0.001 and 0.03, respectively). The rpoB531 mutations were more frequently found in Beijing strains while LAM drug resistant strains mainly harbored rpoB516 and inhA −15 mutations. Taken together with a high rate of multidrug resistance among Beijing strains from new TB cases (79.3% versus 44.4% in LAM), these findings suggest the critical impact of the Beijing genotype on the current situation with MDR-TB in the Pskov region in northwestern Russia. PMID:22844457

Hypervariable and highly divergent intron-exon organizations in the chordate Oikopleura dioica.

PubMed

Edvardsen, Rolf B; Lerat, Emmanuelle; Maeland, Anne Dorthea; Flåt, Mette; Tewari, Rita; Jensen, Marit F; Lehrach, Hans; Reinhardt, Richard; Seo, Hee-Chan; Chourrout, Daniel

2004-10-01

Oikopleura dioica is a pelagic tunicate with a very small genome and a very short life cycle. In order to investigate the intron-exon organizations in Oikopleura, we have isolated and characterized ribosomal protein EF-1alpha, Hox, and alpha-tubulin genes. Their intron positions have been compared with those of the same genes from various invertebrates and vertebrates, including four species with entirely sequenced genomes. Oikopleura genes, like Caenorhabditis genes, have introns at a large number of nonconserved positions, which must originate from late insertions or intron sliding of ancient insertions. Both species exhibit hypervariable intron-exon organization within their alpha-tubulin gene family. This is due to localization of most nonconserved intron positions in single members of this gene family. The hypervariability and divergence of intron positions in Oikopleura and Caenorhabditis may be related to the predominance of short introns, the processing of which is not very dependent upon the exonic environment compared to large introns. Also, both species have an undermethylated genome, and the control of methylation-induced point mutations imposes a control on exon size, at least in vertebrate genes. That introns placed at such variable positions in Oikopleura or C. elegans may serve a specific purpose is not easy to infer from our current knowledge and hypotheses on intron functions. We propose that new introns are retained in species with very short life cycles, because illegitimate exchanges including gene conversion are repressed. We also speculate that introns placed at gene-specific positions may contribute to suppressing these exchanges and thereby favor their own persistence.

Phylogeographic Differentiation of Mitochondrial DNA in Han Chinese

PubMed Central

Yao, Yong-Gang; Kong, Qing-Peng; Bandelt, Hans-Jürgen; Kivisild, Toomas; Zhang, Ya-Ping

2002-01-01

To characterize the mitochondrial DNA (mtDNA) variation in Han Chinese from several provinces of China, we have sequenced the two hypervariable segments of the control region and the segment spanning nucleotide positions 10171–10659 of the coding region, and we have identified a number of specific coding-region mutations by direct sequencing or restriction-fragment–length–polymorphism tests. This allows us to define new haplogroups (clades of the mtDNA phylogeny) and to dissect the Han mtDNA pool on a phylogenetic basis, which is a prerequisite for any fine-grained phylogeographic analysis, the interpretation of ancient mtDNA, or future complete mtDNA sequencing efforts. Some of the haplogroups under study differ considerably in frequencies across different provinces. The southernmost provinces show more pronounced contrasts in their regional Han mtDNA pools than the central and northern provinces. These and other features of the geographical distribution of the mtDNA haplogroups observed in the Han Chinese make an initial Paleolithic colonization from south to north plausible but would suggest subsequent migration events in China that mainly proceeded from north to south and east to west. Lumping together all regional Han mtDNA pools into one fictive general mtDNA pool or choosing one or two regional Han populations to represent all Han Chinese is inappropriate for prehistoric considerations as well as for forensic purposes or medical disease studies. PMID:11836649

The minisatellite of the GPI/AMF/NLK/MF gene: interspecies conservation and transcriptional activity.

PubMed

Williams, R R; Hassan-Walker, A F; Lavender, F L; Morgan, M; Faik, P; Ragoussis, J

2001-05-16

Minisatellites are tandemly repeated DNA sequences found throughout the genomes of all eukaryotes. They are regions often prone to instability and hence hypervariability; thus repeat unit sequence is generally not conserved beyond closely related species. We have studied the minisatellite located in intron 9 of the human glucose phosphate isomerase (GPI) gene (also known as neuroleukin, autocrine motility factor, maturation and differentiation factor) and have found, by Zoo blotting coupled with PCR amplification and DNA sequencing, that similar repeat units are present in seven other species of mammal. There is also evidence for the presence of the minisatellite in chicken. The repeat unit does not appear to be present at any other locus in these genomes. Minisatellite DNA has been reported to be involved in recombination activity, control of gene expression of nearby gene(s) (both transcriptional and translational), whilst others form protein coding regions. The high level of conservation exhibited by the GPI minisatellite, coupled with the unique location, strongly suggests a functional role. Our results from transient and stable transfections using luciferase reporter constructs have shown that the GPI minisatellite region can act to increase transcription from the SV40 promoter, CMV promoter and the human GPI promoter.

Identification and molecular analysis of infectious bursal disease in broiler farms in the Kurdistan Regional Government of Iraq.

PubMed

Amin, Oumed Gerjis M; Jackwood, Daral J

2014-10-01

The present study was undertaken to characterize field isolates of infectious bursal disease virus (IBDV). The identification was done using reverse transcription-polymerase chain reaction (RT-PCR) and partial sequencing of the VP2 gene. Pooled bursal samples were collected from commercial broiler farms located in the Kurdistan Regional Government (KRG) of Iraq. The genetic material of the IBDV was detected in 10 out of 29 field samples. Sequences of the hypervariable VP2 region were determined for 10 of these viruses. Molecular analysis of the VP2 gene of five IBDVs showed amino acid sequences consistent with the very virulent (vv) IBDV. Two samples were identified as classic vaccine viruses, and three samples were classic vaccine viruses that appear to have mutated during replication in the field. Phylogenetic analysis showed that all five field IBDV strains of the present study were closely related to each other. On the basis of nucleotide sequencing and phylogenetic analysis, it is very likely that IBD-causing viruses in this part of Iraq are of the very virulent type. These IBDVs appear to be evolving relative to their type strains.

Metagenomic Analysis of Hot Springs in Central India Reveals Hydrocarbon Degrading Thermophiles and Pathways Essential for Survival in Extreme Environments

PubMed Central

Saxena, Rituja; Dhakan, Darshan B.; Mittal, Parul; Waiker, Prashant; Chowdhury, Anirban; Ghatak, Arundhuti; Sharma, Vineet K.

2017-01-01

Extreme ecosystems such as hot springs are of great interest as a source of novel extremophilic species, enzymes, metabolic functions for survival and biotechnological products. India harbors hundreds of hot springs, the majority of which are not yet explored and require comprehensive studies to unravel their unknown and untapped phylogenetic and functional diversity. The aim of this study was to perform a large-scale metagenomic analysis of three major hot springs located in central India namely, Badi Anhoni, Chhoti Anhoni, and Tattapani at two geographically distinct regions (Anhoni and Tattapani), to uncover the resident microbial community and their metabolic traits. Samples were collected from seven distinct sites of the three hot spring locations with temperature ranging from 43.5 to 98°C. The 16S rRNA gene amplicon sequencing of V3 hypervariable region and shotgun metagenome sequencing uncovered a unique taxonomic and metabolic diversity of the resident thermophilic microbial community in these hot springs. Genes associated with hydrocarbon degradation pathways, such as benzoate, xylene, toluene, and benzene were observed to be abundant in the Anhoni hot springs (43.5–55°C), dominated by Pseudomonas stutzeri and Acidovorax sp., suggesting the presence of chemoorganotrophic thermophilic community with the ability to utilize complex hydrocarbons as a source of energy. A high abundance of genes belonging to methane metabolism pathway was observed at Chhoti Anhoni hot spring, where methane is reported to constitute >80% of all the emitted gases, which was marked by the high abundance of Methylococcus capsulatus. The Tattapani hot spring, with a high-temperature range (61.5–98°C), displayed a lower microbial diversity and was primarily dominated by a nitrate-reducing archaeal species Pyrobaculum aerophilum. A higher abundance of cell metabolism pathways essential for the microbial survival in extreme conditions was observed at Tattapani. Taken together, the results of this study reveal a novel consortium of microbes, genes, and pathways associated with the hot spring environment. PMID:28105025

Genetic polymorphisms in prehistoric Pacific islanders determined by analysis of ancient bone DNA.

PubMed

Hagelberg, E; Clegg, J B

1993-05-22

A previously characterized Asian-specific mitochondrial DNA (mtDNA) length mutation has been detected in DNA isolated from prehistoric human bones from Polynesia, including Hawaii, Chatham Islands and Society Islands. In contrast, the Asian mutation was absent in skeletal samples from the Melanesian archipelagos of New Britain and Vanuatu and in the oldest samples from Fiji, Tonga and Samoa in the central Pacific (2700-1600 years BP) although it was present in a more recent prehistoric sample from Tonga. These results, augmented by informative DNA sequence data from the hypervariable region of mtDNA, fail to support current views that the central Pacific was settled directly by voyagers from island Southeast Asia, the putative ancestors of modern Polynesians. An earlier occupation by peoples from the neighbouring Melanesian archipelagos seems more likely.

Changes in the respiratory microbiome during acute exacerbations of idiopathic pulmonary fibrosis.

PubMed

Molyneaux, Philip L; Cox, Michael J; Wells, Athol U; Kim, Ho Cheol; Ji, Wonjun; Cookson, William O C; Moffatt, Miriam F; Kim, Dong Soon; Maher, Toby M

2017-02-01

Acute exacerbations of idiopathic pulmonary fibrosis (AE-IPF) have been defined as events of clinically significant respiratory deterioration with an unidentifiable cause. They carry a significant mortality and morbidity and while their exact pathogenesis remains unclear, the possibility remains that hidden infection may play a role. The aim of this pilot study was to determine whether changes in the respiratory microbiota occur during an AE-IPF. Bacterial DNA was extracted from bronchoalveolar lavage from patients with stable IPF and those experiencing an AE-IPF. A hyper-variable region of the 16S ribosomal RNA gene (16S rRNA) was amplified, quantified and pyrosequenced. Culture independent techniques demonstrate AE-IPF is associated with an increased BAL bacterial burden compared to stable disease and highlight shifts in the composition of the respiratory microbiota during an AE-IPF.

Classification of European Mtdnas from an Analysis of Three European Populations

PubMed Central

Torroni, A.; Huoponen, K.; Francalacci, P.; Petrozzi, M.; Morelli, L.; Scozzari, R.; Obinu, D.; Savontaus, M. L.; Wallace, D. C.

1996-01-01

Mitochondrial DNA (mtDNA) sequence variation was examined in Finns, Swedes and Tuscans by PCR amplification and restriction analysis. About 99% of the mtDNAs were subsumed within 10 mtDNA haplogroups (H, I, J, K, M, T, U, V, W, and X) suggesting that the identified haplogroups could encompass virtually all European mtDNAs. Because both hypervariable segments of the mtDNA control region were previously sequenced in the Tuscan samples, the mtDNA haplogroups and control region sequences could be compared. Using a combination of haplogroup-specific restriction site changes and control region nucleotide substitutions, the distribution of the haplogroups was surveyed through the published restriction site polymorphism and control region sequence data of Caucasoids. This supported the conclusion that most haplogroups observed in Europe are Caucasoid-specific, and that at least some of them occur at varying frequencies in different Caucasoid populations. The classification of almost all European mtDNA variation in a number of well defined haplogroups could provide additional insights about the origin and relationships of Caucasoid populations and the process of human colonization of Europe, and is valuable for the definition of the role played by mtDNA backgrounds in the expression of pathological mtDNA mutations PMID:8978068

Reconstruction of structural evolution in the trnL intron P6b loop of symbiotic Nostoc (Cyanobacteria).

PubMed

Olsson, Sanna; Kaasalainen, Ulla; Rikkinen, Jouko

2012-02-01

In this study we reconstruct the structural evolution of the hyper-variable P6b region of the group I trnLeu intron in a monophyletic group of lichen-symbiotic Nostoc strains and establish it as a useful marker in the phylogenetic analysis of these organisms. The studied cyanobacteria occur as photosynthetic and/or nitrogen-fixing symbionts in lichen species of the diverse Nephroma guild. Phylogenetic analyses and secondary structure reconstructions are used to improve the understanding of the replication mechanisms in the P6b stem-loop and to explain the observed distribution patterns of indels. The variants of the P6b region in the Nostoc clade studied consist of different combinations of five sequence modules. The distribution of indels together with the ancestral character reconstruction performed enables the interpretation of the evolution of each sequence module. Our results indicate that the indel events are usually associated with single nucleotide changes in the P6b region and have occurred several times independently. In spite of their homoplasy, they provide phylogenetic information for closely related taxa. Thus we recognize that features of the P6b region can be used as molecular markers for species identification and phylogenetic studies involving symbiotic Nostoc cyanobacteria.

A complex ligase ribozyme evolved in vitro from a group I ribozyme domain

NASA Technical Reports Server (NTRS)

Jaeger, L.; Wright, M. C.; Joyce, G. F.; Bada, J. L. (Principal Investigator)

1999-01-01

Like most proteins, complex RNA molecules often are modular objects made up of distinct structural and functional domains. The component domains of a protein can associate in alternative combinations to form molecules with different functions. These observations raise the possibility that complex RNAs also can be assembled from preexisting structural and functional domains. To test this hypothesis, an in vitro evolution procedure was used to isolate a previously undescribed class of complex ligase ribozymes, starting from a pool of 10(16) different RNA molecules that contained a constant region derived from a large structural domain that occurs within self-splicing group I ribozymes. Attached to this constant region were three hypervariable regions, totaling 85 nucleotides, that gave rise to the catalytic motif within the evolved catalysts. The ligase ribozymes catalyze formation of a 3',5'-phosphodiester linkage between adjacent template-bound oligonucleotides, one bearing a 3' hydroxyl and the other a 5' triphosphate. Ligation occurs in the context of a Watson-Crick duplex, with a catalytic rate of 0.26 min(-1) under optimal conditions. The constant region is essential for catalytic activity and appears to retain the tertiary structure of the group I ribozyme. This work demonstrates that complex RNA molecules, like their protein counterparts, can share common structural domains while exhibiting distinct catalytic functions.

Intrinsic challenges in ancient microbiome reconstruction using 16S rRNA gene amplification.

PubMed

Ziesemer, Kirsten A; Mann, Allison E; Sankaranarayanan, Krithivasan; Schroeder, Hannes; Ozga, Andrew T; Brandt, Bernd W; Zaura, Egija; Waters-Rist, Andrea; Hoogland, Menno; Salazar-García, Domingo C; Aldenderfer, Mark; Speller, Camilla; Hendy, Jessica; Weston, Darlene A; MacDonald, Sandy J; Thomas, Gavin H; Collins, Matthew J; Lewis, Cecil M; Hofman, Corinne; Warinner, Christina

2015-11-13

To date, characterization of ancient oral (dental calculus) and gut (coprolite) microbiota has been primarily accomplished through a metataxonomic approach involving targeted amplification of one or more variable regions in the 16S rRNA gene. Specifically, the V3 region (E. coli 341-534) of this gene has been suggested as an excellent candidate for ancient DNA amplification and microbial community reconstruction. However, in practice this metataxonomic approach often produces highly skewed taxonomic frequency data. In this study, we use non-targeted (shotgun metagenomics) sequencing methods to better understand skewed microbial profiles observed in four ancient dental calculus specimens previously analyzed by amplicon sequencing. Through comparisons of microbial taxonomic counts from paired amplicon (V3 U341F/534R) and shotgun sequencing datasets, we demonstrate that extensive length polymorphisms in the V3 region are a consistent and major cause of differential amplification leading to taxonomic bias in ancient microbiome reconstructions based on amplicon sequencing. We conclude that systematic amplification bias confounds attempts to accurately reconstruct microbiome taxonomic profiles from 16S rRNA V3 amplicon data generated using universal primers. Because in silico analysis indicates that alternative 16S rRNA hypervariable regions will present similar challenges, we advocate for the use of a shotgun metagenomics approach in ancient microbiome reconstructions.

Intrinsic challenges in ancient microbiome reconstruction using 16S rRNA gene amplification

PubMed Central

Ziesemer, Kirsten A.; Mann, Allison E.; Sankaranarayanan, Krithivasan; Schroeder, Hannes; Ozga, Andrew T.; Brandt, Bernd W.; Zaura, Egija; Waters-Rist, Andrea; Hoogland, Menno; Salazar-García, Domingo C.; Aldenderfer, Mark; Speller, Camilla; Hendy, Jessica; Weston, Darlene A.; MacDonald, Sandy J.; Thomas, Gavin H.; Collins, Matthew J.; Lewis, Cecil M.; Hofman, Corinne; Warinner, Christina

2015-01-01

To date, characterization of ancient oral (dental calculus) and gut (coprolite) microbiota has been primarily accomplished through a metataxonomic approach involving targeted amplification of one or more variable regions in the 16S rRNA gene. Specifically, the V3 region (E. coli 341–534) of this gene has been suggested as an excellent candidate for ancient DNA amplification and microbial community reconstruction. However, in practice this metataxonomic approach often produces highly skewed taxonomic frequency data. In this study, we use non-targeted (shotgun metagenomics) sequencing methods to better understand skewed microbial profiles observed in four ancient dental calculus specimens previously analyzed by amplicon sequencing. Through comparisons of microbial taxonomic counts from paired amplicon (V3 U341F/534R) and shotgun sequencing datasets, we demonstrate that extensive length polymorphisms in the V3 region are a consistent and major cause of differential amplification leading to taxonomic bias in ancient microbiome reconstructions based on amplicon sequencing. We conclude that systematic amplification bias confounds attempts to accurately reconstruct microbiome taxonomic profiles from 16S rRNA V3 amplicon data generated using universal primers. Because in silico analysis indicates that alternative 16S rRNA hypervariable regions will present similar challenges, we advocate for the use of a shotgun metagenomics approach in ancient microbiome reconstructions. PMID:26563586

Effects of Physiochemical Factors on Prokaryotic Biodiversity in Malaysian Circumneutral Hot Springs.

PubMed

Chan, Chia S; Chan, Kok-Gan; Ee, Robson; Hong, Kar-Wai; Urbieta, María S; Donati, Edgardo R; Shamsir, Mohd S; Goh, Kian M

2017-01-01

Malaysia has a great number of hot springs, especially along the flank of the Banjaran Titiwangsa mountain range. Biological studies of the Malaysian hot springs are rare because of the lack of comprehensive information on their microbial communities. In this study, we report a cultivation-independent census to describe microbial communities in six hot springs. The Ulu Slim (US), Sungai Klah (SK), Dusun Tua (DT), Sungai Serai (SS), Semenyih (SE), and Ayer Hangat (AH) hot springs exhibit circumneutral pH with temperatures ranging from 43°C to 90°C. Genomic DNA was extracted from environmental samples and the V3-V4 hypervariable regions of 16S rRNA genes were amplified, sequenced, and analyzed. High-throughput sequencing analysis showed that microbial richness was high in all samples as indicated by the detection of 6,334-26,244 operational taxonomy units. In total, 59, 61, 72, 73, 65, and 52 bacterial phyla were identified in the US, SK, DT, SS, SE, and AH hot springs, respectively. Generally, Firmicutes and Proteobacteria dominated the bacterial communities in all hot springs. Archaeal communities mainly consisted of Crenarchaeota, Euryarchaeota, and Parvarchaeota. In beta diversity analysis, the hot spring microbial memberships were clustered primarily on the basis of temperature and salinity. Canonical correlation analysis to assess the relationship between the microbial communities and physicochemical variables revealed that diversity patterns were best explained by a combination of physicochemical variables, rather than by individual abiotic variables such as temperature and salinity.

Large Outbreak of Hepatitis C Virus Associated With Drug Diversion by a Healthcare Technician.

PubMed

Alroy-Preis, Sharon; Daly, Elizabeth R; Adamski, Christine; Dionne-Odom, Jodie; Talbot, Elizabeth A; Gao, Fengxiang; Cavallo, Steffany J; Hansen, Katrina; Mahoney, Jennifer C; Metcalf, Erin; Loring, Carol; Bean, Christine; Drobeniuc, Jan; Xia, Guo-Liang; Kamili, Saleem; Montero, José T

2018-05-14

In May 2012, the New Hampshire (NH) Division of Public Health Services (DPHS) was notified of 4 persons with newly diagnosed hepatitis C virus (HCV) infection at hospital X. Initial investigation suggested a common link to the hospital cardiac catheterization laboratory (CCL) because the infected persons included 3 CCL patients and a CCL technician. NH DPHS initiated an investigation to determine the source and control the outbreak. NH DPHS conducted site visits, case patient and employee interviews, medical record and medication use review, and employee and patient HCV testing using enzyme immunoassay for anti-HCV, reverse-transcription polymerase chain reaction for HCV RNA, nonstructural 5B (NS5B) and hypervariable region 1 (HVR1) sequencing, and quasispecies analysis. HCV HVR1 analysis of the first 4 cases confirmed a common source of infection. HCV testing identified 32 of 1074 CCL patients infected with the outbreak strain, including 3 patients coinfected with >1 HCV strain. The epidemiologic investigation revealed evidence of drug diversion by the HCV-infected technician, evidenced by gaps in controlled medication control, higher fentanyl use during procedures for confirmed cases, and building card key access records documenting the presence of the technician during days when transmission occurred. The employee's status as a traveling technician led to a multistate investigation, which identified additional cases at prior employment sites. This is the largest laboratory-confirmed drug diversion-associated HCV outbreak published to date. Recommendations to reduce drug diversion risk and to conduct outbreak investigations are provided.

Detection and identification of Theileria infection in sika deer ( Cervus nippon ) in China.

PubMed

He, Lan; Khan, Muhanmad Kasib; Zhang, Wen-Jie; Zhang, Qing-Li; Zhou, Yan-Qin; Hu, Min; Zhao, Junlong

2012-06-01

The sika deer ( Cervus nippon ) is a first-grade state-protected animal in China and designated a threatened species by the World Conservation Union. To detect hemoparasite infection of sika deer, blood samples were collected from 24 animals in the Hubei Province Deer Center. Genomic DNA was extracted, and the V4 hypervariable region encoding 18S rRNA was analyzed by reverse line blot hybridization assay. PCR products hybridized with Babesia / Theileria genus-specific probes but failed to hybridize with any of the Babesia or Theileria species-specific probes, suggesting the presence of a novel, or variant, species. Here 18S rRNA and internal transcribed spacer (ITS) genes were amplified, cloned, and sequenced from 7 isolates. Alignment and BlastN of the cloned sequences revealed high similarities to the homologous 18S rRNA genes and ITS genes of Theileria cervi (AY735122), Theileria sp. CNY1A (AB012194), and Theileria sp. ex Yamaguchi (AF529272). Phylogenetic analysis based on the 18S rRNA gene and ITS sequences showed that all cloned sequences were grouped within the Theileria clade. Phylogeny based on the 18S rRNA gene divided the organisms into 2 groups. Group 1 was closest to Theileria sp. ex Yamaguchi (AF529272), and group 2 was distinct from all other identified Theileria and Babesia species. These results suggest the existence of Theileria sp. infection in sika deer in China. To our knowledge, this is the first report of cervine Theileria sp. in China.

Ciliate diversity, community structure, and novel taxa in lakes of the McMurdo Dry Valleys, Antarctica.

PubMed

Xu, Yuan; Vick-Majors, Trista; Morgan-Kiss, Rachael; Priscu, John C; Amaral-Zettler, Linda

2014-10-01

We report an in-depth survey of next-generation DNA sequencing of ciliate diversity and community structure in two permanently ice-covered McMurdo Dry Valley lakes during the austral summer and autumn (November 2007 and March 2008). We tested hypotheses on the relationship between species richness and environmental conditions including environmental extremes, nutrient status, and day length. On the basis of the unique environment that exists in these high-latitude lakes, we expected that novel taxa would be present. Alpha diversity analyses showed that extreme conditions-that is, high salinity, low oxygen, and extreme changes in day length-did not impact ciliate richness; however, ciliate richness was 30% higher in samples with higher dissolved organic matter. Beta diversity analyses revealed that ciliate communities clustered by dissolved oxygen, depth, and salinity, but not by season (i.e., day length). The permutational analysis of variance test indicated that depth, dissolved oxygen, and salinity had significant influences on the ciliate community for the abundance matrices of resampled data, while lake and season were not significant. This result suggests that the vertical trends in dissolved oxygen concentration and salinity may play a critical role in structuring ciliate communities. A PCR-based strategy capitalizing on divergent eukaryotic V9 hypervariable region ribosomal RNA gene targets unveiled two new genera in these lakes. A novel taxon belonging to an unknown class most closely related to Cryptocaryon irritans was also inferred from separate gene phylogenies. © 2014 Marine Biological Laboratory.

Effects of Physiochemical Factors on Prokaryotic Biodiversity in Malaysian Circumneutral Hot Springs

PubMed Central

Chan, Chia S.; Chan, Kok-Gan; Ee, Robson; Hong, Kar-Wai; Urbieta, María S.; Donati, Edgardo R.; Shamsir, Mohd S.; Goh, Kian M.

2017-01-01

Malaysia has a great number of hot springs, especially along the flank of the Banjaran Titiwangsa mountain range. Biological studies of the Malaysian hot springs are rare because of the lack of comprehensive information on their microbial communities. In this study, we report a cultivation-independent census to describe microbial communities in six hot springs. The Ulu Slim (US), Sungai Klah (SK), Dusun Tua (DT), Sungai Serai (SS), Semenyih (SE), and Ayer Hangat (AH) hot springs exhibit circumneutral pH with temperatures ranging from 43°C to 90°C. Genomic DNA was extracted from environmental samples and the V3–V4 hypervariable regions of 16S rRNA genes were amplified, sequenced, and analyzed. High-throughput sequencing analysis showed that microbial richness was high in all samples as indicated by the detection of 6,334–26,244 operational taxonomy units. In total, 59, 61, 72, 73, 65, and 52 bacterial phyla were identified in the US, SK, DT, SS, SE, and AH hot springs, respectively. Generally, Firmicutes and Proteobacteria dominated the bacterial communities in all hot springs. Archaeal communities mainly consisted of Crenarchaeota, Euryarchaeota, and Parvarchaeota. In beta diversity analysis, the hot spring microbial memberships were clustered primarily on the basis of temperature and salinity. Canonical correlation analysis to assess the relationship between the microbial communities and physicochemical variables revealed that diversity patterns were best explained by a combination of physicochemical variables, rather than by individual abiotic variables such as temperature and salinity. PMID:28729863

Emergence of new virulent rabbit hemorrhagic disease virus strains in Saudi Arabia.

PubMed

Ismail, Mahmoud M; Mohamed, Mahmoud H A; El-Sabagh, Ibrahim M; Al-Hammadi, Mohamed A

2017-02-01

Rabbit hemorrhagic disease is an acute fatal highly contagious viral infectious disease that causes high losses among rabbitries. The disease was first reported in China in 1984 and later on in Saudi Arabia in 1996. The aim of this study was to investigate the emergence and pathogenicity of new rabbit hemorrhagic disease virus (RHDV) strains in Saudi Arabia. The pathogenicity was confirmed by inoculation in susceptible rabbits. Three RHDV strains were detected by reverse transcriptase polymerase chain reaction (RT-PCR) using primers targeting VP60 capsid protein gene in infected rabbitries during 2012 and 2013. These strains clustered into two genetically distinct genogroups related to year of isolation (G2 and G3). All new Saudi Arabia viruses clustered with the European strains, while the old strains clustered with strains from China and America. Based on amino acids and nucleotide sequences, the Saudi Arabia strains (RHD/1/SA/2012, RHD/2/SA/2012, and RHD/3/SA /2013) had high identity with Mexico89, Ca11-ITA, and 00-13,FRA virus; on the other hand, there was a relatively high identity with Bahrain strain. The evolutionary relationship of Saudi RHDVs strains revealed significant nucleotides and amino acid substitutions in hypervariable region E, suggesting the emergence of new RHDVs circulating in Saudi Arabia rabbitries. These antigenic changes represented by the antigenic index might be a potential cause of vaccination failure and raises the need to review the vaccination strategies against RHD.

Three Thousand Years of Continuity in the Maternal Lineages of Ancient Sheep (Ovis aries) in Estonia

PubMed Central

Rannamäe, Eve; Lõugas, Lembi; Speller, Camilla F.; Valk, Heiki; Maldre, Liina; Wilczyński, Jarosław; Mikhailov, Aleksandr; Saarma, Urmas

2016-01-01

Although sheep (Ovis aries) have been one of the most exploited domestic animals in Estonia since the Late Bronze Age, relatively little is known about their genetic history. Here, we explore temporal changes in Estonian sheep populations and their mitochondrial genetic diversity over the last 3000 years. We target a 558 base pair fragment of the mitochondrial hypervariable region in 115 ancient sheep from 71 sites in Estonia (c. 1200 BC–AD 1900s), 19 ancient samples from Latvia, Russia, Poland and Greece (6800 BC–AD 1700), as well as 44 samples of modern Kihnu native sheep breed. Our analyses revealed: (1) 49 mitochondrial haplotypes, associated with sheep haplogroups A and B; (2) high haplotype diversity in Estonian ancient sheep; (3) continuity in mtDNA haplotypes through time; (4) possible population expansion during the first centuries of the Middle Ages (associated with the establishment of the new power regime related to 13th century crusades); (5) significant difference in genetic diversity between ancient populations and modern native sheep, in agreement with the beginning of large-scale breeding in the 19th century and population decline in local sheep. Overall, our results suggest that in spite of the observed fluctuations in ancient sheep populations, and changes in the natural and historical conditions, the utilisation of local sheep has been constant in the territory of Estonia, displaying matrilineal continuity from the Middle Bronze Age through the Modern Period, and into modern native sheep. PMID:27732668

Characterization of PhPRP1, a histidine domain arabinogalactan protein from Petunia hybrida pistils.

PubMed

Twomey, Megan C; Brooks, Jenna K; Corey, Jillaine M; Singh-Cundy, Anu

2013-10-15

An arabinogalactan protein, PhPRP1, was purified from Petunia hybrida pistils and shown to be orthologous to TTS-1 and TTS-2 from Nicotiana tabacum and NaTTS from Nicotiana alata. Sequence comparisons among these proteins, and CaPRP1 from Capsicum annuum, reveal a conserved histidine-rich domain and two hypervariable domains. Immunoblots show that TTS-1 and PhPRP1 are also expressed in vegetative tissues of tobacco and petunia respectively. In contrast to the molecular mass heterogeneity displayed by the pistil proteins, the different isoforms found in seedlings, roots, and leaves each has a discrete size (37, 80, 160, and 200 kDa) on SDS-PAGE gels. On the basis of their chemistry, distinctive domain architecture, and the unique pattern of expression, we have named this group of proteins HD-AGPs (histidine domain-arabinogalactan proteins). Copyright © 2013 Elsevier GmbH. All rights reserved.

Phylogenetic Network for European mtDNA

PubMed Central

Finnilä, Saara; Lehtonen, Mervi S.; Majamaa, Kari

2001-01-01

The sequence in the first hypervariable segment (HVS-I) of the control region has been used as a source of evolutionary information in most phylogenetic analyses of mtDNA. Population genetic inference would benefit from a better understanding of the variation in the mtDNA coding region, but, thus far, complete mtDNA sequences have been rare. We determined the nucleotide sequence in the coding region of mtDNA from 121 Finns, by conformation-sensitive gel electrophoresis and subsequent sequencing and by direct sequencing of the D loop. Furthermore, 71 sequences from our previous reports were included, so that the samples represented all the mtDNA haplogroups present in the Finnish population. We found a total of 297 variable sites in the coding region, which allowed the compilation of unambiguous phylogenetic networks. The D loop harbored 104 variable sites, and, in most cases, these could be localized within the coding-region networks, without discrepancies. Interestingly, many homoplasies were detected in the coding region. Nucleotide variation in the rRNA and tRNA genes was 6%, and that in the third nucleotide positions of structural genes amounted to 22% of that in the HVS-I. The complete networks enabled the relationships between the mtDNA haplogroups to be analyzed. Phylogenetic networks based on the entire coding-region sequence in mtDNA provide a rich source for further population genetic studies, and complete sequences make it easier to differentiate between disease-causing mutations and rare polymorphisms. PMID:11349229

Evidence for mitochondrial DNA recombination in a human population of island Melanesia.

PubMed Central

Hagelberg, E; Goldman, N; Lió, P; Whelan, S; Schiefenhövel, W; Clegg, J B; Bowden, D K

1999-01-01

Mitochondrial DNA (mtDNA) analysis has proved useful in studies of recent human evolution and the genetic affinities of human groups of different geographical regions. As part of an extensive survey of mtDNA diversity in present-day Pacific populations, we obtained sequence information of the hypervariable mtDNA control region of 452 individuals from various localities in the western Pacific. The mtDNA types fell into three major groups which reflect the settlement history of the area. Interestingly, we detected an extremely rare point mutation at high frequency in the small island of Nguna in the Melanesian archipelago of Vanuatu. Phylogenetic analysis of the mtDNA data indicated that the mutation was present in individuals of separate mtDNA lineages. We propose that the multiple occurrence of a rare mutation event in one isolated locality is highly improbable, and that recombination between different mtDNA types is a more likely explanation for our observation. If correct, this conclusion has important implications for the use of mtDNA in phylogenetic and evolutionary studies. PMID:10189712

Evidence for mitochondrial DNA recombination in a human population of island Melanesia.

PubMed

Hagelberg, E; Goldman, N; Lió, P; Whelan, S; Schiefenhövel, W; Clegg, J B; Bowden, D K

1999-03-07

Mitochondrial DNA (mtDNA) analysis has proved useful in studies of recent human evolution and the genetic affinities of human groups of different geographical regions. As part of an extensive survey of mtDNA diversity in present-day Pacific populations, we obtained sequence information of the hypervariable mtDNA control region of 452 individuals from various localities in the western Pacific. The mtDNA types fell into three major groups which reflect the settlement history of the area. Interestingly, we detected an extremely rare point mutation at high frequency in the small island of Nguna in the Melanesian archipelago of Vanuatu. Phylogenetic analysis of the mtDNA data indicated that the mutation was present in individuals of separate mtDNA lineages. We propose that the multiple occurrence of a rare mutation event in one isolated locality is highly improbable, and that recombination between different mtDNA types is a more likely explanation for our observation. If correct, this conclusion has important implications for the use of mtDNA in phylogenetic and evolutionary studies.

Forensics and mitochondrial DNA: applications, debates, and foundations.

PubMed

Budowle, Bruce; Allard, Marc W; Wilson, Mark R; Chakraborty, Ranajit

2003-01-01

Debate on the validity and reliability of scientific methods often arises in the courtroom. When the government (i.e., the prosecution) is the proponent of evidence, the defense is obliged to challenge its admissibility. Regardless, those who seek to use DNA typing methodologies to analyze forensic biological evidence have a responsibility to understand the technology and its applications so a proper foundation(s) for its use can be laid. Mitochondrial DNA (mtDNA), an extranuclear genome, has certain features that make it desirable for forensics, namely, high copy number, lack of recombination, and matrilineal inheritance. mtDNA typing has become routine in forensic biology and is used to analyze old bones, teeth, hair shafts, and other biological samples where nuclear DNA content is low. To evaluate results obtained by sequencing the two hypervariable regions of the control region of the human mtDNA genome, one must consider the genetically related issues of nomenclature, reference population databases, heteroplasmy, paternal leakage, recombination, and, of course, interpretation of results. We describe the approaches, the impact some issues may have on interpretation of mtDNA analyses, and some issues raised in the courtroom.

No evidence of radiation effect on mutation rates at hypervariable minisatellite loci in the germ cells of atomic bomb survivors.

PubMed

Kodaira, Mieko; Izumi, Shizue; Takahashi, Norio; Nakamura, Nori

2004-10-01

Human minisatellites consist of tandem arrays of short repeat sequences, and some are highly polymorphic in numbers of repeats among individuals. Since these loci mutate much more frequently than coding sequences, they make attractive markers for screening populations for genetic effects of mutagenic agents. Here we report the results of our analysis of mutations at eight hypervariable minisatellite loci in the offspring (61 from exposed families in 60 of which only one parent was exposed, and 58 from unexposed parents) of atomic bomb survivors with mean doses of >1 Sv. We found 44 mutations in paternal alleles and eight mutations in maternal alleles with no indication that the high doses of acutely applied radiation had caused significant genetic effects. Our finding contrasts with those of some other studies in which much lower radiation doses, applied chronically, caused significantly increased mutation rates. Possible reasons for this discrepancy are discussed.

Comprehensive view of the population history of Arabia as inferred by mtDNA variation.

PubMed

Černý, Viktor; Čížková, Martina; Poloni, Estella S; Al-Meeri, Ali; Mulligan, Connie J

2016-04-01

Genetic and archaeological research supports the theory that Arabia was the first region traversed by modern humans as they left Africa and dispersed throughout Eurasia. However, the role of Arabia from the initial migration out of Africa until more recent times is still unclear. We have generated 379 new hypervariable segment 1 (HVS-1) sequences from a range of geographic locations throughout Yemen. We compare these data to published HVS-1 sequences representing Arabia and neighboring regions to build a unique dataset of 186 populations and 14,290 sequences. We identify 4,563 haplotypes unevenly distributed across Arabia and neighboring regions. Arabia contains higher proportions of shared haplotypes than the regions with which it shares these haplotypes, suggesting high levels of migration through the region. Populations in Arabia show higher levels of population expansion than those in East Africa, but lower levels than the Near East, Middle East or India. Arabian populations also show very high levels of genetic variation that overlaps with variation from most other regions. We take a population genetics approach to provide a comprehensive view of the relationships of Arabian and neighboring populations. We show that Arabian populations share closest links to the Near East and North Africa, but have a more ancient origin with slower demographic growth and/or lower migration rates. Our conclusions are supported by phylogenetic studies but also suggest that recent migrations have erased signals of earlier events. © 2015 Wiley Periodicals, Inc.

DXS10011: studies on structure, allele distribution in three populations and genetic linkage to further q-telomeric chromosome X markers.

PubMed

Hering, Sandra; Brundirs, Nicola; Kuhlisch, Eberhard; Edelmann, Jeanett; Plate, Ines; Benecke, Mark; Van, Pham Hung; Michael, Matthias; Szibor, Reinhard

2004-12-01

The hypervariable tetranucleotide STR polymorphism DXS10011 is a powerful marker for forensic purposes. Investigation of this STR led to an allele nomenclature which is in consensus with the ISFG recommendations. DXS10011 is located at Xq28 and genetically closely linked to DXS7423 and DXS8377 but is unlinked to HPRTB and more distant X-chromosomal STRs. DXS10011 is a very complex marker exhibiting some structural variants within alleles of identical length. Two types of repeat structure (regular and inter-alleles) are known and described as types A and B. Two SNPs which are in strong linkage disequilibrium to the different sequence types were found in the repeat flanking region. The type A sequence consists of a long stretch of uninterrupted homogenous repeats which is highly susceptible to slippage mutation during male meiosis.

Rapid detection and differentiation of avian infectious bronchitis virus: an application of Mass genotype by melting temperature analysis in RT-qPCR using SYBR Green I

PubMed Central

OKINO, Cintia Hiromi; MONTASSIER, Maria de Fátima Silva; de OLIVEIRA, Andressa Peres; MONTASSIER, Helio José

2018-01-01

A method based on Melting Temperature analysis of Hypervariable regions (HVR) of S1 gene within a RT-qPCR was developed to detect different genotypes of avian infectious bronchitis virus (IBV) and identify the Mass genotype. The method was able to rapidly identify the Mass genotype among IBV field isolates, vaccine attenuated strains and reference M41 strain in allantoic liquid and also directly in tissues. The RT-qPCR developed detected the virus in both tracheal and pulmonary samples from M41-infected or H120-infected birds, in a larger post-infection period compared to detection by standard method of virus isolation. RT-qPCR method tested provided a sensitivity and rapid approach for screening on IBV detection and Mass genotyping from IBV isolates. PMID:29491226

Changes in Abundance of Oral Microbiota Associated with Oral Cancer

PubMed Central

Schmidt, Brian L.; Kuczynski, Justin; Bhattacharya, Aditi; Huey, Bing; Corby, Patricia M.; Queiroz, Erica L. S.; Nightingale, Kira; Kerr, A. Ross; DeLacure, Mark D.; Veeramachaneni, Ratna; Olshen, Adam B.; Albertson, Donna G.

2014-01-01

Individual bacteria and shifts in the composition of the microbiome have been associated with human diseases including cancer. To investigate changes in the microbiome associated with oral cancers, we profiled cancers and anatomically matched contralateral normal tissue from the same patient by sequencing 16S rDNA hypervariable region amplicons. In cancer samples from both a discovery and a subsequent confirmation cohort, abundance of Firmicutes (especially Streptococcus) and Actinobacteria (especially Rothia) was significantly decreased relative to contralateral normal samples from the same patient. Significant decreases in abundance of these phyla were observed for pre-cancers, but not when comparing samples from contralateral sites (tongue and floor of mouth) from healthy individuals. Weighted UniFrac principal coordinates analysis based on 12 taxa separated most cancers from other samples with greatest separation of node positive cases. These studies begin to develop a framework for exploiting the oral microbiome for monitoring oral cancer development, progression and recurrence. PMID:24887397

Insights into mechanisms of bacterial antigenic variation derived from the complete genome sequence of Anaplasma marginale.

PubMed

Palmer, Guy H; Futse, James E; Knowles, Donald P; Brayton, Kelly A

2006-10-01

Persistence of Anaplasma spp. in the animal reservoir host is required for efficient tick-borne transmission of these pathogens to animals and humans. Using A. marginale infection of its natural reservoir host as a model, persistent infection has been shown to reflect sequential cycles in which antigenic variants emerge, replicate, and are controlled by the immune system. Variation in the immunodominant outer-membrane protein MSP2 is generated by a process of gene conversion, in which unique hypervariable region sequences (HVRs) located in pseudogenes are recombined into a single operon-linked msp2 expression site. Although organisms expressing whole HVRs derived from pseudogenes emerge early in infection, long-term persistent infection is dependent on the generation of complex mosaics in which segments from different HVRs recombine into the expression site. The resulting combinatorial diversity generates the number of variants both predicted and shown to emerge during persistence.

Mitochondrial DNA typing from human axillary, pubic and head hair shafts - success rates and sequence comparisons.

PubMed

Pfeiffer, H; Hühne, J; Ortmann, C; Waterkamp, K; Brinkmann, B

1999-01-01

The analysis of mitochondrial DNA (mtDNA) from shed hairs has gained high importance in forensic casework since telogen hairs are one of the most common types of evidence left at the crime scene. In this systematic study of hair shafts from 20 individuals, the correlation of mtDNA recovery with hair morphology (length, diameter, volume, colour), with sex, and with body localisation (head, armpit, pubis) was investigated. The highest average success rate of hypervariable region 1 (HV 1) sequencing was found in head hair shafts (75%) followed by pubic (66%) and axillary hair shafts (52%). No statistically significant correlation between morphological parameters or sex and the success rate of sequencing was found. MtDNA sequences of buccal cells, head, pubic and axillary hair shafts did not show intraindividual differences. Heteroplasmic base positions were observed neither in the hair shafts nor in control samples of buccal cells.

Censusing marine eukaryotic diversity in the twenty-first century

PubMed Central

Knowlton, Nancy

2016-01-01

The ocean constitutes one of the vastest and richest biomes on our planet. Most recent estimations, all based on indirect approaches, suggest that there are millions of marine eukaryotic species. Moreover, a large majority of these are small (less than 1 mm), cryptic and still unknown to science. However, this knowledge gap, caused by the lack of diagnostic morphological features in small organisms and the limited sampling of the global ocean, is currently being filled, thanks to new DNA-based approaches. The molecular technique of PCR amplification of homologous gene regions combined with high-throughput sequencing, routinely used to census unculturable prokaryotes, is now also being used to characterize whole communities of marine eukaryotes. Here, we review how this methodological advancement has helped to better quantify the magnitude and patterns of marine eukaryotic diversity, with an emphasis on taxonomic groups previously largely overlooked. We then discuss obstacles remaining to achieve a global understanding of marine eukaryotic diversity. In particular, we argue that 18S variable regions do not provide sufficient taxonomic resolution to census marine life, and suggest combining broad eukaryotic surveys targeting the 18S rRNA region with more taxon-focused analyses of hypervariable regions to improve our understanding of the diversity of species, the functional units of marine ecosystems. This article is part of the themed issue ‘From DNA barcodes to biomes’. PMID:27481783

Sequence variation and phylogenetic analysis of envelope glycoprotein of hepatitis G virus.

PubMed

Lim, M Y; Fry, K; Yun, A; Chong, S; Linnen, J; Fung, K; Kim, J P

1997-11-01

A transfusion-transmissible agent provisionally designated hepatitis G virus (HGV) was recently identified. In this study, we examined the variability of the HGV genome by analysing sequences in the putative envelope region from 72 isolates obtained from diverse geographical sources. The 1561 nucleotide sequence of the E1/E2/NS2a region of HGV was determined from 12 isolates, and compared with three published sequences. The most variability was observed in 400 nucleotides at the N terminus of E2. We next analysed this 400 nucleotide envelope variable region (EV) from an additional 60 HGV isolates. This sequence varied considerably among the 75 isolates, with overall identity ranging from 79.3% to 99.5% at the nucleotide level, and from 83.5% to 100% at the amino acid level. However, hypervariable regions were not identified. Phylogenetic analyses indicated that the 75 HGV isolates belong to a single genotype. A single-tier distribution of evolutionary distances was observed among the 15 E1/E2/NS2a sequences and the 75 EV sequences. In contrast, 11 isolates of HCV were analysed and showed a three-tiered distribution, representing genotypes, subtypes, and isolates. The 75 isolates of HGV fell into four clusters on the phylogenetic tree. Tight geographical clustering was observed among the HGV isolates from Japan and Korea.

Excised leaf method for high volume evaluation of sorghum germplasm for resistance against Colletotrichum sublineolum

USDA-ARS?s Scientific Manuscript database

Foliar phase of anthracnose, caused by Colletotrichum sublineolum is the most important leaf disease of sorghum. Due to the hyper-variable nature of the fungus, continuous evaluation of sorghum germplasm to identify new sources of resistance is imperative. Field and greenhouse evaluations for anth...

Genetic Evidence for Modifying Oceanic Boundaries Relative to Fiji.

PubMed

Shipley, Gerhard P; Taylor, Diana A; N'Yeurt, Antoine D R; Tyagi, Anand; Tiwari, Geetanjali; Redd, Alan J

2016-07-01

We present the most comprehensive genetic characterization to date of five Fijian island populations: Viti Levu, Vanua Levu, Kadavu, the Lau Islands, and Rotuma, including nonrecombinant Y (NRY) chromosome and mitochondrial DNA (mtDNA) haplotypes and haplogroups. As a whole, Fijians are genetically intermediate between Melanesians and Polynesians, but the individual Fijian island populations exhibit significant genetic structure reflecting different settlement experiences in which the Rotumans and the Lau Islanders were more influenced by Polynesians, and the other Fijian island populations were more influenced by Melanesians. In particular, Rotuman and Lau Islander NRY chromosomal and mtDNA haplogroup frequencies and Rotuman mtDNA hypervariable segment 1 region haplotypes more closely resemble those of Polynesians, while genetic markers of the other populations more closely resemble those of the Near Oceanic Melanesians. Our findings provide genetic evidence supportive of modifying regional boundaries relative to Fiji, as has been suggested by others based on a variety of nongenetic evidence. Specifically, for the traditional Melanesia/Polynesia/Micronesia scheme, our findings support moving the Melanesia-Polynesia boundary to include Rotuma and the Lau Islands in Polynesia. For the newer Near/Remote Oceania scheme, our findings support keeping Rotuma and the Lau Islands in Remote Oceania and locating the other Fijian island populations in an intermediate or "Central Oceania" region to better reflect the great diversity of Oceania.

Microbial composition of the Korean traditional food "kochujang" analyzed by a massive sequencing technique.

PubMed

Nam, Young-Do; Park, So-lim; Lim, Seong-Il

2012-04-01

Kochujang is a traditional Korean fermented food that is made with red pepper, glutinous rice, salt, and soybean. Kochujang is fermented by naturally occurring microorganisms through which it obtains various health-promoting properties. In this study, the bacterial diversities of 9 local and 2 commercial brands of kochujang were analyzed with a barcoded pyrosequencing technique targeting the hyper-variable regions V1/V2 of the 16S rRNA gene. Through the analysis of 13524 bacterial pyrosequences, 223 bacterial species were identified, most of which converged on the phylum Firmicutes (average 93.1%). All of the kochujang samples were largely populated (>90.9% of abundance) by 12 bacterial families, and Bacillaceae showed the highest abundance in all but one sample. Bacillus subtilis and B. licheniformis were the most dominant bacterial species and were broadly distributed among the kochujang samples. Each sample contained a high abundance of region-specific bacterial species, such as B. sonorensis, B. pumilus, Weissella salipiscis, and diverse unidentified Bacillus species. Phylotype- and phylogeny-based community comparison analysis showed that the microbial communities of the two commercial brands were different from those of the local brands. Moreover, each local brand kochujang sample had region-specific microbial community reflecting the manufacturing environment. © 2012 Institute of Food Technologists®

[Microbiological Surveillance of Measles and Rubella in Spain. Laboratory Network].

PubMed

Echevarría, Juan Emilio; Fernández García, Aurora; de Ory, Fernando

2015-01-01

The Laboratory is a fundamental component on the surveillance of measles and rubella. Cases need to be properly confirmed to ensure an accurate estimation of the incidence. Strains should be genetically characterized to know the transmission pattern of these viruses and frequently, outbreaks and transmission chains can be totally discriminated only after that. Finally, the susceptibility of the population is estimated on the basis of sero-prevalence surveys. Detection of specific IgM response is the base of the laboratory diagnosis of these diseases. It should be completed with genomic detection by RT-PCR to reach an optimal efficiency, especially when sampling is performed early in the course of the disease. Genotyping is performed by genomic sequencing according to reference protocols of the WHO. Laboratory surveillance of measles and rubella in Spain is organized as a net of regional laboratories with different capabilities. The National Center of Microbiology as National Reference Laboratory (NRL), supports regional laboratories ensuring the availability of all required techniques in the whole country and watching for the quality of the results. The NRL is currently working in the implementation of new molecular techniques based on the analysis of genomic hypervariable regions for the strain characterization at sub-genotypic levels and use them in the surveillance.

Where are the Caribs? Ancient DNA from ceramic period human remains in the Lesser Antilles

PubMed Central

Mendisco, F.; Pemonge, M. H.; Leblay, E.; Romon, T.; Richard, G.; Courtaud, P.; Deguilloux, M. F.

2015-01-01

The identity and history of the indigenous groups who occupied the Lesser Antilles during the ceramic periods remain highly controversial. Although recent archaeological evidence has challenged hypotheses concerning the organization of human groups in this region, more biological data are needed to fully inform the discussion. Our study provides, to our knowledge, the first palaeogenetic data for Late Ceramic groups of the Guadeloupe archipelago, yielding crucial information concerning the identities of these groups. Despite the generally poor DNA preservation in the tested remains, we were able to retrieve Hypervariable Region 1 sequences from 11 individuals and mitochondrial single-nucleotide polymorphisms from 13 individuals. These novel data provide interesting preliminary results in favour of a common origin for all Saladoid Caribbean communities, i.e. the first ceramic groups of the region, as well as for a local continuity between the Saladoid and post-Saladoid groups. A combination of the genetic data obtained and several pieces of cultural evidence allows us to propose that two different groups inhabited the Guadeloupe archipelago during the Late Ceramic period, with the possible occupation of the La Désirade and Marie-Galante islands by groups affiliated with the Taíno communities. The working hypotheses proposed here appear consistent with recent archaeological evidence. PMID:25487339

Variation and Evolution in the Glutamine-Rich Repeat Region of Drosophila Argonaute-2

PubMed Central

Palmer, William H.; Obbard, Darren J.

2016-01-01

RNA interference pathways mediate biological processes through Argonaute-family proteins, which bind small RNAs as guides to silence complementary target nucleic acids . In insects and crustaceans Argonaute-2 silences viral nucleic acids, and therefore acts as a primary effector of innate antiviral immunity. Although the function of the major Argonaute-2 domains, which are conserved across most Argonaute-family proteins, are known, many invertebrate Argonaute-2 homologs contain a glutamine-rich repeat (GRR) region of unknown function at the N-terminus . Here we combine long-read amplicon sequencing of Drosophila Genetic Reference Panel (DGRP) lines with publicly available sequence data from many insect species to show that this region evolves extremely rapidly and is hyper-variable within species. We identify distinct GRR haplotype groups in Drosophila melanogaster, and suggest that one of these haplotype groups has recently risen to high frequency in a North American population. Finally, we use published data from genome-wide association studies of viral resistance in D. melanogaster to test whether GRR haplotypes are associated with survival after virus challenge. We find a marginally significant association with survival after challenge with Drosophila C Virus in the DGRP, but we were unable to replicate this finding using lines from the Drosophila Synthetic Population Resource panel. PMID:27317784

ITS all right mama: investigating the formation of chimeric sequences in the ITS2 region by DNA metabarcoding analyses of fungal mock communities of different complexities.

PubMed

Bjørnsgaard Aas, Anders; Davey, Marie Louise; Kauserud, Håvard

2017-07-01

The formation of chimeric sequences can create significant methodological bias in PCR-based DNA metabarcoding analyses. During mixed-template amplification of barcoding regions, chimera formation is frequent and well documented. However, profiling of fungal communities typically uses the more variable rDNA region ITS. Due to a larger research community, tools for chimera detection have been developed mainly for the 16S/18S markers. However, these tools are widely applied to the ITS region without verification of their performance. We examined the rate of chimera formation during amplification and 454 sequencing of the ITS2 region from fungal mock communities of different complexities. We evaluated the chimera detecting ability of two common chimera-checking algorithms: perseus and uchime. Large proportions of the chimeras reported were false positives. No false negatives were found in the data set. Verified chimeras accounted for only 0.2% of the total ITS2 reads, which is considerably less than what is typically reported in 16S and 18S metabarcoding analyses. Verified chimeric 'parent sequences' had significantly higher per cent identity to one another than to random members of the mock communities. Community complexity increased the rate of chimera formation. GC content was higher around the verified chimeric break points, potentially facilitating chimera formation through base pair mismatching in the neighbouring regions of high similarity in the chimeric region. We conclude that the hypervariable nature of the ITS region seems to buffer the rate of chimera formation in comparison with other, less variable barcoding regions, due to shorter regions of high sequence similarity. © 2016 John Wiley & Sons Ltd.

Molecular Basis for Lytic Bacteriophage Resistance in Enterococci.

PubMed

Duerkop, Breck A; Huo, Wenwen; Bhardwaj, Pooja; Palmer, Kelli L; Hooper, Lora V

2016-08-30

The human intestine harbors diverse communities of bacteria and bacteriophages. Given the specificity of phages for their bacterial hosts, there is growing interest in using phage therapies to combat the rising incidence of multidrug-resistant bacterial infections. A significant barrier to such therapies is the rapid development of phage-resistant bacteria, highlighting the need to understand how bacteria acquire phage resistance in vivo Here we identify novel lytic phages in municipal raw sewage that kill Enterococcus faecalis, a Gram-positive opportunistic pathogen that resides in the human intestine. We show that phage infection of E. faecalis requires a predicted integral membrane protein that we have named PIPEF (for phage infection protein from E. faecalis). We find that PIPEF is conserved in E. faecalis and harbors a 160-amino-acid hypervariable region that determines phage tropism for distinct enterococcal strains. Finally, we use a gnotobiotic mouse model of in vivo phage predation to show that the sewage phages temporarily reduce E. faecalis colonization of the intestine but that E. faecalis acquires phage resistance through mutations in PIPEF Our findings define the molecular basis for an evolutionary arms race between E. faecalis and the lytic phages that prey on them. They also suggest approaches for engineering E. faecalis phages that have altered host specificity and that can subvert phage resistance in the host bacteria. Bacteriophage therapy has received renewed attention as a potential solution to the rise in antibiotic-resistant bacterial infections. However, bacteria can acquire phage resistance, posing a major barrier to phage therapy. To overcome this problem, it is necessary to understand phage resistance mechanisms in bacteria. We have unraveled one such resistance mechanism in Enterococcus faecalis, a Gram-positive natural resident of the human intestine that has acquired antibiotic resistance and can cause opportunistic infections. We have identified a cell wall protein hypervariable region that specifies phage tropism in E. faecalis Using a gnotobiotic mouse model of in vivo phage predation, we show that E. faecalis acquires phage resistance through mutations in this cell wall protein. Our findings define the molecular basis for lytic phage resistance in E. faecalis They also suggest opportunities for engineering E. faecalis phages that circumvent the problem of bacterial phage resistance. Copyright © 2016 Duerkop et al.

The lung tissue microbiota of mild and moderate chronic obstructive pulmonary disease.

PubMed

Pragman, Alexa A; Lyu, Tianmeng; Baller, Joshua A; Gould, Trevor J; Kelly, Rosemary F; Reilly, Cavan S; Isaacson, Richard E; Wendt, Chris H

2018-01-09

Oral taxa are often found in the chronic obstructive pulmonary disease (COPD) lung microbiota, but it is not clear if this is due to a physiologic process such as aspiration or experimental contamination at the time of specimen collection. Microbiota samples were obtained from nine subjects with mild or moderate COPD by swabbing lung tissue and upper airway sites during lung lobectomy. Lung specimens were not contaminated with upper airway taxa since they were obtained surgically. The microbiota were analyzed with 16S rRNA gene qPCR and 16S rRNA gene hypervariable region 3 (V3) sequencing. Data analyses were performed using QIIME, SourceTracker, and R. Streptococcus was the most common genus in the oral, bronchial, and lung tissue samples, and multiple other taxa were present in both the upper and lower airways. Each subject's own bronchial and lung tissue microbiota were more similar to each other than were the bronchial and lung tissue microbiota of two different subjects (permutation test, p = 0.0139), indicating more within-subject similarity than between-subject similarity at these two lung sites. Principal coordinate analysis of all subject samples revealed clustering by anatomic sampling site (PERMANOVA, p = 0.001), but not by subject. SourceTracker analysis found that the sources of the lung tissue microbiota were 21.1% (mean) oral microbiota, 8.7% nasal microbiota, and 70.1% unknown. An analysis using the neutral theory of community ecology revealed that the lung tissue microbiota closely reflects the bronchial, oral, and nasal microbiota (immigration parameter estimates 0.69, 0.62, and 0.74, respectively), with some evidence of ecologic drift occurring in the lung tissue. This is the first study to evaluate the mild-moderate COPD lung tissue microbiota without potential for upper airway contamination of the lung samples. In our small study of subjects with COPD, we found oral and nasal bacteria in the lung tissue microbiota, confirming that aspiration is a source of the COPD lung microbiota.

Whole mitochondrial genome sequencing of domestic horses reveals incorporation of extensive wild horse diversity during domestication

PubMed Central

2011-01-01

Background DNA target enrichment by micro-array capture combined with high throughput sequencing technologies provides the possibility to obtain large amounts of sequence data (e.g. whole mitochondrial DNA genomes) from multiple individuals at relatively low costs. Previously, whole mitochondrial genome data for domestic horses (Equus caballus) were limited to only a few specimens and only short parts of the mtDNA genome (especially the hypervariable region) were investigated for larger sample sets. Results In this study we investigated whole mitochondrial genomes of 59 domestic horses from 44 breeds and a single Przewalski horse (Equus przewalski) using a recently described multiplex micro-array capture approach. We found 473 variable positions within the domestic horses, 292 of which are parsimony-informative, providing a well resolved phylogenetic tree. Our divergence time estimate suggests that the mitochondrial genomes of modern horse breeds shared a common ancestor around 93,000 years ago and no later than 38,000 years ago. A Bayesian skyline plot (BSP) reveals a significant population expansion beginning 6,000-8,000 years ago with an ongoing exponential growth until the present, similar to other domestic animal species. Our data further suggest that a large sample of wild horse diversity was incorporated into the domestic population; specifically, at least 46 of the mtDNA lineages observed in domestic horses (73%) already existed before the beginning of domestication about 5,000 years ago. Conclusions Our study provides a window into the maternal origins of extant domestic horses and confirms that modern domestic breeds present a wide sample of the mtDNA diversity found in ancestral, now extinct, wild horse populations. The data obtained allow us to detect a population expansion event coinciding with the beginning of domestication and to estimate both the minimum number of female horses incorporated into the domestic gene pool and the time depth of the domestic horse mtDNA gene pool. PMID:22082251

Bacterial Diversity and Community Structure of Supragingival Plaques in Adults with Dental Health or Caries Revealed by 16S Pyrosequencing

PubMed Central

Xiao, Cuicui; Ran, Shujun; Huang, Zhengwei; Liang, Jingping

2016-01-01

Dental caries has a polymicrobial etiology within the complex oral microbial ecosystem. However, the overall diversity and structure of supragingival plaque microbiota in adult dental health and caries are not well understood. Here, 160 supragingival plaque samples from patients with dental health and different severities of dental caries were collected for bacterial genomic DNA extraction, pyrosequencing by amplification of the 16S rDNA V1–V3 hypervariable regions, and bioinformatic analysis. High-quality sequences (2,261,700) clustered into 10,365 operational taxonomic units (OTUs; 97% identity), representing 453 independent species belonging to 122 genera, 66 families, 34 orders, 21 classes, and 12 phyla. All groups shared 7522 OTUs, indicating the presence of a core plaque microbiome. α diversity analysis showed that the microbial diversity in healthy plaques exceeded that of dental caries, with the diversity decreasing gradually with the severity of caries. The dominant phyla of plaque microbiota included Bacteroidetes, Actinobacteria, Proteobacteria, Firmicutes, Fusobacteria, and TM7. The dominant genera included Capnocytophaga, Prevotella, Actinomyces, Corynebacterium, Neisseria, Streptococcus, Rothia, and Leptotrichia. β diversity analysis showed that the plaque microbial community structure was similar in all groups. Using LEfSe analysis, 25 differentially abundant taxa were identified as potential biomarkers. Key genera (27) that potentially contributed to the differential distributions of plaque microbiota between groups were identified by PLS-DA analysis. Finally, co-occurrence network analysis and function predictions were performed. Treatment strategies directed toward modulating microbial interactions and their functional output should be further developed. PMID:27499752

Xenoepitope substitution avoids deceptive imprinting and broadens the immune response to foot-and-mouth disease virus.

PubMed

Szczepanek, Steven M; Barrette, Roger W; Rood, Debra; Alejo, Diana; Silbart, Lawrence K

2012-04-01

Many RNA viruses encode error-prone polymerases which introduce mutations into B and T cell epitopes, providing a mechanism for immunological escape. When regions of hypervariability are found within immunodominant epitopes with no known function, they are referred to as "decoy epitopes," which often deceptively imprint the host's immune response. In this work, a decoy epitope was identified in the foot-and-mouth disease virus (FMDV) serotype O VP1 G-H loop after multiple sequence alignment of 118 isolates. A series of chimeric cyclic peptides resembling the type O G-H loop were prepared, each bearing a defined "B cell xenoepitope" from another virus in place of the native decoy epitope. These sequences were derived from porcine respiratory and reproductive syndrome virus (PRRSV), from HIV, or from a presumptively tolerogenic sequence from murine albumin and were subsequently used as immunogens in BALB/c mice. Cross-reactive antibody responses against all peptides were compared to a wild-type peptide and ovalbumin (OVA). A broadened antibody response was generated in animals inoculated with the PRRSV chimeric peptide, in which virus binding of serum antibodies was also observed. A B cell epitope mapping experiment did not reveal recognition of any contiguous linear epitopes, raising the possibility that the refocused response was directed to a conformational epitope. Taken together, these results indicate that xenoepitope substitution is a novel method for immune refocusing against decoy epitopes of RNA viruses such as FMDV as part of the rational design of next-generation vaccines.

Next-generation sequencing (NGS) for assessment of microbial water quality: current progress, challenges, and future opportunities

PubMed Central

Tan, BoonFei; Ng, Charmaine; Nshimyimana, Jean Pierre; Loh, Lay Leng; Gin, Karina Y.-H.; Thompson, Janelle R.

2015-01-01

Water quality is an emergent property of a complex system comprised of interacting microbial populations and introduced microbial and chemical contaminants. Studies leveraging next-generation sequencing (NGS) technologies are providing new insights into the ecology of microbially mediated processes that influence fresh water quality such as algal blooms, contaminant biodegradation, and pathogen dissemination. In addition, sequencing methods targeting small subunit (SSU) rRNA hypervariable regions have allowed identification of signature microbial species that serve as bioindicators for sewage contamination in these environments. Beyond amplicon sequencing, metagenomic and metatranscriptomic analyses of microbial communities in fresh water environments reveal the genetic capabilities and interplay of waterborne microorganisms, shedding light on the mechanisms for production and biodegradation of toxins and other contaminants. This review discusses the challenges and benefits of applying NGS-based methods to water quality research and assessment. We will consider the suitability and biases inherent in the application of NGS as a screening tool for assessment of biological risks and discuss the potential and limitations for direct quantitative interpretation of NGS data. Secondly, we will examine case studies from recent literature where NGS based methods have been applied to topics in water quality assessment, including development of bioindicators for sewage pollution and microbial source tracking, characterizing the distribution of toxin and antibiotic resistance genes in water samples, and investigating mechanisms of biodegradation of harmful pollutants that threaten water quality. Finally, we provide a short review of emerging NGS platforms and their potential applications to the next generation of water quality assessment tools. PMID:26441948

Evolution, phylogeny, and molecular epidemiology of Chlamydia.

PubMed

Nunes, Alexandra; Gomes, João P

2014-04-01

The Chlamydiaceae are a family of obligate intracellular bacteria characterized by a unique biphasic developmental cycle. It encompasses the single genus Chlamydia, which involves nine species that affect a wide range of vertebral hosts, causing infections with serious impact on human health (mainly due to Chlamydia trachomatis infections) and on farming and veterinary industries. It is believed that Chlamydiales originated ∼700mya, whereas C. trachomatis likely split from the other Chlamydiaceae during the last 6mya. This corresponds to the emergence of modern human lineages, with the first descriptions of chlamydial infections as ancient as four millennia. Chlamydiaceae have undergone a massive genome reduction, on behalf of the deletional bias "use it or lose it", stabilizing at 1-1.2Mb and keeping a striking genome synteny. Their phylogeny reveals species segregation according to biological properties, with huge differences in terms of host range, tissue tropism, and disease outcomes. Genome differences rely on the occurrence of mutations in the >700 orthologous genes, as well as on events of recombination, gene loss, inversion, and paralogous expansion, affecting both a hypervariable region named the plasticity zone, and genes essentially encoding polymorphic and transmembrane head membrane proteins, type III secretion effectors and some metabolic pathways. Procedures for molecular typing are still not consensual but have allowed the knowledge of molecular epidemiology patterns for some species as well as the identification of outbreaks and emergence of successful clones for C. trachomatis. This manuscript intends to provide a comprehensive review on the evolution, phylogeny, and molecular epidemiology of Chlamydia. Copyright © 2014 Elsevier B.V. All rights reserved.

Comparative analysis of fecal microbial communities in cattle and Bactrian camels

PubMed Central

Ming, Liang; Yi, Li; Siriguleng; Hasi, Surong; He, Jing; Hai, Le; Wang, Zhaoxia; Guo, Fucheng; Qiao, Xiangyu; Jirimutu

2017-01-01

Bactrian camels may have a unique gastrointestinal (GI) microbiome because of their distinctive digestive systems, unique eating habits and extreme living conditions. However, understanding of the microbial communities in the Bactrian camel GI tract is still limited. In this study, microbial communities were investigated by comparative analyses of 16S rRNA hypervariable region V4 sequences of fecal bacteria sampled from 94 animals in four population groups: Inner Mongolian cattle (IMG-Cattle), Inner Mongolian domestic Bactrian camels (IMG-DBC), Mongolian domestic Bactrian camels (MG-DBC), and Mongolian wild Bactrian camels (MG-WBC). A total of 2,097,985 high-quality reads were obtained and yielded 471,767,607 bases of sequence. Firmicutes was the predominant phylum in the population groups IMG-Cattle, IMG-DBC and MG-WBC, followed (except in the Inner Mongolian cattle) by Verrucomicrobia. Bacteroidetes were abundant in the IMG-DBC and MG-WBC populations. Hierarchical clustered heatmap analysis revealed that the microbial community composition within the three Bactrian camel groups was relatively similar, and somewhat distinct from that in the cattle. A similar result was determined by principal component analysis, in which the camels grouped together. We also found several species-specific differences in microbial communities at the genus level: for example, Desulfovibrio was abundant in the IMG-DBC and MG-WBC groups; Pseudomonas was abundant in the IMG-Cattle group; and Fibrobacter, Coprobacillus, and Paludibacter were scarce in the MG-WBC group. Such differences may be related to different eating habits and living conditions of the cattle and the various camel populations. PMID:28301489

Unraveling the genomic mosaic of a ubiquitous genus of marine cyanobacteria

PubMed Central

Dufresne, Alexis; Ostrowski, Martin; Scanlan, David J; Garczarek, Laurence; Mazard, Sophie; Palenik, Brian P; Paulsen, Ian T; de Marsac, Nicole Tandeau; Wincker, Patrick; Dossat, Carole; Ferriera, Steve; Johnson, Justin; Post, Anton F; Hess, Wolfgang R; Partensky, Frédéric

2008-01-01

Background The picocyanobacterial genus Synechococcus occurs over wide oceanic expanses, having colonized most available niches in the photic zone. Large scale distribution patterns of the different Synechococcus clades (based on 16S rRNA gene markers) suggest the occurrence of two major lifestyles ('opportunists'/'specialists'), corresponding to two distinct broad habitats ('coastal'/'open ocean'). Yet, the genetic basis of niche partitioning is still poorly understood in this ecologically important group. Results Here, we compare the genomes of 11 marine Synechococcus isolates, representing 10 distinct lineages. Phylogenies inferred from the core genome allowed us to refine the taxonomic relationships between clades by revealing a clear dichotomy within the main subcluster, reminiscent of the two aforementioned lifestyles. Genome size is strongly correlated with the cumulative lengths of hypervariable regions (or 'islands'). One of these, encompassing most genes encoding the light-harvesting phycobilisome rod complexes, is involved in adaptation to changes in light quality and has clearly been transferred between members of different Synechococcus lineages. Furthermore, we observed that two strains (RS9917 and WH5701) that have similar pigmentation and physiology have an unusually high number of genes in common, given their phylogenetic distance. Conclusion We propose that while members of a given marine Synechococcus lineage may have the same broad geographical distribution, local niche occupancy is facilitated by lateral gene transfers, a process in which genomic islands play a key role as a repository for transferred genes. Our work also highlights the need for developing picocyanobacterial systematics based on genome-derived parameters combined with ecological and physiological data. PMID:18507822

Water channel in the binding site of a high affinity anti-methotrexate antibody.

PubMed

Gayda, Susan; Longenecker, Kenton L; Manoj, Sharmila; Judge, Russell A; Saldana, Sylvia C; Ruan, Qiaoqiao; Swift, Kerry M; Tetin, Sergey Y

2014-06-17

In the present study, we report the structure of the free and drug-bound Fab fragment of a high affinity anti-methotrexate antibody and perform a thermodynamic analysis of the binding process. The anti-methotrexate Fab fragment features a remarkably rigid tunnel-like binding site that extends into a water channel serving as a specialized route to move solvent out and into the site upon ligand binding and dissociation. This new finding in antibody structure-function relationships directly relates to the fast association (1 × 10⁷ M⁻¹ s⁻¹) and slow dissociation (4 × 10⁻⁵ s⁻¹) rates determined for mAb ADD056, resulting in a very strong binding with a K(D) ~ 3.6 pM at 20 °C. As follows from the X-ray data analysis, the methotrexate-antibody complex is stabilized by an extended network of hydrogen bonds and stacking interactions. The analysis also shows structural involvement of the CDR H3 in formation of the water channel revealing another important role of this hypervariable region. This suggests a new direction in natural affinity maturation and opens a new possibility in antibody engineering. Methotrexate is a widely used therapeutic agent for many malignant diseases and inflammatory disorders. Unfortunately, it may also interfere with central aspects of metabolism and thereby cause inevitable side effects. Therefore, methotrexate therapy requires careful monitoring of drug blood levels, which is traditionally done by immunoassays. An understanding of the structure-function properties of antibodies selected for drug monitoring substantiates the performance and robustness of such tests.

Evaluation of the genetic diversity of Plum pox virus in a single plum tree.

PubMed

Predajňa, Lukáš; Šubr, Zdeno; Candresse, Thierry; Glasa, Miroslav

2012-07-01

Genetic diversity of Plum pox virus (PPV) and its distribution within a single perennial woody host (plum, Prunus domestica) has been evaluated. A plum tree was triply infected by chip-budding with PPV-M, PPV-D and PPV-Rec isolates in 2003 and left to develop untreated under open field conditions. In September 2010 leaf and fruit samples were collected from different parts of the tree canopy. A 745-bp NIb-CP fragment of PPV genome, containing the hypervariable region encoding the CP N-terminal end was amplified by RT-PCR from each sample and directly sequenced to determine the dominant sequence. In parallel, the PCR products were cloned and a total of 105 individual clones were sequenced. Sequence analysis revealed that after 7 years of infection, only PPV-M was still detectable in the tree and that the two other isolates (PPV-Rec and PPV-D) had been displaced. Despite the fact that the analysis targeted a relatively short portion of the genome, a substantial amount of intra-isolate variability was observed for PPV-M. A total of 51 different haplotypes could be identified from the 105 individual sequences, two of which were largely dominant. However, no clear-cut structuration of the viral population by the tree architecture could be highlighted although the results obtained suggest the possibility of intra-leaf/fruit differentiation of the viral population. Comparison of the consensus sequence with the original source isolate showed no difference, suggesting within-plant stability of this original isolate under open field conditions. Copyright © 2012 Elsevier B.V. All rights reserved.

Phylogenetic and phylogeographic evidence for a Pleistocene disjunction between Campanula jacobaea (Cape Verde Islands) and C. balfourii (Socotra).

PubMed

Alarcón, Marisa; Roquet, Cristina; García-Fernández, Alfredo; Vargas, Pablo; Aldasoro, Juan José

2013-12-01

Our understanding of processes that led to biogeographic disjunct patterns of plant lineages in Macaronesia, North Africa and Socotra remains poor. Here, we study a group of Campanula species distributed across these areas integrating morphological and reproductive traits with phylogenetic and phylogeographic data based on the obtention of sequences for 4 highly variable cpDNA regions and AFLP data. The phylogeny obtained shows a sister relationship between Campanula jacobaea (endemic to Cape Verde Islands) and C. balfourii (endemic to Socotra), thus revealing a striking disjunct pattern (8300 km). These species diverged around 1.0 Mya; AFLP and haplotype data suggest that no genetic interchange has occurred since then. Their closest taxon, C. hypocrateriformis, is endemic to SW Morocco. The archipelagos of Macaronesia and Socotra have probably acted as refugia for North-African species, leading to speciation through isolation. Although C. balfourii has a restricted distribution, its genetic variability suggests that its populations have suffered no bottlenecks. C. jacobaea is also genetically rich and its distribution across Cape Verde Islands seems to have been influenced by the NE-SW trade winds, which may also have favoured the admixture found among the populations of the three southern islands. Floral features of the morphologically hypervariable C. jacobaea were also measured to assess whether the taxon C. bravensis, described for some of the southeast populations of C. jacobaea, corresponds to a different evolutionary entity. We show that morphological variation in C. jacobaea does not correspond to any genetic or geographic group. Copyright © 2013 Elsevier Inc. All rights reserved.

The Origins and Genetic Distinctiveness of the Chamorros of the Marianas Islands: An mtDNA Perspective

PubMed Central

VILAR, MIGUEL G.; CHAN, CHIM W; SANTOS, DANA R; LYNCH, DANIEL; SPATHIS, RITA; GARRUTO, RALPH M; LUM, J KOJI

2013-01-01

Background Archaeological and linguistic evidence suggests the Marianas Islands were settled around 3,600 years before present (ybp) from Island Southeast Asia (ISEA). Around 1,000 ybp latte stone pillars and the first evidence of rice cultivation appear in the Marianas. Both traditions are absent in the rest of prehistoric Oceania. Objective To examine the genetic origins and postsettlement gene flow of Chamorros of the Marianas Islands. Methods To infer the origins of the Chamorros we analyzed ~360 base pairs of the hypervariable-region 1 (HVS1) of mitochondrial DNA from 105 Chamorros from Guam, Rota, and Saipan, and the complete mitochondrial genome of 32 Guamanian Chamorros, and compared them to lineages from ISEA and neighboring Pacific archipelagoes from the database. Results Results reveal that 92% of Chamorros belong to haplogroup E, also found in ISEA but rare in Oceania. The two most numerous E lineages were identical to lineages currently found in Indonesia, while the remaining E lineages differed by only one or two mutations and all were unique to the Marianas. Seven percent of the lineages belonged to a single Chamorro-specific lineage within haplogroup B4, common to ISEA as well as Micronesia and Polynesia. Conclusions These patterns suggest a small founding population had reached and settled the Marianas from ISEA by 4,000 ybp, and developed unique mutations in isolation. A second migration from ISEA may have arrived around 1,000 ybp, introducing the latte pillars, rice agriculture and the homogeneous minority B4 lineage. PMID:23180676

The origins and genetic distinctiveness of the Chamorros of the Marianas Islands: an mtDNA perspective.

PubMed

Vilar, Miguel G; Chan, Chim W; Santos, Dana R; Lynch, Daniel; Spathis, Rita; Garruto, Ralph M; Lum, J Koji

2013-01-01

Archaeological and linguistic evidence suggests the Marianas Islands were settled around 3,600 years before present (ybp) from Island Southeast Asia (ISEA). Around 1,000 ybp latte stone pillars and the first evidence of rice cultivation appear in the Marianas. Both traditions are absent in the rest of prehistoric Oceania. To examine the genetic origins and postsettlement gene flow of Chamorros of the Marianas Islands. To infer the origins of the Chamorros we analyzed ∼360 base pairs of the hypervariable-region 1 (HVS1) of mitochondrial DNA from 105 Chamorros from Guam, Rota, and Saipan, and the complete mitochondrial genome of 32 Guamanian Chamorros, and compared them to lineages from ISEA and neighboring Pacific archipelagoes from the database. Results reveal that 92% of Chamorros belong to haplogroup E, also found in ISEA but rare in Oceania. The two most numerous E lineages were identical to lineages currently found in Indonesia, while the remaining E lineages differed by only one or two mutations and all were unique to the Marianas. Seven percent of the lineages belonged to a single Chamorro-specific lineage within haplogroup B4, common to ISEA as well as Micronesia and Polynesia. These patterns suggest a small founding population had reached and settled the Marianas from ISEA by 4,000 ybp, and developed unique mutations in isolation. A second migration from ISEA may have arrived around 1,000 ybp, introducing the latte pillars, rice agriculture and the homogeneous minority B4 lineage. Copyright © 2012 Wiley Periodicals, Inc.

Comparative genomics reveals phylogenetic distribution patterns of secondary metabolites in Amycolatopsis species.

PubMed

Adamek, Martina; Alanjary, Mohammad; Sales-Ortells, Helena; Goodfellow, Michael; Bull, Alan T; Winkler, Anika; Wibberg, Daniel; Kalinowski, Jörn; Ziemert, Nadine

2018-06-01

Genome mining tools have enabled us to predict biosynthetic gene clusters that might encode compounds with valuable functions for industrial and medical applications. With the continuously increasing number of genomes sequenced, we are confronted with an overwhelming number of predicted clusters. In order to guide the effective prioritization of biosynthetic gene clusters towards finding the most promising compounds, knowledge about diversity, phylogenetic relationships and distribution patterns of biosynthetic gene clusters is necessary. Here, we provide a comprehensive analysis of the model actinobacterial genus Amycolatopsis and its potential for the production of secondary metabolites. A phylogenetic characterization, together with a pan-genome analysis showed that within this highly diverse genus, four major lineages could be distinguished which differed in their potential to produce secondary metabolites. Furthermore, we were able to distinguish gene cluster families whose distribution correlated with phylogeny, indicating that vertical gene transfer plays a major role in the evolution of secondary metabolite gene clusters. Still, the vast majority of the diverse biosynthetic gene clusters were derived from clusters unique to the genus, and also unique in comparison to a database of known compounds. Our study on the locations of biosynthetic gene clusters in the genomes of Amycolatopsis' strains showed that clusters acquired by horizontal gene transfer tend to be incorporated into non-conserved regions of the genome thereby allowing us to distinguish core and hypervariable regions in Amycolatopsis genomes. Using a comparative genomics approach, it was possible to determine the potential of the genus Amycolatopsis to produce a huge diversity of secondary metabolites. Furthermore, the analysis demonstrates that horizontal and vertical gene transfer play an important role in the acquisition and maintenance of valuable secondary metabolites. Our results cast light on the interconnections between secondary metabolite gene clusters and provide a way to prioritize biosynthetic pathways in the search and discovery of novel compounds.

The impact of modern migrations on present-day multi-ethnic Argentina as recorded on the mitochondrial DNA genome.

PubMed

Catelli, María Laura; Alvarez-Iglesias, Vanesa; Gómez-Carballa, Alberto; Mosquera-Miguel, Ana; Romanini, Carola; Borosky, Alicia; Amigo, Jorge; Carracedo, Angel; Vullo, Carlos; Salas, Antonio

2011-08-30

The genetic background of Argentineans is a mosaic of different continental ancestries. From colonial to present times, the genetic contribution of Europeans and sub-Saharan Africans has superposed to or replaced the indigenous genetic 'stratum'. A sample of 384 individuals representing different Argentinean provinces was collected and genotyped for the first and the second mitochondrial DNA (mtDNA) hypervariable regions, and selectively genotyped for mtDNA SNPs. This data was analyzed together with additional 440 profiles from rural and urban populations plus 304 from Native American Argentineans, all available from the literature. A worldwide database was used for phylogeographic inferences, inter-population comparisons, and admixture analysis. Samples identified as belonging to hg (hg) H2a5 were sequenced for the entire mtDNA genome. Phylogenetic and admixture analyses indicate that only half of the Native American component in urban Argentineans might be attributed to the legacy of extinct ancestral Argentineans and that the Spanish genetic contribution is slightly higher than the Italian one. Entire H2a5 genomes linked these Argentinean mtDNAs to the Basque Country and improved the phylogeny of this Basque autochthonous clade. The fingerprint of African slaves in urban Argentinean mtDNAs was low and it can be phylogeographically attributed predominantly to western African. The European component is significantly more prevalent in the Buenos Aires province, the main gate of entrance for Atlantic immigration to Argentina, while the Native American component is larger in North and South Argentina. AMOVA, Principal Component Analysis and hgs/haplotype patterns in Argentina revealed an important level of genetic sub-structure in the country. Studies aimed to compare mtDNA frequency profiles from different Argentinean geographical regions (e.g., forensic and case-control studies) should take into account the important genetic heterogeneity of the country in order to prevent false positive claims of association in disease studies or inadequate evaluation of forensic evidence.

Evidence for horizontal gene transfer and separation of effector recognition from effector function revealed by analysis of effector genes shared between cape-gooseberry- and tomato-infecting formae speciales of Fusarium oxysporum.

PubMed

Simbaqueba, Jaime; Catanzariti, Ann-Maree; González, Carolina; Jones, David A

2018-05-22

RNAseq reads from cape-gooseberry plants (Physalis peruviana) infected with Fusarium oxysporum f. sp. physali (Foph) were mapped against the lineage-specific transcriptome of Fusarium oxysporum f. sp. lycopersici (Fol) to look for putative effector genes. Homologues of Fol SIX1 (designated SIX1a and SIX1b), SIX7, SIX10, SIX12, SIX15 and Ave1 were identified. The near identity of the Foph and Fol SIX7, SIX10 and SIX12 genes and their intergenic regions suggest that this gene cluster may have undergone recent lateral transfer. Foph SIX1a and SIX1b were tested for their ability to complement a SIX1 knockout mutant of Fol. This mutant has reduced pathogenicity on susceptible tomato plants, but is able to infect otherwise resistant tomato plants carrying the I-3 gene for Fusarium wilt resistance (SIX1 corresponds to Avr3). Neither, SIX1a nor SIX1b could restore full pathogenicity on susceptible tomato plants, suggesting that any role they may play in pathogenicity is likely to be specific to cape gooseberry. SIX1b, but not SIX1a, was able to restore avirulence on tomato plants carrying I-3. These findings separate the recognition of SIX1 from its role as an effector and suggest direct recognition by I-3. A hypervariable region of SIX1 undergoing diversifying selection within the F. oxysporum species complex is likely to play an important role in SIX1 recognition. These findings also indicate that I-3 could potentially be deployed as a transgene in cape gooseberry to protect this emerging crop from Foph. Alternatively, cape gooseberry germplasm could be explored for I-3 homologues capable of providing resistance to Foph. This article is protected by copyright. All rights reserved. © 2018 BSPP and John Wiley & Sons Ltd.

The impact of modern migrations on present-day multi-ethnic Argentina as recorded on the mitochondrial DNA genome

PubMed Central

2011-01-01

Background The genetic background of Argentineans is a mosaic of different continental ancestries. From colonial to present times, the genetic contribution of Europeans and sub-Saharan Africans has superposed to or replaced the indigenous genetic 'stratum'. A sample of 384 individuals representing different Argentinean provinces was collected and genotyped for the first and the second mitochondrial DNA (mtDNA) hypervariable regions, and selectively genotyped for mtDNA SNPs. This data was analyzed together with additional 440 profiles from rural and urban populations plus 304 from Native American Argentineans, all available from the literature. A worldwide database was used for phylogeographic inferences, inter-population comparisons, and admixture analysis. Samples identified as belonging to hg (hg) H2a5 were sequenced for the entire mtDNA genome. Results Phylogenetic and admixture analyses indicate that only half of the Native American component in urban Argentineans might be attributed to the legacy of extinct ancestral Argentineans and that the Spanish genetic contribution is slightly higher than the Italian one. Entire H2a5 genomes linked these Argentinean mtDNAs to the Basque Country and improved the phylogeny of this Basque autochthonous clade. The fingerprint of African slaves in urban Argentinean mtDNAs was low and it can be phylogeographically attributed predominantly to western African. The European component is significantly more prevalent in the Buenos Aires province, the main gate of entrance for Atlantic immigration to Argentina, while the Native American component is larger in North and South Argentina. AMOVA, Principal Component Analysis and hgs/haplotype patterns in Argentina revealed an important level of genetic sub-structure in the country. Conclusions Studies aimed to compare mtDNA frequency profiles from different Argentinean geographical regions (e.g., forensic and case-control studies) should take into account the important genetic heterogeneity of the country in order to prevent false positive claims of association in disease studies or inadequate evaluation of forensic evidence. PMID:21878127

Microevolution in prehistoric Andean populations: chronologic mtDNA variation in the desert valleys of northern Chile.

PubMed

Moraga, Mauricio; Santoro, Calogero M; Standen, Vivien G; Carvallo, Pilar; Rothhammer, Francisco

2005-06-01

Archeological evidence suggests that the iconographic and technological developments that took place in the highlands around Lake Titicaca in the Central Andean region had an influence on the cultural elaborations of the human groups in the valleys and the Pacific coast of northern Chile. In a previous communication, we were able to show, by means of a distance analysis, that a craniofacial differentiation accompanied the process of cultural evolution in the valleys (Rothhammer and Santoro [2001] Lat. Am. Antiq. 12:59-66). Recently, numerous South Amerindian mtDNA studies were published, and more accurate molecular techniques to study ancient mtDNA are available. In view of these recent developments, we decided 1) to study chronological changes of ancient mtDNA haplogroup frequencies in the nearby Lluta, Azapa, and Camarones Valleys, 2) to identify microevolutionary forces responsible for such changes, and 3) to compare ancient mtDNA haplogroup frequencies with previous data in order to validate craniometrical results and to reconstruct the biological history of the prehistoric valley groups in the context of their interaction with culturally more developed highland populations. From a total of 97 samples from 83 individuals, 68 samples (61 individuals) yielded amplifications for the fragments that harbor classical mtDNA markers. The haplogroup distribution among the total sample was as follows: 26.2%, haplogroup A; 34.4%, haplogroup B; 14.8%, haplogroup C; 3.3%, haplogroup D; and 21.3%, other haplogroups. Haplogroup B tended to increase, and haplogroup A to decrease during a 3,900-year time interval. The sequence data are congruent with the haplogroup analysis. In fact, the sequencing of hypervariable region I of 30 prehistoric individuals revealed 43 polymorphic sites. Sequence alignment and subsequent phylogenetic tree construction showed two major clusters associated with the most common restriction haplogroups. Individuals belonging to haplogroups C and D tended to cluster together with nonclassical lineages. 2004 Wiley-Liss, Inc.

Genetic diversity of Candidatus Liberibacter asiaticus based on two hypervariable effector genes in Thailand

USDA-ARS?s Scientific Manuscript database

Huanglongbing (HLB), also known as citrus greening, is one of the most destructive diseases of citrus worldwide. HLB is associated with three species of ‘Candidatus Liberibacter’ with ‘Ca. L. asiaticus’ (Las) being the most widely distributed around the world, and the only species detected in Thail...

Improving IT Portfolio Management Decision Confidence Using Multi-Criteria Decision Making and Hypervariate Display Techniques

ERIC Educational Resources Information Center

Landmesser, John Andrew

2014-01-01

Information technology (IT) investment decision makers are required to process large volumes of complex data. An existing body of knowledge relevant to IT portfolio management (PfM), decision analysis, visual comprehension of large volumes of information, and IT investment decision making suggest Multi-Criteria Decision Making (MCDM) and…

Hypervariable minisatellites: recombinators or innocent bystanders?

PubMed

Jarman, A P; Wells, R A

1989-11-01

It has become apparent in recent years that unexpectedly large numbers of minisatellites exist within the eukaryotic genome. Their use in genetics is well known, but as with any new class of sequence, there is also much speculation about their involvement in a range of biological processes. How much is known of their biology?

An assessment of the hypervariable domains of the 16S rRNA genes for their value in determining microbial community diversity: the paradox of traditional ecological indices.

PubMed

Mills, DeEtta K; Entry, James A; Voss, Joshua D; Gillevet, Patrick M; Mathee, Kalai

2006-09-01

Amplicon length heterogeneity PCR (LH-PCR) was investigated for its ability to distinguish between microbial community patterns from the same soil type under different land management practices. Natural sagebrush and irrigated mouldboard-ploughed soils from Idaho were queried as to which hypervariable domains, or combinations of 16S rRNA gene domains, were the best molecular markers. Using standard ecological indices to measure richness, diversity and evenness, the combination of three domains, V1, V3 and V1+V2, or the combined V1 and V3 domains were the markers that could best distinguish the undisturbed natural sagebrush communities from the mouldboard-ploughed microbial communities. Bray-Curtis similarity and multidimensional scaling were found to be better metrics to ordinate and cluster the LH-PCR community profiling data. The use/misuse of traditional ecological indices such as diversity and evenness to study microbial community profiles will remain a major point to consider when performing metagenomic studies.

Ribosomal Biosynthesis of the Cyclic Peptide Toxins of Amanita Mushrooms

PubMed Central

Walton, Jonathan D.; Hallen-Adams, Heather E.; Luo, Hong

2014-01-01

Some species of mushrooms in the genus Amanita are extremely poisonous and frequently fatal to mammals including humans and dogs. Their extreme toxicity is due to amatoxins such as α- and β-amanitin. Amanita mushrooms also biosynthesize a chemically related group of toxins, the phallotoxins, such as phalloidin. The amatoxins and phallotoxins (collectively known as the Amanita toxins) are bicyclic octa- and heptapeptides, respectively. Both contain an unusual Trp-Cys cross-bridge known as tryptathionine. We have shown that, in Amanita bisporigera, the amatoxins and phallotoxins are synthesized as proproteins on ribosomes and not by nonribosomal peptide synthetases. The proproteins are 34–35 amino acids in length and have no predicted signal peptides. The genes for α-amanitin (AMA1) and phallacidin (PHA1) are members of a large family of related genes, characterized by highly conserved amino acid sequences flanking a hypervariable “toxin” region. The toxin regions are flanked by invariant proline (Pro) residues. An enzyme that could cleave the proprotein of phalloidin was purified from the phalloidin-producing lawn mushroom Conocybe apala. The enzyme is a serine protease in the prolyl oligopeptidase (POP) subfamily. The same enzyme cuts at both Pro residues to release the linear hepta- or octapeptide. PMID:20564017

A new genome-mining tool redefines the lasso peptide biosynthetic landscape

PubMed Central

Tietz, Jonathan I.; Schwalen, Christopher J.; Patel, Parth S.; Maxson, Tucker; Blair, Patricia M.; Tai, Hua-Chia; Zakai, Uzma I.; Mitchell, Douglas A.

2016-01-01

Ribosomally synthesized and post-translationally modified peptide (RiPP) natural products are attractive for genome-driven discovery and re-engineering, but limitations in bioinformatic methods and exponentially increasing genomic data make large-scale mining difficult. We report RODEO (Rapid ORF Description and Evaluation Online), which combines hidden Markov model-based analysis, heuristic scoring, and machine learning to identify biosynthetic gene clusters and predict RiPP precursor peptides. We initially focused on lasso peptides, which display intriguing physiochemical properties and bioactivities, but their hypervariability renders them challenging prospects for automated mining. Our approach yielded the most comprehensive mapping of lasso peptide space, revealing >1,300 compounds. We characterized the structures and bioactivities of six lasso peptides, prioritized based on predicted structural novelty, including an unprecedented handcuff-like topology and another with a citrulline modification exceptionally rare among bacteria. These combined insights significantly expand the knowledge of lasso peptides, and more broadly, provide a framework for future genome-mining efforts. PMID:28244986

Characterization of Equine Infectious Anemia Virus Long Terminal Repeat Quasispecies In Vitro and In Vivo

PubMed Central

Wang, Xue-Feng; Liu, Qiang; Wang, Yu-Hong; Wang, Shuai; Chen, Jie; Lin, Yue-Zhi; Ma, Jian; Zhou, Jian-Hua

2018-01-01

ABSTRACT The equine infectious anemia virus (EIAV) attenuated vaccine was developed by long-term passaging of a field-isolated virulent strain in cross-species hosts, followed by successive cultivation in cells in vitro. To explore the molecular mechanism underlying the evolution of the EIAV attenuated vaccine, a systematic study focusing on long-terminal-repeat (LTR) variation in numerous virus strains ranging from virulent EIAV to attenuated EIAV was performed over time both in vitro and in vivo. Two hypervariable regions were identified within the U3 region in the enhancer region (EHR) and the negative regulatory element (NRE) and within the R region in the transcription start site (TSS) and the Tat-activating region (TAR). Among these sites, variation in the U3 region resulted in the formation of additional transcription factor binding sites; this variation of the in vitro-adapted strains was consistent with the loss of pathogenicity. Notably, the same LTR variation pattern was observed both in vitro and in vivo. Generally, the LTR variation in both the attenuated virus and the virulent strain fluctuated over time in vivo. Interestingly, the attenuated-virus-specific LTR variation was also detected in horses infected with the virulent strain, supporting the hypothesis that the evolution of an attenuated virus might have involved branching from EIAV quasispecies. This hypothesis was verified by phylogenetic analysis. The present systematic study examining the molecular evolution of attenuated EIAV from EIAV quasispecies may provide an informative model reflecting the evolution of similar lentiviruses. IMPORTANCE The attenuated EIAV vaccine was the first lentiviral vaccine used to successfully control for equine infectious anemia in China. This vaccine provides an important reference for studying the relationship between EIAV gene variation and changes in biological characteristics. Importantly, the vaccine provides a model for the investigation of lentiviral quasispecies evolution. This study followed the “natural” development of the attenuated EIAV vaccine by use of a systematic analysis of LTR evolution in vitro and in vivo. The results revealed that the increase in LTR variation with passaging was accompanied by a decrease in virulence, which indicated that LTR variability might parallel the attenuation of virulence. Interestingly, the attenuated-virus-specific LTR variation was also detected in virulent-strain-infected horses, a finding consistent with those of previous investigations of gp90 and S2 evolution. Therefore, we present a hypothesis that the evolution of the attenuated virus may involve branching from EIAV quasispecies present in vivo. PMID:29386282

Identifying Plants of Stampede Pinto Bean with Resistance to New races of Rust Pathogen

USDA-ARS?s Scientific Manuscript database

The rust disease of dry beans is caused by a hyper-variable fungus that continually produces new virulent strains. Two new strains, known as races, emerged in Michigan and North Dakota in 2007 and 2008, respectively, which surmounted the resistance of a widely used rust-resistance gene known as Ur-...

Interdisciplinary approach to the demography of Jamaica

PubMed Central

2012-01-01

Background The trans-Atlantic slave trade dramatically changed the demographic makeup of the New World, with varying regions of the African coast exploited differently over roughly a 400 year period. When compared to the discrete mitochondrial haplotype distribution of historically appropriate source populations, the unique distribution within a specific source population can prove insightful in estimating the contribution of each population. Here, we analyzed the first hypervariable region of mitochondrial DNA in a sample from the Caribbean island of Jamaica and compared it to aggregated populations in Africa divided according to historiographically defined segments of the continent's coastline. The results from these admixture procedures were then compared to the wealth of historic knowledge surrounding the disembarkation of Africans on the island. Results In line with previous findings, the matriline of Jamaica is almost entirely of West African descent. Results from the admixture analyses suggest modern Jamaicans share a closer affinity with groups from the Gold Coast and Bight of Benin despite high mortality, low fecundity, and waning regional importation. The slaves from the Bight of Biafra and West-central Africa were imported in great numbers; however, the results suggest a deficit in expected maternal contribution from those regions. Conclusions When considering the demographic pressures imposed by chattel slavery on Jamaica during the slave era, the results seem incongruous. Ethnolinguistic and ethnographic evidence, however, may explain the apparent non-random levels of genetic perseverance. The application of genetics may prove useful in answering difficult demographic questions left by historically voiceless groups. PMID:22360861

A South American Prehistoric Mitogenome: Context, Continuity, and the Origin of Haplogroup C1d.

PubMed

Sans, Mónica; Figueiro, Gonzalo; Hughes, Cris E; Lindo, John; Hidalgo, Pedro C; Malhi, Ripan S

2015-01-01

Based on mitochondrial DNA (mtDNA), it has been estimated that at least 15 founder haplogroups peopled the Americas. Subhaplogroup C1d3 was defined based on the mitogenome of a living individual from Uruguay that carried a lineage previously identified in hypervariable region I sequences from ancient and modern Uruguayan individuals. When complete mitogenomes were studied, additional substitutions were found in the coding region of the mitochondrial genome. Using a complete ancient mitogenome and three modern mitogenomes, we aim to clarify the ancestral state of subhaplogroup C1d3 and to better understand the peopling of the region of the Río de la Plata basin, as well as of the builders of the mounds from which the ancient individuals were recovered. The ancient mitogenome, belonging to a female dated to 1,610±46 years before present, was identical to the mitogenome of one of the modern individuals. All individuals share the mutations defining subhaplogroup C1d3. We estimated an age of 8,974 (5,748-12,261) years for the most recent common ancestor of C1d3, in agreement with the initial peopling of the geographic region. No individuals belonging to the defined lineage were found outside of Uruguay, which raises questions regarding the mobility of the prehistoric inhabitants of the country. Moreover, the present study shows the continuity of Native lineages over at least 6,000 years.

A South American Prehistoric Mitogenome: Context, Continuity, and the Origin of Haplogroup C1d

PubMed Central

Sans, Mónica; Figueiro, Gonzalo; Hughes, Cris E.; Lindo, John; Hidalgo, Pedro C.; Malhi, Ripan S.

2015-01-01

Based on mitochondrial DNA (mtDNA), it has been estimated that at least 15 founder haplogroups peopled the Americas. Subhaplogroup C1d3 was defined based on the mitogenome of a living individual from Uruguay that carried a lineage previously identified in hypervariable region I sequences from ancient and modern Uruguayan individuals. When complete mitogenomes were studied, additional substitutions were found in the coding region of the mitochondrial genome. Using a complete ancient mitogenome and three modern mitogenomes, we aim to clarify the ancestral state of subhaplogroup C1d3 and to better understand the peopling of the region of the Río de la Plata basin, as well as of the builders of the mounds from which the ancient individuals were recovered. The ancient mitogenome, belonging to a female dated to 1,610±46 years before present, was identical to the mitogenome of one of the modern individuals. All individuals share the mutations defining subhaplogroup C1d3. We estimated an age of 8,974 (5,748–12,261) years for the most recent common ancestor of C1d3, in agreement with the initial peopling of the geographic region. No individuals belonging to the defined lineage were found outside of Uruguay, which raises questions regarding the mobility of the prehistoric inhabitants of the country. Moreover, the present study shows the continuity of Native lineages over at least 6,000 years. PMID:26509686

Analysis of uniparental lineages in two villages of Santiago Del Estero, Argentina, seat of Pueblos de Indios in colonial times.

PubMed

Pauro, Maia; García, Angelina; Nores, Rodrigo; Demarchi, Darío A

2013-10-01

Based on the analysis of the mitochondrial control region and seven biallelic markers of the Y chromosome, we investigated the genetic composition of two rural populations of southern Santiago del Estero, Argentina, that were seats in colonial times of pueblos de indios, a colonial practice that consisted of concentrating the indigenous populations in organized and accessible settlements, to facilitate Christianizing and policing. We found the Native American Y chromosome haplogroup Q1a3a in only 11% (3 of 27) of the males. Haplogroup R, common in European populations, is the most frequent haplogroup in Santiago del Estero (55%). In contrast, the persistence of Native American maternal lineages is extremely high (95%). This finding is most likely due to the low incidence in that region of the 20th century European wave of migration and to the existence of pueblos de indios from 1612 to the first decades of the 19th century. In contrast to archeological records that suggest Santiago del Estero late pre-Hispanic groups were strongly influenced by the Andean world, we did not find genetic evidence in support of significant gene fl ow. On the other hand, these populations share many mitochondrial DNA hypervariable region I (HVRI) haplotypes with other populations from the Sierras Pampeanas (particularly with Córdoba) and the Gran Chaco regions. Copyright © 2014 Wayne State University Press, Detroit, Michigan 48201-1309.

Interdisciplinary approach to the demography of Jamaica.

PubMed

Deason, Michael L; Salas, Antonio; Newman, Simon P; Macaulay, Vincent A; St A Morrison, Errol Y; Pitsiladis, Yannis P

2012-02-23

The trans-Atlantic slave trade dramatically changed the demographic makeup of the New World, with varying regions of the African coast exploited differently over roughly a 400 year period. When compared to the discrete mitochondrial haplotype distribution of historically appropriate source populations, the unique distribution within a specific source population can prove insightful in estimating the contribution of each population. Here, we analyzed the first hypervariable region of mitochondrial DNA in a sample from the Caribbean island of Jamaica and compared it to aggregated populations in Africa divided according to historiographically defined segments of the continent's coastline. The results from these admixture procedures were then compared to the wealth of historic knowledge surrounding the disembarkation of Africans on the island. In line with previous findings, the matriline of Jamaica is almost entirely of West African descent. Results from the admixture analyses suggest modern Jamaicans share a closer affinity with groups from the Gold Coast and Bight of Benin despite high mortality, low fecundity, and waning regional importation. The slaves from the Bight of Biafra and West-central Africa were imported in great numbers; however, the results suggest a deficit in expected maternal contribution from those regions. When considering the demographic pressures imposed by chattel slavery on Jamaica during the slave era, the results seem incongruous. Ethnolinguistic and ethnographic evidence, however, may explain the apparent non-random levels of genetic perseverance. The application of genetics may prove useful in answering difficult demographic questions left by historically voiceless groups.

A primer on the phylogeography of Lagothrix lagotricha (sensu Fooden) in northern South America.

PubMed

Botero, Sergio; Stevenson, Pablo R; Di Fiore, Anthony

2015-01-01

The taxonomic history of the genus Lagothrix is complex, with molecular and morphological assessments giving conflicting results for the separation between its taxa. Phylogeographic studies of the most widely distributed species, Lagothrix lagotricha, have only been attempted recently and are limited to few individuals per collection site, many of which were captive making their geographical origin dubious. There is debate regarding the possibility of raising subspecies of Lagothrix lagotricha to the species level, therefore the geographical origin of samples is particularly relevant. In the present work we revisit the intraspecific phylogeography of L. lagotricha from northwestern South America, including the subspecies L. l. poeppiggi, L. l. lagotricha and L. l. lugens (sensu Fooden, 1963), using DNA sequence data from hypervariable region I of the mitochondrial control region (D-loop HVI). Our results suggest a complex picture in which there are well delimited evolutionary units that, nonetheless, do not correlate well with the morphological variation used to support the current delimitation of taxa. Additionally, we corroborate previous results showing a lack of reciprocal monophyly between the putative subspecies of Lagothrix lagotricha, and we propose that this may be due to ancestral polymorphism that has been maintained following the recent spread of woolly monkeys throughout the western Amazonian lowlands and into the inter-Andean region of Colombia. Copyright © 2014 Elsevier Inc. All rights reserved.

Multiple point mutations in a shuttle vector propagated in human cells: evidence for an error-prone DNA polymerase activity.

PubMed

Seidman, M M; Bredberg, A; Seetharam, S; Kraemer, K H

1987-07-01

Mutagenesis was studied at the DNA-sequence level in human fibroblast and lymphoid cells by use of a shuttle vector plasmid, pZ189, containing a suppressor tRNA marker gene. In a series of experiments, 62 plasmids were recovered that had two to six base substitutions in the 160-base-pair marker gene. Approximately 20-30% of the mutant plasmids that were recovered after passing ultraviolet-treated pZ189 through a repair-proficient human fibroblast line contained these multiple mutations. In contrast, passage of ultraviolet-treated pZ189 through an excision-repair-deficient (xeroderma pigmentosum) line yielded only 2% multiple base substitution mutants. Introducing a single-strand nick in otherwise unmodified pZ189 adjacent to the marker, followed by passage through the xeroderma pigmentosum cells, resulted in about 66% multiple base substitution mutants. The multiple mutations were found in a 160-base-pair region containing the marker gene but were rarely found in an adjacent 170-base-pair region. Passing ultraviolet-treated or nicked pZ189 through a repair-proficient human B-cell line also yielded multiple base substitution mutations in 20-33% of the mutant plasmids. An explanation for these multiple mutations is that they were generated by an error-prone polymerase while filling gaps. These mutations share many of the properties displayed by mutations in the immunoglobulin hypervariable regions.

Analysis of sequence variation among smeDEF multi drug efflux pump genes and flanking DNA from defined 16S rRNA subgroups of clinical Stenotrophomonas maltophilia isolates.

PubMed

Gould, Virginia C; Okazaki, Aki; Howe, Robin A; Avison, Matthew B

2004-08-01

To determine the level of variation in the smeDEF efflux pump and smeT transcriptional regulator genes among three defined 16S rRNA sequence subgroups of clinical Stenotrophomonas maltophilia isolates. smeDEF sequencing used a PCR genome walking approach. Determination of the sequence surrounding smeDEF used a flanking primer PCR method and specific primers anchored in smeD or smeF together with random primers. smeDEF is chromosomal and located in the same position in the chromosome in all three subgroups of isolates. Flanking smeD is a gene, smeT, encoding a putative transcriptional repressor for smeDEF. Variation at these loci among the isolates is considerably lower (up to 10%) than at intrinsic beta-lactamase loci (up to 30%) in the same isolates, implying greater functional constraint. The smeD-smeT intergenic region contains a highly conserved section, which maps with previously predicted promoter/operator regions, and a hypervariable untranslated region, which can be used to subgroup clinical isolates. These data provide further evidence that it is possible to group clinical isolates of the inherently variable species, S. maltophilia, based on genotypic properties. Isolate D457, in which most work concerning smeDEF expression has been performed, does not fall into S. maltophilia subgroup A, which is the most typical.

A new fungal large subunit ribosomal RNA primer for high throughput sequencing surveys

DOE Office of Scientific and Technical Information (OSTI.GOV)

Mueller, Rebecca C.; Gallegos-Graves, La Verne; Kuske, Cheryl R.

The inclusion of phylogenetic metrics in community ecology has provided insights into important ecological processes, particularly when combined with high-throughput sequencing methods; however, these approaches have not been widely used in studies of fungal communities relative to other microbial groups. Two obstacles have been considered: (1) the internal transcribed spacer (ITS) region has limited utility for constructing phylogenies and (2) most PCR primers that target the large subunit (LSU) ribosomal unit generate amplicons that exceed current limits of high-throughput sequencing platforms. We designed and tested a PCR primer (LR22R) to target approximately 300–400 bp region of the D2 hypervariable regionmore » of the fungal LSU for use with the Illumina MiSeq platform. Both in silico and empirical analyses showed that the LR22R–LR3 pair captured a broad range of fungal taxonomic groups with a small fraction of non-fungal groups. Phylogenetic placement of publically available LSU D2 sequences showed broad agreement with taxonomic classification. Comparisons of the LSU D2 and the ITS2 ribosomal regions from environmental samples and known communities showed similar discriminatory abilities of the two primer sets. Altogether, these findings show that the LR22R–LR3 primer pair has utility for phylogenetic analyses of fungal communities using high-throughput sequencing methods.« less

Analysis of the epitope structure of Plum pox virus coat protein.

PubMed

Candresse, Thierry; Saenz, Pilar; García, Juan Antonio; Boscia, Donato; Navratil, Milan; Gorris, Maria Teresa; Cambra, Mariano

2011-05-01

Typing of the particular Plum pox virus (PPV) strain responsible in an outbreak has important practical implications and is frequently performed using strain-specific monoclonal antibodies (MAbs). Analysis in Western blots of the reactivity of 24 MAbs to a 112-amino-acid N-terminal fragment of the PPV coat protein (CP) expressed in Escherichia coli showed that 21 of the 24 MAbs recognized linear or denaturation-insensitive epitopes. A series of eight C-truncated CP fragments allowed the mapping of the epitopes recognized by the MAbs. In all, 14 of them reacted to the N-terminal hypervariable region, defining a minimum of six epitopes, while 7 reacted to the beginning of the core region, defining a minimum of three epitopes. Sequence comparisons allowed the more precise positioning of regions recognized by several MAbs, including those recognized by the 5B-IVIA universal MAb (amino acids 94 to 100) and by the 4DG5 and 4DG11 D serogroup-specific MAbs (amino acids 43 to 64). A similar approach coupled with infectious cDNA clone mutagenesis showed that a V74T mutation in the N-terminus of the CP abolished the binding of the M serogroup-specific AL MAb. Taken together, these results provide a detailed positioning of the epitopes recognized by the most widely used PPV detection and typing MAbs.

Strong positive selection and recombination drive the antigenic variation of the PilE protein of the human pathogen Neisseria meningitidis.

PubMed

Andrews, T Daniel; Gojobori, Takashi

2004-01-01

The PilE protein is the major component of the Neisseria meningitidis pilus, which is encoded by the pilE/pilS locus that includes an expressed gene and eight homologous silent fragments. The silent gene fragments have been shown to recombine through gene conversion with the expressed gene and thereby provide a means by which novel antigenic variants of the PilE protein can be generated. We have analyzed the evolutionary rate of the pilE gene using the nucleotide sequence of two complete pilE/pilS loci. The very high rate of evolution displayed by the PilE protein appears driven by both recombination and positive selection. Within the semivariable region of the pilE and pilS genes, recombination appears to occur within multiple small sequence blocks that lie between conserved sequence elements. Within the hypervariable region, positive selection was identified from comparison of the silent and expressed genes. The unusual gene conversion mechanism that operates at the pilE/pilS locus is a strategy employed by N. meningitidis to enhance mutation of certain regions of the PilE protein. The silent copies of the gene effectively allow "parallelized" evolution of pilE, thus enabling the encoded protein to rapidly explore a large area of sequence space in an effort to find novel antigenic variants.

Geographic distribution of Theileria sp. (buffalo) and Theileria sp. (bougasvlei) in Cape buffalo (Syncerus caffer) in southern Africa: implications for speciation.

PubMed

Pienaar, Ronel; Latif, Abdalla A; Thekisoe, Oriel M M; Mans, Ben J

2014-03-01

Strict control measures apply to movement of buffalo in South Africa including testing for Theileria parva, the causative agent of Corridor disease in cattle. The official test is a real-time hybridization PCR assay that amplifies the 18S rRNA V4 hyper-variable region of T. parva, T. sp. (buffalo) and T. sp. (bougasvlei). Mixed infections with the latter organisms affect diagnostic sensitivity due to PCR suppression. While the incidence of mixed infections in the Corridor disease endemic region of South Africa is significant, little information is available on the specific distribution and prevalence of T. sp. (buffalo) and T. sp. (bougasvlei). Specific real-time PCR assays were developed and a total of 1211 samples known to harbour these parasites were screened. Both parasites are widely distributed in southern Africa and the incidence of mixed infections with T. parva within the endemic region is similar (∼25-50%). However, a significant discrepancy exists in regard to mixed infections of T. sp. (buffalo) and T. sp. (bougasvlei) (∼10%). Evidence for speciation between T. sp. (buffalo) and T. sp. (bougasvlei) is supported by phylogenetic analysis of the COI gene, and their designation as different species. This suggests mutual exclusion of parasites and the possibility of hybrid sterility in cases of mixed infections.

Mitochondrial Variation among the Aymara and the Signatures of Population Expansion in the Central Andes

PubMed Central

BATAI, KEN; WILLIAMS, SLOAN R.

2015-01-01

Objectives The exploitation of marine resources and intensive agriculture led to a marked population increase early in central Andean prehistory. Constant historic and prehistoric population movements also characterize this region. These features undoubtedly affected regional genetic variation, but the exact nature of these effects remains uncertain. Methods Mitochondrial DNA (mtDNA) hypervariable region I sequence variation in 61 Aymara individuals from La Paz, Bolivia, was analyzed and compared to sequences from 47 other South American populations to test hypotheses of whether increased female effective population size and gene flow influenced the mtDNA variation among central Andean populations. Results The Aymara and Quechua were genetically diverse showing evidence of population expansion and large effective population size, and a demographic expansion model fits the mtDNA variation found among central Andean populations well. Estimated migration rates and the results of AMOVA and multidimensional scaling analysis suggest that female gene flow was also an important factor, influencing genetic variation among the central Andeans as well as lowland populations from western South America. mtDNA variation in south central Andes correlated better with geographic proximity than with language, and fit a population continuity model. Conclusion The mtDNA data suggests that the central Andeans experienced population expansion, most likely because of rapid demographic expansion after introduction of intensive agriculture, but roles of female gene flow need to be further explored. PMID:24449040

A new fungal large subunit ribosomal RNA primer for high throughput sequencing surveys

DOE PAGES

Mueller, Rebecca C.; Gallegos-Graves, La Verne; Kuske, Cheryl R.

2015-12-09

The inclusion of phylogenetic metrics in community ecology has provided insights into important ecological processes, particularly when combined with high-throughput sequencing methods; however, these approaches have not been widely used in studies of fungal communities relative to other microbial groups. Two obstacles have been considered: (1) the internal transcribed spacer (ITS) region has limited utility for constructing phylogenies and (2) most PCR primers that target the large subunit (LSU) ribosomal unit generate amplicons that exceed current limits of high-throughput sequencing platforms. We designed and tested a PCR primer (LR22R) to target approximately 300–400 bp region of the D2 hypervariable regionmore » of the fungal LSU for use with the Illumina MiSeq platform. Both in silico and empirical analyses showed that the LR22R–LR3 pair captured a broad range of fungal taxonomic groups with a small fraction of non-fungal groups. Phylogenetic placement of publically available LSU D2 sequences showed broad agreement with taxonomic classification. Comparisons of the LSU D2 and the ITS2 ribosomal regions from environmental samples and known communities showed similar discriminatory abilities of the two primer sets. Altogether, these findings show that the LR22R–LR3 primer pair has utility for phylogenetic analyses of fungal communities using high-throughput sequencing methods.« less

New Population and Phylogenetic Features of the Internal Variation within Mitochondrial DNA Macro-Haplogroup R0

PubMed Central

Cerezo, Maria; Quintáns, Beatriz; Zarrabeitia, Maria Teresa; Cuscó, Ivon; Lareu, Maria Victoria; García, Óscar; Pérez-Jurado, Luis; Carracedo, Ángel; Salas, Antonio

2009-01-01

Background R0 embraces the most common mitochondrial DNA (mtDNA) lineage in West Eurasia, namely, haplogroup H (∼40%). R0 sub-lineages are badly defined in the control region and therefore, the analysis of diagnostic coding region polymorphisms is needed in order to gain resolution in population and medical studies. Methodology/Principal Findings We sequenced the first hypervariable segment (HVS-I) of 518 individuals from different North Iberian regions. The mtDNAs belonging to R0 (∼57%) were further genotyped for a set of 71 coding region SNPs characterizing major and minor branches of R0. We found that the North Iberian Peninsula shows moderate levels of population stratification; for instance, haplogroup V reaches the highest frequency in Cantabria (north-central Iberia), but lower in Galicia (northwest Iberia) and Catalonia (northeast Iberia). When compared to other European and Middle East populations, haplogroups H1, H3 and H5a show frequency peaks in the Franco-Cantabrian region, declining from West towards the East and South Europe. In addition, we have characterized, by way of complete genome sequencing, a new autochthonous clade of haplogroup H in the Basque country, named H2a5. Its coalescence age, 15.6±8 thousand years ago (kya), dates to the period immediately after the Last Glacial Maximum (LGM). Conclusions/Significance In contrast to other H lineages that experienced re-expansion outside the Franco-Cantabrian refuge after the LGM (e.g. H1 and H3), H2a5 most likely remained confined to this area till present days. PMID:19340307

Ultrasensitive electrochemical biosensor for detection of DNA from Bacillus subtilis by coupling target-induced strand displacement and nicking endonuclease signal amplification.

PubMed

Hu, Yuhua; Xu, Xueqin; Liu, Qionghua; Wang, Ling; Lin, Zhenyu; Chen, Guonan

2014-09-02

A simple, ultrasensitive, and specific electrochemical biosensor was designed to determine the given DNA sequence of Bacillus subtilis by coupling target-induced strand displacement and nicking endonuclease signal amplification. The target DNA (TD, the DNA sequence from the hypervarient region of 16S rDNA of Bacillus subtilis) could be detected by the differential pulse voltammetry (DPV) in a range from 0.1 fM to 20 fM with the detection limit down to 0.08 fM at the 3s(blank) level. This electrochemical biosensor exhibits high distinction ability to single-base mismatch, double-bases mismatch, and noncomplementary DNA sequence, which may be expected to detect single-base mismatch and single nucleotide polymorphisms (SNPs). Moreover, the applicability of the designed biosensor for detecting the given DNA sequence from Bacillus subtilis was investigated. The result obtained by electrochemical method is approximately consistent with that by a real-time quantitative polymerase chain reaction detecting system (QPCR) with SYBR Green.

Dip in the gene pool: metagenomic survey of natural coccolithovirus communities.

PubMed

Pagarete, António; Kusonmano, Kanthida; Petersen, Kjell; Kimmance, Susan A; Martínez Martínez, Joaquín; Wilson, William H; Hehemann, Jan-Hendrik; Allen, Michael J; Sandaa, Ruth-Anne

2014-10-01

Despite the global oceanic distribution and recognised biogeochemical impact of coccolithoviruses (EhV), their diversity remains poorly understood. Here we employed a metagenomic approach to study the occurrence and progression of natural EhV community genomic variability. Analysis of EhV metagenomes from the early and late stages of an induced bloom led to three main discoveries. First, we observed resilient and specific genomic signatures in the EhV community associated with the Norwegian coast, which reinforce the existence of limitations to the capacity of dispersal and genomic exchange among EhV populations. Second, we identified a hyper-variable region (approximately 21kbp long) in the coccolithovirus genome. Third, we observed a clear trend for EhV relative amino-acid diversity to reduce from early to late stages of the bloom. This study validated two new methodological combinations, and proved very useful in the discovery of new genomic features associated with coccolithovirus natural communities. Copyright © 2014 Elsevier Inc. All rights reserved.

Extremophiles in Household Water Heaters

NASA Astrophysics Data System (ADS)

Wilpiszeski, R.; House, C. H.

2016-12-01

A significant fraction of Earth's microbial diversity comes from species living in extreme environments, but natural extreme environments can be difficult to access. Manmade systems like household water heaters serve as an effective proxy for thermophilic environments that are otherwise difficult to sample directly. As such, we are investigating the biogeography, taxonomic distribution, and evolution of thermophiles growing in domestic water heaters. Citizen scientists collected hot tap water culture- and filter- samples from 101 homes across the United States. We recovered a single species of thermophilic heterotroph from culture samples inoculated from water heaters across the United States, Thermus scotoductus. Whole-genome sequencing was conducted to better understand the distribution and evolution of this single species. We have also sequenced hyper-variable regions of the 16S rRNA gene from whole-community filter samples to identify the broad diversity and distribution of microbial cells captured from each water heater. These results shed light on the processes that shape thermophilic populations and genomes at a spatial resolution that is difficult to access in naturally occurring extreme ecosystems.

Hepatitis C Virus Antigenic Convergence

PubMed Central

Campo, David S.; Dimitrova, Zoya; Yokosawa, Jonny; Hoang, Duc; Perez, Nestor O.; Ramachandran, Sumathi; Khudyakov, Yury

2012-01-01

Vaccine development against hepatitis C virus (HCV) is hindered by poor understanding of factors defining cross-immunoreactivity among heterogeneous epitopes. Using synthetic peptides and mouse immunization as a model, we conducted a quantitative analysis of cross-immunoreactivity among variants of the HCV hypervariable region 1 (HVR1). Analysis of 26,883 immunological reactions among pairs of peptides showed that the distribution of cross-immunoreactivity among HVR1 variants was skewed, with antibodies against a few variants reacting with all tested peptides. The HVR1 cross-immunoreactivity was accurately modeled based on amino acid sequence alone. The tested peptides were mapped in the HVR1 sequence space, which was visualized as a network of 11,319 sequences. The HVR1 variants with a greater network centrality showed a broader cross-immunoreactivity. The entire sequence space is explored by each HCV genotype and subtype. These findings indicate that HVR1 antigenic diversity is extensively convergent and effectively limited, suggesting significant implications for vaccine development. PMID:22355779

Ecological succession of the microbial communities of an air-conditioning cooling coil in the tropics.

PubMed

Acerbi, E; Chénard, C; Miller, D; Gaultier, N E; Heinle, C E; Chang, V W-C; Uchida, A; Drautz-Moses, D I; Schuster, S C; Lauro, F M

2017-03-01

Air-conditioning systems harbor microorganisms, potentially spreading them to indoor environments. While air and surfaces in air-conditioning systems are periodically sampled as potential sources of indoor microbes, little is known about the dynamics of cooling coil-associated communities and their effect on the downstream airflow. Here, we conducted a 4-week time series sampling to characterize the succession of an air-conditioning duct and cooling coil after cleaning. Using an universal primer pair targeting hypervariable regions of the 16S/18S ribosomal RNA, we observed a community succession for the condensed water, with the most abundant airborne taxon Agaricomycetes fungi dominating the initial phase and Sphingomonas bacteria becoming the most prevalent taxa toward the end of the experiment. Duplicate air samples collected upstream and downstream of the coil suggest that the system does not act as ecological filter or source/sink for specific microbial taxa during the duration of the experiment. © 2016 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

Molecular characterization of chicken infectious anemia viruses detected from breeder and broiler chickens in South Korea.

PubMed

Kim, H-R; Kwon, Y-K; Bae, Y-C; Oem, J-K; Lee, O-S

2010-11-01

In South Korea, 32 sequences of chicken infectious anemia virus (CIAV) from various flocks of breeder and commercial chickens were genetically characterized for the first time. Phylogenetic analysis of the viral protein 1 gene, including a hypervariable region of the CIAV genome, indicated that Korean CIAV strains were separated into groups II, IIIa, and IIIb. Strains were commonly identified in great-grandparent and grandparent breeder farms as well as commercial chicken farms. In the field, CIAV strains from breeder farms had no clinical effects, but commercial farm strains were associated with depression, growth retardation, and anemia regardless of the group from which the strain originated. In addition, we identified 7 CIAV genomes that were similar to vaccine strains from vaccinated and unvaccinated breeder flocks. These data suggest that further studies on pathogenicity and vaccine efficacy against the different CIAV group are needed, along with continuous CIAV surveillance and genetic analysis at breeder farms.

Isolation of novel variants of infectious bursal disease virus from different outbreaks in Northeast India.

PubMed

Morla, Sudhir; Deka, Pankaj; Kumar, Sachin

2016-04-01

Infectious bursal disease virus (IBDV) is a highly infectious disease of young chicken that predominantly affects the immune system. In the present study, we are reporting first comprehensive study of IBDV outbreaks from the Northeastern part of India. Northeast India shares a porous border with four different countries; and as a rule any outbreak in the neighboring countries substantially affects the poultry population in the adjoining states. Nucleotide sequence analysis of the VP2 gene of the IBDV isolates from the Northeastern part of India suggested the extreme virulent nature of the virus. The virulent marker amino acids (A222, I242, Q253, I256 and S299) in the hypervariable region of the Northeastern isolates were found identical with the reported very virulent strains of IBDV. A unique insertion of I/L294V was recorded in all the isolates of the Northeastern India. The study will be useful in understanding the circulating pathotypes of IBDV in India. Copyright © 2016 Elsevier Ltd. All rights reserved.

DEVELOPMENT OF CRASSPHAGE-BASED QPCR ASSAYS ...

EPA Pesticide Factsheets

A newly discovered bacteriophage, “crAssphage”, is predicted to be both highlyabundant and predominantly human-associated, both ideal characteristics for a human-specific fecal indicator. A total of 384 end-point PCR primers were designed along the length of the crAssphage genome, eliminating regions suspected to be hypervariable or react with other animal sources. The primer pairs were rigorously tested in three rounds of screening for specificity, geographic variability, limit of detection, and environmental water performance. The two best performing assays, crAss056 and crAss064, were adapted to a qPCR platform and exhibited a specificity of 98.0% and 98.9%, respectively. The markers’ abundance was compared with two bacterial based assays and were found at concentrations at or above the bacterial based assays in wastewater influent and impacted environmental waters. This poster will present the methodology of the novel marker development and the potential uses for this technology in maintaining sustainable waterways in the future. To inform the public.

Single substitutions to closely related amino acids contribute to the functional diversification of an insect-inducible, positively selected plant cystatin.

PubMed

Rasoolizadeh, Asieh; Goulet, Marie-Claire; Sainsbury, Frank; Cloutier, Conrad; Michaud, Dominique

2016-04-01

A causal link has been reported between positively selected amino acids in plant cystatins and the inhibitory range of these proteins against insect digestive cysteine (Cys) proteases. Here we assessed the impact of single substitutions to closely related amino acids on the contribution of positive selection to cystatin diversification. Cystatin sequence alignments, while confirming hypervariability, indicated a preference for related amino acids at positively selected sites. For example, the non-polar residues leucine (Leu), isoleucine (Ile) and valine (Val) were shown to predominate at positively selected site 2 in the N-terminal region, unlike selected sites 6 and 10, where polar residues are preferred. The model cystatin SlCYS8 and single variants with Leu, Ile or Val at position 2 were compared with regard to their ability to bind digestive proteases of the coleopteran pest Leptinotarsa decemlineata and to induce compensatory responses in this insect. A functional proteomics procedure to capture target Cys proteases in midgut extracts allowed confirmation of distinct binding profiles for the cystatin variants. A shotgun proteomics procedure to monitor whole Cys protease complements revealed protease family specific compensatory responses in the insect, dependent on the variant ingested. Our data confirm the contribution of closely related amino acids to the functional diversity of positively selected plant cystatins in a broader structure/function context imposing physicochemical constraints to primary structure alterations. They also underline the complexity of protease/inhibitor interactions in plant-insect systems, and the challenges still to be met in order to harness the full potential of ectopically expressed protease inhibitors in crop protection. © 2016 Federation of European Biochemical Societies.

Phylogenetic Characterization of Fecal Microbial Communities of Dogs Fed Diets with or without Supplemental Dietary Fiber Using 454 Pyrosequencing

PubMed Central

Middelbos, Ingmar S.; Vester Boler, Brittany M.; Qu, Ani; White, Bryan A.; Swanson, Kelly S.; Fahey, George C.

2010-01-01

Background Dogs suffer from many of the same maladies as humans that may be affected by the gut microbiome, but knowledge of the canine microbiome is incomplete. This work aimed to use 16S rDNA tag pyrosequencing to phylogenetically characterize hindgut microbiome in dogs and determine how consumption of dietary fiber affects community structure. Principal Findings Six healthy adult dogs were used in a crossover design. A control diet without supplemental fiber and a beet pulp-supplemented (7.5%) diet were fed. Fecal DNA was extracted and the V3 hypervariable region of the microbial 16S rDNA gene amplified using primers suitable for 454-pyrosequencing. Microbial diversity was assessed on random 2000-sequence subsamples of individual and pooled DNA samples by diet. Our dataset comprised 77,771 reads with an average length of 141 nt. Individual samples contained approximately 129 OTU, with Fusobacteria (23 – 40% of reads), Firmicutes (14 – 28% of reads) and Bacteroidetes (31 – 34% of reads) being co-dominant phyla. Feeding dietary fiber generally decreased Fusobacteria and increased Firmicutes, but these changes were not equally apparent in all dogs. UniFrac analysis revealed that structure of the gut microbiome was affected by diet and Firmicutes appeared to play a strong role in by-diet clustering. Conclusions Our data suggest three co-dominant bacterial phyla in the canine hindgut. Furthermore, a relatively small amount of dietary fiber changed the structure of the gut microbiome detectably. Our data are among the first to characterize the healthy canine gut microbiome using pyrosequencing and provide a basis for studies focused on devising dietary interventions for microbiome-associated diseases. PMID:20339542

Genetic variation of the VP1 gene of the virulent duck hepatitis A virus type 1 (DHAV-1) isolates in Shandong province of China.

PubMed

Gao, Jiming; Chen, Junhao; Si, Xingkui; Xie, Zhijing; Zhu, Yanli; Zhang, Xingxiao; Wang, Shujing; Jiang, Shijin

2012-08-01

To investigate the relationship of the variation of virulence and the external capsid proteins of the pandemic duck hepatitis A virus type 1 (DHAV-1) isolates, the virulence, cross neutralization assays and the complete sequence of the virion protein 1 (VP1) gene of nine virulent DHAV-1 strains, which were isolated from infected ducklings with clinical symptoms in Shandong province of China in 2007-2008, were tested. The fifth generation duck embryo allantoic liquids of the 9 isolates were tested on 12-day-old duck embryos and on 7-day-old ducklings for the median embryonal lethal doses (ELD(50)s) and the median lethal doses (LD(50)s), respectively. The results showed that the ELD(50)s of embryonic duck eggs of the 9 DHAV-1 isolates were between 1.9 × 10(6)/mL to 1.44 × 10(7)/mL, while the LD(50)s were 2.39 × 10(5)/mL to 6.15 × 10(6)/mL. Cross-neutralization tests revealed that the 9 DHAV-1 isolates were completely neutralized by the standard serum and the hyperimmune sera against the 9 DHAV-1 isolates, respectively. Compared with other virulent, moderate virulent, attenuated vaccine and mild strains, the VP1 genes of the 9 strains shared 89.8%-99.7% similarity at the nucleotide level and 92.4%-99.6% at amino acid level with other DHAV-1 strains. There were three hypervariable regions at the C-terminus (aa 158-160, 180-193 and 205-219) and other variable points in VP1 protein, but which didn't cause virulence of DHAV-1 change.

Porcine reproductive and respiratory syndrome virus nonstructural protein 2 contributes to NF-κB activation.

PubMed

Fang, Ying; Fang, Liurong; Wang, Yang; Lei, Yingying; Luo, Rui; Wang, Dang; Chen, Huanchun; Xiao, Shaobo

2012-04-30

Nuclear factor-kappaB (NF-κB) is an inducible transcription factor that plays a key role in inflammation and immune responses, as well as in the regulation of cell proliferation and survival. Previous studies by our group and others have demonstrated that porcine reproductive and respiratory syndrome virus (PRRSV) infection could activate NF-κB in MARC-145 cells and alveolar macrophages. The nucleocapsid (N) protein was identified as an NF-κB activator among the structural proteins encoded by PRRSV; however, it remains unclear whether the nonstructural proteins (Nsps) contribute to NF-κB activation. In this study, we identified which Nsps can activate NF-κB and investigated the potential mechanism(s) by which they act. By screening the individual Nsps of PRRSV strain WUH3, Nsp2 exhibited great potential to activate NF-κB in MARC-145 and HeLa cells. Overexpression of Nsp2 induced IκBα degradation and nuclear translocation of NF-κB. Furthermore, Nsp2 also induced NF-κB-dependent inflammatory factors, including interleukin (IL)-6, IL-8, COX-2, and RANTES. Compared with the Nsp2 of the classical PRRSV strain, the Nsp2 of highly pathogenic PRRSV (HP-PRRSV) strains that possess a 30 amino acid (aa) deletion in Nsp2 displayed greater NF-κB activation. However, the 30-aa deletion was demonstrated to not be associated with NF-κB activation. Further functional domain analyses revealed that the hypervariable region (HV) of Nsp2 was essential for NF-κB activation. Taken together, these data indicate that PRRSV Nsp2 is a multifunctional protein participating in the modulation of host inflammatory response, which suggests an important role of Nsp2 in pathogenesis and disease outcomes.

Microbial Diversity in Sediment Ecosystems (Evaporites Domes, Microbial Mats, and Crusts) of Hypersaline Laguna Tebenquiche, Salar de Atacama, Chile.

PubMed

Fernandez, Ana B; Rasuk, Maria C; Visscher, Pieter T; Contreras, Manuel; Novoa, Fernando; Poire, Daniel G; Patterson, Molly M; Ventosa, Antonio; Farias, Maria E

2016-01-01

We combined nucleic acid-based molecular methods, biogeochemical measurements, and physicochemical characteristics to investigate microbial sedimentary ecosystems of Laguna Tebenquiche, Atacama Desert, Chile. Molecular diversity, and biogeochemistry of hypersaline microbial mats, rhizome-associated concretions, and an endoevaporite were compared with: The V4 hypervariable region of the 16S rRNA gene was amplified by pyrosequencing to analyze the total microbial diversity (i.e., bacteria and archaea) in bulk samples, and in addition, in detail on a millimeter scale in one microbial mat and in one evaporite. Archaea were more abundant than bacteria. Euryarchaeota was one of the most abundant phyla in all samples, and particularly dominant (97% of total diversity) in the most lithified ecosystem, the evaporite. Most of the euryarchaeal OTUs could be assigned to the class Halobacteria or anaerobic and methanogenic archaea. Planctomycetes potentially also play a key role in mats and rhizome-associated concretions, notably the aerobic organoheterotroph members of the class Phycisphaerae. In addition to cyanobacteria, members of Chromatiales and possibly the candidate family Chlorotrichaceae contributed to photosynthetic carbon fixation. Other abundant uncultured taxa such as the candidate division MSBL1, the uncultured MBGB, and the phylum Acetothermia potentially play an important metabolic role in these ecosystems. Lithifying microbial mats contained calcium carbonate precipitates, whereas endoevoporites consisted of gypsum, and halite. Biogeochemical measurements revealed that based on depth profiles of O2 and sulfide, metabolic activities were much higher in the non-lithifying mat (peaking in the least lithified systems) than in lithifying mats with the lowest activity in endoevaporites. This trend in decreasing microbial activity reflects the increase in salinity, which may play an important role in the biodiversity.

From Wolves to Dogs, and Back: Genetic Composition of the Czechoslovakian Wolfdog.

PubMed

Smetanová, Milena; Černá Bolfíková, Barbora; Randi, Ettore; Caniglia, Romolo; Fabbri, Elena; Galaverni, Marco; Kutal, Miroslav; Hulva, Pavel

2015-01-01

The Czechoslovakian Wolfdog is a unique dog breed that originated from hybridization between German Shepherds and wild Carpathian wolves in the 1950s as a military experiment. This breed was used for guarding the Czechoslovakian borders during the cold war and is currently kept by civilian breeders all round the world. The aim of our study was to characterize, for the first time, the genetic composition of this breed in relation to its known source populations. We sequenced the hypervariable part of the mtDNA control region and genotyped the Amelogenin gene, four sex-linked microsatellites and 39 autosomal microsatellites in 79 Czechoslovakian Wolfdogs, 20 German Shepherds and 28 Carpathian wolves. We performed a range of population genetic analyses based on both empirical and simulated data. Only two mtDNA and two Y-linked haplotypes were found in Czechoslovakian Wolfdogs. Both mtDNA haplotypes were of domestic origin, while only one of the Y-haplotypes was shared with German Shepherds and the other was unique to Czechoslovakian Wolfdogs. The observed inbreeding coefficient was low despite the small effective population size of the breed, possibly due to heterozygote advantages determined by introgression of wolf alleles. Moreover, Czechoslovakian Wolfdog genotypes were distinct from both parental populations, indicating the role of founder effect, drift and/or genetic hitchhiking. The results revealed the peculiar genetic composition of the Czechoslovakian Wolfdog, showing a limited introgression of wolf alleles within a higher proportion of the dog genome, consistent with the reiterated backcrossing used in the pedigree. Artificial selection aiming to keep wolf-like phenotypes but dog-like behavior resulted in a distinctive genetic composition of Czechoslovakian Wolfdogs, which provides a unique example to study the interactions between dog and wolf genomes.

Insights into the bacterial community and its temporal succession during the fermentation of wine grapes

PubMed Central

Piao, Hailan; Hawley, Erik; Kopf, Scott; DeScenzo, Richard; Sealock, Steven; Henick-Kling, Thomas; Hess, Matthias

2015-01-01

Grapes harbor complex microbial communities. It is well known that yeasts, typically Saccharomyces cerevisiae, and bacteria, commonly the lactic acid fermenting Oenococcus oeni, work sequentially during primary and secondary wine fermentation. In addition to these main players, several microbes, often with undesirable effects on wine quality, have been found in grapes and during wine fermentation. However, still little is known about the dynamics of the microbial community during the fermentation process. In previous studies culture dependent methods were applied to detect and identify microbial organisms associated with grapes and grape products, which resulted in a picture that neglected the non-culturable fraction of the microbes. To obtain a more complete picture of how microbial communities change during grape fermentation and how different fermentation techniques might affect the microbial community composition, we employed next-generation sequencing (NGS)—a culture-independent method. A better understanding of the microbial dynamics and their effect on the final product is of great importance to help winemakers produce wine styles of consistent and high quality. In this study, we focused on the bacterial community dynamics during wine vinification by amplifying and sequencing the hypervariable V1–V3 region of the 16S rRNA gene—a phylogenetic marker gene that is ubiquitous within prokaryotes. Bacterial communities and their temporal succession was observed for communities associated with organically and conventionally produced wines. In addition, we analyzed the chemical characteristics of the grape musts during the organic and conventional fermentation process. These analyses revealed distinct bacterial population with specific temporal changes as well as different chemical profiles for the organically and conventionally produced wines. In summary these results suggest a possible correlation between the temporal succession of the bacterial population and the chemical wine profiles. PMID:26347718

Site-Directed Mutagenesis, in Vivo Electroporation and Mass Spectrometry in Search for Determinants of the Subcellular Targeting of Rab7b Paralogue in the Model Eukaryote Paramecium Octaurelia

PubMed Central

Wyroba, E.; Kwaśniak, P.; Miller, K.; Kobyłecki, K.; Osińska, M.

2016-01-01

Protein products of paralogous genes resulting from whole genome duplication may acquire new functions. The role of post-translational modifications (PTM) in proper targeting of Paramecium Rab7b paralogue (distinct from that of Rab7a directly involved in phagocytosis) was studied using point mutagenesis, proteomic analysis and double immunofluorescence after in vivo electroporation of the mutagenized protein. Here we show that substitution of Thr200 by Ala diminished the incorporation of [P32] by 37% and of [C14-]UDP-glucose by 24% into recombinant Rab7b_200 in comparison to the non-mutagenized control. Double confocal imaging revealed that Rab7b_200 was mistargeted upon electroporation into living cells in contrast to non-mutagenized recombinant Rab7b correctly incorporated in the cytostome area. Using nano LC-MS/MS to compare the peptide map of Rab7b with that after deglycosylation with a mixture of five enzymes of different specificity we identified a peptide ion at m/z=677.63+ representing a glycan group attached to Thr200. Based on its mass and quantitative assays with [P32] and [C14]UDP-glucose, the suggested composition of the adduct attached to Thr200 is (Hex)1(HexNAc)1(Phos)3 or (HexNAc)1 (Deoxyhexose)1 (Phos)1 (HexA)1. These data indicate that PTM of Thr200 located in the hypervariable C-region of Paramecium octaurelia Rab7b is crucial for the proper localization/function of this protein. Moreover, the two Rab7 paralogues differ also in another PTM: substantially more phosphorylated amino acid residues are in Rab7b than in Rab7a. PMID:27349314

Characterization of shed medicinal leech mucus reveals a diverse microbiota

PubMed Central

Ott, Brittany M.; Rickards, Allen; Gehrke, Lauren; Rio, Rita V. M.

2015-01-01

Microbial transmission through mucosal-mediated mechanisms is widespread throughout the animal kingdom. One example of this occurs with Hirudo verbana, the medicinal leech, where host attraction to shed conspecific mucus facilitates horizontal transmission of a predominant gut symbiont, the Gammaproteobacterium Aeromonas veronii. However, whether this mucus may harbor other bacteria has not been examined. Here, we characterize the microbiota of shed leech mucus through Illumina deep sequencing of the V3-V4 hypervariable region of the 16S rRNA gene. Additionally, Restriction Fragment Length Polymorphism (RFLP) typing with subsequent Sanger Sequencing of a 16S rRNA gene clone library provided qualitative confirmation of the microbial composition. Phylogenetic analyses of full-length 16S rRNA sequences were performed to examine microbial taxonomic distribution. Analyses using both technologies indicate the dominance of the Bacteroidetes and Proteobacteria phyla within the mucus microbiota. We determined the presence of other previously described leech symbionts, in addition to a number of putative novel leech-associated bacteria. A second predominant gut symbiont, the Rikenella-like bacteria, was also identified within mucus and exhibited similar population dynamics to A. veronii, suggesting persistence in syntrophy beyond the gut. Interestingly, the most abundant bacterial genus belonged to Pedobacter, which includes members capable of producing heparinase, an enzyme that degrades the anticoagulant, heparin. Additionally, bacteria associated with denitrification and sulfate cycling were observed, indicating an abundance of these anions within mucus, likely originating from the leech excretory system. A diverse microbiota harbored within shed mucus has significant potential implications for the evolution of microbiomes, including opportunities for gene transfer and utility in host capture of a diverse group of symbionts. PMID:25620963

Effects of an EPSPS-transgenic soybean line ZUTS31 on root-associated bacterial communities during field growth

PubMed Central

Cheng, Jing; Wang, Gu-Hao; Zhu, Yin-Ling; Zhang, Li-Ya; Shou, Hui-Xia; Qi, Jin-Liang

2018-01-01

The increased worldwide commercial cultivation of transgenic crops during the past 20 years is accompanied with potential effects on the soil microbial communities, because many rhizosphere and endosphere bacteria play important roles in promoting plant health and growth. Previous studies reported that transgenic plants exert differential effects on soil microbial communities, especially rhizobacteria. Thus, this study compared the soybean root-associated bacterial communities between a 5-enolpyruvylshikimate-3-phosphate synthase -transgenic soybean line (ZUTS31 or simply Z31) and its recipient cultivar (Huachun3 or simply HC3) at the vegetative, flowering, and seed-filling stages. High-throughput sequencing of 16S rRNA gene (16S rDNA) V4 hypervariable region amplicons via Illumina MiSeq and real-time quantitative PCR (qPCR) were performed. Our results revealed no significant differences in the overall alpha diversity of root-associated bacterial communities at the three developmental stages and in the beta diversity of root-associated bacterial communities at the flowering stage between Z31 and HC3 under field growth. However, significant differences in the beta diversity of rhizosphere bacterial communities were found at the vegetative and seed-filling stages between the two groups. Furthermore, the results of next generation sequencing and qPCR showed that the relative abundances of root-associated main nitrogen-fixing bacterial genera, especially Bradyrhizobium in the roots, evidently changed from the flowering stage to the seed-filling stage. In conclusion, Z31 exerts transitory effects on the taxonomic diversity of rhizosphere bacterial communities at the vegetative and seed-filling stages compared to the control under field conditions. In addition, soybean developmental change evidently influences the main symbiotic nitrogen-fixing bacterial genera in the roots from the flowering stage to the seed-filling stage. PMID:29408918

Unbiased Characterization of Anopheles Mosquito Blood Meals by Targeted High-Throughput Sequencing

PubMed Central

Logue, Kyle; Keven, John Bosco; Cannon, Matthew V.; Reimer, Lisa; Siba, Peter; Walker, Edward D.; Zimmerman, Peter A.; Serre, David

2016-01-01

Understanding mosquito host choice is important for assessing vector competence or identifying disease reservoirs. Unfortunately, the availability of an unbiased method for comprehensively evaluating the composition of insect blood meals is very limited, as most current molecular assays only test for the presence of a few pre-selected species. These approaches also have limited ability to identify the presence of multiple mammalian hosts in a single blood meal. Here, we describe a novel high-throughput sequencing method that enables analysis of 96 mosquitoes simultaneously and provides a comprehensive and quantitative perspective on the composition of each blood meal. We validated in silico that universal primers targeting the mammalian mitochondrial 16S ribosomal RNA genes (16S rRNA) should amplify more than 95% of the mammalian 16S rRNA sequences present in the NCBI nucleotide database. We applied this method to 442 female Anopheles punctulatus s. l. mosquitoes collected in Papua New Guinea (PNG). While human (52.9%), dog (15.8%) and pig (29.2%) were the most common hosts identified in our study, we also detected DNA from mice, one marsupial species and two bat species. Our analyses also revealed that 16.3% of the mosquitoes fed on more than one host. Analysis of the human mitochondrial hypervariable region I in 102 human blood meals showed that 5 (4.9%) of the mosquitoes unambiguously fed on more than one person. Overall, analysis of PNG mosquitoes illustrates the potential of this approach to identify unsuspected hosts and characterize mixed blood meals, and shows how this approach can be adapted to evaluate inter-individual variations among human blood meals. Furthermore, this approach can be applied to any disease-transmitting arthropod and can be easily customized to investigate non-mammalian host sources. PMID:26963245

Molecular detection and identification of piroplasms in sika deer (Cervus nippon) from Jilin Province, China.

PubMed

Liu, Junlong; Yang, Jifei; Guan, Guiquan; Liu, Aihong; Wang, Bingjie; Luo, Jianxun; Yin, Hong

2016-03-16

Piroplasmosis is an important disease of domestic animals and wildlife and is caused by organisms from the genera Theileria and Babesia. Wildlife such as sika deer play an important role as reservoir hosts for several species of Theileria and Babesia. Using blood samples collected from sika deer, we investigated the epidemiology of Theileria spp. and Babesia spp. in sika deer from Jilin Province in China and identified those species that cause pathogenic infections in sika deer. Sixty-eight blood samples of sika deer were collected from three areas of the Jilin Province in Northeast China. Genomic DNA was extracted, and the V4 hypervariable region of the 18S rRNA of the piroplasms was amplified using the nested PCR method. The selected positive samples were sequenced to identify species of Babesia and Theileria. PCR detection revealed that 24 samples were positive for Theileria and Babesia spp. (35.29 %, 95 % CI = 11.8-46.8). After alignment, a sequenced fragment for Theileria cervi was found to be the most prevalent from the obtained samples (22.06 %, 95 % CI = 11.8-49.6). Six sika deer samples were identified as being infected with a Theileria sp. that was similar to a Theileria sp. found from spotted deer in India. In addition to the results above, for the first time, we identified T. annulata infection from one sample of sika deer and Babesia sp. from two samples, which showed high identity with Babesia motasi found in sheep from China. The present study offers new data on the pathogens of piroplasmosis in sika deer in northeastern China. For the first time, sika deer was confirmed as a reservoir host for the T. annulata of cattle and the B. motasi of sheep, which was found in China.

Evaluation of composition and individual variability of rumen microbiota in yaks by 16S rRNA high-throughput sequencing technology.

PubMed

Guo, Wei; Li, Ying; Wang, Lizhi; Wang, Jiwen; Xu, Qin; Yan, Tianhai; Xue, Bai

2015-08-01

The Yak (Bos grunniens) is a unique species of ruminant animals that is important to agriculture of the Tibetan plateau, and has a complex intestinal microbial community. The objective of the present study was to characterize the composition and individual variability of microbiota in the rumen of yaks using 16S rRNA gene high-throughput sequencing technique. Rumen samples used in the present study were obtained from grazing adult male yaks (n = 6) in a commercial farm in Ganzi Autonomous Prefecture of Sichuan Province, China. Universal prokaryote primers were used to target the V4-V5 hypervariable region of 16S rRNA gene. A total of 7200 operational taxonomic units (OTUs) were obtained after sequence filtering and chimera removal. Within these OTUs, 0.56% belonged to Archaea (40 OTUs), 7.19% to unassigned species (518 OTUs), and the remaining OTUs (6642) in all samples were of bacterial origin. When examining the community structure of bacteria, we identified 23 phyla within 159 families after taxonomic summarization. Bacteroidetes and Firmicutes were the predominant phyla accounting for 39.68% (SD = 0.05) and 45.90% (SD = 0.06), respectively. Moreover, 3764 OTUs were identified as shared OTUs (i.e. represented in all yaks) and belonged to 35 genera, exhibiting highly variable abundance across individual samples. Phylogenetic placement of these genera across individual samples was examined. In addition, we evaluated the distance among the 6 rumen samples by adding taxon phylogeny using UniFrac, representing 24.1% of average distance. In summary, the current study reveals a shared rumen microbiome and phylogenetic lineage and presents novel information on composition and individual variability of the bacterial community in the rumen of yaks. Copyright © 2015. Published by Elsevier Ltd.

The genome of an ancient Rouran individual reveals an important paternal lineage in the Donghu population.

PubMed

Li, Jiawei; Zhang, Ye; Zhao, Yongbin; Chen, Yongzhi; Ochir, A; Sarenbilige; Zhu, Hong; Zhou, Hui

2018-04-21

Following the Xiongnu and Xianbei, the Rouran Khaganate (Rouran) was the third great nomadic tribe on the Mongolian Steppe. However, few human remains from this tribe are available for archaeologists and geneticists to study, as traces of the tombs of these nomadic people have rarely been found. In 2014, the IA-M1 remains (TL1) at the Khermen Tal site from the Rouran period were found by a Sino-Mongolian joint archaeological team in Mongolia, providing precious material for research into the genetic imprint of the Rouran. The mtDNA hypervariable sequence I (HVS-I) and Y-chromosome SNPs were analyzed, and capture of the paternal non-recombining region of the Y chromosome (NRY) and whole-genome shotgun sequencing of TL1 were performed. The materials from three sites representing the three ancient nationalities (Donghu, Xianbei, and Shiwei) were selected for comparison with the TL1 individual. The mitochondrial haplotype of the TL1 individual was D4b1a2a1. The Y-chromosome haplotype was C2b1a1b/F3830 (ISOGG 2015), which was the same as that of the other two ancient male nomadic samples (ZHS5 and GG3) related to the Xianbei and Shiwei, which were also detected as F3889; this haplotype was reported to be downstream of F3830 by Wei et al. (). We conclude that F3889 downstream of F3830 is an important paternal lineage of the ancient Donghu nomads. The Donghu-Xianbei branch is expected to have made an important paternal genetic contribution to Rouran. This component of gene flow ultimately entered the gene pool of modern Mongolic- and Manchu-speaking populations. © 2018 Wiley Periodicals, Inc.

Molecular Characterization of Human Respiratory Syncytial Virus in the Philippines, 2012-2013.

PubMed

Malasao, Rungnapa; Okamoto, Michiko; Chaimongkol, Natthawan; Imamura, Tadatsugu; Tohma, Kentaro; Dapat, Isolde; Dapat, Clyde; Suzuki, Akira; Saito, Mayuko; Saito, Mariko; Tamaki, Raita; Pedrera-Rico, Gay Anne Granada; Aniceto, Rapunzel; Quicho, Reynaldo Frederick Negosa; Segubre-Mercado, Edelwisa; Lupisan, Socorro; Oshitani, Hitoshi

2015-01-01

Human respiratory syncytial virus (HRSV) is a major cause of acute lower respiratory tract infections in infants and children worldwide. We performed molecular analysis of HRSV among infants and children with clinical diagnosis of severe pneumonia in four study sites in the Philippines, including Biliran, Leyte, Palawan, and Metro Manila from June 2012 to July 2013. Nasopharyngeal swabs were collected and screened for HRSV using real-time polymerase chain reaction (PCR). Positive samples were tested by conventional PCR and sequenced for the second hypervariable region (2nd HVR) of the G gene. Among a total of 1,505 samples, 423 samples were positive for HRSV (28.1%), of which 305 (72.1%) and 118 (27.9%) were identified as HRSV-A and HRSV-B, respectively. Two genotypes of HRSV-A, NA1 and ON1, were identified during the study period. The novel ON1 genotype with a 72-nucleotide duplication in 2nd HVR of the G gene increased rapidly and finally became the predominant genotype in 2013 with an evolutionary rate higher than the NA1 genotype. Moreover, in the ON1 genotype, we found positive selection at amino acid position 274 (p<0.05) and massive O- and N-glycosylation in the 2nd HVR of the G gene. Among HRSV-B, BA9 was the predominant genotype circulating in the Philippines. However, two sporadic cases of GB2 genotype were found, which might share a common ancestor with other Asian strains. These findings suggest that HRSV is an important cause of severe acute respiratory infection among children in the Philippines and revealed the emergence and subsequent predominance of the ON1 genotype and the sporadic detection of the GB2 genotype. Both genotypes were detected for the first time in the Philippines.

Molecular Characterization of Human Respiratory Syncytial Virus in the Philippines, 2012-2013

PubMed Central

Malasao, Rungnapa; Okamoto, Michiko; Chaimongkol, Natthawan; Imamura, Tadatsugu; Tohma, Kentaro; Dapat, Isolde; Dapat, Clyde; Suzuki, Akira; Saito, Mayuko; Saito, Mariko; Tamaki, Raita; Pedrera-Rico, Gay Anne Granada; Aniceto, Rapunzel; Quicho, Reynaldo Frederick Negosa; Segubre-Mercado, Edelwisa; Lupisan, Socorro; Oshitani, Hitoshi

2015-01-01

Human respiratory syncytial virus (HRSV) is a major cause of acute lower respiratory tract infections in infants and children worldwide. We performed molecular analysis of HRSV among infants and children with clinical diagnosis of severe pneumonia in four study sites in the Philippines, including Biliran, Leyte, Palawan, and Metro Manila from June 2012 to July 2013. Nasopharyngeal swabs were collected and screened for HRSV using real-time polymerase chain reaction (PCR). Positive samples were tested by conventional PCR and sequenced for the second hypervariable region (2nd HVR) of the G gene. Among a total of 1,505 samples, 423 samples were positive for HRSV (28.1%), of which 305 (72.1%) and 118 (27.9%) were identified as HRSV-A and HRSV-B, respectively. Two genotypes of HRSV-A, NA1 and ON1, were identified during the study period. The novel ON1 genotype with a 72-nucleotide duplication in 2nd HVR of the G gene increased rapidly and finally became the predominant genotype in 2013 with an evolutionary rate higher than the NA1 genotype. Moreover, in the ON1 genotype, we found positive selection at amino acid position 274 (p<0.05) and massive O- and N-glycosylation in the 2nd HVR of the G gene. Among HRSV-B, BA9 was the predominant genotype circulating in the Philippines. However, two sporadic cases of GB2 genotype were found, which might share a common ancestor with other Asian strains. These findings suggest that HRSV is an important cause of severe acute respiratory infection among children in the Philippines and revealed the emergence and subsequent predominance of the ON1 genotype and the sporadic detection of the GB2 genotype. Both genotypes were detected for the first time in the Philippines. PMID:26540236

The Features of Fecal and Ileal Mucosa-Associated Microbiota in Dairy Calves during Early Infection with Mycobacterium avium Subspecies paratuberculosis.

PubMed

Derakhshani, Hooman; De Buck, Jeroen; Mortier, Rienske; Barkema, Herman W; Krause, Denis O; Khafipour, Ehsan

2016-01-01

Current diagnostic tests for Johne's disease (JD), a chronic granulomatous inflammation of the gastrointestinal tract of ruminants caused by Mycobacterium avium subspecies paratuberculosis (MAP), lack the sensitivity to identify infected animals at early (asymptomatic) stages of the disease. The objective was to determine the pattern of MAP-associated dysbiosis of intestinal microbiota as a potential biomarker for early detection of infected cattle. To that end, genomic DNA was extracted from ileal mucosa and fecal samples collected from 28 MAP-positive and five control calves. High-throughput Illumina sequencing of the V4 hypervariable region of the 16S rRNA gene was used for community profiling of ileal mucosa-associated (MAM) or fecal microbiota. The PERMANOVA analysis of unweighted UniFrac distances revealed distinct clustering of ileal MAM (P = 0.049) and fecal microbiota (P = 0.068) in MAP-infected vs. control cattle. Microbiota profile of MAP-infected animals was further investigated by linear discriminant analysis effective size (LEfSe); several bacterial taxa within the phylum Proteobacteria were overrepresented in ileal MAM of control calves. Moreover, based on reconstructed metagenomes (PICRUSt) of ileal MAM, functional pathways associated with MAP infection were inferred. Enrichment of lysine and histidine metabolism pathways, and underrepresentation of glutathione metabolism and leucine and isoleucine degradation pathways in MAP-infected calves suggested potential contributions of ileal MAM in development of intestinal inflammation. Finally, simultaneous overrepresentation of families Planococcaceae and Paraprevotellaceae, as well as underrepresentation of genera Faecalibacterium and Akkermansia in the fecal microbiota of infected cattle, served as potential biomarker for identifying infected cattle during subclinical stages of JD. Collectively, based on compositional and functional shifts in intestinal microbiota of infected cattle, we inferred that this dynamic network of microorganisms had an active role in intestinal homeostasis.

Molecular Survey of Bacterial Communities Associated with Bacterial Chondronecrosis with Osteomyelitis (BCO) in Broilers

PubMed Central

Jiang, Tieshan; Mandal, Rabindra K.; Wideman, Robert F.; Khatiwara, Anita; Pevzner, Igal; Min Kwon, Young

2015-01-01

Bacterial chondronecrosis with osteomyelitis (BCO) is recognized as an important cause of lameness in commercial broiler chickens (meat-type chickens). Relatively little is known about the microbial communities associated with BCO. This study was conducted to increase our understanding of the microbial factors associated with BCO using a culture-independent approach. Using Illumina sequencing of the hyper-variable region V6 in the 16S rRNA gene, we characterized the bacterial communities in 97 femoral or tibial heads from normal and lame broilers carefully selected to represent diverse variations in age, line, lesion type, floor type, clinical status and bone type. Our in-depth survey based on 14 million assembled sequence reads revealed that complex bacterial communities exist in all samples, including macroscopically normal bones from clinically healthy birds. Overall, Proteobacteria (mean 90.9%) comprised the most common phylum, followed by Firmicutes (6.1%) and Actinobacteria (2.6%), accounting for more than 99% of all reads. Statistical analyses demonstrated that there are differences in bacterial communities in different types of bones (femur vs. tibia), lesion types (macroscopically normal femora or tibiae vs. those with pathognomonic BCO lesions), and among individual birds. This analysis also showed that BCO samples overrepresented genera Staphylococcus, whose species have been frequently isolated in BCO samples in previous studies. Rarefaction analysis demonstrated the general tendency that increased severities of BCO lesions were associated with reduced species diversity in both femoral and tibial samples when compared to macroscopically normal samples. These observations suggest that certain bacterial subgroups are preferentially selected in association with the development of BCO lesions. Understanding the microbial species associated with BCO will identify opportunities for understanding and modulating the pathogenesis of this form of lameness in broilers. PMID:25881241

Mitochondrial DNA perspective of Serbian genetic diversity.

PubMed

Davidovic, Slobodan; Malyarchuk, Boris; Aleksic, Jelena M; Derenko, Miroslava; Topalovic, Vladanka; Litvinov, Andrey; Stevanovic, Milena; Kovacevic-Grujicic, Natasa

2015-03-01

Although south-Slavic populations have been studied to date from various aspects, the population of Serbia, occupying the central part of the Balkan Peninsula, is still genetically understudied at least at the level of mitochondrial DNA (mtDNA) variation. We analyzed polymorphisms of the first and the second mtDNA hypervariable segments (HVS-I and HVS-II) and informative coding-region markers in 139 Serbians to shed more light on their mtDNA variability, and used available data on other Slavic and neighboring non-Slavic populations to assess their interrelations in a broader European context. The contemporary Serbian mtDNA profile is consistent with the general European maternal landscape having a substantial proportion of shared haplotypes with eastern, central, and southern European populations. Serbian population was characterized as an important link between easternmost and westernmost south-Slavic populations due to the observed lack of genetic differentiation with all other south-Slavic populations and its geographical positioning within the Balkan Peninsula. An increased heterogeneity of south Slavs, most likely mirroring turbulent demographic events within the Balkan Peninsula over time (i.e., frequent admixture and differential introgression of various gene pools), and a marked geographical stratification of Slavs to south-, east-, and west-Slavic groups, were also found. A phylogeographic analyses of 20 completely sequenced Serbian mitochondrial genomes revealed not only the presence of mtDNA lineages predominantly found within the Slavic gene pool (U4a2a*, U4a2a1, U4a2c, U4a2g, HV10), supporting a common Slavic origin, but also lineages that may have originated within the southern Europe (H5*, H5e1, H5a1v) and the Balkan Peninsula in particular (H6a2b and L2a1k). © 2014 Wiley Periodicals, Inc.

Prevalence and molecular characterization of avian infectious bronchitis virus in poultry flocks in Morocco from 2010 to 2014 and first detection of Italy 02 in Africa.

PubMed

Fellahi, Siham; Ducatez, Mariette; El Harrak, Mehdi; Guérin, Jean-Luc; Touil, Nadia; Sebbar, Ghizlane; Bouaiti, El Arbi; Khataby, Khadija; Ennaji, My Mustapha; El-Houadfi, Mohammed

2015-01-01

The aim of this study was to investigate the prevalence and diversity of infectious bronchitis virus (IBV) genotypes in poultry flocks in 16 areas of Morocco between 2010 and 2014. A total of 360 chicken flocks suspected of being infected by IBV were screened for the IBV N gene using real-time reverse transcriptase-polymerase chain reaction (RT-PCR). Flocks were classified into four groups according to their IBV vaccination programme. Group 1 contained unvaccinated birds. Group 2 received a single application of live H120 vaccine. Groups 3 and 4 birds received one or two booster vaccination(s), respectively, mostly using the H120 vaccine. The real-time RT-PCR results showed that 51.7% of the flocks were positive for the IBV genome with geographical disparities. Molecular characterization of IBV was performed on 50 RT-PCR positive samples by partially sequencing the S1 gene, including the hypervariable regions (nucleotides 705-1097). Two predominant genotypes were detected, with the Massachusetts type dominating (66%), among which 25% of the samples were identical to the H120 vaccine. The second most common genotype (present in 32% of the flocks) was surprisingly Italy 02, revealing the first detection of this genotype in Morocco and also in Africa. 793B, the predominant genotype in the late 1990s in Morocco, was only detected on one occasion and was identical to the 4/91 vaccine strain. This study highlights the high prevalence of IBV in poultry farms in Morocco and confirms its continuous dynamic changes and evolution.

Microbial Diversity in Sediment Ecosystems (Evaporites Domes, Microbial Mats, and Crusts) of Hypersaline Laguna Tebenquiche, Salar de Atacama, Chile

PubMed Central

Fernandez, Ana B.; Rasuk, Maria C.; Visscher, Pieter T.; Contreras, Manuel; Novoa, Fernando; Poire, Daniel G.; Patterson, Molly M.; Ventosa, Antonio; Farias, Maria E.

2016-01-01

We combined nucleic acid-based molecular methods, biogeochemical measurements, and physicochemical characteristics to investigate microbial sedimentary ecosystems of Laguna Tebenquiche, Atacama Desert, Chile. Molecular diversity, and biogeochemistry of hypersaline microbial mats, rhizome-associated concretions, and an endoevaporite were compared with: The V4 hypervariable region of the 16S rRNA gene was amplified by pyrosequencing to analyze the total microbial diversity (i.e., bacteria and archaea) in bulk samples, and in addition, in detail on a millimeter scale in one microbial mat and in one evaporite. Archaea were more abundant than bacteria. Euryarchaeota was one of the most abundant phyla in all samples, and particularly dominant (97% of total diversity) in the most lithified ecosystem, the evaporite. Most of the euryarchaeal OTUs could be assigned to the class Halobacteria or anaerobic and methanogenic archaea. Planctomycetes potentially also play a key role in mats and rhizome-associated concretions, notably the aerobic organoheterotroph members of the class Phycisphaerae. In addition to cyanobacteria, members of Chromatiales and possibly the candidate family Chlorotrichaceae contributed to photosynthetic carbon fixation. Other abundant uncultured taxa such as the candidate division MSBL1, the uncultured MBGB, and the phylum Acetothermia potentially play an important metabolic role in these ecosystems. Lithifying microbial mats contained calcium carbonate precipitates, whereas endoevoporites consisted of gypsum, and halite. Biogeochemical measurements revealed that based on depth profiles of O2 and sulfide, metabolic activities were much higher in the non-lithifying mat (peaking in the least lithified systems) than in lithifying mats with the lowest activity in endoevaporites. This trend in decreasing microbial activity reflects the increase in salinity, which may play an important role in the biodiversity. PMID:27597845

Structural basis of recognition of farnesylated and methylated KRAS4b by PDEδ.

PubMed

Dharmaiah, Srisathiyanarayanan; Bindu, Lakshman; Tran, Timothy H; Gillette, William K; Frank, Peter H; Ghirlando, Rodolfo; Nissley, Dwight V; Esposito, Dominic; McCormick, Frank; Stephen, Andrew G; Simanshu, Dhirendra K

2016-11-01

Farnesylation and carboxymethylation of KRAS4b (Kirsten rat sarcoma isoform 4b) are essential for its interaction with the plasma membrane where KRAS-mediated signaling events occur. Phosphodiesterase-δ (PDEδ) binds to KRAS4b and plays an important role in targeting it to cellular membranes. We solved structures of human farnesylated-methylated KRAS4b in complex with PDEδ in two different crystal forms. In these structures, the interaction is driven by the C-terminal amino acids together with the farnesylated and methylated C185 of KRAS4b that binds tightly in the central hydrophobic pocket present in PDEδ. In crystal form II, we see the full-length structure of farnesylated-methylated KRAS4b, including the hypervariable region. Crystal form I reveals structural details of farnesylated-methylated KRAS4b binding to PDEδ, and crystal form II suggests the potential binding mode of geranylgeranylated-methylated KRAS4b to PDEδ. We identified a 5-aa-long sequence motif (Lys-Ser-Lys-Thr-Lys) in KRAS4b that may enable PDEδ to bind both forms of prenylated KRAS4b. Structure and sequence analysis of various prenylated proteins that have been previously tested for binding to PDEδ provides a rationale for why some prenylated proteins, such as KRAS4a, RalA, RalB, and Rac1, do not bind to PDEδ. Comparison of all four available structures of PDEδ complexed with various prenylated proteins/peptides shows the presence of additional interactions due to a larger protein-protein interaction interface in KRAS4b-PDEδ complex. This interface might be exploited for designing an inhibitor with minimal off-target effects.

Epidemic history of hepatitis C virus infection in two remote communities in Nigeria, West Africa.

PubMed

Forbi, Joseph C; Purdy, Michael A; Campo, David S; Vaughan, Gilberto; Dimitrova, Zoya E; Ganova-Raeva, Lilia M; Xia, Guo-Liang; Khudyakov, Yury E

2012-07-01

We investigated the molecular epidemiology and population dynamics of HCV infection among indigenes of two semi-isolated communities in North-Central Nigeria. Despite remoteness and isolation, ~15% of the population had serological or molecular markers of hepatitis C virus (HCV) infection. Phylogenetic analysis of the NS5b sequences obtained from 60 HCV-infected residents showed that HCV variants belonged to genotype 1 (n=51; 85%) and genotype 2 (n=9; 15%). All sequences were unique and intermixed in the phylogenetic tree with HCV sequences from people infected from other West African countries. The high-throughput 454 pyrosequencing of the HCV hypervariable region 1 and an empirical threshold error correction algorithm were used to evaluate intra-host heterogeneity of HCV strains of genotype 1 (n=43) and genotype 2 (n=6) from residents of the communities. Analysis revealed a rare detectable intermixing of HCV intra-host variants among residents. Identification of genetically close HCV variants among all known groups of relatives suggests a common intra-familial HCV transmission in the communities. Applying Bayesian coalescent analysis to the NS5b sequences, the most recent common ancestors for genotype 1 and 2 variants were estimated to have existed 675 and 286 years ago, respectively. Bayesian skyline plots suggest that HCV lineages of both genotypes identified in the Nigerian communities experienced epidemic growth for 200-300 years until the mid-20th century. The data suggest a massive introduction of numerous HCV variants to the communities during the 20th century in the background of a dynamic evolutionary history of the hepatitis C epidemic in Nigeria over the past three centuries.

Oral microbial community typing of caries and pigment in primary dentition.

PubMed

Li, Yanhui; Zou, Cheng-Gang; Fu, Yu; Li, Yanhong; Zhou, Qing; Liu, Bo; Zhang, Zhigang; Liu, Juan

2016-08-05

Black extrinsic discoloration in primary dentition is a common clinical and aesthetic problem that can co-occur with dental caries, the most common oral diseases in childhood. Although the role of bacteria in the formation of pigment and caries in primary dentition is important, their basic features still remain a further mystery. Using targeted sequencing of the V1-V3 hypervariable regions of bacterial 16S ribosomal RNA (rRNA) genes, we obtained a dataset consisting of 831,381 sequences from 111 saliva samples and 110 supragingival plaque samples from 40 patients with pigment (black extrinsic stain), 20 with caries (obvious decay), and 25 with both pigment and caries and from 26 healthy individuals. We applied a Dirichlet multinomial mixture (DMM)-based community typing approach to investigate oral microbial community types. Our results revealed significant structural segregation of microbial communities, as indicated by the identification of two plaque community types (A and B) and three saliva community types (C-E). We found that the independent occurrence of the two plaque community types, A and B, was potentially associated with our oral diseases of interest. For type A, three co-occurring bacterial genus pairs could separately play a potential role in the formation of pigment (Leptotrichia and Fusobacterium), caries (unclassified Gemellales and Granulicatella), and mixed caries and pigment (Streptococcus and Mogibacterium). For type B, three co-occurring bacterial genera (unclassified Clostridiaceae, Peptostreptococcus, and Clostridium) were related to mixed pigment and caries. Three dominant bacterial genera (Selenomonas, Gemella, and Streptobacillus) were linked to the presence of caries. Our study demonstrates that plaque-associated oral microbial communities could majorly contribute to the formation of pigment and caries in primary dentition and suggests potential clinical applications of monitoring oral microbiota as an indicator for disease diagnosis and prognosis.

Investigation of paternity establishing without the putative father using hypervariable DNA probes.

PubMed

Yokoi, T; Odaira, T; Nata, M; Sagisaka, K

1990-09-01

Seven kinds of DNA probes which recognize hypervariable loci were applied for paternity test. The putative father was decreased and unavailable for the test. The two legitimate children and their mother (the deceased's wife) and the four illegitimate children and their mother (the deceased's kept mistress) were available for analysis. Paternity index of four illegitimate child was investigated. Allelic frequencies and their confidence intervals among unrelated Japanese individuals were previously reported from our laboratory, and co-dominant segregation of the polymorphism was confirmed in family studies. Cumulative paternity indices of four illegitimate children from 16 kinds of standard blood group markers were 165, 42, 0.09, and 36, respectively. On the other hand, cumulative paternity indices from 7 kinds of DNA probes are 2,363, 4,685, 57,678, and 54,994, respectively, which are 14, 113, 640, 864, and 1,509 times higher than that from standard blood group markers. The DNA analyses gave nearly conclusive evidence that the putative father was the biological father of the children. Especially, the paternity relation of the third illegitimate child could not be established without the DNA analyses. Accordingly, DNA polymorphism is considered to be informative enough for paternity test.

Mitochondrial DNA structure of an isolated Tunisian Berber population and its relationship with Mediterranean populations.

PubMed

Ben Halim, Nizar; Hsouna, Sana; Lasram, Khaled; Chargui, Mariem; Khemira, Laaroussi; Saidane, Rachid; Abdelhak, Sonia; Kefi, Rym

2018-02-01

Douiret is an isolated Berber population from South-Eastern Tunisia. The strong geographic and cultural isolation characterising this population might have contributed to remarkable endogamy and consanguinity, which were practiced for several centuries. The objective of this study is to evaluate the mitochondrial DNA (mtDNA) genetic structure of Douiret and to compare it to other Mediterranean populations with a special focus on major haplogroup T. Genomic DNA was extracted from blood samples of 58 unrelated individuals collected from the different patrilineal lineages of the population. The hypervariable region 1 of the mtDNA was amplified and sequenced. For comparative analyses, additional HVS1 sequences (n = 4857) were compiled from previous studies. The maternal background of the studied sample from Douiret was mainly of Eurasian origin (74%) followed by Sub-Saharan (17%) and North African (3%) lineages. Douiret harbours the highest frequency of haplogroup T in the Mediterranean region, assigned to the unique subclade T1a (38%). Phylogenetic analysis showed an outlier position of Douiret at the Mediterranean level. The genetic structure of Douiret highlights the presence of founders, most likely of Near/Middle Eastern origin, who conquered this area during the Middle/Late Upper Palaeolithic and Neolithic dispersals.

Mitochondrial sequence analysis for forensic identification using pyrosequencing technology.

PubMed

Andréasson, H; Asp, A; Alderborn, A; Gyllensten, U; Allen, M

2002-01-01

Over recent years, requests for mtDNA analysis in the field of forensic medicine have notably increased, and the results of such analyses have proved to be very useful in forensic cases where nuclear DNA analysis cannot be performed. Traditionally, mtDNA has been analyzed by DNA sequencing of the two hypervariable regions, HVI and HVII, in the D-loop. DNA sequence analysis using the conventional Sanger sequencing is very robust but time consuming and labor intensive. By contrast, mtDNA analysis based on the pyrosequencing technology provides fast and accurate results from the human mtDNA present in many types of evidence materials in forensic casework. The assay has been developed to determine polymorphic sites in the mitochondrial D-loop as well as the coding region to further increase the discrimination power of mtDNA analysis. The pyrosequencing technology for analysis of mtDNA polymorphisms has been tested with regard to sensitivity, reproducibility, and success rate when applied to control samples and actual casework materials. The results show that the method is very accurate and sensitive; the results are easily interpreted and provide a high success rate on casework samples. The panel of pyrosequencing reactions for the mtDNA polymorphisms were chosen to result in an optimal discrimination power in relation to the number of bases determined.

Unravelling migrations in the steppe: mitochondrial DNA sequences from ancient central Asians.

PubMed Central

Lalueza-Fox, C.; Sampietro, M. L.; Gilbert, M. T. P.; Castri, L.; Facchini, F.; Pettener, D.; Bertranpetit, J.

2004-01-01

This study helps to clarify the debate on the Western and Eastern genetic influences in Central Asia. Thirty-six skeletal remains from Kazakhstan (Central Asia), excavated from different sites dating between the fifteenth century BC to the fifth century AD, have been analysed for the hypervariable control region (HVR-I) and haplogroup diagnostic single nucleotide polymorphisms (SNPs) of the mitochondrial DNA genome. Standard authentication criteria for ancient DNA studies, including multiple extractions, cloning of PCR products and independent replication, have been followed. The distribution of east and west Eurasian lineages through time in the region is concordant with the available archaeological information: prior to the thirteenth-seventh century BC, all Kazakh samples belong to European lineages; while later an arrival of east Eurasian sequences that coexisted with the previous west Eurasian genetic substratum can be detected. The presence of an ancient genetic substratum of European origin in West Asia may be related to the discovery of ancient mummies with European features in Xinjiang and to the existence of an extinct Indo-European language, Tocharian. This study demonstrates the usefulness of the ancient DNA in unravelling complex patterns of past human migrations so as to help decipher the origin of present-day admixed populations. PMID:15255049

A set of plastid loci for use in multiplex fragment length genotyping for intraspecific variation in Pinus (Pinaceae)1

PubMed Central

Wofford, Austin M.; Finch, Kristen; Bigott, Adam; Willyard, Ann

2014-01-01

• Premise of the study: Recently released Pinus plastome sequences support characterization of 15 plastid simple sequence repeat (cpSSR) loci originally published for P. contorta and P. thunbergii. This allows selection of loci for single-tube PCR multiplexed genotyping in any subsection of the genus. • Methods: Unique placement of primers and primer conservation across the genus were investigated, and a set of six loci were selected for single-tube multiplexing. We compared interspecific variation between cpSSRs and nucleotide sequences of ycf1 and tested intraspecific variation for cpSSRs using 911 samples in the P. ponderosa species complex. • Results: The cpSSR loci contain mononucleotide and complex repeats with additional length variation in flanking regions. They are not located in hypervariable regions, and most primers are conserved across the genus. A single PCR per sample multiplexed for six loci yielded 45 alleles in 911 samples. • Discussion: The protocol allows efficient genotyping of many samples. The cpSSR loci are too variable for Pinus phylogenies but are useful for the study of genetic structure within and among populations. The multiplex method could easily be extended to other plant groups by choosing primers for cpSSR loci in a plastome alignment for the target group. PMID:25202625

Hepatitis C: progress and problems.

PubMed Central

Cuthbert, J A

1994-01-01

The hepatitis C virus (HCV), a single-stranded RNA virus, is the major cause of posttransfusion hepatitis. HCV isolates differ in nucleotide and amino acid sequences. Nucleotide changes are concentrated in hypervariable regions and may be related to immune selection. In most immunocompetent persons, HCV infection is diagnosed serologically, using antigens from conserved regions. Amplification of RNA may be necessary to detect infection in immunosuppressed patients. Transmission by known parenteral routes is frequent; other means of spread are less common and may represent inapparent, percutaneous dissemination. Infection can lead to classical acute hepatitis, but most infected persons have no history of acute disease. Once infected, most individuals apparently remain carriers of the virus, with varying degrees of hepatocyte damage and fibrosis ensuing. Chronic hepatitis may lead to cirrhosis and hepatocellular carcinoma. However, disease progression varies widely, from less than 2 years to cirrhosis in some patients to more than 30 years with only chronic hepatitis in others. Determinants important in deciding outcome are unknown. Alpha interferon, which results in sustained remission in selected patients, is the only available therapy. Long-term benefits from such therapy have not been demonstrated. Prevention of HCV infection by vaccination is likely to be challenging if ongoing viral mutation results in escape from neutralization and clearance. PMID:7834603

Hepatitis B virus genetic diversity and its impact on diagnostic assays.

PubMed

Hollinger, F B

2007-11-01

Hepatitis B virus (HBV) circulates in blood as closely related, but genetically diverse molecules called quasispecies. During replication, HBV production may approach 10(11) molecules/day, although during peak activity this rate may increase 100-1000 times. Generally, DNA polymerases have excellent fidelity in reading DNA templates because they are associated with an exonuclease which removes incorrectly added nucleotides. However, the HBV-DNA polymerase lacks fidelity and proofreading function partly because exonuclease activity is either absent or deficient. Thus, the HBV genome and especially the envelope gene, is mutated with unusually high frequency. These mutations can affect more than one open reading frame because of overlapping genes. The S gene contains an exposed major hydrophilic region (residues 110-155), which encompasses the 'a' determinant that is important for inducing immunity. Nucleotide substitutions in this region are common and result in reduced binding or failure to detect hepatitis B surface antigen (HBsAg) in diagnostic assays. Adaptive immunity also depends on the recognition of HBsAg by specific antibody and variants pose a threat if they interfere with binding to antibody. Finally, genomic hypervariability allows HBV to escape selection pressures imposed by antiviral therapies, vaccines and the host immune system, and is responsible for creating genotypes, subgenotypes and subtypes.

mtDNA variation in the Yanomami: evidence for additional New World founding lineages.

PubMed

Easton, R D; Merriwether, D A; Crews, D E; Ferrell, R E

1996-07-01

Native Americans have been classified into four founding haplogroups with as many as seven founding lineages based on mtDNA RFLPs and DNA sequence data. mtDNA analysis was completed for 83 Yanomami from eight villages in the Surucucu and Catrimani Plateau regions of Roraima in northwestern Brazil. Samples were typed for 15 polymorphic mtDNA sites (14 RFLP sites and 1 deletion site), and a subset was sequenced for both hypervariable regions of the mitochondrial D-loop. Substantial mitochondrial diversity was detected among the Yanomami, five of seven accepted founding haplotypes and three others were observed. Of the 83 samples, 4 (4.8%) were lineage B1, 1 (1.2%) was lineage B2, 31 (37.4%) were lineage C1, 29 (34.9%) were lineage C2, 2 (2.4%) were lineage D1, 6 (7.2%) were lineage D2, 7 (8.4%) were a haplotype we designated "X6," and 3 (3.6%) were a haplotype we designated "X7." Sequence analysis found 43 haplotypes in 50 samples. B2, X6, and X7 are previously unrecognized mitochondrial founding lineage types of Native Americans. The widespread distribution of these haplotypes in the New World and Asia provides support for declaring these lineages to be New World founding types.

mtDNA variation in the Yanomami: evidence for additional New World founding lineages.

PubMed Central

Easton, R. D.; Merriwether, D. A.; Crews, D. E.; Ferrell, R. E.

1996-01-01

Native Americans have been classified into four founding haplogroups with as many as seven founding lineages based on mtDNA RFLPs and DNA sequence data. mtDNA analysis was completed for 83 Yanomami from eight villages in the Surucucu and Catrimani Plateau regions of Roraima in northwestern Brazil. Samples were typed for 15 polymorphic mtDNA sites (14 RFLP sites and 1 deletion site), and a subset was sequenced for both hypervariable regions of the mitochondrial D-loop. Substantial mitochondrial diversity was detected among the Yanomami, five of seven accepted founding haplotypes and three others were observed. Of the 83 samples, 4 (4.8%) were lineage B1, 1 (1.2%) was lineage B2, 31 (37.4%) were lineage C1, 29 (34.9%) were lineage C2, 2 (2.4%) were lineage D1, 6 (7.2%) were lineage D2, 7 (8.4%) were a haplotype we designated "X6," and 3 (3.6%) were a haplotype we designated "X7." Sequence analysis found 43 haplotypes in 50 samples. B2, X6, and X7 are previously unrecognized mitochondrial founding lineage types of Native Americans. The widespread distribution of these haplotypes in the New World and Asia provides support for declaring these lineages to be New World founding types. PMID:8659527

2S Albumin Storage Proteins: What Makes them Food Allergens?

PubMed

Moreno, F Javier; Clemente, Alfonso

2008-01-01

2S albumin storage proteins are becoming of increasing interest in nutritional and clinical studies as they have been reported as major food allergens in seeds of many mono- and di-cotyledonous plants. This review describes the main biochemical, structural and functional properties of these proteins thought to play a role in determining their potential allergenicity. 2S albumins are considered to sensitize directly via the gastrointestinal tract (GIT). The high stability of their intrinsic protein structure, dominated by a well-conserved skeleton of cysteine residues, to the harsh conditions present in the GIT suggests that these proteins are able to cross the gut mucosal barrier to sensitize the mucosal immune system and/or elicit an allergic response. The flexible and solvent-exposed hypervariable region of these proteins is immunodominant and has the ability to bind IgE from allergic patients sera. Several linear IgE-binding epitopes of 2S albumins spanning this region have been described to play a major role in allergenicity; the role of conformational epitopes of these proteins in food allergy is far from being understood and need to be investigated. Finally, the interaction of these proteins with other components of the food matrix might influence the absorption rates of immunologically reactive 2S albumins but also in their immune response.

2S Albumin Storage Proteins: What Makes them Food Allergens?

PubMed Central

Moreno, F. Javier; Clemente, Alfonso

2008-01-01

2S albumin storage proteins are becoming of increasing interest in nutritional and clinical studies as they have been reported as major food allergens in seeds of many mono- and di-cotyledonous plants. This review describes the main biochemical, structural and functional properties of these proteins thought to play a role in determining their potential allergenicity. 2S albumins are considered to sensitize directly via the gastrointestinal tract (GIT). The high stability of their intrinsic protein structure, dominated by a well-conserved skeleton of cysteine residues, to the harsh conditions present in the GIT suggests that these proteins are able to cross the gut mucosal barrier to sensitize the mucosal immune system and/or elicit an allergic response. The flexible and solvent-exposed hypervariable region of these proteins is immunodominant and has the ability to bind IgE from allergic patients´ sera. Several linear IgE-binding epitopes of 2S albumins spanning this region have been described to play a major role in allergenicity; the role of conformational epitopes of these proteins in food allergy is far from being understood and need to be investigated. Finally, the interaction of these proteins with other components of the food matrix might influence the absorption rates of immunologically reactive 2S albumins but also in their immune response. PMID:18949071

Seasonal changes in bacterial communities associated with healthy and diseased Porites coral in southern Taiwan.

PubMed

Lin, Chorng-Horng; Chuang, Chih-Hsiang; Twan, Wen-Hung; Chiou, Shu-Fen; Wong, Tit-Yee; Liu, Jong-Kang; Kao, Chyuan-Yao; Kuo, Jimmy

2016-12-01

We compared the bacterial communities associated with healthy scleractinian coral Porites sp. with those associated with coral infected with pink spot syndrome harvested during summer and winter from waters off the coast of southern Taiwan. Members of the bacterial community associated with the coral were characterized by means of denaturing gradient gel electrophoresis (DGGE) of a short region of the 16S rRNA gene and clone library analysis. Of 5 different areas of the 16S rRNA gene, we demonstrated that the V3 hypervariable region is most suited to represent the coral-associated bacterial community. The DNA sequences of 26 distinct bands extracted from DGGE gels and 269 sequences of the 16S rRNA gene from clone libraries were determined. We found that the communities present in diseased coral were more heterogeneous than the bacterial communities of uninfected coral. In addition, bacterial communities associated with coral harvested in the summer were more diverse than those associated with coral collected in winter, regardless of the health status of the coral. Our study suggested that the compositions of coral-associated bacteria communities are complex, and the population of bacteria varies greatly between seasons and in coral of differing health status.

An in silico argument for mitochondrial microRNA as a determinant of primary non function in liver transplantation.

PubMed

Khorsandi, Shirin Elizabeth; Salehi, Siamak; Cortes, Miriam; Vilca-Melendez, Hector; Menon, Krishna; Srinivasan, Parthi; Prachalias, Andreas; Jassem, Wayel; Heaton, Nigel

2018-02-15

Mitochondria have their own genomic, transcriptomic and proteomic machinery but are unable to be autonomous, needing both nuclear and mitochondrial genomes. The aim of this work was to use computational biology to explore the involvement of Mitochondrial microRNAs (MitomiRs) and their interactions with the mitochondrial proteome in a clinical model of primary non function (PNF) of the donor after cardiac death (DCD) liver. Archival array data on the differential expression of miRNA in DCD PNF was re-analyzed using a number of publically available computational algorithms. 10 MitomiRs were identified of importance in DCD PNF, 7 with predicted interaction of their seed sequence with the mitochondrial transcriptome that included both coding, and non coding areas of the hypervariability region 1 (HVR1) and control region. Considering miRNA regulation of the nuclear encoded mitochondrial proteome, 7 hypothetical small proteins were identified with homolog function that ranged from co-factor for formation of ATP Synthase, REDOX balance and an importin/exportin protein. In silico, unconventional seed interactions, both non canonical and alternative seed sites, appear to be of greater importance in MitomiR regulation of the mitochondrial genome. Additionally, a number of novel small proteins of relevance in transplantation have been identified which need further characterization.

Identifying protist consumers of photosynthetic picoeukaryotes in the surface ocean using stable isotope probing.

PubMed

Orsi, William D; Wilken, Susanne; Del Campo, Javier; Heger, Thierry; James, Erick; Richards, Thomas A; Keeling, Patrick J; Worden, Alexandra Z; Santoro, Alyson E

2018-02-01

Photosynthetic picoeukaryotes contribute a significant fraction of primary production in the upper ocean. Micromonas pusilla is an ecologically relevant photosynthetic picoeukaryote, abundantly and widely distributed in marine waters. Grazing by protists may control the abundance of picoeukaryotes such as M. pusilla, but the diversity of the responsible grazers is poorly understood. To identify protists consuming photosynthetic picoeukaryotes in a productive North Pacific Ocean region, we amended seawater with living 15 N, 13 C-labelled M. pusilla cells in a 24-h replicated bottle experiment. DNA stable isotope probing, combined with high-throughput sequencing of V4 hypervariable regions from 18S rRNA gene amplicons (Tag-SIP), identified 19 operational taxonomic units (OTUs) of microbial eukaryotes that consumed M. pusilla. These OTUs were distantly related to cultured taxa within the dinoflagellates, ciliates, stramenopiles (MAST-1C and MAST-3 clades) and Telonema flagellates, thus, far known only from their environmental 18S rRNA gene sequences. Our discovery of eukaryotic prey consumption by MAST cells confirms that their trophic role in marine microbial food webs includes grazing upon picoeukaryotes. Our study provides new experimental evidence directly linking the genetic identity of diverse uncultivated microbial eukaryotes to the consumption of picoeukaryotic phytoplankton in the upper ocean. © 2017 Society for Applied Microbiology and John Wiley & Sons Ltd.

Myticalins: A Novel Multigenic Family of Linear, Cationic Antimicrobial Peptides from Marine Mussels (Mytilus spp.).

PubMed

Leoni, Gabriele; De Poli, Andrea; Mardirossian, Mario; Gambato, Stefano; Florian, Fiorella; Venier, Paola; Wilson, Daniel N; Tossi, Alessandro; Pallavicini, Alberto; Gerdol, Marco

2017-08-22

The application of high-throughput sequencing technologies to non-model organisms has brought new opportunities for the identification of bioactive peptides from genomes and transcriptomes. From this point of view, marine invertebrates represent a potentially rich, yet largely unexplored resource for de novo discovery due to their adaptation to diverse challenging habitats. Bioinformatics analyses of available genomic and transcriptomic data allowed us to identify myticalins, a novel family of antimicrobial peptides (AMPs) from the mussel Mytilus galloprovincialis , and a similar family of AMPs from Modiolus spp., named modiocalins. Their coding sequence encompasses two conserved N-terminal (signal peptide) and C-terminal (propeptide) regions and a hypervariable central cationic region corresponding to the mature peptide. Myticalins are taxonomically restricted to Mytiloida and they can be classified into four subfamilies. These AMPs are subject to considerable interindividual sequence variability and possibly to presence/absence variation. Functional assays performed on selected members of this family indicate a remarkable tissue-specific expression (in gills) and broad spectrum of activity against both Gram-positive and Gram-negative bacteria. Overall, we present the first linear AMPs ever described in marine mussels and confirm the great potential of bioinformatics tools for the de novo discovery of bioactive peptides in non-model organisms.

Clonal Lineages of Erysipelothrix rhusiopathiae Responsible for Acute Swine Erysipelas in Japan Identified by Using Genome-Wide Single-Nucleotide Polymorphism Analysis.

PubMed

Ogawa, Yohsuke; Shiraiwa, Kazumasa; Ogura, Yoshitoshi; Ooka, Tadasuke; Nishikawa, Sayaka; Eguchi, Masahiro; Hayashi, Tetsuya; Shimoji, Yoshihiro

2017-06-01

Erysipelothrix rhusiopathiae causes swine erysipelas, an important infectious disease in the swine industry. In Japan, the incidence of acute swine erysipelas due to E. rhusiopathiae serovar 1a has recently increased markedly. To study the genetic relatedness of the strains from the recent cases, we analyzed 34 E. rhusiopathiae serovar 1a swine isolates collected between 1990 and 2011 and further investigated the possible association of the live Koganei 65-0.15 vaccine strain (serovar 1a) with the increase in cases. Pulsed-field gel electrophoresis analysis revealed no marked variation among the isolates; however, sequencing analysis of a hypervariable region in the surface-protective antigen A gene ( spaA ) revealed that the strains isolated after 2007 exhibited the same spaA genotype and could be differentiated from older strains. Phylogenetic analysis based on genome-wide single-nucleotide polymorphisms (SNPs) revealed that the Japanese strains examined were closely related, showing a relatively small number of SNPs among them. The strains were classified into four major lineages, with Koganei 65-0.15 (lineage III) being phylogenetically separated from the other three lineages. The strains isolated after 2007 and the two older strains constituted one major lineage (lineage IV) with a specific spaA genotype (M203/I257-SpaA), while the recent isolates were further divided into two geographic groups. The remaining older isolates belonged to either lineage I, with the I203/L257-SpaA type, or lineage II, with the I203/I257-SpaA type. These results indicate that the recent increased incidence of acute swine erysipelas in Japan is associated with two sublineages of lineage IV, which have independently evolved in two different geographic regions. IMPORTANCE Using large-scale whole-genome sequence data from Erysipelothrix rhusiopathiae isolates from a wide range of hosts and geographic origins, a recent study clarified the existence of three distinct clades (clades 1, 2, and 3) that are found across multiple continents and host species, representing both livestock and wildlife, and an "intermediate" clade between clade 2 and the dominant clade 3 within the species. In this study, we found that the E. rhusiopathiae Japanese strains examined exhibited remarkably low levels of genetic diversity and confirmed that all of the Japanese and Chinese swine isolates examined in this study belong to clonal lineages within the intermediate clade. We report that spaA genotyping of E. rhusiopathiae strains is a practical alternative to whole-genome sequencing analysis of the E. rhusiopathiae isolates from eastern Asian countries. Copyright © 2017 American Society for Microbiology.

The C-terminal hypervariable domain targets Aradopsis ROP9 to the invaginated pollen tube plasma membrane

USDA-ARS?s Scientific Manuscript database

Rop9 is a small GTPase of the Type II class, whereas the often studied type I Rops play roles during pollen tube growth. In pollen, Rop9 is located at the invaginated plasma membrane that surrounds the sperm cells, whereas type I Rops are located at the apical membrane of the pollen tube. The C-ter...

Phylogeography of the American woodcock (Scolopax minor): Are management units based on band recovery data reflected in genetically based management units?

USGS Publications Warehouse

Rhymer, J.M.; McAuley, D.G.; Ziel, H.L.

2005-01-01

Information on population connectivity throughout the annual cycle has become more crucial, because populations of many migratory birds are in decline. One such species is the American Woodcock (Scolopax minor), which inhabits early-successional forests in eastern North America. Although band recoveries have proved useful for dividing populations of this game bird species into an Eastern Region and Central Region for management purposes, these data do not provide enough detail to determine the breeding population of origin of birds recovered on stopover and wintering areas. To obtain more fine-scale data, we undertook a phylogeographic study of American Woodcock populations throughout their primary breeding range in the eastern United States and Canada using mitochondrial DNA (mtDNA) sequences from the hypervariable control region I (CRI) and ND6 gene. Despite high haplotype diversity, nucleotide diversity was low and there was no phylogeographic structure among American Woodcock populations across the species range, with birds from many states and provinces in both management regions sharing identical haplotypes. Results suggest recent or ongoing gene flow among populations, with asymmetric movement of birds between migration flyways. As has been demonstrated for several other avian species in North America, American Woodcock appear to have undergone a rapid population expansion following the late Pleistocene glacial retreat. Thus, a combination of historical demographic factors and recent or ongoing gene flow mask any population structure based on mtDNA that might accrue from philopatry to breeding areas observed in studies of marked birds.

The Enigmatic Alphavirus Non-Structural Protein 3 (nsP3) Revealing Its Secrets at Last

PubMed Central

Götte, Benjamin; Liu, Lifeng

2018-01-01

Alphaviruses encode 4 non-structural proteins (nsPs), most of which have well-understood functions in capping and membrane association (nsP1), polyprotein processing and RNA helicase activity (nsP2) and as RNA-dependent RNA polymerase (nsP4). The function of nsP3 has been more difficult to pin down and it has long been referred to as the more enigmatic of the nsPs. The protein comprises three domains, an N-terminal macro domain, a central zinc-binding domain and a C-terminal hypervariable domain (HVD). In this article, we review old and new literature about the functions of the three domains. Much progress in recent years has contributed to a picture of nsP3, particularly through its HVD as a hub for interactions with host cell molecules, with multiple effects on the biology of the host cell at early points in infection. These and many future discoveries will provide targets for anti-viral therapies as well as strategies for modification of vectors for vaccine and oncolytic interventions. PMID:29495654

Worldwide exploration of the microbiome harbored by the cnidarian model, Exaiptasia pallida (Agassiz in Verrill, 1864) indicates a lack of bacterial association specificity at a lower taxonomic rank

PubMed Central

Brown, Tanya; Otero, Christopher; Grajales, Alejandro; Rodriguez, Estefania

2017-01-01

Examination of host-microbe interactions in early diverging metazoans, such as cnidarians, is of great interest from an evolutionary perspective to understand how host-microbial consortia have evolved. To address this problem, we analyzed whether the bacterial community associated with the cosmopolitan and model sea anemone Exaiptasia pallida shows specific patterns across worldwide populations ranging from the Caribbean Sea, and the Atlantic and Pacific oceans. By comparing sequences of the V1–V3 hypervariable regions of the bacterial 16S rRNA gene, we revealed that anemones host a complex and diverse microbial community. When examined at the phylum level, bacterial diversity and abundance associated with E. pallida are broadly conserved across geographic space with samples, containing largely Proteobacteria and Bacteroides. However, the species-level makeup within these phyla differs drastically across space suggesting a high-level core microbiome with local adaptation of the constituents. Indeed, no bacterial OTU was ubiquitously found in all anemones samples. We also revealed changes in the microbial community structure after rearing anemone specimens in captivity within a period of four months. Furthermore, the variation in bacterial community assemblages across geographical locations did not correlate with the composition of microalgal Symbiodinium symbionts. Our findings contrast with the postulation that cnidarian hosts might actively select and maintain species-specific microbial communities that could have resulted from an intimate co-evolution process. The fact that E. pallida is likely an introduced species in most sampled localities suggests that this microbial turnover is a relatively rapid process. Our findings suggest that environmental settings, not host specificity, seem to dictate bacterial community structure associated with this sea anemone. More than maintaining a specific composition of bacterial species some cnidarians associate with a wide range of bacterial species as long as they provide the same physiological benefits towards the maintenance of a healthy host. The examination of the previously uncharacterized bacterial community associated with the cnidarian sea anemone model E. pallida is the first global-scale study of its kind. PMID:28533949

Mutation and Evolutionary Rates in Adélie Penguins from the Antarctic

PubMed Central

Millar, Craig D.; Dodd, Andrew; Anderson, Jennifer; Gibb, Gillian C.; Ritchie, Peter A.; Baroni, Carlo; Woodhams, Michael D.; Hendy, Michael D.; Lambert, David M.

2008-01-01

Precise estimations of molecular rates are fundamental to our understanding of the processes of evolution. In principle, mutation and evolutionary rates for neutral regions of the same species are expected to be equal. However, a number of recent studies have shown that mutation rates estimated from pedigree material are much faster than evolutionary rates measured over longer time periods. To resolve this apparent contradiction, we have examined the hypervariable region (HVR I) of the mitochondrial genome using families of Adélie penguins (Pygoscelis adeliae) from the Antarctic. We sequenced 344 bps of the HVR I from penguins comprising 508 families with 915 chicks, together with both their parents. All of the 62 germline heteroplasmies that we detected in mothers were also detected in their offspring, consistent with maternal inheritance. These data give an estimated mutation rate (μ) of 0.55 mutations/site/Myrs (HPD 95% confidence interval of 0.29–0.88 mutations/site/Myrs) after accounting for the persistence of these heteroplasmies and the sensitivity of current detection methods. In comparison, the rate of evolution (k) of the same HVR I region, determined using DNA sequences from 162 known age sub-fossil bones spanning a 37,000-year period, was 0.86 substitutions/site/Myrs (HPD 95% confidence interval of 0.53 and 1.17). Importantly, the latter rate is not statistically different from our estimate of the mutation rate. These results are in contrast to the view that molecular rates are time dependent. PMID:18833304

Molecular and phylogenetic characterization of bovine coronavirus virus isolated from dairy cattle in Central Region, Thailand.

PubMed

Singasa, Kanokwan; Songserm, Taweesak; Lertwatcharasarakul, Preeda; Arunvipas, Pipat

2017-10-01

Bovine coronavirus (BCoV) is involved mainly in enteric infections in cattle. This study reports the first molecular detection of BCoV in a diarrhea outbreak in dairy cows in the Central Region, Thailand. BCoV was molecularly detected from bloody diarrheic cattle feces by using nested PCR. Agarose gel electrophoresis of three diarrheic fecal samples yielded from the 25 samples desired amplicons that were 488 base pairs and sequencing substantiated that have BCoV. The sequence alignment indicated that nucleotide and amino acid sequences, the three TWD isolated in Thailand, were more quite homologous to each other (amino acid at position 39 of TWD1, TWD3 was proline, but TWD2 was serine) and closely related to OK-0514-3strain (virulent respiratory strain; RBCoV).The amino acid sequencing identities among TWD1, TWD2,TWD3, and OK-0514-3 strain were 96.0 to 96.6%, those at which T3I, H65N, D87G, H127Y, andQ136R were changed. In addition, the phylogenetic tree of the hypervariable region S1subunit spike glycoprotein BCoV gene was composed of three major clades by using the 54 sequences generated and showed that the evolutionally distance, TWD1, TWD2, and TWD3 were the isolated group together and most similar to OK-0514-3 strain (98.2 to 98.5% similarity). Further study will develop ELISA assay for serologic detection of winter dysentery disease.

The influence of habitats on female mobility in Central and Western Africa inferred from human mitochondrial variation

PubMed Central

2013-01-01

Background When studying the genetic structure of human populations, the role of cultural factors may be difficult to ascertain due to a lack of formal models. Linguistic diversity is a typical example of such a situation. Patrilocality, on the other hand, can be integrated into a biological framework, allowing the formulation of explicit working hypotheses. The present study is based on the assumption that patrilocal traditions make the hypervariable region I of the mtDNA a valuable tool for the exploration of migratory dynamics, offering the opportunity to explore the relationships between genetic and linguistic diversity. We studied 85 Niger-Congo-speaking patrilocal populations that cover regions from Senegal to Central African Republic. A total of 4175 individuals were included in the study. Results By combining a multivariate analysis aimed at investigating the population genetic structure, with a Bayesian approach used to test models and extent of migration, we were able to detect a stepping-stone migration model as the best descriptor of gene flow across the region, with the main discontinuities corresponding to forested areas. Conclusions Our analyses highlight an aspect of the influence of habitat variation on human genetic diversity that has yet to be understood. Rather than depending simply on geographic linear distances, patterns of female genetic variation vary substantially between savannah and rainforest environments. Our findings may be explained by the effects of recent gene flow constrained by environmental factors, which superimposes on a background shaped by pre-agricultural peopling. PMID:23360301

Genomic analysis of the Chinese genotype 1F rubella virus that disappeared after 2002 in China.

PubMed

Zhu, Zhen; Chen, Min-Hsin; Abernathy, Emily; Zhou, Shujie; Wang, Changyin; Icenogle, Joseph; Xu, Wenbo

2014-12-01

Genotype 1F was likely localized geographically to China as it has not been reported elsewhere. In this study, whole genome sequences of two rubella 1F virus isolates were completed. Both viruses contained 9,761 nt with a single nucleotide deletion in the intergenic region, compared to the NCBI rubella reference sequence (NC 001545). No evidence of recombination was found between 1F and other rubella viruses. The genetic distance between 1F viruses and 10 other rubella virus genotypes (1a, 1B, 1C, 1D, 1E, 1G, 1J 2A, 2B, and 2C) ranged from 3.9% to 8.6% by pairwise comparison. A region known to be hypervariable in other rubella genotypes was also the most variable region in the 1F genomes. Comparisons to all available rubella virus sequences from GenBank identified 22 nucleotide variations exclusively in 1F viruses. Among these unique variations, C9306U is located within the recommended molecular window for rubella virus genotyping assignment, could be useful to confirm 1F viruses. Using the Bayesian Markov Chain Monte Carlo (MCMC) method, the time of the most recent common ancestor for the genotype 1F was estimated between 1976 and 1995. Recent rubella molecular surveillance suggests that this indigenous strain may have circulated for less than three decades, as it has not been detected since 2002. © 2014 Wiley Periodicals, Inc.

Elucidating geological and biological processes underlying the diversification of Sulawesi tarsiers.

PubMed

Merker, Stefan; Driller, Christine; Perwitasari-Farajallah, Dyah; Pamungkas, Joko; Zischler, Hans

2009-05-26

Because of their exceptionally long independent evolution, a range diminution of their Eocene relatives, and a remarkable subsequent diversification in Southeast Asia, tarsiers are of particular importance to evolutionary primatologists. Little is known, however, on the processes shaping the radiation of these small enigmatic primates-especially on the Indonesian island of Sulawesi, their center of endemism. Geological reconstructions and progress in applying DNA sequence information to divergence dating now provide us with the tools and background to comprehend tarsier dispersal. Here, we describe effects of plate-tectonic movements, Pleistocene sea level changes, and hybridization on the divergence of central Sulawesi tarsiers. We analyzed 12 microsatellites, the cytochrome b gene, the hypervariable region I of the mitochondrial control region, and the sex-determining region on the Y-chromosome from 144 specimens captured along a transect crossing a species boundary and a contact zone between 2 microplates. Based on these differentially inherited genetic markers, geographic information, and recordings of vocalizations, we demonstrate that the species boundary coincides with a tectonic suture. We estimate the most recent common ancestor of the 2 taxa to have lived 1.4 Mya, we describe asymmetrical introgressive hybridization, and we give evidence of unbiased dispersal in one species and male-biased dispersal in another species. This study exemplifies that the distribution of tarsier acoustic forms on Sulawesi is consistent with the allocation of genetic variability and that plate-tectonic and glacial events have left traceable marks in the biogeography of this island's unique fauna.

Revisiting the TALE repeat.

PubMed

Deng, Dong; Yan, Chuangye; Wu, Jianping; Pan, Xiaojing; Yan, Nieng

2014-04-01

Transcription activator-like (TAL) effectors specifically bind to double stranded (ds) DNA through a central domain of tandem repeats. Each TAL effector (TALE) repeat comprises 33-35 amino acids and recognizes one specific DNA base through a highly variable residue at a fixed position in the repeat. Structural studies have revealed the molecular basis of DNA recognition by TALE repeats. Examination of the overall structure reveals that the basic building block of TALE protein, namely a helical hairpin, is one-helix shifted from the previously defined TALE motif. Here we wish to suggest a structure-based re-demarcation of the TALE repeat which starts with the residues that bind to the DNA backbone phosphate and concludes with the base-recognition hyper-variable residue. This new numbering system is consistent with the α-solenoid superfamily to which TALE belongs, and reflects the structural integrity of TAL effectors. In addition, it confers integral number of TALE repeats that matches the number of bound DNA bases. We then present fifteen crystal structures of engineered dHax3 variants in complex with target DNA molecules, which elucidate the structural basis for the recognition of bases adenine (A) and guanine (G) by reported or uncharacterized TALE codes. Finally, we analyzed the sequence-structure correlation of the amino acid residues within a TALE repeat. The structural analyses reported here may advance the mechanistic understanding of TALE proteins and facilitate the design of TALEN with improved affinity and specificity.

Biogeographic Comparison of Lophelia-Associated Bacterial Communities in the Western Atlantic Reveals Conserved Core Microbiome

PubMed Central

Kellogg, Christina A.; Goldsmith, Dawn B.; Gray, Michael A.

2017-01-01

Over the last decade, publications on deep-sea corals have tripled. Most attention has been paid to Lophelia pertusa, a globally distributed scleractinian coral that creates critical three-dimensional habitat in the deep ocean. The bacterial community associated with L. pertusa has been previously described by a number of studies at sites in the Mediterranean Sea, Norwegian fjords, off Great Britain, and in the Gulf of Mexico (GOM). However, use of different methodologies prevents direct comparisons in most cases. Our objectives were to address intra-regional variation and to identify any conserved bacterial core community. We collected samples from three distinct colonies of L. pertusa at each of four locations within the western Atlantic: three sites within the GOM and one off the east coast of the United States. Amplicon libraries of 16S rRNA genes were generated using primers targeting the V4–V5 hypervariable region and 454 pyrosequencing. The dominant phylum was Proteobacteria (75–96%). At the family level, 80–95% of each sample was comprised of five groups: Pirellulaceae, Pseudonocardiaceae, Rhodobacteraceae, Sphingomonadaceae, and unclassified Oceanospirillales. Principal coordinate analysis based on weighted UniFrac distances showed a clear distinction between the GOM and Atlantic samples. Interestingly, the replicate samples from each location did not always cluster together, indicating there is not a strong site-specific influence. The core bacterial community, conserved in 100% of the samples, was dominated by the operational taxonomic units of genera Novosphingobium and Pseudonocardia, both known degraders of aromatic hydrocarbons. The sequence of another core member, Propionibacterium, was also found in prior studies of L. pertusa from Norway and Great Britain, suggesting a role as a conserved symbiont. By examining more than 40,000 sequences per sample, we found that GOM samples were dominated by the identified conserved core sequences, whereas open Atlantic samples had a much higher proportion of locally consistent bacteria. Further, predictive functional profiling highlights the potential for the L. pertusa microbiome to contribute to chemoautotrophy, nutrient cycling, and antibiotic production. PMID:28522997

Biogeographic Comparison of Lophelia-Associated Bacterial Communities in the Western Atlantic Reveals Conserved Core Microbiome.

PubMed

Kellogg, Christina A; Goldsmith, Dawn B; Gray, Michael A

2017-01-01

Over the last decade, publications on deep-sea corals have tripled. Most attention has been paid to Lophelia pertusa , a globally distributed scleractinian coral that creates critical three-dimensional habitat in the deep ocean. The bacterial community associated with L. pertusa has been previously described by a number of studies at sites in the Mediterranean Sea, Norwegian fjords, off Great Britain, and in the Gulf of Mexico (GOM). However, use of different methodologies prevents direct comparisons in most cases. Our objectives were to address intra-regional variation and to identify any conserved bacterial core community. We collected samples from three distinct colonies of L. pertusa at each of four locations within the western Atlantic: three sites within the GOM and one off the east coast of the United States. Amplicon libraries of 16S rRNA genes were generated using primers targeting the V4-V5 hypervariable region and 454 pyrosequencing. The dominant phylum was Proteobacteria (75-96%). At the family level, 80-95% of each sample was comprised of five groups: Pirellulaceae, Pseudonocardiaceae, Rhodobacteraceae, Sphingomonadaceae, and unclassified Oceanospirillales. Principal coordinate analysis based on weighted UniFrac distances showed a clear distinction between the GOM and Atlantic samples. Interestingly, the replicate samples from each location did not always cluster together, indicating there is not a strong site-specific influence. The core bacterial community, conserved in 100% of the samples, was dominated by the operational taxonomic units of genera Novosphingobium and Pseudonocardia , both known degraders of aromatic hydrocarbons. The sequence of another core member, Propionibacterium , was also found in prior studies of L. pertusa from Norway and Great Britain, suggesting a role as a conserved symbiont. By examining more than 40,000 sequences per sample, we found that GOM samples were dominated by the identified conserved core sequences, whereas open Atlantic samples had a much higher proportion of locally consistent bacteria. Further, predictive functional profiling highlights the potential for the L. pertusa microbiome to contribute to chemoautotrophy, nutrient cycling, and antibiotic production.

Biogeographic comparison of Lophelia-associated bacterial communities in the Western Atlantic reveals conserved core microbiome

USGS Publications Warehouse

Kellogg, Christina A.; Goldsmith, Dawn; Gray, Michael A.

2017-01-01

Over the last decade, publications on deep-sea corals have tripled. Most attention has been paid to Lophelia pertusa, a globally distributed scleractinian coral that creates critical three-dimensional habitat in the deep ocean. The bacterial community associated with L. pertusa has been previously described by a number of studies at sites in the Mediterranean Sea, Norwegian fjords, off Great Britain, and in the Gulf of Mexico (GOM). However, use of different methodologies prevents direct comparisons in most cases. Our objectives were to address intra-regional variation and to identify any conserved bacterial core community. We collected samples from three distinct colonies of L. pertusa at each of four locations within the western Atlantic: three sites within the GOM and one off the east coast of the United States. Amplicon libraries of 16S rRNA genes were generated using primers targeting the V4–V5 hypervariable region and 454 pyrosequencing. The dominant phylum was Proteobacteria (75–96%). At the family level, 80–95% of each sample was comprised of five groups: Pirellulaceae, Pseudonocardiaceae, Rhodobacteraceae, Sphingomonadaceae, and unclassified Oceanospirillales. Principal coordinate analysis based on weighted UniFrac distances showed a clear distinction between the GOM and Atlantic samples. Interestingly, the replicate samples from each location did not always cluster together, indicating there is not a strong site-specific influence. The core bacterial community, conserved in 100% of the samples, was dominated by the operational taxonomic units of genera Novosphingobium and Pseudonocardia, both known degraders of aromatic hydrocarbons. The sequence of another core member, Propionibacterium, was also found in prior studies of L. pertusa from Norway and Great Britain, suggesting a role as a conserved symbiont. By examining more than 40,000 sequences per sample, we found that GOM samples were dominated by the identified conserved core sequences, whereas open Atlantic samples had a much higher proportion of locally consistent bacteria. Further, predictive functional profiling highlights the potential for the L. pertusa microbiome to contribute to chemoautotrophy, nutrient cycling, and antibiotic production.

Bacterial diversity in typical Italian salami at different ripening stages as revealed by high-throughput sequencing of 16S rRNA amplicons.

PubMed

Połka, Justyna; Rebecchi, Annalisa; Pisacane, Vincenza; Morelli, Lorenzo; Puglisi, Edoardo

2015-04-01

The bacterial diversity involved in food fermentations is one of the most important factors shaping the final characteristics of traditional foods. Knowledge about this diversity can be greatly improved by the application of high-throughput sequencing technologies (HTS) coupled to the PCR amplification of the 16S rRNA subunit. Here we investigated the bacterial diversity in batches of Salame Piacentino PDO (Protected Designation of Origin), a dry fermented sausage that is typical of a regional area of Northern Italy. Salami samples from 6 different local factories were analysed at 0, 21, 49 and 63 days of ripening; raw meat at time 0 and casing samples at 21 days of ripening where also analysed, and the effect of starter addition was included in the experimental set-up. Culture-based microbiological analyses and PCR-DGGE were carried out in order to be compared with HTS results. A total of 722,196 high quality sequences were obtained after trimming, paired-reads assembly and quality screening of raw reads obtained by Illumina MiSeq sequencing of the two bacterial 16S hypervariable regions V3 and V4; manual curation of 16S database allowed a correct taxonomical classification at the species for 99.5% of these reads. Results confirmed the presence of main bacterial species involved in the fermentation of salami as assessed by PCR-DGGE, but with a greater extent of resolution and quantitative assessments that are not possible by the mere analyses of gel banding patterns. Thirty-two different Staphylococcus and 33 Lactobacillus species where identified in the salami from different producers, while the whole data set obtained accounted for 13 main families and 98 rare ones, 23 of which were present in at least 10% of the investigated samples, with casings being the major sources of the observed diversity. Multivariate analyses also showed that batches from 6 local producers tend to cluster altogether after 21 days of ripening, thus indicating that HTS has the potential for fine scale differentiation of local fermented foods. Copyright © 2014 Elsevier Ltd. All rights reserved.

Identification and Characterisation of a Hyper-Variable Apoplastic Effector Gene Family of the Potato Cyst Nematodes

PubMed Central

Eves-van den Akker, Sebastian; Lilley, Catherine J.; Jones, John T.; Urwin, Peter E.

2014-01-01

Sedentary endoparasitic nematodes are obligate biotrophs that modify host root tissues, using a suite of effector proteins to create and maintain a feeding site that is their sole source of nutrition. Using assumptions about the characteristics of genes involved in plant-nematode biotrophic interactions to inform the identification strategy, we provide a description and characterisation of a novel group of hyper-variable extracellular effectors termed HYP, from the potato cyst nematode Globodera pallida. HYP effectors comprise a large gene family, with a modular structure, and have unparalleled diversity between individuals of the same population: no two nematodes tested had the same genetic complement of HYP effectors. Individuals vary in the number, size, and type of effector subfamilies. HYP effectors are expressed throughout the biotrophic stages in large secretory cells associated with the amphids of parasitic stage nematodes as confirmed by in situ hybridisation. The encoded proteins are secreted into the host roots where they are detectable by immunochemistry in the apoplasm, between the anterior end of the nematode and the feeding site. We have identified HYP effectors in three genera of plant parasitic nematodes capable of infecting a broad range of mono- and dicotyledon crop species. In planta RNAi targeted to all members of the effector family causes a reduction in successful parasitism. PMID:25255291

Identification and characterisation of a hyper-variable apoplastic effector gene family of the potato cyst nematodes.

PubMed

Eves-van den Akker, Sebastian; Lilley, Catherine J; Jones, John T; Urwin, Peter E

2014-09-01

Sedentary endoparasitic nematodes are obligate biotrophs that modify host root tissues, using a suite of effector proteins to create and maintain a feeding site that is their sole source of nutrition. Using assumptions about the characteristics of genes involved in plant-nematode biotrophic interactions to inform the identification strategy, we provide a description and characterisation of a novel group of hyper-variable extracellular effectors termed HYP, from the potato cyst nematode Globodera pallida. HYP effectors comprise a large gene family, with a modular structure, and have unparalleled diversity between individuals of the same population: no two nematodes tested had the same genetic complement of HYP effectors. Individuals vary in the number, size, and type of effector subfamilies. HYP effectors are expressed throughout the biotrophic stages in large secretory cells associated with the amphids of parasitic stage nematodes as confirmed by in situ hybridisation. The encoded proteins are secreted into the host roots where they are detectable by immunochemistry in the apoplasm, between the anterior end of the nematode and the feeding site. We have identified HYP effectors in three genera of plant parasitic nematodes capable of infecting a broad range of mono- and dicotyledon crop species. In planta RNAi targeted to all members of the effector family causes a reduction in successful parasitism.

Median regression spline modeling of longitudinal FEV1 measurements in cystic fibrosis (CF) and chronic obstructive pulmonary disease (COPD) patients.

PubMed

Conrad, Douglas J; Bailey, Barbara A; Hardie, Jon A; Bakke, Per S; Eagan, Tomas M L; Aarli, Bernt B

2017-01-01

Clinical phenotyping, therapeutic investigations as well as genomic, airway secretion metabolomic and metagenomic investigations can benefit from robust, nonlinear modeling of FEV1 in individual subjects. We demonstrate the utility of measuring FEV1 dynamics in representative cystic fibrosis (CF) and chronic obstructive pulmonary disease (COPD) populations. Individual FEV1 data from CF and COPD subjects were modeled by estimating median regression splines and their predicted first and second derivatives. Classes were created from variables that capture the dynamics of these curves in both cohorts. Nine FEV1 dynamic variables were identified from the splines and their predicted derivatives in individuals with CF (n = 177) and COPD (n = 374). Three FEV1 dynamic classes (i.e. stable, intermediate and hypervariable) were generated and described using these variables from both cohorts. In the CF cohort, the FEV1 hypervariable class (HV) was associated with a clinically unstable, female-dominated phenotypes while stable FEV1 class (S) individuals were highly associated with the male-dominated milder clinical phenotype. In the COPD cohort, associations were found between the FEV1 dynamic classes, the COPD GOLD grades, with exacerbation frequency and symptoms. Nonlinear modeling of FEV1 with splines provides new insights and is useful in characterizing CF and COPD clinical phenotypes.

Coastal bacterioplankton community diversity along a latitudinal gradient in Latin America by means of V6 tag pyrosequencing.

PubMed

Thompson, Fabiano L; Bruce, Thiago; Gonzalez, Alessandra; Cardoso, Alexander; Clementino, Maysa; Costagliola, Marcela; Hozbor, Constanza; Otero, Ernesto; Piccini, Claudia; Peressutti, Silvia; Schmieder, Robert; Edwards, Robert; Smith, Mathew; Takiyama, Luis Roberto; Vieira, Ricardo; Paranhos, Rodolfo; Artigas, Luis Felipe

2011-02-01

The bacterioplankton diversity of coastal waters along a latitudinal gradient between Puerto Rico and Argentina was analyzed using a total of 134,197 high-quality sequences from the V6 hypervariable region of the small-subunit ribosomal RNA gene (16S rRNA) (mean length of 60 nt). Most of the OTUs were identified into Proteobacteria, Bacteriodetes, Cyanobacteria, and Actinobacteria, corresponding to approx. 80% of the total number of sequences. The number of OTUs corresponding to species varied between 937 and 1946 in the seven locations. Proteobacteria appeared at high frequency in the seven locations. An enrichment of Cyanobacteria was observed in Puerto Rico, whereas an enrichment of Bacteroidetes was detected in the Argentinian shelf and Uruguayan coastal lagoons. The highest number of sequences of Actinobacteria and Acidobacteria were obtained in the Amazon estuary mouth. The rarefaction curves and Good coverage estimator for species diversity suggested a significant coverage, with values ranging between 92 and 97% for Good coverage. Conserved taxa corresponded to aprox. 52% of all sequences. This study suggests that human-contaminated environments may influence bacterioplankton diversity.

The evolution of the gut microbiota in the giant and the red pandas.

PubMed

Li, Ying; Guo, Wei; Han, Shushu; Kong, Fanli; Wang, Chengdong; Li, Desheng; Zhang, Heming; Yang, Mingyao; Xu, Huailiang; Zeng, Bo; Zhao, Jiangchao

2015-05-18

The independent dietary shift from carnivore to herbivore with over 90% being bamboo in the giant and the red pandas is of great interests to biologists. Although previous studies have shown convergent evolution of the giant and the red pandas at both morphological and molecular level, the evolution of the gut microbiota in these pandas remains largely unknown. The goal of this study was to determine whether the gut microbiota of the pandas converged due to the same diet, or diverged. We characterized the fecal microbiota from these two species by pyrosequencing the 16S V1-V3 hypervariable regions using the 454 GS FLX Titanium platform. We also included fecal samples from Asian black bears, a species phylogenetically closer to the giant panda, in our analyses. By analyzing the microbiota from these 3 species and those from other carnivores reported previously, we found the gut microbiotas of the giant pandas are distinct from those of the red pandas and clustered closer to those of the black bears. Our data suggests the divergent evolution of the gut microbiota in the pandas.

Gene family encoding the major toxins of lethal Amanita mushrooms

PubMed Central

Hallen, Heather E.; Luo, Hong; Scott-Craig, John S.; Walton, Jonathan D.

2007-01-01

Amatoxins, the lethal constituents of poisonous mushrooms in the genus Amanita, are bicyclic octapeptides. Two genes in A. bisporigera, AMA1 and PHA1, directly encode α-amanitin, an amatoxin, and the related bicyclic heptapeptide phallacidin, a phallotoxin, indicating that these compounds are synthesized on ribosomes and not by nonribosomal peptide synthetases. α-Amanitin and phallacidin are synthesized as proproteins of 35 and 34 amino acids, respectively, from which they are predicted to be cleaved by a prolyl oligopeptidase. AMA1 and PHA1 are present in other toxic species of Amanita section Phalloidae but are absent from nontoxic species in other sections. The genomes of A. bisporigera and A. phalloides contain multiple sequences related to AMA1 and PHA1. The predicted protein products of this family of genes are characterized by a hypervariable “toxin” region capable of encoding a wide variety of peptides of 7–10 amino acids flanked by conserved sequences. Our results suggest that these fungi have a broad capacity to synthesize cyclic peptides on ribosomes. PMID:18025465

The evolution of the gut microbiota in the giant and the red pandas

PubMed Central

Li, Ying; Guo, Wei; Han, Shushu; Kong, Fanli; Wang, Chengdong; Li, Desheng; Zhang, Heming; Yang, Mingyao; Xu, Huailiang; Zeng, Bo; Zhao, Jiangchao

2015-01-01

The independent dietary shift from carnivore to herbivore with over 90% being bamboo in the giant and the red pandas is of great interests to biologists. Although previous studies have shown convergent evolution of the giant and the red pandas at both morphological and molecular level, the evolution of the gut microbiota in these pandas remains largely unknown. The goal of this study was to determine whether the gut microbiota of the pandas converged due to the same diet, or diverged. We characterized the fecal microbiota from these two species by pyrosequencing the 16S V1–V3 hypervariable regions using the 454 GS FLX Titanium platform. We also included fecal samples from Asian black bears, a species phylogenetically closer to the giant panda, in our analyses. By analyzing the microbiota from these 3 species and those from other carnivores reported previously, we found the gut microbiotas of the giant pandas are distinct from those of the red pandas and clustered closer to those of the black bears. Our data suggests the divergent evolution of the gut microbiota in the pandas. PMID:25985413

Serratia sp. ZF03: an efficient radium biosorbent isolated from hot-spring waters in high background radiation areas.

PubMed

Zakeri, Farideh; Noghabi, Kambiz Akbari; Sadeghizadeh, Majid; Kardan, Mohammad Reza; Masoomi, Fatemeh; Farshidpour, Mohammad Reza; Atarilar, Ali

2010-12-01

The aim of this study is to isolate and characterize (226)Ra biosorbing indigenous bacterial strains from soils and hot-springs containing high concentrations of (226)Ra by using biochemical and molecular approaches. Fifteen bacteria were isolated and their phylogenetic affiliations were determined based on their 16S rRNA gene and the two most relevant hypervariable regions of this gene; V3 and V6 analysis. A pigmented Serratia sp. ZF03 strain isolated from the water with (226)Ra content of 50471 mBq l(-1), caused 70% removal of (226)Ra at a radioactivity level of 50 Bq ml(-1), after 5 min and 75-80% in equilibrium time of 1 h, depending on the particular biosorption system and experimental conditions studied. The biosorption equilibrium was described by Langmuir and Freundlich isotherm models. Kinetic studies indicated that the biosorption follows pseudo-second-order kinetics. Effect of different physico-chemical parameters on (226)Ra sorption, FTIR, SEM and TEM analysis were also investigated. 2010 Elsevier Ltd. All rights reserved.

Mendelian breeding units versus standard sampling strategies: Mitochondrial DNA variation in southwest Sardinia

PubMed Central

Sanna, Daria; Pala, Maria; Cossu, Piero; Dedola, Gian Luca; Melis, Sonia; Fresu, Giovanni; Morelli, Laura; Obinu, Domenica; Tonolo, Giancarlo; Secchi, Giannina; Triunfo, Riccardo; Lorenz, Joseph G.; Scheinfeldt, Laura; Torroni, Antonio; Robledo, Renato; Francalacci, Paolo

2011-01-01

We report a sampling strategy based on Mendelian Breeding Units (MBUs), representing an interbreeding group of individuals sharing a common gene pool. The identification of MBUs is crucial for case-control experimental design in association studies. The aim of this work was to evaluate the possible existence of bias in terms of genetic variability and haplogroup frequencies in the MBU sample, due to severe sample selection. In order to reach this goal, the MBU sampling strategy was compared to a standard selection of individuals according to their surname and place of birth. We analysed mitochondrial DNA variation (first hypervariable segment and coding region) in unrelated healthy subjects from two different areas of Sardinia: the area around the town of Cabras and the western Campidano area. No statistically significant differences were observed when the two sampling methods were compared, indicating that the stringent sample selection needed to establish a MBU does not alter original genetic variability and haplogroup distribution. Therefore, the MBU sampling strategy can be considered a useful tool in association studies of complex traits. PMID:21734814

Antigenic variation of Anaplasma marginale msp2 occurs by combinatorial gene conversion.

PubMed

Brayton, Kelly A; Palmer, Guy H; Lundgren, Anna; Yi, Jooyoung; Barbet, Anthony F

2002-03-01

The rickettsial pathogen Anaplasma marginale establishes lifelong persistent infection in the mammalian reservoir host, during which time immune escape variants continually arise in part because of variation in the expressed copy of the immunodominant outer membrane protein MSP2. A key question is how the small 1.2 Mb A. marginale genome generates sufficient variants to allow long-term persistence in an immunocompetent reservoir host. The recombination of whole pseudogenes into the single msp2 expression site has been previously identified as one method of generating variants, but is inadequate to generate the number of variants required for persistent infection. In the present study, we demonstrate that recombination of a whole pseudogene is followed by a second level of variation in which small segments of pseudogenes recombine into the expression site by gene conversion. Evidence for four short sequential changes in the hypervariable region of msp2 coupled with the identification of nine pseudogenes from a single strain of A. marginale provides for a combinatorial number of possible expressed MSP2 variants sufficient for lifelong persistence.

Short Communication Quick method for identifying horse (Equus caballus) and donkey (Equus asinus) hybrids.

PubMed

Franco, M M; Santos, J B F; Mendonça, A S; Silva, T C F; Antunes, R C; Melo, E O

2016-09-23

The domestication of the Equus genus 5000-6000 years ago has influenced the history of human civilization. As soon as horse and donkey species had been domesticated, they were crossbred, producing humanity's first documented attempt at animal genome manipulation. Since then, the mule (male donkey x female horse) and the reciprocal cross (the hinny, male horse x female donkey) have been the most common equine hybrids in the world. Due to their hybrid vigor, mules and hinnies have been intensively used for carrying loads and people and for tilling the land. Despite their importance, visual distinction of mules and hinnies is difficult due to high phenotypic resemblance. However, the distinction between these two hybrids is of pivotal importance for equid breeders and ranchers. In this study, an easy, low-cost, effective, and fast multiplex-polymerase chain reaction method was developed to distinguish the maternal origin of mules and hinnies, targeting the hyper-variable mitochondrial DNA D-loop region. This methodology can help breeders, ranchers, animal science professionals, and researchers manage their equine herds with more confidence and precision.

Human and rat gut microbiome composition is maintained following sleep restriction

PubMed Central

Zhang, Shirley L.; Bai, Lei; Goel, Namni; Bailey, Aubrey; Jang, Christopher J.; Bushman, Frederic D.; Meerlo, Peter; Dinges, David F.; Sehgal, Amita

2017-01-01

Insufficient sleep increasingly characterizes modern society, contributing to a host of serious medical problems. Loss of sleep is associated with metabolic diseases such as obesity and diabetes, cardiovascular disorders, and neurological and cognitive impairments. Shifts in gut microbiome composition have also been associated with the same pathologies; therefore, we hypothesized that sleep restriction may perturb the gut microbiome to contribute to a disease state. In this study, we examined the fecal microbiome by using a cross-species approach in both rat and human studies of sleep restriction. We used DNA from hypervariable regions (V1-V2) of 16S bacteria rRNA to define operational taxonomic units (OTUs) of the microbiome. Although the OTU richness of the microbiome is decreased by sleep restriction in rats, major microbial populations are not altered. Only a single OTU, TM7-3a, was found to increase with sleep restriction of rats. In the human microbiome, we find no overt changes in the richness or composition induced by sleep restriction. Together, these results suggest that the microbiome is largely resistant to changes during sleep restriction. PMID:28179566

Human and rat gut microbiome composition is maintained following sleep restriction.

PubMed

Zhang, Shirley L; Bai, Lei; Goel, Namni; Bailey, Aubrey; Jang, Christopher J; Bushman, Frederic D; Meerlo, Peter; Dinges, David F; Sehgal, Amita

2017-02-21

Insufficient sleep increasingly characterizes modern society, contributing to a host of serious medical problems. Loss of sleep is associated with metabolic diseases such as obesity and diabetes, cardiovascular disorders, and neurological and cognitive impairments. Shifts in gut microbiome composition have also been associated with the same pathologies; therefore, we hypothesized that sleep restriction may perturb the gut microbiome to contribute to a disease state. In this study, we examined the fecal microbiome by using a cross-species approach in both rat and human studies of sleep restriction. We used DNA from hypervariable regions (V1-V2) of 16S bacteria rRNA to define operational taxonomic units (OTUs) of the microbiome. Although the OTU richness of the microbiome is decreased by sleep restriction in rats, major microbial populations are not altered. Only a single OTU, TM7-3a, was found to increase with sleep restriction of rats. In the human microbiome, we find no overt changes in the richness or composition induced by sleep restriction. Together, these results suggest that the microbiome is largely resistant to changes during sleep restriction.

The population genetics of a solitary oligolectic sweat bee, Lasioglossum (Sphecodogastra) oenotherae (Hymenoptera: Halictidae).

PubMed

Zayed, A; Packer, L

2007-10-01

Strong evidence exists for global declines in pollinator populations. Data on the population genetics of solitary bees, especially diet specialists, are generally lacking. We studied the population genetics of the oligolectic bee Lasioglossum oenotherae, a specialist on the pollen of evening primrose (Onagraceae), by genotyping 455 females from 15 populations across the bee's North American range at six hyper-variable microsatellite loci. We found significant levels of genetic differentiation between populations, even at small geographic scales, as well as significant patterns of isolation by distance. However, using multilocus genotype assignment tests, we detected 11 first-generation migrants indicating that L. oenotherae's sub-populations are experiencing ongoing gene flow. Southern populations of L. oenotherae were significantly more likely to deviate from Hardy-Weinberg equilibrium and from genotypic equilibrium, suggesting regional differences in gene flow and/or drift and inbreeding. Short-term N(e) estimated using temporal changes in allele frequencies in several populations ranged from approximately 223 to 960. We discuss our findings in terms of the conservation genetics of specialist pollinators, a group of considerable ecological importance.

Molecular characterization of infectious bursal disease viruses from Pakistan.

PubMed

Shabbir, Muhammad Zubair; Ali, Muhammad; Abbas, Muhammad; Chaudhry, Umer Naveed; Zia-Ur-Rehman; Munir, Muhammad

2016-07-01

Since the first report of infectious bursal disease in Pakistan in 1987, outbreaks have been common even in vaccinated flocks. Despite appropriate administration of vaccines, concerns arise if the circulating strains are different from the ones used in the vaccine. Here, we sequenced the hypervariable region (HVR) of the VP2 gene of circulating strains of infectious bursal disease virus (IBDV) originating from outbreaks (n = 4) in broiler flocks in Pakistan. Nucleotide sequencing followed by phylogeny and deduced amino acid sequence analysis showed the circulating strains to be very virulent (vv) and identified characteristic residues at position 222 (A), 242 (I), 256 (I), 294 (I) and 299 (S). In addition, a substitution at positions 221 (Q→H) was found to be exclusive to Pakistani strains in our analysis, although a larger dataset is required to confirm this finding. Compared to vaccine strains that are commonly used in Pakistan, substitution mutations were found at key amino acid positions in VP2 that may be responsible for potential changes in neutralization epitopes and vaccine failure.

Design of sphingomonad-detecting probes for a DNA array, and its application to investigate the behavior, distribution, and source of Rhizospherous sphingomonas and other sphingomonads inhabiting an acid sulfate soil paddock in Kalimantan, Indonesia.

PubMed

Hashidoko, Yasuyuki; Kitagawa, Emiko; Iwahashi, Hitoshi; Purnomo, Erry; Hasegawa, Toshihiro; Tahara, Satoshi

2007-02-01

Throughout Central and South Kalimantan, Indonesia, strongly acidic soil (pH 2.1-3.7) is widely distributed, and the local acidic soil-tolerant plants, including local rice varieties, often possess sphingomonads in their rhizosphere and rhizoplane. To investigate the behavior of sphingomonads inhabiting the rhizosphere of such acid-tolerant plants, we designed 13 different DNA array probes (each of 72 mer) specific to a group of sphingomonads, using a hypervariable V6 region of the 16S rRNA gene. This DNA array system was used preliminarily for an analysis of microfloral dynamisms, particularly of sphingomonads, in acidic paddock ecosystems, and the results suggest that the acid-tolerant local rice shares rhizospherous sphingomonads with wild Juncus sp., a predominant weed that thrives in acidic paddocks during the off-season for rice farming. This tentative conclusion supports the bio-rationality of the traditional rice farming system with respect to functional rhizobacteria.

Identification of clinically relevant Corynebacterium strains by Api Coryne, MALDI-TOF-mass spectrometry and molecular approaches.

PubMed

Alibi, S; Ferjani, A; Gaillot, O; Marzouk, M; Courcol, R; Boukadida, J

2015-09-01

We evaluated the Bruker Biotyper matrix-assisted laser desorption ionization-time of flight (MALDI-TOF) mass spectrometry (MS) for the identification of 97 Corynebacterium clinical in comparison to identification strains by Api Coryne and MALDI-TOF-MS using 16S rRNA gene and hypervariable region of rpoB genes sequencing as a reference method. C. striatum was the predominant species isolated followed by C. amycolatum. There was an agreement between Api Coryne strips and MALDI-TOF-MS identification in 88.65% of cases. MALDI-TOF-MS was unable to differentiate C. aurimucosum from C. minutissimum and C. minutissimum from C. singulare but reliably identify 92 of 97 (94.84%) strains. Two strains remained incompletely identified to the species level by MALDI-TOF-MS and molecular approaches. They belonged to Cellulomonas and Pseudoclavibacter genus. In conclusion, MALDI-TOF-MS is a rapid and reliable method for the identification of Corynebacterium species. However, some limits have been noted and have to be resolved by the application of molecular methods. Copyright © 2015. Published by Elsevier SAS.

Pyrosequencing analysis of oral microbiota in children with severe early childhood dental caries.

PubMed

Jiang, Wen; Zhang, Jie; Chen, Hui

2013-11-01

Severe early childhood caries are a prevalent public health problem among preschool children throughout the world. However, little is known about the microbiota found in association with severe early childhood caries. Our study aimed to explore the bacterial microbiota of dental plaques to study the etiology of severe early childhood caries through pyrosequencing analysis based on 16S rRNA gene V1-V3 hypervariable regions. Forty participants were enrolled in the study, and we obtained twenty samples of supragingival plaque from caries-free subjects and twenty samples from subjects with severe early childhood caries. A total of 175,918 reads met the quality control standards, and the bacteria found belonged to fourteen phyla and sixty-three genera. Our results show the overall structure and microbial composition of oral bacterial communities, and they suggest that these bacteria may present a core microbiome in the dental plaque microbiota. Three genera, Streptococcus, Granulicatella, and Actinomyces, were increased significantly in children with severe dental cavities. These data may facilitate improvements in the prevention and treatment of severe early childhood caries.

Psychrophile spoilers dominate the bacterial microbiome in musculature samples of slaughter pigs.

PubMed

Mann, Evelyne; Wetzels, Stefanie U; Pinior, Beate; Metzler-Zebeli, Barbara U; Wagner, Martin; Schmitz-Esser, Stephan

2016-07-01

The aim of this study was to disentangle the microbial diversity on porcine musculature. The hypervariable V1-V2 region of the 16S rRNA gene was amplified from DNA samples of clinically healthy slaughter pigs (n=8). Pyrosequencing yielded 37,000 quality-controlled reads and a diverse microbiome with 54-159 OTUs per sample was detected. Interestingly, 6 out of 8 samples were strongly dominated by 1-2 highly abundant OTUs (best hits of highly abundant OTUs: Serratia proteamaculans, Pseudomonas syringae, Aeromonas allosaccharophila, Brochothrix thermosphacta, Acidiphilium cryptum and Escherichia coli). In 1g musculature scraping, 3.20E+06 16S rRNA gene copies and 4.45E+01 Enterobacteriaceae rRNA gene copies were detected with qPCR. We conclude that i.) next-generation sequencing technologies help encompass the full content of complex, bacterial contamination, ii.) psychrophile spoilers dominated the microbiota and iii.) E. coli is an effective marker species for pork contamination, as it was one of very few abundant species being present in all samples. Copyright © 2016 Elsevier Ltd. All rights reserved.

Foot-and-mouth disease virus (FMDV) with a stable FLAG epitope in the VP1 G-H loop as a new tool for studying FMDV pathogenesis

USDA-ARS?s Scientific Manuscript database

In this study, we generated a recombinant foot-and-mouth disease virus (FMDV) particle derived from A24 Cruzeiro with a FLAG tag (DYKDDDDK) substitution in the hypervariable antigenic site of the G-H loop of the VP1 capsid protein in an effort to expand the immunogenicity of the virus particle and t...

Characterization of the patterns of polymorphism in a [open quotes]cryptic repeat[close quotes] reveals a novel type of hypervariable sequence

DOE Office of Scientific and Technical Information (OSTI.GOV)

Jacobson, D.P.; Schmeling, P.; Sommer, S.S.

Alternating purine and pyrimidine repeats (RY(i)) are an abundant source of polymorphism. The subset with long tandem repeats of GT or AC (GT(i)) have been studied extensively, but cryptic RY(i) (i.e., no single tandem repeat predominates) have received little attention. The factor IX gene has a polymorphic cryptic RY(i) of 142-216 bp. Previously, there were four known polymorphic alleles, of the form AB, A[sub 2]B, A[sub 2]B[sub 2], and A[sub 3]B[sub 2], where A = (GT)(AC)[sub 3](AT)[sub 3](GT)(AT)[sub 4] and B = A with an additional 3' AT dinucleotide. To further characterize this locus, the authors examined more than 1,700more » additional human chromosomes and determined the sequences of the homologous sites in orangutans and chimpanzees. The novel alleles found in humans expand the repertoire of A/B alleles to A[sub 0-4]B[sub 1] and A[sub 1-3]B[sub 2]. The A[sub n]B[sub 2] series are abundant in Caucasians but are absent in blacks and Asians. Conversely, the A[sub 0]B[sub 1] allele is common in blacks but is not found in more than 1,700 Caucasian chromosomes. The data are compatible with a model in which recombination is more frequent than polymerase slippage at this locus. In orangutans, the RY(i) is present, but the sequence is markedly different. An A/B-type of pattern was discerned in which B differs from A by an additional six (AT) dinucleotides at the 3' end. In chimpanzees, the size of the RY(i) locus was greatly expanded, and the sequence showed a novel pattern of hypervariability in which there are many tandem repeats of the form (GT)[sub n](AC)[sub 0](AT)[sub p](GT)[sub q](AT)[sub s], where n, o, p, q, and s are different integers. The sequences of the factor IX intron 1 cryptic RY(i) in three primates provide perspective on the range of possible patterns of polymorphism. Analysis of the patterns suggests how the RY(i) can be conserved during evolution, while the precise sequence varies. 25 refs., 5 figs., 3 tabs.« less

Selective amplification of T-cell receptor variable region species is demonstrable but not essential in early lesions of psoriasis vulgaris: analysis by anchored polymerase chain reaction and hypervariable region size spectratyping.

PubMed

Vekony, M A; Holder, J E; Lee, A J; Horrocks, C; Eperon, I C; Camp, R D

1997-07-01

Several groups have investigated the role of T cells in the pathogenesis of psoriasis by determination of T-cell receptor (TCR) B-chain variable (V) region usage, both in chronic plaque (psoriasis vulgaris) and guttate forms, with various results. Because there are no data on TCR expression in early psoriasis vulgaris, when specific cellular immune events may be expected to be most pronounced, we have analyzed early lesions (less than 3 wk old) of ten patients, with highly reproducible results. We have developed a highly controlled anchored polymerase chain reaction (PCR) method in which TCR beta chain species are all amplified with the same primer pair and products are quantified by dot blot hybridization with BV family-specific oligonucleotide probes. Overexpression of certain TCR BV genes was observed in the majority of lesional biopsies, but in samples in which the expanded BV family formed more than 10% of total lesional BV (half of the samples analyzed), BV2 and BV6 predominated. The consistency of overexpression of these BV species between patients was much less than in previous studies of TCRBV usage in established chronic plaque psoriasis lesions. Complementarity-determining region 3 (CDR3) size spectratyping demonstrated evidence for selective clonal T cell accumulation in less than half of the lesional samples showing BV expansion. These results indicate that selective amplification of TCRBV species occurs in early psoriasis vulgaris but is not essential to the pathogenic process and may be more important in the maintenance or expansion of chronic lesions.

Metabarcoding Baseline for the Sargasso Sea Zooplankton Community

NASA Astrophysics Data System (ADS)

Blanco-Bercial, L.; Alam, S.

2016-02-01

Understanding the responses and evolution of any community over space and time requires a deep knowledge of the species present at each location and their interactions. Where taxonomy turns out to be challenging, as it is in the case of zooplankton, supra-species grouping is a common resort in community characterization. Although this makes morphological identification manageable, there is the associated price of a limited depth of study and the risk of mixing different species' organismal responses. As global change begins to influence species distributions and physiologies, it becomes ever more important to discriminate at a species specific level. The development of DNA-based identification protocols during the last decades are rapidly driving these limitations away, increasing our understanding of the existing complexity of even very close taxa to different stressors or environmental conditions. Beyond the mere taxonomic discrimination of the analyzed community, the use of DNA sequences allows for the rapid integration of phylogenetic measurements and related indexes. In this presentation, we show our first results tackling one of the regions with the highest zooplankton diversity, the Subtropical North Atlantic at the Bermuda Atlantic Time-Series Study (BATS) site. The chosen metabarcoding region was the hypervariable V9 region of the 18S rRNA gene. In this first investigation, we establish the baseline information needed for further and more comprehensive analyses on the time series: minimum coverage depth per sample, taxonomic and phylogenetic diversity of the community and effect of the Diel Vertical Migration in the epipelagic community. We also analyze the limitations of the species identification in relation to the variability of the V9 region within and between species.

Individual Signatures Define Canine Skin Microbiota Composition and Variability

PubMed Central

Cuscó, Anna; Sánchez, Armand; Altet, Laura; Ferrer, Lluís; Francino, Olga

2017-01-01

Dogs present almost all their skin sites covered by hair, but canine skin disorders are more common in certain skin sites and breeds. The goal of our study is to characterize the composition and variability of the skin microbiota in healthy dogs and to evaluate the effect of the breed, the skin site, and the individual. We have analyzed eight skin sites of nine healthy dogs from three different breeds by massive sequencing of 16S rRNA gene V1–V2 hypervariable regions. The main phyla inhabiting the skin microbiota in healthy dogs are Proteobacteria, Firmicutes, Fusobacteria, Actinobacteria, and Bacteroidetes. Our results suggest that skin microbiota composition pattern is individual specific, with some dogs presenting an even representation of the main phyla and other dogs with only a major phylum. The individual is the main force driving skin microbiota composition and diversity rather than the skin site or the breed. The individual is explaining 45% of the distances among samples, whereas skin site explains 19% and breed 9%. Moreover, analysis of similarities suggests a strong dissimilarity among individuals (R = 0.79, P = 0.001) that is mainly explained by low-abundant species in each dog. Skin site also plays a role: inner pinna presents the highest diversity value, whereas perianal region presents the lowest one and the most differentiated microbiota composition. PMID:28220148

Individual Signatures Define Canine Skin Microbiota Composition and Variability.

PubMed

Cuscó, Anna; Sánchez, Armand; Altet, Laura; Ferrer, Lluís; Francino, Olga

2017-01-01

Dogs present almost all their skin sites covered by hair, but canine skin disorders are more common in certain skin sites and breeds. The goal of our study is to characterize the composition and variability of the skin microbiota in healthy dogs and to evaluate the effect of the breed, the skin site, and the individual. We have analyzed eight skin sites of nine healthy dogs from three different breeds by massive sequencing of 16S rRNA gene V1-V2 hypervariable regions. The main phyla inhabiting the skin microbiota in healthy dogs are Proteobacteria, Firmicutes, Fusobacteria, Actinobacteria, and Bacteroidetes. Our results suggest that skin microbiota composition pattern is individual specific, with some dogs presenting an even representation of the main phyla and other dogs with only a major phylum. The individual is the main force driving skin microbiota composition and diversity rather than the skin site or the breed. The individual is explaining 45% of the distances among samples, whereas skin site explains 19% and breed 9%. Moreover, analysis of similarities suggests a strong dissimilarity among individuals ( R  = 0.79, P  = 0.001) that is mainly explained by low-abundant species in each dog. Skin site also plays a role: inner pinna presents the highest diversity value, whereas perianal region presents the lowest one and the most differentiated microbiota composition.

Ecology of the microbiome of the infected root canal system: a comparison between apical and coronal root segments

PubMed Central

Özok, A.R.; Persoon, I.F.; Huse, S.M.; Keijser, B.J.F.; Wesselink, P.R.; Crielaard, W.; Zaura, E.

2016-01-01

Aim To evaluate the microbial ecology of the coronal and apical segments of infected root canal systems using a complete sampling technique and next-generation sequencing. Methodology The roots of 23 extracted teeth with apical periodontitis were sectioned in half, horizontally, and cryo-pulverized. Bacterial communities were profiled using tagged 454 pyrosequencing of the 16S rDNA hypervariable V5–V6 region. Results The sequences were classified into 606 taxa (species or higher taxon), representing 24 bacterial phyla or candidate divisions and one archaeal phylum. Proteobacteria were more abundant in the apical samples (p<0.05), while Actinobacteria were in significantly higher proportions in the coronal samples. The apical samples harbored statistically significantly more taxa than the coronal samples (p=0.01), and showed a higher microbial diversity. Several taxa belonging to fastidious obligate anaerobes were significantly more abundant in the apical segments of the roots compared to their coronal counterparts. Conclusions Endodontic infections are more complex than reported previously. The apical part of the root canal system drives the selection of a more diverse and more anaerobe community than the coronal part. The presence of a distinct ecological niche in the apical region explains the difficulty of eradication of the infection, and emphasizes the need that new treatment approaches should be developed. PMID:22251411

Marked population structure and recent migration in the critically endangered Sumatran orangutan (Pongo abelii).

PubMed

Nater, Alexander; Arora, Natasha; Greminger, Maja P; van Schaik, Carel P; Singleton, Ian; Wich, Serge A; Fredriksson, Gabriella; Perwitasari-Farajallah, Dyah; Pamungkas, Joko; Krützen, Michael

2013-01-01

A multitude of factors influence how natural populations are genetically structured, including dispersal barriers, inhomogeneous habitats, and social organization. Such population subdivision is of special concern in endangered species, as it may lead to reduced adaptive potential and inbreeding in local subpopulations, thus increasing the risk of future extinctions. With only 6600 animals left in the wild, Sumatran orangutans (Pongo abelii) are among the most endangered, but also most enigmatic, great ape species. In order to infer the fine-scale population structure and connectivity of Sumatran orangutans, we analyzed the most comprehensive set of samples to date, including mitochondrial hyper-variable region I haplotypes for 123 individuals and genotypes of 27 autosomal microsatellite markers for 109 individuals. For both mitochondrial and autosomal markers, we found a pronounced population structure, caused by major rivers, mountain ridges, and the Toba caldera. We found that genetic diversity and corresponding long-term effective population size estimates vary strongly among sampling regions for mitochondrial DNA, but show remarkable similarity for autosomal markers, hinting at male-driven long-distance gene flow. In support of this, we identified several individuals that were most likely sired by males originating from other genetic clusters. Our results highlight the effect of natural barriers in shaping the genetic structure of great ape populations, but also point toward important dispersal corridors on northern Sumatra that allow for genetic exchange.

Population structure of plasmid-containing strains of Streptococcus mutans, a member of the human indigenous biota.

PubMed

Caufield, Page W; Saxena, Deepak; Fitch, David; Li, Yihong

2007-02-01

There are suggestions that the phylogeny of Streptococcus mutans, a member of the human indigenous biota that is transmitted mostly mother to child, might parallel the evolutionary history of its human host. The relatedness and phylogeny of plasmid-containing strains of S. mutans were examined based on chromosomal DNA fingerprints (CDF), a hypervariable region (HVR) of a 5.6-kb plasmid, the rRNA gene intergenic spacer region (IGSR), serotypes, and the genotypes of mutacin I and II. Plasmid-containing strains were studied because their genetic diversity was twice as great as that of plasmid-free strains. The CDF of S. mutans from unrelated human hosts were unique, except those from Caucasians, which were essentially identical. The evolutionary history of the IGSR, with or without the serotype and mutacin characters, clearly delineated an Asian clade. Also, a continuous association with mutacin II could be reconstructed through an evolutionary lineage with the IGSR, but not for serotype e. DNA sequences from the HVR of the plasmid produced a well-resolved phylogeny that differed from the chromosomal phylogeny, indicating that the horizontal transfer of the plasmid may have occurred multiple times. The plasmid phylogeny was more congruent with serotype e than with mutacin II evolution, suggesting a possible functional correlation. Thus, the history of this three-tiered relationship between human, bacterium, and plasmid supported both coevolution and independent evolution.

Community Structures of Fecal Bacteria in Cattle from Different Animal Feeding Operations▿†

PubMed Central

Shanks, Orin C.; Kelty, Catherine A.; Archibeque, Shawn; Jenkins, Michael; Newton, Ryan J.; McLellan, Sandra L.; Huse, Susan M.; Sogin, Mitchell L.

2011-01-01

The fecal microbiome of cattle plays a critical role not only in animal health and productivity but also in food safety, pathogen shedding, and the performance of fecal pollution detection methods. Unfortunately, most published molecular surveys fail to provide adequate detail about variability in the community structures of fecal bacteria within and across cattle populations. Using massively parallel pyrosequencing of a hypervariable region of the rRNA coding region, we profiled the fecal microbial communities of cattle from six different feeding operations where cattle were subjected to consistent management practices for a minimum of 90 days. We obtained a total of 633,877 high-quality sequences from the fecal samples of 30 adult beef cattle (5 individuals per operation). Sequence-based clustering and taxonomic analyses indicate less variability within a population than between populations. Overall, bacterial community composition correlated significantly with fecal starch concentrations, largely reflected in changes in the Bacteroidetes, Proteobacteria, and Firmicutes populations. In addition, network analysis demonstrated that annotated sequences clustered by management practice and fecal starch concentration, suggesting that the structures of bovine fecal bacterial communities can be dramatically different in different animal feeding operations, even at the phylum and family taxonomic levels, and that the feeding operation is a more important determinant of the cattle microbiome than is the geographic location of the feedlot. PMID:21378055

Bacterial Community Composition of South China Sea Sediments through Pyrosequencing-Based Analysis of 16S rRNA Genes

PubMed Central

Zhu, Daochen; Tanabe, Shoko-Hosoi; Yang, Chong; Zhang, Weimin; Sun, Jianzhong

2013-01-01

Background Subseafloor sediments accumulate large amounts of organic and inorganic materials that contain a highly diverse microbial ecosystem. The aim of this study was to survey the bacterial community of subseafloor sediments from the South China Sea. Methodology/Principal Findings Pyrosequencing of over 265,000 amplicons of the V3 hypervariable region of the 16S ribosomal RNA gene was performed on 16 sediment samples collected from multiple locations in the northern region of the South China Sea from depths ranging from 35 to 4000 m. A total of 9,726 operational taxonomic units (OTUs; between 695 and 2819 unique OTUs per sample) at 97% sequence similarity level were generated. In total, 40 bacterial phyla including 22 formally described phyla and 18 candidate phyla, with Proteobacteria, Firmicutes, Planctomycetes, Actinobacteria and Chloroflexi being most diverse, were identified. The most abundant phylotype, accounting for 42.6% of all sequences, belonged to Gammaproteobacteria, which possessed absolute predominance in the samples analyzed. Among the 18 candidate phyla, 12 were found for the first time in the South China Sea. Conclusions This study provided a novel insight into the composition of bacterial communities of the South China Sea subseafloor. Furthermore, abundances and community similarity analysis showed that the compositions of the bacterial communities are very similar at phylum level at different depths from 35-4000 m. PMID:24205246

Bacterial community composition of South China Sea sediments through pyrosequencing-based analysis of 16S rRNA genes.

PubMed

Zhu, Daochen; Tanabe, Shoko-Hosoi; Yang, Chong; Zhang, Weimin; Sun, Jianzhong

2013-01-01

Subseafloor sediments accumulate large amounts of organic and inorganic materials that contain a highly diverse microbial ecosystem. The aim of this study was to survey the bacterial community of subseafloor sediments from the South China Sea. Pyrosequencing of over 265,000 amplicons of the V3 hypervariable region of the 16S ribosomal RNA gene was performed on 16 sediment samples collected from multiple locations in the northern region of the South China Sea from depths ranging from 35 to 4000 m. A total of 9,726 operational taxonomic units (OTUs; between 695 and 2819 unique OTUs per sample) at 97% sequence similarity level were generated. In total, 40 bacterial phyla including 22 formally described phyla and 18 candidate phyla, with Proteobacteria, Firmicutes, Planctomycetes, Actinobacteria and Chloroflexi being most diverse, were identified. The most abundant phylotype, accounting for 42.6% of all sequences, belonged to Gammaproteobacteria, which possessed absolute predominance in the samples analyzed. Among the 18 candidate phyla, 12 were found for the first time in the South China Sea. This study provided a novel insight into the composition of bacterial communities of the South China Sea subseafloor. Furthermore, abundances and community similarity analysis showed that the compositions of the bacterial communities are very similar at phylum level at different depths from 35-4000 m.

Unexpected convergence of fungal and bacterial communities during fermentation of traditional Korean alcoholic beverages inoculated with various natural starters.

PubMed

Jung, Mi-Ja; Nam, Young-Do; Roh, Seong Woon; Bae, Jin-Woo

2012-05-01

Makgeolli is a traditional Korean alcoholic beverage manufactured with a natural starter, called nuruk, and grains. Nuruk is a starchy disk or tablet formed from wheat or grist containing various fungal and bacterial strains from the surrounding environment that are allowed to incorporate naturally into the starter, each of which simultaneously participates in the makgeolli fermentation process. In the current study, changes in microbial dynamics during laboratory-scale fermentation of makgeolli inoculated with six different kinds of nuruk were evaluated by barcoded pyrosequencing using fungal- and bacterial-specific primers targeting the internal transcribed spacer 2 region and hypervariable regions V1 to V3 of the 16S rRNA gene, respectively. A total of 61,571 fungal and 68,513 bacterial sequences were used for the analysis of microbial diversity in ferment samples. During fermentation, the proportion of fungal microorganisms belonging to the family Saccharomycetaceae increased significantly, and the major bacterial phylum of the samples shifted from γ-Proteobacteria to Firmicutes. The results of quantitative PCR indicated that the bacterial content in the final ferments was higher than in commercial rice beers, while total fungi appeared similar. This is the first report of a comparative analysis of bacterial and fungal dynamics in parallel during the fermentation of Korean traditional alcoholic beverage using barcoded pyrosequencing. Copyright © 2011 Elsevier Ltd. All rights reserved.

Next-Generation Sequencing Analyses of Bacterial Community Structures in Soybean Pastes Produced in Northeast China.

PubMed

Lee, Mi-Hwa; Li, Fan-Zhu; Lee, Jiyeon; Kang, Jisu; Lim, Seong-Il; Nam, Young-Do

2017-04-01

Fermented soybean foods contain nutritional components including easily digestible peptides, cholesterol-free oils, minerals, and vitamins. Various fermented soybean foods have been developed and are consumed as flavoring condiments in Asian regions. While the quality of fermented soybean foods is largely affected by microorganisms that participate in the fermentation process, our knowledge about the microorganisms in soybean pastes manufactured in Northeast China is limited. The current study used a culture-independent barcoded pyrosequencing method targeting hypervariable V1/V2 regions of the 16S rRNA gene to evaluate Korean doenjang and soybean pastes prepared by the Hun Chinese (SPHC) and Korean minority (SPKM) populations in Northeast China. In total, 63399 high-quality sequences were derived from 16 soybean paste samples collected in Northeast China. Each bacterial species-level taxon of SPHC, SPKM, and Korean doenjang was clustered separately. Each paste contained representative bacterial species that could be distinguished from each other: Bacillus subtilis in SPKM, Tetragenococcus halophilus in SPHC, and Enterococcus durans in Korean doenjang. This is the 1st massive sequencing-based study analyzing microbial communities in soybean pastes manufactured in Northeast China, compared to Korean doenjang. Our results clearly showed that each soybean paste contained unique microbial communities that varied depending on the manufacturing process and location. © 2017 Institute of Food Technologists®.

Dissecting the genetic make-up of North-East Sardinia using a large set of haploid and autosomal markers.

PubMed

Pardo, Luba M; Piras, Giovanna; Asproni, Rosanna; van der Gaag, Kristiaan J; Gabbas, Attilio; Ruiz-Linares, Andres; de Knijff, Peter; Monne, Maria; Rizzu, Patrizia; Heutink, Peter

2012-09-01

Sardinia has been used for genetic studies because of its historical isolation, genetic homogeneity and increased prevalence of certain rare diseases. Controversy remains concerning the genetic substructure and the extent of genetic homogeneity, which has implications for the design of genome-wide association studies (GWAS). We revisited this issue by examining the genetic make-up of a sample from North-East Sardinia using a dense set of autosomal, Y chromosome and mitochondrial markers to assess the potential of the sample for GWAS and fine mapping studies. We genotyped individuals for 500K single-nucleotide polymorphisms, Y chromosome markers and sequenced the mitochondrial hypervariable (HVI-HVII) regions. We identified major haplogroups and compared these with other populations. We estimated linkage disequilibrium (LD) and haplotype diversity across autosomal markers, and compared these with other populations. Our results show that within Sardinia there is no major population substructure and thus it can be considered a genetically homogenous population. We did not find substantial differences in the extent of LD in Sardinians compared with other populations. However, we showed that at least 9% of genomic regions in Sardinians differed in LD structure, which is helpful for identifying functional variants using fine mapping. We concluded that Sardinia is a powerful setting for genetic studies including GWAS and other mapping approaches.

Biological properties of purified recombinant HCV particles with an epitope-tagged envelope

DOE Office of Scientific and Technical Information (OSTI.GOV)

Takahashi, Hitoshi; Akazawa, Daisuke; Toray Industries, Inc., Kanagawa

2010-05-14

To establish a simple system for purification of recombinant infectious hepatitis C virus (HCV) particles, we designed a chimeric J6/JFH-1 virus with a FLAG (FL)-epitope-tagged sequence at the N-terminal region of the E2 hypervariable region-1 (HVR1) gene (J6/JFH-1/1FL). We found that introduction of an adaptive mutation at the potential N-glycosylation site (E2N151K) leads to efficient production of the chimeric virus. This finding suggests the involvement of glycosylation at Asn within the envelope protein(s) in HCV morphogenesis. To further analyze the biological properties of the purified recombinant HCV particles, we developed a strategy for large-scale production and purification of recombinant J6/JFH-1/1FL/E2N151K.more » Infectious particles were purified from the culture medium of J6/JFH-1/1FL/E2N151K-infected Huh-7 cells using anti-FLAG affinity chromatography in combination with ultrafiltration. Electron microscopy of the purified particles using negative staining showed spherical particle structures with a diameter of 40-60 nm and spike-like projections. Purified HCV particle-immunization induced both an anti-E2 and an anti-FLAG antibody response in immunized mice. This strategy may contribute to future detailed analysis of HCV particle structure and to HCV vaccine development.« less

[Identification of new conserved and variable regions in the 16S rRNA gene of acetic acid bacteria and acetobacteraceae family].

PubMed

Chakravorty, S; Sarkar, S; Gachhui, R

2015-01-01

The Acetobacteraceae family of the class Alpha Proteobacteria is comprised of high sugar and acid tolerant bacteria. The Acetic Acid Bacteria are the economically most significant group of this family because of its association with food products like vinegar, wine etc. Acetobacteraceae are often hard to culture in laboratory conditions and they also maintain very low abundances in their natural habitats. Thus identification of the organisms in such environments is greatly dependent on modern tools of molecular biology which require a thorough knowledge of specific conserved gene sequences that may act as primers and or probes. Moreover unconserved domains in genes also become markers for differentiating closely related genera. In bacteria, the 16S rRNA gene is an ideal candidate for such conserved and variable domains. In order to study the conserved and variable domains of the 16S rRNA gene of Acetic Acid Bacteria and the Acetobacteraceae family, sequences from publicly available databases were aligned and compared. Near complete sequences of the gene were also obtained from Kombucha tea biofilm, a known Acetobacteraceae family habitat, in order to corroborate the domains obtained from the alignment studies. The study indicated that the degree of conservation in the gene is significantly higher among the Acetic Acid Bacteria than the whole Acetobacteraceae family. Moreover it was also observed that the previously described hypervariable regions V1, V3, V5, V6 and V7 were more or less conserved in the family and the spans of the variable regions are quite distinct as well.

Identification of different lineages of measles virus strains circulating in Uttar Pradesh, North India.

PubMed

Shakya, Akhalesh Kumar; Shukla, Vibha; Maan, Harjeet Singh; Dhole, Tapan N

2012-10-16

Genetic analysis of measles viruses associated with recent cases and outbreaks has proven to bridge information gaps in routine outbreak investigations and has made a substantial contribution to measles control efforts by helping to identify the transmission pathways of the virus. The present study describes the genetic characterization of wild type measles viruses from Uttar Pradesh, India isolated between January 2008 and January 2011. In the study, 526 suspected measles cases from 15 outbreaks were investigated. Blood samples were collected from suspected measles outbreaks and tested for the presence of measles specific IgM; throat swab and urine samples were collected for virus isolation and RT-PCR. Genotyping of circulating measles viruses in Uttar Pradesh was performed by sequencing a 450-bp region encompassing the nucleoprotein hypervariable region and phylogenetic analysis. Based on serological results, all the outbreaks were confirmed as measles. Thirty eight strains were obtained. Genetic analysis of circulating measles strains (n = 38) in Uttar Pradesh from 235 cases of laboratory-confirmed cases from 526 suspected measles cases between 2008 and 2011 showed that all viruses responsible for outbreaks were within clade D and all were genotype D8.Analysis of this region showed that it is highly divergent (up to 3.4% divergence in the nucleotide sequence and 4.1% divergence in the amino acid sequence between most distant strains). Considerable genetic heterogeneity was observed in the MV genotype D8 viruses in North India and underscores the need for continued surveillance and in particular increases in vaccination levels to decrease morbidity and mortality attributable to measles.

The broad-band SEDs of four `hypervariable' AGN

NASA Astrophysics Data System (ADS)

Collinson, James S.; Ward, Martin J.; Lawrence, Andy; Bruce, Alastair; MacLeod, Chelsea L.; Elvis, Martin; Gezari, Suvi; Marshall, Philip J.; Done, Chris

2018-03-01

We present an optical-to-X-ray spectral analysis of four `hypervariable' AGN (HVAs) discovered by comparing Pan-STARRS data to that from the Sloan Digital Sky Survey over a 10 yr baseline (Lawrence et al.). There is some evidence that these objects are X-ray loud for their corresponding UV luminosities, but given that we measured them in a historic high state, it is not clear whether to take the high state or low state as typical of the properties of these HVAs. We estimate black hole masses based on Mg II and H α emission line profiles, and either the high- or low-state luminosities, finding mass ranges log (MBH/M⊙) = 8.2-8.8 and log (MBH/M⊙) = 7.9-8.3, respectively. We then fit energy-conserving models to the spectral energy distributions (SEDs), obtaining strong constraints on the bolometric luminosity and αOX. We compare the SED properties with a larger, X-ray selected AGN sample for both of these scenarios, and observe distinct groupings in spectral shape versus luminosity parameter space. In general, the SED properties are closer to normal if we assume that the low state is representative. This supports the idea that the large slow outbursts may be due to extrinsic effects (for example microlensing) as opposed to accretion rate changes, but a larger sample of HVAs is needed to be confident of this conclusion.

Subsurface metagenomes uncover a vast repertoire of hypervariable proteins encoded by genetic elements in uncultivated organisms and viruses

NASA Astrophysics Data System (ADS)

Paul, B. G.; Burstein, D.; Castelle, C. J.; Banfield, J. F.; Valentine, D. L.; Miller, J. F.; Ghosh, P.; Handa, S.; Arambula, D.; Czornyj, E.; Thomas, B. C.

2016-12-01

Uncultivated microorganisms primarily account for the remarkable diversity harbored in subsurface environments and represent an expansive subset of the current Tree of Life. Recent metagenomic efforts to investigate subsurface biomes have unveiled an array of bacterial and archaeal candidate phyla, whose members have minimal genomes and an apparent host-dependent existence. Still, little is known about the adaptive strategies that mediate host interactions in these organisms or their viruses. Genomic features known as diversity-generating retroelements (DGRs), which guide variability into targeted genes, were recently discovered in two single-cell genomes of uncultivated nanoarchaea, and independently in the genome of a marine virus from methane seep sediments. These prodigious drivers of protein hypervariability were first identified as the key force behind phage tail fiber diversification for binding different host receptors. Since their discovery, approximately 500 new DGRs have been found across a wide range of bacterial genomes representing various niches. We identified an unexpected 1136 distinct diversifiers from a single groundwater environment in reconstructed microbial genomes and genome fragments. The newly detected DGRs - predominantly linked to members of the candidate phyla radiation (CPR) - appear to target genes associated with cell-cell attachment, signaling, and transcription regulation. These findings suggest that targeted protein diversification may have an important role in regulating symbiotic or parasitic associations in groundwater microbiomes.

Short hypervariable microhaplotypes: A novel set of very short high discriminating power loci without stutter artefacts.

PubMed

van der Gaag, Kristiaan J; de Leeuw, Rick H; Laros, Jeroen F J; den Dunnen, Johan T; de Knijff, Peter

2018-07-01

Since two decades, short tandem repeats (STRs) are the preferred markers for human identification, routinely analysed by fragment length analysis. Here we present a novel set of short hypervariable autosomal microhaplotypes (MH) that have four or more SNPs in a span of less than 70 nucleotides (nt). These MHs display a discriminating power approaching that of STRs and provide a powerful alternative for the analysis;1;is of forensic samples that are problematic when the STR fragment size range exceeds the integrity range of severely degraded DNA or when multiple donors contribute to an evidentiary stain and STR stutter artefacts complicate profile interpretation. MH typing was developed using the power of massively parallel sequencing (MPS) enabling new powerful, fast and efficient SNP-based approaches. MH candidates were obtained from queries in data of the 1000 Genomes, and Genome of the Netherlands (GoNL) projects. Wet-lab analysis of 276 globally dispersed samples and 97 samples of nine large CEPH families assisted locus selection and corroboration of informative value. We infer that MHs represent an alternative marker type with good discriminating power per locus (allowing the use of a limited number of loci), small amplicon sizes and absence of stutter artefacts that can be especially helpful when unbalanced mixed samples are submitted for human identification. Copyright © 2018 The Authors. Published by Elsevier B.V. All rights reserved.

Bacterial RecA Protein Promotes Adenoviral Recombination during In Vitro Infection

PubMed Central

Lee, Jeong Yoon; Lee, Ji Sun; Materne, Emma C.; Rajala, Rahul; Ismail, Ashrafali M.; Seto, Donald; Dyer, David W.

2018-01-01

ABSTRACT Adenovirus infections in humans are common and sometimes lethal. Adenovirus-derived vectors are also commonly chosen for gene therapy in human clinical trials. We have shown in previous work that homologous recombination between adenoviral genomes of human adenovirus species D (HAdV-D), the largest and fastest growing HAdV species, is responsible for the rapid evolution of this species. Because adenovirus infection initiates in mucosal epithelia, particularly at the gastrointestinal, respiratory, genitourinary, and ocular surfaces, we sought to determine a possible role for mucosal microbiota in adenovirus genome diversity. By analysis of known recombination hot spots across 38 human adenovirus genomes in species D (HAdV-D), we identified nucleotide sequence motifs similar to bacterial Chi sequences, which facilitate homologous recombination in the presence of bacterial Rec enzymes. These motifs, referred to here as ChiAD, were identified immediately 5′ to the sequence encoding penton base hypervariable loop 2, which expresses the arginine-glycine-aspartate moiety critical to adenoviral cellular entry. Coinfection with two HAdV-Ds in the presence of an Escherichia coli lysate increased recombination; this was blocked in a RecA mutant strain, E. coli DH5α, or upon RecA depletion. Recombination increased in the presence of E. coli lysate despite a general reduction in viral replication. RecA colocalized with viral DNA in HAdV-D-infected cell nuclei and was shown to bind specifically to ChiAD sequences. These results indicate that adenoviruses may repurpose bacterial recombination machinery, a sharing of evolutionary mechanisms across a diverse microbiota, and unique example of viral commensalism. IMPORTANCE Adenoviruses are common human mucosal pathogens of the gastrointestinal, respiratory, and genitourinary tracts and ocular surface. Here, we report finding Chi-like sequences in adenovirus recombination hot spots. Adenovirus coinfection in the presence of bacterial RecA protein facilitated homologous recombination between viruses. Genetic recombination led to evolution of an important external feature on the adenoviral capsid, namely, the penton base protein hypervariable loop 2, which contains the arginine-glycine-aspartic acid motif critical to viral internalization. We speculate that free Rec proteins present in gastrointestinal secretions upon bacterial cell death facilitate the evolution of human adenoviruses through homologous recombination, an example of viral commensalism and the complexity of virus-host interactions, including regional microbiota. PMID:29925671

Fast discovery and visualization of conserved regions in DNA sequences using quasi-alignment

PubMed Central

2013-01-01

Background Next Generation Sequencing techniques are producing enormous amounts of biological sequence data and analysis becomes a major computational problem. Currently, most analysis, especially the identification of conserved regions, relies heavily on Multiple Sequence Alignment and its various heuristics such as progressive alignment, whose run time grows with the square of the number and the length of the aligned sequences and requires significant computational resources. In this work, we present a method to efficiently discover regions of high similarity across multiple sequences without performing expensive sequence alignment. The method is based on approximating edit distance between segments of sequences using p-mer frequency counts. Then, efficient high-throughput data stream clustering is used to group highly similar segments into so called quasi-alignments. Quasi-alignments have numerous applications such as identifying species and their taxonomic class from sequences, comparing sequences for similarities, and, as in this paper, discovering conserved regions across related sequences. Results In this paper, we show that quasi-alignments can be used to discover highly similar segments across multiple sequences from related or different genomes efficiently and accurately. Experiments on a large number of unaligned 16S rRNA sequences obtained from the Greengenes database show that the method is able to identify conserved regions which agree with known hypervariable regions in 16S rRNA. Furthermore, the experiments show that the proposed method scales well for large data sets with a run time that grows only linearly with the number and length of sequences, whereas for existing multiple sequence alignment heuristics the run time grows super-linearly. Conclusion Quasi-alignment-based algorithms can detect highly similar regions and conserved areas across multiple sequences. Since the run time is linear and the sequences are converted into a compact clustering model, we are able to identify conserved regions fast or even interactively using a standard PC. Our method has many potential applications such as finding characteristic signature sequences for families of organisms and studying conserved and variable regions in, for example, 16S rRNA. PMID:24564200

Fast discovery and visualization of conserved regions in DNA sequences using quasi-alignment.

PubMed

Nagar, Anurag; Hahsler, Michael

2013-01-01

Next Generation Sequencing techniques are producing enormous amounts of biological sequence data and analysis becomes a major computational problem. Currently, most analysis, especially the identification of conserved regions, relies heavily on Multiple Sequence Alignment and its various heuristics such as progressive alignment, whose run time grows with the square of the number and the length of the aligned sequences and requires significant computational resources. In this work, we present a method to efficiently discover regions of high similarity across multiple sequences without performing expensive sequence alignment. The method is based on approximating edit distance between segments of sequences using p-mer frequency counts. Then, efficient high-throughput data stream clustering is used to group highly similar segments into so called quasi-alignments. Quasi-alignments have numerous applications such as identifying species and their taxonomic class from sequences, comparing sequences for similarities, and, as in this paper, discovering conserved regions across related sequences. In this paper, we show that quasi-alignments can be used to discover highly similar segments across multiple sequences from related or different genomes efficiently and accurately. Experiments on a large number of unaligned 16S rRNA sequences obtained from the Greengenes database show that the method is able to identify conserved regions which agree with known hypervariable regions in 16S rRNA. Furthermore, the experiments show that the proposed method scales well for large data sets with a run time that grows only linearly with the number and length of sequences, whereas for existing multiple sequence alignment heuristics the run time grows super-linearly. Quasi-alignment-based algorithms can detect highly similar regions and conserved areas across multiple sequences. Since the run time is linear and the sequences are converted into a compact clustering model, we are able to identify conserved regions fast or even interactively using a standard PC. Our method has many potential applications such as finding characteristic signature sequences for families of organisms and studying conserved and variable regions in, for example, 16S rRNA.

Characterization and Exploitation of CRISPR Loci in Bifidobacterium longum

PubMed Central

Hidalgo-Cantabrana, Claudio; Crawley, Alexandra B.; Sanchez, Borja; Barrangou, Rodolphe

2017-01-01

Diverse CRISPR-Cas systems provide adaptive immunity in many bacteria and most archaea, via a DNA-encoded, RNA-mediated, nucleic-acid targeting mechanism. Over time, CRISPR loci expand via iterative uptake of invasive DNA sequences into the CRISPR array during the adaptation process. These genetic vaccination cards thus provide insights into the exposure of strains to phages and plasmids in space and time, revealing the historical predatory exposure of a strain. These genetic loci thus constitute a unique basis for genotyping of strains, with potential of resolution at the strain-level. Here, we investigate the occurrence and diversity of CRISPR-Cas systems in the genomes of various Bifidobacterium longum strains across three sub-species. Specifically, we analyzed the genomic content of 66 genomes belonging to B. longum subsp. longum, B. longum subsp. infantis and B. longum subsp. suis, and identified 25 strains that carry 29 total CRISPR-Cas systems. We identify various Type I and Type II CRISPR-Cas systems that are widespread in this species, notably I-C, I-E, and II-C. Noteworthy, Type I-C systems showed extended CRISPR arrays, with extensive spacer diversity. We show how these hypervariable loci can be used to gain insights into strain origin, evolution and phylogeny, and can provide discriminatory sequences to distinguish even clonal isolates. By investigating CRISPR spacer sequences, we reveal their origin and implicate phages and prophages as drivers of CRISPR immunity expansion in this species, with redundant targeting of select prophages. Analysis of CRISPR spacer origin also revealed novel PAM sequences. Our results suggest that CRISPR-Cas immune systems are instrumental in mounting diversified viral resistance in B. longum, and show that these sequences are useful for typing across three subspecies. PMID:29033911

Characterization and Exploitation of CRISPR Loci in Bifidobacterium longum.

PubMed

Hidalgo-Cantabrana, Claudio; Crawley, Alexandra B; Sanchez, Borja; Barrangou, Rodolphe

2017-01-01

Diverse CRISPR-Cas systems provide adaptive immunity in many bacteria and most archaea, via a DNA-encoded, RNA-mediated, nucleic-acid targeting mechanism. Over time, CRISPR loci expand via iterative uptake of invasive DNA sequences into the CRISPR array during the adaptation process. These genetic vaccination cards thus provide insights into the exposure of strains to phages and plasmids in space and time, revealing the historical predatory exposure of a strain. These genetic loci thus constitute a unique basis for genotyping of strains, with potential of resolution at the strain-level. Here, we investigate the occurrence and diversity of CRISPR-Cas systems in the genomes of various Bifidobacterium longum strains across three sub-species. Specifically, we analyzed the genomic content of 66 genomes belonging to B. longum subsp. longum, B. longum subsp. infantis and B. longum subsp. suis , and identified 25 strains that carry 29 total CRISPR-Cas systems. We identify various Type I and Type II CRISPR-Cas systems that are widespread in this species, notably I-C, I-E, and II-C. Noteworthy, Type I-C systems showed extended CRISPR arrays, with extensive spacer diversity. We show how these hypervariable loci can be used to gain insights into strain origin, evolution and phylogeny, and can provide discriminatory sequences to distinguish even clonal isolates. By investigating CRISPR spacer sequences, we reveal their origin and implicate phages and prophages as drivers of CRISPR immunity expansion in this species, with redundant targeting of select prophages. Analysis of CRISPR spacer origin also revealed novel PAM sequences. Our results suggest that CRISPR-Cas immune systems are instrumental in mounting diversified viral resistance in B. longum , and show that these sequences are useful for typing across three subspecies.

Domain Organization and Evolution of the Highly Divergent 5′ Coding Region of Genomes of Arteriviruses, Including the Novel Possum Nidovirus

PubMed Central

Gulyaeva, Anastasia; Hoogendoorn, Erik; Giles, Julia; Samborskiy, Dmitry

2017-01-01

ABSTRACT In five experimentally characterized arterivirus species, the 5′-end genome coding region encodes the most divergent nonstructural proteins (nsp's), nsp1 and nsp2, which include papain-like proteases (PLPs) and other poorly characterized domains. These are involved in regulation of transcription, polyprotein processing, and virus-host interaction. Here we present results of a bioinformatics analysis of this region of 14 arterivirus species, including that of the most distantly related virus, wobbly possum disease virus (WPDV), determined by a modified 5′ rapid amplification of cDNA ends (RACE) protocol. By combining profile-profile comparisons and phylogeny reconstruction, we identified an association of the four distinct domain layouts of nsp1-nsp2 with major phylogenetic lineages, implicating domain gain, including duplication, and loss in the early nsp1 evolution. Specifically, WPDV encodes highly divergent homologs of PLP1a, PLP1b, PLP1c, and PLP2, with PLP1a lacking the catalytic Cys residue, but does not encode nsp1 Zn finger (ZnF) and “nuclease” domains, which are conserved in other arteriviruses. Unexpectedly, our analysis revealed that the only catalytically active nsp1 PLP of equine arteritis virus (EAV), known as PLP1b, is most similar to PLP1c and thus is likely to be a PLP1b paralog. In all non-WPDV arteriviruses, PLP1b/c and PLP1a show contrasting patterns of conservation, with the N- and C-terminal subdomains, respectively, being enriched with conserved residues, which is indicative of different functional specializations. The least conserved domain of nsp2, the hypervariable region (HVR), has its size varied 5-fold and includes up to four copies of a novel PxPxPR motif that is potentially recognized by SH3 domain-containing proteins. Apparently, only EAV lacks the signal that directs −2 ribosomal frameshifting in the nsp2 coding region. IMPORTANCE Arteriviruses comprise a family of mammalian enveloped positive-strand RNA viruses that include some of the most economically important pathogens of swine. Most of our knowledge about this family has been obtained through characterization of viruses from five species: Equine arteritis virus, Simian hemorrhagic fever virus, Lactate dehydrogenase-elevating virus, Porcine respiratory and reproductive syndrome virus 1, and Porcine respiratory and reproductive syndrome virus 2. Here we present the results of comparative genomics analyses of viruses from all known 14 arterivirus species, including the most distantly related virus, WPDV, whose genome sequence was completed in this study. Our analysis focused on the multifunctional 5′-end genome coding region that encodes multidomain nonstructural proteins 1 and 2. Using diverse bioinformatics techniques, we identified many patterns of evolutionary conservation that are specific to members of distinct arterivirus species, both characterized and novel, or their groups. They are likely associated with structural and functional determinants important for virus replication and virus-host interaction. PMID:28053107

The molecular identification of Streptococcus equi subsp. equi strains isolated within New Zealand.

PubMed

Patty, O A; Cursons, R T M

2014-03-01

To identify Streptococcus equi subsp. equi (S. equi) by PCR analysis and obtain isolates by culture, in order to investigate the strains of S. equi infecting horses within New Zealand. A diagnostic PCR, based on the amplification of the seeI gene for S. equi, was used on 168 samples submitted from horses with and without clinical signs of strangles. Samples were also processed and cultured on selective media for the isolation of β-haemolytic colonies. In addition, the hypervariable region of the seM gene of S. equi was amplified and then sequenced for strain typing purposes. Of the 168 samples, 35 tested positive for S. equi using PCR. Thirty-two confirmed samples were from horses with a clinical diagnosis of strangles and three were from horses where clinical information was unavailable. Only 22/35 (63%) confirmed S. equi samples were successfully isolated following culture. Strain typing demonstrated that two novel seM alleles of S. equi were found in New Zealand with SeM-99 strains being restricted to the North Island while SeM-100 strains were found in both North and South Islands. The application of PCR for the laboratory confirmation of strangles allowed for a rapid and sensitive identification of S. equi. Moreover, seM typing revealed that within the samples examined two strains of S. equi co-circulated within the North Island of New Zealand but only one strain in the South Island. PCR reduces the time required to obtain laboratory confirmation of strangles compared with culture methods. It also has greater sensitivity in detecting S. equi infections, which is of particular importance in the detection of carrier animals which normally shed low numbers of bacteria. Additionally, seM molecular typing can differentiate between bacterial strains, assisting in the monitoring of local strains of S. equi subsp. equi causing disease.

Recovering mitochondrial DNA lineages of extinct Amerindian nations in extant homopatric Brazilian populations.

PubMed

Gonçalves, Vanessa F; Parra, Flavia C; Gonçalves-Dornelas, Higgor; Rodrigues-Carvalho, Claudia; Silva, Hilton P; Pena, Sergio Dj

2010-12-01

Brazilian Amerindians have experienced a drastic population decrease in the past 500 years. Indeed, many native groups from eastern Brazil have vanished. However, their mitochondrial mtDNA haplotypes, still persist in Brazilians, at least 50 million of whom carry Amerindian mitochondrial lineages. Our objective was to test whether, by analyzing extant rural populations from regions anciently occupied by specific Amerindian groups, we could identify potentially authentic mitochondrial lineages, a strategy we have named 'homopatric targeting'. We studied 173 individuals from Queixadinha, a small village located in a territory previously occupied by the now extinct Botocudo Amerindian nation. Pedigree analysis revealed 74 unrelated matrilineages, which were screened for Amerindian mtDNA lineages by restriction fragment length polymorphism. A cosmopolitan control group was composed of 100 individuals from surrounding cities. All Amerindian lineages identified had their hypervariable segment HVSI sequenced, yielding 13 Amerindian haplotypes in Queixadinha, nine of which were not present in available databanks or in the literature. Among these haplotypes, there was a significant excess of haplogroup C (70%) and absence of haplogroup A lineages, which were the most common in the control group. The novelty of the haplotypes and the excess of the C haplogroup suggested that we might indeed have identified Botocudo lineages. To validate our strategy, we studied teeth extracted from 14 ancient skulls of Botocudo Amerindians from the collection of the National Museum of Rio de Janeiro. We recovered mtDNA sequences from all the teeth, identifying only six different haplotypes (a low haplotypic diversity of 0.8352 ± 0.0617), one of which was present among the lineages observed in the extant individuals studied. These findings validate the technique of homopatric targeting as a useful new strategy to study the peopling and colonization of the New World, especially when direct analysis of genetic material is not possible.

Plaque bacterial microbiome diversity in children younger than 30 months with or without caries prior to eruption of second primary molars.

PubMed

Xu, He; Hao, Wenjing; Zhou, Qiong; Wang, Wenhong; Xia, Zhongkui; Liu, Chuan; Chen, Xiaochi; Qin, Man; Chen, Feng

2014-01-01

Our primary objective is to phylogenetically characterize the supragingival plaque bacterial microbiome of children prior to eruption of second primary molars by pyrosequencing method for studying etiology of early childhood caries. Supragingival plaque samples were collected from 10 caries children and 9 caries-free children. Plaque DNA was extracted, used to generate DNA amplicons of the V1-V3 hypervariable region of the bacterial 16S rRNA gene, and subjected to 454-pyrosequencing. On average, over 22,000 sequences per sample were generated. High bacterial diversity was noted in the plaque of children with caries [170 operational taxonomical units (OTU) at 3% divergence] and caries-free children (201 OTU at 3% divergence) with no significant difference. A total of 8 phyla, 15 classes, 21 orders, 30 families, 41 genera and 99 species were represented. In addition, five predominant phyla (Firmicute, Fusobacteria, Proteobacteria, Bacteroidetes and Actinobacteria) and seven genera (Leptotrichia, Streptococcus, Actinomyces, Prevotella, Porphyromonas, Neisseria, and Veillonella) constituted a majority of contents of the total microbiota, independent of the presence or absence of caries. Principal Component Analysis (PCA) presented that caries-related genera included Streptococcus and Veillonella; while Leptotrichia, Selenomonas, Fusobacterium, Capnocytophaga and Porphyromonas were more related to the caries-free samples. Neisseria and Prevotella presented approximately in between. In both groups, the degree of shared organism lineages (as defined by species-level OTUs) among individual supragingival plaque microbiomes was minimal. Our study represented for the first time using pyrosequencing to elucidate and monitor supragingival plaque bacterial diversity at such young age with second primary molar unerrupted. Distinctions were revealed between caries and caries-free microbiomes in terms of microbial community structure. We observed differences in abundance for several microbial groups between the caries and caries-free host populations, which were consistent with the ecological plaque hypothesis. Our approach and findings could be extended to correlating microbiomic changes after occlusion establishment and caries treatment.

Site-directed mutagenesis, in vivo electroporation and mass spectrometry in search for determinants of the subcellular targeting of Rab7b paralogue in the model eukaryote Paramecium octaurelia.

PubMed

Wyroba, E; Kwaśniak, P; Miller, K; Kobyłecki, K; Osińska, M

2016-04-11

Protein products of the paralogous genes resulting from the whole genome duplication may acquire new function. The role of post-translational modifications (PTM) in proper targeting of Paramecium Rab7b paralogue - distinct from that of Rab7a directly involved in phagocytosis - was studied using point mutagenesis, proteomic analysis and double immunofluorescence after in vivo electroporation of the mutagenized protein. Here we show that substitution of Thr200 by Ala200 resulted in diminished incorporation of [P32] by 37.4% and of 32 [C14-]UDP-glucose by 24%, respectively, into recombinant Rab7b_200 in comparison to the non-mutagenized control. Double confocal imaging revealed that Rab7b_200 was mistargeted upon electroporation into living cells contrary to non- mutagenized recombinant Rab7b correctly incorporated in the cytostome area. We identified the peptide ion at m/z=677.63+ characteristic for the glycan group attached to Thr200 in Rab7b using nano LC-MS/MS and comparing the peptide map of this protein with that after deglycosylation with the mixture of five enzymes of different specificity. Based on the mass of this peptide ion and quantitative radioactive assays with [P32]and  [C14-]UDP- glucose, the suggested composition of the adduct attached to Thr200 might be (Hex)1(HexNAc)1(Phos)3 or (HexNAc)1 (Deoxyhexose)1 (Phos)1 (HexA)1. These data indicate that PTM of Thr200 located in the hypervariable C-region of Rab7b in Paramecium is crucial for the proper localization/function of this protein. Moreover, these proteins differ also in other PTM: the number of phosphorylated amino acids in Rab7b is much higher than in Rab7a.

De novo assembly and phasing of a Korean human genome.

PubMed

Seo, Jeong-Sun; Rhie, Arang; Kim, Junsoo; Lee, Sangjin; Sohn, Min-Hwan; Kim, Chang-Uk; Hastie, Alex; Cao, Han; Yun, Ji-Young; Kim, Jihye; Kuk, Junho; Park, Gun Hwa; Kim, Juhyeok; Ryu, Hanna; Kim, Jongbum; Roh, Mira; Baek, Jeonghun; Hunkapiller, Michael W; Korlach, Jonas; Shin, Jong-Yeon; Kim, Changhoon

2016-10-13

Advances in genome assembly and phasing provide an opportunity to investigate the diploid architecture of the human genome and reveal the full range of structural variation across population groups. Here we report the de novo assembly and haplotype phasing of the Korean individual AK1 (ref. 1) using single-molecule real-time sequencing, next-generation mapping, microfluidics-based linked reads, and bacterial artificial chromosome (BAC) sequencing approaches. Single-molecule sequencing coupled with next-generation mapping generated a highly contiguous assembly, with a contig N50 size of 17.9 Mb and a scaffold N50 size of 44.8 Mb, resolving 8 chromosomal arms into single scaffolds. The de novo assembly, along with local assemblies and spanning long reads, closes 105 and extends into 72 out of 190 euchromatic gaps in the reference genome, adding 1.03 Mb of previously intractable sequence. High concordance between the assembly and paired-end sequences from 62,758 BAC clones provides strong support for the robustness of the assembly. We identify 18,210 structural variants by direct comparison of the assembly with the human reference, identifying thousands of breakpoints that, to our knowledge, have not been reported before. Many of the insertions are reflected in the transcriptome and are shared across the Asian population. We performed haplotype phasing of the assembly with short reads, long reads and linked reads from whole-genome sequencing and with short reads from 31,719 BAC clones, thereby achieving phased blocks with an N50 size of 11.6 Mb. Haplotigs assembled from single-molecule real-time reads assigned to haplotypes on phased blocks covered 89% of genes. The haplotigs accurately characterized the hypervariable major histocompatability complex region as well as demonstrating allele configuration in clinically relevant genes such as CYP2D6. This work presents the most contiguous diploid human genome assembly so far, with extensive investigation of unreported and Asian-specific structural variants, and high-quality haplotyping of clinically relevant alleles for precision medicine.

Recovering mitochondrial DNA lineages of extinct Amerindian nations in extant homopatric Brazilian populations

PubMed Central

2010-01-01

Background Brazilian Amerindians have experienced a drastic population decrease in the past 500 years. Indeed, many native groups from eastern Brazil have vanished. However, their mitochondrial mtDNA haplotypes, still persist in Brazilians, at least 50 million of whom carry Amerindian mitochondrial lineages. Our objective was to test whether, by analyzing extant rural populations from regions anciently occupied by specific Amerindian groups, we could identify potentially authentic mitochondrial lineages, a strategy we have named 'homopatric targeting'. Results We studied 173 individuals from Queixadinha, a small village located in a territory previously occupied by the now extinct Botocudo Amerindian nation. Pedigree analysis revealed 74 unrelated matrilineages, which were screened for Amerindian mtDNA lineages by restriction fragment length polymorphism. A cosmopolitan control group was composed of 100 individuals from surrounding cities. All Amerindian lineages identified had their hypervariable segment HVSI sequenced, yielding 13 Amerindian haplotypes in Queixadinha, nine of which were not present in available databanks or in the literature. Among these haplotypes, there was a significant excess of haplogroup C (70%) and absence of haplogroup A lineages, which were the most common in the control group. The novelty of the haplotypes and the excess of the C haplogroup suggested that we might indeed have identified Botocudo lineages. To validate our strategy, we studied teeth extracted from 14 ancient skulls of Botocudo Amerindians from the collection of the National Museum of Rio de Janeiro. We recovered mtDNA sequences from all the teeth, identifying only six different haplotypes (a low haplotypic diversity of 0.8352 ± 0.0617), one of which was present among the lineages observed in the extant individuals studied. Conclusions These findings validate the technique of homopatric targeting as a useful new strategy to study the peopling and colonization of the New World, especially when direct analysis of genetic material is not possible. PMID:21122100

The Features of Fecal and Ileal Mucosa-Associated Microbiota in Dairy Calves during Early Infection with Mycobacterium avium Subspecies paratuberculosis

PubMed Central

Derakhshani, Hooman; De Buck, Jeroen; Mortier, Rienske; Barkema, Herman W.; Krause, Denis O.; Khafipour, Ehsan

2016-01-01

Current diagnostic tests for Johne's disease (JD), a chronic granulomatous inflammation of the gastrointestinal tract of ruminants caused by Mycobacterium avium subspecies paratuberculosis (MAP), lack the sensitivity to identify infected animals at early (asymptomatic) stages of the disease. The objective was to determine the pattern of MAP-associated dysbiosis of intestinal microbiota as a potential biomarker for early detection of infected cattle. To that end, genomic DNA was extracted from ileal mucosa and fecal samples collected from 28 MAP-positive and five control calves. High-throughput Illumina sequencing of the V4 hypervariable region of the 16S rRNA gene was used for community profiling of ileal mucosa-associated (MAM) or fecal microbiota. The PERMANOVA analysis of unweighted UniFrac distances revealed distinct clustering of ileal MAM (P = 0.049) and fecal microbiota (P = 0.068) in MAP-infected vs. control cattle. Microbiota profile of MAP-infected animals was further investigated by linear discriminant analysis effective size (LEfSe); several bacterial taxa within the phylum Proteobacteria were overrepresented in ileal MAM of control calves. Moreover, based on reconstructed metagenomes (PICRUSt) of ileal MAM, functional pathways associated with MAP infection were inferred. Enrichment of lysine and histidine metabolism pathways, and underrepresentation of glutathione metabolism and leucine and isoleucine degradation pathways in MAP-infected calves suggested potential contributions of ileal MAM in development of intestinal inflammation. Finally, simultaneous overrepresentation of families Planococcaceae and Paraprevotellaceae, as well as underrepresentation of genera Faecalibacterium and Akkermansia in the fecal microbiota of infected cattle, served as potential biomarker for identifying infected cattle during subclinical stages of JD. Collectively, based on compositional and functional shifts in intestinal microbiota of infected cattle, we inferred that this dynamic network of microorganisms had an active role in intestinal homeostasis. PMID:27065983

Characterization of the core rumen microbiome in cattle during transition from forage to concentrate as well as during and after an acidotic challenge.

PubMed

Petri, Renee M; Schwaiger, Tyler; Penner, Greg B; Beauchemin, Karen A; Forster, Robert J; McKinnon, John J; McAllister, Tim A

2013-01-01

This study investigated the effect of diet and host on the rumen bacterial microbiome and the impact of an acidotic challenge on its composition. Using parallel pyrosequencing of the V3 hypervariable region of 16S rRNA gene, solid and liquid associated bacterial communities of 8 heifers were profiled. Heifers were exclusively fed forage, before being transitioned to a concentrate diet, subjected to an acidotic challenge and allowed to recover. Samples of rumen digesta were collected when heifers were fed forage, mixed forage, high grain, during challenge (4 h and 12 h) and recovery. A total of 560,994 high-quality bacterial sequences were obtained from the solid and liquid digesta. Using cluster analysis, prominent bacterial populations differed (P≤0.10) in solid and liquid fractions between forage and grain diets. Differences among hosts and diets were not revealed by DGGE, but real time qPCR showed that several bacteria taxon were impacted by changes in diet, with the exception of Streptococcus bovis. Analysis of the core rumen microbiome identified 32 OTU's representing 10 distinct bacterial taxa including Bacteroidetes (32.8%), Firmicutes (43.2%) and Proteobacteria (14.3%). Diversity of OTUs was highest with forage with 38 unique OTUs identified as compared to only 11 with the high grain diet. Comparison of the microbial profiles of clincial vs. subclinical acidotic heifers found a increases in the relative abundances of Acetitomaculum, Lactobacillus, Prevotella, and Streptococcus. Increases in Streptococcus and Lactobacillus likely reflect the tolerance of these species to low pH and their ability to proliferate on surplus fermentable carbohydrate. The acetogen, Acetitomaculum may thereforeplay a role in the conversion of lactate to acetate in acidotic animals. Further profiling of the bacterial populations associated with subclinical and clinical acidosis could establish a microbial fingerprint for these disorders and provide insight into whether there are causative microbial populations that could potentially be therapeutically manipulated.

Next-generation sequencing sheds light on the natural history of hepatitis C infection in patients who fail treatment.

PubMed

Abdelrahman, Tamer; Hughes, Joseph; Main, Janice; McLauchlan, John; Thursz, Mark; Thomson, Emma

2015-01-01

High rates of sexually transmitted infection and reinfection with hepatitis C virus (HCV) have recently been reported in human immunodeficiency virus (HIV)-infected men who have sex with men and reinfection has also been described in monoinfected injecting drug users. The diagnosis of reinfection has traditionally been based on direct Sanger sequencing of samples pre- and posttreatment, but not on more sensitive deep sequencing techniques. We studied viral quasispecies dynamics in patients who failed standard of care therapy in a high-risk HIV-infected cohort of patients with early HCV infection to determine whether treatment failure was associated with reinfection or recrudescence of preexisting infection. Paired sequences (pre- and posttreatment) were analyzed. The HCV E2 hypervariable region-1 was amplified using nested reverse-transcription polymerase chain reaction (RT-PCR) with indexed genotype-specific primers and the same products were sequenced using both Sanger and 454 pyrosequencing approaches. Of 99 HIV-infected patients with acute HCV treated with 24-48 weeks of pegylated interferon alpha and ribavirin, 15 failed to achieve a sustained virological response (six relapsed, six had a null response, and three had a partial response). Using direct sequencing, 10/15 patients (66%) had evidence of a previously undetected strain posttreatment; in many studies, this is interpreted as reinfection. However, pyrosequencing revealed that 15/15 (100%) of patients had evidence of persisting infection; 6/15 (40%) patients had evidence of a previously undetected variant present in the posttreatment sample in addition to a variant that was detected at baseline. This could represent superinfection or a limitation of the sensitivity of pyrosequencing. In this high-risk group, the emergence of new viral strains following treatment failure is most commonly associated with emerging dominance of preexisting minority variants rather than reinfection. Superinfection may occur in this cohort but reinfection is overestimated by Sanger sequencing. © 2014 The Authors. Hepatology published by Wiley on behalf of the American Association for the Study of Liver Diseases.

Genetic variation and population structure in Jamunapari goats using microsatellites, mitochondrial DNA, and milk protein genes.

PubMed

Rout, P K; Thangraj, K; Mandal, A; Roy, R

2012-01-01

Jamunapari, a dairy goat breed of India, has been gradually declining in numbers in its home tract over the years. We have analysed genetic variation and population history in Jamunapari goats based on 17 microsatellite loci, 2 milk protein loci, mitochondrial hypervariable region I (HVRI) sequencing, and three Y-chromosomal gene sequencing. We used the mitochondrial DNA (mtDNA) mismatch distribution, microsatellite data, and bottleneck tests to infer the population history and demography. The mean number of alleles per locus was 9.0 indicating that the allelic variation was high in all the loci and the mean heterozygosity was 0.769 at nuclear loci. Although the population size is smaller than 8,000 individuals, the amount of variability both in terms of allelic richness and gene diversity was high in all the microsatellite loci except ILST 005. The gene diversity and effective number of alleles at milk protein loci were higher than the 10 other Indian goat breeds that they were compared to. Mismatch analysis was carried out and the analysis revealed that the population curve was unimodal indicating the expansion of population. The genetic diversity of Y-chromosome genes was low in the present study. The observed mean M ratio in the population was above the critical significance value (Mc) and close to one indicating that it has maintained a slowly changing population size. The mode-shift test did not detect any distortion of allele frequency and the heterozygosity excess method showed that there was no significant departure from mutation-drift equilibrium detected in the population. However, the effects of genetic bottlenecks were observed in some loci due to decreased heterozygosity and lower level of M ratio. There were two observed genetic subdivisions in the population supporting the observations of farmers in different areas. This base line information on genetic diversity, bottleneck analysis, and mismatch analysis was obtained to assist the conservation decision and management of the breed.

Genetic Variation and Population Structure in Jamunapari Goats Using Microsatellites, Mitochondrial DNA, and Milk Protein Genes

PubMed Central

Rout, P. K.; Thangraj, K.; Mandal, A.; Roy, R.

2012-01-01

Jamunapari, a dairy goat breed of India, has been gradually declining in numbers in its home tract over the years. We have analysed genetic variation and population history in Jamunapari goats based on 17 microsatellite loci, 2 milk protein loci, mitochondrial hypervariable region I (HVRI) sequencing, and three Y-chromosomal gene sequencing. We used the mitochondrial DNA (mtDNA) mismatch distribution, microsatellite data, and bottleneck tests to infer the population history and demography. The mean number of alleles per locus was 9.0 indicating that the allelic variation was high in all the loci and the mean heterozygosity was 0.769 at nuclear loci. Although the population size is smaller than 8,000 individuals, the amount of variability both in terms of allelic richness and gene diversity was high in all the microsatellite loci except ILST 005. The gene diversity and effective number of alleles at milk protein loci were higher than the 10 other Indian goat breeds that they were compared to. Mismatch analysis was carried out and the analysis revealed that the population curve was unimodal indicating the expansion of population. The genetic diversity of Y-chromosome genes was low in the present study. The observed mean M ratio in the population was above the critical significance value (Mc) and close to one indicating that it has maintained a slowly changing population size. The mode-shift test did not detect any distortion of allele frequency and the heterozygosity excess method showed that there was no significant departure from mutation-drift equilibrium detected in the population. However, the effects of genetic bottlenecks were observed in some loci due to decreased heterozygosity and lower level of M ratio. There were two observed genetic subdivisions in the population supporting the observations of farmers in different areas. This base line information on genetic diversity, bottleneck analysis, and mismatch analysis was obtained to assist the conservation decision and management of the breed. PMID:22606053

Characterization of the Core Rumen Microbiome in Cattle during Transition from Forage to Concentrate as Well as during and after an Acidotic Challenge

PubMed Central

Petri, Renee M.; Schwaiger, Tyler; Penner, Greg B.; Beauchemin, Karen A.; Forster, Robert J.; McKinnon, John J.; McAllister, Tim A.

2013-01-01

This study investigated the effect of diet and host on the rumen bacterial microbiome and the impact of an acidotic challenge on its composition. Using parallel pyrosequencing of the V3 hypervariable region of 16S rRNA gene, solid and liquid associated bacterial communities of 8 heifers were profiled. Heifers were exclusively fed forage, before being transitioned to a concentrate diet, subjected to an acidotic challenge and allowed to recover. Samples of rumen digesta were collected when heifers were fed forage, mixed forage, high grain, during challenge (4 h and 12 h) and recovery. A total of 560,994 high-quality bacterial sequences were obtained from the solid and liquid digesta. Using cluster analysis, prominent bacterial populations differed (P≤0.10) in solid and liquid fractions between forage and grain diets. Differences among hosts and diets were not revealed by DGGE, but real time qPCR showed that several bacteria taxon were impacted by changes in diet, with the exception of Streptococcus bovis. Analysis of the core rumen microbiome identified 32 OTU's representing 10 distinct bacterial taxa including Bacteroidetes (32.8%), Firmicutes (43.2%) and Proteobacteria (14.3%). Diversity of OTUs was highest with forage with 38 unique OTUs identified as compared to only 11 with the high grain diet. Comparison of the microbial profiles of clincial vs. subclinical acidotic heifers found a increases in the relative abundances of Acetitomaculum, Lactobacillus, Prevotella, and Streptococcus. Increases in Streptococcus and Lactobacillus likely reflect the tolerance of these species to low pH and their ability to proliferate on surplus fermentable carbohydrate. The acetogen, Acetitomaculum may thereforeplay a role in the conversion of lactate to acetate in acidotic animals. Further profiling of the bacterial populations associated with subclinical and clinical acidosis could establish a microbial fingerprint for these disorders and provide insight into whether there are causative microbial populations that could potentially be therapeutically manipulated. PMID:24391765

Opc expression, LPS immunotype switch and pilin conversion contribute to serum resistance of unencapsulated meningococci.

PubMed

Hubert, Kerstin; Pawlik, Marie-Christin; Claus, Heike; Jarva, Hanna; Meri, Seppo; Vogel, Ulrich

2012-01-01

Neisseria meningitidis employs polysaccharides and outer membrane proteins to cope with human serum complement attack. To screen for factors influencing serum resistance, an assay was developed based on a colorimetric serum bactericidal assay. The screening used a genetically modified sequence type (ST)-41/44 clonal complex (cc) strain lacking LPS sialylation, polysaccharide capsule, the factor H binding protein (fHbp) and MutS, a protein of the DNA repair mechanism. After killing of >99.9% of the bacterial cells by serum treatment, the colorimetric assay was used to screen 1000 colonies, of which 35 showed enhanced serum resistance. Three mutant classes were identified. In the first class of mutants, enhanced expression of Opc was identified. Opc expression was associated with vitronectin binding and reduced membrane attack complex deposition confirming recent observations. Lipopolysaccharide (LPS) immunotype switch from immunotype L3 to L8/L1 by lgtA and lgtC phase variation represented the second class. Isogenic mutant analysis demonstrated that in ST-41/44 cc strains the L8/L1 immunotype was more serum resistant than the L3 immunotype. Consecutive analysis revealed that the immunotypes L8 and L1 were frequently observed in ST-41/44 cc isolates from both carriage and disease. Immunotype switch to L8/L1 is therefore suggested to contribute to the adaptive capacity of this meningococcal lineage. The third mutant class displayed a pilE allelic exchange associated with enhanced autoaggregation. The mutation of the C terminal hypervariable region D of PilE included a residue previously associated with increased pilus bundle formation. We suggest that autoaggregation reduced the surface area accessible to serum complement and protected from killing. The study highlights the ability of meningococci to adapt to environmental stress by phase variation and intrachromosomal recombination affecting subcapsular antigens.

Opc Expression, LPS Immunotype Switch and Pilin Conversion Contribute to Serum Resistance of Unencapsulated Meningococci

PubMed Central

Hubert, Kerstin; Pawlik, Marie-Christin; Claus, Heike; Jarva, Hanna; Meri, Seppo; Vogel, Ulrich

2012-01-01

Neisseria meningitidis employs polysaccharides and outer membrane proteins to cope with human serum complement attack. To screen for factors influencing serum resistance, an assay was developed based on a colorimetric serum bactericidal assay. The screening used a genetically modified sequence type (ST)-41/44 clonal complex (cc) strain lacking LPS sialylation, polysaccharide capsule, the factor H binding protein (fHbp) and MutS, a protein of the DNA repair mechanism. After killing of >99.9% of the bacterial cells by serum treatment, the colorimetric assay was used to screen 1000 colonies, of which 35 showed enhanced serum resistance. Three mutant classes were identified. In the first class of mutants, enhanced expression of Opc was identified. Opc expression was associated with vitronectin binding and reduced membrane attack complex deposition confirming recent observations. Lipopolysaccharide (LPS) immunotype switch from immunotype L3 to L8/L1 by lgtA and lgtC phase variation represented the second class. Isogenic mutant analysis demonstrated that in ST-41/44 cc strains the L8/L1 immunotype was more serum resistant than the L3 immunotype. Consecutive analysis revealed that the immunotypes L8 and L1 were frequently observed in ST-41/44 cc isolates from both carriage and disease. Immunotype switch to L8/L1 is therefore suggested to contribute to the adaptive capacity of this meningococcal lineage. The third mutant class displayed a pilE allelic exchange associated with enhanced autoaggregation. The mutation of the C terminal hypervariable region D of PilE included a residue previously associated with increased pilus bundle formation. We suggest that autoaggregation reduced the surface area accessible to serum complement and protected from killing. The study highlights the ability of meningococci to adapt to environmental stress by phase variation and intrachromosomal recombination affecting subcapsular antigens. PMID:23028802

High-throughput sequencing technology to reveal the composition and function of cecal microbiota in Dagu chicken.

PubMed

Xu, Yunhe; Yang, Huixin; Zhang, Lili; Su, Yuhong; Shi, Donghui; Xiao, Haidi; Tian, Yumin

2016-11-04

The chicken gut microbiota is an important and complicated ecosystem for the host. They play an important role in converting food into nutrient and energy. The coding capacity of microbiome vastly surpasses that of the host's genome, encoding biochemical pathways that the host has not developed. An optimal gut microbiota can increase agricultural productivity. This study aims to explore the composition and function of cecal microbiota in Dagu chicken under two feeding modes, free-range (outdoor, OD) and cage (indoor, ID) raising. Cecal samples were collected from 24 chickens across 4 groups (12-w OD, 12-w ID, 18-w OD, and 18-w ID). We performed high-throughput sequencing of the 16S rRNA genes V4 hypervariable regions to characterize the cecal microbiota of Dagu chicken and compare the difference of cecal microbiota between free-range and cage raising chickens. It was found that 34 special operational taxonomic units (OTUs) in OD groups and 4 special OTUs in ID groups. 24 phyla were shared by the 24 samples. Bacteroidetes was the most abundant phylum with the largest proportion, followed by Firmicutes and Proteobacteria. The OD groups showed a higher proportion of Bacteroidetes (>50 %) in cecum, but a lower Firmicutes/Bacteroidetes ratio in both 12-w old (0.42, 0.62) and 18-w old groups (0.37, 0.49) compared with the ID groups. Cecal microbiota in the OD groups have higher abundance of functions involved in amino acids and glycan metabolic pathway. The composition and function of cecal microbiota in Dagu chicken under two feeding modes, free-range and cage raising are different. The cage raising mode showed a lower proportion of Bacteroidetes in cecum, but a higher Firmicutes/Bacteroidetes ratio compared with free-range mode. Cecal microbiota in free-range mode have higher abundance of functions involved in amino acids and glycan metabolic pathway.

Independent studies using deep sequencing resolve the same set of core bacterial species dominating gut communities of honey bees.

PubMed

Sabree, Zakee L; Hansen, Allison K; Moran, Nancy A

2012-01-01

Starting in 2003, numerous studies using culture-independent methodologies to characterize the gut microbiota of honey bees have retrieved a consistent and distinctive set of eight bacterial species, based on near identity of the 16S rRNA gene sequences. A recent study [Mattila HR, Rios D, Walker-Sperling VE, Roeselers G, Newton ILG (2012) Characterization of the active microbiotas associated with honey bees reveals healthier and broader communities when colonies are genetically diverse. PLoS ONE 7(3): e32962], using pyrosequencing of the V1-V2 hypervariable region of the 16S rRNA gene, reported finding entirely novel bacterial species in honey bee guts, and used taxonomic assignments from these reads to predict metabolic activities based on known metabolisms of cultivable species. To better understand this discrepancy, we analyzed the Mattila et al. pyrotag dataset. In contrast to the conclusions of Mattila et al., we found that the large majority of pyrotag sequences belonged to clusters for which representative sequences were identical to sequences from previously identified core species of the bee microbiota. On average, they represent 95% of the bacteria in each worker bee in the Mattila et al. dataset, a slightly lower value than that found in other studies. Some colonies contain small proportions of other bacteria, mostly species of Enterobacteriaceae. Reanalysis of the Mattila et al. dataset also did not support a relationship between abundances of Bifidobacterium and of putative pathogens or a significant difference in gut communities between colonies from queens that were singly or multiply mated. Additionally, consistent with previous studies, the dataset supports the occurrence of considerable strain variation within core species, even within single colonies. The roles of these bacteria within bees, or the implications of the strain variation, are not yet clear.

A Mainly Circum-Mediterranean Origin for West Eurasian and North African mtDNAs in Puerto Rico with Strong Contributions from the Canary Islands and West Africa.

PubMed

Díaz-Zabala, Héctor J; Nieves-Colón, María A; Martínez-Cruzado, Juan C

2017-04-01

Maternal lineages of West Eurasian and North African origin account for 11.5% of total mitochondrial ancestry in Puerto Rico. Historical sources suggest that this ancestry arrived mostly from European migrations that took place during the four centuries of the Spanish colonization of Puerto Rico. This study analyzed 101 mitochondrial control region sequences and diagnostic coding region variants from a sample set randomly and systematically selected using a census-based sampling frame to be representative of the Puerto Rican population, with the goal of defining West Eurasian-North African maternal clades and estimating their possible geographical origin. Median-joining haplotype networks were constructed using hypervariable regions 1 and 2 sequences from various reference populations in search of shared haplotypes. A posterior probability analysis was performed to estimate the percentage of possible origins across wide geographic regions for the entire sample set and for the most common haplogroups on the island. Principal component analyses were conducted to place the Puerto Rican mtDNA set within the variation present among all reference populations. Our study shows that up to 38% of West Eurasian and North African mitochondrial ancestry in Puerto Rico most likely migrated from the Canary Islands. However, most of those haplotypes had previously migrated to the Canary Islands from elsewhere, and there are substantial contributions from various populations across the circum-Mediterranean region and from West African populations related to the modern Wolof and Serer peoples from Senegal and the nomad Fulani who extend up to Cameroon. In conclusion, the West Eurasian mitochondrial ancestry in Puerto Ricans is geographically diverse. However, haplotype diversity seems to be low, and frequencies have been shaped by population bottlenecks, migration waves, and random genetic drift. Consequently, approximately 47% of mtDNAs of West Eurasian and North African ancestry in Puerto Rico probably arrived early in its colonial history.

Geographically widespread swordfish barcode stock identification: a case study of its application.

PubMed

Pappalardo, Anna Maria; Guarino, Francesca; Reina, Simona; Messina, Angela; De Pinto, Vito

2011-01-01

The swordfish (Xiphias gladius) is a cosmopolitan large pelagic fish inhabiting tempered and tropical waters and it is a target species for fisheries all around the world. The present study investigated the ability of COI barcoding to reliably identify swordfish and particularly specific stocks of this commercially important species. We applied the classical DNA barcoding technology, upon a 682 bp segment of COI, and compared swordfish sequences from different geographical sources (Atlantic, Indian Oceans and Mediterranean Sea). The sequences of the 5' hyper-variable fragment of the control region (5'dloop), were also used to validate the efficacy of COI as a stock-specific marker. This information was successfully applied to the discrimination of unknown samples from the market, detecting in some cases mislabeled seafood products. The NJ distance-based phenogram (K2P model) obtained with COI sequences allowed us to correlate the swordfish haplotypes to the different geographical stocks. Similar results were obtained with 5'dloop. Our preliminary data in swordfish Xiphias gladius confirm that Cytochrome Oxidase I can be proposed as an efficient species-specific marker that has also the potential to assign geographical provenance. This information might speed the samples analysis in commercial application of barcoding.

Detection of iron-depositing Pedomicrobium species in native biofilms from the Odertal National Park by a new, specific FISH probe.

PubMed

Braun, Burga; Richert, Inga; Szewzyk, Ulrich

2009-10-01

Iron-depositing bacteria play an important role in technical water systems (water wells, distribution systems) due to their intense deposition of iron oxides and resulting clogging effects. Pedomicrobium is known as iron- and manganese-oxidizing and accumulating bacterium. The ability to detect and quantify members of this species in biofilm communities is therefore desirable. In this study the fluorescence in situ hybridization (FISH) method was used to detect Pedomicrobium in iron and manganese incrusted biofilms. Based on comparative sequence analysis, we designed and evaluated a specific oligonucleotide probe (Pedo 1250) complementary to the hypervariable region 8 of the 16S rRNA gene for Pedomicrobium. Probe specificities were tested against 3 different strains of Pedomicrobium and Sphingobium yanoikuyae as non-target organism. Using optimized conditions the probe hybridized with all tested strains of Pedomicrobium with an efficiency of 80%. The non-target organism showed no hybridization signals. The new FISH probe was applied successfully for the in situ detection of Pedomicrobium in different native, iron-depositing biofilms. The hybridization results of native bioflims using probe Pedo_1250 agreed with the results of the morphological structure of Pedomicrobium bioflims based on scanning electron microscopy.

Generation of a Kupffer Cell-evading Adenovirus for Systemic and Liver-directed Gene Transfer

PubMed Central

Khare, Reeti; May, Shannon M; Vetrini, Francesco; Weaver, Eric A; Palmer, Donna; Rosewell, Amanda; Grove, Nathan; Ng, Philip; Barry, Michael A

2011-01-01

As much as 90% of an intravenously (i.v.) injected dose of adenovirus serotype 5 (Ad5) is absorbed and destroyed by liver Kupffer cells. Viruses that escape these cells can then transduce hepatocytes after binding factor X (FX). Given that interactions with FX and Kupffer cells are thought to occur on the Ad5 hexon protein, we replaced its exposed hypervariable regions (HVR) with those from Ad6. When tested in vivo in BALB/c mice and in hamsters, the Ad5/6 chimera mediated >10 times higher transduction in the liver. This effect was not due to changes in FX binding. Rather, Ad5/6 appeared to escape Kupffer cell uptake as evidenced by producing no Kupffer cell death in vivo, not requiring predosing in vivo, and being phagocytosed less efficiently by macrophages in vitro compared to Ad5. When tested as a helper-dependent adenovirus (Ad) vector, Ad5/6 mediated higher luciferase and factor IX transgene expression than either helper-dependent adenoviral 5 (HD-Ad5) or HD-Ad6 vectors. These data suggest that the Ad5/6 hexon-chimera evades Kupffer cells and may have utility for systemic and liver-directed therapies. PMID:21505422

Generation of a Kupffer cell-evading adenovirus for systemic and liver-directed gene transfer.

PubMed

Khare, Reeti; May, Shannon M; Vetrini, Francesco; Weaver, Eric A; Palmer, Donna; Rosewell, Amanda; Grove, Nathan; Ng, Philip; Barry, Michael A

2011-07-01

As much as 90% of an intravenously (i.v.) injected dose of adenovirus serotype 5 (Ad5) is absorbed and destroyed by liver Kupffer cells. Viruses that escape these cells can then transduce hepatocytes after binding factor X (FX). Given that interactions with FX and Kupffer cells are thought to occur on the Ad5 hexon protein, we replaced its exposed hypervariable regions (HVR) with those from Ad6. When tested in vivo in BALB/c mice and in hamsters, the Ad5/6 chimera mediated >10 times higher transduction in the liver. This effect was not due to changes in FX binding. Rather, Ad5/6 appeared to escape Kupffer cell uptake as evidenced by producing no Kupffer cell death in vivo, not requiring predosing in vivo, and being phagocytosed less efficiently by macrophages in vitro compared to Ad5. When tested as a helper-dependent adenovirus (Ad) vector, Ad5/6 mediated higher luciferase and factor IX transgene expression than either helper-dependent adenoviral 5 (HD-Ad5) or HD-Ad6 vectors. These data suggest that the Ad5/6 hexon-chimera evades Kupffer cells and may have utility for systemic and liver-directed therapies.

A Portable, Shock-Proof, Surface-Heated Droplet PCR System for Escherichia coli Detection

PubMed Central

Angus, Scott V.; Cho, Soohee; Harshman, Dustin K.; Song, Jae-Young; Yoon, Jeong-Yeol

2015-01-01

A novel polymerase chain reaction (PCR) device was developed that uses wire-guided droplet manipulation (WDM) to guide a droplet over three different heating chambers. After PCR amplification, end-point detection is achieved using a smartphone-based fluorescence microscope. The device was tested for identification of the 16S rRNA gene V3 hypervariable region from Escherichia coli genomic DNA. The lower limit of detection was 103 genome copies per sample. The device is portable with smartphone-based end-point detection and provides the assay results quickly (15 min for a 30-cycle amplification) and accurately. The system is also shock and vibration resistant, due to the multiple points of contact between the droplet and the thermocouple and the Teflon film on the heater surfaces. The thermocouple also provides realtime droplet temperature feedback to ensure it reaches the set temperature before moving to the next chamber/step in PCR. The device is equipped to use either silicone oil or coconut oil. Coconut oil provides additional portability and ease of transportation by eliminating spilling because its high melting temperature means it is solid at room temperature. PMID:26164008

Complex sputum microbial composition in patients with pulmonary tuberculosis

PubMed Central

2012-01-01

Background An increasing number of studies have implicated the microbiome in certain diseases, especially chronic diseases. In this study, the bacterial communities in the sputum of pulmonary tuberculosis patients were explored. Total DNA was extracted from sputum samples from 31 pulmonary tuberculosis patients and respiratory secretions of 24 healthy participants. The 16S rRNA V3 hyper-variable regions were amplified using bar-coded primers and pyro-sequenced using Roche 454 FLX. Results The results showed that the microbiota in the sputum of pulmonary tuberculosis patients were more diverse than those of healthy participants (p<0.05). The sequences were classified into 24 phyla, all of which were found in pulmonary tuberculosis patients and 17 of which were found in healthy participants. Furthermore, many foreign bacteria, such as Stenotrophomonas, Cupriavidus, Pseudomonas, Thermus, Sphingomonas, Methylobacterium, Diaphorobacter, Comamonas, and Mobilicoccus, were unique to pulmonary tuberculosis patients. Conclusions This study concluded that the microbial composition of the respiratory tract of pulmonary tuberculosis patients is more complicated than that of healthy participants, and many foreign bacteria were found in the sputum of pulmonary tuberculosis patients. The roles of these foreign bacteria in the onset or development of pulmonary tuberculosis shoud be considered by clinicians. PMID:23176186

Whole-loop mitochondrial DNA D-loop sequence variability in Egyptian Arabian equine matrilines

PubMed Central

Hudson, William

2017-01-01

Background Egyptian Arabian horses have been maintained in a state of genetic isolation for over a hundred years. There is only limited genetic proof that the studbook records of female lines of Egyptian Arabian pedigrees are reliable. This study characterized the mitochondrial DNA (mtDNA) signatures of 126 horses representing 14 matrilines in the Egyptian Agricultural Organization (EAO) horse-breeding program. Findings Analysis of the whole D-loop sequence yielded additional information compared to hypervariable region-1 (HVR1) analysis alone, with 42 polymorphic sites representing ten haplotypes compared to 16 polymorphic sites representing nine haplotypes, respectively. Most EAO haplotypes belonged to ancient haplogroups, suggesting origin from a wide geographical area over many thousands of years, although one haplotype was novel. Conclusions Historical families share haplotypes and some individuals from different strains belonged to the same haplogroup: the classical EAO strain designation is not equivalent to modern monophyletic matrilineal groups. Phylogenetic inference showed that the foundation mares of the historical haplotypes were highly likely to have the same haplotypes as the animals studied (p > 0.998 in all cases), confirming the reliability of EAO studbook records and providing the opportunity for breeders to confirm the ancestry of their horses. PMID:28859174

Evidence for a genetic discontinuity between Neandertals and 24,000-year-old anatomically modern Europeans.

PubMed

Caramelli, David; Lalueza-Fox, Carles; Vernesi, Cristiano; Lari, Martina; Casoli, Antonella; Mallegni, Francesco; Chiarelli, Brunetto; Dupanloup, Isabelle; Bertranpetit, Jaume; Barbujani, Guido; Bertorelle, Giorgio

2003-05-27

During the late Pleistocene, early anatomically modern humans coexisted in Europe with the anatomically archaic Neandertals for some thousand years. Under the recent variants of the multiregional model of human evolution, modern and archaic forms were different but related populations within a single evolving species, and both have contributed to the gene pool of current humans. Conversely, the Out-of-Africa model considers the transition between Neandertals and anatomically modern humans as the result of a demographic replacement, and hence it predicts a genetic discontinuity between them. Following the most stringent current standards for validation of ancient DNA sequences, we typed the mtDNA hypervariable region I of two anatomically modern Homo sapiens sapiens individuals of the Cro-Magnon type dated at about 23 and 25 thousand years ago. Here we show that the mtDNAs of these individuals fall well within the range of variation of today's humans, but differ sharply from the available sequences of the chronologically closer Neandertals. This discontinuity is difficult to reconcile with the hypothesis that both Neandertals and early anatomically modern humans contributed to the current European gene pool.

PIGSPro: prediction of immunoGlobulin structures v2.

PubMed

Lepore, Rosalba; Olimpieri, Pier P; Messih, Mario A; Tramontano, Anna

2017-07-03

PIGSpro is a significant upgrade of the popular PIGS server for the prediction of the structure of immunoglobulins. The software has been completely rewritten in python following a similar pipeline as in the original method, but including, at various steps, relevant modifications found to improve its prediction accuracy, as demonstrated here. The steps of the pipeline include the selection of the appropriate framework for predicting the conserved regions of the molecule by homology; the target template alignment for this portion of the molecule; the selection of the main chain conformation of the hypervariable loops according to the canonical structure model, the prediction of the third loop of the heavy chain (H3) for which complete canonical structures are not available and the packing of the light and heavy chain if derived from different templates. Each of these steps has been improved including updated methods developed along the years. Last but not least, the user interface has been completely redesigned and an automatic monthly update of the underlying database has been implemented. The method is available as a web server at http://biocomputing.it/pigspro. © The Author(s) 2017. Published by Oxford University Press on behalf of Nucleic Acids Research.

TCR hypervariable regions expressed by T cells that respond to effective tumor vaccines.

PubMed

Jordan, Kimberly R; Buhrman, Jonathan D; Sprague, Jonathan; Moore, Brandon L; Gao, Dexiang; Kappler, John W; Slansky, Jill E

2012-10-01

A major goal of immunotherapy for cancer is the activation of T cell responses against tumor-associated antigens (TAAs). One important strategy for improving antitumor immunity is vaccination with peptide variants of TAAs. Understanding the mechanisms underlying the expansion of T cells that respond to the native tumor antigen is an important step in developing effective peptide-variant vaccines. Using an immunogenic mouse colon cancer model, we compare the binding properties and the TCR genes expressed by T cells elicited by peptide variants that elicit variable antitumor immunity directly ex vivo. The steady-state affinity of the natural tumor antigen for the T cells responding to effective peptide vaccines was higher relative to ineffective peptides, consistent with their improved function. Ex vivo analysis showed that T cells responding to the effective peptides expressed a CDR3β motif, which was also shared by T cells responding to the natural antigen and not those responding to the less effective peptide vaccines. Importantly, these data demonstrate that peptide vaccines can expand T cells that naturally respond to tumor antigens, resulting in more effective antitumor immunity. Future immunotherapies may require similar stringent analysis of the responding T cells to select optimal peptides as vaccine candidates.

Forensic and phylogeographic characterisation of mtDNA lineages from Somalia.

PubMed

Mikkelsen, Martin; Fendt, Liane; Röck, Alexander W; Zimmermann, Bettina; Rockenbauer, Eszter; Hansen, Anders J; Parson, Walther; Morling, Niels

2012-07-01

The African mitochondrial (mt) phylogeny is coarsely resolved but the majority of population data generated so far is limited to the analysis of the first hypervariable segment (HVS-1) of the control region (CR). Therefore, this study aimed on the investigation of the entire CR of 190 unrelated Somali individuals to enrich the severely underrepresented African mtDNA pool. The majority (60.5 %) of the haplotypes were of sub-Saharan origin with L0a1d, L2a1h and L3f being the most frequently observed haplogroups. This is in sharp contrast to previous data reported from the Y-chromosome, where only about 5 % of the observed haplogroups were of sub-Saharan provenance. We compared the genetic distances based on population pairwise F (st) values between 11 published East, Central and North African as well as western Asian populations and the Somali sequences and displayed them in a multi-dimensional scaling plot. Genetic proximity evidenced by clustering roughly reflected the relative geographic location of the populations. The sequences will be included in the EMPOP database ( www.empop.org ) under accession number EMP00397 upon publication (Parson and Dür Forensic Sci Int Genet 1:88-92, 2007).

Phylogeographic distribution of very virulent infectious bursal disease virus isolates in the Iberian Peninsula.

PubMed

Cortey, Martí; Bertran, Kateri; Toskano, Jennifer; Majó, Natàlia; Dolz, Roser

2012-01-01

Viral population dynamics of very virulent infectious bursal disease virus (vvIBDV) field strains isolated in the Iberian Peninsula since the first outbreak in the 1990s have been analysed. Low levels of genetic variability and a global purification selection pattern were reported in 480 base pairs of the hypervariable region of the VP2 gene, indicating a lack of a selection-driven immune escape in the evolutive pathway of the virus. The viral population structure of vvIBDV strains in the Iberian Peninsula showed a strong relationship between geography and phylogeny, with two main groups observed. A global comparison among vvIBDV strains also showed an association with sequences from the same country. The low variability, the strong purifying selection and the geographical pattern observed point to a picture where the virus evolves slowly, occupying the same geographical niche for a long time. The scenario depicted fits well with the biological features of the virus: being able to remain viable for long periods of time due to a strong environmental resistance, and as an immunosuppressive agent, capable per se of annihilating temporally the immune system of the host.

SLFL Genes Participate in the Ubiquitination and Degradation Reaction of S-RNase in Self-compatible Peach.

PubMed

Chen, Qiuju; Meng, Dong; Gu, Zhaoyu; Li, Wei; Yuan, Hui; Duan, Xuwei; Yang, Qing; Li, Yang; Li, Tianzhong

2018-01-01

It has been proved that the gametophytic self-incompatibility (GSI), mainly exists in Rosaceae and Solanaceae, is controlled by S genes, which are two tightly linked genes located at highly polymorphic S -locus: the S-RNase for pistil specificity and the F-box gene ( SFB/SLF ) for pollen specificity, respectively. However, the roles of those genes in SI of peach are still a subject of extensive debate. In our study, we selected 37 representative varieties according to the evolution route of peach and identified their S genotypes. We cloned pollen determinant genes mutated PperSFB1m, PperSFB2m, PperSFB4m , and normal PperSFB2 , and style determinant genes PperS1-RNase, PperS2-RNase, PperS2m-RNase , and PperS4-RNase . The mutated PperSFBs encode truncated SFB proteins due to a fragment insertion. The truncated PperSFBs and normal PperSFB2 interacted with PperS-RNases demonstrated by Y2H. Normal PperSFB2 was divided into four parts: box, box-V1, V1-V2, and HVa-HVb. The box domain of PperSFB2 did not interact with PperS-RNases, both of the box-V1 and V1-V2 had interactions with PperS-RNases, while the hypervariable region of PperSFB2 HVa-HVb only interacted with PperS2-RNase showed by Y2H and BiFC assay. Bioinformatics analysis of peach genome revealed that there were other F-box genes located at S-locus, and of which three F-box genes were specifically expressed in pollen, named as PperSLFL1, PperSLFL2 , and PperSLFL3 , respectively. In phylogenetic analysis PperSLFLs clustered with Maloideae SFBB genes, and PperSFB genes were clustered into the other group with other SFB genes of Prunus . Protein interaction analysis revealed that the three PperSLFLs interacted with PperSSK1 and PperS-RNases with no allelic specificity. In vitro ubiquitination assay showed that PperSLFLs could tag ubiquitin molecules onto PperS-RNases. The above results suggest that three PperSLFLs are the appropriate candidates for the "general inhibitor," which would inactivate the S-RNases in pollen tubes, involved in the self-incompatibility of peach.

SLFL Genes Participate in the Ubiquitination and Degradation Reaction of S-RNase in Self-compatible Peach

PubMed Central

Chen, Qiuju; Meng, Dong; Gu, Zhaoyu; Li, Wei; Yuan, Hui; Duan, Xuwei; Yang, Qing; Li, Yang; Li, Tianzhong

2018-01-01

It has been proved that the gametophytic self-incompatibility (GSI), mainly exists in Rosaceae and Solanaceae, is controlled by S genes, which are two tightly linked genes located at highly polymorphic S-locus: the S-RNase for pistil specificity and the F-box gene (SFB/SLF) for pollen specificity, respectively. However, the roles of those genes in SI of peach are still a subject of extensive debate. In our study, we selected 37 representative varieties according to the evolution route of peach and identified their S genotypes. We cloned pollen determinant genes mutated PperSFB1m, PperSFB2m, PperSFB4m, and normal PperSFB2, and style determinant genes PperS1-RNase, PperS2-RNase, PperS2m-RNase, and PperS4-RNase. The mutated PperSFBs encode truncated SFB proteins due to a fragment insertion. The truncated PperSFBs and normal PperSFB2 interacted with PperS-RNases demonstrated by Y2H. Normal PperSFB2 was divided into four parts: box, box-V1, V1-V2, and HVa-HVb. The box domain of PperSFB2 did not interact with PperS-RNases, both of the box-V1 and V1-V2 had interactions with PperS-RNases, while the hypervariable region of PperSFB2 HVa-HVb only interacted with PperS2-RNase showed by Y2H and BiFC assay. Bioinformatics analysis of peach genome revealed that there were other F-box genes located at S-locus, and of which three F-box genes were specifically expressed in pollen, named as PperSLFL1, PperSLFL2, and PperSLFL3, respectively. In phylogenetic analysis PperSLFLs clustered with Maloideae SFBB genes, and PperSFB genes were clustered into the other group with other SFB genes of Prunus. Protein interaction analysis revealed that the three PperSLFLs interacted with PperSSK1 and PperS-RNases with no allelic specificity. In vitro ubiquitination assay showed that PperSLFLs could tag ubiquitin molecules onto PperS-RNases. The above results suggest that three PperSLFLs are the appropriate candidates for the “general inhibitor,” which would inactivate the S-RNases in pollen tubes, involved in the self-incompatibility of peach. PMID:29520292

Pyrosequencing Reveals the Predominance of Pseudomonadaceae in Gut Microbiome of a Gall Midge

PubMed Central

Bansal, Raman; Hulbert, Scot H.; Reese, John C.; Whitworth, Robert J.; Stuart, Jeffrey J.; Chen, Ming-Shun

2014-01-01

Gut microbes are known to play various roles in insects such as digestion of inaccessible nutrients, synthesis of deficient amino acids, and interaction with ecological environments, including host plants. Here, we analyzed the gut microbiome in Hessian fly, a serious pest of wheat. A total of 3,654 high quality sequences of the V3 hypervariable region of the 16S rRNA gene were obtained through 454-pyrosequencing. From these sequences, 311 operational taxonomic units (OTUs) were obtained at the ≥97% similarity cutoff. In the gut of 1st instar, otu01, a member of Pseudomonas, was predominant, representing 90.2% of total sequences. otu13, an unidentified genus in the Pseudomonadaceae family, represented 1.9% of total sequences. The remaining OTUs were each less than 1%. In the gut of the 2nd instar, otu01 and otu13 decreased to 85.5% and 1.5%, respectively. otu04, a member of Buttiauxella, represented 9.7% of total sequences. The remaining OTUs were each less than 1%. In the gut of the 3rd instar, otu01 and otu13 further decreased to 29.0% and 0%, respectively. otu06, otu08, and otu16, also three members of the Pseudomonadaceae family were 13.2%, 8.6%, and 2.3%, respectively. In addition, otu04 and otu14, two members of the Enterobacteriaceae family, were 4.7% and 2.5%; otu18 and otu20, two members of the Xanthomonadaceae family, were 1.3% and 1.2%, respectively; otu12, a member of Achromobacter, was 4.2%; otu19, a member of Undibacterium, was 1.4%; and otu9, otu10, and otu15, members of various families, were 6.1%, 6.3%, and 1.9%, respectively. The investigation into dynamics of Pseudomonas, the most abundant genera, revealed that its population level was at peak in freshly hatched or 1 day larvae as well as in later developmental stages, thus suggesting a prominent role for this bacterium in Hessian fly development and in its interaction with host plants. This study is the first comprehensive survey on bacteria associated with the gut of a gall midge, and provides a foundation for future studies to elucidate the roles of gut microbes in Hessian fly virulence and biology. PMID:25437809

Genetic analysis of ancestry, admixture and selection in Bolivian and Totonac populations of the New World.

PubMed

Watkins, W Scott; Xing, Jinchuan; Huff, Chad; Witherspoon, David J; Zhang, Yuhua; Perego, Ugo A; Woodward, Scott R; Jorde, Lynn B

2012-05-20

Populations of the Americas were founded by early migrants from Asia, and some have experienced recent genetic admixture. To better characterize the native and non-native ancestry components in populations from the Americas, we analyzed 815,377 autosomal SNPs, mitochondrial hypervariable segments I and II, and 36 Y-chromosome STRs from 24 Mesoamerican Totonacs and 23 South American Bolivians. We analyzed common genomic regions from native Bolivian and Totonac populations to identify 324 highly predictive Native American ancestry informative markers (AIMs). As few as 40-50 of these AIMs perform nearly as well as large panels of random genome-wide SNPs for predicting and estimating Native American ancestry and admixture levels. These AIMs have greater New World vs. Old World specificity than previous AIMs sets. We identify highly-divergent New World SNPs that coincide with high-frequency haplotypes found at similar frequencies in all populations examined, including the HGDP Pima, Maya, Colombian, Karitiana, and Surui American populations. Some of these regions are potential candidates for positive selection. European admixture in the Bolivian sample is approximately 12%, though individual estimates range from 0-48%. We estimate that the admixture occurred ~360-384 years ago. Little evidence of European or African admixture was found in Totonac individuals. Bolivians with pre-Columbian mtDNA and Y-chromosome haplogroups had 5-30% autosomal European ancestry, demonstrating the limitations of Y-chromosome and mtDNA haplogroups and the need for autosomal ancestry informative markers for assessing ancestry in admixed populations.

The relation between oral Candida load and bacterial microbiome profiles in Dutch older adults.

PubMed

Kraneveld, Eefje A; Buijs, Mark J; Bonder, Marc J; Visser, Marjolein; Keijser, Bart J F; Crielaard, Wim; Zaura, Egija

2012-01-01

Currently there are no evidence-based ecological measures for prevention of overgrowth and subsequent infection by fungi in the oral cavity. The aim of this study was to increase our knowledge on fungal-bacterial ecological interactions. Salivary Candida abundance of 82 Dutch adults aged 58-80 years was established relative to the bacterial load by quantitative PCR analysis of the Internal Transcribed (ITS) region (Candida) and 16S rDNA gene (bacteria). The salivary microbiome was assessed using barcoded pyrosequencing of the bacterial hypervariable regions V5-V7 of 16S rDNA. Sequencing data was preprocessed by denoising and chimera removal, clustered in Operational Taxonomic Units (OTUs) and assigned to taxonomy. Both OTU-based (PCA, diversity statistics) and phylogeny-based analyses (UniFrac, PCoA) were performed. Saliva of Dutch older adults contained 0-4 × 10(8) CFU/mL Candida with a median Candida load of 0.06%. With increased Candida load the diversity of the salivary microbiome decreased significantly (p<0.001). Increase in the Candida load correlated positively with class Bacilli, and negatively with class Fusobacteria, Flavobacteria, and Bacteroidia. Microbiomes with high Candida load were less diverse and had a distinct microbial composition towards dominance by saccharolytic and acidogenic bacteria--streptococci. The control of the acidification of the oral environment may be a potential preventive measure for Candida outgrowth that should be evaluated in longitudinal clinical intervention trials.

Influence of Electron–Holes on DNA Sequence-Specific Mutation Rates

PubMed Central

Suárez-Villagrán, Martha Y; Azevedo, Ricardo B R; Miller, John H

2018-01-01

Abstract Biases in mutation rate can influence molecular evolution, yielding rates of evolution that vary widely in different parts of the genome and even among neighboring nucleotides. Here, we explore one possible mechanism of influence on sequence-specific mutation rates, the electron–hole, which can localize and potentially trigger a replication mismatch. A hole is a mobile site of positive charge created during one-electron oxidation by, for example, radiation, contact with a mutagenic agent, or oxidative stress. Its quantum wavelike properties cause it to localize at various sites with probabilities that vary widely, by orders of magnitude, and depend strongly on the local sequence. We find significant correlations between hole probabilities and mutation rates within base triplets, observed in published mutation accumulation experiments on four species of bacteria. We have also computed hole probability spectra for hypervariable segment I of the human mtDNA control region, which contains several mutational hotspots, and for heptanucleotides in noncoding regions of the human genome, whose polymorphism levels have recently been reported. We observe significant correlations between hole probabilities, and context-specific mutation and substitution rates. The correlation with hole probability cannot be explained entirely by CpG methylation in the heptanucleotide data. Peaks in hole probability tend to coincide with mutational hotspots, even in mtDNA where CpG methylation is rare. Our results suggest that hole-enhanced mutational mechanisms, such as oxidation-stabilized tautomerization and base deamination, contribute to molecular evolution. PMID:29617801

Molecular and antigenic characterization of bovine Coronavirus circulating in Argentinean cattle during 1994-2010.

PubMed

Bok, M; Miño, S; Rodriguez, D; Badaracco, A; Nuñes, I; Souza, S P; Bilbao, G; Louge Uriarte, E; Galarza, R; Vega, C; Odeon, A; Saif, L J; Parreño, V

2015-12-31

Bovine coronavirus (BCoV) is an important viral pathogen associated with neonatal calf diarrhea. Our aim was to investigate the incidence of BCoV in diarrhea outbreaks in beef and dairy herds from Argentina during 1994-2010. A total of 5.365 fecal samples from diarrheic calves were screened for BCoV diagnosis by ELISA. The virus was detected in 1.71% (92/5365) of the samples corresponding to 5.95% (63/1058) of the diarrhea cases in 239 beef and 324 dairy farms. The detection rate of BCoV was significantly higher in dairy than in beef herds: 12.13% (29/239) vs. 4.32% (14/324) respectively. Phylogenetic analysis of the hypervariable S1 region of seven representative samples (from different husbandry systems, farm locations and years of sampling) indicated that BCoV strains circulating in Argentinean beef and dairy herds formed a cluster distinct from other geographical regions. Interestingly, Argentinean strains are distantly related (at both the nucleotide and amino acid levels) with the Mebus historic reference BCoV strain included in the vaccines currently available in Argentina. However, Mebus-induced antibodies were capable of neutralizing the BCoV Arg95, a field strain adapted to grow in vitro, and vice versa, indicating that both strains belong to the same CoV serotype reported in cattle. This work represents the first large survey describing BCoV circulation in Argentinean cattle. Copyright © 2015. Published by Elsevier B.V.

The Relation between Oral Candida Load and Bacterial Microbiome Profiles in Dutch Older Adults

PubMed Central

Kraneveld, Eefje A.; Buijs, Mark J.; Bonder, Marc J.; Visser, Marjolein; Keijser, Bart J. F.; Crielaard, Wim; Zaura, Egija

2012-01-01

Currently there are no evidence-based ecological measures for prevention of overgrowth and subsequent infection by fungi in the oral cavity. The aim of this study was to increase our knowledge on fungal–bacterial ecological interactions. Salivary Candida abundance of 82 Dutch adults aged 58–80 years was established relative to the bacterial load by quantitative PCR analysis of the Internal Transcribed (ITS) region (Candida) and 16S rDNA gene (bacteria). The salivary microbiome was assessed using barcoded pyrosequencing of the bacterial hypervariable regions V5–V7 of 16S rDNA. Sequencing data was preprocessed by denoising and chimera removal, clustered in Operational Taxonomic Units (OTUs) and assigned to taxonomy. Both OTU-based (PCA, diversity statistics) and phylogeny-based analyses (UniFrac, PCoA) were performed. Saliva of Dutch older adults contained 0–4 × 108 CFU/mL Candida with a median Candida load of 0.06%. With increased Candida load the diversity of the salivary microbiome decreased significantly (p<0.001). Increase in the Candida load correlated positively with class Bacilli, and negatively with class Fusobacteria, Flavobacteria, and Bacteroidia. Microbiomes with high Candida load were less diverse and had a distinct microbial composition towards dominance by saccharolytic and acidogenic bacteria - streptococci. The control of the acidification of the oral environment may be a potential preventive measure for Candida outgrowth that should be evaluated in longitudinal clinical intervention trials. PMID:22900048

mtDNA history of the Cayapa Amerinds of Ecuador: detection of additional founding lineages for the Native American populations.

PubMed Central

Rickards, O; Martínez-Labarga, C; Lum, J K; De Stefano, G F; Cann, R L

1999-01-01

mtDNA variation in the Cayapa, an Ecuadorian Amerindian tribe belonging to the Chibcha-Paezan linguistic branch, was analyzed by use of hypervariable control regions I and II along with two linked regions undergoing insertion/deletion mutations. Three major maternal lineage clusters fit into the A, B, and C founding groups first described by Schurr and colleagues in 1990, whereas a fourth lineage, apparently unique to the Cayapa, has ambiguous affinity to known clusters. The time of divergence from a common maternal ancestor of the four lineage groups is of sufficient age that it indicates an origin in Asia and supports the hypothesis that the degree of variability carried by the Asian ancestral populations into the New World was rather high. Spatial autocorrelation analysis points out (a) statistically significant nonrandom distributions of the founding lineages in the Americas, because of north-south population movements that have occurred since the first Asian migrants spread through Beringia into the Americas, and (b) an unusual pattern associated with the D lineage cluster. The values of haplotype and nucleotide diversity that are displayed by the Cayapa appear to differ from those observed in other Chibchan populations but match those calculated for South American groups belonging to various linguistic stocks. These data, together with the results of phylogenetic analysis performed with the Amerinds of Central and South America, highlight the difficulty in the identification of clear coevolutionary patterns between linguistic and genetic relationships in particular human populations. PMID:10417294

Phylogeographic Analysis of Mitochondrial DNA in Northern Asian Populations

PubMed Central

Derenko, Miroslava ; Malyarchuk, Boris ; Grzybowski, Tomasz ; Denisova, Galina ; Dambueva, Irina ; Perkova, Maria ; Dorzhu, Choduraa ; Luzina, Faina ; Lee, Hong Kyu ; Vanecek, Tomas ; Villems, Richard ; Zakharov, Ilia 

2007-01-01

To elucidate the human colonization process of northern Asia and human dispersals to the Americas, a diverse subset of 71 mitochondrial DNA (mtDNA) lineages was chosen for complete genome sequencing from the collection of 1,432 control-region sequences sampled from 18 autochthonous populations of northern, central, eastern, and southwestern Asia. On the basis of complete mtDNA sequencing, we have revised the classification of haplogroups A, D2, G1, M7, and I; identified six new subhaplogroups (I4, N1e, G1c, M7d, M7e, and J1b2a); and fully characterized haplogroups N1a and G1b, which were previously described only by the first hypervariable segment (HVS1) sequencing and coding-region restriction-fragment–length polymorphism analysis. Our findings indicate that the southern Siberian mtDNA pool harbors several lineages associated with the Late Upper Paleolithic and/or early Neolithic dispersals from both eastern Asia and southwestern Asia/southern Caucasus. Moreover, the phylogeography of the D2 lineages suggests that southern Siberia is likely to be a geographical source for the last postglacial maximum spread of this subhaplogroup to northern Siberia and that the expansion of the D2b branch occurred in Beringia ∼7,000 years ago. In general, a detailed analysis of mtDNA gene pools of northern Asians provides the additional evidence to rule out the existence of a northern Asian route for the initial human colonization of Asia. PMID:17924343

Phylogeographic analysis of mitochondrial DNA in northern Asian populations.

PubMed

Derenko, Miroslava; Malyarchuk, Boris; Grzybowski, Tomasz; Denisova, Galina; Dambueva, Irina; Perkova, Maria; Dorzhu, Choduraa; Luzina, Faina; Lee, Hong Kyu; Vanecek, Tomas; Villems, Richard; Zakharov, Ilia

2007-11-01

To elucidate the human colonization process of northern Asia and human dispersals to the Americas, a diverse subset of 71 mitochondrial DNA (mtDNA) lineages was chosen for complete genome sequencing from the collection of 1,432 control-region sequences sampled from 18 autochthonous populations of northern, central, eastern, and southwestern Asia. On the basis of complete mtDNA sequencing, we have revised the classification of haplogroups A, D2, G1, M7, and I; identified six new subhaplogroups (I4, N1e, G1c, M7d, M7e, and J1b2a); and fully characterized haplogroups N1a and G1b, which were previously described only by the first hypervariable segment (HVS1) sequencing and coding-region restriction-fragment-length polymorphism analysis. Our findings indicate that the southern Siberian mtDNA pool harbors several lineages associated with the Late Upper Paleolithic and/or early Neolithic dispersals from both eastern Asia and southwestern Asia/southern Caucasus. Moreover, the phylogeography of the D2 lineages suggests that southern Siberia is likely to be a geographical source for the last postglacial maximum spread of this subhaplogroup to northern Siberia and that the expansion of the D2b branch occurred in Beringia ~7,000 years ago. In general, a detailed analysis of mtDNA gene pools of northern Asians provides the additional evidence to rule out the existence of a northern Asian route for the initial human colonization of Asia.

Molecular analysis of transmission of hepatitis C virus in a nurse who acquired acute hepatitis C after caring for a viremic patient with epistaxis.

PubMed

Toda, Takayuki; Mitsui, Takehiro; Tsukamoto, Yukie; Ebara, Takeshi; Hirose, Akinori; Masuko, Kazuo; Nagashima, Shigeo; Takahashi, Masaharu; Okamoto, Hiroaki

2009-08-01

A 23-year-old nurse (HC-IP) developed acute hepatitis C. Intrafamilial transmission of hepatitis C virus (HCV) was suspected initially because her parents were carriers of HCV of the same genotype (1b) as that of Patient HC-IP. However, the HCV isolate from Patient HC-IP and those from her parents shared identities of only 92.4-92.7% in the 1,087-nucleotide (nt) sequence within the NS5B region. It was then suspected that she contracted HCV infection during medical practice. Sixteen patients with antibodies to HCV (anti-HCV) were hospitalized 1-3 months before she became positive for anti-HCV. Upon analysis of stored serum samples, 14 of the 16 patients were found to be positive for HCV RNA, and 9 of the 14 viremic patients had genotype 1b HCV. Although the shared identities between the HCV isolate from Patient HC-IP and those from eight of the nine patients were merely 90.6-93.9% within the 1,087-nt NS5B sequence, the HCV isolate from the remaining one patient (HC-P12) was 99.7% identical to that from Patient HC-IP. Upon analysis of the E1 and E2 junctional region including hypervariable region 1 (283 nt), there was a close relationship (99.3-100%) between clones obtained from Patients HC-IP and HC-P12. Although the nurse HC-IP had a finger injury, she took care of Patient HC-P12, a 70-year-old man with HCV-related cirrhosis and recurrent epistaxis, occasionally without wearing protective gloves. This study indicates the occurrence of HCV transmission by exposure of nonintact skin to blood in health care settings. 2009 Wiley-Liss, Inc.

Genome skimming identifies polymorphism in tern populations and species

PubMed Central

2012-01-01

Background Terns (Charadriiformes: Sterninae) are a lineage of cosmopolitan shorebirds with a disputed evolutionary history that comprises several species of conservation concern. As a non-model system in genetics, previous study has left most of the nuclear genome unexplored, and population-level studies are limited to only 15% of the world's species of terns and noddies. Screening of polymorphic nuclear sequence markers is needed to enhance genetic resolution because of supposed low mitochondrial mutation rate, documentation of nuclear insertion of hypervariable mitochondrial regions, and limited success of microsatellite enrichment in terns. Here, we investigated the phylogenetic and population genetic utility for terns and relatives of a variety of nuclear markers previously developed for other birds and spanning the nuclear genome. Markers displaying a variety of mutation rates from both the nuclear and mitochondrial genome were tested and prioritized according to optimal cross-species amplification and extent of genetic polymorphism between (1) the main tern clades and (2) individual Royal Terns (Thalasseus maxima) breeding on the US East Coast. Results Results from this genome skimming effort yielded four new nuclear sequence-based markers for tern phylogenetics and 11 intra-specific polymorphic markers. Further, comparison between the two genomes indicated a phylogenetic conflict at the base of terns, involving the inclusion (mitochondrial) or exclusion (nuclear) of the Angel Tern (Gygis alba). Although limited mitochondrial variation was confirmed, both nuclear markers and a short tandem repeat in the mitochondrial control region indicated the presence of considerable genetic variation in Royal Terns at a regional scale. Conclusions These data document the value of intronic markers to the study of terns and allies. We expect that these and additional markers attained through next-generation sequencing methods will accurately map the genetic origin and species history of this group of birds. PMID:22333071

Mitochondrial phylogeography of moose (Alces alces): Late Pleistocene divergence and population expansion

USGS Publications Warehouse

Hundertmark, Kris J.; Shields, Gerald F.; Udina, Irina G.; Bowyer, R. Terry; Danilkin, Alexei A.; Schwartz, Charles C.

2002-01-01

We examined phylogeographic relationships of moose (Alces alces) worldwide to test the proposed existence of two geographic races and to infer the timing and extent of demographic processes underpinning the expansion of this species across the Northern Hemisphere in the late Pleistocene. Sequence variation within the left hypervariable domain of the control region occurred at low or moderate levels worldwide and was structured geographically. Partitioning of genetic variance among regions indicated that isolation by distance was the primary agent for differentiation of moose populations but does not support the existence of distinct eastern and western races. Levels of genetic variation and structure of phylogenetic trees identify Asia as the origin of all extant mitochondrial lineages. A recent coalescence is indicated, with the most recent common ancestor dating to the last ice age. Moose have undergone two episodes of population expansion, likely corresponding to the final interstade of the most recent ice age and the onset of the current interglacial. Timing of expansion for the population in the Yakutia–Manchuria region of eastern Asia indicates that it is one of the oldest populations of moose and may represent the source of founders of extant populations in North America, which were colonized within the last 15,000 years. Our data suggest an extended period of low population size or a severe bottleneck prior to the divergence and expansion of extant lineages and a recent, less-severe bottleneck among European lineages. Climate change during the last ice age, acting through contraction and expansion of moose habitat and the flooding of the Bering land bridge, undoubtedly was a key factor influencing the divergence and expansion of moose populations.

Five Complete Chloroplast Genome Sequences from Diospyros: Genome Organization and Comparative Analysis.

PubMed

Fu, Jianmin; Liu, Huimin; Hu, Jingjing; Liang, Yuqin; Liang, Jinjun; Wuyun, Tana; Tan, Xiaofeng

2016-01-01

Diospyros is the largest genus in Ebenaceae, comprising more than 500 species with remarkable economic value, especially Diospyros kaki Thunb., which has traditionally been an important food resource in China, Korea, and Japan. Complete chloroplast (cp) genomes from D. kaki, D. lotus L., D. oleifera Cheng., D. glaucifolia Metc., and Diospyros 'Jinzaoshi' were sequenced using Illumina sequencing technology. This is the first cp genome reported in Ebenaceae. The cp genome sequences of Diospyros ranged from 157,300 to 157,784 bp in length, presenting a typical quadripartite structure with two inverted repeats each separated by one large and one small single-copy region. For each cp genome, 134 genes were annotated, including 80 protein-coding, 31 tRNA, and 4 rRNA unique genes. In all, 179 repeats and 283 single sequence repeats were identified. Four hypervariable regions, namely, intergenic region of trnQ_rps16, trnV_ndhC, and psbD_trnT, and intron of ndhA, were identified in the Diospyros genomes. Phylogenetic analyses based on the whole cp genome, protein-coding, and intergenic and intron sequences indicated that D. oleifera is closely related to D. kaki and could be used as a model plant for future research on D. kaki; to our knowledge, this is proposed for the first time. Further, these analyses together with two large deletions (301 and 140 bp) in the cp genome of D. 'Jinzaoshi', support its placement as a new species in Diospyros. Both maximum parsimony and likelihood analyses for 19 taxa indicated the basal position of Ericales in asterids and suggested that Ebenaceae is monophyletic in Ericales.

Five Complete Chloroplast Genome Sequences from Diospyros: Genome Organization and Comparative Analysis

PubMed Central

Hu, Jingjing; Liang, Yuqin; Liang, Jinjun; Wuyun, Tana; Tan, Xiaofeng

2016-01-01

Diospyros is the largest genus in Ebenaceae, comprising more than 500 species with remarkable economic value, especially Diospyros kaki Thunb., which has traditionally been an important food resource in China, Korea, and Japan. Complete chloroplast (cp) genomes from D. kaki, D. lotus L., D. oleifera Cheng., D. glaucifolia Metc., and Diospyros ‘Jinzaoshi’ were sequenced using Illumina sequencing technology. This is the first cp genome reported in Ebenaceae. The cp genome sequences of Diospyros ranged from 157,300 to 157,784 bp in length, presenting a typical quadripartite structure with two inverted repeats each separated by one large and one small single-copy region. For each cp genome, 134 genes were annotated, including 80 protein-coding, 31 tRNA, and 4 rRNA unique genes. In all, 179 repeats and 283 single sequence repeats were identified. Four hypervariable regions, namely, intergenic region of trnQ_rps16, trnV_ndhC, and psbD_trnT, and intron of ndhA, were identified in the Diospyros genomes. Phylogenetic analyses based on the whole cp genome, protein-coding, and intergenic and intron sequences indicated that D. oleifera is closely related to D. kaki and could be used as a model plant for future research on D. kaki; to our knowledge, this is proposed for the first time. Further, these analyses together with two large deletions (301 and 140 bp) in the cp genome of D. ‘Jinzaoshi’, support its placement as a new species in Diospyros. Both maximum parsimony and likelihood analyses for 19 taxa indicated the basal position of Ericales in asterids and suggested that Ebenaceae is monophyletic in Ericales. PMID:27442423

[Experimental study of candidate vaccines against variable or quasi-species pathogenes: multiepitopic synthetic peptide antigenes and new receptor-guiding adjuvants].

PubMed

Ignat'eva, G A; Maksiutov, A Z; L'vov, V L; Kolobov, A A; Ignat'ev, T I

2011-01-01

The short multiepitopic synthetic peptides from the sequences of hypervariable area of V3-loope of gp120 of HIV don't induce anti-peptides antibodies production in mice themselves. We prepared the potent immunogen by noncovalent conjugations of the multitude peptides with pure peptidoglycans from cell wall of Salmonella typhi. The sera from immunized mice have the anti-peptides antibody titers (3-5) x 10(5) in ELISA, as high as Freund's adjuvant is of use.

Effect of glycosylation on an immunodominant region in the V1V2 variable domain of the HIV-1 envelope gp120 protein

DOE PAGES

Tian, Jianhui; Lopez, Cesar Augusto; Derdeyn, Cynthia A.; ...

2016-10-07

Heavy glycosylation of the envelope (Env) surface subunit, gp120, is a key adaptation of HIV-1; however, the precise effects of glycosylation on the folding, conformation and dynamics of this protein are poorly understood. Here we explore the patterns of HIV-1 Env gp120 glycosylation, and particularly the enrichment in glycosylation sites proximal to the disulfide linkages at the base of the surface-exposed variable domains. To dissect the influence of glycans on the conformation these regions, we focused on an antigenic peptide fragment from a disulfide bridge-bounded region spanning the V1 and V2 hyper-variable domains of HIV-1 gp120. We used replica exchangemore » molecular dynamics (MD) simulations to investigate how glycosylation influences its conformation and stability. Simulations were performed with and without N-linked glycosylation at two sites that are highly conserved across HIV-1 isolates (N156 and N160); both are contacts for recognition by V1V2-targeted broadly neutralizing antibodies against HIV-1. Glycosylation stabilized the pre-existing conformations of this peptide construct, reduced its propensity to adopt other secondary structures, and provided resistance against thermal unfolding. Simulations performed in the context of the Env trimer also indicated that glycosylation reduces flexibility of the V1V2 region, and provided insight into glycan-glycan interactions in this region. These stabilizing effects were influenced by a combination of factors, including the presence of a disulfide bond between the Cysteines at 131 and 157, which increased the formation of beta-strands. Together, these results provide a mechanism for conservation of disulfide linkage proximal glycosylation adjacent to the variable domains of gp120 and begin to explain how this could be exploited to enhance the immunogenicity of those regions. Furthermore, these studies suggest that glycopeptide immunogens can be designed to stabilize the most relevant Env conformations to focus the immune response on key neutralizing epitopes.« less

Effect of glycosylation on an immunodominant region in the V1V2 variable domain of the HIV-1 envelope gp120 protein

DOE Office of Scientific and Technical Information (OSTI.GOV)

Tian, Jianhui; Lopez, Cesar Augusto; Derdeyn, Cynthia A.

Heavy glycosylation of the envelope (Env) surface subunit, gp120, is a key adaptation of HIV-1; however, the precise effects of glycosylation on the folding, conformation and dynamics of this protein are poorly understood. Here we explore the patterns of HIV-1 Env gp120 glycosylation, and particularly the enrichment in glycosylation sites proximal to the disulfide linkages at the base of the surface-exposed variable domains. To dissect the influence of glycans on the conformation these regions, we focused on an antigenic peptide fragment from a disulfide bridge-bounded region spanning the V1 and V2 hyper-variable domains of HIV-1 gp120. We used replica exchangemore » molecular dynamics (MD) simulations to investigate how glycosylation influences its conformation and stability. Simulations were performed with and without N-linked glycosylation at two sites that are highly conserved across HIV-1 isolates (N156 and N160); both are contacts for recognition by V1V2-targeted broadly neutralizing antibodies against HIV-1. Glycosylation stabilized the pre-existing conformations of this peptide construct, reduced its propensity to adopt other secondary structures, and provided resistance against thermal unfolding. Simulations performed in the context of the Env trimer also indicated that glycosylation reduces flexibility of the V1V2 region, and provided insight into glycan-glycan interactions in this region. These stabilizing effects were influenced by a combination of factors, including the presence of a disulfide bond between the Cysteines at 131 and 157, which increased the formation of beta-strands. Together, these results provide a mechanism for conservation of disulfide linkage proximal glycosylation adjacent to the variable domains of gp120 and begin to explain how this could be exploited to enhance the immunogenicity of those regions. Furthermore, these studies suggest that glycopeptide immunogens can be designed to stabilize the most relevant Env conformations to focus the immune response on key neutralizing epitopes.« less

Structural models of antibody variable fragments: A method for investigating binding mechanisms

NASA Astrophysics Data System (ADS)

Petit, Samuel; Brard, Frédéric; Coquerel, Gérard; Perez, Guy; Tron, François

1998-03-01

The value of comparative molecular modeling for elucidating structure-function relationships was demonstrated by analyzing six anti-nucleosome autoantibody variable fragments. Structural models were built using the automated procedure developed in the COMPOSER software, subsequently minimized with the AMBER force field, and validated according to several standard geometric and chemical criteria. Canonical class assignment from Chothia and Lesk's [Chottin and Lesk, J. Mol. Biol., 196 (1987) 901; Chothia et al., Nature, 342 (1989) 877] work was used as a supplementary validation tool for five of the six hypervariable loops. The analysis, based on the hypothesis that antigen binding could occur through electrostatic interactions, reveals a diversity of possible binding mechanisms of anti-nucleosome or anti-histone antibodies to their cognate antigen. These results lead us to postulate that anti-nucleosome autoantibodies could have different origins. Since both anti-DNA and anti-nculeosome autoantibodies are produced during the course of systemic lupus erythematosus, a non-organ specific autoimmune disease, a comparative structural and electrostatic analysis of the two populations of autoantibodies may constitute a way to elucidate their origin and the role of the antigen in tolerance breakdown. The present study illustrates some interests, advantages and limits of a methodology based on the use of comparative modeling and analysis of molecular surface properties.

Little genetic differentiation as assessed by uniparental markers in the presence of substantial language variation in peoples of the Cross River region of Nigeria

PubMed Central

2010-01-01

Background The Cross River region in Nigeria is an extremely diverse area linguistically with over 60 distinct languages still spoken today. It is also a region of great historical importance, being a) adjacent to the likely homeland from which Bantu-speaking people migrated across most of sub-Saharan Africa 3000-5000 years ago and b) the location of Calabar, one of the largest centres during the Atlantic slave trade. Over 1000 DNA samples from 24 clans representing speakers of the six most prominent languages in the region were collected and typed for Y-chromosome (SNPs and microsatellites) and mtDNA markers (Hypervariable Segment 1) in order to examine whether there has been substantial gene flow between groups speaking different languages in the region. In addition the Cross River region was analysed in the context of a larger geographical scale by comparison to bordering Igbo speaking groups as well as neighbouring Cameroon populations and more distant Ghanaian communities. Results The Cross River region was shown to be extremely homogenous for both Y-chromosome and mtDNA markers with language spoken having no noticeable effect on the genetic structure of the region, consistent with estimates of inter-language gene flow of 10% per generation based on sociological data. However the groups in the region could clearly be differentiated from others in Cameroon and Ghana (and to a lesser extent Igbo populations). Significant correlations between genetic distance and both geographic and linguistic distance were observed at this larger scale. Conclusions Previous studies have found significant correlations between genetic variation and language in Africa over large geographic distances, often across language families. However the broad sampling strategies of these datasets have limited their utility for understanding the relationship within language families. This is the first study to show that at very fine geographic/linguistic scales language differences can be maintained in the presence of substantial gene flow over an extended period of time and demonstrates the value of dense sampling strategies and having DNA of known and detailed provenance, a practice that is generally rare when investigating sub-Saharan African demographic processes using genetic data. PMID:20356404

A variable region within the genome of Streptococcus pneumoniae contributes to strain-strain variation in virulence.

PubMed

Harvey, Richard M; Stroeher, Uwe H; Ogunniyi, Abiodun D; Smith-Vaughan, Heidi C; Leach, Amanda J; Paton, James C

2011-05-05

The bacterial factors responsible for the variation in invasive potential between different clones and serotypes of Streptococcus pneumoniae are largely unknown. Therefore, the isolation of rare serotype 1 carriage strains in Indigenous Australian communities provided a unique opportunity to compare the genomes of non-invasive and invasive isolates of the same serotype in order to identify such factors. The human virulence status of non-invasive, intermediately virulent and highly virulent serotype 1 isolates was reflected in mice and showed that whilst both human non-invasive and highly virulent isolates were able to colonize the murine nasopharynx equally, only the human highly virulent isolates were able to invade and survive in the murine lungs and blood. Genomic sequencing comparisons between these isolates identified 8 regions >1 kb in size that were specific to only the highly virulent isolates, and included a version of the pneumococcal pathogenicity island 1 variable region (PPI-1v), phage-associated adherence factors, transporters and metabolic enzymes. In particular, a phage-associated endolysin, a putative iron/lead permease and an operon within PPI-1v exhibited niche-specific changes in expression that suggest important roles for these genes in the lungs and blood. Moreover, in vivo competition between pneumococci carrying PPI-1v derivatives representing the two identified versions of the region showed that the version of PPI-1v in the highly virulent isolates was more competitive than the version from the less virulent isolates in the nasopharyngeal tissue, blood and lungs. This study is the first to perform genomic comparisons between serotype 1 isolates with distinct virulence profiles that correlate between mice and humans, and has highlighted the important role that hypervariable genomic loci, such as PPI-1v, play in pneumococcal disease. The findings of this study have important implications for understanding the processes that drive progression from colonization to invasive disease and will help direct the development of novel therapeutic strategies.

Decreased mutation frequencies among immunoglobulin G variable region genes during viremic HIV-1 infection.

PubMed

Bowers, Elisabeth; Scamurra, Ronald W; Asrani, Anil; Beniguel, Lydie; MaWhinney, Samantha; Keays, Kathryne M; Thurn, Joseph R; Janoff, Edward N

2014-01-01

HIV-1 infection is complicated by high rates of opportunistic infections against which specific antibodies contribute to immune defense. Antibody function depends on somatic hypermutation (SHM) of variable regions of immunoglobulin heavy chain genes (VH-D-J). We characterized the frequency of SHM in expressed IgG mRNA immunoglobulin transcripts from control and HIV-1-infected patients. We compared utilization of genes in the most prominent VH family (VH3) and mutation frequencies and patterns of cDNA from VH3-IgG genes from 10 seronegative control subjects and 21 patients with HIV-1 infection (6 without and 15 patients with detectable plasma viremia). Unique IgG VH3 family cDNA sequences (n = 1,565) were PCR amplified, cloned, and sequenced from blood. Sequences were analyzed using online (Vbase) and in-house immunoglobulin alignment resources. Mutation frequencies in the antigen-binding hypervariable complementarity determining regions (CDR1/2) of IgG class-switched B cells were lower among viremic HIV-1-infected patients vs. controls for nucleotides (CDR1/2: 10±5% vs. 13.5±6%, p = 0.03) and amino acids (CDR: 20%±10 vs. 25%±12, p = 0.02) and in structural framework regions. Mutation patterns were similar among groups. The most common VH3 gene, VH3-23, was utilized less frequently among viremic HIV-1-infected patients (p = 0.03), and overall, mutation frequencies were decreased in nearly all VH3 genes compared with controls. B cells from HIV-1-infected patients show decreased mutation frequencies, especially in antigen-binding VH3 CDR genes, and selective defects in gene utilization. Similar mutation patterns suggest defects in the quantity, but not quality, of mutator activity. Lower levels of SHM in IgG class-switched B cells from HIV-1-infected patients may contribute to the increased risk of opportunistic infections and impaired humoral responses to preventative vaccines.

Slow-blue nuclear hypervariables in PanSTARRS-1

DOE PAGES

Lawrence, A.; Bruce, A. G.; MacLeod, C.; ...

2016-09-08

We discuss 76 large amplitude transients (Δm > 1.5) occurring in the nuclei of galaxies, nearly all with no previously known active galactic nucleus (AGN). They have been discovered as part of the Pan-STARRS1 (PS1) 3π survey, by comparison with Sloan Digital Sky Survey (SDSS) photometry a decade earlier, and then monitored with the Liverpool Telescope, and studied spectroscopically with the William Herschel Telescope (WHT). Based on colours, light-curve shape, and spectra, these transients fall into four groups. A few are misclassified stars or objects of unknown type. Some are red/fast transients and are known or likely nuclear supernovae. Amore » few are either radio sources or erratic variables and so likely blazars. However the majority (~66 per cent) are blue and evolve slowly, on a time-scale of years. Spectroscopy shows them to be AGN at z ~0.3 – 1.4, which must have brightened since the SDSS photometry by around an order of magnitude. It is likely that these objects were in fact AGN a decade ago, but too weak to be recognized by SDSS; they could then be classed as ‘hypervariable’ AGN. By searching the SDSS Stripe 82 quasar database, we find 15 similar objects. We discuss several possible explanations for these slow-blue hypervariables – (i) unusually luminous tidal disruption events; (ii) extinction events; (iii) changes in accretion state; and (iv) large amplitude microlensing by stars in foreground galaxies. A mixture of explanations (iii) and (iv) seems most likely. As a result, both hold promise of considerable new insight into the AGN phenomenon.« less

Mutation of CD2AP and SH3KBP1 Binding Motif in Alphavirus nsP3 Hypervariable Domain Results in Attenuated Virus.

PubMed

Mutso, Margit; Morro, Ainhoa Moliner; Smedberg, Cecilia; Kasvandik, Sergo; Aquilimeba, Muriel; Teppor, Mona; Tarve, Liisi; Lulla, Aleksei; Lulla, Valeria; Saul, Sirle; Thaa, Bastian; McInerney, Gerald M; Merits, Andres; Varjak, Margus

2018-04-27

Infection by Chikungunya virus (CHIKV) of the Old World alphaviruses (family Togaviridae) in humans can cause arthritis and arthralgia. The virus encodes four non-structural proteins (nsP) (nsP1, nsp2, nsP3 and nsP4) that act as subunits of the virus replicase. These proteins also interact with numerous host proteins and some crucial interactions are mediated by the unstructured C-terminal hypervariable domain (HVD) of nsP3. In this study, a human cell line expressing EGFP tagged with CHIKV nsP3 HVD was established. Using quantitative proteomics, it was found that CHIKV nsP3 HVD can bind cytoskeletal proteins, including CD2AP, SH3KBP1, CAPZA1, CAPZA2 and CAPZB. The interaction with CD2AP was found to be most evident; its binding site was mapped to the second SH3 ligand-like element in nsP3 HVD. Further assessment indicated that CD2AP can bind to nsP3 HVDs of many different New and Old World alphaviruses. Mutation of the short binding element hampered the ability of the virus to establish infection. The mutation also abolished ability of CD2AP to co-localise with nsP3 and replication complexes of CHIKV; the same was observed for Semliki Forest virus (SFV) harbouring a similar mutation. Similar to CD2AP, its homolog SH3KBP1 also bound the identified motif in CHIKV and SFV nsP3.

Slow-blue nuclear hypervariables in PanSTARRS-1

DOE Office of Scientific and Technical Information (OSTI.GOV)

Lawrence, A.; Bruce, A. G.; MacLeod, C.

We discuss 76 large amplitude transients (Δm > 1.5) occurring in the nuclei of galaxies, nearly all with no previously known active galactic nucleus (AGN). They have been discovered as part of the Pan-STARRS1 (PS1) 3π survey, by comparison with Sloan Digital Sky Survey (SDSS) photometry a decade earlier, and then monitored with the Liverpool Telescope, and studied spectroscopically with the William Herschel Telescope (WHT). Based on colours, light-curve shape, and spectra, these transients fall into four groups. A few are misclassified stars or objects of unknown type. Some are red/fast transients and are known or likely nuclear supernovae. Amore » few are either radio sources or erratic variables and so likely blazars. However the majority (~66 per cent) are blue and evolve slowly, on a time-scale of years. Spectroscopy shows them to be AGN at z ~0.3 – 1.4, which must have brightened since the SDSS photometry by around an order of magnitude. It is likely that these objects were in fact AGN a decade ago, but too weak to be recognized by SDSS; they could then be classed as ‘hypervariable’ AGN. By searching the SDSS Stripe 82 quasar database, we find 15 similar objects. We discuss several possible explanations for these slow-blue hypervariables – (i) unusually luminous tidal disruption events; (ii) extinction events; (iii) changes in accretion state; and (iv) large amplitude microlensing by stars in foreground galaxies. A mixture of explanations (iii) and (iv) seems most likely. As a result, both hold promise of considerable new insight into the AGN phenomenon.« less

Genetic analysis of ancestry, admixture and selection in Bolivian and Totonac populations of the New World

PubMed Central

2012-01-01

Background Populations of the Americas were founded by early migrants from Asia, and some have experienced recent genetic admixture. To better characterize the native and non-native ancestry components in populations from the Americas, we analyzed 815,377 autosomal SNPs, mitochondrial hypervariable segments I and II, and 36 Y-chromosome STRs from 24 Mesoamerican Totonacs and 23 South American Bolivians. Results and Conclusions We analyzed common genomic regions from native Bolivian and Totonac populations to identify 324 highly predictive Native American ancestry informative markers (AIMs). As few as 40–50 of these AIMs perform nearly as well as large panels of random genome-wide SNPs for predicting and estimating Native American ancestry and admixture levels. These AIMs have greater New World vs. Old World specificity than previous AIMs sets. We identify highly-divergent New World SNPs that coincide with high-frequency haplotypes found at similar frequencies in all populations examined, including the HGDP Pima, Maya, Colombian, Karitiana, and Surui American populations. Some of these regions are potential candidates for positive selection. European admixture in the Bolivian sample is approximately 12%, though individual estimates range from 0–48%. We estimate that the admixture occurred ~360–384 years ago. Little evidence of European or African admixture was found in Totonac individuals. Bolivians with pre-Columbian mtDNA and Y-chromosome haplogroups had 5–30% autosomal European ancestry, demonstrating the limitations of Y-chromosome and mtDNA haplogroups and the need for autosomal ancestry informative markers for assessing ancestry in admixed populations. PMID:22606979

Mitochondrial DNA diversity in two ethnic groups in southeastern Kenya: perspectives from the northeastern periphery of the Bantu expansion

PubMed Central

Batai, Ken; Babrowski, Kara B.; Arroyo, Juan Pablo; Kusimba, Chapurukha M.; Williams, Sloan R.

2013-01-01

The Bantu languages are widely distributed throughout sub-Saharan Africa. Genetic research supports linguists and historians who argue that migration played an important role in the spread of this language family, but the genetic data also indicates a more complex process involving substantial gene flow with resident populations. In order to understand the Bantu expansion process in east Africa, mtDNA hypervariable region I variation in 352 individuals from the Taita and Mijikenda ethnic groups was analyzed, and we evaluated the interactions that took place between the Bantu- and non-Bantu-speaking populations in east Africa. The Taita and Mijikenda are Bantu-speaking agropastoralists from southeastern Kenya, at least some of whose ancestors probably migrated into the area as part of Bantu migrations that began around 3,000 BCE. Our analyses indicate that they show some distinctive differences that reflect their unique cultural histories. The Taita are genetically more diverse than the Mijikenda with larger estimates of genetic diversity. The Taita cluster with other east African groups, having high frequencies of haplogroups from that region, while the Mijikenda have high frequencies of central African haplogroups and cluster more closely with central African Bantu-speaking groups. The non-Bantu speakers who lived in southeastern Kenya before Bantu speaking groups arrived were at least partially incorporated into what are now Bantu-speaking Taita groups. In contrast, gene flow from non-Bantu speakers into the Mijikenda was more limited. These results suggest a more complex demographic history where the nature of Bantu and non-Bantu interactions varied throughout the area. PMID:23382080

BCR CDR3 length distributions differ between blood and spleen and between old and young patients, and TCR distributions can be used to detect myelodysplastic syndrome

NASA Astrophysics Data System (ADS)

Pickman, Yishai; Dunn-Walters, Deborah; Mehr, Ramit

2013-10-01

Complementarity-determining region 3 (CDR3) is the most hyper-variable region in B cell receptor (BCR) and T cell receptor (TCR) genes, and the most critical structure in antigen recognition and thereby in determining the fates of developing and responding lymphocytes. There are millions of different TCR Vβ chain or BCR heavy chain CDR3 sequences in human blood. Even now, when high-throughput sequencing becomes widely used, CDR3 length distributions (also called spectratypes) are still a much quicker and cheaper method of assessing repertoire diversity. However, distribution complexity and the large amount of information per sample (e.g. 32 distributions of the TCRα chain, and 24 of TCRβ) calls for the use of machine learning tools for full exploration. We have examined the ability of supervised machine learning, which uses computational models to find hidden patterns in predefined biological groups, to analyze CDR3 length distributions from various sources, and distinguish between experimental groups. We found that (a) splenic BCR CDR3 length distributions are characterized by low standard deviations and few local maxima, compared to peripheral blood distributions; (b) healthy elderly people's BCR CDR3 length distributions can be distinguished from those of the young; and (c) a machine learning model based on TCR CDR3 distribution features can detect myelodysplastic syndrome with approximately 93% accuracy. Overall, we demonstrate that using supervised machine learning methods can contribute to our understanding of lymphocyte repertoire diversity.

Genetic origin, admixture, and asymmetry in maternal and paternal human lineages in Cuba

PubMed Central

2008-01-01

Background Before the arrival of Europeans to Cuba, the island was inhabited by two Native American groups, the Tainos and the Ciboneys. Most of the present archaeological, linguistic and ancient DNA evidence indicates a South American origin for these populations. In colonial times, Cuban Native American people were replaced by European settlers and slaves from Africa. It is still unknown however, to what extent their genetic pool intermingled with and was 'diluted' by the arrival of newcomers. In order to investigate the demographic processes that gave rise to the current Cuban population, we analyzed the hypervariable region I (HVS-I) and five single nucleotide polymorphisms (SNPs) in the mitochondrial DNA (mtDNA) coding region in 245 individuals, and 40 Y-chromosome SNPs in 132 male individuals. Results The Native American contribution to present-day Cubans accounted for 33% of the maternal lineages, whereas Africa and Eurasia contributed 45% and 22% of the lineages, respectively. This Native American substrate in Cuba cannot be traced back to a single origin within the American continent, as previously suggested by ancient DNA analyses. Strikingly, no Native American lineages were found for the Y-chromosome, for which the Eurasian and African contributions were around 80% and 20%, respectively. Conclusion While the ancestral Native American substrate is still appreciable in the maternal lineages, the extensive process of population admixture in Cuba has left no trace of the paternal Native American lineages, mirroring the strong sexual bias in the admixture processes taking place during colonial times. PMID:18644108

Mitochondrial DNA variability in the Titicaca basin: Matches and mismatches with linguistics and ethnohistory.

PubMed

Barbieri, Chiara; Heggarty, Paul; Castrì, Loredana; Luiselli, Donata; Pettener, Davide

2011-01-01

The Titicaca basin was the cradle of some of the major complex societies of pre-Columbian South America and is today home to three surviving native languages: Quechua, Aymara, and Uro. This study seeks to contribute to reconstructing the population prehistory of the region, by providing a first genetic profile of its inhabitants, set also into the wider context of South American genetic background. We report the first mitochondrial DNA first hypervariable segment sequences of native populations of the environs of Lake Titicaca: speakers of Aymara and Quechua, and the "Uros" of the Lake's floating islands. We sampled Aymara speakers from a locality where the Uro language was formerly documented, to check for possible language shift patterns. These data are compared with those for other Amerindian populations, collated from already published sources. Our results uncover the genetic distinctiveness of our formerly Uro but now Aymara-speaking sample, in contrast with a relative homogeneity for all the other Central Andean samples. The genetic affinities that characterize Central Andean populations are highly consistent with the succession of expansive polities in the region, culminating with the Incas. In the environs of Lake Titicaca, however, one subset of the present day Aymara-speaking population exhibits a peculiar position: perhaps a genetic correlate to their original Uro linguistic lineage (now extinct in the area), tallying with ethnohistorical claims for the distinctiveness of the Uro population. Our results emphasize the need for genetic descriptions to consider the widespread phenomenon of language shift. © 2010 Wiley-Liss, Inc.

Bacterial community comparisons by taxonomy-supervised analysis independent of sequence alignment and clustering

PubMed Central

Sul, Woo Jun; Cole, James R.; Jesus, Ederson da C.; Wang, Qiong; Farris, Ryan J.; Fish, Jordan A.; Tiedje, James M.

2011-01-01

High-throughput sequencing of 16S rRNA genes has increased our understanding of microbial community structure, but now even higher-throughput methods to the Illumina scale allow the creation of much larger datasets with more samples and orders-of-magnitude more sequences that swamp current analytic methods. We developed a method capable of handling these larger datasets on the basis of assignment of sequences into an existing taxonomy using a supervised learning approach (taxonomy-supervised analysis). We compared this method with a commonly used clustering approach based on sequence similarity (taxonomy-unsupervised analysis). We sampled 211 different bacterial communities from various habitats and obtained ∼1.3 million 16S rRNA sequences spanning the V4 hypervariable region by pyrosequencing. Both methodologies gave similar ecological conclusions in that β-diversity measures calculated by using these two types of matrices were significantly correlated to each other, as were the ordination configurations and hierarchical clustering dendrograms. In addition, our taxonomy-supervised analyses were also highly correlated with phylogenetic methods, such as UniFrac. The taxonomy-supervised analysis has the advantages that it is not limited by the exhaustive computation required for the alignment and clustering necessary for the taxonomy-unsupervised analysis, is more tolerant of sequencing errors, and allows comparisons when sequences are from different regions of the 16S rRNA gene. With the tremendous expansion in 16S rRNA data acquisition underway, the taxonomy-supervised approach offers the potential to provide more rapid and extensive community comparisons across habitats and samples. PMID:21873204

Oligonucleotide probes to the 16S ribosomal RNA: implications of sequence homology and secondary structure with particular reference to the oral species Prevotella intermedia and Prevotella nigrescens.

PubMed

Shah, H N; Gharbia, S E; Scully, C; Finegold, S M

1995-03-01

Eight oligonucleotides based upon regions of the small subunit 16S ribosomal RNA gene sequences were analysed against a background of their position within the molecule and their two-dimensional structure to rationalise their use in recognising Prevotella intermedia and Prevotella nigrescens. The 41 clinical isolates from both oral and respiratory sites and two reference strains were subjected to DNA-DNA hybridisation and multilocus enzyme electrophoresis to confirm their identity. Alignment of oligonucleotide probes designated I Bi-2 to I Bi-6 (for P. intermedia) and 2Bi-2 (for P. nigrescens) with the 16S rRNA suggested that these probes lacked specificity or were constructed from hypervariable regions. A 52-mer oligonucleotide (designated Bi) reliably detected both species. Because of the high degree of concordance between the 16S rRNAs of both species, it was necessary to vary the stringency of hybridisation conditions for detection of both species. Thus probe I Bi-I recognised P. intermedia while I Bi-I detected both P. intermedia and P. nigrescens at low stringency. However, under conditions of high stringency only P. nigrescens was recognised by probe 2Bi-I. These probes were highly specific and did not hybridise with DNA from the closely related P. corporis, nor other periodontal pathogens such as Fusobacterium nucleatum, Actinobacillus actinomycetemcomitans, Treponema denticola and several pigmented species such as Prevotella melaninogenica, P. denticola, P. loescheii, Porphyromonas asaccharolytica, Py. endodontalis, Py. gingivalis, Py. levii, and Py. macacae.

Glaciation Effects on the Phylogeographic Structure of Oligoryzomys longicaudatus (Rodentia: Sigmodontinae) in the Southern Andes

PubMed Central

Palma, R. Eduardo; Boric-Bargetto, Dusan; Torres-Pérez, Fernando; Hernández, Cristián E.; Yates, Terry L.

2012-01-01

The long-tailed pygmy rice rat Oligoryzomys longicaudatus (Sigmodontinae), the major reservoir of Hantavirus in Chile and Patagonian Argentina, is widely distributed in the Mediterranean, Temperate and Patagonian Forests of Chile, as well as in adjacent areas in southern Argentina. We used molecular data to evaluate the effects of the last glacial event on the phylogeographic structure of this species. We examined if historical Pleistocene events had affected genetic variation and spatial distribution of this species along its distributional range. We sampled 223 individuals representing 47 localities along the species range, and sequenced the hypervariable domain I of the mtDNA control region. Aligned sequences were analyzed using haplotype network, Bayesian population structure and demographic analyses. Analysis of population structure and the haplotype network inferred three genetic clusters along the distribution of O. longicaudatus that mostly agreed with the three major ecogeographic regions in Chile: Mediterranean, Temperate Forests and Patagonian Forests. Bayesian Skyline Plots showed constant population sizes through time in all three clusters followed by an increase after and during the Last Glacial Maximum (LGM; between 26,000–13,000 years ago). Neutrality tests and the “g” parameter also suggest that populations of O. longicaudatus experienced demographic expansion across the species entire range. Past climate shifts have influenced population structure and lineage variation of O. longicaudatus. This species remained in refugia areas during Pleistocene times in southern Temperate Forests (and adjacent areas in Patagonia). From these refugia, O. longicaudatus experienced demographic expansions into Patagonian Forests and central Mediterranean Chile using glacial retreats. PMID:22396751

Exploring prehistory in the North American southwest with mitochondrial DNA diversity exhibited by Yumans and Athapaskans.

PubMed

Monroe, Cara; Kemp, Brian M; Smith, David Glenn

2013-04-01

A recent study of mitochondrial DNA variation in Native American populations from the American Southwest detected signatures of a population expansion of subhaplogroup B2a, dated to 2,105 years before present (99.5% confidence interval, 1,273-3,773 YBP), following the introduction and intensification of maize agriculture in the region. Only one Yuman group and no Athapaskan speakers were analyzed in previous studies. Here we report mtDNA haplogroup and hypervariable region (HVR I, and II) sequence data from 263 extant Yuman speakers, representing the major branches of the Yuman language family, in addition to the Western Apache (Athapaskan) to further investigate the demographic context and geographic extent of this expansion. Data presented indicate that the expansion of B2a is only slightly older [2,410 YBP (99.5% CI: 1,458-4,320 YBP)] than previously estimated and not significantly. Despite large confidence intervals there are implications for the origin and expansion of the Yuman language family. Cultural transformations due to the inundation and draining of Lake Cahuilla may explain in part the frequencies of this lineage among the Kumeyaay and other Yuman and Takic groups in Southern California. This may have been the result of group fissions and fusions followed by migration and interaction that included expanded trade networks and intermarriage among Yuman speakers. In addition, a series of in-situ genetic bottlenecks is proposed to have occurred among the Western Apache leading to increasing homogeneity within haplogroup A, culminating in an admixture event with the Yavapai. Copyright © 2013 Wiley Periodicals, Inc.

mtDNA diversity in Azara's owl monkeys (Aotus azarai azarai) of the Argentinean Chaco.

PubMed

Babb, Paul L; Fernandez-Duque, Eduardo; Baiduc, Caitlin A; Gagneux, Pascal; Evans, Sian; Schurr, Theodore G

2011-10-01

Owl monkeys (Aotus spp.) inhabit much of South America yet represent an enigmatic evolutionary branch among primates. While morphological, cytogenetic, and immunological evidence suggest that owl monkey populations have undergone isolation and diversification since their emergence in the New World, problems with adjacent species ranges, and sample provenance have complicated efforts to characterize genetic variation within the genus. As a result, the phylogeographic history of owl monkey species and subspecies remains unclear, and the extent of genetic diversity at the population level is unknown. To explore these issues, we analyzed mitochondrial DNA (mt DNA) variation in a population of wild Azara's owl monkeys (Aotus azarai azarai) living in the Gran Chaco region of Argentina. We sequenced the complete mitochondrial genome from one individual (16,585 base pairs (bp)) and analyzed 1,099 bp of the hypervariable control region (CR) and 696 bp of the cytochrome oxidase II (COII) gene in 117 others. In addition, we sequenced the mitochondrial genome (16,472 bp) of one Nancy Ma's owl monkey (A. nancymaae). Based on the whole mtDNA and COII data, we observed an ancient phylogeographic discontinuity among Aotus species living north, south, and west of the Amazon River that began more than eight million years ago. Our population analyses identified three major CR lineages and detected a high level of haplotypic diversity within A. a. azarai. These data point to a recent expansion of Azara's owl monkeys into the Argentinean Chaco. Overall, we provide a detailed view of owl monkey mtDNA variation at genus, species, and population levels. Copyright © 2011 Wiley-Liss, Inc.

Hexon-modified recombinant E1-deleted adenoviral vectors as bivalent vaccine carriers for Coxsackievirus A16 and Enterovirus 71.

PubMed

Zhang, Chao; Yang, Yong; Chi, Yudan; Yin, Jieyun; Yan, Lijun; Ku, Zhiqiang; Liu, Qingwei; Huang, Zhong; Zhou, Dongming

2015-09-22

Hand, foot and mouth disease (HFMD) is a major public health concern in Asia; more efficient vaccines against HFMD are urgently required. Adenoviral (Ad) capsids have been used widely for the presentation of foreign antigens to induce specific immune responses in the host. Here, we describe a novel bivalent vaccine for HFMD based on the hexon-modified, E1-deleted chimpanzee adenovirus serotype 68 (AdC68). The novel vaccine candidate was generated by incorporating the neutralising epitope of Coxsackievirus A16 (CA16), PEP71, into hypervariable region 1 (HVR1), and a shortened neutralising epitope of Enterovirus 71 (EV71), sSP70, into HVR2 of the AdC68 hexon. In order to enhance the immunogenicity of EV71, VP1 of EV71 was cloned into the E1-region of the AdC68 vectors. The results demonstrated that these two epitopes were well presented on the virion surface and had high affinity towards specific antibodies, and VP1 of EV71 was also significantly expressed. In pre-clinical mouse models, the hexon-modified AdC68 elicited neutralising antibodies against both CA16 and EV71, which conferred protection to suckling mice against a lethal challenge of CA16 and EV71. In summary, this study demonstrates that the hexon-modified AdC68 may represent a promising bivalent vaccine carrier against EV71 and CA16 and an epitope-display platform for other pathogens. Copyright © 2015 Elsevier Ltd. All rights reserved.

Molecular Identification of Unusual Pathogenic Yeast Isolates by Large Ribosomal Subunit Gene Sequencing: 2 Years of Experience at the United Kingdom Mycology Reference Laboratory▿

PubMed Central

Linton, Christopher J.; Borman, Andrew M.; Cheung, Grace; Holmes, Ann D.; Szekely, Adrien; Palmer, Michael D.; Bridge, Paul D.; Campbell, Colin K.; Johnson, Elizabeth M.

2007-01-01

Rapid identification of yeast isolates from clinical samples is particularly important given their innately variable antifungal susceptibility profiles. We present here an analysis of the utility of PCR amplification and sequence analysis of the hypervariable D1/D2 region of the 26S rRNA gene for the identification of yeast species submitted to the United Kingdom Mycology Reference Laboratory over a 2-year period. A total of 3,033 clinical isolates were received from 2004 to 2006 encompassing 50 different yeast species. While more than 90% of the isolates, corresponding to the most common Candida species, could be identified by using the AUXACOLOR2 yeast identification kit, 153 isolates (5%), comprised of 47 species, could not be identified by using this system and were subjected to molecular identification via 26S rRNA gene sequencing. These isolates included some common species that exhibited atypical biochemical and phenotypic profiles and also many rarer yeast species that are infrequently encountered in the clinical setting. All 47 species requiring molecular identification were unambiguously identified on the basis of D1/D2 sequences, and the molecular identities correlated well with the observed biochemical profiles of the various organisms. Together, our data underscore the utility of molecular techniques as a reference adjunct to conventional methods of yeast identification. Further, we show that PCR amplification and sequencing of the D1/D2 region reliably identifies more than 45 species of clinically significant yeasts and can also potentially identify new pathogenic yeast species. PMID:17251397

Glaciation effects on the phylogeographic structure of Oligoryzomys longicaudatus (Rodentia: Sigmodontinae) in the southern Andes.

PubMed

Palma, R Eduardo; Boric-Bargetto, Dusan; Torres-Pérez, Fernando; Hernández, Cristián E; Yates, Terry L

2012-01-01

The long-tailed pygmy rice rat Oligoryzomys longicaudatus (Sigmodontinae), the major reservoir of Hantavirus in Chile and Patagonian Argentina, is widely distributed in the Mediterranean, Temperate and Patagonian Forests of Chile, as well as in adjacent areas in southern Argentina. We used molecular data to evaluate the effects of the last glacial event on the phylogeographic structure of this species. We examined if historical Pleistocene events had affected genetic variation and spatial distribution of this species along its distributional range. We sampled 223 individuals representing 47 localities along the species range, and sequenced the hypervariable domain I of the mtDNA control region. Aligned sequences were analyzed using haplotype network, bayesian population structure and demographic analyses. Analysis of population structure and the haplotype network inferred three genetic clusters along the distribution of O. longicaudatus that mostly agreed with the three major ecogeographic regions in Chile: Mediterranean, Temperate Forests and Patagonian Forests. Bayesian Skyline Plots showed constant population sizes through time in all three clusters followed by an increase after and during the Last Glacial Maximum (LGM; between 26,000-13,000 years ago). Neutrality tests and the "g" parameter also suggest that populations of O. longicaudatus experienced demographic expansion across the species entire range. Past climate shifts have influenced population structure and lineage variation of O. longicaudatus. This species remained in refugia areas during Pleistocene times in southern Temperate Forests (and adjacent areas in Patagonia). From these refugia, O. longicaudatus experienced demographic expansions into Patagonian Forests and central Mediterranean Chile using glacial retreats.

Artemisia supplementation differentially affects the mucosal and luminal ileal microbiota of diet-induced obese mice

PubMed Central

Shawna, Wicks; M., Taylor Christopher; Meng, Luo; Eugene, Blanchard IV; David, Ribnicky; T., Cefalu William; L., Mynatt Randall; A., Welsh David

2014-01-01

Objective The gut microbiome has been implicated in obesity and metabolic syndrome; however, most studies have focused on fecal or colonic samples. Several species of Artemisia have been reported to ameliorate insulin signaling both in vitro and in vivo. The aim of this study was to characterize the mucosal and luminal bacterial populations in the terminal ileum with or without supplementation with Artemisia extracts. Materials/Methods Following 4 weeks of supplementation with different Artemisia extracts (PMI 5011, Santa or Scopa), diet-induced obese mice were sacrificed and luminal and mucosal samples of terminal ileum were used to evaluate microbial community composition by pyrosequencing of 16S rDNA hypervariable regions. Results Significant differences in community structure and membership were observed between luminal and mucosal samples, irrespective of diet group. All Artemisia extracts increased the Bacteroidetes:Firmicutes ratio in mucosal samples. This effect was not observed in the luminal compartment. There was high inter-individual variability in the phylogenetic assessments of the ileal microbiota, limiting the statistical power of this pilot investigation. Conclusions Marked differences in bacterial communities exist dependent upon the biogeographic compartment in the terminal ileum. Future studies testing the effects of Artemisia or other botanical supplements require larger sample sizes for adequate statistical power. PMID:24985102

A 28,000 Years Old Cro-Magnon mtDNA Sequence Differs from All Potentially Contaminating Modern Sequences

PubMed Central

Caramelli, David; Milani, Lucio; Vai, Stefania; Modi, Alessandra; Pecchioli, Elena; Girardi, Matteo; Pilli, Elena; Lari, Martina; Lippi, Barbara; Ronchitelli, Annamaria; Mallegni, Francesco; Casoli, Antonella; Bertorelle, Giorgio; Barbujani, Guido

2008-01-01

Background DNA sequences from ancient speciments may in fact result from undetected contamination of the ancient specimens by modern DNA, and the problem is particularly challenging in studies of human fossils. Doubts on the authenticity of the available sequences have so far hampered genetic comparisons between anatomically archaic (Neandertal) and early modern (Cro-Magnoid) Europeans. Methodology/Principal Findings We typed the mitochondrial DNA (mtDNA) hypervariable region I in a 28,000 years old Cro-Magnoid individual from the Paglicci cave, in Italy (Paglicci 23) and in all the people who had contact with the sample since its discovery in 2003. The Paglicci 23 sequence, determined through the analysis of 152 clones, is the Cambridge reference sequence, and cannot possibly reflect contamination because it differs from all potentially contaminating modern sequences. Conclusions/Significance: The Paglicci 23 individual carried a mtDNA sequence that is still common in Europe, and which radically differs from those of the almost contemporary Neandertals, demonstrating a genealogical continuity across 28,000 years, from Cro-Magnoid to modern Europeans. Because all potential sources of modern DNA contamination are known, the Paglicci 23 sample will offer a unique opportunity to get insight for the first time into the nuclear genes of early modern Europeans. PMID:18628960

High mitochondrial mutation rates estimated from deep-rooting Costa Rican pedigrees

PubMed Central

Madrigal, Lorena; Melendez-Obando, Mauricio; Villegas-Palma, Ramon; Barrantes, Ramiro; Raventos, Henrieta; Pereira, Reynaldo; Luiselli, Donata; Pettener, Davide; Barbujani, Guido

2012-01-01

Estimates of mutation rates for the noncoding hypervariable Region I (HVR-I) of mitochondrial DNA (mtDNA) vary widely, depending on whether they are inferred from phylogenies (assuming that molecular evolution is clock-like) or directly from pedigrees. All pedigree-based studies so far were conducted on populations of European origin. In this paper we analyzed 19 deep-rooting pedigrees in a population of mixed origin in Costa Rica. We calculated two estimates of the HVR-I mutation rate, one considering all apparent mutations, and one disregarding changes at sites known to be mutational hot spots and eliminating genealogy branches which might be suspected to include errors, or unrecognized adoptions along the female lines. At the end of this procedure, we still observed a mutation rate equal to 1.24 × 10−6, per site per year, i.e., at least three-fold as high as estimates derived from phylogenies. Our results confirm that mutation rates observed in pedigrees are much higher than estimated assuming a neutral model of long-term HVRI evolution. We argue that, until the cause of these discrepancies will be fully understood, both lower estimates (i.e., those derived from phylogenetic comparisons) and higher, direct estimates such as those obtained in this study, should be considered when modeling evolutionary and demographic processes. PMID:22460349

Benthic protists: the under-charted majority.

PubMed

Forster, Dominik; Dunthorn, Micah; Mahé, Fréderic; Dolan, John R; Audic, Stéphane; Bass, David; Bittner, Lucie; Boutte, Christophe; Christen, Richard; Claverie, Jean-Michel; Decelle, Johan; Edvardsen, Bente; Egge, Elianne; Eikrem, Wenche; Gobet, Angélique; Kooistra, Wiebe H C F; Logares, Ramiro; Massana, Ramon; Montresor, Marina; Not, Fabrice; Ogata, Hiroyuki; Pawlowski, Jan; Pernice, Massimo C; Romac, Sarah; Shalchian-Tabrizi, Kamran; Simon, Nathalie; Richards, Thomas A; Santini, Sébastien; Sarno, Diana; Siano, Raffaele; Vaulot, Daniel; Wincker, Patrick; Zingone, Adriana; de Vargas, Colomban; Stoeck, Thorsten

2016-08-01

Marine protist diversity inventories have largely focused on planktonic environments, while benthic protists have received relatively little attention. We therefore hypothesize that current diversity surveys have only skimmed the surface of protist diversity in marine sediments, which may harbor greater diversity than planktonic environments. We tested this by analyzing sequences of the hypervariable V4 18S rRNA from benthic and planktonic protist communities sampled in European coastal regions. Despite a similar number of OTUs in both realms, richness estimations indicated that we recovered at least 70% of the diversity in planktonic protist communities, but only 33% in benthic communities. There was also little overlap of OTUs between planktonic and benthic communities, as well as between separate benthic communities. We argue that these patterns reflect the heterogeneity and diversity of benthic habitats. A comparison of all OTUs against the Protist Ribosomal Reference database showed that a higher proportion of benthic than planktonic protist diversity is missing from public databases; similar results were obtained by comparing all OTUs against environmental references from NCBI's Short Read Archive. We suggest that the benthic realm may therefore be the world's largest reservoir of marine protist diversity, with most taxa at present undescribed. © FEMS 2016. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.

Narrowing the position of the Treacher Collins syndrome locus to a small interval between three new microsatellite markers at 5q32-33. 1

DOE Office of Scientific and Technical Information (OSTI.GOV)

Dixon, M.J.; Dixon, J.; Houseal, T.

Treacher Collins syndrome (TCOF1) is an autosomal dominant disorder of craniofacial development, the features of which include conductive hearing loss and cleft palate. The TCOF1 locus has been localized to chromosome 5q32-33.2. In the present study the authors have used the combined techniques of genetic linkage analysis and fluorescence in situ hybridization (FISH) to more accurately define the TCOF1 critical region. Cosmids IG90 and SPARC, which map to distal 5q, encompass two and one hypervariable microsatellite markers, respectively. The heterozygosity values of these three markers range from .72 to .81. Twenty-two unrelated TCOF1 families have been analyzed for linkage tomore » these markers. There is strong evidence demonstrating linkage to all three markers, the strongest support for positive linkage being provided by haplotyping those markers at the locus encompassed by the cosmid IG90 (Z[sub max]= 19.65; 0 = .010). FISH to metaphase chromosomes and interphase nuclei established that IG90 lies centromeric to SPARC. This information combined with the data generated by genetic linkage analysis demonstrated that the TCOF1 locus is closely flanked proximally by IG90 and distally by SPARC. 30 refs., 2 figs., 4 tabs.« less

Ecological factors rather than temporal factors dominate the evolution of vesicular stomatitis virus.

PubMed

Rodríguez, L L; Fitch, W M; Nichol, S T

1996-11-12

Vesicular stomatitis New Jersey virus (VSV-NJ) is a rhabdovirus that causes economically important disease in cattle and other domestic animals in endemic areas from southeastern United States to northern South America. Its negatively stranded RNA genome is capable of undergoing rapid evolution, which allows phylogenetic analysis and molecular epidemiology studies to be performed. Previous epidemiological studies in Costa Rica showed the existence of at least two distinct ecological zones of high VSV-NJ activity, one located in the highlands (premontane tropical moist forest) and the other in the lowlands (tropical dry forest). We wanted to test the hypothesis that the viruses circulating in these ecological zones were genetically distinct. For this purpose, we sequenced the hypervariable region of the phosphoprotein gene for 50 VSV-NJ isolates from these areas. Phylogenetic analysis showed that viruses from each ecological zone had distinct genotypes. These genotypes were maintained in each area for periods of up to 8 years. This evolutionary pattern of VSV-NJ suggests an adaptation to ecological factors that could exert selective pressure on the virus. As previous data indicated an absence of virus adaptation to factors related to the bovine host (including immunological pressure), it appears that VSV genetic divergence represents positive selection to adapt to specific vectors and/or reservoirs at each ecological zone.

Dysbiosis of oral buccal mucosa microbiota in patients with oral lichen planus.

PubMed

He, Y; Gong, D; Shi, C; Shao, F; Shi, J; Fei, J

2017-07-01

The bacterial community structure of buccal mucosa in patients with oral lichen planus was evaluated and compared with healthy control. Buccal scraping samples have been taken on 43 oral lichen planus patients (21 erosive and 22 non-erosive) and 21 mucosal healthy volunteers. The V3 hypervariable 16S rDNA region was amplified and sequenced by high-throughput 454 pyrosequencing. 94.26% of the total buccal bacteria were classified into 15 abundant genera. Eight of these abundant genera could be detected in all cases, namely Streptococcus, Prevotella, Haemophilu, Neisseria, Fusobacterium, Leptotrichia, Veillonella and Actinomyces. Four abundant bacteria showed significantly different prevalence at the genus level: Streptococcus was more abundant (P < 0.01) in healthy control group, while Fusobacterium (P < 0.01), Leptotrichia (P < 0.001) and Lautropia (P < 0.001) showed higher abundance in OLP group. Few differences can be found between erosive and non-erosive OLP. In general, 19 'core' OTUs at three levels were defined to be OLP specific, comparing with healthy control group. These results suggest that OLP is associated with dysbiosis of the oral microbiome. Further studies should be taken to elucidate the inner relationship between these observed changes and OLP development. © 2017 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

Occurrence of potentially pathogenic nontuberculous mycobacteria in Mexican household potable water: a pilot study.

PubMed

Perez-Martinez, Iza; Aguilar-Ayala, Diana A; Fernandez-Rendon, Elizabeth; Carrillo-Sanchez, Alma K; Helguera-Repetto, Addy C; Rivera-Gutierrez, Sandra; Estrada-Garcia, Teresa; Cerna-Cortes, Jorge F; Gonzalez-Y-Merchand, Jorge A

2013-12-11

Nontuberculous mycobacteria (NTM) are environmental opportunistic pathogens found in natural and human-engineered waters, including drinking water distribution systems and household plumbing. This pilot study examined the frequency of occurrence of NTM in household potable water samples in Mexico City. Potable water samples were collected from the "main house faucet" and kitchen faucet. The presence of aerobic-mesophilic bacteria (AMB), total coliforms (TC), fecal coliforms (FC) and NTM species were determined. Mycobacteria species were identified by PCR restriction enzyme pattern analysis (PRA) of the 65-kDa heat shock protein gene (hsp65) and sequencing of the hypervariable region 2 (V2) of the 16S rRNA gene and of the rpoB gene. AMB (<100 CFU/ml) were present in 118 out of 120 samples; only two samples were outside guidelines ranges (>100 CFU/ml). TC and FC were detected in four and one samples, respectively. NTM species were recovered from 16% samples (19/120) and included M. mucogenicum (nine), M. porcinum (three), M. avium (three), M. gordonae (one), M. cosmeticum (one), M. fortuitum (one), and Mycobacterium sp (one). All household water samples that contained NTM complied with the standards required to grade the water as "good quality" potable water. Household potable water may be a potential source of NTM infection in Mexico City.

Origins and evolution of the Etruscans' mtDNA.

PubMed

Ghirotto, Silvia; Tassi, Francesca; Fumagalli, Erica; Colonna, Vincenza; Sandionigi, Anna; Lari, Martina; Vai, Stefania; Petiti, Emmanuele; Corti, Giorgio; Rizzi, Ermanno; De Bellis, Gianluca; Caramelli, David; Barbujani, Guido

2013-01-01

The Etruscan culture is documented in Etruria, Central Italy, from the 8(th) to the 1(st) century BC. For more than 2,000 years there has been disagreement on the Etruscans' biological origins, whether local or in Anatolia. Genetic affinities with both Tuscan and Anatolian populations have been reported, but so far all attempts have failed to fit the Etruscans' and modern populations in the same genealogy. We extracted and typed the hypervariable region of mitochondrial DNA of 14 individuals buried in two Etruscan necropoleis, analyzing them along with other Etruscan and Medieval samples, and 4,910 contemporary individuals from the Mediterranean basin. Comparing ancient (30 Etruscans, 27 Medieval individuals) and modern DNA sequences (370 Tuscans), with the results of millions of computer simulations, we show that the Etruscans can be considered ancestral, with a high degree of confidence, to the current inhabitants of Casentino and Volterra, but not to the general contemporary population of the former Etruscan homeland. By further considering two Anatolian samples (35 and 123 individuals) we could estimate that the genetic links between Tuscany and Anatolia date back to at least 5,000 years ago, strongly suggesting that the Etruscan culture developed locally, and not as an immediate consequence of immigration from the Eastern Mediterranean shores.

Mapping the Laminin Receptor Binding Domains of Neisseria meningitidis PorA and Haemophilus influenzae OmpP2

PubMed Central

Mahdavi, Jafar; Oldfield, Neil J.; Wheldon, Lee M.; Wooldridge, Karl G.; Ala'Aldeen, Dlawer A. A.

2012-01-01

Neisseria meningitidis, Haemophilus influenzae and Streptococcus pneumoniae are major bacterial agents of meningitis. They each bind the 37/67-kDa laminin receptor (LamR) via the surface protein adhesins: meningococcal PilQ and PorA, H. influenzae OmpP2 and pneumococcal CbpA. We have previously reported that a surface-exposed loop of the R2 domain of CbpA mediates LamR-binding. Here we have identified the LamR-binding regions of PorA and OmpP2. Using truncated recombinant proteins we show that binding is dependent on amino acids 171–240 and 91–99 of PorA and OmpP2, respectively, which are predicted to localize to the fourth and second surface-exposed loops, respectively, of these proteins. Synthetic peptides corresponding to the loops bound LamR and could block LamR-binding to bacterial ligands in a dose dependant manner. Meningococci expressing PorA lacking the apex of loop 4 and H. influenzae expressing OmpP2 lacking the apex of loop 2 showed significantly reduced LamR binding. Since both loops are hyper-variable, our data may suggest a molecular basis for the range of LamR-binding capabilities previously reported among different meningococcal and H. influenzae strains. PMID:23049988

Molecular characterization of allelic variants of (GATA)n microsatellite loci in parthenogenetic lizards Darevskia unisexualis (Lacertidae).

PubMed

Korchagin, V I; Badaeva, T N; Tokarskaya, O N; Martirosyan, I A; Darevsky, I S; Ryskov, A P

2007-05-01

Populations of parthenogenetic lizards of the genus Darevskia consist of genetically identical animals, and represent a unique model for studying the molecular mechanisms underlying the variability and evolution of hypervariable DNA repeats. As unisexual lineages, parthenogenetic lizards are characterized by some level of genetic diversity at microsatellite loci. We cloned and sequenced a number of (GATA)n microsatellite loci of Darevskia unisexualis. PCR products from these loci were also sequenced and the degree of intraspecific polymorphism was assessed. Among the five (GATA)n loci analysed, two (Du215 and Du281) were polymorphic. Cross-species analysis of Du215 and Du281 indicate that the priming sites at the D. unisexualis loci are conserved in the bisexual parental species, D. raddei and D. valentini. Sequencing the PCR products amplified from Du215 and Du281 and from monomorphic Du323 showed that allelic differences at the polymorphic loci are caused by microsatellite mutations and by point mutations in the flanking regions. The haplotypes identified among the allelic variants of Du281 and among its orthologues in the parental species provide new evidence of the cross-species origin of D. unisexualis. To our knowledge, these data are the first to characterize the nucleotide sequences of allelic variants at microsatellite loci within parthenogenetic vertebrate animals.

Common genomic features of Campylobacter jejuni subsp. doylei strains distinguish them from C. jejuni subsp. jejuni

PubMed Central

Parker, Craig T; Miller, William G; Horn, Sharon T; Lastovica, Albert J

2007-01-01

Background Campylobacter jejuni has been divided into two subspecies: C. jejuni subsp. jejuni (Cjj) and C. jejuni subsp. doylei (Cjd). Nearly all of the C. jejuni strains isolated are Cjj; nevertheless, although Cjd strains are isolated infrequently, they differ from Cjj in two key aspects: they are obtained primarily from human clinical samples and are associated often with bacteremia, in addition to gastroenteritis. In this study, we utilized multilocus sequence typing (MLST) and a DNA microarray-based comparative genomic indexing (CGI) approach to examine the genomic diversity and gene content of Cjd strains. Results A geographically diverse collection of eight Cjd strains was examined by MLST and determined to be phylogenetically distinct from Cjj strains. Microarray-based CGI approach also supported this. We were able to demonstrate that Cjd strains exhibited divergence from Cjj strains NCTC 11168 and RM1221 in many of the intraspecies hypervariable regions. Moreover, multiple metabolic, transport and virulence functions (e.g. cytolethal distending toxin) were shown to be absent in the Cjd strains examined. Conclusion Our data demonstrate that Cjd are phylogenetically distinct from Cjj strains. Using the CGI approach, we identified subsets of absent genes from amongst the C. jejuni genes that provide clues as to the potential evolutionary origin and unusual pathogenicity of Cjd. PMID:17535437

Maternal Genetic Ancestry and Legacy of 10th Century AD Hungarians

NASA Astrophysics Data System (ADS)

Csősz, Aranka; Szécsényi-Nagy, Anna; Csákyová, Veronika; Langó, Péter; Bódis, Viktória; Köhler, Kitti; Tömöry, Gyöngyvér; Nagy, Melinda; Mende, Balázs Gusztáv

2016-09-01

The ancient Hungarians originated from the Ural region in today’s central Russia and migrated across the Eastern European steppe, according to historical sources. The Hungarians conquered the Carpathian Basin 895-907 AD, and admixed with the indigenous communities. Here we present mitochondrial DNA results from three datasets: one from the Avar period (7th-9th centuries) of the Carpathian Basin (n = 31) one from the Hungarian conquest-period (n = 76) and a completion of the published 10th-12th century Hungarian-Slavic contact zone dataset by four samples. We compare these mitochondrial DNA hypervariable segment sequences and haplogroup results with published ancient and modern Eurasian data. Whereas the analyzed Avars represents a certain group of the Avar society that shows East and South European genetic characteristics, the Hungarian conquerors’ maternal gene pool is a mixture of West Eurasian and Central and North Eurasian elements. Comprehensively analyzing the results, both the linguistically recorded Finno-Ugric roots and historically documented Turkic and Central Asian influxes had possible genetic imprints in the conquerors’ genetic composition. Our data allows a complex series of historic and population genetic events before the formation of the medieval population of the Carpathian Basin, and the maternal genetic continuity between 10th-12th century and modern Hungarians.

[Application of multiple polymorphism genetic markers in determination of half sibling sharing a same mother].

PubMed

Que, Ting-zhi; Zhao, Shu-min; Li, Cheng-tao

2010-08-01

Determination strategies for half sibling sharing a same mother were investigated through the detection of autosomal and X-chromosomal STR (X-STR) loci and polymorphisms on hypervariable (HV) region of mitochondrial DNA (mtDNA). Genomic DNA were extracted from blood stain samples of the 3 full siblings and one dubious half sibling sharing the same mother with them. Fifteen autosomal STR loci were genotyped by Sinofiler kit, and 19 X-STR loci were genotyped by Mentype Argus X-8 kit and 16 plex in-house system. Polymorphisms of mtDNA HV-I and HV-II were also detected with sequencing technology. Full sibling relationship between the dubious half sibling and each of the 3 full siblings were excluded based on the results of autosomal STR genotyping and calculation of full sibling index (FSI) and half sibling index (HIS). Results of sequencing for mtDNA HV-I and HV-II showed that all of the 4 samples came from a same maternal line. X-STR genotyping results determined that the dubious half sibling shared a same mother with the 3 full siblings. It is reliable to combine three different genotyping technologies including autosomal STR, X-STR and sequencing of mtDNA HV-I and HV-II for determination of half sibling sharing a same mother.

Maternal Genetic Ancestry and Legacy of 10(th) Century AD Hungarians.

PubMed

Csősz, Aranka; Szécsényi-Nagy, Anna; Csákyová, Veronika; Langó, Péter; Bódis, Viktória; Köhler, Kitti; Tömöry, Gyöngyvér; Nagy, Melinda; Mende, Balázs Gusztáv

2016-09-16

The ancient Hungarians originated from the Ural region in today's central Russia and migrated across the Eastern European steppe, according to historical sources. The Hungarians conquered the Carpathian Basin 895-907 AD, and admixed with the indigenous communities. Here we present mitochondrial DNA results from three datasets: one from the Avar period (7(th)-9(th) centuries) of the Carpathian Basin (n = 31); one from the Hungarian conquest-period (n = 76); and a completion of the published 10(th)-12(th) century Hungarian-Slavic contact zone dataset by four samples. We compare these mitochondrial DNA hypervariable segment sequences and haplogroup results with published ancient and modern Eurasian data. Whereas the analyzed Avars represents a certain group of the Avar society that shows East and South European genetic characteristics, the Hungarian conquerors' maternal gene pool is a mixture of West Eurasian and Central and North Eurasian elements. Comprehensively analyzing the results, both the linguistically recorded Finno-Ugric roots and historically documented Turkic and Central Asian influxes had possible genetic imprints in the conquerors' genetic composition. Our data allows a complex series of historic and population genetic events before the formation of the medieval population of the Carpathian Basin, and the maternal genetic continuity between 10(th)-12(th) century and modern Hungarians.

Intracellular mature IL-37 suppresses tumor metastasis via inhibiting Rac1 activation.

PubMed

Li, Y; Zhao, M; Guo, C; Chu, H; Li, W; Chen, X; Wang, X; Li, Y; Jia, Y; Koussatidjoa, S; Zhu, F; Wang, J; Wang, X; Wang, Q; Zhao, W; Shi, Y; Chen, W; Zhang, L

2018-02-22

IL-37, a newly found anti-inflammatory cytokine of the IL-1 family, has both extracellular and intracellular functions. Accumulating evidences indicate that it is also involved in tumor progression. However, the mechanism and its intracellular target are unclear. In this study, clinical data from 84 patients showed that loss or reduced expression of IL-37 in lung adenocarcinoma tissues was significantly associated with tumor metastasis. We further provided evidence that IL-37 inhibited effectively tumor metastasis in vitro and in vivo. Moreover, we uncovered a novel mechanism by which IL-37 suppressed tumor cell migration via its intracellular mature form (amino acids 46-218). Intracellular mature form of IL-37, but not its extracellular form, markedly inhibited migration of multiple kinds of tumor cells through inhibiting Rac1 activation. Mechanistically, intracellular mature IL-37 directly bound to the CAAX motif in the C-terminal hypervariable region of Rac1, and then inhibited Rac1 membrane translocation and subsequent downstream signaling. Our research identifies intracellular mature IL-37 as a novel endogenous inhibitor of Rac1. Given the crucial roles of Rac1 in tumor angiogenesis and metastasis, intracellular mature IL-37 might serve as a potential strategy for the control of Rac1 activity and tumor progression.

Nanoparticle-based strategy for personalized B-cell lymphoma therapy

PubMed Central

Martucci, Nicola M; Migliaccio, Nunzia; Ruggiero, Immacolata; Albano, Francesco; Calì, Gaetano; Romano, Simona; Terracciano, Monica; Rea, Ilaria; Arcari, Paolo; Lamberti, Annalisa

2016-01-01

B-cell lymphoma is associated with incomplete response to treatment, and the development of effective strategies targeting this disease remains challenging. A new personalized B-cell lymphoma therapy, based on a site-specific receptor-mediated drug delivery system, was developed in this study. Specifically, natural silica-based nanoparticles (diatomite) were modified to actively target the antiapoptotic factor B-cell lymphoma/leukemia 2 (Bcl2) with small interfering RNA (siRNA). An idiotype-specific peptide (Id-peptide) specifically recognized by the hypervariable region of surface immunoglobulin B-cell receptor was exploited as a homing device to ensure specific targeting of lymphoma cells. Specific nanoparticle uptake, driven by the Id-peptide, was evaluated by flow cytometry and confocal microscopy and was increased by approximately threefold in target cells compared with nonspecific myeloma cells and when a random control peptide was used instead of Id-peptide. The specific internalization efficiency was increased by fourfold when siRNA was also added to the modified nanoparticles. The modified diatomite particles were not cytotoxic and their effectiveness in downregulation of gene expression was explored using siRNA targeting Bcl2 and evaluated by quantitative real-time polymerase chain reaction and Western blot analyses. The resulting gene silencing observed is of significant biological importance and opens new possibilities for the personalized treatment of lymphomas. PMID:27895482

High Rhodotorula sequences in skin transcriptome of patients with diffuse systemic sclerosis

PubMed Central

Arron, Sarah T.; Dimon, Michelle T.; Li, Zhenghui; Johnson, Michael E.; Wood, Tammara; Feeney, Luzviminda; Angeles, Jorge Gil; Lafyatis, Robert; Whitfield, Michael L.

2014-01-01

Previous studies have suggested a role for pathogens as a trigger of systemic sclerosis (SSc), though neither a pathogen nor a mechanism of pathogenesis is known. Here we show enrichment of Rhodotorula sequences in the skin of patients with early, diffuse SSc compared to normal controls. RNA-seq was performed on four SSc and four controls, to a depth of 200 million reads per patient. Data were analyzed to quantify the non-human sequence reads in each sample. We found little difference between bacterial microbiome and viral read counts, but found a significant difference between the read counts for a mycobiome component, R. glutinis. Normal samples contained almost no detected R. glutinis or other Rhodotorula sequence reads (mean score 0.021 for R. glutinis, 0.024 for all Rhodotorula). In contrast, SSc samples had a mean score of 5.039 for R. glutinis (5.232 for Rhodotorula). We were able to assemble the D1–D2 hypervariable region of the 28S rRNA of R. glutinis from each of the SSc samples. Taken together, these results suggest R. glutinis may be present in the skin of early SSc patients at higher levels than normal skin, raising the possibility that it may be triggering the inflammatory response found in SSc. PMID:24608988

High Rhodotorula sequences in skin transcriptome of patients with diffuse systemic sclerosis.

PubMed

Arron, Sarah T; Dimon, Michelle T; Li, Zhenghui; Johnson, Michael E; Wood, Tammara A; Feeney, Luzviminda; Angeles, Jorge G; Lafyatis, Robert; Whitfield, Michael L

2014-08-01

Previous studies have suggested a role for pathogens as a trigger of systemic sclerosis (SSc), although neither a pathogen nor a mechanism of pathogenesis is known. Here we show enrichment of Rhodotorula sequences in the skin of patients with early, diffuse SSc compared with that in normal controls. RNA-seq was performed on four SSc patients and four controls, to a depth of 200 million reads per patient. Data were analyzed to quantify the nonhuman sequence reads in each sample. We found little difference between bacterial microbiome and viral read counts, but found a significant difference between the read counts for a mycobiome component, R. glutinis. Normal samples contained almost no detected R. glutinis or other Rhodotorula sequence reads (mean score 0.021 for R. glutinis, 0.024 for all Rhodotorula). In contrast, SSc samples had a mean score of 5.039 for R. glutinis (5.232 for Rhodotorula). We were able to assemble the D1-D2 hypervariable region of the 28S ribosomal RNA (rRNA) of R. glutinis from each of the SSc samples. Taken together, these results suggest that R. glutinis may be present in the skin of early SSc patients at higher levels than in normal skin, raising the possibility that it may be triggering the inflammatory response found in SSc.

Mapping the laminin receptor binding domains of Neisseria meningitidis PorA and Haemophilus influenzae OmpP2.

PubMed

Abouseada, Noha M; Assafi, Mahde Saleh A; Mahdavi, Jafar; Oldfield, Neil J; Wheldon, Lee M; Wooldridge, Karl G; Ala'Aldeen, Dlawer A A

2012-01-01

Neisseria meningitidis, Haemophilus influenzae and Streptococcus pneumoniae are major bacterial agents of meningitis. They each bind the 37/67-kDa laminin receptor (LamR) via the surface protein adhesins: meningococcal PilQ and PorA, H. influenzae OmpP2 and pneumococcal CbpA. We have previously reported that a surface-exposed loop of the R2 domain of CbpA mediates LamR-binding. Here we have identified the LamR-binding regions of PorA and OmpP2. Using truncated recombinant proteins we show that binding is dependent on amino acids 171-240 and 91-99 of PorA and OmpP2, respectively, which are predicted to localize to the fourth and second surface-exposed loops, respectively, of these proteins. Synthetic peptides corresponding to the loops bound LamR and could block LamR-binding to bacterial ligands in a dose dependant manner. Meningococci expressing PorA lacking the apex of loop 4 and H. influenzae expressing OmpP2 lacking the apex of loop 2 showed significantly reduced LamR binding. Since both loops are hyper-variable, our data may suggest a molecular basis for the range of LamR-binding capabilities previously reported among different meningococcal and H. influenzae strains.

Maternal Genetic Ancestry and Legacy of 10th Century AD Hungarians

PubMed Central

Csősz, Aranka; Szécsényi-Nagy, Anna; Csákyová, Veronika; Langó, Péter; Bódis, Viktória; Köhler, Kitti; Tömöry, Gyöngyvér; Nagy, Melinda; Mende, Balázs Gusztáv

2016-01-01

The ancient Hungarians originated from the Ural region in today’s central Russia and migrated across the Eastern European steppe, according to historical sources. The Hungarians conquered the Carpathian Basin 895–907 AD, and admixed with the indigenous communities. Here we present mitochondrial DNA results from three datasets: one from the Avar period (7th–9th centuries) of the Carpathian Basin (n = 31); one from the Hungarian conquest-period (n = 76); and a completion of the published 10th–12th century Hungarian-Slavic contact zone dataset by four samples. We compare these mitochondrial DNA hypervariable segment sequences and haplogroup results with published ancient and modern Eurasian data. Whereas the analyzed Avars represents a certain group of the Avar society that shows East and South European genetic characteristics, the Hungarian conquerors’ maternal gene pool is a mixture of West Eurasian and Central and North Eurasian elements. Comprehensively analyzing the results, both the linguistically recorded Finno-Ugric roots and historically documented Turkic and Central Asian influxes had possible genetic imprints in the conquerors’ genetic composition. Our data allows a complex series of historic and population genetic events before the formation of the medieval population of the Carpathian Basin, and the maternal genetic continuity between 10th–12th century and modern Hungarians. PMID:27633963

Illumina MiSeq Sequencing for Preliminary Analysis of Microbiome Causing Primary Endodontic Infections in Egypt

PubMed Central

Azab, Marwa Mohamed; Fayyad, Dalia Mukhtar

2018-01-01

The use of high throughput next generation technologies has allowed more comprehensive analysis than traditional Sanger sequencing. The specific aim of this study was to investigate the microbial diversity of primary endodontic infections using Illumina MiSeq sequencing platform in Egyptian patients. Samples were collected from 19 patients in Suez Canal University Hospital (Endodontic Department) using sterile # 15K file and paper points. DNA was extracted using Mo Bio power soil DNA isolation extraction kit followed by PCR amplification and agarose gel electrophoresis. The microbiome was characterized on the basis of the V3 and V4 hypervariable region of the 16S rRNA gene by using paired-end sequencing on Illumina MiSeq device. MOTHUR software was used in sequence filtration and analysis of sequenced data. A total of 1858 operational taxonomic units at 97% similarity were assigned to 26 phyla, 245 families, and 705 genera. Four main phyla Firmicutes, Bacteroidetes, Proteobacteria, and Synergistetes were predominant in all samples. At genus level, Prevotella, Bacillus, Porphyromonas, Streptococcus, and Bacteroides were the most abundant. Illumina MiSeq platform sequencing can be used to investigate oral microbiome composition of endodontic infections. Elucidating the ecology of endodontic infections is a necessary step in developing effective intracanal antimicrobials. PMID:29849646

Efficient affinity maturation of antibody variable domains requires co-selection of compensatory mutations to maintain thermodynamic stability

PubMed Central

Julian, Mark C.; Li, Lijuan; Garde, Shekhar; Wilen, Rebecca; Tessier, Peter M.

2017-01-01

The ability of antibodies to accumulate affinity-enhancing mutations in their complementarity-determining regions (CDRs) without compromising thermodynamic stability is critical to their natural function. However, it is unclear if affinity mutations in the hypervariable CDRs generally impact antibody stability and to what extent additional compensatory mutations are required to maintain stability during affinity maturation. Here we have experimentally and computationally evaluated the functional contributions of mutations acquired by a human variable (VH) domain that was evolved using strong selections for enhanced stability and affinity for the Alzheimer’s Aβ42 peptide. Interestingly, half of the key affinity mutations in the CDRs were destabilizing. Moreover, the destabilizing effects of these mutations were compensated for by a subset of the affinity mutations that were also stabilizing. Our findings demonstrate that the accumulation of both affinity and stability mutations is necessary to maintain thermodynamic stability during extensive mutagenesis and affinity maturation in vitro, which is similar to findings for natural antibodies that are subjected to somatic hypermutation in vivo. These findings for diverse antibodies and antibody fragments specific for unrelated antigens suggest that the formation of the antigen-binding site is generally a destabilizing process and that co-enrichment for compensatory mutations is critical for maintaining thermodynamic stability. PMID:28349921

Diversity and Assembling Processes of Bacterial Communities in Cryoconite Holes of a Karakoram Glacier.

PubMed

Ambrosini, Roberto; Musitelli, Federica; Navarra, Federico; Tagliaferri, Ilario; Gandolfi, Isabella; Bestetti, Giuseppina; Mayer, Christoph; Minora, Umberto; Azzoni, Roberto Sergio; Diolaiuti, Guglielmina; Smiraglia, Claudio; Franzetti, Andrea

2017-05-01

Cryoconite holes are small ponds that form on the surface of glaciers that contain a dark debris, the cryoconite, at the bottom and host active ecological communities. Differences in the structure of bacterial communities have been documented among Arctic and mountain glaciers, and among glaciers in different areas of the world. In this study, we investigated the structure of bacterial communities of cryoconite holes of Baltoro Glacier, a large (62 km in length and 524 km 2 of surface) glacier of the Karakoram, by high-throughput sequencing of the V5-V6 hypervariable regions of the 16S rRNA gene. We found that Betaproteobacteria dominated bacterial communities, with large abundance of genera Polaromonas, probably thanks to its highly versatile metabolism, and Limnohabitans, which may have been favoured by the presence of supraglacial lakes in the area where cryoconite holes were sampled. Variation in bacterial communities among different sampling areas of the glacier could be explained by divergent selective processes driven by variation in environmental conditions, particularly pH, which was the only environmental variable that significantly affected the structure of bacterial communities. This variability may be due to both temporal and spatial patterns of variation in environmental conditions.

Adenoviral vectors elicit humoral immunity against variable loop 2 of clade C HIV-1 gp120 via "Antigen Capsid-Incorporation" strategy.

PubMed

Gu, Linlin; Krendelchtchikova, Valentina; Krendelchtchikov, Alexandre; Farrow, Anitra L; Derdeyn, Cynthia A; Matthews, Qiana L

2016-01-01

Adenoviral (Ad) vectors in combination with the "Antigen Capsid-Incorporation" strategy have been applied in developing HIV-1 vaccines, due to the vectors׳ abilities in incorporating and inducing immunity of capsid-incorporated antigens. Variable loop 2 (V2)-specific antibodies were suggested in the RV144 trial to correlate with reduced HIV-1 acquisition, which highlights the importance of developing novel HIV-1 vaccines by targeting the V2 loop. Therefore, the V2 loop of HIV-1 has been incorporated into the Ad capsid protein. We generated adenovirus serotype 5 (Ad5) vectors displaying variable loop 2 (V2) of HIV-1 gp120, with the "Antigen Capsid-Incorporation" strategy. To assess the incorporation capabilities on hexon hypervariable region1 (HVR1) and protein IX (pIX), 20aa or full length (43aa) of V2 and V1V2 (67aa) were incorporated, respectively. Immunizations with the recombinant vectors significantly generated antibodies against both linear and discontinuous V2 epitopes. The immunizations generated durable humoral immunity against V2. This study will lead to more stringent development of various serotypes of adenovirus-vectored V2 vaccine candidates, based on breakthroughs regarding the immunogenicity of V2. Copyright © 2015. Published by Elsevier Inc.

Polymorphism of the MHC class II Eb gene determines the protection against collagen-induced arthritis

DOE Office of Scientific and Technical Information (OSTI.GOV)

Gonzalez-Gay, M.A.; Zanelli, E.; Krco, C.J.

1995-05-01

Collagen-induced arthritis (CIA) is an animal model of auto immune polyarthritis, sharing similarities with rheumatoid arthritis (RA). Paradoxally, susceptibility to mouse CIA is controlled by the H2A loci (DQ homologous) while RA is linked to HLA.DR genes (H2E homologous). We recently showed that the E{beta}{sup d} molecule prevents CIA development in susceptible H2{sup q} mice. We addressed the question of whether H2Eb polymorphism will influence CIA incidence as HLA.DRB1 polymorphism does in RA. In F{sub 1} mice, only H2Eb{sup d} and H2Eb{sup s} molecules showed protection. Using recombinant B10.RDD (Eb{sup d/b}) mice, we found that CIA protection was mediated bymore » the first domain of the E{beta}{sup d} molecule. Using peptides covering the third hypervariable region of the E{beta} chain, we found a perfect correlation between presentation of E{beta} peptides by the H2A{sup q} molecule and protection on CIA. Therefore, the mechanism by which H2Eb protects against CIA seems to rely on the affinity of E{beta} peptides for the H2A{sup q} molecule. 35 refs., 2 figs., 3 tabs.« less

Diversity of halophilic bacteria isolated from Rambla Salada, Murcia (Spain).

PubMed

Luque, Rocío; Béjar, Victoria; Quesada, Emilia; Llamas, Inmaculada

2014-12-01

In this study we analyzed the diversity of the halophilic bacteria community from Rambla Salada during the years 2006 and 2007. We collected a total of 364 strains, which were then identified by means of phenotypic tests and by the hypervariable V1-V3 region of the 16S rRNA sequences (around 500 bp). The ribosomal data showed that the isolates belonged to Proteobacteria (72.5%), Firmicutes (25.8%), Actinobacteria (1.4%), and Bacteroidetes (0.3%) phyla, with Gammaproteobacteria the predominant class. Halomonas was the most abundant genus (41.2% isolates) followed by Marinobacter (12.9% isolates) and Bacillus (12.6% isolates). In addition, 9 strains showed <97% sequence identity with validly described species and may well represent new taxa. The diversity of the bacterial community analyzed with the DOTUR package determined 139 operational taxonomic units at 3% genetic distance level. Rarefaction curves and diversity indexes demonstrated that our collection of isolates adequately represented all the bacterial community at Rambla Salada that can be grown under the conditions used in this work. We found that the sampling season influenced the composition of the bacterial community, and bacterial diversity was higher in 2007; this fact could be related to lower salinity at this sampling time.