Identification of Variable-Number Tandem-Repeat (VNTR) Sequences in Acinetobacter baumannii and Interlaboratory Validation of an Optimized Multiple-Locus VNTR Analysis Typing Scheme▿†

PubMed Central

Pourcel, Christine; Minandri, Fabrizia; Hauck, Yolande; D'Arezzo, Silvia; Imperi, Francesco; Vergnaud, Gilles; Visca, Paolo

2011-01-01

Acinetobacter baumannii is an important opportunistic pathogen responsible for nosocomial outbreaks, mostly occurring in intensive care units. Due to the multiplicity of infection sources, reliable molecular fingerprinting techniques are needed to establish epidemiological correlations among A. baumannii isolates. Multiple-locus variable-number tandem-repeat analysis (MLVA) has proven to be a fast, reliable, and cost-effective typing method for several bacterial species. In this study, an MLVA assay compatible with simple PCR- and agarose gel-based electrophoresis steps as well as with high-throughput automated methods was developed for A. baumannii typing. Preliminarily, 10 potential polymorphic variable-number tandem repeats (VNTRs) were identified upon bioinformatic screening of six annotated genome sequences of A. baumannii. A collection of 7 reference strains plus 18 well-characterized isolates, including unique types and representatives of the three international A. baumannii lineages, was then evaluated in a two-center study aimed at validating the MLVA assay and comparing it with other genotyping assays, namely, macrorestriction analysis with pulsed-field gel electrophoresis (PFGE) and PCR-based sequence group (SG) profiling. The results showed that MLVA can discriminate between isolates with identical PFGE types and SG profiles. A panel of eight VNTR markers was selected, all showing the ability to be amplified and good amounts of polymorphism in the majority of strains. Independently generated MLVA profiles, composed of an ordered string of allele numbers corresponding to the number of repeats at each VNTR locus, were concordant between centers. Typeability, reproducibility, stability, discriminatory power, and epidemiological concordance were excellent. A database containing information and MLVA profiles for several A. baumannii strains is available from http://mlva.u-psud.fr/. PMID:21147956

Allele frequency distribution for the variable number of tandem repeat locus D10S28 in Tamil Nadu (south India) population.

PubMed

Pandian, S K; Kumar, S; Krishnan, M; Dharmalingam, K; Damodaran, C

1995-09-01

Allele frequencies were determined in unrelated individuals of Tamil speaking population from the Madras City (Tamil Nadu, South India) area for the polymorphic DNA locus D10S28 using the probe TBQ7. Membranes hybridized with the probe YNH24 were subjected to deprobing and were subsequently hybridized with random priming - labeled, purified inserts of TBQ7. The sizes of the fragments were grouped to 100 bp as well as to arbitrary fixed bins (Federal Bureau of Investigation / Royal Canadian Mounted Police). There were 14 bins in the latter with the most common bin being 11 (1789-1924 bp) with a frequency of 9.8%. We observed a heterozygosity of 92% comparable to Caucasian populations. The data presented here can be used as the basis for utilizing this variable number of tandem repeats (TNTR) DNA marker for paternity determinations and forensic investigations.

Antibiotic Susceptibility and Molecular Diversity of Bacillus anthracis Strains in Chad: Detection of a New Phylogenetic Subgroup

PubMed Central

Maho, Angaya; Rossano, Alexandra; Hächler, Herbert; Holzer, Anita; Schelling, Esther; Zinsstag, Jakob; Hassane, Mahamat H.; Toguebaye, Bhen S.; Akakpo, Ayayi J.; Van Ert, Matthew; Keim, Paul; Kenefic, Leo; Frey, Joachim; Perreten, Vincent

2006-01-01

We genotyped 15 Bacillus anthracis isolates from Chad, Africa, using multiple-locus variable-number tandem repeat analysis and three additional direct-repeat markers. We identified two unique genotypes that represent a novel genetic lineage in the A cluster. Chadian isolates were susceptible to 11 antibiotics and free of 94 antibiotic resistance genes. PMID:16954291

Second generation subtyping: a proposed PulseNet protocol for multiple-locus variable-number tandem repeat analysis of Shiga toxin-producing Escherichia coli O157 (STEC O157).

PubMed

Hyytiä-Trees, Eija; Smole, Sandra C; Fields, Patricia A; Swaminathan, Bala; Ribot, Efrain M

2006-01-01

Most bacterial genomes contain tandem duplications of short DNA sequences, termed "variable-number tandem repeats" (VNTR). A subtyping method targeting these repeats, multiple-locus VNTR analysis (MLVA), has emerged as a powerful tool for characterization of clonal organisms such as Shiga toxin-producing Escherichia coli O157 (STEC O157). We modified and optimized a recently published MLVA scheme targeting 29 polymorphic VNTR regions of STEC O157 to render it suitable for routine use by public health laboratories that participate in PulseNet, the national and international molecular subtyping network for foodborne disease surveillance. Nine VNTR loci were included in the final protocol. They were amplified in three PCR reactions, after which the PCR products were sized using capillary electrophoresis. Two hundred geographically diverse, sporadic and outbreak- related STEC O157 isolates were characterized by MLVA and the results were compared with data obtained by pulsed-field gel electrophoresis (PFGE) using XbaI macrorestriction of genomic DNA. A total of 139 unique XbaI PFGE patterns and 162 MLVA types were identified. A subset of 100 isolates characterized by both XbaI and BlnI macrorestriction had 62 unique PFGE and MLVA types. Although the clustering of isolates by the two subtyping systems was generally in agreement, some discrepancies were observed. Importantly, MLVA was able to discriminate among some epidemiologically unrelated isolates which were indistinguishable by PFGE. However, among strains from three of the eight outbreaks included in the study, two single locus MLVA variants and one double locus variant were detected among epidemiologically implicated isolates that were indistinguishable by PFGE. Conversely, in three other outbreaks, isolates that were indistinguishable by MLVA displayed multiple PFGE types. An additional more extensive multi-laboratory validation of the MLVA protocol is in progress in order to address critical issues such as establishing epidemiologically relevant interpretation guidelines for the MLVA data.

An Undergraduate Laboratory Experiment for Upper-Level Forensic Science, Biochemistry, or Molecular Biology Courses: Human DNA Amplification Using STR Single Locus Primers by Real-Time PCR with SYBR Green Detection

ERIC Educational Resources Information Center

Elkins, Kelly M.; Kadunc, Raelynn E.

2012-01-01

In this laboratory experiment, real-time polymerase chain reaction (real-time PCR) was conducted using published human TPOX single-locus DNA primers for validation and various student-designed short tandem repeat (STR) primers for Combined DNA Index System (CODIS) loci. SYBR Green was used to detect the amplification of the expected amplicons. The…

Multiple-Locus Variable-Number Tandem-Repeats Analysis of Escherichia coli O157 using PCR multiplexing and multi-colored capillary electrophoresis.

PubMed

Lindstedt, Bjørn-Arne; Vardund, Traute; Kapperud, Georg

2004-08-01

The Multiple-Locus Variable-Number Tandem-Repeats Analysis (MLVA) method is currently being used as the primary typing tool for Shiga-toxin-producing Escherichia coli (STEC) O157 isolates in our laboratory. The initial assay was performed using a single fluorescent dye and the different patterns were assigned using a gel image. Here, we present a significantly improved assay using multiple dye colors and enhanced PCR multiplexing to increase speed, and ease the interpretation of the results. The different MLVA patterns are now based on allele sizes entered as character values, thus removing the uncertainties introduced when analyzing band patterns from the gel image. We additionally propose an easy numbering scheme for the identification of separate isolates that will facilitate exchange of typing data. Seventy-two human and animal strains of Shiga-toxin-producing E. coli O157 were used for the development of the improved MLVA assay. The method is based on capillary separation of multiplexed PCR products of VNTR loci in the E. coli O157 genome labeled with multiple fluorescent dyes. The different alleles at each locus were then assigned to allele numbers, which were used for strain comparison.

Resurgence of Pertussis and Emergence of the Ptxp3 Toxin Promoter Allele in South Italy.

PubMed

Loconsole, Daniela; De Robertis, Anna Lisa; Morea, Anna; Metallo, Angela; Lopalco, Pier Luigi; Chironna, Maria

2018-05-01

Despite universal immunization programs, pertussis remains a major public health concern. This study aimed to describe the pertussis epidemiology in the Puglia region in 2006-2015 and to identify recent polymorphisms in Bordetella pertussis virulence-associated genes. The pertussis cases in 2006-2015 were identified from the National Hospital Discharge Database and the Information System of Infectious Diseases. Samples of pertussis cases in 2014-2016 that were confirmed by the Regional Reference Laboratory were subjected to ptxA, ptxP and prn gene sequencing and, in 10 cases, multiple-locus variable-number tandem repeat analysis. In Puglia in 2006-2015, the pertussis incidence rose from an average of 1.39/100,000 inhabitants in 2006-2013 to 2.56-2.54/100,000 in 2014-2015. In infants <1 year of age, the incidence rose from an average of 60.4/100,000 infants in 2006-2013 to 149.9/100,000 in 2015. Of the 661 cases recorded in 2006-2015, 80.3% required hospitalization; of these, 45.4% were <1 year of age. Of the 80 sequenced samples, the allelic profile ptxA1-ptxP3-prn2 was detected in 74. This variant was detected in both vaccinated and unvaccinated people. Six Bordetella pertussis samples were prn deficient. The multiple-locus variable-number tandem repeat analysis cases exhibited multiple-locus variable-number tandem repeat analysis-type 27. The pertussis incidence in Puglia has risen. The hypervirulent strain was also found in vaccinated people. This suggests bacterial adaptation to the vaccine and raises questions about acellular vaccine effectiveness. Prevention of infant pertussis cases is best achieved by immunizing the pregnant mother. Enhanced surveillance and systematic laboratory confirmation of pertussis should be improved in Italy.

Locus-specific mutational events in a multilocus variable-number tandem repeat analysis of Escherichia coli O157:H7.

PubMed

Noller, Anna C; McEllistrem, M Catherine; Shutt, Kathleen A; Harrison, Lee H

2006-02-01

Multilocus variable-number tandem repeat analysis (MLVA) is a validated molecular subtyping method for detecting and evaluating Escherichia coli O157:H7 outbreaks. In a previous study, five outbreaks with a total of 21 isolates were examined by MLVA. Nearly 20% of the epidemiologically linked strains were single-locus variants (SLV) of their respective predominant outbreak clone. This result prompted an investigation into the mutation rates of the seven MLVA loci (TR1 to TR7). With an outbreak strain that was an SLV at the TR1 locus of the predominant clone, parallel and serial batch culture experiments were performed. In a parallel experiment, none (0/384) of the strains analyzed had mutations at the seven MLVA loci. In contrast, in the two 5-day serial experiments, 4.3% (41/960) of the strains analyzed had a significant variation in at least one of these loci (P < 0.001). The TR2 locus accounted for 85.3% (35/41) of the mutations, with an average mutation rate of 3.5 x 10(-3); the mutations rates for TR1 and TR5 were 10-fold lower. Single additions accounted for 77.1% (27/35) of the mutation events in TR2 and all (6/6) of the additions in TR1 and TR5. The remaining four loci had no slippage events detected. The mutation rates were locus specific and may impact the interpretation of MLVA data for epidemiologic investigations.

Is Tobacco Smoke a Germ-Cell Mutagen?

EPA Science Inventory

Although no international organization exists to declare whether an agent is a germ-cell mutagen, tobacco smoke may be a human germ-cell mutagen. In the mouse, tobacco smoke induces a significant increase in the mutation frequency at an expanded simple tandem repeat (ESTR) locus....

Developmental Validation of Short Tandem Repeat Reagent Kit for Forensic DNA Profiling of Canine Biological Materials

PubMed Central

Dayton, Melody; Koskinen, Mikko T; Tom, Bradley K; Mattila, Anna-Maria; Johnston, Eric; Halverson, Joy; Fantin, Dennis; DeNise, Sue; Budowle, Bruce; Smith, David Glenn; Kanthaswamy, Sree

2009-01-01

Aim To develop a reagent kit that enables multiplex polymerase chain reaction (PCR) amplification of 18 short tandem repeats (STR) and the canine sex-determining Zinc Finger marker. Methods Validation studies to determine the robustness and reliability in forensic DNA typing of this multiplex assay included sensitivity testing, reproducibility studies, intra- and inter-locus color balance studies, annealing temperature and cycle number studies, peak height ratio determination, characterization of artifacts such as stutter percentages and dye blobs, mixture analyses, species-specificity, case type samples analyses and population studies. Results The kit robustly amplified domesticated dog samples and consistently generated full 19-locus profiles from as little as 125 pg of dog DNA. In addition, wolf DNA samples could be analyzed with the kit. Conclusion The kit, which produces robust, reliable, and reproducible results, will be made available for the forensic research community after modifications based on this study’s evaluation to comply with the quality standards expected for forensic casework. PMID:19480022

Multiple-locus variable number of tandem repeat analysis (MLVA) of Irish verocytotoxigenic Escherichia coli O157 from feedlot cattle: uncovering strain dissemination routes.

PubMed

Murphy, Mary; Minihan, Donal; Buckley, James F; O'Mahony, Micheál; Whyte, Paul; Fanning, Séamus

2008-01-24

The identification of the routes of dissemination of Escherichia coli (E. coli) O157 through a cohort of cattle is a critical step to control this pathogen at farm level. The aim of this study was to identify potential routes of dissemination of E. coli O157 using Multiple-Locus Variable number of tandem repeat Analysis (MLVA). Thirty-eight environmental and sixteen cattle faecal isolates, which were detected in four adjacent pens over a four-month period were sub-typed. MLVA could separate these isolates into broadly defined clusters consisting of twelve MLVA types. Strain diversity was observed within pens, individual cattle and the environment. Application of MLVA is a broadly useful and convenient tool when applied to uncover the dissemination of E. coli O157 in the environment and in supporting improved on-farm management of this important pathogen. These data identified diverse strain types based on amplification of VNTR markers in each case.

Characterization of Escherichia coli O157:H7 in New Zealand using multiple-locus variable-number tandem-repeat analysis.

PubMed

Dyet, K H; Robertson, I; Turbitt, E; Carter, P E

2011-03-01

Recently, multiple-locus variable-number tandem-repeat analysis (MLVA) has been proposed as an alternative to pulsed-field gel electrophoresis (PFGE) for characterization of Escherichia coli O157:H7. In this study we characterized 118 E. coli O157:H7 isolates from cases of gastrointestinal disease in New Zealand using XbaI PFGE profiles and a MLVA scheme that assessed variability in eight polymorphic loci. The 118 isolates characterized included all 80 E. coli O157:H7 referred to New Zealand's Enteric Reference Laboratory in 2006 and 29 phage-type 2 isolates from 2005. When applied to these isolates the discriminatory power of PFGE and MLVA was not significantly different. However, MLVA data may be more epidemiologically relevant as isolates from family clusters of disease had identical MLVA profiles, even when the XbaI PFGE profiles differed slightly. Furthermore, most isolates with indistinguishable XbaI PFGE profiles that did not appear to be epidemiologically related had distinct MLVA profiles.

A multiple-locus variable-number tandem repeat analysis (MLVA) of Listeria monocytogenes isolated from Norwegian salmon-processing factories and from listeriosis patients.

PubMed

Lunestad, B T; Truong, T T T; Lindstedt, B-A

2013-10-01

The objective of this study was to characterize Listeria monocytogenes isolated from farmed Atlantic salmon (Salmo salar) and the processing environment in three different Norwegian factories, and compare these to clinical isolates by multiple-locus variable-number tandem repeat analysis (MLVA). The 65 L. monocytogenes isolates obtained gave 15 distinct MLVA profiles. There was great heterogeneity in the distribution of MLVA profiles in factories and within each factory. Nine of the 15 MLVA profiles found in the fish-associated isolates were found to match human profiles. The MLVA profile 07-07-09-10-06 was the most common strain in Norwegian listeriosis patients. L. monocytogenes with this profile has previously been associated with at least two known listeriosis outbreaks in Norway, neither determined to be due to fish consumption. However, since this profile was also found in fish and in the processing environment, fish should be considered as a possible food vehicle during sporadic cases and outbreaks of listeriosis.

STRBase: a short tandem repeat DNA database for the human identity testing community

PubMed Central

Ruitberg, Christian M.; Reeder, Dennis J.; Butler, John M.

2001-01-01

The National Institute of Standards and Technology (NIST) has compiled and maintained a Short Tandem Repeat DNA Internet Database (http://www.cstl.nist.gov/biotech/strbase/) since 1997 commonly referred to as STRBase. This database is an information resource for the forensic DNA typing community with details on commonly used short tandem repeat (STR) DNA markers. STRBase consolidates and organizes the abundant literature on this subject to facilitate on-going efforts in DNA typing. Observed alleles and annotated sequence for each STR locus are described along with a review of STR analysis technologies. Additionally, commercially available STR multiplex kits are described, published polymerase chain reaction (PCR) primer sequences are reported, and validation studies conducted by a number of forensic laboratories are listed. To supplement the technical information, addresses for scientists and hyperlinks to organizations working in this area are available, along with the comprehensive reference list of over 1300 publications on STRs used for DNA typing purposes. PMID:11125125

A Trio of Human Molecular Genetics PCR Assays

ERIC Educational Resources Information Center

Reinking, Jeffrey L.; Waldo, Jennifer T.; Dinsmore, Jannett

2013-01-01

This laboratory exercise demonstrates three different analytical forms of the polymerase chain reaction (PCR) that allow students to genotype themselves at four different loci. Here, we present protocols to allow students to a) genotype a non-coding polymorphic Variable Number of Tandem Repeat (VNTR) locus on human chromosome 5 using conventional…

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 Effective Mutation Rate at Y Chromosome Short Tandem Repeats, with Application to Human Population-Divergence Time

PubMed Central

Zhivotovsky, Lev A.; Underhill, Peter A.; Cinnioğlu, Cengiz; Kayser, Manfred; Morar, Bharti; Kivisild, Toomas; Scozzari, Rosaria; Cruciani, Fulvio; Destro-Bisol, Giovanni; Spedini, Gabriella; Chambers, Geoffrey K.; Herrera, Rene J.; Yong, Kiau Kiun; Gresham, David; Tournev, Ivailo; Feldman, Marcus W.; Kalaydjieva, Luba

2004-01-01

We estimate an effective mutation rate at an average Y chromosome short-tandem repeat locus as 6.9×10-4 per 25 years, with a standard deviation across loci of 5.7×10-4, using data on microsatellite variation within Y chromosome haplogroups defined by unique-event polymorphisms in populations with documented short-term histories, as well as comparative data on worldwide populations at both the Y chromosome and various autosomal loci. This value is used to estimate the times of the African Bantu expansion, the divergence of Polynesian populations (the Maoris, Cook Islanders, and Samoans), and the origin of Gypsy populations from Bulgaria. PMID:14691732

Concerted evolution of the tandemly repeated genes encoding primate U2 small nuclear RNA (the RNU2 locus) does not prevent rapid diversification of the (CT){sub n} {center_dot} (GA){sub n} microsatellite embedded within the U2 repeat unit

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

Liao, D.; Weiner, A.M.

1995-12-10

The RNU2 locus encoding human U2 small nuclear RNA (snRNA) is organized as a nearly perfect tandem array containing 5 to 22 copies of a 5.8-kb repeat unit. Just downstream of the U2 snRNA gene in each 5.8-kb repeat unit lies a large (CT){sub n}{center_dot}(GA){sub n} dinucleotide repeat (n {approx} 70). This form of genomic organization, in which one repeat is embedded within another, provides an unusual opportunity to study the balance of forces maintaining the homogeneity of both kinds of repeats. Using a combination of field inversion gel electrophoresis and polymerase chain reaction, we have been able to studymore » the CT microsatellites within individual U2 tandem arrays. We find that the CT microsatellites within an RNU2 allele exhibit significant length polymorphism, despite the remarkable homogeneity of the surrounding U2 repeat units. Length polymorphism is due primarily to loss or gain of CT dinucleotide repeats, but other types of deletions, insertions, and substitutions are also frequent. Polymorphism is greatly reduced in regions where pure (CT){sub n} tracts are interrupted by occasional G residues, suggesting that irregularities stabilize both the length and the sequence of the dinucleotide repeat. We further show that the RNU2 loci of other catarrhine primates (gorilla, chimpanzee, ogangutan, and baboon) contain orthologous CT microsatellites; these also exhibit length polymorphism, but are highly divergent from each other. Thus, although the CT microsatellite is evolving far more rapidly than the rest of the U2 repeat unit, it has persisted through multiple speciation events spanning >35 Myr. The persistence of the CT microsatellite, despite polymorphism and rapid evolution, suggests that it might play a functional role in concerted evolution of the RNU2 loci, perhaps as an initiation site for recombination and/or gene conversion. 70 refs., 5 figs.« less

Evolution and selection of Rhg1, a copy-number variant nematode-resistance locus

PubMed Central

Lee, Tong Geon; Kumar, Indrajit; Diers, Brian W; Hudson, Matthew E

2015-01-01

The soybean cyst nematode (SCN) resistance locus Rhg1 is a tandem repeat of a 31.2 kb unit of the soybean genome. Each 31.2-kb unit contains four genes. One allele of Rhg1, Rhg1-b, is responsible for protecting most US soybean production from SCN. Whole-genome sequencing was performed, and PCR assays were developed to investigate allelic variation in sequence and copy number of the Rhg1 locus across a population of soybean germplasm accessions. Four distinct sequences of the 31.2-kb repeat unit were identified, and some Rhg1 alleles carry up to three different types of repeat unit. The total number of copies of the repeat varies from 1 to 10 per haploid genome. Both copy number and sequence of the repeat correlate with the resistance phenotype, and the Rhg1 locus shows strong signatures of selection. Significant linkage disequilibrium in the genome outside the boundaries of the repeat allowed the Rhg1 genotype to be inferred using high-density single nucleotide polymorphism genotyping of 15 996 accessions. Over 860 germplasm accessions were found likely to possess Rhg1 alleles. The regions surrounding the repeat show indications of non-neutral evolution and high genetic variability in populations from different geographic locations, but without evidence of fixation of the resistant genotype. A compelling explanation of these results is that balancing selection is in operation at Rhg1. PMID:25735447

High Genetic Diversity Revealed by Variable-Number Tandem Repeat Genotyping and Analysis of hsp65 Gene Polymorphism in a Large Collection of “Mycobacterium canettii” Strains Indicates that the M. tuberculosis Complex Is a Recently Emerged Clone of “M. canettii”

PubMed Central

Fabre, Michel; Koeck, Jean-Louis; Le Flèche, Philippe; Simon, Fabrice; Hervé, Vincent; Vergnaud, Gilles; Pourcel, Christine

2004-01-01

We have analyzed, using complementary molecular methods, the diversity of 43 strains of “Mycobacterium canettii” originating from the Republic of Djibouti, on the Horn of Africa, from 1998 to 2003. Genotyping by multiple-locus variable-number tandem repeat analysis shows that all the strains belong to a single but very distant group when compared to strains of the Mycobacterium tuberculosis complex (MTBC). Thirty-one strains cluster into one large group with little variability and five strains form another group, whereas the other seven are more diverged. In total, 14 genotypes are observed. The DR locus analysis reveals additional variability, some strains being devoid of a direct repeat locus and others having unique spacers. The hsp65 gene polymorphism was investigated by restriction enzyme analysis and sequencing of PCR amplicons. Four new single nucleotide polymorphisms were discovered. One strain was characterized by three nucleotide changes in 441 bp, creating new restriction enzyme polymorphisms. As no sequence variability was found for hsp65 in the whole MTBC, and as a single point mutation separates M. tuberculosis from the closest “M. canettii” strains, this diversity within “M. canettii” subspecies strongly suggests that it is the most probable source species of the MTBC rather than just another branch of the MTBC. PMID:15243089

Highly diverse variable number tandem repeat loci in the E. coli O157:H7 and O55:H7 genomes for high-resolution molecular typing.

PubMed

Keys, C; Kemper, S; Keim, P

2005-01-01

Evaluation of the Escherichia coli genome for variable number tandem repeat (VNTR) loci in order to provide a subtyping tool with greater discrimination and more efficient capacity. Twenty-nine putative VNTR loci were identified from the E. coli genomic sequence. Their variability was validated by characterizing the number of repeats at each locus in a set of 56 E. coli O157:H7/HN and O55:H7 isolates. An optimized multiplex assay system was developed to facility high capacity analysis. Locus diversity values ranged from 0.23 to 0.95 while the number of alleles ranged from two to 29. This multiple-locus VNTR analysis (MLVA) data was used to describe genetic relationships among these isolates and was compared with PFGE (pulse field gel electrophoresis) data from a subset of the same strains. Genetic similarity values were highly correlated between the two approaches, through MLVA was capable of discrimination amongst closely related isolates when PFGE similar values were equal to 1.0. Highly variable VNTR loci exist in the E. coli O157:H7 genome and are excellent estimators of genetic relationships, in particular for closely related isolates. Escherichia coli O157:H7 MLVA offers a complimentary analysis to the more traditional PFGE approach. Application of MLVA to an outbreak cluster could generate superior molecular epidemiology and result in a more effective public health response.

Intergenic Variable-Number Tandem-Repeat Polymorphism Upstream of rocA Alters Toxin Production and Enhances Virulence in Streptococcus pyogenes.

PubMed

Zhu, Luchang; Olsen, Randall J; Horstmann, Nicola; Shelburne, Samuel A; Fan, Jia; Hu, Ye; Musser, James M

2016-07-01

Variable-number tandem-repeat (VNTR) polymorphisms are ubiquitous in bacteria. However, only a small fraction of them has been functionally studied. Here, we report an intergenic VNTR polymorphism that confers an altered level of toxin production and increased virulence in Streptococcus pyogenes The nature of the polymorphism is a one-unit deletion in a three-tandem-repeat locus upstream of the rocA gene encoding a sensor kinase. S. pyogenes strains with this type of polymorphism cause human infection and produce significantly larger amounts of the secreted cytotoxins S. pyogenes NADase (SPN) and streptolysin O (SLO). Using isogenic mutant strains, we demonstrate that deleting one or more units of the tandem repeats abolished RocA production, reduced CovR phosphorylation, derepressed multiple CovR-regulated virulence factors (such as SPN and SLO), and increased virulence in a mouse model of necrotizing fasciitis. The phenotypic effect of the VNTR polymorphism was nearly the same as that of inactivating the rocA gene. In summary, we identified and characterized an intergenic VNTR polymorphism in S. pyogenes that affects toxin production and virulence. These new findings enhance understanding of rocA biology and the function of VNTR polymorphisms in S. pyogenes. Copyright © 2016, American Society for Microbiology. All Rights Reserved.

Molecular typing of Argentinian Mycobacterium avium subsp. paratuberculosis isolates by multiple-locus variable number-tandem repeat analysis

PubMed Central

Gioffré, Andrea; Correa Muñoz, Magnolia; Alvarado Pinedo, María F.; Vaca, Roberto; Morsella, Claudia; Fiorentino, María Andrea; Paolicchi, Fernando; Ruybal, Paula; Zumárraga, Martín; Travería, Gabriel E.; Romano, María Isabel

2015-01-01

Multiple-locus variable number-tandem repeat analysis (MLVA) of Mycobacterium avium subspecies paratuberculosis (MAP) isolates may contribute to the knowledge of strain diversity in Argentina. Although the diversity of MAP has been previously investigated in Argentina using IS900-RFLP, a small number of isolates were employed, and a low discriminative power was reached. The aim of the present study was to test the genetic diversity among MAP isolates using an MLVA approach based on 8 repetitive loci. We studied 97 isolates from cattle, goat and sheep and could describe 7 different patterns: INMV1, INMV2, INMV11, INMV13, INMV16, INMV33 and one incomplete pattern. INMV1 and INMV2 were the most frequent patterns, grouping 76.3% of the isolates. We were also able to demonstrate the coexistence of genotypes in herds and co-infection at the organism level. This study shows that all the patterns described are common to those described in Europe, suggesting an epidemiological link between the continents. PMID:26273274

Molecular characterization of Shiga-toxigenic Escherichia coli isolated from diverse sources from India by multi-locus variable number tandem repeat analysis (MLVA).

PubMed

Kumar, A; Taneja, N; Sharma, R K; Sharma, H; Ramamurthy, T; Sharma, M

2014-12-01

In a first study from India, a diverse collection of 140 environmental and clinical non-O157 Shiga-toxigenic Escherichia coli strains from a large geographical area in north India was typed by multi-locus variable number tandem repeat analysis (MLVA). The distribution of major virulence genes stx1, stx2 and eae was found to be 78%, 70% and 10%, respectively; 15 isolates were enterohaemorrhagic E. coli (stx1 +/stx2 + and eae +). By MLVA analysis, 44 different alleles were obtained. Dendrogram analysis revealed 104 different genotypes and 19 MLVA-type complexes divided into two main lineages, i.e. mutton and animal stool. Human isolates presented a statistically significant greater odds ratio for clustering with mutton samples compared to animal stool isolates. Five human isolates clustered with animal stool strains suggesting that some of the human infections may be from cattle, perhaps through milk, contact or the environment. Further epidemiological studies are required to explore these sources in context with occurrence of human cases.

DXYS156: a multi-purpose short tandem repeat locus for determination of sex, paternal and maternal geographic origins and DNA fingerprinting.

PubMed

Calì, Francesco; Forster, P; Kersting, Christian; Mirisola, Mario G; D'Anna, Rosalba; De Leo, Giacomo; Romano, Valentino

2002-06-01

In forensic science and in legal medicine Y chromosomal typing is indispensable for sex determination, for paternity testing in the absence of the father and for distinguishing males in multiple rape cases. Another potential application is the estimation of paternal geographic origin or family name from a crime stain to narrow down the range of suspects and thus reduce costs of mass screenings. However, Y typing alone cannot provide a sufficiently resolved DNA fingerprint as required for court convictions. Thus, there is a dilemma whether or not to sacrifice valuable material for the sake of extensive Y chromosomal investigations when stain DNA is limited (typically allowing only few PCR amplifications). We here describe a Y-chromosome-specific nucleotide insertion in the duplicate short tandem repeat (STR) locus DXYS156 which allows us to distinguish males from females as does the commonly used amelogenin system, but with the advantage that this locus is multi-allelic, thus substantially contributing towards DNA fingerprinting of a sample and furthermore enabling the detection of sample contamination. Yet another bonus is that both the X and the Y copies of DXYS156 have alleles specific to different parts of the world, offering separate estimates of maternal and paternal descent of that sample. We therefore recommend the inclusion of DXYS156 in standard multiplexing kits for forensic, archaeological and genealogical applications.

Investigation of Salmonella Enteritidis outbreaks in South Africa using multi-locus variable-number tandem-repeats analysis, 2013-2015.

PubMed

Muvhali, Munyadziwa; Smith, Anthony Marius; Rakgantso, Andronica Moipone; Keddy, Karen Helena

2017-10-02

Salmonella enterica serovar Enteritidis (Salmonella Enteritidis) has become a significant pathogen in South Africa, and the need for improved molecular surveillance of this pathogen has become important. Over the years, multi-locus variable-number tandem-repeats analysis (MLVA) has become a valuable molecular subtyping technique for Salmonella, particularly for highly homogenic serotypes such as Salmonella Enteritidis. This study describes the use of MLVA in the molecular epidemiological investigation of outbreak isolates in South Africa. Between the years 2013 and 2015, the Centre for Enteric Diseases (CED) received 39 Salmonella Enteritidis isolates from seven foodborne illness outbreaks, which occurred in six provinces. MLVA was performed on all isolates. Three MLVA profiles (MLVA profiles 21, 22 and 28) were identified among the 39 isolates. MLVA profile 28 accounted for 77% (30/39) of the isolates. Isolates from a single outbreak were grouped into a single MLVA profile. A minimum spanning tree (MST) created from the MLVA data showed a close relationship between MLVA profiles 21, 22 and 28, with a single VNTR locus difference between them. MLVA has proven to be a reliable method for the molecular epidemiological investigation of Salmonella Enteritidis outbreaks in South Africa. These foodborne outbreaks emphasize the importance of the One Health approach as an essential component for combating the spread of zoonotic pathogens such as Salmonella Enteritidis.

Application of multilocus variable number tandem repeat analysis to monitor Verocytotoxin-producing Escherichia coli O157 phage type 8 in England and Wales: emergence of a profile associated with a national outbreak.

PubMed

Perry, N; Cheasty, T; Dallman, T; Launders, N; Willshaw, G

2013-10-01

Evaluation of multilocus variable number tandem repeat analysis (MLVA) to subtype all isolates of Vero cytotoxin-producing Escherichia coli O157 phage type 8 in England and Wales. Over a 13 month period from December 2010, 483 isolates of VTEC O157 PT8 were tested by MLVA; 39% were received in the first 4 months of 2011, when infections are generally low. One profile, or single locus variants of it, was present in 249 (52%) isolates but was not common previously. These cases represented a national increase in PT8, associated epidemiologically with soil-contaminated vegetables. Most of the 177 other MLVA profiles were unique to a single isolate. Profiles shared by >1 isolate included cases from two small community, food-borne outbreaks and 11 households. Several shared profiles were found among 23 isolates without known links. Apart from one group, isolates linked to travel abroad had very diverse profiles. Multilocus variable number tandem repeat analysis discriminated apparent sporadic isolates of the same PT and assisted in detection of cases in an emerging national outbreak. Multilocus variable number tandem repeat analysis is an epidemiologically valid complement to surveillance and applicable as a rapid, practical test for large numbers of isolates. © 2013 The Society for Applied Microbiology.

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

Transferability of short tandem repeat markers for two wild Canid species inhabiting the Brazilian Cerrado.

PubMed

Rodrigues, F M; Telles, M P C; Resende, L V; Soares, T N; Diniz-Filho, J A F; Jácomo, A T A; Silveira, L

2006-12-13

The maned wolf (Chrysocyon brachyurus) and the crab-eating fox (Cerdocyon thous) are two wild-canid species found in the Brazilian Cerrado. We tested cross-amplification and transferability of 29 short tandem repeat primers originally developed for cattle and domestic dogs and cats on 38 individuals of each of these two species, collected in the Emas National Park, which is the largest national park in the Cerrado region. Six of these primers were successfully transferred (CSSM-038, PEZ-05, PEZ-12, LOCO-13, LOCO-15, and PEZ-20); five of which were found to be polymorphic. Genetic parameter values (number of alleles per locus, observed and expected heterozygosities, and fixation indices) were within the expected range reported for canid populations worldwide.

Population genetic study for 24 STR loci and Y indel (GlobalFiler™ PCR Amplification kit and PowerPlex® Fusion system) in 1000 Korean individuals.

PubMed

Park, Hyun-Chul; Kim, Kicheol; Nam, Younhyoung; Park, Jihye; Lee, Jinmyung; Lee, Hyehyeon; Kwon, Hansol; Jin, Hanjun; Kim, Wook; Kim, Won; Lim, Sikeun

2016-07-01

Allele frequencies for 23 autosomal short tandem repeat loci (D3S1358, vWA, D16S539, CSF1PO, TPOX, D8S1179, D21S11, D18S51, TH01, FGA, D5S818, D13S317, D7S820, D2S441, D19S433, D22S1045, D10S1248, D1S1656, D12S391, D2S1338, SE33, Penta D, Penta E), 1 Y-chromosome short tandem repeat locus (DYS391) and Y indel were obtained from 1000 unrelated individuals of the Korean population. Copyright © 2016 Elsevier Ireland Ltd. All rights reserved.

Multiple-locus variable-number tandem-repeats analysis of Listeria monocytogenes using multicolour capillary electrophoresis and comparison with pulsed-field gel electrophoresis typing.

PubMed

Lindstedt, Bjørn-Arne; Tham, Wilhelm; Danielsson-Tham, Marie-Louise; Vardund, Traute; Helmersson, Seved; Kapperud, Georg

2008-02-01

The multiple-locus variable-number tandem-repeats analysis (MLVA) method for genotyping has proven to be a fast and reliable typing tool in several bacterial species. MLVA is in our laboratory the routine typing method for Salmonella enterica subsp. enterica serovar Typhimurium and Escherichia coli O157. The gram-positive bacteria Listeria monocytogenes, while not isolated as frequent as S. Typhimurium and E. coli, causes severe illness with an overall mortality rate of 30%. Thus, it is important that any outbreak of this pathogen is detected early and a fast trace to the source can be performed. In view of this, we have used the information provided by two fully sequenced L. monocytogenes strains to develop a MLVA assay coupled with high-resolution capillary electrophoresis and compared it to pulsed-field gel electrophoresis (PFGE) in two sets of isolates, one Norwegian (79 isolates) and one Swedish (61 isolates) set. The MLVA assay could resolve all of the L. monocytogenes serotypes tested, and was slightly more discriminatory than PFGE for the Norwegian isolates (28 MLVA profiles and 24 PFGE profiles) and opposite for the Swedish isolates (42 MLVA profiles and 43 PFGE profiles).

Multiple-locus variable-number tandem repeat analysis for strain discrimination of non-O157 Shiga toxin-producing Escherichia coli.

PubMed

Timmons, Chris; Trees, Eija; Ribot, Efrain M; Gerner-Smidt, Peter; LaFon, Patti; Im, Sung; Ma, Li Maria

2016-06-01

Non-O157 Shiga toxin-producing Escherichia coli (STEC) are foodborne pathogens of growing concern worldwide that have been associated with several recent multistate and multinational outbreaks of foodborne illness. Rapid and sensitive molecular-based bacterial strain discrimination methods are critical for timely outbreak identification and contaminated food source traceback. One such method, multiple-locus variable-number tandem repeat analysis (MLVA), is being used with increasing frequency in foodborne illness outbreak investigations to augment the current gold standard bacterial subtyping technique, pulsed-field gel electrophoresis (PFGE). The objective of this study was to develop a MLVA assay for intra- and inter-serogroup discrimination of six major non-O157 STEC serogroups-O26, O111, O103, O121, O45, and O145-and perform a preliminary internal validation of the method on a limited number of clinical isolates. The resultant MLVA scheme consists of ten variable number tandem repeat (VNTR) loci amplified in three multiplex PCR reactions. Sixty-five unique MLVA types were obtained among 84 clinical non-O157 STEC strains comprised of geographically diverse sporadic and outbreak related isolates. Compared to PFGE, the developed MLVA scheme allowed similar discrimination among serogroups O26, O111, O103, and O121 but not among O145 and O45. To more fully compare the discriminatory power of this preliminary MLVA method to PFGE and to determine its epidemiological congruence, a thorough internal and external validation needs to be performed on a carefully selected large panel of strains, including multiple isolates from single outbreaks. Copyright © 2016. Published by Elsevier B.V.

Application of Molecular Typing Results in Source Attribution Models: The Case of Multiple Locus Variable Number Tandem Repeat Analysis (MLVA) of Salmonella Isolates Obtained from Integrated Surveillance in Denmark.

PubMed

de Knegt, Leonardo V; Pires, Sara M; Löfström, Charlotta; Sørensen, Gitte; Pedersen, Karl; Torpdahl, Mia; Nielsen, Eva M; Hald, Tine

2016-03-01

Salmonella is an important cause of bacterial foodborne infections in Denmark. To identify the main animal-food sources of human salmonellosis, risk managers have relied on a routine application of a microbial subtyping-based source attribution model since 1995. In 2013, multiple locus variable number tandem repeat analysis (MLVA) substituted phage typing as the subtyping method for surveillance of S. Enteritidis and S. Typhimurium isolated from animals, food, and humans in Denmark. The purpose of this study was to develop a modeling approach applying a combination of serovars, MLVA types, and antibiotic resistance profiles for the Salmonella source attribution, and assess the utility of the results for the food safety decisionmakers. Full and simplified MLVA schemes from surveillance data were tested, and model fit and consistency of results were assessed using statistical measures. We conclude that loci schemes STTR5/STTR10/STTR3 for S. Typhimurium and SE9/SE5/SE2/SE1/SE3 for S. Enteritidis can be used in microbial subtyping-based source attribution models. Based on the results, we discuss that an adjustment of the discriminatory level of the subtyping method applied often will be required to fit the purpose of the study and the available data. The issues discussed are also considered highly relevant when applying, e.g., extended multi-locus sequence typing or next-generation sequencing techniques. © 2015 Society for Risk Analysis.

Clostridium botulinum Group I Strain Genotyping by 15-Locus Multilocus Variable-Number Tandem-Repeat Analysis ▿ †

PubMed Central

Fillo, Silvia; Giordani, Francesco; Anniballi, Fabrizio; Gorgé, Olivier; Ramisse, Vincent; Vergnaud, Gilles; Riehm, Julia M.; Scholz, Holger C.; Splettstoesser, Wolf D.; Kieboom, Jasper; Olsen, Jaran-Strand; Fenicia, Lucia; Lista, Florigio

2011-01-01

Clostridium botulinum is a taxonomic designation that encompasses a broad variety of spore-forming, Gram-positive bacteria producing the botulinum neurotoxin (BoNT). C. botulinum is the etiologic agent of botulism, a rare but severe neuroparalytic disease. Fine-resolution genetic characterization of C. botulinum isolates of any BoNT type is relevant for both epidemiological studies and forensic microbiology. A 10-locus multiple-locus variable-number tandem-repeat analysis (MLVA) was previously applied to isolates of C. botulinum type A. The present study includes five additional loci designed to better address proteolytic B and F serotypes. We investigated 79 C. botulinum group I strains isolated from human and food samples in several European countries, including types A (28), B (36), AB (4), and F (11) strains, and 5 nontoxic Clostridium sporogenes. Additional data were deduced from in silico analysis of 10 available fully sequenced genomes. This 15-locus MLVA (MLVA-15) scheme identified 86 distinct genotypes that clustered consistently with the results of amplified fragment length polymorphism (AFLP) and MLVA genotyping in previous reports. An MLVA-7 scheme, a subset of the MLVA-15, performed on a lab-on-a-chip device using a nonfluorescent subset of primers, is also proposed as a first-line assay. The phylogenetic grouping obtained with the MLVA-7 does not differ significantly from that generated by the MLVA-15. To our knowledge, this report is the first to analyze genetic variability among all of the C. botulinum group I serotypes by MLVA. Our data provide new insights into the genetic variability of group I C. botulinum isolates worldwide and demonstrate that this group is genetically highly diverse. PMID:22012011

Clostridium botulinum group I strain genotyping by 15-locus multilocus variable-number tandem-repeat analysis.

PubMed

Fillo, Silvia; Giordani, Francesco; Anniballi, Fabrizio; Gorgé, Olivier; Ramisse, Vincent; Vergnaud, Gilles; Riehm, Julia M; Scholz, Holger C; Splettstoesser, Wolf D; Kieboom, Jasper; Olsen, Jaran-Strand; Fenicia, Lucia; Lista, Florigio

2011-12-01

Clostridium botulinum is a taxonomic designation that encompasses a broad variety of spore-forming, Gram-positive bacteria producing the botulinum neurotoxin (BoNT). C. botulinum is the etiologic agent of botulism, a rare but severe neuroparalytic disease. Fine-resolution genetic characterization of C. botulinum isolates of any BoNT type is relevant for both epidemiological studies and forensic microbiology. A 10-locus multiple-locus variable-number tandem-repeat analysis (MLVA) was previously applied to isolates of C. botulinum type A. The present study includes five additional loci designed to better address proteolytic B and F serotypes. We investigated 79 C. botulinum group I strains isolated from human and food samples in several European countries, including types A (28), B (36), AB (4), and F (11) strains, and 5 nontoxic Clostridium sporogenes. Additional data were deduced from in silico analysis of 10 available fully sequenced genomes. This 15-locus MLVA (MLVA-15) scheme identified 86 distinct genotypes that clustered consistently with the results of amplified fragment length polymorphism (AFLP) and MLVA genotyping in previous reports. An MLVA-7 scheme, a subset of the MLVA-15, performed on a lab-on-a-chip device using a nonfluorescent subset of primers, is also proposed as a first-line assay. The phylogenetic grouping obtained with the MLVA-7 does not differ significantly from that generated by the MLVA-15. To our knowledge, this report is the first to analyze genetic variability among all of the C. botulinum group I serotypes by MLVA. Our data provide new insights into the genetic variability of group I C. botulinum isolates worldwide and demonstrate that this group is genetically highly diverse.

Rapid carrier screening using short tandem repeats in the phenylalanine hydroxylase gene.

PubMed

Shawky, R M; el-Aleem, K A; Rifaat, M M; el-Naggar, R L; Marzouk, G M

2002-01-01

Phenylketonuria (PKU) is an autosomal recessive genetic disorder caused by defects in the phenylalanine hydroxylase (PAH) system. Our work aimed to screen the PAH locus for the presence of potentially useful short tandem repeats (STR) as markers for carrier detection in PKU families in Egypt, and to determine the level of PAH heterozygosity within the Egyptian population. The system contains at least eight independent alleles in the Egyptian population, transmitted in a Mendelian fashion. Variations in the number of STR in the 16 families studied gave rise to polymorphisms that proved to be suitable markers for PKU carrier detection and prenatal diagnosis. The most frequent allelic fragment size in PKU patients was 246 bp (35.7%), which together with a fragment of 254 bp accounted for 60.7% of the mutant chromosomes.

Determination of Sources of Escherichia coli on Beef by Multiple-Locus Variable-Number Tandem Repeat Analysis.

PubMed

Yang, Xianqin; Tran, Frances; Youssef, Mohamed K; Gill, Colin O

2015-07-01

The possible origin of Escherichia coli found on cuts and trimmings in the breaking facility of a beef packing plant was examined using multiple-locus variable-number tandem repeat analysis. Coliforms and E. coli were enumerated in samples obtained from 160 carcasses that would enter the breaking facility when work commenced and after each of the three production breaks throughout the day, from the conveyor belt before work and after each break, and from cuts and trimmings when work commenced and after each break. Most samples yielded no E. coli, irrespective of the surface types. E. coli was recovered from 7 (<5%) carcasses, at numbers mostly ≤1.0 log CFU/160,000 cm(2). The log total numbers of E. coli recovered from the conveyor belt, cuts, and trimmings were mostly between 1 and 2 log CFU/80,000 cm(2). A total of 554 E. coli isolates were recovered. Multiple-locus variable-number tandem repeat analysis of 327 selected isolates identified 80 distinct genotypes, with 37 (46%) each containing one isolate. However, 28% of the isolates were of genotypes that were recovered from more than one sampling day. Of the 80 genotypes, 65 and 2% were found in one or all four sampling periods throughout the day. However, they represented 23 and 14% of the isolates, respectively. Of the genotypes identified for each surface type, at least one contained ≥9 isolates. No unique genotypes were associated with carcasses, but 10, 17, and 19 were uniquely associated with cuts, trimmings, and the belt, respectively. Of the isolates recovered from cuts, 49, 3, and 19% were of genotypes that were found among isolates recovered from the belt, carcasses, or both the belt and carcasses, respectively. A similar composition was found for isolates recovered from trimmings. These findings show that the E. coli found on cuts and trimmings at this beef packing plant mainly originated from the conveyor belt and that small number of E. coli strains survived the daily cleaning and sanitation process, thus persisting in the plant.

Genetic data and de novo mutation rates in father-son pairs of 23 Y-STR loci in Southern Brazil population.

PubMed

Da Fré, Nicole Nascimento; Rodenbusch, Rodrigo; Gastaldo, André Zoratto; Hanson, Erin; Ballantyne, Jack; Alho, Clarice Sampaio

2015-11-01

We evaluated haplotype and allele frequencies, as well as statistical forensic parameters, for 23 Y-chromosome short tandem repeats (STRs) loci of the PowerPlex®Y23 system (DYS19, DYS385a/b, DYS389I/II, DYS390, DYS391, DYS392, DYS393, DYS437, DYS438, DYS439, DYS448, DYS456, DYS458, DYS635, Y-GATA-H4, DYS481, DYS533, DYS549, DYS570, DYS576, DYS643) in a sample of 150 apparently healthy males, resident in South Brazil. A total of 150 different haplotypes were identified. The highest gene diversity (GD) was observed for the single locus marker DYS570 (GD = 0.7888) and for a two-locus system DYS385 (GD = 0.9009). We also examined 150 father-son pairs by the same system, and a total of 13 mutations were identified in the 3450 father-son allelic transfers, with an overall mutation rate across the 23 loci of 3.768 × 10(-3) (95% CI: 3.542 × 10(-3) to 3.944 × 10(-3)). In all cases there was only one locus mutated with gain/loss of repeats in the son (5 one-repeat gains, and 7 one-repeat and 1 two-repeat losses); we observed no instances of mutations involving a non-integral number of repeats.

Multiple-Locus Variable-Number Tandem Repeat Analysis of Dutch Bordetella pertussis Strains Reveals Rapid Genetic Changes with Clonal Expansion during the Late 1990s

PubMed Central

Schouls, Leo M.; van der Heide, Han G. J.; Vauterin, Luc; Vauterin, Paul; Mooi, Frits R.

2004-01-01

Bordetella pertussis, the causative agent of whooping cough, has remained endemic in The Netherlands despite extensive nationwide vaccination since 1953. In the 1990s, several epidemic periods have resulted in many cases of pertussis. We have proposed that strain variation has played a major role in the upsurges of this disease in The Netherlands. Therefore, molecular characterization of strains is important in identifying the causes of pertussis epidemiology. For this reason, we have developed a multiple-locus variable-number tandem repeat analysis (MLVA) typing system for B. pertussis. By combining the MLVA profile with the allelic profile based on multiple-antigen sequence typing, we were able to further differentiate strains. The relationships between the various genotypes were visualized by constructing a minimum spanning tree. MLVA of Dutch strains of B. pertussis revealed that the genotypes of the strains isolated in the prevaccination period were diverse and clearly distinct from the strains isolated in the 1990s. Furthermore, there was a decrease in diversity in the strains from the late 1990s, with a remarkable clonal expansion that coincided with the epidemic periods. Using this genotyping, we have been able to show that B. pertussis is much more dynamic than expected. PMID:15292152

Molecular characterization of enterohemorrhagic Escherichia coli O157:H7 isolates dispersed across Japan by pulsed-field gel electrophoresis and multiple-locus variable-number tandem repeat analysis.

PubMed

Pei, Yingxin; Terajima, Jun; Saito, Yasunori; Suzuki, Reiko; Takai, Nobuko; Izumiya, Hidemasa; Morita-Ishihara, Tomoko; Ohnishi, Makoto; Miura, Masashi; Iyoda, Sunao; Mitobe, Jiro; Wang, Binyou; Watanabe, Haruo

2008-01-01

We identified seven distinct subtypes of enterohemorrhagic Escherichia coli (EHEC) O157:H7 isolates that were derived from sporadic cases and outbreaks from multiple prefectures in Japan in 2005. A surveillance system utilizing pulsed-field gel electrophoresis (PFGE), PulseNet Japan, was used. Some strains showed indistinguishable PFGE patterns using another restriction enzyme (BlnI or SpeI) in each subtype of EHEC O157:H7 isolates that were routinely subtyped by the XbaI PFGE pattern. In order to examine the genotypic relatedness of these strains, we carried out a multiple-locus variable-number tandem repeat (VNTR) analysis (MLVA). By using the MLVA system, we found that three of seven subtypes of EHEC O157:H7 strains that were isolated from sporadic cases dispersed across multiple prefectures within a few months showed indistinguishable PFGE patterns and identical MLVA types. Strains belonging to the other four subtypes of EHEC O157:H7 in the PFGE analysis were further classified into different clusters of EHEC O157:H7. Therefore, compared to PFGE, MLVA showed greater discriminatory power with respect to analysis of the isolates in this study.

Multi-locus variable number tandem repeat analysis of 7th pandemic Vibrio cholerae

PubMed Central

2012-01-01

Background Seven pandemics of cholera have been recorded since 1817, with the current and ongoing pandemic affecting almost every continent. Cholera remains endemic in developing countries and is still a significant public health issue. In this study we use multilocus variable number of tandem repeats (VNTRs) analysis (MLVA) to discriminate between isolates of the 7th pandemic clone of Vibrio cholerae. Results MLVA of six VNTRs selected from previously published data distinguished 66 V. cholerae isolates collected between 1961–1999 into 60 unique MLVA profiles. Only 4 MLVA profiles consisted of more than 2 isolates. The discriminatory power was 0.995. Phylogenetic analysis showed that, except for the closely related profiles, the relationships derived from MLVA profiles were in conflict with that inferred from Single Nucleotide Polymorphism (SNP) typing. The six SNP groups share consensus VNTR patterns and two SNP groups contained isolates which differed by only one VNTR locus. Conclusions MLVA is highly discriminatory in differentiating 7th pandemic V. cholerae isolates and MLVA data was most useful in resolving the genetic relationships among isolates within groups previously defined by SNPs. Thus MLVA is best used in conjunction with SNP typing in order to best determine the evolutionary relationships among the 7th pandemic V. cholerae isolates and for longer term epidemiological typing. PMID:22624829

Capillary sample introduction of polymerase chain reaction (PCR) products separated in ultrathin slab gels.

PubMed

Bullard, K M; Hietpas, P B; Ewing, A G

1998-01-01

Polymerase chain reaction (PCR) amplified short tandem repeat (STR) samples from the HUMVWF locus have been analyzed using a unique sample introduction and separation technique. A single capillary is used to transfer samples onto an ultrathin slab gel (57 microm thin). This ultrathin nondenaturing polyacrylamide gel is used to separate the amplified fragments, and laser-induced fluorescence with ethidium bromide is used for detection. The feasibility of performing STR analysis using this system has been investigated by examining the reproducibility for repeated samples. Reproducibility is examined by comparing the migration of the 14 and 17 HUMVWF alleles on three consecutive separations on the ultrathin slab gel. Using one locus, separations match in migration time with the two alleles 42 s apart for each of the three consecutive separations. This technique shows potential to increase sample throughput in STR analysis techniques although separation resolution still needs to be improved.

Interpreting short tandem repeat variations in humans using mutational constraint

PubMed Central

Gymrek, Melissa; Willems, Thomas; Reich, David; Erlich, Yaniv

2017-01-01

Identifying regions of the genome that are depleted of mutations can reveal potentially deleterious variants. Short tandem repeats (STRs), also known as microsatellites, are among the largest contributors of de novo mutations in humans. However, per-locus studies of STR mutations have been limited to highly ascertained panels of several dozen loci. Here, we harnessed bioinformatics tools and a novel analytical framework to estimate mutation parameters for each STR in the human genome by correlating STR genotypes with local sequence heterozygosity. We applied our method to obtain robust estimates of the impact of local sequence features on mutation parameters and used this to create a framework for measuring constraint at STRs by comparing observed vs. expected mutation rates. Constraint scores identified known pathogenic variants with early onset effects. Our metric will provide a valuable tool for prioritizing pathogenic STRs in medical genetics studies. PMID:28892063

Sub-typing of extended-spectrum-β-lactamase-producing isolates from a nosocomial outbreak: application of a 10-loci generic Escherichia coli multi-locus variable number tandem repeat analysis.

PubMed

Karami, Nahid; Helldal, Lisa; Welinder-Olsson, Christina; Ahrén, Christina; Moore, Edward R B

2013-01-01

Extended-spectrum β-lactamase producing Escherichia coli (ESBL-E. coli) were isolated from infants hospitalized in a neonatal, post-surgery ward during a four-month-long nosocomial outbreak and six-month follow-up period. A multi-locus variable number tandem repeat analysis (MLVA), using 10 loci (GECM-10), for 'generic' (i.e., non-STEC) E. coli was applied for sub-species-level (i.e., sub-typing) delineation and characterization of the bacterial isolates. Ten distinct GECM-10 types were detected among 50 isolates, correlating with the types defined by pulsed-field gel electrophoresis (PFGE), which is recognized to be the 'gold-standard' method for clinical epidemiological analyses. Multi-locus sequence typing (MLST), multiplex PCR genotyping of bla CTX-M, bla TEM, bla OXA and bla SHV genes and antibiotic resistance profiling, as well as a PCR assay specific for detecting isolates of the pandemic O25b-ST131 strain, further characterized the outbreak isolates. Two clusters of isolates with distinct GECM-10 types (G06-04 and G07-02), corresponding to two major PFGE types and the MLST-based sequence types (STs) 131 and 1444, respectively, were confirmed to be responsible for the outbreak. The application of GECM-10 sub-typing provided reliable, rapid and cost-effective epidemiological characterizations of the ESBL-producing isolates from a nosocomial outbreak that correlated with and may be used to replace the laborious PFGE protocol for analyzing generic E. coli.

Comprehensive mutation analysis of 17 Y-chromosomal short tandem repeat polymorphisms included in the AmpFlSTR Yfiler PCR amplification kit.

PubMed

Goedbloed, Miriam; Vermeulen, Mark; Fang, Rixun N; Lembring, Maria; Wollstein, Andreas; Ballantyne, Kaye; Lao, Oscar; Brauer, Silke; Krüger, Carmen; Roewer, Lutz; Lessig, Rüdiger; Ploski, Rafal; Dobosz, Tadeusz; Henke, Lotte; Henke, Jürgen; Furtado, Manohar R; Kayser, Manfred

2009-11-01

The Y-chromosomal short tandem repeat (Y-STR) polymorphisms included in the AmpFlSTR Yfiler polymerase chain reaction amplification kit have become widely used for forensic and evolutionary applications where a reliable knowledge on mutation properties is necessary for correct data interpretation. Therefore, we investigated the 17 Yfiler Y-STRs in 1,730-1,764 DNA-confirmed father-son pairs per locus and found 84 sequence-confirmed mutations among the 29,792 meiotic transfers covered. Of the 84 mutations, 83 (98.8%) were single-repeat changes and one (1.2%) was a double-repeat change (ratio, 1:0.01), as well as 43 (51.2%) were repeat gains and 41 (48.8%) repeat losses (ratio, 1:0.95). Medians from Bayesian estimation of locus-specific mutation rates ranged from 0.0003 for DYS448 to 0.0074 for DYS458, with a median rate across all 17 Y-STRs of 0.0025. The mean age (at the time of son's birth) of fathers with mutations was with 34.40 (+/-11.63) years higher than that of fathers without ones at 30.32 (+/-10.22) years, a difference that is highly statistically significant (p < 0.001). A Poisson-based modeling revealed that the Y-STR mutation rate increased with increasing father's age on a statistically significant level (alpha = 0.0294, 2.5% quantile = 0.0001). From combining our data with those previously published, considering all together 135,212 meiotic events and 331 mutations, we conclude for the Yfiler Y-STRs that (1) none had a mutation rate of >1%, 12 had mutation rates of >0.1% and four of <0.1%, (2) single-repeat changes were strongly favored over multiple-repeat ones for all loci but 1 and (3) considerable variation existed among loci in the ratio of repeat gains versus losses. Our finding of three Y-STR mutations in one father-son pair (and two pairs with two mutations each) has consequences for determining the threshold of allelic differences to conclude exclusion constellations in future applications of Y-STRs in paternity testing and pedigree analyses.

Rapid and high resolution genotyping of all Escherichia coli serotypes using 10 genomic repeat-containing loci.

PubMed

Løbersli, Inger; Haugum, Kjersti; Lindstedt, Bjørn-Arne

2012-01-01

Our laboratory has previously published two multiple-locus variable-number tandem-repeats analysis (MLVA) methods for rapid genotyping of Escherichia coli (E. coli), which are now in routine use for surveillance and outbreak detection. The first assay developed was specific for E. coli O157:H7; however this assay was not suitable for genotyping other E. coli serotypes. A new generic MLVA-assay was then developed with the capability of genotyping all E. coli serotypes. This generic E. coli MLVA (GECM7) was based on polymorphism in seven variable number of tandem repeats (VNTR) loci. GECM7 worked well with the majority of E. coli serotypes; however we wanted to increase the resolution for this method based in part of comparison with PFGE typing of E. coli O26:H11, where PFGE appeared to display higher resolution. The GECM7 method was improved by adding three new repeat-loci to a total of ten (GECM10), and a considerable increase in resolution was observed (from 296 to 507 genotypes on the same set of strains). Copyright © 2011 Elsevier B.V. All rights reserved.

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

Aplin, H.M.; Hirst, K.L.; Crosby, A.H.

Dentinogenesis imperfecta type II (DGI1) is an autosomal dominant disorder of dentin formation, which has been mapped to human chromosome 4q12-q21. The region most likely to contain the DGI1 locus is a 3.2-cM region surrounding the osteopontin (SPP1) locus. Recently, a novel dentin-specific acidic phosphoprotein (dmp1) has been cloned in the rat and mapped to mouse chromosome 5q21. In the current investigation, we have isolated a cosmid containing the human DMP1 gene. The isolation of a short tandem repeat polymorphism at this locus has allowed us to map the DMP1 locus to human chromosome 4q21 and demonstrate that it ismore » tightly linked to DGI1 in two families (Z{sub max} = 11.01, {theta} = 0.001). The creation of a yeast artificial chromosome contig around SPP1 has further allowed us to demonstrate that DMP1 is located within 150 kb of the bone sialoprotein and 490 kb of the SPP1 loci, respectively. DMP1 is therefore a strong candidate for the DGI1 locus. 12 refs., 2 figs., 1 tab.« less

Diversity among French Bacillus anthracis isolates.

PubMed

Fouet, Agnès; Smith, Kimothy L; Keys, Chris; Vaissaire, Josée; Le Doujet, Claudine; Lévy, Martine; Mock, Michèle; Keim, Paul

2002-12-01

While outbreaks of animal anthrax zoonoses still regularly occur in France, little is known about the epidemiology links between them. We have used the eight-locus multilocus variable-number tandem repeat analysis typing technique against a collection of 50 Bacillus anthracis isolates from France. There were eight distinct genotypes belonging to two dissimilar genetic clusters. Regional strain patterns were observed, with the B2 genotypes prevalent in southern France and the A1a genotypes found only in northern France.

Genetic variation in a compound short tandem repeat/Alu haplotype system at the SB19.3 locus: properties and interpretation.

PubMed

Gaspar, Paulo; Seixas, Susana; Rocha, Jorge

2004-04-01

The genetic variation at a compound nonrecombining haplotype system, consisting of the previously reported SB19.3 Alu insertion polymorphism and a newly identified adjacent short tandem repeat (STR), was studied in population samples from Portugal and São Tomé (Gulf of Guinea, West Africa). Age estimates based on the linked microsatellite variation suggest that the Alu insertion occurred about 190,000 years ago. In accordance with the global patterns of distribution of human genetic variation, the highest haplotype diversity was found in the African sample. This excess in African diversity was due to both a substantial reduction in heterozygosity at the Alu polymorphism and a lower STR variability associated with the predominant Alu insertion allele in the Portuguese sample. The high level of interpopulation differentiation observed at the Alu locus (F(ST) = 0.43) was interpreted under alternative selective and demographic scenarios. The need for compatibility between patterns of variation at the STR and Alu loci could be used to restrict the range of selection coefficients in selection-driven genetic hitchhiking frameworks and to favor demographic scenarios dominated by larger pre-expansion African population sizes. Taken together, the data show that the SB19.3 Alu-STR system is an informative marker that can be included in more extended batteries of compound haplotypes used in human evolutionary studies.

Multicolor-based discrimination of 21 short tandem repeats and amelogenin using four fluorescent universal primers.

PubMed

Asari, Masaru; Okuda, Katsuhiro; Hoshina, Chisato; Omura, Tomohiro; Tasaki, Yoshikazu; Shiono, Hiroshi; Matsubara, Kazuo; Shimizu, Keiko

2016-02-01

The aim of this study was to develop a cost-effective genotyping method using high-quality DNA for human identification. A total of 21 short tandem repeats (STRs) and amelogenin were selected, and fluorescent fragments at 22 loci were simultaneously amplified in a single-tube reaction using locus-specific primers with 24-base universal tails and four fluorescent universal primers. Several nucleotide substitutions in universal tails and fluorescent universal primers enabled the detection of specific fluorescent fragments from the 22 loci. Multiplex polymerase chain reaction (PCR) produced intense FAM-, VIC-, NED-, and PET-labeled fragments ranging from 90 to 400 bp, and these fragments were discriminated using standard capillary electrophoretic analysis. The selected 22 loci were also analyzed using two commercial kits (the AmpFLSTR Identifiler Kit and the PowerPlex ESX 17 System), and results for two loci (D19S433 and D16S539) were discordant between these kits due to mutations at the primer binding sites. All genotypes from the 100 samples were determined using 2.5 ng of DNA by our method, and the expected alleles were completely recovered. Multiplex 22-locus genotyping using four fluorescent universal primers effectively reduces the costs to less than 20% of genotyping using commercial kits, and our method would be useful to detect silent alleles from commercial kit analysis. Copyright © 2015 Elsevier Inc. All rights reserved.

Multiple locus VNTR analysis highlights that geographical clustering and distribution of Dichelobacter nodosus, the causal agent of footrot in sheep, correlates with inter-country movements☆

PubMed Central

Russell, Claire L.; Smith, Edward M.; Calvo-Bado, Leonides A.; Green, Laura E.; Wellington, Elizabeth M.H.; Medley, Graham F.; Moore, Lynda J.; Grogono-Thomas, Rosemary

2014-01-01

Dichelobacter nodosus is a Gram-negative, anaerobic bacterium and the causal agent of footrot in sheep. Multiple locus variable number tandem repeat (VNTR) analysis (MLVA) is a portable technique that involves the identification and enumeration of polymorphic tandem repeats across the genome. The aims of this study were to develop an MLVA scheme for D. nodosus suitable for use as a molecular typing tool, and to apply it to a global collection of isolates. Seventy-seven isolates selected from regions with a long history of footrot (GB, Australia) and regions where footrot has recently been reported (India, Scandinavia), were characterised. From an initial 61 potential VNTR regions, four loci were identified as usable and in combination had the attributes required of a typing method for use in bacterial epidemiology: high discriminatory power (D > 0.95), typeability and reproducibility. Results from the analysis indicate that D. nodosus appears to have evolved via recombinational exchanges and clonal diversification. This has resulted in some clonal complexes that contain isolates from multiple countries and continents; and others that contain isolates from a single geographic location (country or region). The distribution of alleles between countries matches historical accounts of sheep movements, suggesting that the MLVA technique is sufficiently specific and sensitive for an epidemiological investigation of the global distribution of D. nodosus. PMID:23748018

Characterization of the differentially methylated region of the Impact gene that exhibits Glires-specific imprinting.

PubMed

Okamura, Kohji; Wintle, Richard F; Scherer, Stephen W

2008-01-01

Imprinted genes are exclusively expressed from one of the two parental alleles in a parent-of-origin-specific manner. In mammals, nearly 100 genes are documented to be imprinted. To understand the mechanism behind this gene regulation and to identify novel imprinted genes, common features of DNA sequences have been analyzed; however, the general features required for genomic imprinting have not yet been identified, possibly due to variability in underlying molecular mechanisms from locus to locus. We performed a thorough comparative genomic analysis of a single locus, Impact, which is imprinted only in Glires (rodents and lagomorphs). The fact that Glires and primates diverged from each other as recent as 70 million years ago makes comparisons between imprinted and non-imprinted orthologues relatively reliable. In species from the Glires clade, Impact bears a differentially methylated region, whereby the maternal allele is hypermethylated. Analysis of this region demonstrated that imprinting was not associated with the presence of direct tandem repeats nor with CpG dinucleotide density. In contrast, a CpG periodicity of 8 bp was observed in this region in species of the Glires clade compared to those of carnivores, artiodactyls, and primates. We show that tandem repeats are dispensable, establishment of the differentially methylated region does not rely on G+C content and CpG density, and the CpG periodicity of 8 bp is meaningful to the imprinting. This interval has recently been reported to be optimal for de novo methylation by the Dnmt3a-Dnmt3L complex, suggesting its importance in the establishment of imprinting in Impact and other genes.

Neutral polymorphisms in putative housekeeping genes and tandem repeats unravels the population genetics and evolutionary history of Plasmodium vivax in India.

PubMed

Prajapati, Surendra K; Joshi, Hema; Carlton, Jane M; Rizvi, M Alam

2013-01-01

The evolutionary history and age of Plasmodium vivax has been inferred as both recent and ancient by several studies, mainly using mitochondrial genome diversity. Here we address the age of P. vivax on the Indian subcontinent using selectively neutral housekeeping genes and tandem repeat loci. Analysis of ten housekeeping genes revealed a substantial number of SNPs (n = 75) from 100 P. vivax isolates collected from five geographical regions of India. Neutrality tests showed a majority of the housekeeping genes were selectively neutral, confirming the suitability of housekeeping genes for inferring the evolutionary history of P. vivax. In addition, a genetic differentiation test using housekeeping gene polymorphism data showed a lack of geographical structuring between the five regions of India. The coalescence analysis of the time to the most recent common ancestor estimate yielded an ancient TMRCA (232,228 to 303,030 years) and long-term population history (79,235 to 104,008) of extant P. vivax on the Indian subcontinent. Analysis of 18 tandem repeat loci polymorphisms showed substantial allelic diversity and heterozygosity per locus, and analysis of potential bottlenecks revealed the signature of a stable P. vivax population, further corroborating our ancient age estimates. For the first time we report a comparable evolutionary history of P. vivax inferred by nuclear genetic markers (putative housekeeping genes) to that inferred from mitochondrial genome diversity.

A Large Population Genetic Study of 15 Autosomal Short Tandem Repeat Loci for Establishment of Korean DNA Profile Database

PubMed Central

Yoo, Seong Yeon; Cho, Nam Soo; Park, Myung Jin; Seong, Ki Min; Hwang, Jung Ho; Song, Seok Bean; Han, Myun Soo; Lee, Won Tae; Chung, Ki Wha

2011-01-01

Genotyping of highly polymorphic short tandem repeat (STR) markers is widely used for the genetic identification of individuals in forensic DNA analyses and in paternity disputes. The National DNA Profile Databank recently established by the DNA Identification Act in Korea contains the computerized STR DNA profiles of individuals convicted of crimes. For the establishment of a large autosomal STR loci population database, 1805 samples were obtained at random from Korean individuals and 15 autosomal STR markers were analyzed using the AmpFlSTR Identifiler PCR Amplification kit. For the 15 autosomal STR markers, no deviations from the Hardy-Weinberg equilibrium were observed. The most informative locus in our data set was the D2S1338 with a discrimination power of 0.9699. The combined matching probability was 1.521 × 10-17. This large STR profile dataset including atypical alleles will be important for the establishment of the Korean DNA database and for forensic applications. PMID:21597912

A large population genetic study of 15 autosomal short tandem repeat loci for establishment of Korean DNA profile database.

PubMed

Yoo, Seong Yeon; Cho, Nam Soo; Park, Myung Jin; Seong, Ki Min; Hwang, Jung Ho; Song, Seok Bean; Han, Myun Soo; Lee, Won Tae; Chung, Ki Wha

2011-07-01

Genotyping of highly polymorphic short tandem repeat (STR) markers is widely used for the genetic identification of individuals in forensic DNA analyses and in paternity disputes. The National DNA Profile Databank recently established by the DNA Identification Act in Korea contains the computerized STR DNA profiles of individuals convicted of crimes. For the establishment of a large autosomal STR loci population database, 1805 samples were obtained at random from Korean individuals and 15 autosomal STR markers were analyzed using the AmpFlSTR Identifiler PCR Amplification kit. For the 15 autosomal STR markers, no deviations from the Hardy-Weinberg equilibrium were observed. The most informative locus in our data set was the D2S1338 with a discrimination power of 0.9699. The combined matching probability was 1.521 × 10(-17). This large STR profile dataset including atypical alleles will be important for the establishment of the Korean DNA database and for forensic applications.

Stability of Tandem Repeats in the Drosophila Melanogaster HSR-Omega Nuclear RNA

PubMed Central

Hogan, N. C.; Slot, F.; Traverse, K. L.; Garbe, J. C.; Bendena, W. G.; Pardue, M. L.

1995-01-01

The Drosophila melanogaster Hsr-omega locus produces a nuclear RNA containing >5 kb of tandem repeat sequences. These repeats are unique to Hsr-omega and show concerted evolution similar to that seen with classical satellite DNAs. In D. melanogaster the monomer is ~280 bp. Sequences of 191/2 monomers differ by 8 +/- 5% (mean +/- SD), when all pairwise comparisons are considered. Differences are single nucleotide substitutions and 1-3 nucleotide deletions/insertions. Changes appear to be randomly distributed over the repeat unit. Outer repeats do not show the decrease in monomer homogeneity that might be expected if homogeneity is maintained by recombination. However, just outside the last complete repeat at each end, there are a few fragments of sequence similar to the monomer. The sequences in these flanking regions are not those predicted for sequences decaying in the absence of recombination. Instead, the fragmentation of the sequence homology suggests that flanking regions have undergone more severe disruptions, possibly during an insertion or amplification event. Hsr-omega alleles differing in the number of repeats are detected and appear to be stable over a few thousand generations; however, both increases and decreases in repeat numbers have been observed. The new alleles appear to be as stable as their predecessors. No alleles of less than ~5 kb nor more than ~16 kb of repeats were seen in any stocks examined. The evidence that there is a limit on the minimum number of repeats is consistent with the suggestion that these repeats are important in the function of the unusual Hsr-omega nuclear RNA. PMID:7540581

[Family-based association study of a variable number of tandem repeat polymorphism of DAT1 gene with Tourette syndrome in a Chinese Han population].

PubMed

Zheng, Lanlan; Han, Zhen-liang; Zhang, Xin-hua; Wang, Xue-qin; Jiang, Wei-hua; Yi, Ming-ji; Liu, Shi-guo

2013-10-01

To assess the association of a 40 bp variable number of tandem repeat (VNTR) polymorphism within 3 untranslated region of dopamine transporter gene (DAT1) with Tourette syndrome (TS) in a Chinese Han population. A total of 160 TS patients and their parents were recruited. The VNTR polymorphism was detected with polymerase chain reaction-VNTR analysis, and its association with TS and its subtypes were assessed through a family-based association study comprising transmission disequilibrium test (TDT) and haplotype relative risk (HRR) analysis. The repeat numbers at the DAT1 40 bp locus were 11, 10, 9, 7.5 and 7 among the patients and their parents, with the most common type being a 10-repeat allele. No significant association was detected between the polymorphism and TS (TDT: X ² = 0.472, df = 1, P = 0.583; HRR: X ² = 0.313, P = 0.576, OR = 0.855, 95%CI: 0.493-1.481). Our data suggested that the VNTR polymorphism of DAT1 gene is not associated with susceptibility to TS in Chinese Han population. However, our results are to be validated in larger sets of patients collected from other populations.

Mutation rates at 42 Y chromosomal short tandem repeats in Chinese Han population in Eastern China.

PubMed

Wu, Weiwei; Ren, Wenyan; Hao, Honglei; Nan, Hailun; He, Xin; Liu, Qiuling; Lu, Dejian

2018-01-31

Mutation analysis of 42 Y chromosomal short tandem repeats (Y-STRs) loci was performed using a sample of 1160 father-son pairs from the Chinese Han population in Eastern China. The results showed that the average mutation rate across the 42 Y-STR loci was 0.0041 (95% CI 0.0036-0.0047) per locus per generation. The locus-specific mutation rates varied from 0.000 to 0.0190. No mutation was found at DYS388, DYS437, DYS448, DYS531, and GATA_H4. DYS627, DYS570, DYS576, and DYS449 could be classified as rapidly mutating Y-STRs, with mutation rates higher than 1.0 × 10 -2 . DYS458, DYS630, and DYS518 were moderately mutating Y-STRs, with mutation rates ranging from 8 × 10 -3 to 1 × 10 -2 . Although the characteristics of the Y-STR mutations were consistent with those in previous studies, mutation rate differences between our data and previous published data were found at some rapidly mutating Y-STRs. The single-copy loci located on the short arm of the Y chromosome (Yp) showed relatively higher mutation rates more frequently than the multi-copy loci. These results will not only extend the data for Y-STR mutations but also be important for kinship analysis, paternal lineage identification, and family relationship reconstruction in forensic Y-STR analysis.

Genetic characterization of non-O157 verocytotoxigenic Escherichia coli isolated from raw beef products using multiple-locus variable-number tandem repeat analysis.

PubMed

Franci, Tomás; Sanso, A Mariel; Bustamante, Ana V; Lucchesi, Paula M A; Parma, Alberto E

2011-09-01

Verocytotoxigenic Escherichia coli (VTEC) can produce serious human illness linked to the consumption of contaminated food, mainly of bovine origin. There is growing concern about non-O157 VTEC serotypes, which in some countries cause severe infections in a proportion similar to O157:H7 strains. As several epidemiological studies indicated the important role of meat as the major vehicle in the transmission of this pathogen to human consumers, our aim was to investigate the genetic diversity among non-O157:H7 VTEC isolated from raw beef products. We performed a multiple-locus variable-number tandem repeat (VNTR) analysis (MLVA), and to our knowledge, this is the first time that VTEC serotypes O8:H19, O112:H2, O113:NM, O171:NM, ONT:H7, ONT:H19, and ONT:H21 were typed by this method. MLVA typing grouped the total number of strains from this study (51) into 21 distinct genotypes, and 11 of them were unique. Several MLVA profiles were found in different serotypes, O178:H19 being the most variable. The isolates could be principally discriminated by alleles of three of seven loci studied (CVN001, CVN004, and CVN014), and on the other hand, CVN003 rendered null alleles in all the isolates. As some VNTR markers might be serotype specific, it is possible that the implementation of new VNTR loci will increase intraserotype discrimination.

Multiple-locus variable-nucleotide tandem repeat subtype analysis implicates European starlings as biological vectors for Escherichia coli O157:H7 in Ohio, USA.

PubMed

Williams, M L; Pearl, D L; Lejeune, J T

2011-10-01

To provide molecular epidemiological evidence of avian transmission of Escherichia coli O157:H7 between dairy farms in Ohio, this study was designed to identify genetic relatedness between isolates originating from bovine faecal samples and intestinal contents of European starlings captured on these farms. During a three-year period (2007-2009), cattle (n = 9000) and starlings (n = 430) on 150 different dairy farms in northern Ohio were sampled for the presence of E. coli O157:H7. Isolates were subjected to multiple-locus variable-nucleotide tandem repeat analysis (MLVA). Distinct allelic groups were identified on most farms; however, isolates clustering into three MLVA groups originated from both cattle and birds on different farms. Sharing of indistinguishable epidemiologically linked E. coli O157 MLVA subtypes between starlings and cattle on different farms supports the hypothesis that these birds contribute to the transmission of E. coli O157:H7 between dairy farms. A continued need exists to identify and to improve preharvest measures for controlling E. coli O157:H7. Controlling wildlife intrusion, particularly European starlings, on livestock operations, may be an important strategy for reducing dissemination of E. coli O157:H7 between farms and thereby potentially decreasing the on-farm prevalence of E. coli O157:H7 and enhancing the safety of the food supply. © 2011 The Authors. Journal of Applied Microbiology © 2011 The Society for Applied Microbiology.

CRISPR: A Useful Genetic Feature to Follow Vaginal Carriage of Group B Streptococcus

PubMed Central

Beauruelle, Clémence; Pastuszka, Adeline; Horvath, Philippe; Perrotin, Franck; Mereghetti, Laurent; Lanotte, Philippe

2017-01-01

Clustered regularly interspaced short palindromic repeats (CRISPR) and Cas (CRISPR-associated proteins) play a critical role in adaptive immunity against mobile genetic elements, especially phages, through their ability to acquire novel spacer sequences. Polarized spacer acquisition results in spacer polymorphism and temporal organization of CRISPR loci, making them attractive epidemiological markers. Group B Streptococcus (GBS), a genital commensal for 10 to 30% of healthy women and a major neonatal pathogen, possesses a ubiquitous and functional CRISPR1 locus. Our aim was to assess the CRISPR1 locus as an epidemiological marker to follow vaginal carriage of GBS in women. This study also allowed us to observe the evolution of the CRISPR1 locus in response to probable phage infection occurring in vivo. We followed carriage of GBS among 100 women over an 11-year period, with a median duration of approximately 2 years. The CRISPR1 locus was highly conserved over time. The isolates that show the same CRISPR1 genotype were collected from 83% of women. There was an agreement between CRISPR genotyping and other typing methods [MLVA (multilocus variable number of tandem repeat Analysis) and MLST (multilocus sequence typing)] for 94% of the cases. The CRISPR1 locus of the isolates from 18 women showed modifications, four of which acquired polarized spacer, highlighting the in vivo functionality of the system. The novel spacer of one isolate had sequence similarity with phage, suggesting that phage infection occurred during carriage. These findings improve our understanding of CRISPR-Cas evolution in GBS and provide a glimpse of host-phage dynamics in vivo. PMID:29075246

Multilocus variable-number tandem repeat analysis distinguishes outbreak and sporadic Escherichia coli O157:H7 isolates.

PubMed

Noller, Anna C; McEllistrem, M Catherine; Pacheco, Antonio G F; Boxrud, David J; Harrison, Lee H

2003-12-01

Escherichia coli O157:H7 is a major cause of food-borne illness in the United States. Outbreak detection involves traditional epidemiological methods and routine molecular subtyping by pulsed-field gel electrophoresis (PFGE). PFGE is labor-intensive, and the results are difficult to analyze and not easily transferable between laboratories. Multilocus variable-number tandem repeat (VNTR) analysis (MLVA) is a fast, portable method that analyzes multiple VNTR loci, which are areas of the bacterial genome that evolve quickly. Eighty isolates, including 21 isolates from five epidemiologically well-characterized outbreaks from Pennsylvania and Minnesota, were analyzed by PFGE and MLVA. Strains in PFGE clusters were defined as strains that differed by less than or equal to one band by using XbaI and the confirmatory enzyme SpeI. MLVA was performed by comparing the number of tandem repeats at seven loci. From 6 to 30 alleles were found at the seven loci, resulting in 64 MLVA types among the 80 isolates. MLVA correctly identified the isolates from all five outbreaks if only a single-locus variant was allowed. MLVA differentiated strains with unique PFGE types. Additionally, MLVA discriminated strains within PFGE-defined clusters that were not known to be part of an outbreak. In addition to being a simple and validated method for E. coli O157:H7 outbreak detection, MLVA appears to have a sensitivity equal to that of PFGE and a specificity superior to that of PFGE.

Genetic distribution of 15 autosomal STR markers in the Punjabi population of Pakistan.

PubMed

Shan, Muhammad Adnan; Hussain, Manzoor; Shafique, Muhammad; Shahzad, Muhammad; Perveen, Rukhsana; Idrees, Muhammad

2016-11-01

Genetic diversity of 15 autosomal short tandem repeat (STR) loci was evaluated in 713 unrelated individual samples of a Punjabi population of Pakistan. These loci were scrutinized to establish allelic frequencies and statistical parameters of forensic and paternity interests. A total of 165 alleles were observed with the corresponding allele frequencies ranging from 0.001 to 0.446. D2S1338 was found as the most informative locus while TPOX (0.611) was the least discriminating locus. The combined power of discrimination (CPD), the combined probability of exclusion (CPE), and cumulative probability of matching (CPM) were found equaled to 0.999999999999999998606227424808, 0.999995777557989, and 1.37543 × 10-18, respectively. All the loci followed the Hardy-Weinberg equilibrium after the Bonferroni correction (p < 0.0033) except one locus D3S1358. The study revealed that these STR loci are highly polymorphic, suitable for forensic and parentage analyses. In comparison to different populations (Asians and non-Asians), significant differences were recorded for these loci.

Identification and characterization of tandem repeats in exon III of dopamine receptor D4 (DRD4) genes from different mammalian species.

PubMed

Larsen, Svend Arild; Mogensen, Line; Dietz, Rune; Baagøe, Hans Jørgen; Andersen, Mogens; Werge, Thomas; Rasmussen, Henrik Berg

2005-12-01

In this study we have identified and characterized dopamine receptor D4 (DRD4) exon III tandem repeats in 33 public available nucleotide sequences from different mammalian species. We found that the tandem repeat in canids could be described in a novel and simple way, namely, as a structure composed of 15- and 12- bp modules. Tandem repeats composed of 18-bp modules were found in sequences from the horse, zebra, onager, and donkey, Asiatic bear, polar bear, common raccoon, dolphin, harbor porpoise, and domestic cat. Several of these sequences have been analyzed previously without a tandem repeat being found. In the domestic cow and gray seal we identified tandem repeats composed of 36-bp modules, each consisting of two closely related 18-bp basic units. A tandem repeat consisting of 9-bp modules was identified in sequences from mink and ferret. In the European otter we detected an 18-bp tandem repeat, while a tandem repeat consisting of 27-bp modules was identified in a sequence from European badger. Both these tandem repeats were composed of 9-bp basic units, which were closely related with the 9-bp repeat modules identified in the mink and ferret. Tandem repeats could not be identified in sequences from rodents. All tandem repeats possessed a high GC content with a strong bias for C. On phylogenetic analysis of the tandem repeats evolutionary related species were clustered into the same groups. The degree of conservation of the tandem repeats varied significantly between species. The deduced amino acid sequences of most of the tandem repeats exhibited a high propensity for disorder. This was also the case with an amino acid sequence of the human DRD4 exon III tandem repeat, which was included in the study for comparative purposes. We identified proline-containing motifs for SH3 and WW domain binding proteins, potential phosphorylation sites, PDZ domain binding motifs, and FHA domain binding motifs in the amino acid sequences of the tandem repeats. The numbers of potential functional sites varied pronouncedly between species. Our observations provide a platform for future studies of the architecture and evolution of the DRD4 exon III tandem repeat, and they suggest that differences in the structure of this tandem repeat contribute to specialization and generation of diversity in receptor function.

Escherichia coli O157:H7 in Ecuador: animal reservoirs, yet no human disease.

PubMed

Trueba, Gabriel; Garcés, Verónica; V, Verónica Barragan; Colman, Rebecca E; Seymour, Meagan; Vogler, Amy J; Keim, Paul

2013-05-01

Escherichia coli O157:H7 is frequently isolated from cases of diarrhea in many industrialized countries; however, it is seldom found in developing countries. The present manuscript reports the presence of E. coli O157:H7 in Ecuadorian livestock, a country where enterohemorrhagic E. coli disease in humans has never been reported. The Ecuadorian isolates were genetically related to some strains linked to clinical cases in the United States as assessed by multiple-locus variable number tandem repeat (VNTR) analysis.

CRISPRcompar: a website to compare clustered regularly interspaced short palindromic repeats.

PubMed

Grissa, Ibtissem; Vergnaud, Gilles; Pourcel, Christine

2008-07-01

Clustered regularly interspaced short palindromic repeat (CRISPR) elements are a particular family of tandem repeats present in prokaryotic genomes, in almost all archaea and in about half of bacteria, and which participate in a mechanism of acquired resistance against phages. They consist in a succession of direct repeats (DR) of 24-47 bp separated by similar sized unique sequences (spacers). In the large majority of cases, the direct repeats are highly conserved, while the number and nature of the spacers are often quite diverse, even among strains of a same species. Furthermore, the acquisition of new units (DR + spacer) was shown to happen almost exclusively on one side of the locus. Therefore, the CRISPR presents an interesting genetic marker for comparative and evolutionary analysis of closely related bacterial strains. CRISPRcompar is a web service created to assist biologists in the CRISPR typing process. Two tools facilitates the in silico investigation: CRISPRcomparison and CRISPRtionary. This website is freely accessible at http://crispr.u-psud.fr/CRISPRcompar/.

Evaluation of a highly discriminating multiplex multi-locus variable-number of tandem-repeats (MLVA) analysis for Vibrio cholerae.

PubMed

Olsen, Jaran S; Aarskaug, Tone; Skogan, Gunnar; Fykse, Else Marie; Ellingsen, Anette Bauer; Blatny, Janet M

2009-09-01

Vibrio cholerae is the etiological agent of cholera and may be used in bioterror actions due to the easiness of its dissemination, and the public fear for acquiring the cholera disease. A simple and highly discriminating method for connecting clinical and environmental isolates of V. cholerae is needed in microbial forensics. Twelve different loci containing variable numbers of tandem-repeats (VNTRs) were evaluated in which six loci were polymorphic. Two multiplex reactions containing PCR primers targeting these six VNTRs resulted in successful DNA amplification of 142 various environmental and clinical V. cholerae isolates. The genetic distribution inside the V. cholerae strain collection was used to evaluate the discriminating power (Simpsons Diversity Index=0.99) of this new MLVA analysis, showing that the assay have a potential to differentiate between various strains, but also to identify those isolates which are collected from a common V. cholerae outbreak. This work has established a rapid and highly discriminating MLVA assay useful for track back analyses and/or forensic studies of V. cholerae infections.

Short tandem repeat DNA typing provides an international reference standard for authentication of human cell lines.

PubMed

Dirks, Wilhelm Gerhard; Faehnrich, Silke; Estella, Isabelle Annick Janine; Drexler, Hans Guenter

2005-01-01

Cell lines have wide applications as model systems in the medical and pharmaceutical industry. Much drug and chemical testing is now first carried out exhaustively on in vitro systems, reducing the need for complicated and invasive animal experiments. The basis for any research, development or production program involving cell lines is the choice of an authentic cell line. Microsatellites in the human genome that harbour short tandem repeat (STR) DNA markers allow individualisation of established cell lines at the DNA level. Fluorescence polymerase chain reaction amplification of eight highly polymorphic microsatellite STR loci plus gender determination was found to be the best tool to screen the uniqueness of DNA profiles in a fingerprint database. Our results demonstrate that cross-contamination and misidentification remain chronic problems in the use of human continuous cell lines. The combination of rapidly generated DNA types based on single-locus STR and their authentication or individualisation by screening the fingerprint database constitutes a highly reliable and robust method for the identification and verification of cell lines.

Variable-number-of-tandem-repeats analysis of genetic diversity in Pasteuria ramosa.

PubMed

Mouton, L; Ebert, D

2008-05-01

Variable-number-of-tandem-repeats (VNTR) markers are increasingly being used in population genetic studies of bacteria. They were recently developed for Pasteuria ramosa, an endobacterium that infects Daphnia species. In the present study, we genotyped P. ramosa in 18 infected hosts from the United Kingdom, Belgium, and two lakes in the United States using seven VNTR markers. Two Daphnia species were collected: D. magna and D. dentifera. Six loci showed length polymorphism, with as many as five alleles identified for a single locus. Similarity coefficient calculations showed that the extent of genetic variation between pairs of isolates within populations differed according to the population, but it was always less than the genetic distances among populations. Analysis of the genetic distances performed using principal component analysis revealed strong clustering by location of origin, but not by host Daphnia species. Our study demonstrated that the VNTR markers available for P. ramosa are informative in revealing genetic differences within and among populations and may therefore become an important tool for providing detailed analysis of population genetics and epidemiology.

DNA fingerprinting of Shiga-toxin producing Escherichia coli O157 based on Multiple-Locus Variable-Number Tandem-Repeats Analysis (MLVA)

PubMed Central

Lindstedt, Bjørn-Arne; Heir, Even; Gjernes, Elisabet; Vardund, Traute; Kapperud, Georg

2003-01-01

Background The ability to react early to possible outbreaks of Escherichia coli O157:H7 and to trace possible sources relies on the availability of highly discriminatory and reliable techniques. The development of methods that are fast and has the potential for complete automation is needed for this important pathogen. Methods In all 73 isolates of shiga-toxin producing E. coli O157 (STEC) were used in this study. The two available fully sequenced STEC genomes were scanned for tandem repeated stretches of DNA, which were evaluated as polymorphic markers for isolate identification. Results The 73 E. coli isolates displayed 47 distinct patterns and the MLVA assay was capable of high discrimination between the E. coli O157 strains. The assay was fast and all the steps can be automated. Conclusion The findings demonstrate a novel high discriminatory molecular typing method for the important pathogen E. coli O157 that is fast, robust and offers many advantages compared to current methods. PMID:14664722

Tracing Staphylococcus aureus in small and medium-sized food-processing factories on the basis of molecular sub-species typing.

PubMed

Koreňová, Janka; Rešková, Zuzana; Véghová, Adriana; Kuchta, Tomáš

2015-01-01

Contamination by Staphylococcus aureus of the production environment of three small or medium-sized food-processing factories in Slovakia was investigated on the basis of sub-species molecular identification by multiple locus variable number of tandem repeats analysis (MLVA). On the basis of MLVA profiling, bacterial isolates were assigned to 31 groups. Data from repeated samplings over a period of 3 years facilitated to draw spatial and temporal maps of the contamination routes for individual factories, as well as identification of potential persistent strains. Information obtained by MLVA typing allowed to identify sources and routes of contamination and, subsequently, will allow to optimize the technical and sanitation measures to ensure hygiene.

Not all predicted CRISPR-Cas systems are equal: isolated cas genes and classes of CRISPR like elements.

PubMed

Zhang, Quan; Ye, Yuzhen

2017-02-06

The CRISPR-Cas systems in prokaryotes are RNA-guided immune systems that target and deactivate foreign nucleic acids. A typical CRISPR-Cas system consists of a CRISPR array of repeat and spacer units, and a locus of cas genes. The CRISPR and the cas locus are often located next to each other in the genomes. However, there is no quantitative estimate of the co-location. In addition, ad-hoc studies have shown that some non-CRISPR genomic elements contain repeat-spacer-like structures and are mistaken as CRISPRs. Using available genome sequences, we observed that a significant number of genomes have isolated cas loci and/or CRISPRs. We found that 11%, 22% and 28% of the type I, II and III cas loci are isolated (without CRISPRs in the same genomes at all or with CRISPRs distant in the genomes), respectively. We identified a large number of genomic elements that superficially reassemble CRISPRs but don't contain diverse spacers and have no companion cas genes. We called these elements false-CRISPRs and further classified them into groups, including tandem repeats and Staphylococcus aureus repeat (STAR)-like elements. This is the first systematic study to collect and characterize false-CRISPR elements. We demonstrated that false-CRISPRs could be used to reduce the false annotation of CRISPRs, therefore showing them to be useful for improving the annotation of CRISPR-Cas systems.

Sex-Linked Pheromone Receptor Genes of the European Corn Borer, Ostrinia nubilalis, Are in Tandem Arrays

PubMed Central

Yasukochi, Yuji; Miura, Nami; Nakano, Ryo; Sahara, Ken; Ishikawa, Yukio

2011-01-01

Background Tuning of the olfactory system of male moths to conspecific female sex pheromones is crucial for correct species recognition; however, little is known about the genetic changes that drive speciation in this system. Moths of the genus Ostrinia are good models to elucidate this question, since significant differences in pheromone blends are observed within and among species. Odorant receptors (ORs) play a critical role in recognition of female sex pheromones; eight types of OR genes expressed in male antennae were previously reported in Ostrinia moths. Methodology/Principal Findings We screened an O. nubilalis bacterial artificial chromosome (BAC) library by PCR, and constructed three contigs from isolated clones containing the reported OR genes. Fluorescence in situ hybridization (FISH) analysis using these clones as probes demonstrated that the largest contig, which contained eight OR genes, was located on the Z chromosome; two others harboring two and one OR genes were found on two autosomes. Sequence determination of BAC clones revealed the Z-linked OR genes were closely related and tandemly arrayed; moreover, four of them shared 181-bp direct repeats spanning exon 7 and intron 7. Conclusions/Significance This is the first report of tandemly arrayed sex pheromone receptor genes in Lepidoptera. The localization of an OR gene cluster on the Z chromosome agrees with previous findings for a Z-linked locus responsible for O. nubilalis male behavioral response to sex pheromone. The 181-bp direct repeats might enhance gene duplications by unequal crossovers. An autosomal locus responsible for male response to sex pheromone in Heliothis virescens and H. subflexa was recently reported to contain at least four OR genes. Taken together, these findings support the hypothesis that generation of additional copies of OR genes can increase the potential for male moths to acquire altered specificity for pheromone components, and accordingly, facilitate differentiation of sex pheromones. PMID:21526121

Prolonged and mixed non-O157 Escherichia coli infection in an Australian household.

PubMed

Staples, M; Graham, R M A; Doyle, C J; Smith, H V; Jennison, A V

2012-05-01

An Australian family was identified through a Public Health follow up on a Shiga-toxigenic Escherichia coli (STEC) positive bloody diarrhoea case, with three of the four family members experiencing either symptomatic or asymptomatic STEC shedding. Bacterial isolates were submitted to stx sequence sub-typing, multi-locus variable number tandem repeat analysis (MLVA), multi-locus sequence typing (MLST) and binary typing. The analysis revealed that there were multiple strains of STEC being shed by the family members, with similar virulence gene profiles and the same serogroup but differing in their MLVA and MLST profiles. This study illustrates the potentially complicated nature of non-O157 STEC infections and the importance of molecular epidemiology in understanding disease clusters. © 2012 QUEENSLAND HEALTH. Clinical Microbiology and Infection © 2012 European Society of Clinical Microbiology and Infectious Diseases.

Length Variation of Cag/Caa Trinucleotide Repeats in Natural Populations of Drosophila Melanogaster and Its Relation to the Recombination Rate

PubMed Central

Michalakis, Y.; Veuille, M.

1996-01-01

Eleven genes distributed along the Drosophila melanogaster chromosome 2 and showing exonic tandem repeats of glutamine codons (CAG or CAA) were surveyed for length variation in a sample of four European and African populations. Only one gene was monomorphic. Eight genes were polymorphic in all populations, with a total number of alleles varying between five and 12 for 120 chromosomes. The average heterozygozity per locus and population was 0.41. Selective neutrality in length variation could not be rejected under the assumptions of the infinite allele model. Significant population subdivision was found though no geographical pattern emerged, all populations being equally different. Significant linkage disequilibrium was found in four out of seven cases where the genetic distance between loci was <1 cM and was negligible when the distance was larger. There is evidence that these associations were established after the populations separated. An unexpected result was that variation at each locus was independent of the coefficient of exchange, although the latter ranged from zero to the relatively high value of 6.7%. This would indicate that background selection and selective hitchhiking, which are thought to affect levels of nucleotide substitution polymorphism, have no effect on trinucleotide repeat variation. PMID:8844158

Multiple-locus variable-number tandem-repeat analysis of the swine dysentery pathogen, Brachyspira hyodysenteriae.

PubMed

Hidalgo, Alvaro; Carvajal, Ana; La, Tom; Naharro, Germán; Rubio, Pedro; Phillips, Nyree D; Hampson, David J

2010-08-01

The spirochete Brachyspira hyodysenteriae is the causative agent of swine dysentery, a severe colonic infection of pigs that has a considerable economic impact in many swine-producing countries. In spite of its importance, knowledge about the global epidemiology and population structure of B. hyodysenteriae is limited. Progress in this area has been hampered by the lack of a low-cost, portable, and discriminatory method for strain typing. The aim of the current study was to develop and test a multiple-locus variable-number tandem-repeat analysis (MLVA) method that could be used in basic veterinary diagnostic microbiology laboratories equipped with PCR technology or in more advanced laboratories with access to capillary electrophoresis. Based on eight loci, and when performed on isolates from different farms in different countries, as well as type and reference strains, the MLVA technique developed was highly discriminatory (Hunter and Gaston discriminatory index, 0.938 [95% confidence interval, 0.9175 to 0.9584]) while retaining a high phylogenetic value. Using the technique, the species was shown to be diverse (44 MLVA types from 172 isolates and strains), although isolates were stable in herds over time. The population structure appeared to be clonal. The finding of B. hyodysenteriae MLVA type 3 in piggeries in three European countries, as well as other, related, strains in different countries, suggests that spreading of the pathogen via carrier pigs is likely. MLVA overcame drawbacks associated with previous typing techniques for B. hyodysenteriae and was a powerful method for epidemiologic and population structure studies on this important pathogenic spirochete.

Use of multiple-locus variable-number tandem repeat analysis to evaluate Escherichia coli O157 subtype distribution and transmission dynamics following natural exposure on a closed beef feedlot facility.

PubMed

Williams, Michele L; Pearl, David L; Bishop, Katherine E; Lejeune, Jeffrey T

2013-10-01

To better understand the epizootiology of Escherichia coli O157:H7 among cattle, all E. coli O157 isolates recovered on a research feedlot during a single feeding period were characterized by multiple-locus variable-number tandem repeat analysis (MLVA). Three distinct MLVA subtypes (A, B, C), accounting for 24%, 15%, and 64% of total isolates, respectively, were identified. Subtypes A and B were isolated at the initiation of sampling, but their prevalence waned and subtype C, first isolated on the third sampling date, became the predominant subtype on the feedlot. Supershedding events, however, occurred with equal frequency for all three MLVA-types. Using a multilevel logistic regression model, we investigated whether the odds of shedding subtype C relative to subtypes A or B were associated with time, diet, or the presence of a penmate shedding high numbers of subtype C. Only time and exposure to an animal shedding MLVA-type C at 10³ colony-forming units or greater in the pen at the time of sampling were significantly associated with increased shedding of subtype C. High-level shedding of those E. coli O157 subtypes better suited for survival in the environment and/or in the host appear to play a significant role in the development of predominant E. coli O157 subtypes. Supershedding events alone are neither required nor sufficient to drive the epidemiology of specific E. coli O157 subtypes. Additional factors are necessary to direct successful on-farm transmission of E. coli O157.

Genetic diversity of O157:H7 and non-O157 verocytotoxigenic Escherichia coli from Argentina inferred from multiple-locus variable-number tandem repeat analysis (MLVA).

PubMed

Bustamante, Ana V; Sanso, A Mariel; Lucchesi, Paula M A; Parma, Alberto E

2010-04-01

Although serotype O157:H7 has been implicated in most cases of haemolytic-uraemic syndrome (HUS), there is growing concern about non-O157 serotypes of verocytotoxigenic Escherichia coli (VTEC). Multiple-locus variable-number tandem repeat analysis (MLVA) has been focused on the specific typing of O157:H7 isolates, but recently, a generic MLVA assay for E. coli and Shigella has been developed. We performed a study of the polymorphism in 7 generic VNTR loci both in VTEC O157:H7 and non-O157 isolates from Argentina, in order to asses the ability of the method to type this group of isolates and to get insight into their genetic diversity. Sixty-four isolates from cattle, patients with diarrhoea, and contaminated food belonging to 8 different serotypes were studied. All of them could be typed by this method and revealed 41 different MLVA genotypes. The MLVA dendrogram showed 2 main clusters which corresponded to O157:H7 and non-O157, respectively. Our results confirm the suitability of this MLVA method for analyzing VTEC isolates belonging to several serotypes, both O157:H7 as well as non-O157, highlight the genetic variability of the O157:H7 serotype and the need of additional research in order to find more VNTR loci that could allow a higher discrimination among non-O157 VTEC. (c) 2009 Elsevier GmbH. All rights reserved.

Characterization of a highly polymorphic region 5′ to JH in the human immunoglobulin heavy chain

PubMed Central

Silva, Alcino J.; Johnson, John P.; White, Raymond L.

1987-01-01

A cloned DNA segment 1.25 kilobases (kb) upstream from the joining segments of the human heavy chain immunoglobulin gene revealed extensive polymorphic variation at this locus, and the polymorphic pattern was stably transmitted to the next generation. Genomic restriction analysis showed that the polymorphism was caused by insertions/deletions within an MspI/BamHI fragment. Sequencing of one allele, 848 base pairs (bp) long, revealed eleven 50-base-pair tandem repeats. A second allele, 648 bp long, was cloned from a human genomic cosmid library, sequenced, and found to contain four fewer repeats than the first allele. A survey of 186 chromosomes from unrelated individuals of primarily northern European descent revealed at least six alleles. Images PMID:2884636

An Outbreak of Food-Borne Typhoid Fever Due to Salmonella enterica Serotype Typhi in Japan Reported for the First Time in 16 Years

PubMed Central

Kobayashi, Tetsuro; Kutsuna, Satoshi; Hayakawa, Kayoko; Kato, Yasuyuki; Ohmagari, Norio; Uryu, Hideko; Yamada, Ritsuko; Kashiwa, Naoyuki; Nei, Takahito; Ehara, Akihito; Takei, Reiko; Mori, Nobuaki; Yamada, Yasuhiro; Hayasaka, Tomomi; Kagawa, Narito; Sugawara, Momoko; Suzaki, Ai; Takahashi, Yuno; Nishiyama, Hiroyuki; Morita, Masatomo; Izumiya, Hidemasa; Ohnishi, Makoto

2016-01-01

For the first time in 16 years, a food-borne outbreak of typhoid fever due to Salmonella enterica serotype Typhi was reported in Japan. Seven patients consumed food in an Indian buffet at a restaurant in the center of Tokyo, while one was a Nepali chef in the restaurant, an asymptomatic carrier and the implicated source of this outbreak. The multiple-locus variable-number tandem repeat analysis showed 100% consistency in the genomic sequence for five of the eight cases. PMID:26621565

DXS106 and DXSW559 flank the X-linked dystonia-parkisonism syndrome locus (DYT3)

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

Mueller, U.; Haberhausen, G.; Wagner, T.

1994-09-01

The locus (DYT3) underlying the X-linked dystonia-parkinsonism syndrome (XDP) was delineated within proximal Xq12-Xq13.1 by analysis of linkage, allelic association, and haplotypes. Short tandem repeat polymorphism at loci DXS227, DXS559, DXS453, DXS106, DXS339, and DXS135 were studied. The occurrence of a recombination within a three-generation family established DXS559 as the distal flanking marker of DYT3. /{phi}/and /{Delta}/ values were determined as indicators of the degree of allelic association between DYT3 and the six marker loci. In addition, haplotype analysis was performed at the loci studied. The findings establish DXS106 as the proximal flanking marker of DYT3. Given an approximate distancemore » between DXS106 and DXS559 of 3.0 Mb, isolation of DYT3 is now feasible by positional cloning techniques. 21 refs., 2 figs., 3 tabs.« less

Multi-locus variable-number tandem repeat analysis for outbreak studies of Salmonella enterica serotype Enteritidis

PubMed Central

Malorny, Burkhard; Junker, Ernst; Helmuth, Reiner

2008-01-01

Background Salmonella enterica subsp. enterica serotype Enteritidis is known as an important and pathogenic clonal group which continues to cause worldwide sporadic cases and outbreaks in humans. Here a new multiple-locus variable-number tandem repeat analysis (MLVA) method is reported for highly-discriminative subtyping of Salmonella Enteritidis. Emphasis was given on the most predominant phage types PT4 and PT8. The method comprises multiplex PCR specifically amplifying repeated sequences from nine different loci followed by an automatic fragment size analysis using a multicolor capillary electrophoresis instrument. A total of 240 human, animal, food and environmental isolates of S. Enteritidis including 23 definite phage types were used for development and validation. Furthermore, the MLVA types were compared to the phage types of several isolates from two recent outbreaks to determine the concordance between both methods and to estimate their in vivo stability. The in vitro stability of the two MLVA types specifically for PT4 and PT8 strains were determined by multiple freeze-thaw cycles. Results Seventy-nine different MLVA types were identified in 240 S. Enteritidis strains. The Simpson's diversity index for the MLVA method was 0.919 and Nei diversity values for the nine VNTR loci ranged from 0.07 to 0.65. Twenty-four MLVA types could be assigned to 62 PT4 strains and 21 types to 81 PT8 strains. All outbreak isolates had an indistinguishable outbreak specific MLVA type. The in vitro stability experiments showed no changes of the MLVA type compared to the original isolate. Conclusion This MLVA method is useful to discriminate S. Enteritidis strains even within a single phage type. It is easy in use, fast, and cheap compared to other high-resolution molecular methods and therefore an important tool for surveillance and outbreak studies for S. Enteritidis. PMID:18513386

VNTR internal structure mapping at the {alpha}-globin 3{prime}HVR locus reveals a hierachy of related lineages in oceania

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

Martinson, J.J.; Clegg, J.B.; Boyce, A.J.

1994-09-01

Analysis of the {alpha}-globin gene complex in Oceania has revealed many different rearrangements which remove one of the adult globin genes. Frequencies of these deletion chromosomes are elevated by malarial resistance conferred by the resulting {alpha}-thalassaemia. One particular deletion chromosome, designated -{alpha}{sup 3.7}III, is found at high levels in Melanesia and Polynesia: RFLP haplotype analysis shows that this deletion is always found on chromosomes bearing the IIIa haplotype and is likely to be the product of one single rearrangement event. A subset of the -{alpha}{sup 3.7}III chromosomes carries a more recent mutation which generates the haemoglobin variant HbJ{sup Tongariki}. Wemore » have characterized the allelic variation at the 3{prime}HVR VNTR locus located 6 kb from the globin genes in each of these groups of chromosomes. We have determined the internal structure of these alleles by RFLP mapping of PCR-amplified DNA: within each group, the allelic diversity results from the insertion and/or deletion of small {open_quotes}motifs{close_quotes} of up to 6 adjacent repeats. Mapping of 3{prime}HVR alleles associated with other haplotypes reveals that these are composed of repeat arrays that are substantially different to those derived from IIIa chromosomes, indicating that interchromosomal recombination between heterologous haplotypes does not account for any of the diversity seen to date. We have recently shown that allelic size variation at the two VNTR loci flanking the {alpha}-globin complex is very closely linked to the haplotypes known to be present at this locus. Here we show that, within a haplotype, VNTR alleles are very closely related to each other on the basis of internal structure and demonstrate that intrachromosomal mutation processes involving small numbers of tandem repeats are the main cause of variation at this locus.« less

A Dual Origin of the Xist Gene from a Protein-Coding Gene and a Set of Transposable Elements

PubMed Central

Elisaphenko, Eugeny A.; Kolesnikov, Nikolay N.; Shevchenko, Alexander I.; Rogozin, Igor B.; Nesterova, Tatyana B.; Brockdorff, Neil; Zakian, Suren M.

2008-01-01

X-chromosome inactivation, which occurs in female eutherian mammals is controlled by a complex X-linked locus termed the X-inactivation center (XIC). Previously it was proposed that genes of the XIC evolved, at least in part, as a result of pseudogenization of protein-coding genes. In this study we show that the key XIC gene Xist, which displays fragmentary homology to a protein-coding gene Lnx3, emerged de novo in early eutherians by integration of mobile elements which gave rise to simple tandem repeats. The Xist gene promoter region and four out of ten exons found in eutherians retain homology to exons of the Lnx3 gene. The remaining six Xist exons including those with simple tandem repeats detectable in their structure have similarity to different transposable elements. Integration of mobile elements into Xist accompanies the overall evolution of the gene and presumably continues in contemporary eutherian species. Additionally we showed that the combination of remnants of protein-coding sequences and mobile elements is not unique to the Xist gene and is found in other XIC genes producing non-coding nuclear RNA. PMID:18575625

Multi-locus variable-number tandem repeat analysis of Bordetella pertussis isolates circulating in Poland in the period 1959-2013.

PubMed

Mosiej, Ewa; Krysztopa-Grzybowska, Katarzyna; Polak, Maciej; Prygiel, Marta; Lutyńska, Anna

2017-06-01

Despite the long history of pertussis vaccination and high vaccination coverage in Poland and many other developed countries, pertussis incidence rates have increased substantially, making whooping cough one of the most prevalent vaccine-preventable diseases. Among the factors potentially involved in pertussis resurgence, the adaptation of the Bordetella pertussis population to country-specific vaccine-induced immunity through selection of non-vaccine-type strains still needs detailed studies. Multi-locus variable-number tandem repeat analysis (MLVA), also linked to MLST and PFGE profiling, was applied to trace the genetic changes in the B. pertussis population circulating in Poland in the period 1959-2013 versus country-specific vaccine strains. Generally, among 174 B. pertussis isolates, 31 MLVA types were detected, of which 11 were not described previously. The predominant MLVA types of recent isolates in Poland were different from those of the typical isolates circulating in other European countries. The MT27 type, currently predominant in Europe, was rarely seen and detected in only five isolates among all studied. The features of the vaccine strains used for production of the pertussis component of a national whole-cell diphtheria-tetanus-pertussis (DTP) vaccine, as studied by MLVA and MLST tools, were found to not match those observed in the currently circulating B. pertussis isolates in Poland. Differences traced by MLVA in relation to the MLST and PFGE profiling confirmed that the B. pertussis strain types currently observed elsewhere in Europe, even if appearing in Poland, were not able to successfully disseminate within a human population in Poland that has been vaccinated with a whole-cell pertussis vaccine not used in other countries.

Multiple-locus variable number of tandem repeats fingerprinting (MLVF) and virulence factor analysis of methicillin resistant Staphylococcus aureus SCCmec type III.

PubMed

Emaneini, Mohammad; Jabalameli, Leila; Iman-Eini, Hossein; Aligholi, Marzieh; Ghasemi, Amir; Nakhjavani, Farrokh Akbari; Taherikalani, Morovat; Khoramian, Babak; Asadollahi, Parisa; Jabalameli, Fereshteh

2011-01-01

Methicillin resistant Staphylococcus aureus (MRSA), particularly strains with type III staphylococcal cassette chromosome mec (SCCmec), represent a serious human pathogen in Tehran, Iran. The disease-causing capability depends on their ability to produce a wide variety of virulent factors. The prevalence of exotoxin genes and multiple-locus variable number of tandem repeats fingerprinting (MLVF) profile among MRSA isolates, from patients in Tehran, was evaluated by PCR and Multiplex-PCR. The MLVF typing of 144 MRSA isolates with type III SCCmec produced 5 different MLVF types. Generally, 97.2% (140/144) of all the isolates were positive for at least one of the tested exotoxin genes. The most prevalent genes were hld, found in 87.5% (126/144) of the isolates followed by lukE-lukD and hla found in 72.9% (105/144) and 70.1% (101/144) of the isolates, respectively. The tst gene, belonging to MLVF types I, IV and V, was found among three of the isolates from blood and wound samples. The sea gene was detected in 58.3% (84/144) of the isolates and the sed and see genes were found in one isolate with MLVF type V. The coexistence of genes was observed in the 87.5% (126/144) of the isolates. The rate of coexistence of hld with lukE-lukD, hla with lukE-lukD and sea with lukE-lukD were 66.7% (96/144), 44.4% (64/144) and 44.4% (64/144), respectively. The present study demonstrated that MRSA strains with type III SCCmec show different MLVF patterns and exotoxin profiles.

Leptospira interrogans serovars Bratislava and Muenchen animal infections: Implications for epidemiology and control.

PubMed

Arent, Z; Frizzell, C; Gilmore, C; Allen, A; Ellis, W A

2016-07-15

Strains of Leptospira interrogans belonging to two very closely related serovars - Bratislava and Muenchen - have been associated with disease in domestic animals, in particular pigs, but also in horses and dogs. Similar strains have also been recovered from various wildlife species. Their epidemiology is poorly understood. Two hundred and forty seven such isolates, from UK domestic animal and wildlife species, were examined by restriction endonuclease analysis in an attempt to elucidate their epidemiology. A representative sub-sample of 65 of these isolates was further examined by multiple-locus variable-number tandem repeat analysis and 22 by secY sequencing. Ten restriction pattern types were identified. The majority of isolates fell into one of three restriction endonuclease analysis pattern types designated B2a, B2b and M2a. B2a was ubiquitous and was isolated from 10 species and represented the majority of the horse and all dog isolates. B2b was very different, being isolated only from pigs, indicating that this type was maintained by pigs. The pattern M2a was reported for the majority of isolates from pigs but also was common in small rodents isolates. Five restriction pattern types were found only in wildlife suggesting that they are unlikely to pose a disease threat to domestic animals. Multiple-locus variable-number tandem repeat analysis identified six clusters. The REA types B2a and B2b were all found in one MLVA cluster while the majority of the M2a strains examined occurred in another cluster. The secY sequencing detected only one sequence type, clustered with other serovars of Leptospira interrogans. Copyright © 2016 Elsevier B.V. All rights reserved.

Multi-locus variable-number tandem repeat analysis of Chinese Brucella strains isolated from 1953 to 2013.

PubMed

Tian, Guo-Zhong; Cui, Bu-Yun; Piao, Dong-Ri; Zhao, Hong-Yan; Li, Lan-Yu; Liu, Xi; Xiao, Pei; Zhao, Zhong-Zhi; Xu, Li-Qing; Jiang, Hai; Li, Zhen-Jun

2017-05-02

Brucellosis was a common human and livestock disease caused by Brucella strains, the category B priority pathogens by the US Center for Disease Control (CDC). Identified as a priority disease in human and livestock populations, the increasing incidence in recent years in China needs urgent control measures for this disease but the molecular background important for monitoring the epidemiology of Brucella strains at the national level is still lacking. A total of 600 Brucella isolates collected during 60 years (from 1953 to 2013) in China were genotyped by multiple locus variable-number tandem repeat analysis (MLVA) and the variation degree of MLVA11 loci was calculated by the Hunter Gaston Diversity Index (HGDI) values. The charts and map were processed by Excel 2013, and cluster analysis and epidemiological distribution was performed using BioNumerics (version 5.1). The 600 representative Brucella isolates fell into 104 genotypes with 58 singleton genotypes by the MLVA11 assay, including B. melitensis biovars 2 and 3 (five main genotypes), B. abortus biovars 1 and 3 (two main genotypes), B. suis biovars 1 and 3 (three main genotypes), and B. canis (two main genotypes) respectively. While most B. suis biovar 1 and biovar 3 were respectively found in northern provinces and southern provinces, B. melitensis and B. abortus strains were dominant in China. Canine Brucellosis was only found in animals without any human cases reported. Eight Brucellosis epidemic peaks emerged during the 60 years between 1953 and 2013: 1955 - 1959, 1962 - 1969, 1971 - 1975, 1977 - 1983, 1985 - 1989, 1992 - 1997, 2000 - 2008 and 2010 - 2013 in China. Brucellosis has its unique molecular epidemiological patterns with specific spatial and temporal distribution according to MLVA. IDOP-D-16-00101.

Analysis of the 9p21.3 sequence associated with coronary artery disease reveals a tendency for duplication in a CAD patient

PubMed Central

Kouprina, Natalay; Noskov, Vladimir N.; Waterfall, Joshua J.; Walker, Robert L.; Meltzer, Paul S.; Topol, Eric J.; Larionov, Vladimir

2018-01-01

Tandem segmental duplications (SDs) greater than 10 kb are widespread in complex genomes. They provide material for gene divergence and evolutionary adaptation, while formation of specific de novo SDs is a hallmark of cancer and some human diseases. Most SDs map to distinct genomic regions termed ‘duplication blocks’. SDs organization within these blocks is often poorly characterized as they are mosaics of ancestral duplicons juxtaposed with younger duplicons arising from more recent duplication events. Structural and functional analysis of SDs is further hampered as long repetitive DNA structures are underrepresented in existing BAC and YAC libraries. We applied Transformation-Associated Recombination (TAR) cloning, a versatile technique for large DNA manipulation, to selectively isolate the coronary artery disease (CAD) interval sequence within the 9p21.3 chromosome locus from a patient with coronary artery disease and normal individuals. Four tandem head-to-tail duplicons, each ∼50 kb long, were recovered in the patient but not in normal individuals. Sequence analysis revealed that the repeats varied by 10-15 SNPs between each other and by 82 SNPs between the human genome sequence (version hg19). SNPs polymorphism within the junctions between repeats allowed two junction types to be distinguished, Type 1 and Type 2, which were found at a 2:1 ratio. The junction sequences contained an Alu element, a sequence previously shown to play a role in duplication. Knowledge of structural variation in the CAD interval from more patients could help link this locus to cardiovascular diseases susceptibility, and maybe relevant to other cases of regional amplification, including cancer. PMID:29632643

Genotyping of Brucella melitensis strains from dromedary camels (Camelus dromedarius) from the United Arab Emirates with multiple-locus variable-number tandem repeat analysis.

PubMed

Gyuranecz, Miklós; Wernery, Ulli; Kreizinger, Zsuzsa; Juhász, Judit; Felde, Orsolya; Nagy, Péter

2016-04-15

Camel brucellosis is a widespread zoonotic disease in camel-rearing countries caused by Brucella melitensis and Brucella abortus. The aim of this study was the first genetic analysis of B. melitensis strains isolated from dromedary camels (Camelus dromedarius) using multiple-locus variable-number tandem repeat analysis (MLVA). MLVA 16 and its MLVA 8 and MLVA11 subsets were used to determine the genotypes of 15 B. melitensis isolates from dromedary camels (11 strains) and other host species (4 strains) from the United Arab Emirates and the results were then compared to B. melitensis MLVA genotypes from other parts of the world. Five, including two novel genotypes were identified with MLVA 8. MLVA 16 further discriminated these five genotypes to ten variants. The eleven camel isolates clustered into four main genetic groups within the East-Mediterranean and African clades and this clustering correlated with the geographic origin of the hosts (United Arab Emirates, Kingdom of Saudi Arabia and Sudan) and the date of their isolation. The camel strains were also genetically related to strains isolated from wild and domestic ruminants from their close habitat or from other parts of the world. Although limited number of strains were analysed, based on our data imported animals from foreign countries, local small ruminants and wildlife species are hypothesized to be the main sources of camel brucellosis in the United Arab Emirates. MLVA was successfully applied to determine the epidemiological links between the different camel B. melitensis infections in the United Arab Emirates and it can be a beneficial tool in future disease control programs. Copyright © 2016 Elsevier B.V. All rights reserved.

Preliminary Investigation on Multiple-Locus Variable Number Tandem Repeat Analysis Profiles of Listeria Monocytogenes Isolates from Pork Meat Tested from Packaging to Fork

PubMed Central

Parisi, Antonio; Caruso, Marta; Pasquali, Frédérique; Manfreda, Gerardo

2014-01-01

Listeria monocytogenes is recognised as a public health issue and a serious challenge for the food industry. L. monocytogenes strain characterisation on the basis of serotyping and molecular typing methods is used for surveillance, epidemiological tracking and outbreak investigation purposes. Genetic variants of L. monocytogenes have diversified into four major phylogenetic lineages, with lineages 1 and 2 each containing multiple clonal groups of public health importance. Standardised tools for easy identification of clonal groups are needed to trace such groups and determine their presence in a large variety of sources. Given the current limitations of available methods for L. monocytogenes strain typing, a potentially useful approach is multiple locus variable number of tandem repeats (VNTR) analysis (MLVA). In this study, MLVA has been applied to a random group of 82 L. monocytogenes strains isolated from 8 different batches of loin chops obtained from the same facility and tested between packaging and consumption time. The strains typed were classified into 10 MLVA profiles containing a number of isolates ranging between 1 to 20. According to the identified MLVA profiles, 75.6% of the pork isolates belonged to the phylogenetic lineage 2 and serotype 1/2c, frequently associated to food isolates. However, 3 pork strains belonged to the phylogenetic lineage 1 and serotype 4b. Moreover, 17 isolates were classified in the phylogenetic lineages 2 and serotype 1/2a. Both serotypes 4b and 1/2a are frequently associated to human isolates of L. monocytogenes. These preliminary results show how the MLVA profiles can support the assessment of the risk profile of food products based on the contaminating L. monocytogenes strain types. PMID:27800312

Recent transmission of drug-resistant Mycobacterium tuberculosis in a prison population in southern Brazil.

PubMed

Reis, Ana Julia; David, Simone Maria Martini de; Nunes, Luciana de Souza; Valim, Andreia Rosane de Moura; Possuelo, Lia Gonçalves

2016-01-01

We conducted a cross-sectional, retrospective study, characterized by classical and molecular epidemiology, involving M. tuberculosis isolates from a regional prison in southern Brazil. Between January of 2011 and August of 2014, 379 prisoners underwent sputum smear microscopy and culture; 53 (13.9%) were diagnosed with active tuberculosis. Of those, 8 (22.9%) presented with isoniazid-resistant tuberculosis. Strain genotyping was carried out by 15-locus mycobacterial interspersed repetitive unit-variable-number tandem-repeat analysis; 68.6% of the patients were distributed into five clusters, and 87.5% of the resistant cases were in the same cluster. The frequency of drug-resistant tuberculosis cases and the rate of recent transmission were high. Our data suggest the need to implement an effective tuberculosis control program within the prison system. RESUMO Estudo transversal, retrospectivo, com isolados de M. tuberculosis de pacientes de um presídio regional no sul do Brasil, caracterizado através de epidemiologia clássica e molecular. Entre janeiro de 2011 e agosto de 2014, 379 detentos foram submetidos a baciloscopia e cultura, sendo 53 (13,9%) diagnosticados com tuberculose ativa. Desses, 8 (22,9%) apresentavam tuberculose resistente a isoniazida. A genotipagem das cepas foi realizada por 15-locus mycobacterial interspersed repetitive units-variable number of tandem repeat analysis; 68,6% dos pacientes estavam distribuídos em cinco clusters, e 87,5% dos casos resistentes estavam em um mesmo cluster. Verificou-se uma frequência elevada de casos de resistência e alta taxa de transmissão recente. Estes dados sugerem a necessidade da implantação de um programa efetivo de controle da tuberculose no sistema prisional.

Longitudinal prevalence and molecular typing of Escherichia coli O157:H7 by use of multiple-locus variable-number tandem-repeat analysis and pulsed-field gel electrophoresis in fecal samples collected from a range-based herd of beef cattle in California.

PubMed

Kondo, Sonoko; Hoar, Bruce R; Villanueva, Veronica; Mandrell, Robert E; Atwill, Edward R

2010-11-01

To evaluate seasonal patterns and risk factors for Escherichia coli O157:H7 in feces in a beef cattle herd and determine strain diversity and transition in E coli over time by use of multiple-locus variable-number tandem-repeat analysis (MLVA) and pulsed-field gel electrophoresis (PFGE). 456 samples of freshly passed feces collected over a 1-year period from cattle in a range-based cow-calf operation located in the foothills of the Sierra Nevada Mountains in California. E coli O157:H7 was recovered from feces by use of immunomagnetic separation and 2 selective media. Virulence factors were detected via reverse transcriptase-PCR assay. Escherichia coli O157:H7 isolates were subtyped with MLVA and PFGE. Prevalence estimates were calculated and significant risk factors determined. A dendrogram was constructed on the basis of results of MLVA typing. Overall prevalence estimate for E coli O157:H7 was 10.5%, with the prevalence lowest during the winter. Mean temperature during the 30 days before collection of samples was significantly associated with prevalence of E coli O157:H7 in feces. Nineteen MLVA and 12 PFGE types were identified. A seasonal pattern was detected for prevalence of E coli O157:H7 in feces collected from beef cattle in California. Subtyping via MLVA and PFGE revealed a diversity of E coli O157:H7 strains in a cow-calf operation and noteworthy turnover of predominant types. Given the importance of accurately determining sources of contamination in investigations of disease outbreaks in humans, MLVA combined with PFGE should be powerful tools for epidemiologists.

Verocytotoxigenic Escherichia coli O157 in beef and sheep abattoirs in Ireland and characterisation of isolates by Pulsed-Field Gel Electrophoresis and Multi-Locus Variable Number of Tandem Repeat Analysis.

PubMed

Prendergast, Deirdre M; Lendrum, Lynsey; Pearce, Rachel; Ball, Caroline; McLernon, Joanne; O'Grady, Don; Scott, Lourda; Fanning, Seamus; Egan, John; Gutierrez, Montserrat

2011-01-05

This study aimed to investigate verocytotoxigenic Escherichia coli O157 in the largest beef and sheep slaughter plants in Ireland over a one-year period. Samples consisted of pooled rectal swabs (n=407) and pooled carcass swabs (n=407) from 5 animals belonging to the same herd or flock and minced meat (n=91) from the same sampling date. E. coli O157 isolates were characterised using PCR for a range of genes, i.e. 16S, rfbE, fliC, vtx1, vtx2, eaeA and confirmed VTEC O157 isolates were tested for antimicrobial susceptibility and typed using Pulsed-Field Gel Electrophoresis (PFGE) and Multi-Locus Variable Number of Tandem Repeat Analysis (MLVA). VTEC O157 was isolated from 7.6% and 3.9% of bovine rectal and carcass swab samples and from 5.8% and 2.9% of ovine rectal and carcass swab samples respectively. None of the bovine minced meat samples (n=77) and only one of the 14 ovine minced meat samples was positive for VTEC O157. Following PFGE and MLVA, cross contamination from faeces to carcasses was identified. While PFGE and MLVA identified the same clusters for highly related strains, MLVA discriminated better than PFGE in addition to being more rapid and less labour intensive. Results showed that cattle and sheep presented for slaughter in Ireland harbour VTEC O157, and although the levels entering the food chain are low, this should not be overlooked as possible sources of zoonotic infection; molecular typing was able to demonstrate relationships among strains and could be used to elucidate the sources of human infection. Copyright © 2010 Elsevier B.V. All rights reserved.

TRedD—A database for tandem repeats over the edit distance

PubMed Central

Sokol, Dina; Atagun, Firat

2010-01-01

A ‘tandem repeat’ in DNA is a sequence of two or more contiguous, approximate copies of a pattern of nucleotides. Tandem repeats are common in the genomes of both eukaryotic and prokaryotic organisms. They are significant markers for human identity testing, disease diagnosis, sequence homology and population studies. In this article, we describe a new database, TRedD, which contains the tandem repeats found in the human genome. The database is publicly available online, and the software for locating the repeats is also freely available. The definition of tandem repeats used by TRedD is a new and innovative definition based upon the concept of ‘evolutive tandem repeats’. In addition, we have developed a tool, called TandemGraph, to graphically depict the repeats occurring in a sequence. This tool can be coupled with any repeat finding software, and it should greatly facilitate analysis of results. Database URL: http://tandem.sci.brooklyn.cuny.edu/ PMID:20624712

Genome Dynamics and Evolution of the Mla (Powdery Mildew) Resistance Locus in BarleyW⃞

PubMed Central

Wei, Fusheng; Wing, Rod A.; Wise, Roger P.

2002-01-01

Genes that confer defense against pathogens often are clustered in the genome and evolve via diverse mechanisms. To evaluate the organization and content of a major defense gene complex in cereals, we determined the complete sequence of a 261-kb BAC contig from barley cv Morex that spans the Mla (powdery mildew) resistance locus. Among the 32 predicted genes on this contig, 15 are associated with plant defense responses; 6 of these are associated with defense responses to powdery mildew disease but function in different signaling pathways. The Mla region is organized as three gene-rich islands separated by two nested complexes of transposable elements and a 45-kb gene-poor region. A heterochromatic-like region is positioned directly proximal to Mla and is composed of a gene-poor core with 17 families of diverse tandem repeats that overlap a hypermethylated, but transcriptionally active, gene-dense island. Paleontology analysis of long terminal repeat retrotransposons indicates that the present Mla region evolved over a period of >7 million years through a variety of duplication, inversion, and transposon-insertion events. Sequence-based recombination estimates indicate that R genes positioned adjacent to nested long terminal repeat retrotransposons, such as Mla, do not favor recombination as a means of diversification. We present a model for the evolution of the Mla region that encompasses several emerging features of large cereal genomes. PMID:12172030

Tandem repeats analysis for the high resolution phylogenetic analysis of Yersinia pestis

PubMed Central

Pourcel, C; André-Mazeaud, F; Neubauer, H; Ramisse, F; Vergnaud, G

2004-01-01

Background Yersinia pestis, the agent of plague, is a young and highly monomorphic species. Three biovars, each one thought to be associated with the last three Y. pestis pandemics, have been defined based on biochemical assays. More recently, DNA based assays, including DNA sequencing, IS typing, DNA arrays, have significantly improved current knowledge on the origin and phylogenetic evolution of Y. pestis. However, these methods suffer either from a lack of resolution or from the difficulty to compare data. Variable number of tandem repeats (VNTRs) provides valuable polymorphic markers for genotyping and performing phylogenetic analyses in a growing number of pathogens and have given promising results for Y. pestis as well. Results In this study we have genotyped 180 Y. pestis isolates by multiple locus VNTR analysis (MLVA) using 25 markers. Sixty-one different genotypes were observed. The three biovars were distributed into three main branches, with some exceptions. In particular, the Medievalis phenotype is clearly heterogeneous, resulting from different mutation events in the napA gene. Antiqua strains from Asia appear to hold a central position compared to Antiqua strains from Africa. A subset of 7 markers is proposed for the quick comparison of a new strain with the collection typed here. This can be easily achieved using a Web-based facility, specifically set-up for running such identifications. Conclusion Tandem-repeat typing may prove to be a powerful complement to the existing phylogenetic tools for Y. pestis. Typing can be achieved quickly at a low cost in terms of consumables, technical expertise and equipment. The resulting data can be easily compared between different laboratories. The number and selection of markers will eventually depend upon the type and aim of investigations. PMID:15186506

The immunoglobulin heavy chain locus of the duck. Genomic organization and expression of D, J, and C region genes.

PubMed

Lundqvist, M L; Middleton, D L; Hazard, S; Warr, G W

2001-12-14

The region of the duck IgH locus extending from upstream of the proximal diversity (D) segment to downstream of the constant gene cluster has been cloned and mapped. A sequence contig of 48,796 base pairs established that the organization of the genes is D-J(H)-mu-alpha-upsilon. No evidence for a functional homologue (or remnant) of a delta gene was found. The alpha gene is in inverted transcriptional orientation; class switch to IgA expression thus requires inversion of the approximately 27-kilobase pair region that includes both mu and alpha genes. The secreted forms of duck alpha and mu are each encoded by 4 constant region exons, and the hydrophobic C-terminal regions of the membrane receptor forms of alpha and mu are encoded by one and two transmembrane exons, respectively. Putative switch (S) regions were identified for duck mu and upsilon by comparison with chicken Smu and Supsilon sequences and for duck alpha by comparison with mouse Salpha. The duck IgH locus is rich in complex variable number tandem repeats, which occupy approximately 60% of the sequenced region, and occur at a much higher frequency in the IgH locus than in other sequenced regions of the duck genome.

Bardet-Biedl syndrome: Mapping of a new locus to chromosome 3 and fine-mapping of the chromosome 16 linked locus

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

Kwitek-Black, A.E.; Rokhlina, T.; Nishimura, D.Y.

Bardet-Biedl syndrome (BBS) is a heterogeneous autosomal recessive disorder characterized by mental retardation, post-axial polydactyly, obesity, retinitis pigmentosa, and hypogonadism. Other features of this disease include renal and cardiovascular abnormalities and an increased incidence of hypertension and diabetes mellitus. The molecular etiology for BBS is not known. We previously linked BBS to chromosome 16q13 in a large inbred Bedouin family, and excluded this locus in a second large inbred Bedouin family. We now report linkage of this second family to markers on chromosome 3q, proving non-allelic, genetic heterogeneity in the Bedouin population. A third large inbred Bedouin family was excludedmore » from the 3q and 16q BBS loci. In addition to the identification of a new BBS locus on chromosome 3, we have identified and utilized additional short tandem repeat polymorphisms (STRPs) in the 16q BBS region to narrow the candidate interval to 3 cM. Additional recombinant individuals will allow further refinement of the interval. Identification of genes causing BBS has the potential to provide insight into diverse genetic traits and disease processes including obesity, hypertension, diabetes, retinal degeneration, and abnormal limb, renal and cardiac development.« less

Comparative analysis of tandem repeats from hundreds of species reveals unique insights into centromere evolution.

PubMed

Melters, Daniël P; Bradnam, Keith R; Young, Hugh A; Telis, Natalie; May, Michael R; Ruby, J Graham; Sebra, Robert; Peluso, Paul; Eid, John; Rank, David; Garcia, José Fernando; DeRisi, Joseph L; Smith, Timothy; Tobias, Christian; Ross-Ibarra, Jeffrey; Korf, Ian; Chan, Simon W L

2013-01-30

Centromeres are essential for chromosome segregation, yet their DNA sequences evolve rapidly. In most animals and plants that have been studied, centromeres contain megabase-scale arrays of tandem repeats. Despite their importance, very little is known about the degree to which centromere tandem repeats share common properties between different species across different phyla. We used bioinformatic methods to identify high-copy tandem repeats from 282 species using publicly available genomic sequence and our own data. Our methods are compatible with all current sequencing technologies. Long Pacific Biosciences sequence reads allowed us to find tandem repeat monomers up to 1,419 bp. We assumed that the most abundant tandem repeat is the centromere DNA, which was true for most species whose centromeres have been previously characterized, suggesting this is a general property of genomes. High-copy centromere tandem repeats were found in almost all animal and plant genomes, but repeat monomers were highly variable in sequence composition and length. Furthermore, phylogenetic analysis of sequence homology showed little evidence of sequence conservation beyond approximately 50 million years of divergence. We find that despite an overall lack of sequence conservation, centromere tandem repeats from diverse species showed similar modes of evolution. While centromere position in most eukaryotes is epigenetically determined, our results indicate that tandem repeats are highly prevalent at centromeres of both animal and plant genomes. This suggests a functional role for such repeats, perhaps in promoting concerted evolution of centromere DNA across chromosomes.

Comparative analysis of tandem repeats from hundreds of species reveals unique insights into centromere evolution

PubMed Central

2013-01-01

Background Centromeres are essential for chromosome segregation, yet their DNA sequences evolve rapidly. In most animals and plants that have been studied, centromeres contain megabase-scale arrays of tandem repeats. Despite their importance, very little is known about the degree to which centromere tandem repeats share common properties between different species across different phyla. We used bioinformatic methods to identify high-copy tandem repeats from 282 species using publicly available genomic sequence and our own data. Results Our methods are compatible with all current sequencing technologies. Long Pacific Biosciences sequence reads allowed us to find tandem repeat monomers up to 1,419 bp. We assumed that the most abundant tandem repeat is the centromere DNA, which was true for most species whose centromeres have been previously characterized, suggesting this is a general property of genomes. High-copy centromere tandem repeats were found in almost all animal and plant genomes, but repeat monomers were highly variable in sequence composition and length. Furthermore, phylogenetic analysis of sequence homology showed little evidence of sequence conservation beyond approximately 50 million years of divergence. We find that despite an overall lack of sequence conservation, centromere tandem repeats from diverse species showed similar modes of evolution. Conclusions While centromere position in most eukaryotes is epigenetically determined, our results indicate that tandem repeats are highly prevalent at centromeres of both animal and plant genomes. This suggests a functional role for such repeats, perhaps in promoting concerted evolution of centromere DNA across chromosomes. PMID:23363705

Distinct Copy Number, Coding Sequence, and Locus Methylation Patterns Underlie Rhg1-Mediated Soybean Resistance to Soybean Cyst Nematode1[W][OPEN

PubMed Central

Cook, David E.; Bayless, Adam M.; Wang, Kai; Guo, Xiaoli; Song, Qijian; Jiang, Jiming; Bent, Andrew F.

2014-01-01

Copy number variation of kilobase-scale genomic DNA segments, beyond presence/absence polymorphisms, can be an important driver of adaptive traits. Resistance to Heterodera glycines (Rhg1) is a widely utilized quantitative trait locus that makes the strongest known contribution to resistance against soybean cyst nematode (SCN), Heterodera glycines, the most damaging pathogen of soybean (Glycine max). Rhg1 was recently discovered to be a complex locus at which resistance-conferring haplotypes carry up to 10 tandem repeat copies of a 31-kb DNA segment, and three disparate genes present on each repeat contribute to SCN resistance. Here, we use whole-genome sequencing, fiber-FISH (fluorescence in situ hybridization), and other methods to discover the genetic variation at Rhg1 across 41 diverse soybean accessions. Based on copy number variation, transcript abundance, nucleic acid polymorphisms, and differentially methylated DNA regions, we find that SCN resistance is associated with multicopy Rhg1 haplotypes that form two distinct groups. The tested high-copy-number Rhg1 accessions, including plant introduction (PI) 88788, contain a flexible number of copies (seven to 10) of the 31-kb Rhg1 repeat. The identified low-copy-number Rhg1 group, including PI 548402 (Peking) and PI 437654, contains three copies of the Rhg1 repeat and a newly identified allele of Glyma18g02590 (a predicted α-SNAP [α-soluble N-ethylmaleimide–sensitive factor attachment protein]). There is strong evidence for a shared origin of the two resistance-conferring multicopy Rhg1 groups and subsequent independent evolution. Differentially methylated DNA regions also were identified within Rhg1 that correlate with SCN resistance. These data provide insights into copy number variation of multigene segments, using as the example a disease resistance trait of high economic importance. PMID:24733883

TRAP: automated classification, quantification and annotation of tandemly repeated sequences.

PubMed

Sobreira, Tiago José P; Durham, Alan M; Gruber, Arthur

2006-02-01

TRAP, the Tandem Repeats Analysis Program, is a Perl program that provides a unified set of analyses for the selection, classification, quantification and automated annotation of tandemly repeated sequences. TRAP uses the results of the Tandem Repeats Finder program to perform a global analysis of the satellite content of DNA sequences, permitting researchers to easily assess the tandem repeat content for both individual sequences and whole genomes. The results can be generated in convenient formats such as HTML and comma-separated values. TRAP can also be used to automatically generate annotation data in the format of feature table and GFF files.

Isolation of human simple repeat loci by hybridization selection.

PubMed

Armour, J A; Neumann, R; Gobert, S; Jeffreys, A J

1994-04-01

We have isolated short tandem repeat arrays from the human genome, using a rapid method involving filter hybridization to enrich for tri- or tetranucleotide tandem repeats. About 30% of clones from the enriched library cross-hybridize with probes containing trimeric or tetrameric tandem arrays, facilitating the rapid isolation of large numbers of clones. In an initial analysis of 54 clones, 46 different tandem arrays were identified. Analysis of these tandem repeat loci by PCR showed that 24 were polymorphic in length; substantially higher levels of polymorphism were displayed by the tetrameric repeat loci isolated than by the trimeric repeats. Primary mapping of these loci by linkage analysis showed that they derive from 17 chromosomes, including the X chromosome. We anticipate the use of this strategy for the efficient isolation of tandem repeats from other sources of genomic DNA, including DNA from flow-sorted chromosomes, and from other species.

Short Tandem Repeat DNA Internet Database

National Institute of Standards and Technology Data Gateway

SRD 130 Short Tandem Repeat DNA Internet Database (Web, free access)   Short Tandem Repeat DNA Internet Database is intended to benefit research and application of short tandem repeat DNA markers for human identity testing. Facts and sequence information on each STR system, population data, commonly used multiplex STR systems, PCR primers and conditions, and a review of various technologies for analysis of STR alleles have been included.

Whole-genome sequencing reveals a coding non-pathogenic variant tagging a non-coding pathogenic hexanucleotide repeat expansion in C9orf72 as cause of amyotrophic lateral sclerosis.

PubMed

Herdewyn, Sarah; Zhao, Hui; Moisse, Matthieu; Race, Valérie; Matthijs, Gert; Reumers, Joke; Kusters, Benno; Schelhaas, Helenius J; van den Berg, Leonard H; Goris, An; Robberecht, Wim; Lambrechts, Diether; Van Damme, Philip

2012-06-01

Motor neuron degeneration in amyotrophic lateral sclerosis (ALS) has a familial cause in 10% of patients. Despite significant advances in the genetics of the disease, many families remain unexplained. We performed whole-genome sequencing in five family members from a pedigree with autosomal-dominant classical ALS. A family-based elimination approach was used to identify novel coding variants segregating with the disease. This list of variants was effectively shortened by genotyping these variants in 2 additional unaffected family members and 1500 unrelated population-specific controls. A novel rare coding variant in SPAG8 on chromosome 9p13.3 segregated with the disease and was not observed in controls. Mutations in SPAG8 were not encountered in 34 other unexplained ALS pedigrees, including 1 with linkage to chromosome 9p13.2-23.3. The shared haplotype containing the SPAG8 variant in this small pedigree was 22.7 Mb and overlapped with the core 9p21 linkage locus for ALS and frontotemporal dementia. Based on differences in coverage depth of known variable tandem repeat regions between affected and non-affected family members, the shared haplotype was found to contain an expanded hexanucleotide (GGGGCC)(n) repeat in C9orf72 in the affected members. Our results demonstrate that rare coding variants identified by whole-genome sequencing can tag a shared haplotype containing a non-coding pathogenic mutation and that changes in coverage depth can be used to reveal tandem repeat expansions. It also confirms (GGGGCC)n repeat expansions in C9orf72 as a cause of familial ALS.

Dynamic of mutational events in variable number tandem repeats of Escherichia coli O157:H7.

PubMed

Bustamante, A V; Sanso, A M; Segura, D O; Parma, A E; Lucchesi, P M A

2013-01-01

VNTRs regions have been successfully used for bacterial subtyping; however, the hypervariability in VNTR loci is problematic when trying to predict the relationships among isolates. Since few studies have examined the mutation rate of these markers, our aim was to estimate mutation rates of VNTRs specific for verotoxigenic E. coli O157:H7. The knowledge of VNTR mutational rates and the factors affecting them would make MLVA more effective for epidemiological or microbial forensic investigations. For this purpose, we analyzed nine loci performing parallel, serial passage experiments (PSPEs) on 9 O157:H7 strains. The combined 9 PSPE population rates for the 8 mutating loci ranged from 4.4 × 10(-05) to 1.8 × 10(-03) mutations/generation, and the combined 8-loci mutation rate was of 2.5 × 10(-03) mutations/generation. Mutations involved complete repeat units, with only one point mutation detected. A similar proportion between single and multiple repeat changes was detected. Of the 56 repeat mutations, 59% were insertions and 41% were deletions, and 72% of the mutation events corresponded to O157-10 locus. For alleles with up to 13 UR, a constant and low mutation rate was observed; meanwhile longer alleles were associated with higher and variable mutation rates. Our results are useful to interpret data from microevolution and population epidemiology studies and particularly point out that the inclusion or not of O157-10 locus or, alternatively, a differential weighting data according to the mutation rates of loci must be evaluated in relation with the objectives of the proposed study.

Repeat-mediated epigenetic dysregulation of the FMR1 gene in the fragile X-related disorders.

PubMed

Usdin, Karen; Kumari, Daman

2015-01-01

The fragile X-related disorders are members of the Repeat Expansion Diseases, a group of genetic conditions resulting from an expansion in the size of a tandem repeat tract at a specific genetic locus. The repeat responsible for disease pathology in the fragile X-related disorders is CGG/CCG and the repeat tract is located in the 5' UTR of the FMR1 gene, whose protein product FMRP, is important for the proper translation of dendritic mRNAs in response to synaptic activation. There are two different pathological FMR1 allele classes that are distinguished only by the number of repeats. Premutation alleles have 55-200 repeats and confer risk of fragile X-associated tremor/ataxia syndrome and fragile X-associated primary ovarian insufficiency. Full mutation alleles on the other hand have >200 repeats and result in fragile X syndrome, a disorder that affects learning and behavior. Different symptoms are seen in carriers of premutation and full mutation alleles because the repeat number has paradoxical effects on gene expression: Epigenetic changes increase transcription from premutation alleles and decrease transcription from full mutation alleles. This review will cover what is currently known about the mechanisms responsible for these changes in FMR1 expression and how they may relate to other Repeat Expansion Diseases that also show repeat-mediated changes in gene expression.

Programmable DNA-binding proteins from Burkholderia provide a fresh perspective on the TALE-like repeat domain

PubMed Central

de Lange, Orlando; Wolf, Christina; Dietze, Jörn; Elsaesser, Janett; Morbitzer, Robert; Lahaye, Thomas

2014-01-01

The tandem repeats of transcription activator like effectors (TALEs) mediate sequence-specific DNA binding using a simple code. Naturally, TALEs are injected by Xanthomonas bacteria into plant cells to manipulate the host transcriptome. In the laboratory TALE DNA binding domains are reprogrammed and used to target a fused functional domain to a genomic locus of choice. Research into the natural diversity of TALE-like proteins may provide resources for the further improvement of current TALE technology. Here we describe TALE-like proteins from the endosymbiotic bacterium Burkholderia rhizoxinica, termed Bat proteins. Bat repeat domains mediate sequence-specific DNA binding with the same code as TALEs, despite less than 40% sequence identity. We show that Bat proteins can be adapted for use as transcription factors and nucleases and that sequence preferences can be reprogrammed. Unlike TALEs, the core repeats of each Bat protein are highly polymorphic. This feature allowed us to explore alternative strategies for the design of custom Bat repeat arrays, providing novel insights into the functional relevance of non-RVD residues. The Bat proteins offer fertile grounds for research into the creation of improved programmable DNA-binding proteins and comparative insights into TALE-like evolution. PMID:24792163

Genetic Diversity among Bacillus anthracis Soil Isolates at Fine Geographic Scales

PubMed Central

Bader, Douglas E.

2012-01-01

Environmental samples were collected from carcass sites during and after anthrax outbreaks in 2000 and 2001 in the bison (Bison bison) population within Wood Buffalo National Park and the Hook Lake Region north of Wood Buffalo National Park. Bacillus anthracis spores were isolated from these samples and confirmed using phenotypic characterization and real-time PCR. Confirmed B. anthracis isolates were typed using multiple-locus variable-number tandem repeat analysis (MLVA15) and single-nucleotide-repeat analysis (SNRA). B. anthracis isolates split into two clades based on MLVA15, while SNRA allowed some isolates between carcass sites to be distinguished from each other. SNRA polymorphisms were also present within a single carcass site. Some isolates from different carcass sites having the same SNRA type had divergent MLVA types; this finding leads to questions about hierarchical typing methods and the robustness of the fine-scale typing of Bacillus anthracis. PMID:22773624

[Polymorphic loci and polymorphism analysis of short tandem repeats within XNP gene].

PubMed

Liu, Qi-Ji; Gong, Yao-Qin; Guo, Chen-Hong; Chen, Bing-Xi; Li, Jiang-Xia; Guo, Yi-Shou

2002-01-01

To select polymorphic short tandem repeat markers within X-linked nuclear protein (XNP) gene, genomic clones which contain XNP gene were recognized by homologous analysis with XNP cDNA. By comparing the cDNA with genomic DNA, non-exonic sequences were identified, and short tandem repeats were selected from non-exonic sequences by using BCM search Launcher. Polymorphisms of the short tandem repeats in Chinese population were evaluated by PCR amplification and PAGE. Five short tandem repeats were identified from XNP gene, two of which were polymorphic. Four and 11 alleles were observed in Chinese population for XNPSTR1 and XNPSTR4, respectively. Heterozygosities were 47% for XNPSTR1 and 70% for XNPSTR4. XNPSTR1 and XNPSTR4 localized within 3' end and intron 10, respectively. Two polymorphic short tandem repeats have been identified within XNP gene and will be useful for linkage analysis and gene diagnosis of XNP gene.

Core genome conservation of Staphylococcus haemolyticus limits sequence based population structure analysis.

PubMed

Cavanagh, Jorunn Pauline; Klingenberg, Claus; Hanssen, Anne-Merethe; Fredheim, Elizabeth Aarag; Francois, Patrice; Schrenzel, Jacques; Flægstad, Trond; Sollid, Johanna Ericson

2012-06-01

The notoriously multi-resistant Staphylococcus haemolyticus is an emerging pathogen causing serious infections in immunocompromised patients. Defining the population structure is important to detect outbreaks and spread of antimicrobial resistant clones. Currently, the standard typing technique is pulsed-field gel electrophoresis (PFGE). In this study we describe novel molecular typing schemes for S. haemolyticus using multi locus sequence typing (MLST) and multi locus variable number of tandem repeats (VNTR) analysis. Seven housekeeping genes (MLST) and five VNTR loci (MLVF) were selected for the novel typing schemes. A panel of 45 human and veterinary S. haemolyticus isolates was investigated. The collection had diverse PFGE patterns (38 PFGE types) and was sampled over a 20 year-period from eight countries. MLST resolved 17 sequence types (Simpsons index of diversity [SID]=0.877) and MLVF resolved 14 repeat types (SID=0.831). We found a low sequence diversity. Phylogenetic analysis clustered the isolates in three (MLST) and one (MLVF) clonal complexes, respectively. Taken together, neither the MLST nor the MLVF scheme was suitable to resolve the population structure of this S. haemolyticus collection. Future MLVF and MLST schemes will benefit from addition of more variable core genome sequences identified by comparing different fully sequenced S. haemolyticus genomes. Copyright © 2012 Elsevier B.V. All rights reserved.

Whole genome sequencing of Salmonella Typhimurium illuminates distinct outbreaks caused by an endemic multi-locus variable number tandem repeat analysis type in Australia, 2014.

PubMed

Phillips, Anastasia; Sotomayor, Cristina; Wang, Qinning; Holmes, Nadine; Furlong, Catriona; Ward, Kate; Howard, Peter; Octavia, Sophie; Lan, Ruiting; Sintchenko, Vitali

2016-09-15

Salmonella Typhimurium (STM) is an important cause of foodborne outbreaks worldwide. Subtyping of STM remains critical to outbreak investigation, yet current techniques (e.g. multilocus variable number tandem repeat analysis, MLVA) may provide insufficient discrimination. Whole genome sequencing (WGS) offers potentially greater discriminatory power to support infectious disease surveillance. We performed WGS on 62 STM isolates of a single, endemic MLVA type associated with two epidemiologically independent, food-borne outbreaks along with sporadic cases in New South Wales, Australia, during 2014. Genomes of case and environmental isolates were sequenced using HiSeq (Illumina) and the genetic distance between them was assessed by single nucleotide polymorphism (SNP) analysis. SNP analysis was compared to the epidemiological context. The WGS analysis supported epidemiological evidence and genomes of within-outbreak isolates were nearly identical. Sporadic cases differed from outbreak cases by a small number of SNPs, although their close relationship to outbreak cases may represent an unidentified common food source that may warrant further public health follow up. Previously unrecognised mini-clusters were detected. WGS of STM can discriminate foodborne community outbreaks within a single endemic MLVA clone. Our findings support the translation of WGS into public health laboratory surveillance of salmonellosis.

Evaluation of advanced multiplex short tandem repeat systems in pairwise kinship analysis.

PubMed

Tamura, Tomonori; Osawa, Motoki; Ochiai, Eriko; Suzuki, Takanori; Nakamura, Takashi

2015-09-01

The AmpFLSTR Identifiler Kit, comprising 15 autosomal short tandem repeat (STR) loci, is commonly employed in forensic practice for calculating match probabilities and parentage testing. The conventional system exhibits insufficient estimation for kinship analysis such as sibship testing because of shortness of examined loci. This study evaluated the power of the PowerPlex Fusion System, GlobalFiler Kit, and PowerPlex 21 System, which comprise more than 20 autosomal STR loci, to estimate pairwise blood relatedness (i.e., parent-child, full siblings, second-degree relatives, and first cousins). The genotypes of all 24 STR loci in 10,000 putative pedigrees were constructed by simulation. The likelihood ratio for each locus was calculated from joint probabilities for relatives and non-relatives. The combined likelihood ratio was calculated according to the product rule. The addition of STR loci improved separation between relatives and non-relatives. However, these systems were less effectively extended to the inference for first cousins. In conclusion, these advanced systems will be useful in forensic personal identification, especially in the evaluation of full siblings and second-degree relatives. Moreover, the additional loci may give rise to two major issues of more frequent mutational events and several pairs of linked loci on the same chromosome. Copyright © 2015 Elsevier Ireland Ltd. All rights reserved.

Genetic diversity and antimicrobial resistance pattern of Salmonella enterica serovar Enteritidis clinical isolates in Thailand.

PubMed

Utrarachkij, Fuangfa; Nakajima, Chie; Siripanichgon, Kanokrat; Changkaew, Kanjana; Thongpanich, Yuwanda; Pornraungwong, Srirat; Suthienkul, Orasa; Suzuki, Yasuhiko

2016-04-01

To trace the history of antimicrobial resistance in Salmonella enterica serovar Enteritidis (S. Enteritidis, SE) circulating in Thailand, we characterised clinical isolates obtained during 2004-2007. Antimicrobial resistance profiles, multi-locus variable number tandem repeat analysis (MLVA) types and 3 representative virulence determinants (spvA, sodCI and sopE) were established from SE isolates (n = 192) collected from stool and blood of patients throughout Thailand during the period 2004-2007. Resistance was found in SE against 10 out of 11 antimicrobials studied. The highest resistance ratios were observed for nalidixic acid (83.2%), ciprofloxacin (51.1%) and ampicillin (50.5%), and 25.5% were multidrug resistant. Based on five polymorphic tandem repeat loci analysis, MLVA identified 20 distinct types with three closely related predominant types. A significant increase of AMP resistance from 2004 to 2006 was strongly correlated with that of a MLVA type, 5-5-11-7-3. The usage of antimicrobials in human medicine or farm settings might act as selective pressures and cause the spread of resistant strains. Hence, a strict policy on antimicrobial usage needs to be implemented to achieve the control of resistant SE in Thailand. Copyright © 2015 Japanese Society of Chemotherapy and The Japanese Association for Infectious Diseases. Published by Elsevier Ltd. All rights reserved.

Molecular characterization and distribution of a 145-bp tandem repeat family in the genus Populus.

PubMed

Rajagopal, J; Das, S; Khurana, D K; Srivastava, P S; Lakshmikumaran, M

1999-10-01

This report aims to describe the identification and molecular characterization of a 145-bp tandem repeat family that accounts for nearly 1.5% of the Populus genome. Three members of this repeat family were cloned and sequenced from Populus deltoides and P. ciliata. The dimers of the repeat were sequenced in order to confirm the head-to-tail organization of the repeat. Hybridization-based analysis using the 145-bp tandem repeat as a probe on genomic DNA gave rise to ladder patterns which were identified to be a result of methylation and (or) sequence heterogeneity. Analysis of the methylation pattern of the repeat family using methylation-sensitive isoschizomers revealed variable methylation of the C residues and lack of methylation of the A residues. Sequence comparisons between the monomers revealed a high degree of sequence divergence that ranged between 6% and 11% in P. deltoides and between 4.2% and 8.3% in P. ciliata. This indicated the presence of sub-families within the 145-bp tandem family of repeats. Divergence was mainly due to the accumulation of point mutations and was concentrated in the central region of the repeat. The 145-bp tandem repeat family did not show significant homology to known tandem repeats from plants. A short stretch of 36 bp was found to show homology of 66.7% to a centromeric repeat from Chironomus plumosus. Dot-blot analysis and Southern hybridization data revealed the presence of the repeat family in 13 of the 14 Populus species examined. The absence of the 145-bp repeat from P. euphratica suggested that this species is relatively distant from other members of the genus, which correlates with taxonomic classifications. The widespread occurrence of the tandem family in the genus indicated that this family may be of ancient origin.

Dynamic of Mutational Events in Variable Number Tandem Repeats of Escherichia coli O157:H7

PubMed Central

Bustamante, A. V.; Sanso, A. M.; Segura, D. O.; Parma, A. E.; Lucchesi, P. M. A.

2013-01-01

VNTRs regions have been successfully used for bacterial subtyping; however, the hypervariability in VNTR loci is problematic when trying to predict the relationships among isolates. Since few studies have examined the mutation rate of these markers, our aim was to estimate mutation rates of VNTRs specific for verotoxigenic E. coli O157:H7. The knowledge of VNTR mutational rates and the factors affecting them would make MLVA more effective for epidemiological or microbial forensic investigations. For this purpose, we analyzed nine loci performing parallel, serial passage experiments (PSPEs) on 9 O157:H7 strains. The combined 9 PSPE population rates for the 8 mutating loci ranged from 4.4 × 10−05 to 1.8 × 10−03 mutations/generation, and the combined 8-loci mutation rate was of 2.5 × 10−03 mutations/generation. Mutations involved complete repeat units, with only one point mutation detected. A similar proportion between single and multiple repeat changes was detected. Of the 56 repeat mutations, 59% were insertions and 41% were deletions, and 72% of the mutation events corresponded to O157-10 locus. For alleles with up to 13 UR, a constant and low mutation rate was observed; meanwhile longer alleles were associated with higher and variable mutation rates. Our results are useful to interpret data from microevolution and population epidemiology studies and particularly point out that the inclusion or not of O157-10 locus or, alternatively, a differential weighting data according to the mutation rates of loci must be evaluated in relation with the objectives of the proposed study. PMID:24093095

Comparative analysis of tandem repeats from hundreds of species reveals unique insights into centromere evolution

USDA-ARS?s Scientific Manuscript database

Centromeres are essential for chromosome segregation, yet their DNA sequences evolve rapidly. In most animals and plants that have been studied, centromeres comprise of megabase-scale arrays of tandem repeats. The true prevalence of centromere tandem repeats, and whether they exhibit conserved seque...

Diversity among strains of Pseudomonas aeruginosa from manure and soil, evaluated by multiple locus variable number tandem repeat analysis and antibiotic resistance profiles.

PubMed

Youenou, Benjamin; Brothier, Elisabeth; Nazaret, Sylvie

2014-01-01

The results of a multiple locus variable number of tandem repeat (VNTR) analysis (MLVA)-based study designed to understand the genetic diversity of soil and manure-borne Pseudomonas aeruginosa isolates, and the relationship between these isolates and a set of clinical and environmental isolates, are hereby reported. Fifteen described VNTR markers were first selected, and 62 isolates recovered from agricultural and industrial soils in France and Burkina Faso, and from cattle and horse manure, along with 26 snake-related isolates and 17 environmental and clinical isolates from international collections, were genotyped. Following a comparison with previously published 9-marker MLVA schemes, an optimal 13-marker MLVA scheme (MLVA13-Lyon) was identified that was found to be the most efficient, as it showed high typability (90%) and high discriminatory power (0.987). A comparison of MLVA with PFGE for typing of the snake-related isolates confirmed the MLVA13-Lyon scheme to be a robust method for quickly discriminating and inferring genetic relatedness among environmental isolates. The 62 isolates displayed wide diversity, since 41 MLVA types (i.e. MTs) were observed, with 26 MTs clustered in 10 MLVA clonal complexes (MCs). Three and eight MCs were found among soil and manure isolates, respectively. Only one MC contained both soil and manure-borne isolates. No common MC was observed between soil and manure-borne isolates and the snake-related or environmental and clinical isolates. Antibiotic resistance profiles were performed to determine a potential link between resistance properties and the selective pressure that might be present in the various habitats. Except for four soil and manure isolates resistant to ticarcillin and ticarcillin/clavulanic acid and one isolate from a hydrocarbon-contaminated soil resistant to imipenem, all environmental isolates showed wild-type antibiotic profiles. Copyright © 2013 Institut Pasteur. Published by Elsevier Masson SAS. All rights reserved.

[Use of multiple locus variable number tandem repeats analysis for the Brucella systematization].

PubMed

Kulakov, Iu K; Kovalev, D A; Misetova, E N; Golovneva, S I; Liapustina, L V; Zheludkov, M M

2012-01-01

The methods of molecular-genetic differentiation to strain level acquire increasing significance in the current system of struggle with brucellosis. MLVA (multiple locus variable number tandem repeats analysis) was selected for molecular-genetic differentiation to strain level and simultaneous establishment of the genetic relationship of investigated Brucella strains. The goal of this work was MLVA typing of three pathogenic Brucella species strains with the analysis of stability of chosen loci, discrimination power and concordance to conventional phenotypic methods of the Brucella differentiation for use in systematization of brucellosis causing agents. Twenty six Brucella strains representing reference (n = 15), vaccine (n = 2) and field strains of three pathogenic Brucella species were tested: B. melitensis (n = 3), B. abortus (n = 2), B. suis (n = 2), and isolates (n = 2) with unidentified taxonomic position using MLVA with 9 pairs primers on known variable loci of Brucella genome. The analysis of the stability of chosen loci, discrimination power on Hunter-Gaston discrimination index (HGDI) and consistency to phenotypic methods of identification was performed. MLVA was confirmed for the results of phenotypic methods of identification, stability of the chosen loci in majority reference, and vaccine strains with a high index of variability HGDI 0.9969 for all loci. A dendrogram was plotted on the basis of MLVA data on distributed Brucella strains in related clusters according to its taxonomic species and biovar positions and construction of 25 genotypes. B. melitensis strains formed cluster related to the reference strain of B. melitensis 63/9 biovar 2. Australian isolates of Brucella 83-4 and Brucella 83-6 isolated from rodents formed a cluster distant from other strains of Brucella. MLVA is a promising method for differentiation of Brucella strains with known and unresolved taxonomic status for their systematization and creation of MLVA genotype catalogue that will promote qualitative improvement of brucellosis surveillance system in Russia.

Genetic stability of Brucella abortus isolates from an outbreak by multiple-locus variable-number tandem repeat analysis (MLVA16)

PubMed Central

2014-01-01

Background Brucellosis caused by Brucella abortus is one of the most important zoonoses in the world. Multiple-locus variable-number tandem repeat analysis (MLVA16) has been shown be a useful tool to epidemiological traceback studies in B. abortus infection. Thus, the present study aimed (i) to evaluate the genetic diversity of B. abortus isolates from a brucellosis outbreak, and (ii) to investigate the in vivo stability of the MLVA16 markers. Results Three-hundred and seventy-five clinical samples, including 275 vaginal swabs and 100 milk samples, were cultured from a brucellosis outbreak in a cattle herd, which adopted RB51 vaccination and test-and-slaughter policies. Thirty-seven B. abortus isolates were obtained, eight from milk and twenty-nine from post-partum/abortion vaginal swabs, which were submitted to biotyping and genotyping by MLVA16. Twelve B. abortus isolates obtained from vaginal swabs were identified as RB51. Twenty four isolates, seven obtained from milk samples and seventeen from vaginal swabs, were identified as B. abortus biovar 3, while one isolate from vaginal swabs was identified as B. abortus biovar 1. Three distinct genotypes were observed during the brucellosis outbreak: RB observed in all isolates identified as RB51; W observed in all B. abortus biovar 3 isolates; and Z observed in the single B. abortus biovar 1 isolate. Epidemiological and molecular data show that the B. abortus biovar 1 genotype Z strain is not related to the B. abortus biovar 3 genotype W isolates, and represents a new introduction B. abortus during the outbreak. Conclusions The results of the present study on typing of multiple clinical B. abortus isolates from the same outbreak over a sixteen month period indicate the in vivo stability of MLVA16 markers, a low genetic diversity among B. abortus isolates and the usefulness of MLVA16 for epidemiological studies of bovine brucellosis. PMID:25015840

Subtyping of Canadian isolates of Salmonella Enteritidis using Multiple Locus Variable Number Tandem Repeat Analysis (MLVA) alone and in combination with Pulsed-Field Gel Electrophoresis (PFGE) and phage typing.

PubMed

Ziebell, Kim; Chui, Linda; King, Robin; Johnson, Suzanne; Boerlin, Patrick; Johnson, Roger P

2017-08-01

Salmonella enterica subspecies enterica serovar Enteritidis (SE) is one of the most common causes of human salmonellosis and in Canada currently accounts for over 40% of human cases. Reliable subtyping of isolates is required for outbreak detection and source attribution. However, Pulsed-Field Gel Electrophoresis (PFGE), the current standard subtyping method for Salmonella spp., is compromised by the high genetic homogeneity of SE. Multiple Locus Variable Number Tandem Repeat Analysis (MLVA) was introduced to supplement PFGE, although there is a lack of data on the ability of MLVA to subtype Canadian isolates of SE. Three subtyping methods, PFGE, MLVA and phage typing were compared for their discriminatory power when applied to three panels of Canadian SE isolates: Panel 1: 70 isolates representing the diversity of phage types (PTs) and PFGE subtypes within these PTs; Panel 2: 214 apparently unrelated SE isolates of the most common PTs; and Panel 3: 27 isolates from 10 groups of epidemiologically related strains. For Panel 2 isolates, four MLVA subtypes were shared among 74% of unrelated isolates and in Panel 3 isolates, one MLVA subtype accounted for 62% of the isolates. For all panels, combining results from PFGE, MLVA and PT gave the best discrimination, except in Panel 1, where the combination of PT and PFGE was equally as high, due to the selection criteria for this panel. However, none of these methods is sufficiently discriminatory alone for reliable outbreak detection or source attribution, and must be applied together to achieve sufficient discrimination for practical purposes. Even then, some large clusters were not differentiated adequately. More discriminatory methods are required for reliable subtyping of this genetically highly homogeneous serovar. This need will likely be met by whole genome sequence analysis given the recent promising reports and as more laboratories implement this tool for outbreak response and surveillance. Copyright © 2017 Elsevier B.V. All rights reserved.

Multiple-locus variable-number tandem repeat analysis for molecular typing of Aspergillus fumigatus

PubMed Central

2010-01-01

Background Multiple-locus variable-number tandem repeat (VNTR) analysis (MLVA) is a prominent subtyping method to resolve closely related microbial isolates to provide information for establishing genetic patterns among isolates and to investigate disease outbreaks. The usefulness of MLVA was recently demonstrated for the avian major pathogen Chlamydophila psittaci. In the present study, we developed a similar method for another pathogen of birds: the filamentous fungus Aspergillus fumigatus. Results We selected 10 VNTR markers located on 4 different chromosomes (1, 5, 6 and 8) of A. fumigatus. These markers were tested with 57 unrelated isolates from different hosts or their environment (53 isolates from avian species in France, China or Morocco, 3 isolates from humans collected at CHU Henri Mondor hospital in France and the reference strain CBS 144.89). The Simpson index for individual markers ranged from 0.5771 to 0.8530. A combined loci index calculated with all the markers yielded an index of 0.9994. In a second step, the panel of 10 markers was used in different epidemiological situations and tested on 277 isolates, including 62 isolates from birds in Guangxi province in China, 95 isolates collected in two duck farms in France and 120 environmental isolates from a turkey hatchery in France. A database was created with the results of the present study http://minisatellites.u-psud.fr/MLVAnet/. Three major clusters of isolates were defined by using the graphing algorithm termed Minimum Spanning Tree (MST). The first cluster comprised most of the avian isolates collected in the two duck farms in France, the second cluster comprised most of the avian isolates collected in poultry farms in China and the third one comprised most of the isolates collected in the turkey hatchery in France. Conclusions MLVA displayed excellent discriminatory power. The method showed a good reproducibility. MST analysis revealed an interesting clustering with a clear separation between isolates according to their geographic origin rather than their respective hosts. PMID:21143842

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.

Molecular characterization and physical localization of highly repetitive DNA sequences from Brazilian Alstroemeria species.

PubMed

Kuipers, A G J; Kamstra, S A; de Jeu, M J; Visser, R G F

2002-01-01

Highly repetitive DNA sequences were isolated from genomic DNA libraries of Alstroemeria psittacina and A. inodora. Among the repetitive sequences that were isolated, tandem repeats as well as dispersed repeats could be discerned. The tandem repeats belonged to a family of interlinked Sau3A subfragments with sizes varying from 68-127 bp, and constituted a larger HinfI repeat of approximately 400 bp. Southern hybridization showed a similar molecular organization of the tandem repeats in each of the Brazilian Alstroemeria species tested. None of the repeats hybridized with DNA from Chilean Alstroemeria species, which indicates that they are specific for the Brazilian species. In-situ localization studies revealed the tandem repeats to be localized in clusters on the chromosomes of A. inodora and A. psittacina: distal hybridization sites were found on chromosome arms 2PS, 6PL, 7PS, 7PL and 8PL, interstitial sites on chromosome arms 2PL, 3PL, 4PL and 5PL. The applicability of the tandem repeats for cytogenetic analysis of interspecific hybrids and their role in heterochromatin organization are discussed.

Programmable DNA-binding proteins from Burkholderia provide a fresh perspective on the TALE-like repeat domain.

PubMed

de Lange, Orlando; Wolf, Christina; Dietze, Jörn; Elsaesser, Janett; Morbitzer, Robert; Lahaye, Thomas

2014-06-01

The tandem repeats of transcription activator like effectors (TALEs) mediate sequence-specific DNA binding using a simple code. Naturally, TALEs are injected by Xanthomonas bacteria into plant cells to manipulate the host transcriptome. In the laboratory TALE DNA binding domains are reprogrammed and used to target a fused functional domain to a genomic locus of choice. Research into the natural diversity of TALE-like proteins may provide resources for the further improvement of current TALE technology. Here we describe TALE-like proteins from the endosymbiotic bacterium Burkholderia rhizoxinica, termed Bat proteins. Bat repeat domains mediate sequence-specific DNA binding with the same code as TALEs, despite less than 40% sequence identity. We show that Bat proteins can be adapted for use as transcription factors and nucleases and that sequence preferences can be reprogrammed. Unlike TALEs, the core repeats of each Bat protein are highly polymorphic. This feature allowed us to explore alternative strategies for the design of custom Bat repeat arrays, providing novel insights into the functional relevance of non-RVD residues. The Bat proteins offer fertile grounds for research into the creation of improved programmable DNA-binding proteins and comparative insights into TALE-like evolution. © The Author(s) 2014. Published by Oxford University Press on behalf of Nucleic Acids Research.

A novel typing method for Listeria monocytogenes using high-resolution melting analysis (HRMA) of tandem repeat regions.

PubMed

Ohshima, Chihiro; Takahashi, Hajime; Iwakawa, Ai; Kuda, Takashi; Kimura, Bon

2017-07-17

Listeria monocytogenes, which is responsible for causing food poisoning known as listeriosis, infects humans and animals. Widely distributed in the environment, this bacterium is known to contaminate food products after being transmitted to factories via raw materials. To minimize the contamination of products by food pathogens, it is critical to identify and eliminate factory entry routes and pathways for the causative bacteria. High resolution melting analysis (HRMA) is a method that takes advantage of differences in DNA sequences and PCR product lengths that are reflected by the disassociation temperature. Through our research, we have developed a multiple locus variable-number tandem repeat analysis (MLVA) using HRMA as a simple and rapid method to differentiate L. monocytogenes isolates. While evaluating our developed method, the ability of MLVA-HRMA, MLVA using capillary electrophoresis, and multilocus sequence typing (MLST) was compared for their ability to discriminate between strains. The MLVA-HRMA method displayed greater discriminatory ability than MLST and MLVA using capillary electrophoresis, suggesting that the variation in the number of repeat units, along with mutations within the DNA sequence, was accurately reflected by the melting curve of HRMA. Rather than relying on DNA sequence analysis or high-resolution electrophoresis, the MLVA-HRMA method employs the same process as PCR until the analysis step, suggesting a combination of speed and simplicity. The result of MLVA-HRMA method is able to be shared between different laboratories. There are high expectations that this method will be adopted for regular inspections at food processing facilities in the near future. Copyright © 2017. Published by Elsevier B.V.

Allele Frequencies for 15 Short Tandem Repeat Loci in Representative Sample of Croatian Population

PubMed Central

Projić, Petar; Škaro, Vedrana; Šamija, Ivana; Pojskić, Naris; Durmić-Pašić, Adaleta; Kovačević, Lejla; Bakal, Narcisa; Primorac, Dragan; Marjanović, Damir

2007-01-01

Aim To study the distribution of allele frequencies of 15 short tandem repeat (STR) loci in a representative sample of the Croatian population. Methods A total of 195 unrelated Caucasian individuals born in Croatia, from 14 counties and the City of Zagreb, were sampled for the analysis. All the tested individuals were voluntary donors. Buccal swab was used as the DNA source. AmpFlSTR® Identifiler® was applied to simultaneously amplify 15 STR loci. Total reaction volume was 12.5 μL. The polymerase chain reaction (PCR) amplification was carried out in PE Gene Amp PCR System Thermal Cycler. Electrophoresis of the amplification products was preformed on an ABI PRISM 3130 Genetic Analyzer. After PCR amplification and separation by electrophoresis, raw data were compiled, analyzed, and numerical allele designations of the profiles were obtained. Deviation from Hardy-Weinberg equilibrium, observed and expected heterozygosity, power of discrimination, and power of exclusion were calculated. Bonferroni’s correction was used before each comparative analysis. Results We compared Croatian data with those obtained from geographically neighboring European populations. The significant difference (at P<0.01) in allele frequencies was recorded only between the Croatian and Slovenian populations for vWA locus. There was no significant deviation from Hardy-Weinberg equilibrium for all the observed loci. Conclusion Obtained population data concurred with the expected “STR data frame” for this part of Europe. PMID:17696301

Parthenogenesis in a Brazilian rainbow boa (Epicrates cenchria cenchria).

PubMed

Kinney, Matthew E; Wack, Raymund F; Grahn, Robert A; Lyons, Leslie

2013-03-01

A 22-year-old captive Brazilian rainbow boa (Epicrates cenchria cenchria) gave birth to four offspring after being housed with a vasectomized male. Sexual reproduction as a result of failed prior vasectomy, recanalization of the vas deferens, or prolonged sperm storage was ruled out using the clinical history, histopathology, and gross necropsy. Short tandem repeat (STR) DNA markers were genotyped in the male, female, and four offspring. None of the offspring possessed a diagnostic STR allele present in the potential sire. In addition, all offspring were homozygous at each STR locus evaluated, supporting parthenogenetic reproduction. This is the first report of parthenogenesis in a Brazilian rainbow boa and has implications for the conservation of reptiles maintained in captive breeding programs. © 2012 Wiley Periodicals, Inc.

Comparison of semi-automated commercial rep-PCR fingerprinting, spoligotyping, 12-locus MIRU-VNTR typing and single nucleotide polymorphism analysis of the embB gene as molecular typing tools for Mycobacterium bovis.

PubMed

Armas, Federica; Camperio, Cristina; Coltella, Luana; Selvaggini, Serena; Boniotti, Maria Beatrice; Pacciarini, Maria Lodovica; Di Marco Lo Presti, Vincenzo; Marianelli, Cinzia

2017-08-04

Highly discriminatory genotyping strategies are essential in molecular epidemiological studies of tuberculosis. In this study we evaluated, for the first time, the efficacy of the repetitive sequence-based PCR (rep-PCR) DiversiLab Mycobacterium typing kit over spoligotyping, 12-locus mycobacterial interspersed repetitive unit-variable number tandem repeat (MIRU-VNTR) typing and embB single nucleotide polymorphism (SNP) analysis for Mycobacterium bovis typing. A total of 49 M. bovis animal isolates were used. DNA was extracted and genomic DNA was amplified using the DiversiLab Mycobacterium typing kit. The amplified fragments were separated and detected using a microfluidics chip with Agilent 2100. The resulting rep-PCR-based DNA fingerprints were uploaded to and analysed using web-based DiversiLab software through Pearson's correlation coefficient. Rep-PCR DiversiLab grouped M. bovis isolates into ten different clusters. Most isolates sharing identical spoligotype, MIRU-VNTR profile or embB gene polymorphism were grouped into different rep-PCR clusters. Rep-PCR DiversiLab displayed greater discriminatory power than spoligotyping and embB SNP analysis but a lower resolution power than the 12-locus MIRU-VNTR analysis. MIRU-VNTR confirmed that it is superior to the other PCR-based methods tested here. In combination with spoligotyping and 12-locus MIRU-VNTR analysis, rep-PCR improved the discriminatory power for M. bovis typing.

Analysis of the NMI01 marker for a population database of cannabis seeds.

PubMed

Shirley, Nicholas; Allgeier, Lindsay; Lanier, Tommy; Coyle, Heather Miller

2013-01-01

We have analyzed the distribution of genotypes at a single hexanucleotide short tandem repeat (STR) locus in a Cannabis sativa seed database along with seed-packaging information. This STR locus is defined by the polymerase chain reaction amplification primers CS1F and CS1R and is referred to as NMI01 (for National Marijuana Initiative) in our study. The population database consists of seed seizures of two categories: seed samples from labeled and unlabeled packages regarding seed bank source. Of a population database of 93 processed seeds including 12 labeled Cannabis varieties, the observed genotypes generated from single seeds exhibited between one and three peaks (potentially six alleles if in homozygous state). The total number of observed genotypes was 54 making this marker highly specific and highly individualizing even among seeds of common lineage. Cluster analysis associated many but not all of the handwritten labeled seed varieties tested to date as well as the National Park seizure to our known reference database containing Mr. Nice Seedbank and Sensi Seeds commercially packaged reference samples. © 2012 American Academy of Forensic Sciences.

The Evolution of Advanced Molecular Diagnostics for the Detection and Characterization of Mycoplasma pneumoniae.

PubMed

Diaz, Maureen H; Winchell, Jonas M

2016-01-01

Over the past decade there have been significant advancements in the methods used for detecting and characterizing Mycoplasma pneumoniae, a common cause of respiratory illness and community-acquired pneumonia worldwide. The repertoire of available molecular diagnostics has greatly expanded from nucleic acid amplification techniques (NAATs) that encompass a variety of chemistries used for detection, to more sophisticated characterizing methods such as multi-locus variable-number tandem-repeat analysis (MLVA), Multi-locus sequence typing (MLST), matrix-assisted laser desorption ionization-time-of-flight mass spectrometry (MALDI-TOF MS), single nucleotide polymorphism typing, and numerous macrolide susceptibility profiling methods, among others. These many molecular-based approaches have been developed and employed to continually increase the level of discrimination and characterization in order to better understand the epidemiology and biology of M. pneumoniae. This review will summarize recent molecular techniques and procedures and lend perspective to how each has enhanced the current understanding of this organism and will emphasize how Next Generation Sequencing may serve as a resource for researchers to gain a more comprehensive understanding of the genomic complexities of this insidious pathogen.

ST proteins, a new family of plant tandem repeat proteins with a DUF2775 domain mainly found in Fabaceae and Asteraceae.

PubMed

Albornos, Lucía; Martín, Ignacio; Iglesias, Rebeca; Jiménez, Teresa; Labrador, Emilia; Dopico, Berta

2012-11-07

Many proteins with tandem repeats in their sequence have been described and classified according to the length of the repeats: I) Repeats of short oligopeptides (from 2 to 20 amino acids), including structural cell wall proteins and arabinogalactan proteins. II) Repeats that range in length from 20 to 40 residues, including proteins with a well-established three-dimensional structure often involved in mediating protein-protein interactions. (III) Longer repeats in the order of 100 amino acids that constitute structurally and functionally independent units. Here we analyse ShooT specific (ST) proteins, a family of proteins with tandem repeats of unknown function that were first found in Leguminosae, and their possible similarities to other proteins with tandem repeats. ST protein sequences were only found in dicotyledonous plants, limited to several plant families, mainly the Fabaceae and the Asteraceae. ST mRNAs accumulate mainly in the roots and under biotic interactions. Most ST proteins have one or several Domain(s) of Unknown Function 2775 (DUF2775). All deduced ST proteins have a signal peptide, indicating that these proteins enter the secretory pathway, and the mature proteins have tandem repeat oligopeptides that share a hexapeptide (E/D)FEPRP followed by 4 partially conserved amino acids, which could determine a putative N-glycosylation signal, and a fully conserved tyrosine. In a phylogenetic tree, the sequences clade according to taxonomic group. A possible involvement in symbiosis and abiotic stress as well as in plant cell elongation is suggested, although different STs could play different roles in plant development. We describe a new family of proteins called ST whose presence is limited to the plant kingdom, specifically to a few families of dicotyledonous plants. They present 20 to 40 amino acid tandem repeat sequences with different characteristics (signal peptide, DUF2775 domain, conservative repeat regions) from the described group of 20 to 40 amino acid tandem repeat proteins and also from known cell wall proteins with repeat sequences. Several putative roles in plant physiology can be inferred from the characteristics found.

ST proteins, a new family of plant tandem repeat proteins with a DUF2775 domain mainly found in Fabaceae and Asteraceae

PubMed Central

2012-01-01

Background Many proteins with tandem repeats in their sequence have been described and classified according to the length of the repeats: I) Repeats of short oligopeptides (from 2 to 20 amino acids), including structural cell wall proteins and arabinogalactan proteins. II) Repeats that range in length from 20 to 40 residues, including proteins with a well-established three-dimensional structure often involved in mediating protein-protein interactions. (III) Longer repeats in the order of 100 amino acids that constitute structurally and functionally independent units. Here we analyse ShooT specific (ST) proteins, a family of proteins with tandem repeats of unknown function that were first found in Leguminosae, and their possible similarities to other proteins with tandem repeats. Results ST protein sequences were only found in dicotyledonous plants, limited to several plant families, mainly the Fabaceae and the Asteraceae. ST mRNAs accumulate mainly in the roots and under biotic interactions. Most ST proteins have one or several Domain(s) of Unknown Function 2775 (DUF2775). All deduced ST proteins have a signal peptide, indicating that these proteins enter the secretory pathway, and the mature proteins have tandem repeat oligopeptides that share a hexapeptide (E/D)FEPRP followed by 4 partially conserved amino acids, which could determine a putative N-glycosylation signal, and a fully conserved tyrosine. In a phylogenetic tree, the sequences clade according to taxonomic group. A possible involvement in symbiosis and abiotic stress as well as in plant cell elongation is suggested, although different STs could play different roles in plant development. Conclusions We describe a new family of proteins called ST whose presence is limited to the plant kingdom, specifically to a few families of dicotyledonous plants. They present 20 to 40 amino acid tandem repeat sequences with different characteristics (signal peptide, DUF2775 domain, conservative repeat regions) from the described group of 20 to 40 amino acid tandem repeat proteins and also from known cell wall proteins with repeat sequences. Several putative roles in plant physiology can be inferred from the characteristics found. PMID:23134664

Triplet repeat expansion at the FRAXE locus and X-linked mild mental handicap.

PubMed Central

Knight, S. J.; Voelckel, M. A.; Hirst, M. C.; Flannery, A. V.; Moncla, A.; Davies, K. E.

1994-01-01

We have recently shown that the expression of the FRAXE fragile site in Xq28 is associated with the expansion of a GCC trinucleotide repeat. In the families studied, FRAXE expression is also associated with mild mental handicap. Here we present data on families that previously had been diagnosed as having the fragile X syndrome but that later were found to be negative for trinucleotide repeat expansion at the FRAXA locus. In these families we demonstrate the presence of a GCC trinucleotide repeat expansion at the FRAXE locus. Studies of the FRAXE locus of normal individuals show that they have 6-25 copies of the repeat, whereas affected individuals have > 200 copies. As in the fragile X syndrome, the amplified CpG residues are methylated in affected males. Images Figure 2 Figure 3 Figure 4 PMID:8023854

Small tandemly repeated DNA sequences of higher plants likely originate from a tRNA gene ancestor.

PubMed Central

Benslimane, A A; Dron, M; Hartmann, C; Rode, A

1986-01-01

Several monomers (177 bp) of a tandemly arranged repetitive nuclear DNA sequence of Brassica oleracea have been cloned and sequenced. They share up to 95% homology between one another and up to 80% with other satellite DNA sequences of Cruciferae, suggesting a common ancestor. Both strands of these monomers show more than 50% homology with many tRNA genes; the best homologies have been obtained with Lys and His yeast mitochondrial tRNA genes (respectively 64% and 60%). These results suggest that small tandemly repeated DNA sequences of plants may have evolved from a tRNA gene ancestor. These tandem repeats have probably arisen via a process involving reverse transcription of polymerase III RNA intermediates, as is the case for interspersed DNA sequences of mammalians. A model is proposed to explain the formation of such small tandemly repeated DNA sequences. Images PMID:3774553

Identification of the Rare, Four Repeat Allele of IL-4 Intron-3 VNTR Polymorphism in Indian Populations.

PubMed

Verma, Henu Kumar; Jha, Aditya Nath; Khodiar, Prafulla Kumar; Patra, Pradeep Kumar; Bhaskar, Lakkakula Venkata Kameswara Subrahmanya

2016-06-01

Cytokines are cell signaling molecules which upon release by cells facilitate the recruitment of immune-modulatory cells towards the sites of inflammation. Genetic variations in cytokine genes are shown to regulate their production and affect the risk of infectious as well as autoimmune diseases. Intron-3 of interleukin-4 gene (IL-4) harbors 70-bp variable number of tandem repeats (VNTR) that may alter the expression level of IL-4 gene. To determine the distribution of IL-4 70-bp VNTR polymorphism in seven genetically heterogeneous populations of Chhattisgarh, India and their comparison with the finding of other Indian and world populations. A total of 371 healthy unrelated individuals from 5 caste and 2 tribal populations were included in the present study. The IL-4 70-bp VNTR genotyping was carried out using PCR and electrophoresis. Overall, 3 alleles of IL-4 70-bp VNTR (a2, a3 and a4) were detected. The results demonstrated the variability of the IL-4 70-bp VNTR polymorphism in Chhattisgarh populations. Allele a3 was the most common allele at the 70-bp VNTR locus in all populations followed by a2 allele. This study reports the presence four repeat allele a4 at a low frequency in the majority of the Chhattisgarh populations studied. Further, the frequency of the minor allele (a2) in Chhattisgarh populations showed similarity with the frequencies of European populations but not with the East Asian populations where the a2 allele is a major allele. Our study provides a baseline for future research into the role of the IL-4 locus in diseases linked to inflammation in Indian populations.

Molecular characteristics of "Mycobacterium canettii" the smooth Mycobacterium tuberculosis bacilli.

PubMed

Fabre, Michel; Hauck, Yolande; Soler, Charles; Koeck, Jean-Louis; van Ingen, Jakko; van Soolingen, Dick; Vergnaud, Gilles; Pourcel, Christine

2010-12-01

Since the first discovery of the smooth tubercle (SmTB) bacilli "Mycobacterium canettii" less than 60 isolates have been reported, all but one originating from a limited geographical location, the Horn of Africa. In spite of its rarity, the SmTB lineage deserves special attention. Previous investigations suggested that SmTB isolates represent an ancestral lineage of the Mycobacterium tuberculosis complex (MTBC) and that consequently they might provide essential clues on the origin and evolution of the MTBC. There is evidence that unlike the rest of the MTBC, SmTB strains recombine chromosomal sequences with a yet unknown Mycobacterium species. This behavior contributes to the much larger genetic heterogeneity observed in the SmTB isolates compared to the other members of the MTBC. We have collected 59 SmTB isolates of which 14 were newly recovered since previous reports, and performed extensive phenotypical and genotypical characterization. We take advantage of these investigations to review the current knowledge of "M. canettii". Their characteristics and the apparent lack of human to human transmission are consistent with the previously proposed existence of non-human sources of infection. SmTB strains show remarkably common features together with secondary and taxonomically minor genetic differences such as the presence or absence of the CRISPR (Clustered Regularly Interspersed Palindromic Repeat) locus (usually called Direct Repeat or DR region) or number of IS sequences. Multiple Locus Variable number of tandem repeat Analysis (MLVA) and DR region analyses reveal one predominant clone, one minor clone and a number of more distantly related strains. This suggests that the two most frequent clones may represent successfully emerging lineages. Copyright © 2010 Elsevier B.V. All rights reserved.

The role of the environment in transmission of Dichelobacter nodosus between ewes and their lambs

PubMed Central

Muzafar, Mohd; Calvo-Bado, Leo A.; Green, Laura E.; Smith, Edward M.; Russell, Claire L.; Grogono-Thomas, Rose; Wellington, Elizabeth M.H.

2015-01-01

Dichelobacter nodosus (D. nodosus) is the essential causative agent of footrot in sheep. The current study investigated when D. nodosus was detectable on newborn lambs and possible routes of transmission. Specific qPCR was used to detect and quantify the load of D. nodosus in foot swabs of lambs at birth and 5–13 h post-partum, and their mothers 5–13 h post-partum; and in samples of bedding, pasture, soil and faeces. D. nodosus was not detected on the feet of newborn lambs swabbed at birth, but was detected 5–13 h after birth, once they had stood on bedding containing naturally occurring D. nodosus. Multiple genotypes identified by cloning and sequencing a marker gene, pgrA, and by multi locus variable number tandem repeat analysis (MLVA) of community DNA from swabs on individual feet indicated a mixed population of D. nodosus was present on the feet of both ewes and lambs. There was high variation in pgrA tandem repeat number (between 3 and 21 repeats), and multiple MLVA types. The overall similarity index between the populations on ewes and lambs was 0.45, indicating moderate overlap. Mother offspring pairs shared some alleles but not all, suggesting lambs were infected from sources(s) other than just their mother's feet. We hypothesise that D. nodosus is transferred to the feet of lambs via bedding containing naturally occurring populations of D. nodosus, probably as a result of transfer from the feet of the group of housed ewes. The results support the hypothesis that the environment plays a key role in the transmission of D. nodosus between ewes and lambs. PMID:25953734

Genotype Analysis of Bacillus anthracis Strains Circulating in Bangladesh.

PubMed

Rume, Farzana Islam; Affuso, Alessia; Serrecchia, Luigina; Rondinone, Valeria; Manzulli, Viviana; Campese, Emanuele; Di Taranto, Pietro; Biswas, Paritosh Kumar; Ahsan, Chowdhury Rafiqul; Yasmin, Mahmuda; Fasanella, Antonio; Hugh-Jones, Martin

2016-01-01

In Bangladesh, anthrax, caused by the bacterium Bacillus anthracis, is considered an endemic disease affecting ruminants with sporadic zoonotic occurrences in humans. Due to the lack of knowledge about risks from an incorrect removal of infected carcasses, the disease is not properly monitored, and because of the socio-economic conditions, the situation is under-reported and under-diagnosed. For sensitive species, anthrax represents a fatal outcome with sudden death and sometimes bleeding from natural orifices. The most common source of infection for ruminants is ingestion of spores during grazing in contaminated pastures or through grass and water contaminated with anthrax spores. Domestic cattle, sheep and goats can also become infected through contaminated bone meal (used as feed) originating from anthrax-infected carcasses. The present investigation was conducted to isolate B. anthracis organisms from 169 samples (73 soil, 1 tissue, 4 bone and 91 bone meal samples) collected from 12 different districts of Bangladesh. The sampling was carried out from 2012 to 2015. Twelve samples resulted positive for B. anthracis. Biomolecular analyses were conducted starting from the Canonical Single Nucleotide Polymorphism (CanSNP) to analyze the phylogenetic origin of strains. The analysis of genotype, obtained through the Multiple Locus Variable Number Tandem Repeat Analysis (MLVA) with the analysis of 15 Variable Number Tandem Repeats (VNTR), demonstrated four different genotypes: two of them were previously identified in the district of Sirajganj. The sub-genotyping, conducted with Single Nucleotide Repeats analysis, revealed the presence of eight subgenotypes. The data of the present study concluded that there was no observed correlation between imported cattle feed and anthrax occurrence in Bangladesh and that the remarkable genetic variations of B. anthracis were found in the soil of numerous outbreaks in this country.

Genotype Analysis of Bacillus anthracis Strains Circulating in Bangladesh

PubMed Central

Rume, Farzana Islam; Affuso, Alessia; Serrecchia, Luigina; Rondinone, Valeria; Manzulli, Viviana; Campese, Emanuele; Di Taranto, Pietro; Biswas, Paritosh Kumar; Ahsan, Chowdhury Rafiqul; Yasmin, Mahmuda; Fasanella, Antonio; Hugh-Jones, Martin

2016-01-01

In Bangladesh, anthrax, caused by the bacterium Bacillus anthracis, is considered an endemic disease affecting ruminants with sporadic zoonotic occurrences in humans. Due to the lack of knowledge about risks from an incorrect removal of infected carcasses, the disease is not properly monitored, and because of the socio-economic conditions, the situation is under-reported and under-diagnosed. For sensitive species, anthrax represents a fatal outcome with sudden death and sometimes bleeding from natural orifices. The most common source of infection for ruminants is ingestion of spores during grazing in contaminated pastures or through grass and water contaminated with anthrax spores. Domestic cattle, sheep and goats can also become infected through contaminated bone meal (used as feed) originating from anthrax-infected carcasses. The present investigation was conducted to isolate B. anthracis organisms from 169 samples (73 soil, 1 tissue, 4 bone and 91 bone meal samples) collected from 12 different districts of Bangladesh. The sampling was carried out from 2012 to 2015. Twelve samples resulted positive for B. anthracis. Biomolecular analyses were conducted starting from the Canonical Single Nucleotide Polymorphism (CanSNP) to analyze the phylogenetic origin of strains. The analysis of genotype, obtained through the Multiple Locus Variable Number Tandem Repeat Analysis (MLVA) with the analysis of 15 Variable Number Tandem Repeats (VNTR), demonstrated four different genotypes: two of them were previously identified in the district of Sirajganj. The sub-genotyping, conducted with Single Nucleotide Repeats analysis, revealed the presence of eight subgenotypes. The data of the present study concluded that there was no observed correlation between imported cattle feed and anthrax occurrence in Bangladesh and that the remarkable genetic variations of B. anthracis were found in the soil of numerous outbreaks in this country. PMID:27082248

Population sub-structuring among Trypanosoma evansi stocks.

PubMed

Njiru, Z K; Constantine, C C

2007-10-01

To investigate the population genetic structure of Trypanosoma evansi from domesticated animals, we have analysed 112 stocks from camels, buffaloes, cattle and horses using the tandemly repeated coding sequence (MORF2) and minisatellite markers 292 and cysteine-rich acidic integral membrane protein (CRAM). We recorded a total of six alleles at the MORF2 locus, seven at 292 and 12 at the CRAM loci. Nei's genetic distance showed reduced allelic diversity between buffaloes and cattle stocks (1.2) as compared to the diversity between camels and buffaloes (3.75) and camels and cattle stock (1.69). The mean index of association (IA=0.92) significantly deviated from zero, and the average number of multilocus genotypes (G/N ratio) was 0.21. Twenty-four multilocus genotypes were defined from the combination of alleles at the three loci. The Kenyan sub-populations showed Fst=0.28 and analysis of molecular variance showed significant divergence (22.7%) between the Laikipia, Kulal and Galana regions. The regional and host distribution of multi-locus genotypes significant population differentiation and high Nei's genetic distances suggest existence of genetic sub-structuring within T. evansi stocks while the few multi-locus genotypes and deviation of association index from zero indicate the lack of recombination. In conclusion, this study reveals that some genetic sub-structuring does occur within T. evansi, which has a clonal population structure.

Investigating a tuberculosis cluster among Filipino health care workers in a low-incidence country.

PubMed

Davidson, J A; Fulton, N; Thomas, H L; Lalor, M K; Zenner, D; Brown, T; Murphy, S; Anderson, L F

2018-03-01

Nearly 8% of adult tuberculosis (TB) cases in England, Wales and Northern Ireland (EW&NI) occur among health care workers (HCWs), the majority of whom are from high TB incidence countries. To determine if a TB cluster containing multiple HCWs was due to nosocomial transmission. A cluster of TB cases notified in EW&NI from 2009 to 2014, with indistinguishable 24-locus mycobacterial interspersed repetitive unit-variable number of tandem repeats (MIRU-VNTR) profiles, was identified through routine national cluster review. Cases were investigated to identify epidemiological links, and occupational health (OH) information was collected for HCW cases. To further discriminate strains, typing of eight additional loci was conducted. Of the 53 cases identified, 22 were HCWs. The majority (n = 43), including 21 HCWs, were born in the Philippines. Additional typing split the cluster into three subclusters and seven unique strains. No epidemiological links were identified beyond one household and a common residential area. HCWs in this cluster received no or inadequate OH assessment. The MIRU-VNTR profile of this cluster probably reflects common endemic strains circulating in the Philippines, with reactivation occurring in the UK. Furthermore, 32-locus typing showed that 24-locus MIRU-VNTR failed to distinguish strain diversity. The lack of OH assessment indicates that latent tuberculous infection could have been identified and treated, thereby preventing active cases from occurring.

Genetic and epigenetic variation in 5S ribosomal RNA genes reveals genome dynamics in Arabidopsis thaliana

PubMed Central

Simon, Lauriane; Rabanal, Fernando A; Dubos, Tristan; Oliver, Cecilia; Lauber, Damien; Poulet, Axel; Vogt, Alexander; Mandlbauer, Ariane; Le Goff, Samuel; Sommer, Andreas; Duborjal, Hervé; Tatout, Christophe

2018-01-01

Abstract Organized in tandem repeat arrays in most eukaryotes and transcribed by RNA polymerase III, expression of 5S rRNA genes is under epigenetic control. To unveil mechanisms of transcriptional regulation, we obtained here in depth sequence information on 5S rRNA genes from the Arabidopsis thaliana genome and identified differential enrichment in epigenetic marks between the three 5S rDNA loci situated on chromosomes 3, 4 and 5. We reveal the chromosome 5 locus as the major source of an atypical, long 5S rRNA transcript characteristic of an open chromatin structure. 5S rRNA genes from this locus translocated in the Landsberg erecta ecotype as shown by linkage mapping and chromosome-specific FISH analysis. These variations in 5S rDNA locus organization cause changes in the spatial arrangement of chromosomes in the nucleus. Furthermore, 5S rRNA gene arrangements are highly dynamic with alterations in chromosomal positions through translocations in certain mutants of the RNA-directed DNA methylation pathway and important copy number variations among ecotypes. Finally, variations in 5S rRNA gene sequence, chromatin organization and transcripts indicate differential usage of 5S rDNA loci in distinct ecotypes. We suggest that both the usage of existing and new 5S rDNA loci resulting from translocations may impact neighboring chromatin organization. PMID:29518237

Genetic and epigenetic variation in 5S ribosomal RNA genes reveals genome dynamics in Arabidopsis thaliana.

PubMed

Simon, Lauriane; Rabanal, Fernando A; Dubos, Tristan; Oliver, Cecilia; Lauber, Damien; Poulet, Axel; Vogt, Alexander; Mandlbauer, Ariane; Le Goff, Samuel; Sommer, Andreas; Duborjal, Hervé; Tatout, Christophe; Probst, Aline V

2018-04-06

Organized in tandem repeat arrays in most eukaryotes and transcribed by RNA polymerase III, expression of 5S rRNA genes is under epigenetic control. To unveil mechanisms of transcriptional regulation, we obtained here in depth sequence information on 5S rRNA genes from the Arabidopsis thaliana genome and identified differential enrichment in epigenetic marks between the three 5S rDNA loci situated on chromosomes 3, 4 and 5. We reveal the chromosome 5 locus as the major source of an atypical, long 5S rRNA transcript characteristic of an open chromatin structure. 5S rRNA genes from this locus translocated in the Landsberg erecta ecotype as shown by linkage mapping and chromosome-specific FISH analysis. These variations in 5S rDNA locus organization cause changes in the spatial arrangement of chromosomes in the nucleus. Furthermore, 5S rRNA gene arrangements are highly dynamic with alterations in chromosomal positions through translocations in certain mutants of the RNA-directed DNA methylation pathway and important copy number variations among ecotypes. Finally, variations in 5S rRNA gene sequence, chromatin organization and transcripts indicate differential usage of 5S rDNA loci in distinct ecotypes. We suggest that both the usage of existing and new 5S rDNA loci resulting from translocations may impact neighboring chromatin organization.

[Forensic hematology genetics--paternity testing].

PubMed

Kratzer, A; Bär, W

1997-05-01

In Switzerland paternity investigations are carried out using DNA analysis only since 1991. DNA patterns are inherited and only with the exception of genetically identical twins they are different in everyone and therefore unique to an individual. Hence DNA-systems are an excellent tool to resolve paternity disputes. DNA polymorphisms used for paternity diagnosis are length polymorphisms of the highly polymorphic VNTR loci [variable number of tandem repeats]. The most frequently applied systems are the DNA single locus systems. In addition to the DNA single locus systems the application of PCR (PCR = polymerase chain reaction) based DNA systems has increased particularly in difficult deficiency cases or in cases where only small evidential samples or partially degraded DNA are available. Normally four independent DNA single probes are used to produce a DNA profile from the mother, the child and the alleged father. A child inherits half the DNA patterns from its mother and the other half from its true biological father. If an alleged father doesn't possess the paternal specific DNA pattern in his DNA profile he is excluded from the paternity. In case of non-exclusion the probability for paternity is calculated according to Essen-Möller. When applying four highly polymorphic DNA single locus systems the biostatistical evaluation leads always to W-values exceeding 99.8% [= required value for positive proof of paternity]. DNA analysis is currently the best available method to achieve such effective conclusions in paternity investigations.

A new hybrid approach for MHC genotyping: high-throughput NGS and long read MinION nanopore sequencing, with application to the non-model vertebrate Alpine chamois (Rupicapra rupicapra).

PubMed

Fuselli, S; Baptista, R P; Panziera, A; Magi, A; Guglielmi, S; Tonin, R; Benazzo, A; Bauzer, L G; Mazzoni, C J; Bertorelle, G

2018-03-24

The major histocompatibility complex (MHC) acts as an interface between the immune system and infectious diseases. Accurate characterization and genotyping of the extremely variable MHC loci are challenging especially without a reference sequence. We designed a combination of long-range PCR, Illumina short-reads, and Oxford Nanopore MinION long-reads approaches to capture the genetic variation of the MHC II DRB locus in an Italian population of the Alpine chamois (Rupicapra rupicapra). We utilized long-range PCR to generate a 9 Kb fragment of the DRB locus. Amplicons from six different individuals were fragmented, tagged, and simultaneously sequenced with Illumina MiSeq. One of these amplicons was sequenced with the MinION device, which produced long reads covering the entire amplified fragment. A pipeline that combines short and long reads resolved several short tandem repeats and homopolymers and produced a de novo reference, which was then used to map and genotype the short reads from all individuals. The assembled DRB locus showed a high level of polymorphism and the presence of a recombination breakpoint. Our results suggest that an amplicon-based NGS approach coupled with single-molecule MinION nanopore sequencing can efficiently achieve both the assembly and the genotyping of complex genomic regions in multiple individuals in the absence of a reference sequence.

GENETIC DIVERSITY OF TYPHA LATIFOLIA (TYPHACEAE) AND THE IMPACT OF POLLUTANTS EXAMINED WITH TANDEM-REPETITIVE DNA PROBES

EPA Science Inventory

Genetic diversity at variable-number-tandem-repeat (VNTR) loci was examined in the common cattail, Typha latifolia (Typhaceae), using three synthetic DNA probes composed of tandemly repeated "core" sequences (GACA, GATA, and GCAC). The principal objectives of this investigation w...

Report for the NGFA-5 project.

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

Jaing, C; Jackson, P; Thissen, J

The objective of this project is to provide DHS a comprehensive evaluation of the current genomic technologies including genotyping, TaqMan PCR, multiple locus variable tandem repeat analysis (MLVA), microarray and high-throughput DNA sequencing in the analysis of biothreat agents from complex environmental samples. To effectively compare the sensitivity and specificity of the different genomic technologies, we used SNP TaqMan PCR, MLVA, microarray and high-throughput illumine and 454 sequencing to test various strains from B. anthracis, B. thuringiensis, BioWatch aerosol filter extracts or soil samples that were spiked with B. anthracis, and samples that were previously collected during DHS and EPAmore » environmental release exercises that were known to contain B. thuringiensis spores. The results of all the samples against the various assays are discussed in this report.« less

An examination of the origin and evolution of additional tandem repeats in the mitochondrial DNA control region of Japanese sika deer (Cervus Nippon).

PubMed

Ba, Hengxing; Wu, Lang; Liu, Zongyue; Li, Chunyi

2016-01-01

Tandem repeat units are only detected in the left domain of the mitochondrial DNA control region in sika deer. Previous studies showed that Japanese sika deer have more tandem repeat units than its cousins from the Asian continent and Taiwan, which often have only three repeat units. To determine the origin and evolution of these additional repeat units in Japanese sika deer, we obtained the sequence of repeat units from an expanded dataset of the control region from all sika deer lineages. The functional constraint is inferred to act on the first repeat unit because this repeat has the least sequence divergence in comparison to the other units. Based on slipped-strand mispairing mechanisms, the illegitimate elongation model could account for the addition or deletion of these additional repeat units in the Japanese sika deer population. We also report that these additional repeat units could be occurring in the internal positions of tandem repeat regions, possibly via coupling with a homogenization mechanism within and among these lineages. Moreover, the increased number of repeat units in the Japanese sika deer population could reflect a balance between mutation and selection, as well as genetic drift.

A novel species-specific tandem repeat DNA family from Sinapis arvensis: detection of telomere-like sequences.

PubMed

Kapila, R; Das, S; Srivastava, P S; Lakshmikumaran, M

1996-08-01

DNA sequences representing a tandemly repeated DNA family of the Sinapis arvensis genome were cloned and characterized. The 700-bp tandem repeat family is represented by two clones, pSA35 and pSA52, which are 697 and 709 bp in length, respectively. Dot matrix analysis of the sequences indicates the presence of repeated elements within each monomeric unit. Sequence analysis of the repetitive region of clones pSA35 and pSA52 shows that there are several copies of a 7-bp repeat element organized in tandem. The consensus sequence of this repeat element is 5'-TTTAGGG-3'. These elements are highly mutated and the difference in length between the two clones is due to different copy numbers of these elements. The repetitive region of clone pSA35 has 26 copies of the element TTTAGGG, whereas clone pSA52 has 28 copies. The repetitive region in both clones is flanked on either side by inverted repeats that may be footprints of a transposition event. Sequence comparison indicates that the element TTTAGGG is identical to telomeric repeats present in Arabidopsis, maize, tomato, and other plants. However, Bal31 digestion kinetics indicates non-telomeric localization of the 700-bp tandem repeats. The clones represent a novel repeat family as (i) they contain telomere-like motifs as subrepeats within each unit; and (ii) they do not hybridize to related crucifers and are species-specific in nature.

5meCpG epigenetic marks neighboring a primate-conserved core promoter short tandem repeat indicate X-chromosome inactivation.

PubMed

Machado, Filipe Brum; Machado, Fabricio Brum; Faria, Milena Amendro; Lovatel, Viviane Lamim; Alves da Silva, Antonio Francisco; Radic, Claudia Pamela; De Brasi, Carlos Daniel; Rios, Álvaro Fabricio Lopes; de Sousa Lopes, Susana Marina Chuva; da Silveira, Leonardo Serafim; Ruiz-Miranda, Carlos Ramon; Ramos, Ester Silveira; Medina-Acosta, Enrique

2014-01-01

X-chromosome inactivation (XCI) is the epigenetic transcriptional silencing of an X-chromosome during the early stages of embryonic development in female eutherian mammals. XCI assures monoallelic expression in each cell and compensation for dosage-sensitive X-linked genes between females (XX) and males (XY). DNA methylation at the carbon-5 position of the cytosine pyrimidine ring in the context of a CpG dinucleotide sequence (5meCpG) in promoter regions is a key epigenetic marker for transcriptional gene silencing. Using computational analysis, we revealed an extragenic tandem GAAA repeat 230-bp from the landmark CpG island of the human X-linked retinitis pigmentosa 2 RP2 promoter whose 5meCpG status correlates with XCI. We used this RP2 onshore tandem GAAA repeat to develop an allele-specific 5meCpG-based PCR assay that is highly concordant with the human androgen receptor (AR) exonic tandem CAG repeat-based standard HUMARA assay in discriminating active (Xa) from inactive (Xi) X-chromosomes. The RP2 onshore tandem GAAA repeat contains neutral features that are lacking in the AR disease-linked tandem CAG repeat, is highly polymorphic (heterozygosity rates approximately 0.8) and shows minimal variation in the Xa/Xi ratio. The combined informativeness of RP2/AR is approximately 0.97, and this assay excels at determining the 5meCpG status of alleles at the Xp (RP2) and Xq (AR) chromosome arms in a single reaction. These findings are relevant and directly translatable to nonhuman primate models of XCI in which the AR CAG-repeat is monomorphic. We conducted the RP2 onshore tandem GAAA repeat assay in the naturally occurring chimeric New World monkey marmoset (Callitrichidae) and found it to be informative. The RP2 onshore tandem GAAA repeat will facilitate studies on the variable phenotypic expression of dominant and recessive X-linked diseases, epigenetic changes in twins, the physiology of aging hematopoiesis, the pathogenesis of age-related hematopoietic malignancies and the clonality of cancers in human and nonhuman primates.

5meCpG Epigenetic Marks Neighboring a Primate-Conserved Core Promoter Short Tandem Repeat Indicate X-Chromosome Inactivation

PubMed Central

Machado, Filipe Brum; Machado, Fabricio Brum; Faria, Milena Amendro; Lovatel, Viviane Lamim; Alves da Silva, Antonio Francisco; Radic, Claudia Pamela; De Brasi, Carlos Daniel; Rios, Álvaro Fabricio Lopes; de Sousa Lopes, Susana Marina Chuva; da Silveira, Leonardo Serafim; Ruiz-Miranda, Carlos Ramon; Ramos, Ester Silveira; Medina-Acosta, Enrique

2014-01-01

X-chromosome inactivation (XCI) is the epigenetic transcriptional silencing of an X-chromosome during the early stages of embryonic development in female eutherian mammals. XCI assures monoallelic expression in each cell and compensation for dosage-sensitive X-linked genes between females (XX) and males (XY). DNA methylation at the carbon-5 position of the cytosine pyrimidine ring in the context of a CpG dinucleotide sequence (5meCpG) in promoter regions is a key epigenetic marker for transcriptional gene silencing. Using computational analysis, we revealed an extragenic tandem GAAA repeat 230-bp from the landmark CpG island of the human X-linked retinitis pigmentosa 2 RP2 promoter whose 5meCpG status correlates with XCI. We used this RP2 onshore tandem GAAA repeat to develop an allele-specific 5meCpG-based PCR assay that is highly concordant with the human androgen receptor (AR) exonic tandem CAG repeat-based standard HUMARA assay in discriminating active (Xa) from inactive (Xi) X-chromosomes. The RP2 onshore tandem GAAA repeat contains neutral features that are lacking in the AR disease-linked tandem CAG repeat, is highly polymorphic (heterozygosity rates approximately 0.8) and shows minimal variation in the Xa/Xi ratio. The combined informativeness of RP2/AR is approximately 0.97, and this assay excels at determining the 5meCpG status of alleles at the Xp (RP2) and Xq (AR) chromosome arms in a single reaction. These findings are relevant and directly translatable to nonhuman primate models of XCI in which the AR CAG-repeat is monomorphic. We conducted the RP2 onshore tandem GAAA repeat assay in the naturally occurring chimeric New World monkey marmoset (Callitrichidae) and found it to be informative. The RP2 onshore tandem GAAA repeat will facilitate studies on the variable phenotypic expression of dominant and recessive X-linked diseases, epigenetic changes in twins, the physiology of aging hematopoiesis, the pathogenesis of age-related hematopoietic malignancies and the clonality of cancers in human and nonhuman primates. PMID:25078280

Triplet repeat expansion at the FRAXE locus and x-linked mild mental handicap

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

Knight, S.J.L.; Hirst, M.C.; Flannery, A.V.

1994-07-01

The authors have recently shown that the expression of the FRAXE fragile site in Xq28 is associated with expansion of a GCC trinucleotide repeat. In the families studied, FRAXE expression is also associated with mild mental handicap. Here they present data on families that previously had been diagnosed as having the fragile X syndrome but that later were found to be negative for trinucleotide repeat expansion at the FRAXA locus. In these families they demonstrate the presence of a GCC trinucleotide repeat expansion at the FRAXE locus. Studies of the FRAXE locus of normal individuals show that they have 6-25more » copies of the repeat, whereas affected individuals have >200 copies. As in the fragile X syndrome, the amplified CpG residues are methylated in affected males. 19 refs., 4 figs., 1 tab.« less

First Worldwide Proficiency Study on Variable-Number Tandem-Repeat Typing of Mycobacterium tuberculosis Complex Strains

PubMed Central

de Beer, Jessica L.; Kremer, Kristin; Ködmön, Csaba; Supply, Philip

2012-01-01

Although variable-number tandem-repeat (VNTR) typing has gained recognition as the new standard for the DNA fingerprinting of Mycobacterium tuberculosis complex (MTBC) isolates, external quality control programs have not yet been developed. Therefore, we organized the first multicenter proficiency study on 24-locus VNTR typing. Sets of 30 DNAs of MTBC strains, including 10 duplicate DNA samples, were distributed among 37 participating laboratories in 30 different countries worldwide. Twenty-four laboratories used an in-house-adapted method with fragment sizing by gel electrophoresis or an automated DNA analyzer, nine laboratories used a commercially available kit, and four laboratories used other methods. The intra- and interlaboratory reproducibilities of VNTR typing varied from 0% to 100%, with averages of 72% and 60%, respectively. Twenty of the 37 laboratories failed to amplify particular VNTR loci; if these missing results were ignored, the number of laboratories with 100% interlaboratory reproducibility increased from 1 to 5. The average interlaboratory reproducibility of VNTR typing using a commercial kit was better (88%) than that of in-house-adapted methods using a DNA analyzer (70%) or gel electrophoresis (50%). Eleven laboratories using in-house-adapted manual typing or automated typing scored inter- and intralaboratory reproducibilities of 80% or higher, which suggests that these approaches can be used in a reliable way. In conclusion, this first multicenter study has documented the worldwide quality of VNTR typing of MTBC strains and highlights the importance of international quality control to improve genotyping in the future. PMID:22170917

Linkage study of nonsyndromic cleft lip with or without cleft palate using candidate genes and mapped polymorphic markers

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

Stein, J.D.; Nelson, L.D.; Conner, B.J.

1994-09-01

Nonsyndromic cleft lip with or without cleft palate (CL(P)) involves fusion or growth failure of facial primordia during development. Complex segregation analysis of clefting populations suggest that an autosomal dominant gene may play a role in this common craniofacial disorder. We have ascertained 16 multigenerational families with CL(P) and tested linkage to 29 candidate genes and 139 mapped short tandem repeat markers. The candidate genes were selected based on their expression in craniofacial development or were identified through murine models. These include: TGF{alpha}, TGF{beta}1, TGF{beta}2, TGF{beta}3, EGF, EGFR, GRAS, cMyc, FGFR, Jun, JunB, PDFG{alpha}, PDGF{beta}, IGF2R, GCR Hox7, Hox8, Hox2B,more » twirler, 5 collagen and 3 extracellular matrix genes. Linkage was tested assuming an autosomal dominant model with sex-specific decreased penetrance. Linkage to all of the candidate loci was excluded in 11 families. RARA was tested and was not informative. However, haplotype analysis of markers flanking RARA on 17q allowed exclusion of this candidate locus. We have previously excluded linkage to 61 STR markers in 11 families. Seventy-eight mapped short tandem repeat markers have recently been tested in 16 families and 30 have been excluded. The remaining are being analyzed and an exclusion map is being developed based on the entire study results.« less

Application of Short Tandem Repeat markers in diagnosis of chromosomal aneuploidies and forensic DNA investigation in Pakistan.

PubMed

Chishti, Hafsah Muhammad; Ansar, Muhammad; Ajmal, Muhammad; Hameed, Abdul

2014-09-15

Short Tandem Repeat (STR) genetic markers hold great potential in forensic investigations, molecular diagnostics and molecular genetics research. AmpFlSTR® Identifiler™ PCR amplification kit is a multiplex system for co-amplification of 15 STR markers used worldwide in forensic investigations. This study attempts to assess forensic validity of these STRs in Pakistani population and to investigate its applicability in quick and simultaneous diagnosis and tracing parental source of common chromosomal aneuploidies. Samples from 554 healthy Pakistani individuals from 5 different ethnicities were analyzed for forensic parameters using Identifiler STRs and 74 patients' samples with different aneuploidies were evaluated for diagnostic strengths of these markers. All STRs hold sufficient forensic applicability in Pakistani population with paternity index between 1.5 and 3.5, polymorphic information content from 0.63 to 0.87 and discrimination power ≥0.9 (except TPOX locus). Variation from Hardy-Weinberg equilibrium was observed at some loci reflecting selective breeding and intermarriages trend in Pakistan. Among aneuploidic samples, all trisomies were precisely detectable while aneuploidies involving sex chromosomes or missing chromosomes were not clearly detectable using Identifiler STRs. Parental origin of aneuploidy was traceable in 92.54% patients. The studied STR markers are valuable tools for forensic application in Pakistan and utilizable for quick and simultaneous identification of some common trisomic conditions. Adding more sex chromosome specific STR markers can immensely increase the diagnostic and forensic potential of this system. Copyright © 2014 Elsevier B.V. All rights reserved.

Genetic analysis results of mature cystic teratomas of the ovary in Taiwan disagree with the previous origin theory of this tumor.

PubMed

Wang, Wen-Chung; Lai, Yen-Chein

2016-06-01

The most accepted theory regarding mature cystic teratomas of the ovary is that they are of parthenogenetic origin from oocyte after the completion of first division. Our previous study demonstrated that the origin of mature cystic teratoma of the uterus is not related to the parthenogenetic process, but is most likely pluripotential stem cell or primordial germ cell before meiosis I. Further studies are needed to clarify the origin of benign mature cystic teratomas of the ovary in Taiwan. In the present study, we investigated the DNA profiles of 9 mature cystic teratomas of the ovary using short tandem repeat analysis with AmpFLSTR SGM Plus, Profiler PCR amplification kits. The methylation statuses of the HhaI sites in the SNRPN, H19DMR, and KvDMR regions were determined on methylation-sensitive multiplex ligation-dependent probe amplification analysis. DNA profiling data from the 9 mature cystic teratomas of the ovary excluded parthenogenetic origin, as most of the 15 short tandem repeat loci were heterozygous on genotyping. There were varying degrees of hypermethylation of SNRPN gene and KvDMR locus in the presence of maternal uniparental disomy in all 9 mature cystic teratomas of the ovary. In light of these results, we further postulated that the origin of these mature cystic teratomas of the ovary is oogonia or primary oocyte before germinal vesicle stage failure of meiosis I. Copyright © 2016 Elsevier Inc. All rights reserved.

Genetic analysis of haplotype data for 23 Y-chromosome short tandem repeat loci in the Turkish population recently settled in Sarajevo, Bosnia and Herzegovina.

PubMed

Dogan, Serkan; Primorac, Dragan; Marjanović, Damir

2014-10-01

To explore the distribution and polymorphisms of 23 short tandem repeat (STR) loci on the Y chromosome in the Turkish population recently settled in Sarajevo, Bosnia and Herzegovina and to investigate its genetic relationships with the homeland Turkish population and neighboring populations. This study included 100 healthy unrelated male individuals from the Turkish population living in Sarajevo. Buccal swab samples were collected as a DNA source. Genomic DNA was extracted using the salting out method and amplification was performed using PowerPlex Y 23 amplification kit. The studied population was compared to other populations using pairwise genetic distances, which were represented with a multi-dimensional scaling plot. Haplotype and allele frequencies of the sample population were calculated and the results showed that all 100 samples had unique haplotypes. The most polymorphic locus was DYS458, and the least polymorphic DYS391. The observed haplotype diversity was 1.0000 ± 0.0014, with a discrimination capacity of 1.00 and the match probability of 0.01. Rst values showed that our sample population was closely related in both dimensions to the Lebanese and Iraqi populations, while it was more distant from Bosnian, Croatian, and Macedonian populations. Turkish population residing in Sarajevo could be observed as a representative Turkish population, since our results were consistent with those previously published for the homeland Turkish population. Also, this study once again proved that geographically close populations were genetically more related to each other.

Ca2+-stabilized adhesin helps an Antarctic bacterium reach out and bind ice.

PubMed

Vance, Tyler D R; Olijve, Luuk L C; Campbell, Robert L; Voets, Ilja K; Davies, Peter L; Guo, Shuaiqi

2014-07-04

The large size of a 1.5-MDa ice-binding adhesin [MpAFP (Marinomonas primoryensis antifreeze protein)] from an Antarctic Gram-negative bacterium, M. primoryensis, is mainly due to its highly repetitive RII (Region II). MpAFP_RII contains roughly 120 tandem copies of an identical 104-residue repeat. We have previously determined that a single RII repeat folds as a Ca2+-dependent immunoglobulin-like domain. Here, we solved the crystal structure of RII tetra-tandemer (four tandem RII repeats) to a resolution of 1.8 Å. The RII tetra-tandemer reveals an extended (~190-Å × ~25-Å), rod-like structure with four RII-repeats aligned in series with each other. The inter-repeat regions of the RII tetra-tandemer are strengthened by Ca2+ bound to acidic residues. SAXS (small-angle X-ray scattering) profiles indicate the RII tetra-tandemer is significantly rigidified upon Ca2+ binding, and that the protein's solution structure is in excellent agreement with its crystal structure. We hypothesize that >600 Ca2+ help rigidify the chain of ~120 104-residue repeats to form a ~0.6 μm rod-like structure in order to project the ice-binding domain of MpAFP away from the bacterial cell surface. The proposed extender role of RII can help the strictly aerobic, motile bacterium bind ice in the upper reaches of the Antarctic lake where oxygen and nutrients are most abundant. Ca2+-induced rigidity of tandem Ig-like repeats in large adhesins might be a general mechanism used by bacteria to bind to their substrates and help colonize specific niches.

Rational design of alpha-helical tandem repeat proteins with closed architectures

PubMed Central

Doyle, Lindsey; Hallinan, Jazmine; Bolduc, Jill; Parmeggiani, Fabio; Baker, David; Stoddard, Barry L.; Bradley, Philip

2015-01-01

Tandem repeat proteins, which are formed by repetition of modular units of protein sequence and structure, play important biological roles as macromolecular binding and scaffolding domains, enzymes, and building blocks for the assembly of fibrous materials1,2. The modular nature of repeat proteins enables the rapid construction and diversification of extended binding surfaces by duplication and recombination of simple building blocks3,4. The overall architecture of tandem repeat protein structures – which is dictated by the internal geometry and local packing of the repeat building blocks – is highly diverse, ranging from extended, super-helical folds that bind peptide, DNA, and RNA partners5–9, to closed and compact conformations with internal cavities suitable for small molecule binding and catalysis10. Here we report the development and validation of computational methods for de novo design of tandem repeat protein architectures driven purely by geometric criteria defining the inter-repeat geometry, without reference to the sequences and structures of existing repeat protein families. We have applied these methods to design a series of closed alpha-solenoid11 repeat structures (alpha-toroids) in which the inter-repeat packing geometry is constrained so as to juxtapose the N- and C-termini; several of these designed structures have been validated by X-ray crystallography. Unlike previous approaches to tandem repeat protein engineering12–20, our design procedure does not rely on template sequence or structural information taken from natural repeat proteins and hence can produce structures unlike those seen in nature. As an example, we have successfully designed and validated closed alpha-solenoid repeats with a left-handed helical architecture that – to our knowledge – is not yet present in the protein structure database21. PMID:26675735

Typing Clostridium difficile strains based on tandem repeat sequences

PubMed Central

2009-01-01

Background Genotyping of epidemic Clostridium difficile strains is necessary to track their emergence and spread. Portability of genotyping data is desirable to facilitate inter-laboratory comparisons and epidemiological studies. Results This report presents results from a systematic screen for variation in repetitive DNA in the genome of C. difficile. We describe two tandem repeat loci, designated 'TR6' and 'TR10', which display extensive sequence variation that may be useful for sequence-based strain typing. Based on an investigation of 154 C. difficile isolates comprising 75 ribotypes, tandem repeat sequencing demonstrated excellent concordance with widely used PCR ribotyping and equal discriminatory power. Moreover, tandem repeat sequences enabled the reconstruction of the isolates' largely clonal population structure and evolutionary history. Conclusion We conclude that sequence analysis of the two repetitive loci introduced here may be highly useful for routine typing of C. difficile. Tandem repeat sequence typing resolves phylogenetic diversity to a level equivalent to PCR ribotypes. DNA sequences may be stored in databases accessible over the internet, obviating the need for the exchange of reference strains. PMID:19133124

GENETIC VARIATION IN RED RASPBERRIES (RUBUS IDAEUS L.; ROSACEAE) FROM SITES DIFFERING IN ORGANIC POLLUTANTS COMPARED WITH SYNTHETIC TANDEM REPEAT DNA PROBES

EPA Science Inventory

Two synthetic tandem repetitive DNA probes were used to compare genetic variation at variable-number-tandem-repeat (VNTR) loci among Rubus idaeus L. var. strigosus (Michx.) Maxim. (Rosaceae) individuals sampled at eight sites contaminated by pollutants (N = 39) and eight adjacent...

Variable Number Of Tandem Repeats (VNTR) and its application in bacterial epidemiology.

PubMed

Ramazanzadeh, Rashid; McNerney, Ruth

2007-08-15

Molecular epidemiology is the using of molecular techniques to study bacterial distribution in human populations. Recently molecular epidemiologist benefit from several techniques such as Variable Number Tandem Repeat (VNTR) typing method to typing bacterial strains. Variable Number Tandem Repeat (VNTR) typing is a tool for genotyping and provides data in a simple and numeric format based on the number of repetitive sequences. VNTR for first time identified in M. tuberculosis as Mycobacterial Interspersed Repeat Units (MIRUs). General terms of VNTR have now been reported in Bacillus anthracis, Legionella pneumophila, Pseudomonas aeruginosa, Salmonella enterica and Escherichia coli O157.

Conservation of human chromosome 13 polymorphic microsatellite (CA){sub n} repeats in chimpanzees

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

Deka, R.; Shriver, M.D.; Yu, L.M.

Tandemly repeated (dC-dA){sub n} {center_dot} (dG-dT){sub n} sequences occur abundantly and are found in most eukaryotic genomes. To investigate the level of conservation of these repeat sequences in nonhuman primates, the authors have analyzed seven human chromosome 13 dinucleotide (CA){sub n} repeat loci in chimpanzees by DNA amplification using primers designed for analysis of human loci. Comparable levels of polymorphism at these loci in the two species, revealed by the number of alleles, heterozygosity, and allele sizes, suggest that the (CA){sub n} repeat arrays and their genomic locations are highly conserved. Even though the proportion of shared alleles between themore » two species varies enormously and the modal alleles are not the same, allelic lengths at each locus in the chimpanzees are detected within the bounds of the allele size range observed in humans. A similar observation has been noted in a limited number of gorillas and orangutans. Using a new measure of genetic distance that takes into account the size of alleles, they have compared the genetic distance between humans and chimpanzees. The genetic distance between these two species was found to be ninefold smaller than expected assuming there is no selection or mutational bias toward retention of (CA){sub n} repeat arrays. These findings suggest a functional significance for these microsatellite loci. 34 refs., 1 fig., 2 tabs.« less

Dog leukocyte antigen class II-associated genetic risk testing for immune disorders of dogs: simplified approaches using Pug dog necrotizing meningoencephalitis as a model.

PubMed

Pedersen, Niels; Liu, Hongwei; Millon, Lee; Greer, Kimberly

2011-01-01

A significantly increased risk for a number of autoimmune and infectious diseases in purebred and mixed-breed dogs has been associated with certain alleles or allele combinations of the dog leukocyte antigen (DLA) class II complex containing the DRB1, DQA1, and DQB1 genes. The exact level of risk depends on the specific disease, the alleles in question, and whether alleles exist in a homozygous or heterozygous state. The gold standard for identifying high-risk alleles and their zygosity has involved direct sequencing of the exon 2 regions of each of the 3 genes. However, sequencing and identification of specific alleles at each of the 3 loci are relatively expensive and sequencing techniques are not ideal for additional parentage or identity determination. However, it is often possible to get the same information from sequencing only 1 gene given the small number of possible alleles at each locus in purebred dogs, extensive homozygosity, and tendency for disease-causing alleles at each of the 3 loci to be strongly linked to each other into haplotypes. Therefore, genetic testing in purebred dogs with immune diseases can be often simplified by sequencing alleles at 1 rather than 3 loci. Further simplification of genetic tests for canine immune diseases can be achieved by the use of alternative genetic markers in the DLA class II region that are also strongly linked with the disease genotype. These markers consist of either simple tandem repeats or single nucleotide polymorphisms that are also in strong linkage with specific DLA class II genotypes and/or haplotypes. The current study uses necrotizing meningoencephalitis of Pug dogs as a paradigm to assess simple alternative genetic tests for disease risk. It was possible to attain identical necrotizing meningoencephalitis risk assessments to 3-locus DLA class II sequencing by sequencing only the DQB1 gene, using 3 DLA class II-linked simple tandem repeat markers, or with a small single nucleotide polymorphism array designed to identify breed-specific DQB1 alleles.

Multiple-locus variable-number tandem repeat analysis potentially reveals the existence of two groups of Anaplasma phagocytophilum circulating in cattle in France with different wild reservoirs.

PubMed

Dugat, Thibaud; Zanella, Gina; Véran, Luc; Lesage, Céline; Girault, Guillaume; Durand, Benoît; Lagrée, Anne-Claire; Boulouis, Henri-Jean; Haddad, Nadia

2016-11-22

Anaplasma phagocytophilum is the causative agent of tick-borne fever, a disease with high economic impact for domestic ruminants in Europe. Epidemiological cycles of this species are complex, and involve different ecotypes circulating in various host species. To date, these epidemiological cycles are poorly understood, especially in Europe, as European reservoir hosts (i.e. vertebrate hosts enabling long-term maintenance of the bacterium in the ecosystem), of the bacterium have not yet been clearly identified. In this study, our objective was to explore the presence, the prevalence, and the genetic diversity of A. phagocytophilum in wild animals, in order to better understand their implications as reservoir hosts of this pathogen. The spleens of 101 wild animals were collected from central France and tested for the presence of A. phagocytophilum DNA by msp2 qPCR. Positive samples were then typed by multi-locus variable-number tandem repeat (VNTR) analysis (MLVA), and compared to 179 previously typed A. phagocytophilum samples. Anaplasma phagocytophilum DNA was detected in 82/101 (81.2%) animals including 48/49 red deer (98%), 20/21 roe deer (95.2%), 13/29 wild boars (44.8%), and 1/1 red fox. MLVA enabled the discrimination of two A. phagocytophilum groups: group A contained the majority of A. phagocytophilum from red deer and two thirds of those from cattle, while group B included a human strain and variants from diverse animal species, i.e. sheep, dogs, a horse, the majority of variants from roe deer, and the remaining variants from cattle and red deer. Our results suggest that red deer and roe deer are promising A. phagocytophilum reservoir host candidates. Moreover, we also showed that A. phagocytophilum potentially circulates in at least two epidemiological cycles in French cattle. The first cycle may involve red deer as reservoir hosts and cattle as accidental hosts for Group A strains, whereas the second cycle could involve roe deer as reservoir hosts and at least domestic ruminants, dogs, horses, and humans as accidental hosts for Group B strains.

Prospective Genotyping of Mycobacterium tuberculosis from Fresh Clinical Samples

PubMed Central

Bidovec-Stojkovič, Urška; Seme, Katja; Žolnir-Dovč, Manca; Supply, Philip

2014-01-01

Shorter time-to-result is key for improving molecular-guided epidemiological investigation of tuberculosis (TB) cases. We performed a prospective study to evaluate the use of standardized MIRU-VNTR (mycobacterial interspersed repetitive-unit-variable-number tandem-repeat) typing of Mycobacterium tuberculosis directly on 79 fresh clinical samples from 26 TB patients consecutively enrolled over a 17-month period. Overall, complete 24-locus types were obtained for 18 out of the 26 (69.2%) patients and 14 of the 16 grade 3+ and grade 2+ samples (87.5%). The degree of completion of the genotypes obtained significantly correlated with smear microscopy grade both for 26 first samples (p = 0.0003) and for 53 follow-up samples (p = 0.002). For 20 of the 26 patients for whom complete or even incomplete M. tuberculosis isolate genotypes were obtained, typing applied to the clinical samples allowed the same unambiguous conclusions regarding case clustering or uniqueness as those that could have been drawn based on the corresponding cultured isolates. Standard 24 locus MIRU-VNTR typing of M. tuberculosis can be applied directly to fresh clinical samples, with typeability depending on the bacterial load in the sample. PMID:25313883

Slipped-strand mispairing at noncontiguous repeats in Poecilia reticulata: a model for minisatellite birth.

PubMed Central

Taylor, J S; Breden, F

2000-01-01

The standard slipped-strand mispairing (SSM) model for the formation of variable number tandem repeats (VNTRs) proposes that a few tandem repeats, produced by chance mutations, provide the "raw material" for VNTR expansion. However, this model is unlikely to explain the formation of VNTRs with long motifs (e.g., minisatellites), because the likelihood of a tandem repeat forming by chance decreases rapidly as the length of the repeat motif increases. Phylogenetic reconstruction of the birth of a mitochondrial (mt) DNA minisatellite in guppies suggests that VNTRs with long motifs can form as a consequence of SSM at noncontiguous repeats. VNTRs formed in this manner have motifs longer than the noncontiguous repeat originally formed by chance and are flanked by one unit of the original, noncontiguous repeat. SSM at noncontiguous repeats can therefore explain the birth of VNTRs with long motifs and the "imperfect" or "short direct" repeats frequently observed adjacent to both mtDNA and nuclear VNTRs. PMID:10880490

Clonal structure in Ichthyobacterium seriolicida, the causative agent of bacterial haemolytic jaundice in yellowtail, Seriola quinqueradiata, inferred from molecular epidemiological analysis.

PubMed

Matsuyama, T; Fukuda, Y; Sakai, T; Tanimoto, N; Nakanishi, M; Nakamura, Y; Takano, T; Nakayasu, C

2017-08-01

Bacterial haemolytic jaundice caused by Ichthyobacterium seriolicida has been responsible for mortality in farmed yellowtail, Seriola quinqueradiata, in western Japan since the 1980s. In this study, polymorphic analysis of I. seriolicida was performed using three molecular methods: amplified fragment length polymorphism (AFLP) analysis, multilocus sequence typing (MLST) and multiple-locus variable-number tandem repeat analysis (MLVA). Twenty-eight isolates were analysed using AFLP, while 31 isolates were examined by MLST and MLVA. No polymorphisms were identified by AFLP analysis using EcoRI and MseI, or by MLST of internal fragments of eight housekeeping genes. However, MLVA revealed variation in repeat numbers of three elements, allowing separation of the isolates into 16 sequence types. The unweighted pair group method using arithmetic averages cluster analysis of the MLVA data identified four major clusters, and all isolates belonged to clonal complexes. It is likely that I. seriolicida populations share a common ancestor, which may be a recently introduced strain. © 2016 John Wiley & Sons Ltd.

Genetic variability in maned wolf based on heterologous short-tandem repeat markers from domestic dog.

PubMed

Salim, D C; Akimoto, A A; Carvalho, C B; Oliveira, S F; Grisolia, C K; Moreira, J R; Klautau-Guimarães, M N

2007-06-20

The maned wolf (Chrysocyon brachyurus) is the largest South American canid. Habitat loss and fragmentation, due to agricultural expansion and predatory hunting, are the main threats to this species. It is included in the official list of threatened wildlife species in Brazil, and is also protected by IUCN and CITES. Highly variable genetic markers such as microsatellites have the potential to resolve genetic relationships at all levels of the population structure (among individuals, demes or metapopulations) and also to identify the evolutionary unit for strategies for the conservation of the species. Tests were carried out to verify whether a class of highly polymorphic tetranucleotide repeats described for the domestic dog effectively amplifies DNA in the maned wolf. All five loci studied were amplified; however, one of these, was shown to be monomorphic in 69 maned wolf samples. The average allele number and estimated heterozygosity per polymorphic locus were 4.3 and 67%, respectively. The genetic variability found for this species, which is considered threatened with extinction, showed similar results when compared to studies of other canids.

Diverse Genotypes of Yersinia pestis Caused Plague in Madagascar in 2007.

PubMed

Riehm, Julia M; Projahn, Michaela; Vogler, Amy J; Rajerison, Minoaerisoa; Andersen, Genevieve; Hall, Carina M; Zimmermann, Thomas; Soanandrasana, Rahelinirina; Andrianaivoarimanana, Voahangy; Straubinger, Reinhard K; Nottingham, Roxanne; Keim, Paul; Wagner, David M; Scholz, Holger C

2015-06-01

Yersinia pestis is the causative agent of human plague and is endemic in various African, Asian and American countries. In Madagascar, the disease represents a significant public health problem with hundreds of human cases a year. Unfortunately, poor infrastructure makes outbreak investigations challenging. DNA was extracted directly from 93 clinical samples from patients with a clinical diagnosis of plague in Madagascar in 2007. The extracted DNAs were then genotyped using three molecular genotyping methods, including, single nucleotide polymorphism (SNP) typing, multi-locus variable-number tandem repeat analysis (MLVA), and Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) analysis. These methods provided increasing resolution, respectively. The results of these analyses revealed that, in 2007, ten molecular groups, two newly described here and eight previously identified, were responsible for causing human plague in geographically distinct areas of Madagascar. Plague in Madagascar is caused by numerous distinct types of Y. pestis. Genotyping method choice should be based upon the discriminatory power needed, expense, and available data for any desired comparisons. We conclude that genotyping should be a standard tool used in epidemiological investigations of plague outbreaks.

The genome and variation of Bacillus anthracis

PubMed Central

Keim, Paul; Gruendike, Jeffrey M.; Klevytska, Alexandra M.; Schupp, James M.; Challacombe, Jean; Okinaka, Richard

2009-01-01

The Bacillus anthracis genome reflects its close genetic ties to B. cereus and B. thuringiensis but has been shaped by its own unique biology and evolutionary forces. The genome is comprised of a chromosome and two large virulence plasmids, pXO1 and pXO2. The chromosome is mostly co-linear among B. anthracis strains and even with the closest near neighbor strains. An exception to this pattern has been observed in a large inversion in an attenuated strain suggesting that chromosome co-linearity is important to the natural biology of this pathogen. In general, there are few polymorphic nucleotides among B. anthracis strains reflecting the short evolutionary time since its derivation from a B. cereus-like ancestor. The exceptions to this lack of diversity are the variable number tandem repeat (VNTR) loci that exist in genic and non genic regions of the chromosome and both plasmids. Their variation is associated with high mutability that is driven by rapid insertion and deletion of the repeats within an array. A notable example is found in the vrrC locus which is homologous to known DNA translocase genes from other bacteria. PMID:19729033

A Mitochondrial Genome of Rhyparochromidae (Hemiptera: Heteroptera) and a Comparative Analysis of Related Mitochondrial Genomes.

PubMed

Li, Teng; Yang, Jie; Li, Yinwan; Cui, Ying; Xie, Qiang; Bu, Wenjun; Hillis, David M

2016-10-19

The Rhyparochromidae, the largest family of Lygaeoidea, encompasses more than 1,850 described species, but no mitochondrial genome has been sequenced to date. Here we describe the first mitochondrial genome for Rhyparochromidae: a complete mitochondrial genome of Panaorus albomaculatus (Scott, 1874). This mitochondrial genome is comprised of 16,345 bp, and contains the expected 37 genes and control region. The majority of the control region is made up of a large tandem-repeat region, which has a novel pattern not previously observed in other insects. The tandem-repeats region of P. albomaculatus consists of 53 tandem duplications (including one partial repeat), which is the largest number of tandem repeats among all the known insect mitochondrial genomes. Slipped-strand mispairing during replication is likely to have generated this novel pattern of tandem repeats. Comparative analysis of tRNA gene families in sequenced Pentatomomorpha and Lygaeoidea species shows that the pattern of nucleotide conservation is markedly higher on the J-strand. Phylogenetic reconstruction based on mitochondrial genomes suggests that Rhyparochromidae is not the sister group to all the remaining Lygaeoidea, and supports the monophyly of Lygaeoidea.

Metallo-β-lactamase-producing Pseudomonas aeruginosa in the Netherlands: the nationwide emergence of a single sequence type.

PubMed

Van der Bij, A K; Van der Zwan, D; Peirano, G; Severin, J A; Pitout, J D D; Van Westreenen, M; Goessens, W H F

2012-09-01

Recently, the first outbreak of clonally related VIM-2 metallo-β-lactamase (MBL)-producing Pseudomonas aeruginosa in a Dutch tertiary-care centre was described. Subsequently, a nationwide surveillance study was performed in 2010-2011, which identified the presence of VIM-2 MBL-producing P. aeruginosa in 11 different hospitals. Genotyping by multiple-locus variable-number tandem-repeat analysis (MLVA) showed that the majority of the 82 MBL-producing isolates found belonged to a single MLVA type (n = 70, 85%), identified as ST111 by multilocus sequence typing (MLST). As MBL-producing isolates cause serious infections that are difficult to treat, the presence of clonally related isolates in various hospitals throughout the Netherlands is of nationwide concern. © 2012 The Authors. Clinical Microbiology and Infection © 2012 European Society of Clinical Microbiology and Infectious Diseases.

19-VNTR loci used in genotyping Chinese clinical Mycobacterium tuberculosis complex strains and in association with spoligotyping.

PubMed

Jiang, Yi; Liu, Hai-can; Zheng, Huajun; Dou, Xiangfeng; Tang, Biao; Zhao, Xiu-qin; Zhu, Yongqiang; Lu, Bing; Wang, Shengyue; Dong, Hai-yan; Zhang, Yuan-yuan; Zhao, Guoping; Wan, Kanglin

2013-07-01

Recently, tandem repeat typing has emerged as a rapid and easy method for the molecular epidemiology of the Mycobacterium tuberculosis (M. tuberculosis) complex. In this study, a collection of 19 VNTRs incorporating 15 previously described loci and 4 newly evaluated markers were used to genotype 206 Chinese M. tuberculosis isolates and 9 BCG strains. The discriminatory power was evaluated and compared with that obtained by Spoligotyping. It turned out that 15-locus VNTR could be very useful in M. tuberculosis complex strains genotyping in China. The 4 newly evaluated loci were proved informative and could be useful for future epidemiology studies, especially in Beijing family strains. In addition, a unique pattern of the latter 4 loci were found in Chinese BCG strains. © 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Tandemly repeated sequences in mtDNA control region of whitefish, Coregonus lavaretus.

PubMed

Brzuzan, P

2000-06-01

Length variation of the mitochondrial DNA control region was observed with PCR amplification of a sample of 138 whitefish (Coregonus lavaretus). Nucleotide sequences of representative PCR products showed that the variation was due to the presence of an approximately 100-bp motif tandemly repeated two, three, or five times in the region between the conserved sequence block-3 (CSB-3) and the gene for phenylalanine tRNA. This is the first report on the tandem array composed of long repeat units in mitochondrial DNA of salmonids.

[Analysis on genetic polymorphism of 5 STR loci selected from X chromosome].

PubMed

Liu, Qi-ji; Gong, Yao-qin; Zhang, Xi-yu; Gao, Gui-min; Li, Jiang-xia; Guo, Yi-shou

2005-02-01

To select short tandem repeats(STR) from X chromosome. STR is a universal genetic marker that has changeable polymorphism and stable heredity in human genome. It is a specific DNA segment composed of 2-6 base pairs as its core sequence. It is an ideal DNA marker used in linkage analysis and gene mapping. In this study, 8 short tandem repeats were selected from two genomic clones on X chromosome by using BCM Search Launcher. Primers amplifying the STR loci were designed by using Primer 3.0 according to the unique sequence flanking the STRs. Polymorphisms of the short tandem repeats in Chinese population were evaluated by PCR amplification and PAGE. Five of these STRs were polymorphic. Chi-square test indicated that the distribution of genotypes agreed with Hardy-Weinberg equilibrium (P>0.05). Five polymorphic short tandem repeats have been identified on chromosome X and will be useful for linkage analysis and gene mapping.

Construction of a self-cloning sake yeast that overexpresses alcohol acetyltransferase gene by a two-step gene replacement protocol.

PubMed

Hirosawa, I; Aritomi, K; Hoshida, H; Kashiwagi, S; Nishizawa, Y; Akada, R

2004-07-01

The commercial application of genetically modified industrial microorganisms has been problematic due to public concerns. We constructed a "self-cloning" sake yeast strain that overexpresses the ATF1 gene encoding alcohol acetyltransferase, to improve the flavor profile of Japanese sake. A constitutive yeast overexpression promoter, TDH3p, derived from the glyceraldehyde-3-phosphate dehydrogenase gene from sake yeast was fused to ATF1; and the 5' upstream non-coding sequence of ATF1 was further fused to TDH3p-ATF1. The fragment was placed on a binary vector, pGG119, containing a drug-resistance marker for transformation and a counter-selection marker for excision of unwanted DNA. The plasmid was integrated into the ATF1 locus of a sake yeast strain. This integration constructed tandem repeats of ATF1 and TDH3p-ATF1 sequences, between which the plasmid was inserted. Loss of the plasmid, which occurs through homologous recombination between either the TDH3p downstream ATF1 repeats or the TDH3p upstream repeat sequences, was selected by growing transformants on counter-selective medium. Recombination between the downstream repeats led to reversion to a wild type strain, but that between the upstream repeats resulted in a strain that possessed TDH3p-ATF1 without the extraneous DNA sequences. The self-cloning TDH3p-ATF1 yeast strain produced a higher amount of isoamyl acetate. This is the first expression-controlled self-cloning industrial yeast.

Origin of the CMS gene locus in rapeseed cybrid mitochondria: active and inactive recombination produces the complex CMS gene region in the mitochondrial genomes of Brassicaceae.

PubMed

Oshima, Masao; Kikuchi, Rie; Imamura, Jun; Handa, Hirokazu

2010-01-01

CMS (cytoplasmic male sterile) rapeseed is produced by asymmetrical somatic cell fusion between the Brassica napus cv. Westar and the Raphanus sativus Kosena CMS line (Kosena radish). The CMS rapeseed contains a CMS gene, orf125, which is derived from Kosena radish. Our sequence analyses revealed that the orf125 region in CMS rapeseed originated from recombination between the orf125/orfB region and the nad1C/ccmFN1 region by way of a 63 bp repeat. A precise sequence comparison among the related sequences in CMS rapeseed, Kosena radish and normal rapeseed showed that the orf125 region in CMS rapeseed consisted of the Kosena orf125/orfB region and the rapeseed nad1C/ccmFN1 region, even though Kosena radish had both the orf125/orfB region and the nad1C/ccmFN1 region in its mitochondrial genome. We also identified three tandem repeat sequences in the regions surrounding orf125, including a 63 bp repeat, which were involved in several recombination events. Interestingly, differences in the recombination activity for each repeat sequence were observed, even though these sequences were located adjacent to each other in the mitochondrial genome. We report results indicating that recombination events within the mitochondrial genomes are regulated at the level of specific repeat sequences depending on the cellular environment.

Effects of Transposable Elements on the Expression of the Forked Gene of Drosophila Melanogaster

PubMed Central

Hoover, K. K.; Chien, A. J.; Corces, V. G.

1993-01-01

The products of the forked gene are involved in the formation and/or maintenance of a temporary fibrillar structure within the developing bristle rudiment of Drosophila melanogaster. Mutations in the forked locus alter this structure and result in aberrant development of macrochaetae, microchaetae and trichomes. The locus has been characterized at the molecular level by walking, mutant characterization and transcript analysis. Expression of the six forked transcripts is temporally restricted to midlate pupal development. At this time, RNAs of 6.4, 5.6, 5.4, 2.5, 1.9 and 1.1 kilobases (kb) are detected by Northern analysis. The coding region of these RNAs has been found to be within a 21-kb stretch of genomic DNA. The amino terminus of the proteins encoded by the 5.4- and 5.6-kb forked transcripts contain tandem copies of ankyrin-like repeats that may play an important role in the function of forked-encoded products. The profile of forked RNA expression is altered in seven spontaneous mutations characterized during this study. Three forked mutations induced by the insertion of the gypsy retrotransposon contain a copy of this element inserted into an intron of the gene. In these mutants, the 5.6-, 5.4- and 2.5-kb forked mRNAs are truncated via recognition of the polyadenylation site in the 5' long terminal repeat of the gypsy retrotransposon. These results help explain the role of the forked gene in fly development and further our understanding of the role of transposable elements in mutagenesis. PMID:8244011

Stabilization of perfect and imperfect tandem repeats by single-strand DNA exonucleases

PubMed Central

Feschenko, Vladimir V.; Rajman, Luis A.; Lovett, Susan T.

2003-01-01

Rearrangements between tandemly repeated DNA sequences are a common source of genetic instability. Such rearrangements underlie several human genetic diseases. In many organisms, the mismatch-repair (MMR) system functions to stabilize repeats when the repeat unit is short or when sequence imperfections are present between the repeats. We show here that the action of single-stranded DNA (ssDNA) exonucleases plays an additional, important role in stabilizing tandem repeats, independent of their role in MMR. For perfect repeats of ≈100 bp in Escherichia coli that are not susceptible to MMR, exonuclease (Exo)-I, ExoX, and RecJ exonuclease redundantly inhibit deletion. Our data suggest that >90% of potential deletion events are avoided by the combined action of these three exonucleases. Imperfect tandem repeats, less prone to rearrangements, are stabilized by both the MMR-pathway and ssDNA-specific exonucleases. For 100-bp repeats containing four mispairs, ExoI alone aborts most deletion events, even in the presence of a functional MMR system. By genetic analysis, we show that the inhibitory effect of ssDNA exonucleases on deletion formation is independent of the MutS and UvrD proteins. Exonuclease degradation of DNA displaced during the deletion process may abort slipped misalignment. Exonuclease action is therefore a significant force in genetic stabilization of many forms of repetitive DNA. PMID:12538867

Phylogeography and population structure of the biologically invasive phytopathogen Erwinia amylovora inferred using minisatellites.

PubMed

Bühlmann, Andreas; Dreo, Tanja; Rezzonico, Fabio; Pothier, Joël F; Smits, Theo H M; Ravnikar, Maja; Frey, Jürg E; Duffy, Brion

2014-07-01

Erwinia amylovora causes a major disease of pome fruit trees worldwide, and is regulated as a quarantine organism in many countries. While some diversity of isolates has been observed, molecular epidemiology of this bacterium is hindered by a lack of simple molecular typing techniques with sufficiently high resolution. We report a molecular typing system of E. amylovora based on variable number of tandem repeats (VNTR) analysis. Repeats in the E. amylovora genome were identified with comparative genomic tools, and VNTR markers were developed and validated. A Multiple-Locus VNTR Analysis (MLVA) was applied to E. amylovora isolates from bacterial collections representing global and regional distribution of the pathogen. Based on six repeats, MLVA allowed the distinction of 227 haplotypes among a collection of 833 isolates of worldwide origin. Three geographically separated groups were recognized among global isolates using Bayesian clustering methods. Analysis of regional outbreaks confirmed presence of diverse haplotypes but also high representation of certain haplotypes during outbreaks. MLVA analysis is a practical method for epidemiological studies of E. amylovora, identifying previously unresolved population structure within outbreaks. Knowledge of such structure can increase our understanding on how plant diseases emerge and spread over a given geographical region. © 2013 Society for Applied Microbiology and John Wiley & Sons Ltd.

Genetic analysis of haplotype data for 23 Y-chromosome short tandem repeat loci in the Turkish population recently settled in Sarajevo, Bosnia and Herzegovina

PubMed Central

Dogan, Serkan; Primorac, Dragan; Marjanović, Damir

2014-01-01

Aim To explore the distribution and polymorphisms of 23 short tandem repeat (STR) loci on the Y chromosome in the Turkish population recently settled in Sarajevo, Bosnia and Herzegovina and to investigate its genetic relationships with the homeland Turkish population and neighboring populations. Methods This study included 100 healthy unrelated male individuals from the Turkish population living in Sarajevo. Buccal swab samples were collected as a DNA source. Genomic DNA was extracted using the salting out method and amplification was performed using PowerPlex Y 23 amplification kit. The studied population was compared to other populations using pairwise genetic distances, which were represented with a multi-dimensional scaling plot. Results Haplotype and allele frequencies of the sample population were calculated and the results showed that all 100 samples had unique haplotypes. The most polymorphic locus was DYS458, and the least polymorphic DYS391. The observed haplotype diversity was 1.0000 ± 0.0014, with a discrimination capacity of 1.00 and the match probability of 0.01. Rst values showed that our sample population was closely related in both dimensions to the Lebanese and Iraqi populations, while it was more distant from Bosnian, Croatian, and Macedonian populations. Conclusion Turkish population residing in Sarajevo could be observed as a representative Turkish population, since our results were consistent with those previously published for the homeland Turkish population. Also, this study once again proved that geographically close populations were genetically more related to each other. PMID:25358886

Staphylococcus aureus from 152 cases of bovine, ovine and caprine mastitis investigated by Multiple-locus variable number of tandem repeat analysis (MLVA).

PubMed

Bergonier, Dominique; Sobral, Daniel; Feßler, Andrea T; Jacquet, Eric; Gilbert, Florence B; Schwarz, Stefan; Treilles, Michaël; Bouloc, Philippe; Pourcel, Christine; Vergnaud, Gilles

2014-10-02

Staphylococcus aureus is one of the main etiological agents of mastitis in ruminants. In the present retrospective study, we evaluated the potential interest of a previously described automated multiple loci Variable Number of Tandem Repeats (VNTR) Assay (MLVA) comprising 16 loci as a first line tool to investigate the population structure of S. aureus from mastitis. We determined the genetic diversity of S. aureus strains from cases of clinical and subclinical mastitis in dairy cattle (n = 118, of which 16 were methicillin-resistant), sheep (n = 18) and goats (n = 16). The 152 strains could be subdivided into 115 MLVA genotypes (including 14 genotypes for the ovine strains and 15 genotypes for the caprine strains). This corresponds to a discriminatory index (D) value of 0.9936. Comparison with published MLVA data obtained using the same protocol applied to strains from diverse human and animal origins revealed a low number (8.5%) of human-related MLVA genotypes among the present collection. Eighteen percent of the S. aureus mastitis collection belonged to clonal complexes apparently not associated with other pathological conditions. Some of them displayed a relatively low level of diversity in agreement with a restricted ecological niche. These findings provide arguments suggesting that specific S. aureus lineages particularly adapted to ruminant mammary glands have emerged and that MLVA is a convenient tool to provide a broad overview of the population, owing to the availability via internet of databases compiling published MLVA genotypes.

Evaluating the Use of Multilocus Variable Number Tandem Repeat Analysis of Shiga Toxin-Producing Escherichia coli O157 as a Routine Public Health Tool in England

PubMed Central

Byrne, Lisa; Elson, Richard; Dallman, Timothy J.; Perry, Neil; Ashton, Philip; Wain, John; Adak, Goutam K.; Grant, Kathie A.; Jenkins, Claire

2014-01-01

Multilocus variable number tandem repeat analysis (MLVA) provides microbiological support for investigations of clusters of cases of infection with Shiga toxin-producing E. coli (STEC) O157. All confirmed STEC O157 isolated in England and submitted to the Gastrointestinal Bacteria Reference Unit (GBRU) during a six month period were typed using MLVA, with the aim of assessing the impact of this approach on epidemiological investigations. Of 539 cases investigated, 341 (76%) had unique (>2 single locus variants) MLVA profiles, 12% of profiles occurred more than once due to known household transmission and 12% of profiles occurred as part of 41 clusters, 21 of which were previously identified through routine public health investigation of cases. The remaining 20 clusters were not previously detected and STEC enhanced surveillance data for associated cases were retrospectively reviewed for epidemiological links including shared exposures, geography and/or time. Additional evidence of a link between cases was found in twelve clusters. Compared to phage typing, the number of sporadic cases was reduced from 69% to 41% and the diversity index for MLVA was 0.996 versus 0.782 for phage typing. Using MLVA generates more data on the spatial and temporal dispersion of cases, better defining the epidemiology of STEC infection than phage typing. The increased detection of clusters through MLVA typing highlights the challenges to health protection practices, providing a forerunner to the advent of whole genome sequencing as a diagnostic tool. PMID:24465775

Evaluating the use of multilocus variable number tandem repeat analysis of Shiga toxin-producing Escherichia coli O157 as a routine public health tool in England.

PubMed

Byrne, Lisa; Elson, Richard; Dallman, Timothy J; Perry, Neil; Ashton, Philip; Wain, John; Adak, Goutam K; Grant, Kathie A; Jenkins, Claire

2014-01-01

Multilocus variable number tandem repeat analysis (MLVA) provides microbiological support for investigations of clusters of cases of infection with Shiga toxin-producing E. coli (STEC) O157. All confirmed STEC O157 isolated in England and submitted to the Gastrointestinal Bacteria Reference Unit (GBRU) during a six month period were typed using MLVA, with the aim of assessing the impact of this approach on epidemiological investigations. Of 539 cases investigated, 341 (76%) had unique (>2 single locus variants) MLVA profiles, 12% of profiles occurred more than once due to known household transmission and 12% of profiles occurred as part of 41 clusters, 21 of which were previously identified through routine public health investigation of cases. The remaining 20 clusters were not previously detected and STEC enhanced surveillance data for associated cases were retrospectively reviewed for epidemiological links including shared exposures, geography and/or time. Additional evidence of a link between cases was found in twelve clusters. Compared to phage typing, the number of sporadic cases was reduced from 69% to 41% and the diversity index for MLVA was 0.996 versus 0.782 for phage typing. Using MLVA generates more data on the spatial and temporal dispersion of cases, better defining the epidemiology of STEC infection than phage typing. The increased detection of clusters through MLVA typing highlights the challenges to health protection practices, providing a forerunner to the advent of whole genome sequencing as a diagnostic tool.

Long interspersed repeated DNA (LINE) causes polymorphism at the rat insulin 1 locus.

PubMed

Lakshmikumaran, M S; D'Ambrosio, E; Laimins, L A; Lin, D T; Furano, A V

1985-09-01

The insulin 1, but not the insulin 2, locus is polymorphic (i.e., exhibits allelic variation) in rats. Restriction enzyme analysis and hybridization studies showed that the polymorphic region is 2.2 kilobases upstream of the insulin 1 coding region and is due to the presence or absence of an approximately 2.7-kilobase repeated DNA element. DNA sequence determination showed that this DNA element is a member of a long interspersed repeated DNA family (LINE) that is highly repeated (greater than 50,000 copies) and highly transcribed in the rat. Although the presence or absence of LINE sequences at the insulin 1 locus occurs in both the homozygous and heterozygous states, LINE-containing insulin 1 alleles are more prevalent in the rat population than are alleles without LINEs. Restriction enzyme analysis of the LINE-containing alleles indicated that at least two versions of the LINE sequence may be present at the insulin 1 locus in different rats. Either repeated transposition of LINE sequences or gene conversion between the resident insulin 1 LINE and other sequences in the genome are possible explanations for this.

Tandem-repeat protein domains across the tree of life.

PubMed

Jernigan, Kristin K; Bordenstein, Seth R

2015-01-01

Tandem-repeat protein domains, composed of repeated units of conserved stretches of 20-40 amino acids, are required for a wide array of biological functions. Despite their diverse and fundamental functions, there has been no comprehensive assessment of their taxonomic distribution, incidence, and associations with organismal lifestyle and phylogeny. In this study, we assess for the first time the abundance of armadillo (ARM) and tetratricopeptide (TPR) repeat domains across all three domains in the tree of life and compare the results to our previous analysis on ankyrin (ANK) repeat domains in this journal. All eukaryotes and a majority of the bacterial and archaeal genomes analyzed have a minimum of one TPR and ARM repeat. In eukaryotes, the fraction of ARM-containing proteins is approximately double that of TPR and ANK-containing proteins, whereas bacteria and archaea are enriched in TPR-containing proteins relative to ARM- and ANK-containing proteins. We show in bacteria that phylogenetic history, rather than lifestyle or pathogenicity, is a predictor of TPR repeat domain abundance, while neither phylogenetic history nor lifestyle predicts ARM repeat domain abundance. Surprisingly, pathogenic bacteria were not enriched in TPR-containing proteins, which have been associated within virulence factors in certain species. Taken together, this comparative analysis provides a newly appreciated view of the prevalence and diversity of multiple types of tandem-repeat protein domains across the tree of life. A central finding of this analysis is that tandem repeat domain-containing proteins are prevalent not just in eukaryotes, but also in bacterial and archaeal species.

Ten tandem repeats of {beta}-hCG 109-118 enhance immunogenicity and anti-tumor effects of {beta}-hCG C-terminal peptide carried by mycobacterial heat-shock protein HSP65

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

Zhang Yankai; Yan Rong; He Yi

2006-07-14

The {beta}-subunit of human chorionic gonadotropin ({beta}-hCG) is secreted by many kinds of tumors and it has been used as an ideal target antigen to develop vaccines against tumors. In view of the low immunogenicity of this self-peptide,we designed a method based on isocaudamer technique to repeat tandemly the 10-residue sequence X of {beta}-hCG (109-118), then 10 tandemly repeated copies of the 10-residue sequence combined with {beta}-hCG C-terminal 37 peptides were fused to mycobacterial heat-shock protein 65 to construct a fusion protein HSP65-X10-{beta}hCGCTP37 as an immunogen. In this study, we examined the effect of the tandem repeats of this 10-residuemore » sequence in eliciting an immune by comparing the immunogenicity and anti-tumor effects of the two immunogens, HSP65-X10-{beta}hCGCTP37 and HSP65-{beta}hCGCTP37 (without the 10 tandem repeats). Immunization of mice with the fusion protein HSP65-X10-{beta}hCGCTP37 elicited much higher levels of specific anti-{beta}-hCG antibodies and more effectively inhibited the growth of Lewis lung carcinoma (LLC) in vivo than with HSP65-{beta}hCGCTP37, which should suggest that HSP65-X10-{beta}hCGCTP37 may be an effective protein vaccine for the treatment of {beta}-hCG-dependent tumors and multiple tandem repeats of a certain epitope are an efficient method to overcome the low immunogenicity of self-peptide antigens.« less

The central domain of bovine submaxillary mucin consists of over 50 tandem repeats of 329 amino acids. Chromosomal localization of the BSM1 gene and relations to ovine and porcine counterparts.

PubMed

Jiang, W; Gupta, D; Gallagher, D; Davis, S; Bhavanandan, V P

2000-04-01

We previously elucidated five distinct protein domains (I-V) for bovine submaxillary mucin, which is encoded by two genes, BSM1 and BSM2. Using Southern blot analysis, genomic cloning and sequencing of the BSM1 gene, we now show that the central domain (V) consists of approximately 55 tandem repeats of 329 amino acids and that domains III-V are encoded by a 58.4-kb exon, the largest exon known for all genes to date. The BSM1 gene was mapped by fluorescence in situ hybridization to the proximal half of chromosome 5 at bands q2. 2-q2.3. The amino-acid sequence of six tandem repeats (two full and four partial) were found to have only 92-94% identities. We propose that the variability in the amino-acid sequences of the mucin tandem repeat is important for generating the combinatorial library of saccharides that are necessary for the protective function of mucins. The deduced peptide sequences of the central domain match those determined from the purified bovine submaxillary mucin and also show 68-94% identity to published peptide sequences of ovine submaxillary mucin. This indicates that the core protein of ovine submaxillary mucin is closely related to that of bovine submaxillary mucin and contains similar tandem repeats in the central domain. In contrast, the central domain of porcine submaxillary mucin is reported to consist of 81-amino-acid tandem repeats. However, both bovine submaxillary mucin and porcine submaxillary mucin contain similar N-terminal and C-terminal domains and the corresponding genes are in the conserved linkage regions of the respective genomes.

Gene-for-genes interactions between cotton R genes and Xanthomonas campestris pv. malvacearum avr genes.

PubMed

De Feyter, R; Yang, Y; Gabriel, D W

1993-01-01

Six plasmid-borne avirulence (avr) genes were previously cloned from strain XcmH of the cotton pathogen, Xanthomonas campestris pv. malvacearum. We have now localized all six avr genes on the cloned fragments by subcloning and Tn5-gusA insertional mutagenesis. None of these avr genes appeared to exhibit exclusively gene-for-gene patterns of interactions with cotton R genes, and avrB4 was demonstrated to confer avr gene-for-R genes (plural) avirulence to X. c. pv. malvacearum on congenic cotton lines carrying either of two different resistance loci, B1 or B4. Furthermore, the B1 locus appeared to confer R gene-for-avr genes resistance to cotton against isogenic X. c. pv. malvacearum strains carrying any one of three avr genes: avrB4, avrb6, or avrB102. Restriction enzyme, Southern blot hybridization, and DNA sequence analyses showed that the XcmH avr genes are all highly similar to each other, to avrBs3 and avrBsP from the pepper pathogen X. c. pv. vesicatoria, and to the host-specific virulence gene pthA from the citrus pathogen X. citri. The XcmH avr genes differed primarily in the multiplicity of a tandemly repeated 102-base pair motif within the central portions of the genes, repeated from 14 to 23 times in members of this gene family. The complete nucleotide sequence of avrb6 revealed that it is 97% identical in DNA sequence to avrB4, avrBs3, avrBsP, and pthA and that 62-bp inverted terminal repeats mark the boundaries of homology between avrb6 and all members of this Xanthomonas virulence/avirulence gene family sequenced to date. The terminal 38 bp of both inverted repeats are highly similar to the 38-bp consensus terminal sequence of the Tn3 family of transposons. Up to 11 members of the avr gene family appear to be present in North American strains of X. c. pv. malvacearum, including XcmH. The high level of homology observed among these avr genes and their presence in multiple copies may explain the gene-for-genes interactions and also the observed high frequencies (10(-3) to 10(-4) per locus) of X. c. pv. malvacearum race change mutations. Five spontaneous race change mutants of XcmH suffered avr locus deletions, strongly indicating intergenic recombination as the primary mechanism for generating new races in X. c. pv. malvacearum.

Dicentric chromosome formation and epigenetics of centromere formation in plants.

PubMed

Fu, Shulan; Gao, Zhi; Birchler, James; Han, Fangpu

2012-03-20

Plant centromeres are generally composed of tandem arrays of simple repeats that form a complex chromosome locus where the kinetochore forms and microtubules attach during mitosis and meiosis. Each chromosome has one centromere region, which is essential for accurate division of the genetic material. Recently, chromosomes containing two centromere regions (called dicentric chromosomes) have been found in maize and wheat. Interestingly, some dicentric chromosomes are stable because only one centromere is active and the other one is inactivated. Because such arrays maintain their typical structure for both active and inactive centromeres, the specification of centromere activity has an epigenetic component independent of the DNA sequence. Under some circumstances, the inactive centromeres may recover centromere function, which is called centromere reactivation. Recent studies have highlighted the important changes, such as DNA methylation and histone modification, that occur during centromere inactivation and reactivation. Copyright © 2012. Published by Elsevier Ltd.

Persistence of the same genetic type of Mycoplasma hyopneumoniae in a closed herd for at least two years.

PubMed

Rebaque, Florencia; Camacho, Pablo; Parada, Julián; Lucchesi, Paula; Ambrogi, Arnaldo; Tamiozzo, Pablo

2017-10-20

Two cross-sectional studies were carried out in 2013 and 2015 monitoring for Mycoplasma hyopneumoniae presence in a swine farm. In these studies, the genetic diversity of M. hyopneumoniae was assessed in clinical specimens using a Multiple Locus Variable-number tandem repeat Analysis (MLVA) targeting P97 R1, P146 R3 and H4 loci. The samples from August 2015 showed the MLVA profile prevalent in June 2013, therefore it can be concluded that a same genetic type of M. hyopneumoniae can persist for at least two years in a closed herd. In addition, the nested PCR reactions implemented in this study showed to be useful for MLVA typing in non-invasive clinical samples. Copyright © 2017 Asociación Argentina de Microbiología. Publicado por Elsevier España, S.L.U. All rights reserved.

[Paternity study in Chilean families using DNA fingerprints and erythrocyte blood markers].

PubMed

Aguirre, R; Blanco, R; Cifuentes, L; Chiffelle, I; Armanet, L; Vargas, J; Jara, L

1992-10-01

In the last decade, the electromorphic phenotype corresponding to extremely polymorphic zones of DNA, that include variable number of tandem repeat loci (VNTR) of oligonucleotide sequences, have been added to classical markers to elucidate the problems of parenthood identification and ascription in human beings. Using VNTR of several loci, a band profile practically unique for each individual is obtained (DNA-fingerprints). Since the pattern of VNTR electrophoretic bands is inherited from parents in a proportion of 50% from each one, this system is extremely useful for paternity ascription or exclusion. Nine nuclear families were studied, randomly selected from a group of 170 families that were analyzed using 5 erythrocyte genetic markers and with VNTRs detected using the multi locus probe (CAC)5, aiming to explore the concordance of both methods. Results were similar for both methods; however for VNTR, there is no information available on population frequency of polymorphisms.

Comparison of 2 proposed MLVA protocols for subtyping non-O157:H7 verotoxigenic Escherichia coli.

PubMed

González, Juliana; Sanso, Andrea Mariel; Lucchesi, Paula María Alejandra; Bustamante, Ana Victoria

2014-04-01

Multiple locus variable number tandem repeats (VNTRs) analysis (MLVA) has become a reliable tool, able to establish genetic relationships for epidemiological surveillance and molecular subtyping of pathogens such as verotoxigenic Escherichia coli (VTEC). This emerging pathogen whose primary reservoir is the cattle causes severe disease in humans, such as hemorrhagic colitis and hemolytic uremic syndrome. With the aim of comparing a recently proposed MLVA assay with that used routinely in our laboratory, we analyzed a set of VTEC isolates (n = 72) obtained from meat using both assays. All samples could be typed by the new MLVA assay, and an increase in the number of distinct profiles (31-43) was observed. However, intraserotype resolution was not significantly enhanced; thus, the incorporation of more VNTR loci is still needed to achieve a greater discrimination among non-O157:H7 serotypes. Copyright © 2014 Elsevier Inc. All rights reserved.

A YAC contig encompassing the Treacher Collins syndrome critical region at 5q31. 3-32

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

Dixon, J.; Gladwin, A.J.; Perveen, R.

Treacher Collins syndrome (TCOF1) is an autosomal dominant disorder of craniofacial development the features of which include conductive hearing loss and cleft palate. Previous studies have localized the TCOF1 locus between D5S519 (proximal) and SPARC (distal), a region of 22 centirays as estimated by radiation hybrid mapping. In the current investigation the authors have created a contig across the TCOF1 critical region, using YAC clones. Isolation of a novel short tandem repeat polymorphism corresponding to the end of one of the YACs has allowed reduction of the size of the critical region to [approximately] 840 kb, which has been coveredmore » with three nonchimeric YACs. Restriction mapping has revealed that the region contains a high density of clustered rare-cutter restriction sites, suggesting that it may contain a number of different genes. The results of the present investigation have further allowed confirmation that the RPS14 locus lies proximal to the critical region and can thereby be excluded from a role in the pathogenesis of TCOF1, while ANX6 lies within the TCOF1 critical region and remains a potential candidate for the mutated gene. 26 refs., 4 figs., 1 tab.« less

A global analysis of Y-chromosomal haplotype diversity for 23 STR loci

PubMed Central

Purps, Josephine; Siegert, Sabine; Willuweit, Sascha; Nagy, Marion; Alves, Cíntia; Salazar, Renato; Angustia, Sheila M.T.; Santos, Lorna H.; Anslinger, Katja; Bayer, Birgit; Ayub, Qasim; Wei, Wei; Xue, Yali; Tyler-Smith, Chris; Bafalluy, Miriam Baeta; Martínez-Jarreta, Begoña; Egyed, Balazs; Balitzki, Beate; Tschumi, Sibylle; Ballard, David; Court, Denise Syndercombe; Barrantes, Xinia; Bäßler, Gerhard; Wiest, Tina; Berger, Burkhard; Niederstätter, Harald; Parson, Walther; Davis, Carey; Budowle, Bruce; Burri, Helen; Borer, Urs; Koller, Christoph; Carvalho, Elizeu F.; Domingues, Patricia M.; Chamoun, Wafaa Takash; Coble, Michael D.; Hill, Carolyn R.; Corach, Daniel; Caputo, Mariela; D’Amato, Maria E.; Davison, Sean; Decorte, Ronny; Larmuseau, Maarten H.D.; Ottoni, Claudio; Rickards, Olga; Lu, Di; Jiang, Chengtao; Dobosz, Tadeusz; Jonkisz, Anna; Frank, William E.; Furac, Ivana; Gehrig, Christian; Castella, Vincent; Grskovic, Branka; Haas, Cordula; Wobst, Jana; Hadzic, Gavrilo; Drobnic, Katja; Honda, Katsuya; Hou, Yiping; Zhou, Di; Li, Yan; Hu, Shengping; Chen, Shenglan; Immel, Uta-Dorothee; Lessig, Rüdiger; Jakovski, Zlatko; Ilievska, Tanja; Klann, Anja E.; García, Cristina Cano; de Knijff, Peter; Kraaijenbrink, Thirsa; Kondili, Aikaterini; Miniati, Penelope; Vouropoulou, Maria; Kovacevic, Lejla; Marjanovic, Damir; Lindner, Iris; Mansour, Issam; Al-Azem, Mouayyad; Andari, Ansar El; Marino, Miguel; Furfuro, Sandra; Locarno, Laura; Martín, Pablo; Luque, Gracia M.; Alonso, Antonio; Miranda, Luís Souto; Moreira, Helena; Mizuno, Natsuko; Iwashima, Yasuki; Neto, Rodrigo S. Moura; Nogueira, Tatiana L.S.; Silva, Rosane; Nastainczyk-Wulf, Marina; Edelmann, Jeanett; Kohl, Michael; Nie, Shengjie; Wang, Xianping; Cheng, Baowen; Núñez, Carolina; Pancorbo, Marian Martínez de; Olofsson, Jill K.; Morling, Niels; Onofri, Valerio; Tagliabracci, Adriano; Pamjav, Horolma; Volgyi, Antonia; Barany, Gusztav; Pawlowski, Ryszard; Maciejewska, Agnieszka; Pelotti, Susi; Pepinski, Witold; Abreu-Glowacka, Monica; Phillips, Christopher; Cárdenas, Jorge; Rey-Gonzalez, Danel; Salas, Antonio; Brisighelli, Francesca; Capelli, Cristian; Toscanini, Ulises; Piccinini, Andrea; Piglionica, Marilidia; Baldassarra, Stefania L.; Ploski, Rafal; Konarzewska, Magdalena; Jastrzebska, Emila; Robino, Carlo; Sajantila, Antti; Palo, Jukka U.; Guevara, Evelyn; Salvador, Jazelyn; Ungria, Maria Corazon De; Rodriguez, Jae Joseph Russell; Schmidt, Ulrike; Schlauderer, Nicola; Saukko, Pekka; Schneider, Peter M.; Sirker, Miriam; Shin, Kyoung-Jin; Oh, Yu Na; Skitsa, Iulia; Ampati, Alexandra; Smith, Tobi-Gail; Calvit, Lina Solis de; Stenzl, Vlastimil; Capal, Thomas; Tillmar, Andreas; Nilsson, Helena; Turrina, Stefania; De Leo, Domenico; Verzeletti, Andrea; Cortellini, Venusia; Wetton, Jon H.; Gwynne, Gareth M.; Jobling, Mark A.; Whittle, Martin R.; Sumita, Denilce R.; Wolańska-Nowak, Paulina; Yong, Rita Y.Y.; Krawczak, Michael; Nothnagel, Michael; Roewer, Lutz

2014-01-01

In a worldwide collaborative effort, 19,630 Y-chromosomes were sampled from 129 different populations in 51 countries. These chromosomes were typed for 23 short-tandem repeat (STR) loci (DYS19, DYS389I, DYS389II, DYS390, DYS391, DYS392, DYS393, DYS385ab, DYS437, DYS438, DYS439, DYS448, DYS456, DYS458, DYS635, GATAH4, DYS481, DYS533, DYS549, DYS570, DYS576, and DYS643) and using the PowerPlex Y23 System (PPY23, Promega Corporation, Madison, WI). Locus-specific allelic spectra of these markers were determined and a consistently high level of allelic diversity was observed. A considerable number of null, duplicate and off-ladder alleles were revealed. Standard single-locus and haplotype-based parameters were calculated and compared between subsets of Y-STR markers established for forensic casework. The PPY23 marker set provides substantially stronger discriminatory power than other available kits but at the same time reveals the same general patterns of population structure as other marker sets. A strong correlation was observed between the number of Y-STRs included in a marker set and some of the forensic parameters under study. Interestingly a weak but consistent trend toward smaller genetic distances resulting from larger numbers of markers became apparent. PMID:24854874

Molecular epidemiological characteristics of Salmonella enterica serovars Enteritidis, Typhimurium and Livingstone strains isolated in a Tunisian university hospital.

PubMed

Ktari, Sonia; Ksibi, Boutheina; Gharsallah, Houda; Mnif, Basma; Maalej, Sonda; Rhimi, Fouzia; Hammami, Adnene

2016-03-01

Enteritidis, Typhimurium and Livingstone are the main Salmonella enterica serovars recovered in Tunisia. Here, we aimed to assess the genetic diversity of fifty-seven Salmonella enterica strains from different sampling periods, origins and settings using pulsed-field gel electrophoresis (PFGE), multi-locus sequence typing (MLST) and multi-locus variable-number tandem repeat analysis (MLVA). Salmonella Enteritidis, isolated from human and food sources from two regions in Sfax in 2007, were grouped into one cluster using PFGE. However, using MLVA these strains were divided into two clusters. Salmonella Typhimurium strains, recovered in 2012 and represent sporadic cases of human clinical isolates, were included in one PFGE cluster. Nevertheless, the MLVA technique, divided Salmonella Typhimurium isolates into six clusters with diversity index reaching (DI = 0.757). For Salmonella Livingstone which was responsible of two nosocomial outbreaks during 2000-2003, the PFGE and MLVA methods showed that these strains were genetically closely related. Salmonella Enteritidis and Salmonella Livingstone populations showed a single ST lineage ST11 and ST543 respectively. For Salmonella Typhimurium, two MLST sequence types ST19 and ST328 were defined. Salmonella Enteritidis and Salmonella Typhimurium strains were clearly differentiated by MLVA which was not the case using PFGE. © 2015 APMIS. Published by John Wiley & Sons Ltd.

Tandem-repeat protein domains across the tree of life

PubMed Central

Jernigan, Kristin K.

2015-01-01

Tandem-repeat protein domains, composed of repeated units of conserved stretches of 20–40 amino acids, are required for a wide array of biological functions. Despite their diverse and fundamental functions, there has been no comprehensive assessment of their taxonomic distribution, incidence, and associations with organismal lifestyle and phylogeny. In this study, we assess for the first time the abundance of armadillo (ARM) and tetratricopeptide (TPR) repeat domains across all three domains in the tree of life and compare the results to our previous analysis on ankyrin (ANK) repeat domains in this journal. All eukaryotes and a majority of the bacterial and archaeal genomes analyzed have a minimum of one TPR and ARM repeat. In eukaryotes, the fraction of ARM-containing proteins is approximately double that of TPR and ANK-containing proteins, whereas bacteria and archaea are enriched in TPR-containing proteins relative to ARM- and ANK-containing proteins. We show in bacteria that phylogenetic history, rather than lifestyle or pathogenicity, is a predictor of TPR repeat domain abundance, while neither phylogenetic history nor lifestyle predicts ARM repeat domain abundance. Surprisingly, pathogenic bacteria were not enriched in TPR-containing proteins, which have been associated within virulence factors in certain species. Taken together, this comparative analysis provides a newly appreciated view of the prevalence and diversity of multiple types of tandem-repeat protein domains across the tree of life. A central finding of this analysis is that tandem repeat domain-containing proteins are prevalent not just in eukaryotes, but also in bacterial and archaeal species. PMID:25653910

A TALE-inspired computational screen for proteins that contain approximate tandem repeats.

PubMed

Perycz, Malgorzata; Krwawicz, Joanna; Bochtler, Matthias

2017-01-01

TAL (transcription activator-like) effectors (TALEs) are bacterial proteins that are secreted from bacteria to plant cells to act as transcriptional activators. TALEs and related proteins (RipTALs, BurrH, MOrTL1 and MOrTL2) contain approximate tandem repeats that differ in conserved positions that define specificity. Using PERL, we screened ~47 million protein sequences for TALE-like architecture characterized by approximate tandem repeats (between 30 and 43 amino acids in length) and sequence variability in conserved positions, without requiring sequence similarity to TALEs. Candidate proteins were scored according to their propensity for nuclear localization, secondary structure, repeat sequence complexity, as well as covariation and predicted structural proximity of variable residues. Biological context was tentatively inferred from co-occurrence of other domains and interactome predictions. Approximate repeats with TALE-like features that merit experimental characterization were found in a protein of chestnut blight fungus, a eukaryotic plant pathogen.

A TALE-inspired computational screen for proteins that contain approximate tandem repeats

PubMed Central

Krwawicz, Joanna

2017-01-01

TAL (transcription activator-like) effectors (TALEs) are bacterial proteins that are secreted from bacteria to plant cells to act as transcriptional activators. TALEs and related proteins (RipTALs, BurrH, MOrTL1 and MOrTL2) contain approximate tandem repeats that differ in conserved positions that define specificity. Using PERL, we screened ~47 million protein sequences for TALE-like architecture characterized by approximate tandem repeats (between 30 and 43 amino acids in length) and sequence variability in conserved positions, without requiring sequence similarity to TALEs. Candidate proteins were scored according to their propensity for nuclear localization, secondary structure, repeat sequence complexity, as well as covariation and predicted structural proximity of variable residues. Biological context was tentatively inferred from co-occurrence of other domains and interactome predictions. Approximate repeats with TALE-like features that merit experimental characterization were found in a protein of chestnut blight fungus, a eukaryotic plant pathogen. PMID:28617832

Direct mapping of symbolic DNA sequence into frequency domain in global repeat map algorithm

PubMed Central

Glunčić, Matko; Paar, Vladimir

2013-01-01

The main feature of global repeat map (GRM) algorithm (www.hazu.hr/grm/software/win/grm2012.exe) is its ability to identify a broad variety of repeats of unbounded length that can be arbitrarily distant in sequences as large as human chromosomes. The efficacy is due to the use of complete set of a K-string ensemble which enables a new method of direct mapping of symbolic DNA sequence into frequency domain, with straightforward identification of repeats as peaks in GRM diagram. In this way, we obtain very fast, efficient and highly automatized repeat finding tool. The method is robust to substitutions and insertions/deletions, as well as to various complexities of the sequence pattern. We present several case studies of GRM use, in order to illustrate its capabilities: identification of α-satellite tandem repeats and higher order repeats (HORs), identification of Alu dispersed repeats and of Alu tandems, identification of Period 3 pattern in exons, implementation of ‘magnifying glass’ effect, identification of complex HOR pattern, identification of inter-tandem transitional dispersed repeat sequences and identification of long segmental duplications. GRM algorithm is convenient for use, in particular, in cases of large repeat units, of highly mutated and/or complex repeats, and of global repeat maps for large genomic sequences (chromosomes and genomes). PMID:22977183

Long interspersed repeated DNA (LINE) causes polymorphism at the rat insulin 1 locus.

PubMed Central

Lakshmikumaran, M S; D'Ambrosio, E; Laimins, L A; Lin, D T; Furano, A V

1985-01-01

The insulin 1, but not the insulin 2, locus is polymorphic (i.e., exhibits allelic variation) in rats. Restriction enzyme analysis and hybridization studies showed that the polymorphic region is 2.2 kilobases upstream of the insulin 1 coding region and is due to the presence or absence of an approximately 2.7-kilobase repeated DNA element. DNA sequence determination showed that this DNA element is a member of a long interspersed repeated DNA family (LINE) that is highly repeated (greater than 50,000 copies) and highly transcribed in the rat. Although the presence or absence of LINE sequences at the insulin 1 locus occurs in both the homozygous and heterozygous states, LINE-containing insulin 1 alleles are more prevalent in the rat population than are alleles without LINEs. Restriction enzyme analysis of the LINE-containing alleles indicated that at least two versions of the LINE sequence may be present at the insulin 1 locus in different rats. Either repeated transposition of LINE sequences or gene conversion between the resident insulin 1 LINE and other sequences in the genome are possible explanations for this. Images PMID:3016521

Tandem Repeated Irritation Test (TRIT) Studies and Clinical Relevance: Post 2006.

PubMed

Reddy, Rasika; Maibach, Howard

2018-06-11

Single or multiple applications of irritants can lead to occupational contact dermatitis, and most commonly irritant contact dermatitis (ICD). Tandem irritation, the sequential application of two irritants to a target skin area, has been studied using the Tandem Repeated Irritation Test (TRIT) to provide a more accurate representation of skin irritation. Here we present an update to Kartono's review on tandem irritation studies since 2006 [1]. We surveyed the literature available on PubMed, Embase, Google Scholar, and the UCSF Dermatology library databases since 2006. The studies included discuss the tandem effects of common chemical irritants, organic solvents, occlusion as well as clinical relevance - and enlarge our ability to discern whether multiple chemical exposures are more or less likely to enhance irritation.

Integration of E. coli aroG-pheA tandem genes into Corynebacterium glutamicum tyrA locus and its effect on L-phenylalanine biosynthesis

PubMed Central

Liu, Dong-Xin; Fan, Chang-Sheng; Tao, Ju-Hong; Liang, Guo-Xin; Gao, Shan-E; Wang, Hai-Jiao; Li, Xin; Song, Da-Xin

2004-01-01

AIM: To study the effect of integration of tandem aroG-pheA genes into the tyrA locus of Corynebacterium glutamicum (C. glutamicum) on the production of L-phenylalanine. METHODS: By nitrosoguanidine mutagenesis, five p-fluorophenylalanine (FP)-resistant mutants of C.glutamicum FP were selected. The tyrA gene encoding prephenate dehydrogenase (PDH) of C.glutamicum was amplified by polymerase chain reaction (PCR) and cloned on the plasmid pPR. Kanamycin resistance gene (Km) and the PBF-aroG-pheA-T (GA) fragment of pGA were inserted into tyrA gene to form targeting vectors pTK and pTGAK, respectively. Then, they were transformed into C.glutamicum FP respectively by electroporation. Cultures were screened by a medium containing kanamycin and detected by PCR and phenotype analysis. The transformed strains were used for L-phenylalanine fermentation and enzyme assays. RESULTS: Engineering strains of C.glutamicum (Tyr-) were obtained. Compared with the original strain, the transformed strain C. glutamicum GAK was observed to have the highest elevation of L-phenylalanine production by a 1.71-fold, and 2.9-, 3.36-, and 3.0-fold in enzyme activities of chorismate mutase, prephenate dehydratase and 3-deoxy-D-arabinoheptulosonate-7-phosphate synthase, respectively. CONCLUSION: Integration of tandem aroG-pheA genes into tyrA locus of C. glutamicum chromosome can disrupt tyrA gene and increase the yield of L-phenylalanine production. PMID:15534933

Simple sequence repeats in Escherichia coli: abundance, distribution, composition, and polymorphism.

PubMed

Gur-Arie, R; Cohen, C J; Eitan, Y; Shelef, L; Hallerman, E M; Kashi, Y

2000-01-01

Computer-based genome-wide screening of the DNA sequence of Escherichia coli strain K12 revealed tens of thousands of tandem simple sequence repeat (SSR) tracts, with motifs ranging from 1 to 6 nucleotides. SSRs were well distributed throughout the genome. Mononucleotide SSRs were over-represented in noncoding regions and under-represented in open reading frames (ORFs). Nucleotide composition of mono- and dinucleotide SSRs, both in ORFs and in noncoding regions, differed from that of the genomic region in which they occurred, with 93% of all mononucleotide SSRs proving to be of A or T. Computer-based analysis of the fine position of every SSR locus in the noncoding portion of the genome relative to downstream ORFs showed SSRs located in areas that could affect gene regulation. DNA sequences at 14 arbitrarily chosen SSR tracts were compared among E. coli strains. Polymorphisms of SSR copy number were observed at four of seven mononucleotide SSR tracts screened, with all polymorphisms occurring in noncoding regions. SSR polymorphism could prove important as a genome-wide source of variation, both for practical applications (including rapid detection, strain identification, and detection of loci affecting key phenotypes) and for evolutionary adaptation of microbes.

Genome complexity in the coelacanth is reflected in its adaptive immune system

USGS Publications Warehouse

Saha, Nil Ratan; Ota, Tatsuya; Litman, Gary W.; Hansen, John; Parra, Zuly; Hsu, Ellen; Buonocore, Francesco; Canapa, Adriana; Cheng, Jan-Fang; Amemiya, Chris T.

2014-01-01

We have analyzed the available genome and transcriptome resources from the coelacanth in order to characterize genes involved in adaptive immunity. Two highly distinctive IgW-encoding loci have been identified that exhibit a unique genomic organization, including a multiplicity of tandemly repeated constant region exons. The overall organization of the IgW loci precludes typical heavy chain class switching. A locus encoding IgM could not be identified either computationally or by using several different experimental strategies. Four distinct sets of genes encoding Ig light chains were identified. This includes a variant sigma-type Ig light chain previously identified only in cartilaginous fishes and which is now provisionally denoted sigma-2. Genes encoding α/β and γ/δ T-cell receptors, and CD3, CD4, and CD8 co-receptors also were characterized. Ig heavy chain variable region genes and TCR components are interspersed within the TCR α/δ locus; this organization previously was reported only in tetrapods and raises questions regarding evolution and functional cooption of genes encoding variable regions. The composition, organization and syntenic conservation of the major histocompatibility complex locus have been characterized. We also identified large numbers of genes encoding cytokines and their receptors, and other genes associated with adaptive immunity. In terms of sequence identity and organization, the adaptive immune genes of the coelacanth more closely resemble orthologous genes in tetrapods than those in teleost fishes, consistent with current phylogenomic interpretations. Overall, the work reported described herein highlights the complexity inherent in the coelacanth genome and provides a rich catalog of immune genes for future investigations.

Genetic linkage analysis using pooled DNA and infrared detection of tailed STRP primer patterns

NASA Astrophysics Data System (ADS)

Oetting, William S.; Wildenberg, Scott C.; King, Richard A.

1996-04-01

The mapping of a disease locus to a specific chromosomal region is an important step in the eventual isolation and analysis of a disease causing gene. Conventional mapping methods analyze large multiplex families and/or smaller nuclear families to find linkage between the disease and a chromosome marker that maps to a known chromosomal region. This analysis is time consuming and tedious, typically requiring the determination of 30,000 genotypes or more. For appropriate populations, we have instead utilized pooled DNA samples for gene mapping which greatly reduces the amount of time necessary for an initial chromosomal screen. This technique assumes a common founder for the disease locus of interest and searches for a region of a chromosome shared between affected individuals. Our analysis involves the PCR amplification of short tandem repeat polymorphisms (STRP) to detect these shared regions. In order to reduce the cost of genotyping, we have designed unlabeled tailed PCR primers which, when combined with a labeled universal primer, provides for an alternative to synthesizing custom labeled primers. The STRP pattern is visualized with an infrared fluorescence based automated DNA sequencer and the patterns quantitated by densitometric analysis of the allele pattern. Differences in the distribution of alleles between pools of affected and unaffected individuals, including a reduction in the number of alleles in the affected pool, indicate the sharing of a region of a chromosome. We have found this method effective for markers 10 - 15 cM away from the disease locus for a recessive genetic disease.

Validation of a combined autosomal/Y-chromosomal STR approach for analyzing typical biological stains in sexual-assault cases.

PubMed

Purps, Josephine; Geppert, Maria; Nagy, Marion; Roewer, Lutz

2015-11-01

DNA testing is an established part of the investigation and prosecution of sexual assault. The primary purpose of DNA evidence is to identify a suspect and/or to demonstrate sexual contact. However, due to highly uneven proportions of female and male DNA in typical stains, routine autosomal analysis often fails to detect the DNA of the assailant. To evaluate the forensic efficiency of the combined application of autosomal and Y-chromosomal short tandem repeat (STR) markers, we present a large retrospective casework study of probative evidence collected in sexual-assault cases. We investigated up to 39 STR markers by testing combinations of the 16-locus NGMSElect kit with both the 23-locus PowerPlex Y23 and the 17-locus Yfiler kit. Using this dual approach we analyzed DNA extracts from 2077 biological stains collected in 287 cases over 30 months. To assess the outcome of the combined approach in comparison to stand-alone autosomal analysis we evaluated informative DNA profiles. Our investigation revealed that Y-STR analysis added up to 21% additional, highly informative (complete, single-source) profiles to the set of reportable autosomal STR profiles for typical stains collected in sexual-assault cases. Detection of multiple male contributors was approximately three times more likely with Y-chromosomal profiling than with autosomal STR profiling. In summary, 1/10 cases would have remained inconclusive (and could have been dismissed) if Y-STR analysis had been omitted from DNA profiling in sexual-assault cases. Copyright © 2015 The Authors. Published by Elsevier Ireland Ltd.. All rights reserved.

Significant Admixture Linkage Disequilibrium across 30 cM around the FY Locus in African Americans

PubMed Central

Lautenberger, James A.; Stephens, J. Claiborne; O'Brien, Stephen J.; Smith, Michael W.

2000-01-01

Scientists, to understand the importance of allelic polymorphisms on phenotypes that are quantitative and environmentally interacting, are now turning to population-association screens, especially in instances in which pedigree analysis is difficult. Because association screens require linkage disequilibrium between markers and disease loci, maximizing the degree of linkage disequilibrium increases the chances of discovering functional gene-marker associations. One theoretically valid approach—mapping by admixture linkage disequilibrium (MALD), using recently admixed African Americans—is empirically evaluated here by measurement of marker associations with 15 short tandem repeats (STRs) and an insertion/deletion polymorphism of the AT3 locus in a 70-cM segment at 1q22-23, around the FY (Duffy) locus. The FY polymorphism (−46T→C) disrupts the GATA promoter motif, specifically blocking FY erythroid expression and has a nearly fixed allele-frequency difference between European Americans and native Africans that is likely a consequence of a selective advantage of FY−/− in malaria infections. Analysis of linkage disequilibrium around the FY gene has indicated that there is strong and consistent linkage disequilibrium between FY and three flanking loci (D1S303, SPTA1, and D1S484) spanning 8 cM. We observed significant linkage-disequilibrium signals over a 30-cM region from −4.4 to 16.3 cM (from D1S2777 to D1S196) for STRs and at 26.4 cM (AT3), which provided quantitative estimates of centimorgan limits, by MALD assessment in African American population-association analyses, of 5–10 cM. PMID:10712211

[Multiple-locus variable number tandem repeat analysis of Bordetella pertussis strains collected in the Czech Republic in 1967-2015: spread of a variant adapted to the population with a high vaccination coverage].

PubMed

Lžičařová, D; Zavadilová, J; Musílek, M; Jandová, Z; Křížová, P; Fabiánová, K

To perform multiple-locus variable number tandem repeat analysis (MLVA) of B. pertussis strains from the collection of the National Reference Laboratory for Diphtheria and Pertussis (NRL/DIPE), National Institute of Public Health (NIPH), Prague. The study strains were isolated from clinical specimens collected mostly in the Czech Republic over a nearly 50-year period from 1967 to 2015 (June). The isolates from three periods characterized by different vaccination strategies and trends in pertussis are compared for genetic diversity and distribution of MLVA types (MT). Based on the results obtained, the suitability for use of MLVA in the analysis of epidemic outbreaks of B. pertussis in the Czech Republic is considered. DNA samples extracted from B. pertussis strains included in the present study were examined by MLVA using the standard protocol. Data were processed by means of the eBURST algorithm and the calculation of the Simpson diversity index (DI) was used for the statistical analysis. Data were analyzed as a whole and also separately for strains from the three periods: 1967-1980, 1990-2007, and 2008-2015 (June). Fourteen different MT were detected in the study strains, with three of them not being reported before. The most common MTs were MT27 and MT29. MT29 was predominant in 1967-1980 while MT27 was the most prevalent in 1990-2007 and 2008-2015 (June). The DI was the lowest (0.49) in 2008-2015 (June), and comparably higher DIs were calculated for the two previous periods (i.e. 0.667 for 1967-1980 and 0.654 for 1990-2007). MLVA revealed a decrease in genetic diversity and shifts in MT distribution of B. pertussis strains isolated from clinical specimens in the Czech Republic from 1967 to 2015 (June). These shifts in the Czech Republic can be characterized as a progressive increase in global MTs at the expense of the locally unique ones. The most common MT, similarly to other geographical areas with long-term high vaccination coverage, is MT27. The results of MLVA of 136 B. pertussis strains can provide a background for using this method in molecular epidemiological analysis of smaller groups of strains.

The evolution of filamin – A protein domain repeat perspective

PubMed Central

Light, Sara; Sagit, Rauan; Ithychanda, Sujay S.; Qin, Jun; Elofsson, Arne

2013-01-01

Particularly in higher eukaryotes, some protein domains are found in tandem repeats, performing broad functions often related to cellular organization. For instance, the eukaryotic protein filamin interacts with many proteins and is crucial for the cytoskeleton. The functional properties of long repeat domains are governed by the specific properties of each individual domain as well as by the repeat copy number. To provide better understanding of the evolutionary and functional history of repeating domains, we investigated the mode of evolution of the filamin domain in some detail. Among the domains that are common in long repeat proteins, sushi and spectrin domains evolve primarily through cassette tandem duplications while scavenger and immunoglobulin repeats appear to evolve through clustered tandem duplications. Additionally, immunoglobulin and filamin repeats exhibit a unique pattern where every other domain shows high sequence similarity. This pattern may be the result of tandem duplications, serve to avert aggregation between adjacent domains or it is the result of functional constraints. In filamin, our studies confirm the presence of interspersed integrin binding domains in vertebrates, while invertebrates exhibit more varied patterns, including more clustered integrin binding domains. The most notable case is leech filamin, which contains a 20 repeat expansion and exhibits unique dimerization topology. Clearly, invertebrate filamins are varied and contain examples of similar adjacent integrin-binding domains. Given that invertebrate integrin shows more similarity to the weaker filamin binder, integrin β3, it is possible that the distance between integrin-binding domains is not as crucial for invertebrate filamins as for vertebrates. PMID:22414427

The evolution of filamin-a protein domain repeat perspective.

PubMed

Light, Sara; Sagit, Rauan; Ithychanda, Sujay S; Qin, Jun; Elofsson, Arne

2012-09-01

Particularly in higher eukaryotes, some protein domains are found in tandem repeats, performing broad functions often related to cellular organization. For instance, the eukaryotic protein filamin interacts with many proteins and is crucial for the cytoskeleton. The functional properties of long repeat domains are governed by the specific properties of each individual domain as well as by the repeat copy number. To provide better understanding of the evolutionary and functional history of repeating domains, we investigated the mode of evolution of the filamin domain in some detail. Among the domains that are common in long repeat proteins, sushi and spectrin domains evolve primarily through cassette tandem duplications while scavenger and immunoglobulin repeats appear to evolve through clustered tandem duplications. Additionally, immunoglobulin and filamin repeats exhibit a unique pattern where every other domain shows high sequence similarity. This pattern may be the result of tandem duplications, serve to avert aggregation between adjacent domains or it is the result of functional constraints. In filamin, our studies confirm the presence of interspersed integrin binding domains in vertebrates, while invertebrates exhibit more varied patterns, including more clustered integrin binding domains. The most notable case is leech filamin, which contains a 20 repeat expansion and exhibits unique dimerization topology. Clearly, invertebrate filamins are varied and contain examples of similar adjacent integrin-binding domains. Given that invertebrate integrin shows more similarity to the weaker filamin binder, integrin β3, it is possible that the distance between integrin-binding domains is not as crucial for invertebrate filamins as for vertebrates. Copyright © 2012 Elsevier Inc. All rights reserved.

Hypothalamic expression of Peg3 gene is associated with maternal care differences between SM/J and LG/J mouse strains

PubMed Central

Chiavegatto, Silvana; Sauce, Bruno; Ambar, Guilherme; Cheverud, James M; Peripato, Andrea C

2012-01-01

Maternal care is essential in mammals, and variations in the environment provided by mothers may directly influence the viability of newborns and emotional behavior later in life. A previous study investigated genetic variations associated with maternal care in an intercross of LG/J and SM/J inbred mouse strains and identified two single-locus QTLs (quantitative trait loci). Here, we selected three candidate genes located within these QTLs intervals; Oxt on chromosome 2, and FosB and Peg3 on chromosome 7 and tested their association with maternal care. LG/J females showed impaired postpartum nest building and pup retrieval, a one-day delay in milk ejection, reduced exploratory activity, and higher anxiety-like behavior when compared to SM/J females. The nucleotide sequences of Oxt and FosB were similar between strains, as were their hypothalamic expression levels. Conversely, Peg3 nucleotide sequences showed four nonsynonymous replacement substitutions on LG/J dams, T11062G, G13744A, A13808G, and G13813A, and a 30 base pair (10 aa) in tandem repeat in the coding region with three copies in SM/J and five copies in LG/J. Maternal care impaired LG/J mothers express 37% lower Peg3 mRNA levels in the hypothalamus on the second postpartum day. We also found an association of the Peg3 repeat-variant and poor maternal care in F2 heterozygote females derived from a LG/J × SM/J intercross. These results may suggest that the maternally imprinted Peg3 gene is responsible for the single-locus QTL on chromosome 7 that has been shown to influence maternal care in these strains. Furthermore, these data provide additional support for an epigenetic regulation of maternal behavior. PMID:22950040

Comparative Study of IS6110 Restriction Fragment Length Polymorphism and Variable-Number Tandem-Repeat Typing of Mycobacterium tuberculosis Isolates in the Netherlands, Based on a 5-Year Nationwide Survey

PubMed Central

de Beer, Jessica L.; van Ingen, Jakko; de Vries, Gerard; Erkens, Connie; Sebek, Maruschka; Mulder, Arnout; Sloot, Rosa; van den Brandt, Anne-Marie; Enaimi, Mimount; Kremer, Kristin; Supply, Philip

2013-01-01

In order to switch from IS6110 and polymorphic GC-rich repetitive sequence (PGRS) restriction fragment length polymorphism (RFLP) to 24-locus variable-number tandem-repeat (VNTR) typing of Mycobacterium tuberculosis complex isolates in the national tuberculosis control program in The Netherlands, a detailed evaluation on discriminatory power and agreement with findings in a cluster investigation was performed on 3,975 tuberculosis cases during the period of 2004 to 2008. The level of discrimination of the two typing methods did not differ substantially: RFLP typing yielded 2,733 distinct patterns compared to 2,607 in VNTR typing. The global concordance, defined as isolates labeled unique or identically distributed in clusters by both methods, amounted to 78.5% (n = 3,123). Of the remaining 855 cases, 12% (n = 479) of the cases were clustered only by VNTR, 7.7% (n = 305) only by RFLP typing, and 1.8% (n = 71) revealed different cluster compositions in the two approaches. A cluster investigation was performed for 87% (n = 1,462) of the cases clustered by RFLP. For the 740 cases with confirmed or presumed epidemiological links, 92% were concordant with VNTR typing. In contrast, only 64% of the 722 cases without an epidemiological link but clustered by RFLP typing were also clustered by VNTR typing. We conclude that VNTR typing has a discriminatory power equal to IS6110 RFLP typing but is in better agreement with findings in a cluster investigation performed on an RFLP-clustering-based cluster investigation. Both aspects make VNTR typing a suitable method for tuberculosis surveillance systems. PMID:23363841

Ruminant Rhombencephalitis-Associated Listeria monocytogenes Alleles Linked to a Multilocus Variable-Number Tandem-Repeat Analysis Complex ▿ †

PubMed Central

Balandyté, Lina; Brodard, Isabelle; Frey, Joachim; Oevermann, Anna; Abril, Carlos

2011-01-01

Listeria monocytogenes is among the most important food-borne pathogens and is well adapted to persist in the environment. To gain insight into the genetic relatedness and potential virulence of L. monocytogenes strains causing central nervous system (CNS) infections, we used multilocus variable-number tandem-repeat analysis (MLVA) to subtype 183 L. monocytogenes isolates, most from ruminant rhombencephalitis and some from human patients, food, and the environment. Allelic-profile-based comparisons grouped L. monocytogenes strains mainly into three clonal complexes and linked single-locus variants (SLVs). Clonal complex A essentially consisted of isolates from human and ruminant brain samples. All but one rhombencephalitis isolate from cattle were located in clonal complex A. In contrast, food and environmental isolates mainly clustered into clonal complex C, and none was classified as clonal complex A. Isolates of the two main clonal complexes (A and C) obtained by MLVA were analyzed by PCR for the presence of 11 virulence-associated genes (prfA, actA, inlA, inlB, inlC, inlD, inlE, inlF, inlG, inlJ, and inlC2H). Virulence gene analysis revealed significant differences in the actA, inlF, inlG, and inlJ allelic profiles between clinical isolates (complex A) and nonclinical isolates (complex C). The association of particular alleles of actA, inlF, and newly described alleles of inlJ with isolates from CNS infections (particularly rhombencephalitis) suggests that these virulence genes participate in neurovirulence of L. monocytogenes. The overall absence of inlG in clinical complex A and its presence in complex C isolates suggests that the InlG protein is more relevant for the survival of L. monocytogenes in the environment. PMID:21984240

Occurrence and Nature of Double Alleles in Variable-Number Tandem-Repeat Patterns of More than 8,000 Mycobacterium tuberculosis Complex Isolates in The Netherlands

PubMed Central

Kamst, Miranda; van Hunen, Rianne; de Zwaan, Carolina Catherina; Mulder, Arnout; Supply, Philip; Anthony, Richard; van der Hoek, Wim; van Soolingen, Dick

2017-01-01

ABSTRACT Since 2004, variable-number tandem-repeat (VNTR) typing of Mycobacterium tuberculosis complex isolates has been applied on a structural basis in The Netherlands to study the epidemiology of tuberculosis (TB). Although this technique is faster and technically less demanding than the previously used restriction fragment length polymorphism (RFLP) typing, reproducibility remains a concern. In the period from 2004 to 2015, 8,532 isolates were subjected to VNTR typing in The Netherlands, with 186 (2.2%) of these exhibiting double alleles at one locus. Double alleles were most common in loci 4052 and 2163b. The variables significantly associated with double alleles were urban living (odds ratio [OR], 1.503; 95% confidence interval [CI], 1.084 to 2.084; P = 0.014) and pulmonary TB (OR, 1.703; 95% CI, 1.216 to 2.386; P = 0.002). Single-colony cultures of double-allele strains were produced and revealed single-allele profiles; a maximum of five single nucleotide polymorphisms (SNPs) was observed between the single- and double-allele isolates from the same patient when whole-genome sequencing (WGS) was applied. This indicates the presence of two bacterial populations with slightly different VNTR profiles in the parental population, related to genetic drift. This observation is confirmed by the fact that secondary cases from TB source cases with double-allele isolates sometimes display only one of the two alleles present in the source case. Double alleles occur at a frequency of 2.2% in VNTR patterns in The Netherlands. They are caused by biological variation rather than by technical aberrations and can be transmitted either as single- or double-allele variants. PMID:29142049

Tissue identity testing of cancer by short tandem repeat polymorphism: pitfalls of interpretation in the presence of microsatellite instability.

PubMed

Much, Melissa; Buza, Natalia; Hui, Pei

2014-03-01

Tissue identity testing by short tandem repeat (STR) polymorphism offers discriminating power in resolving tissue mix-up or contamination. However, one caveat is the presence of microsatellite unstable tumors, in which genetic alterations may drastically change the STR wild-type polymorphism leading to unexpected allelic discordance. We examined how tissue identity testing results can be altered by the presence of microsatellite instability (MSI). Eleven cases of MSI-unstable (9 intestinal and 2 endometrial adenocarcinomas) and 10 cases of MSI-stable tumors (all colorectal adenocarcinomas) were included. All had been previously tested by polymerase chain reaction testing at 5 National Cancer Institute (NCI) recommended MSI loci and/or immunohistochemistry for DNA mismatch repair proteins (MLH1, MSH2, MSH6, and PMS2). Tissue identity testing targeting 15 STR loci was performed using AmpF/STR Identifiler Amplification. Ten of 11 MSI-unstable tumors demonstrated novel alleles at 5 to 12 STR loci per case and frequently with 3 or more allelic peaks. However, all affected loci showed identifiable germline allele(s) in MSI-high tumors. A wild-type allelic profile was seen in 7 of 10 MSI-stable tumors. In the remaining 3 cases, isolated novel alleles were present at a unique single locus in addition to germline alleles. Loss of heterozygosity was observed frequently in both MSI-stable (6/11 cases) and MSI-unstable tumors (8/10 cases). In conclusion, MSI may significantly alter the wild-type allelic polymorphism, leading to potential interpretation errors of STR genotyping. Careful examination of the STR allelic pattern, high index of suspicion, and follow-up MSI testing are crucial to avoid erroneous conclusions and subsequent clinical and legal consequences. Copyright © 2014 Elsevier Inc. All rights reserved.

Subtyping of Clostridium difficile PCR ribotypes 591, 106 and 002, the dominant strain types circulating in Medellin, Colombia.

PubMed

Salazar, Clara Lina; Reyes, Catalina; Cienfuegos-Gallet, Astrid Vanessa; Best, Emma; Atehortua, Santiago; Sierra, Patricia; Correa, Margarita M; Fawley, Warren N; Paredes-Sabja, Daniel; Wilcox, Mark; Gonzalez, Angel

2018-01-01

We aimed to achieve a higher typing resolution within the three dominant Clostridium difficile ribotypes (591,106 and 002) circulating in Colombia. A total of 50 C. difficile isolates we had previously typed by PCR-ribotyping, representing the major three ribotypes circulating in Colombia, were analyzed. Twenty-seven isolates of ribotype 591, 12 of ribotype 106 and 11 of ribotype 002 were subtyped by multiple locus variable-number tandem-repeat analysis (MLVA). The presence of the PaLoc genes (tcdA/tcdB), toxin production in culture and antimicrobial susceptibility were also determined. From the total C. difficile ribotypes analyzed, 20 isolates (74%) of ribotype 591, nine (75%) of ribotype 106 and five (45.5%) of ribotype 002 were recovered from patients with Clostridium difficile infection (CDI). MLVA allowed us to recognize four and two different clonal complexes for ribotypes 591 and 002, respectively, having a summed tandem-repeat difference (STRD) <2, whereas none of the ribotype 106 isolates were grouped in a cluster or clonal complex having a STRD >10. Six ribotype 591 and three ribotype 002 isolates belonging to a defined clonal complex were isolated on the same week in two different hospitals. All ribotypes harbored either tcdA+/tcdB+ or tcdA-/tcdB+ PaLoc genes. Moreover, 94% of the isolates were positive for toxin in culture. All isolates were susceptible to vancomycin and metronidazole, while 75% to 100% of the isolates were resistant to clindamycin, and less than 14.8% of ribotype 591 isolates were resistant to moxifloxacina. No significant differences were found among ribotypes with respect to demographic and clinical patients' data; however, our results demonstrated a high molecular heterogeneity of C. difficile strains circulating in Colombia.

Subtyping of Clostridium difficile PCR ribotypes 591, 106 and 002, the dominant strain types circulating in Medellin, Colombia

PubMed Central

Salazar, Clara Lina; Reyes, Catalina; Cienfuegos-Gallet, Astrid Vanessa; Best, Emma; Atehortua, Santiago; Sierra, Patricia; Correa, Margarita M.; Fawley, Warren N.; Paredes-Sabja, Daniel; Wilcox, Mark

2018-01-01

We aimed to achieve a higher typing resolution within the three dominant Clostridium difficile ribotypes (591,106 and 002) circulating in Colombia. A total of 50 C. difficile isolates we had previously typed by PCR-ribotyping, representing the major three ribotypes circulating in Colombia, were analyzed. Twenty-seven isolates of ribotype 591, 12 of ribotype 106 and 11 of ribotype 002 were subtyped by multiple locus variable-number tandem-repeat analysis (MLVA). The presence of the PaLoc genes (tcdA/tcdB), toxin production in culture and antimicrobial susceptibility were also determined. From the total C. difficile ribotypes analyzed, 20 isolates (74%) of ribotype 591, nine (75%) of ribotype 106 and five (45.5%) of ribotype 002 were recovered from patients with Clostridium difficile infection (CDI). MLVA allowed us to recognize four and two different clonal complexes for ribotypes 591 and 002, respectively, having a summed tandem-repeat difference (STRD) <2, whereas none of the ribotype 106 isolates were grouped in a cluster or clonal complex having a STRD >10. Six ribotype 591 and three ribotype 002 isolates belonging to a defined clonal complex were isolated on the same week in two different hospitals. All ribotypes harbored either tcdA+/tcdB+ or tcdA-/tcdB+ PaLoc genes. Moreover, 94% of the isolates were positive for toxin in culture. All isolates were susceptible to vancomycin and metronidazole, while 75% to 100% of the isolates were resistant to clindamycin, and less than 14.8% of ribotype 591 isolates were resistant to moxifloxacina. No significant differences were found among ribotypes with respect to demographic and clinical patients’ data; however, our results demonstrated a high molecular heterogeneity of C. difficile strains circulating in Colombia. PMID:29649308

Molecular typing of Brucella melitensis endemic strains and differentiation from the vaccine strain Rev-1.

PubMed

Noutsios, Georgios T; Papi, Rigini M; Ekateriniadou, Loukia V; Minas, Anastasios; Kyriakidis, Dimitrios A

2012-03-01

In the present study forty-four Greek endemic strains of Br. melitensis and three reference strains were genotyped by Multi locus Variable Number Tandem Repeat (ML-VNTR) analysis based on an eight-base pair tandem repeat sequence that was revealed in eight loci of Br. melitensis genome. The forty-four strains were discriminated from the vaccine strain Rev-1 by Restriction Fragment Length Polymorphism (RFLP) and Denaturant Gradient Gel Electrophoresis (DGGE). The ML-VNTR analysis revealed that endemic, reference and vaccine strains are genetically closely related, while most of the loci tested (1, 2, 4, 5 and 7) are highly polymorphic with Hunter-Gaston Genetic Diversity Index (HGDI) values in the range of 0.939 to 0.775. Analysis of ML-VNTRs loci stability through in vitro passages proved that loci 1 and 5 are non stable. Therefore, vaccine strain can be discriminated from endemic strains by allele's clusters of loci 2, 4, 6 and 7. RFLP and DGGE were also employed to analyse omp2 gene and reveled different patterns among Rev-1 and endemic strains. In RFLP, Rev-1 revealed three fragments (282, 238 and 44 bp), while endemic strains two fragments (238 and 44 bp). As for DGGE, the electrophoretic mobility of Rev-1 is different from the endemic strains due to heterologous binding of DNA chains of omp2a and omp2b gene. Overall, our data show clearly that it is feasible to genotype endemic strains of Br. melitensis and differentiate them from vaccine strain Rev-1 with ML-VNTR, RFLP and DGGE techniques. These tools can be used for conventional investigations in brucellosis outbreaks.

Genotyping of Mycobacterium leprae strains from a region of high endemic leprosy prevalence in India.

PubMed

Lavania, Mallika; Jadhav, Rupendra; Turankar, Ravindra P; Singh, Itu; Nigam, Astha; Sengupta, U

2015-12-01

Leprosy is still a major health problem in India which has the highest number of cases. Multiple locus variable number of tandem repeat analysis (MLVA) and single nucleotide polymorphism (SNP) have been proposed as tools of strain typing for tracking the transmission of leprosy. However, empirical data for a defined population from scale and duration were lacking for studying the transmission chain of leprosy. Seventy slit skin scrapings were collected from Purulia (West Bengal), Miraj (Maharashtra), Shahdara (Delhi), and Naini (UP) hospitals of The Leprosy Mission (TLM). SNP subtyping and MLVA on 10 VNTR loci were applied for the strain typing of Mycobacterium leprae. Along with the strain typing conventional epidemiological investigation was also performed to trace the transmission chain. In addition, phylogenetic analysis was done on variable number of tandem repeat (VNTR) data sets using sequence type analysis and recombinational tests (START) software. START software performs analyses to aid in the investigation of bacterial population structure using multilocus sequence data. These analyses include data summary, lineage assignment, and tests for recombination and selection. Diversity was observed in the cross-sectional survey of isolates obtained from 70 patients. Similarity in fingerprinting profiles observed in specimens of cases from the same family or neighborhood locations indicated a possible common source of infection. The data suggest that these VNTRs including subtyping of SNPs can be used to study the sources and transmission chain in leprosy, which could be very important in monitoring of the disease dynamics in high endemic foci. The present study strongly indicates that multi-case families might constitute epidemic foci and the main source of M. leprae in villages, causing the predominant strain or cluster infection leading to the spread of leprosy in the community. Copyright © 2015 Elsevier B.V. All rights reserved.

Variable Number of Tandem Repeats in Salmonella enterica subsp. enterica for Typing Purposes

PubMed Central

Ramisse, Vincent; Houssu, Perrine; Hernandez, Eric; Denoeud, France; Hilaire, Valérie; Lisanti, Olivier; Ramisse, Françoise; Cavallo, Jean-Didier; Vergnaud, Gilles

2004-01-01

The genomic sequences of Salmonella enterica subsp. enterica strains CT18, Ty2 (serovar Typhi), and LT2 (serovar Typhimurium) were analyzed for potential variable number tandem repeats (VNTRs). A multiple-locus VNTR analysis (MLVA) of 99 strains of S. enterica supsp. enterica based on 10 VNTRs distinguished 52 genotypes and placed them into four groups. All strains tested were independent human isolates from France and did not reflect isolates from outbreak episodes. Of these 10 VNTRs, 7 showed variability within serovar Typhi, whereas 1 showed variability within serovar Typhimurium. Four VNTRs showed high Nei's diversity indices (DIs) of 0.81 to 0.87 within serovar Typhi (n = 27). Additionally, three of these more variable VNTRs showed DIs of 0.18 to 0.58 within serovar Paratyphi A (n = 10). The VNTR polymorphic site within multidrug-resistant (MDR) serovar Typhimurium isolates (n = 39; resistance to ampicillin, chloramphenicol, spectinomycin, sulfonamides, and tetracycline) showed a DI of 0.81. Cluster analysis not only identified three genetically distinct groups consistent with the present serovar classification of salmonellae (serovars Typhi, Paratyphi A, and Typhimurium) but also discriminated 25 subtypes (93%) within serovar Typhi isolates. The analysis discriminated only eight subtypes within serovar Typhimurium isolates resistant to ampicillin, chloramphenicol, spectinomycin, sulfonamides, and tetracycline, possibly reflecting the emergence in the mid-1990s of the DT104 phage type, which often displays such an MDR spectrum. Coupled with the ongoing improvements in automated procedures offered by capillary electrophoresis, use of these markers is proposed in further investigations of the potential of MLVA in outbreaks of salmonellosis, especially outbreaks of typhoid fever. PMID:15583305

A MLVA Genotyping Scheme for Global Surveillance of the Citrus Pathogen Xanthomonas citri pv. citri Suggests a Worldwide Geographical Expansion of a Single Genetic Lineage

PubMed Central

Boyer, Karine; Leduc, Alice; Tourterel, Christophe; Drevet, Christine; Ravigné, Virginie; Gagnevin, Lionel; Guérin, Fabien; Chiroleu, Frédéric; Koebnik, Ralf; Verdier, Valérie; Vernière, Christian

2014-01-01

MultiLocus Variable number of tandem repeat Analysis (MLVA) has been extensively used to examine epidemiological and evolutionary issues on monomorphic human pathogenic bacteria, but not on bacterial plant pathogens of agricultural importance albeit such tools would improve our understanding of their epidemiology, as well as of the history of epidemics on a global scale. Xanthomonas citri pv. citri is a quarantine organism in several countries and a major threat for the citrus industry worldwide. We screened the genomes of Xanthomonas citri pv. citri strain IAPAR 306 and of phylogenetically related xanthomonads for tandem repeats. From these in silico data, an optimized MLVA scheme was developed to assess the global diversity of this monomorphic bacterium. Thirty-one minisatellite loci (MLVA-31) were selected to assess the genetic structure of 129 strains representative of the worldwide pathological and genetic diversity of X. citri pv. citri. Based on Discriminant Analysis of Principal Components (DAPC), four pathotype-specific clusters were defined. DAPC cluster 1 comprised strains that were implicated in the major geographical expansion of X. citri pv. citri during the 20th century. A subset of 12 loci (MLVA-12) resolved 89% of the total diversity and matched the genetic structure revealed by MLVA-31. MLVA-12 is proposed for routine epidemiological identification of X. citri pv. citri, whereas MLVA-31 is proposed for phylogenetic and population genetics studies. MLVA-31 represents an opportunity for international X. citri pv. citri genotyping and data sharing. The MLVA-31 data generated in this study was deposited in the Xanthomonas citri genotyping database (http://www.biopred.net/MLVA/). PMID:24897119

Analysis of Salmonella enterica Serovar Typhimurium Variable-Number Tandem-Repeat Data for Public Health Investigation Based on Measured Mutation Rates and Whole-Genome Sequence Comparisons

PubMed Central

Dimovski, Karolina; Cao, Hanwei; Wijburg, Odilia L. C.; Strugnell, Richard A.; Mantena, Radha K.; Whipp, Margaret; Hogg, Geoff

2014-01-01

Variable-number tandem repeats (VNTRs) mutate rapidly and can be useful markers for genotyping. While multilocus VNTR analysis (MLVA) is increasingly used in the detection and investigation of food-borne outbreaks caused by Salmonella enterica serovar Typhimurium (S. Typhimurium) and other bacterial pathogens, MLVA data analysis usually relies on simple clustering approaches that may lead to incorrect interpretations. Here, we estimated the rates of copy number change at each of the five loci commonly used for S. Typhimurium MLVA, during in vitro and in vivo passage. We found that loci STTR5, STTR6, and STTR10 changed during passage but STTR3 and STTR9 did not. Relative rates of change were consistent across in vitro and in vivo growth and could be accurately estimated from diversity measures of natural variation observed during large outbreaks. Using a set of 203 isolates from a series of linked outbreaks and whole-genome sequencing of 12 representative isolates, we assessed the accuracy and utility of several alternative methods for analyzing and interpreting S. Typhimurium MLVA data. We show that eBURST analysis was accurate and informative. For construction of MLVA-based trees, a novel distance metric, based on the geometric model of VNTR evolution coupled with locus-specific weights, performed better than the commonly used simple or categorical distance metrics. The data suggest that, for the purpose of identifying potential transmission clusters for further investigation, isolates whose profiles differ at one of the rapidly changing STTR5, STTR6, and STTR10 loci should be collapsed into the same cluster. PMID:24957617

Variable-number tandem repeats as molecular markers for biotypes of Pasteuria ramosa in Daphnia spp.

PubMed

Mouton, Laurence; Nong, Guang; Preston, James F; Ebert, Dieter

2007-06-01

Variable-number tandem repeats (VNTRs) have been identified in populations of Pasteuria ramosa, a castrating endobacterium of Daphnia species. The allelic polymorphisms at 14 loci in laboratory and geographically diverse soil samples showed that VNTRs may serve as biomarkers for the genetic characterization of P. ramosa isolates.

Multiple independent insertions of 5S rRNA genes in the spliced-leader gene family of trypanosome species.

PubMed

Beauparlant, Marc A; Drouin, Guy

2014-02-01

Analyses of the 5S rRNA genes found in the spliced-leader (SL) gene repeat units of numerous trypanosome species suggest that such linkages were not inherited from a common ancestor, but were the result of independent 5S rRNA gene insertions. In trypanosomes, 5S rRNA genes are found either in the tandemly repeated units coding for SL genes or in independent tandemly repeated units. Given that trypanosome species where 5S rRNA genes are within the tandemly repeated units coding for SL genes are phylogenetically related, one might hypothesize that this arrangement is the result of an ancestral insertion of 5S rRNA genes into the tandemly repeated SL gene family of trypanosomes. Here, we use the types of 5S rRNA genes found associated with SL genes, the flanking regions of the inserted 5S rRNA genes and the position of these insertions to show that most of the 5S rRNA genes found within SL gene repeat units of trypanosome species were not acquired from a common ancestor but are the results of independent insertions. These multiple 5S rRNA genes insertion events in trypanosomes are likely the result of frequent founder events in different hosts and/or geographical locations in species having short generation times.

Genome-wide analysis of tandem repeats in plants and green algae

Treesearch

Zhixin Zhao; Cheng Guo; Sreeskandarajan Sutharzan; Pei Li; Craig Echt; Jie Zhang; Chun Liang

2014-01-01

Tandem repeats (TRs) extensively exist in the genomes of prokaryotes and eukaryotes. Based on the sequenced genomes and gene annotations of 31 plant and algal species in Phytozome version 8.0 (http://www.phytozome.net/), we examined TRs in a genome-wide scale, characterized their distributions and motif features, and explored their putative biological functions. Among...

Versatile communication strategies among tandem WW domain repeats

PubMed Central

Dodson, Emma Joy; Fishbain-Yoskovitz, Vered; Rotem-Bamberger, Shahar

2015-01-01

Interactions mediated by short linear motifs in proteins play major roles in regulation of cellular homeostasis since their transient nature allows for easy modulation. We are still far from a full understanding and appreciation of the complex regulation patterns that can be, and are, achieved by this type of interaction. The fact that many linear-motif-binding domains occur in tandem repeats in proteins indicates that their mutual communication is used extensively to obtain complex integration of information toward regulatory decisions. This review is an attempt to overview, and classify, different ways by which two and more tandem repeats cooperate in binding to their targets, in the well-characterized family of WW domains and their corresponding polyproline ligands. PMID:25710931

QTL Mapping of Sex Determination Loci Supports an Ancient Pathway in Ants and Honey Bees.

PubMed

Miyakawa, Misato O; Mikheyev, Alexander S

2015-11-01

Sex determination mechanisms play a central role in life-history characteristics, affecting mating systems, sex ratios, inbreeding tolerance, etc. Downstream components of sex determination pathways are highly conserved, but upstream components evolve rapidly. Evolutionary dynamics of sex determination remain poorly understood, particularly because mechanisms appear so diverse. Here we investigate the origins and evolution of complementary sex determination (CSD) in ants and bees. The honey bee has a well-characterized CSD locus, containing tandemly arranged homologs of the transformer gene [complementary sex determiner (csd) and feminizer (fem)]. Such tandem paralogs appear frequently in aculeate hymenopteran genomes. However, only comparative genomic, but not functional, data support a broader role for csd/fem in sex determination, and whether species other than the honey bee use this pathway remains controversial. Here we used a backcross to test whether csd/fem acts as a CSD locus in an ant (Vollenhovia emeryi). After sequencing and assembling the genome, we computed a linkage map, and conducted a quantitative trait locus (QTL) analysis of diploid male production using 68 diploid males and 171 workers. We found two QTLs on separate linkage groups (CsdQTL1 and CsdQTL2) that jointly explained 98.0% of the phenotypic variance. CsdQTL1 included two tandem transformer homologs. These data support the prediction that the same CSD mechanism has indeed been conserved for over 100 million years. CsdQTL2 had no similarity to CsdQTL1 and included a 236-kb region with no obvious CSD gene candidates, making it impossible to conclusively characterize it using our data. The sequence of this locus was conserved in at least one other ant genome that diverged >75 million years ago. By applying QTL analysis to ants for the first time, we support the hypothesis that elements of hymenopteran CSD are ancient, but also show that more remains to be learned about the diversity of CSD mechanisms.

Cis-acting regulatory sequences promote high-frequency gene conversion between repeated sequences in mammalian cells.

PubMed

Raynard, Steven J; Baker, Mark D

2004-01-01

In mammalian cells, little is known about the nature of recombination-prone regions of the genome. Previously, we reported that the immunoglobulin heavy chain (IgH) mu locus behaved as a hotspot for mitotic, intrachromosomal gene conversion (GC) between repeated mu constant (Cmu) regions in mouse hybridoma cells. To investigate whether elements within the mu gene regulatory region were required for hotspot activity, gene targeting was used to delete a 9.1 kb segment encompassing the mu gene promoter (Pmu), enhancer (Emu) and switch region (Smu) from the locus. In these cell lines, GC between the Cmu repeats was significantly reduced, indicating that this 'recombination-enhancing sequence' (RES) is necessary for GC hotspot activity at the IgH locus. Importantly, the RES fragment stimulated GC when appended to the same Cmu repeats integrated at ectopic genomic sites. We also show that deletion of Emu and flanking matrix attachment regions (MARs) from the RES abolishes GC hotspot activity at the IgH locus. However, no stimulation of ectopic GC was observed with the Emu/MARs fragment alone. Finally, we provide evidence that no correlation exists between the level of transcription and GC promoted by the RES. We suggest a model whereby Emu/MARS enhances mitotic GC at the endogenous IgH mu locus by effecting chromatin modifications in adjacent DNA.

Identification of the third/extra allele for forensic application in cases with TPOX tri-allelic pattern.

PubMed

Picanço, Juliane Bentes; Raimann, Paulo Eduardo; Motta, Carlos Henrique Ares Silveira da; Rodenbusch, Rodrigo; Gusmão, Leonor; Alho, Clarice Sampaio

2015-05-01

Genotyping of polymorphic short tandem repeats (STRs) loci is widely used in forensic DNA analysis. STR loci eventually present tri-allelic pattern as a genotyping irregularity and, in that situation, the doubt about the tri-allele locus frequency calculation can reduce the analysis strength. In the TPOX human STR locus, tri-allelic genotypes have been reported with a widely varied frequency among human populations. We investigate whether there is a single extra allele (the third allele) in the TPOX tri-allelic pattern, what it is, and where it is, aiming to understand its genomic anatomy and to propose the knowledge of this TPOX extra allele from genetic profile, thus preserving the two standard TPOX alleles in forensic analyses. We looked for TPOX tri-allelic subjects in 75,113 Brazilian families. Considering only the parental generation (mother+father) we had 150,226 unrelated subjects evaluated. From this total, we found 88 unrelated subjects with tri-allelic pattern in the TPOX locus (0.06%; 88/150,226). Seventy three of these 88 subjects (73/88; 83%) had the Clayton's original Type 2 tri-allelic pattern (three peaks of even intensity). The remaining 17% (15/88) show a new Type 2 derived category with heterozygote peak imbalance (one double dose peak plus one regular sized peak). In this paper we present detailed data from 66 trios (mother+father+child) with true biological relationships. In 39 of these families (39/66; 59%) the extra TPOX allele was transmitted either from the mother or from the father to the child. Evidences indicated the allele 10 as the extra TPOX allele, and it is on the X chromosome. The present data, which support the previous Lane hypothesis, improve the knowledge about tri-allelic pattern of TPOX CODIS' locus allowing the use of TPOX profile in forensic analyses even when with tri-allelic pattern. This evaluation is now available for different forensic applications. Copyright © 2014 Elsevier Ireland Ltd. All rights reserved.

Sunflower centromeres consist of a centromere-specific LINE and a chromosome-specific tandem repeat.

PubMed

Nagaki, Kiyotaka; Tanaka, Keisuke; Yamaji, Naoki; Kobayashi, Hisato; Murata, Minoru

2015-01-01

The kinetochore is a protein complex including kinetochore-specific proteins that plays a role in chromatid segregation during mitosis and meiosis. The complex associates with centromeric DNA sequences that are usually species-specific. In plant species, tandem repeats including satellite DNA sequences and retrotransposons have been reported as centromeric DNA sequences. In this study on sunflowers, a cDNA-encoding centromere-specific histone H3 (CENH3) was isolated from a cDNA pool from a seedling, and an antibody was raised against a peptide synthesized from the deduced cDNA. The antibody specifically recognized the sunflower CENH3 (HaCENH3) and showed centromeric signals by immunostaining and immunohistochemical staining analysis. The antibody was also applied in chromatin immunoprecipitation (ChIP)-Seq to isolate centromeric DNA sequences and two different types of repetitive DNA sequences were identified. One was a long interspersed nuclear element (LINE)-like sequence, which showed centromere-specific signals on almost all chromosomes in sunflowers. This is the first report of a centromeric LINE sequence, suggesting possible centromere targeting ability. Another type of identified repetitive DNA was a tandem repeat sequence with a 187-bp unit that was found only on a pair of chromosomes. The HaCENH3 content of the tandem repeats was estimated to be much higher than that of the LINE, which implies centromere evolution from LINE-based centromeres to more stable tandem-repeat-based centromeres. In addition, the epigenetic status of the sunflower centromeres was investigated by immunohistochemical staining and ChIP, and it was found that centromeres were heterochromatic.

Evolution of short inverted repeat in cupressophytes, transfer of accD to nucleus in Sciadopitys verticillata and phylogenetic position of Sciadopityaceae.

PubMed

Li, Jia; Gao, Lei; Chen, Shanshan; Tao, Ke; Su, Yingjuan; Wang, Ting

2016-02-11

Sciadopitys verticillata is an evergreen conifer and an economically valuable tree used in construction, which is the only member of the family Sciadopityaceae. Acquisition of the S. verticillata chloroplast (cp) genome will be useful for understanding the evolutionary mechanism of conifers and phylogenetic relationships among gymnosperm. In this study, we have first reported the complete chloroplast genome of S. verticillata. The total genome is 138,284 bp in length, consisting of 118 unique genes. The S. verticillata cp genome has lost one copy of the canonical inverted repeats and shown distinctive genomic structure comparing with other cupressophytes. Fifty-three simple sequence repeat loci and 18 forward tandem repeats were identified in the S. verticillata cp genome. According to the rearrangement of cupressophyte cp genome, we proposed one mechanism for the formation of inverted repeat: tandem repeat occured first, then rearrangement divided the tandem repeat into inverted repeats located at different regions. Phylogenetic estimates inferred from 59-gene sequences and cpDNA organizations have both shown that S. verticillata was sister to the clade consisting of Cupressaceae, Taxaceae, and Cephalotaxaceae. Moreover, accD gene was found to be lost in the S. verticillata cp genome, and a nucleus copy was identified from two transcriptome data.

Application of mixsep software package: Performance verification of male-mixed DNA analysis

PubMed Central

HU, NA; CONG, BIN; GAO, TAO; CHEN, YU; SHEN, JUNYI; LI, SHUJIN; MA, CHUNLING

2015-01-01

An experimental model of male-mixed DNA (n=297) was constructed according to the mixed DNA construction principle. This comprised the use of the Applied Biosystems (ABI) 7500 quantitative polymerase chain reaction system, with scientific validation of mixture proportion (Mx; root-mean-square error ≤0.02). Statistical analysis was performed on locus separation accuracy using mixsep, a DNA mixture separation R-package, and the analytical performance of mixsep was assessed by examining the data distribution pattern of different mixed gradients, short tandem repeat (STR) loci and mixed DNA types. The results showed that locus separation accuracy had a negative linear correlation with the mixed gradient (R2=−0.7121). With increasing mixed gradient imbalance, locus separation accuracy first increased and then decreased, with the highest value detected at a gradient of 1:3 (≥90%). The mixed gradient, which is the theoretical Mx, was one of the primary factors that influenced the success of mixed DNA analysis. Among the 16 STR loci detected by Identifiler®, the separation accuracy was relatively high (>88%) for loci D5S818, D8S1179 and FGA, whereas the median separation accuracy value was lowest for the D7S820 locus. STR loci with relatively large numbers of allelic drop-out (ADO; >15) were all located in the yellow and red channels, including loci D18S51, D19S433, FGA, TPOX and vWA. These five loci featured low allele peak heights, which was consistent with the low sensitivity of the ABI 3130xl Genetic Analyzer to yellow and red fluorescence. The locus separation accuracy of the mixsep package was substantially different with and without the inclusion of ADO loci; inclusion of ADO significantly reduced the analytical performance of the mixsep package, which was consistent with the lack of an ADO functional module in this software. The present study demonstrated that the mixsep software had a number of advantages and was recommended for analysis of mixed DNA. This software was easy to operate and produced understandable results with a degree of controllability. PMID:25936428

Molecular Typing of Mycobacterium Tuberculosis Complex by 24-Locus Based MIRU-VNTR Typing in Conjunction with Spoligotyping to Assess Genetic Diversity of Strains Circulating in Morocco

PubMed Central

Bouklata, Nada; Supply, Philip; Jaouhari, Sanae; Charof, Reda; Seghrouchni, Fouad; Sadki, Khalid; El Achhab, Youness; Nejjari, Chakib; Filali-Maltouf, Abdelkarim

2015-01-01

Background Standard 24-locus Mycobacterial Interspersed Repetitive Unit Variable Number Tandem Repeat (MIRU-VNTR) typing allows to get an improved resolution power for tracing TB transmission and predicting different strain (sub) lineages in a community. Methodology During 2010–2012, a total of 168 Mycobacterium tuberculosis Complex (MTBC) isolates were collected by cluster sampling from 10 different Moroccan cities, and centralized by the National Reference Laboratory of Tuberculosis over the study period. All isolates were genotyped using spoligotyping, and a subset of 75 was genotyped using 24-locus based MIRU-VNTR typing, followed by first line drug susceptibility testing. Corresponding strain lineages were predicted using MIRU-VNTRplus database. Principal Findings Spoligotyping resulted in 137 isolates in 18 clusters (2–50 isolates per cluster: clustering rate of 81.54%) corresponding to a SIT number in the SITVIT database, while 31(18.45%) patterns were unique of which 10 were labelled as “unknown” according to the same database. The most prevalent spoligotype family was LAM; (n = 81 or 48.24% of isolates, dominated by SIT42, n = 49), followed by Haarlem (23.80%), T superfamily (15.47%), >Beijing (2.97%), > U clade (2.38%) and S clade (1.19%). Subsequent 24-Locus MIRU-VNTR typing identified 64 unique types and 11 isolates in 5 clusters (2 to 3isolates per cluster), substantially reducing clusters defined by spoligotyping only. The single cluster of three isolates corresponded to two previously treated MDR-TB cases and one new MDR-TB case known to be contact a same index case and belonging to a same family, albeit residing in 3 different administrative regions. MIRU-VNTR loci 4052, 802, 2996, 2163b, 3690, 1955, 424, 2531, 2401 and 960 were highly discriminative in our setting (HGDI >0.6). Conclusions 24-locus MIRU-VNTR typing can substantially improve the resolution of large clusters initially defined by spoligotyping alone and predominating in Morocco, and could therefore be used to better study tuberculosis transmission in a population-based, multi-year sample context. PMID:26285026

Whole genome evaluation of tandem repeat polymorphisms between two pathogenically similar strains of Xylella fastidiosa isolated from almond and grape in California

USDA-ARS?s Scientific Manuscript database

Whole genome tandem repeat polymorphisms were evaluated between two closely related Xylella fastidiosa strains, M23 and Temecula1, both cause almond leaf scorch disease (ALSD) and grape Pierce’s disease (PD) in California. Strain M23 was isolated from almond and the genome was sequenced in this stu...

DNA Fingerprint Analysis of Three Short Tandem Repeat (STR) Loci for Biochemistry and Forensic Science Laboratory Courses

ERIC Educational Resources Information Center

McNamara-Schroeder, Kathleen; Olonan, Cheryl; Chu, Simon; Montoya, Maria C.; Alviri, Mahta; Ginty, Shannon; Love, John J.

2006-01-01

We have devised and implemented a DNA fingerprinting module for an upper division undergraduate laboratory based on the amplification and analysis of three of the 13 short tandem repeat loci that are required by the Federal Bureau of Investigation Combined DNA Index System (FBI CODIS) data base. Students first collect human epithelial (cheek)…

Two different size classes of 5S rDNA units coexisting in the same tandem array in the razor clam Ensis macha: is this region suitable for phylogeographic studies?

PubMed

Fernández-Tajes, Juan; Méndez, Josefina

2009-12-01

For a study of 5S ribosomal genes (rDNA) in the razor clam Ensis macha, the 5S rDNA region was amplified and sequenced. Two variants, so-called type I or short repeat (approximately 430 bp) and type II or long repeat (approximately 735 bp), appeared to be the main components of the 5S rDNA of this species. Their spacers differed markedly, both in length and nucleotide composition. The organization of the two variants was investigated by amplifying the genomic DNA with primers based on the sequence of the type I and type II spacers. PCR amplification products with primers EMLbF and EMSbR showed that the long and short repeats are associated within the same tandem array, suggesting an intermixed arrangement of both spacers. Nevertheless, amplifications carried out with inverse primers EMSinvF/R and EMLinvF/R revealed that some short and long repeats are contiguous in the same tandem array. This is the first report of the coexistence of two variable spacers in the same tandem array in bivalve mollusks.

The osteoporosis-pseudoglioma syndrome locus is on chromosome 11q

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

Gong, Y.; Vikkula, M.; Boon, L.M.

The osteoporosis-pseudoglioma syndrome (OPS), is a rare autosomal recessive disorder characterized by severe osteoporosis with multiple fractures and blindness, both occurring in childhood. The precise pathogenic mechanism for OPS is unknown. Insights into its cause may be useful towards understanding the pathophysiology of more common disorders, such as senile osteoporosis, persistent hyperplasia of the primary vitreous, and retinopathy of prematurity, whose features have some similarity with OPS. As a first step in determining the cause of OPS, we have mapped the locus of the disorder to chromosome 11q. This was accomplished by assuming genetic homogeneity and by performing linkage analysismore » with homozygosity mapping in 18 individuals (7 patients, 5 unaffected siblings, and 7 parents) from 3 different consanguineous kindreds. Since the condition could be caused by an abnormal extracellular matrix component, we began by testing several candidate genes (e.g., COL1A1, COL1A2, Osteopontin, Osteonectin) distributed on 12 different chromosomes. We also initiated a systematic search at 20 cM intervals with highly polymorphic simple sequence tandem repeats. Linkage and homozygosity was detected with marker D11S913 (LOD score 3.8 at {theta} = 0). Additional markers are being tested to confirm this observation. The fibroblast collagenase, fibronectin-like-2 gene and rod outer segment protein-1 (ROM 1) also map to chromosome 11q and are candidate genes.« less

Distribution of Bartonella henselae Variants in Patients, Reservoir Hosts and Vectors in Spain

PubMed Central

Gil, Horacio; Escudero, Raquel; Pons, Inmaculada; Rodríguez-Vargas, Manuela; García-Esteban, Coral; Rodríguez-Moreno, Isabel; García-Amil, Cristina; Lobo, Bruno; Valcárcel, Félix; Pérez, Azucena; Jiménez, Santos; Jado, Isabel; Juste, Ramón; Segura, Ferrán; Anda, Pedro

2013-01-01

We have studied the diversity of B. henselae circulating in patients, reservoir hosts and vectors in Spain. In total, we have fully characterized 53 clinical samples from 46 patients, as well as 78 B. henselae isolates obtained from 35 cats from La Rioja and Catalonia (northeastern Spain), four positive cat blood samples from which no isolates were obtained, and three positive fleas by Multiple Locus Sequence Typing and Multiple Locus Variable Number Tandem Repeats Analysis. This study represents the largest series of human cases characterized with these methods, with 10 different sequence types and 41 MLVA profiles. Two of the sequence types and 35 of the profiles were not described previously. Most of the B. henselae variants belonged to ST5. Also, we have identified a common profile (72) which is well distributed in Spain and was found to persist over time. Indeed, this profile seems to be the origin from which most of the variants identified in this study have been generated. In addition, ST5, ST6 and ST9 were found associated with felines, whereas ST1, ST5 and ST8 were the most frequent sequence types found infecting humans. Interestingly, some of the feline associated variants never found on patients were located in a separate clade, which could represent a group of strains less pathogenic for humans. PMID:23874563

Genetic zygosity of mature ovarian teratomas, struma ovarii, and ovarian carcinoids.

PubMed

Kato, Noriko; Sakamoto, Kazuhiro; Murakami, Koutaro; Iwasaki, Yuri; Kamataki, Akihisa; Kurose, Akira

2018-02-17

Although ovarian monodermal teratomas, including struma ovarii and carcinoids, are closely associated with mature teratomas, their genetic basis is poorly understood. A series of mature and monodermal ovarian teratomas were analyzed by short tandem repeat genotyping to evaluate their genetic zygosity and its associations. Informative DNA genotyping data were obtained for ten mature teratomas, six struma ovarii, and three carcinoids (one insular, one trabecular, and one mucinous). A homozygous genotype was present in five of the ten (50%) mature teratomas, three of the six (50%) struma ovarii, and one of the three (33%) ovarian carcinoids. There was no significant difference in genetic zygosity between mature and monodermal teratomas. Patients' age was not correlated with the genetic zygosity: the youngest age in the homozygous tumor group of patients was 4 years. It is suggested that an oocyte after meiosis I, which has escaped from meiotic arrest, is a significant cause of these tumors. Although one mature teratoma was a rare case with lactating adenoma-like breast tissue, its genetic zygosity was concordant with that of the surrounding teratomatous tissue. In one ovarian carcinoid, the carcinoid and accompanying teratomatous components showed matching zygosity at all but one locus: the carcinoid was heterozygous but teratoma was homozygous at one pericentromeric locus. This suggests that not all carcinoids are secondary neoplasms arising from a fully developed mature teratoma: some are neoplasms deviating from a developing mature teratoma.

A global analysis of Y-chromosomal haplotype diversity for 23 STR loci.

PubMed

Purps, Josephine; Siegert, Sabine; Willuweit, Sascha; Nagy, Marion; Alves, Cíntia; Salazar, Renato; Angustia, Sheila M T; Santos, Lorna H; Anslinger, Katja; Bayer, Birgit; Ayub, Qasim; Wei, Wei; Xue, Yali; Tyler-Smith, Chris; Bafalluy, Miriam Baeta; Martínez-Jarreta, Begoña; Egyed, Balazs; Balitzki, Beate; Tschumi, Sibylle; Ballard, David; Court, Denise Syndercombe; Barrantes, Xinia; Bäßler, Gerhard; Wiest, Tina; Berger, Burkhard; Niederstätter, Harald; Parson, Walther; Davis, Carey; Budowle, Bruce; Burri, Helen; Borer, Urs; Koller, Christoph; Carvalho, Elizeu F; Domingues, Patricia M; Chamoun, Wafaa Takash; Coble, Michael D; Hill, Carolyn R; Corach, Daniel; Caputo, Mariela; D'Amato, Maria E; Davison, Sean; Decorte, Ronny; Larmuseau, Maarten H D; Ottoni, Claudio; Rickards, Olga; Lu, Di; Jiang, Chengtao; Dobosz, Tadeusz; Jonkisz, Anna; Frank, William E; Furac, Ivana; Gehrig, Christian; Castella, Vincent; Grskovic, Branka; Haas, Cordula; Wobst, Jana; Hadzic, Gavrilo; Drobnic, Katja; Honda, Katsuya; Hou, Yiping; Zhou, Di; Li, Yan; Hu, Shengping; Chen, Shenglan; Immel, Uta-Dorothee; Lessig, Rüdiger; Jakovski, Zlatko; Ilievska, Tanja; Klann, Anja E; García, Cristina Cano; de Knijff, Peter; Kraaijenbrink, Thirsa; Kondili, Aikaterini; Miniati, Penelope; Vouropoulou, Maria; Kovacevic, Lejla; Marjanovic, Damir; Lindner, Iris; Mansour, Issam; Al-Azem, Mouayyad; Andari, Ansar El; Marino, Miguel; Furfuro, Sandra; Locarno, Laura; Martín, Pablo; Luque, Gracia M; Alonso, Antonio; Miranda, Luís Souto; Moreira, Helena; Mizuno, Natsuko; Iwashima, Yasuki; Neto, Rodrigo S Moura; Nogueira, Tatiana L S; Silva, Rosane; Nastainczyk-Wulf, Marina; Edelmann, Jeanett; Kohl, Michael; Nie, Shengjie; Wang, Xianping; Cheng, Baowen; Núñez, Carolina; Pancorbo, Marian Martínez de; Olofsson, Jill K; Morling, Niels; Onofri, Valerio; Tagliabracci, Adriano; Pamjav, Horolma; Volgyi, Antonia; Barany, Gusztav; Pawlowski, Ryszard; Maciejewska, Agnieszka; Pelotti, Susi; Pepinski, Witold; Abreu-Glowacka, Monica; Phillips, Christopher; Cárdenas, Jorge; Rey-Gonzalez, Danel; Salas, Antonio; Brisighelli, Francesca; Capelli, Cristian; Toscanini, Ulises; Piccinini, Andrea; Piglionica, Marilidia; Baldassarra, Stefania L; Ploski, Rafal; Konarzewska, Magdalena; Jastrzebska, Emila; Robino, Carlo; Sajantila, Antti; Palo, Jukka U; Guevara, Evelyn; Salvador, Jazelyn; Ungria, Maria Corazon De; Rodriguez, Jae Joseph Russell; Schmidt, Ulrike; Schlauderer, Nicola; Saukko, Pekka; Schneider, Peter M; Sirker, Miriam; Shin, Kyoung-Jin; Oh, Yu Na; Skitsa, Iulia; Ampati, Alexandra; Smith, Tobi-Gail; Calvit, Lina Solis de; Stenzl, Vlastimil; Capal, Thomas; Tillmar, Andreas; Nilsson, Helena; Turrina, Stefania; De Leo, Domenico; Verzeletti, Andrea; Cortellini, Venusia; Wetton, Jon H; Gwynne, Gareth M; Jobling, Mark A; Whittle, Martin R; Sumita, Denilce R; Wolańska-Nowak, Paulina; Yong, Rita Y Y; Krawczak, Michael; Nothnagel, Michael; Roewer, Lutz

2014-09-01

In a worldwide collaborative effort, 19,630 Y-chromosomes were sampled from 129 different populations in 51 countries. These chromosomes were typed for 23 short-tandem repeat (STR) loci (DYS19, DYS389I, DYS389II, DYS390, DYS391, DYS392, DYS393, DYS385ab, DYS437, DYS438, DYS439, DYS448, DYS456, DYS458, DYS635, GATAH4, DYS481, DYS533, DYS549, DYS570, DYS576, and DYS643) and using the PowerPlex Y23 System (PPY23, Promega Corporation, Madison, WI). Locus-specific allelic spectra of these markers were determined and a consistently high level of allelic diversity was observed. A considerable number of null, duplicate and off-ladder alleles were revealed. Standard single-locus and haplotype-based parameters were calculated and compared between subsets of Y-STR markers established for forensic casework. The PPY23 marker set provides substantially stronger discriminatory power than other available kits but at the same time reveals the same general patterns of population structure as other marker sets. A strong correlation was observed between the number of Y-STRs included in a marker set and some of the forensic parameters under study. Interestingly a weak but consistent trend toward smaller genetic distances resulting from larger numbers of markers became apparent. Copyright © 2014 The Authors. Published by Elsevier Ireland Ltd.. All rights reserved.

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.

Repeated human leukocyte antigen mismatches in lung re-transplantation.

PubMed

Sommer, Wiebke; Hallensleben, Michael; Ius, Fabio; Kühn, Christian; Tudorache, Igor; Avsar, Murat; Salman, Jawad; Siemeni, Thierry; Greer, Mark; Gottlieb, Jens; Boethig, Dietmar; Blasczyk, Rainer; Haverich, Axel; Warnecke, Gregor

2017-02-01

The role of HLA-sensitization in the absence of detectable DSA in lung re-transplantation is unclear. Antigens of the second donor matching the HLA typing of the first donor are considered 'unacceptable', by some tissue typing laboratories, especially in kidney re-transplantation. Thus, we performed a retrospective analysis of all lung re-transplantations focussing on the impact of HLA-homologies between the first and the second donor ('unacceptable' antigens; repeated HLA mismatch) on patient and graft survival. A total of 132 lung re-transplantations were performed at our centre between 1985 and 2014, of which 120 with complete HLA data were analysed. 55.8% of the recipients received re-transplants with repeated HLA mismatched antigens whereas 43.2% of the re-transplants were transplanted without repeated HLA mismatched antigens. Postoperative survival showed no difference between re-transplant procedures with or without repeated HLA mismatches (p=0.99). While neither homologies on the HLA-A, -B, -C, or -DR locus, nor the addition of several locus homologies (p=0.72) had an impact on survival, unexpectedly, repeated HLA mismatching on the HLA-DQ locus was correlated with better survival. Re-transplantations with repeated HLA mismatches did not result in more development of CLAD as compared to recipients without repeated HLA mismatches (p=0.99). Neither the number of repeated HLA mismatched antigens (p=0.52) nor the HLA locus (HLA-A(p=0.34), HLA-B(p=0.97), HLA-C (p=0.80), HLA-DR(p=0.49) and HLA-DQ(p=0.07)) had an impact on the development of CLAD after re-transplantation. Transplantation with repeated HLA mismatches due to sensitization by a previous transplantation in the absence of detectable HLA-antibodies does not have a negative impact on patient or graft survival. Copyright © 2016 Elsevier B.V. All rights reserved.

Trends and differences in tuberculosis incidences and clustering among natives in Denmark, Sweden and Finland: comparison of native incidences and molecular epidemiology among three low-incidence countries.

PubMed

Pedersen, M K; Lillebaek, T; Andersen, A B; Soini, H; Haanperä, M; Groenheit, R; Jonsson, J; Svensson, E

2018-07-01

To compare the epidemiology of tuberculosis (TB) in Denmark, Sweden and Finland, by focusing on the native population in order to identify epidemiologic differences and thus indirectly possible differences in TB control. TB incidence trends from 1990 through 2015 were compared among the countries. In addition, for the periods 2012-2013 and 2014-2015, genotyping data were compared. Genotyping was performed using the 24-locus mycobacterial interspersed repetitive unit-variable number of tandem repeat (MIRU-VNTR) method in Denmark and Sweden. For Finland, spoligotyping in conjunction with the 15-locus MIRU-VNTR method was used for 2012-2013 and translated into the 24-locus MIRU-VNTR when feasible, and for 2014-2015 only MIRU-VNTR was used. Both incidence trends and molecular epidemiology were assessed for native cases. The average annual rate of change in TB incidence for native Danes was -2.4% vs. -6.1% and -6.9% for native Swedes and Finns respectively. In 2012-2013 Denmark had 52 native cases in the largest transmission chain vs. three cases in Sweden and ten in Finland, and during the same period the clustering rate for native Danes was 48.8% vs. 6.5% and 18.2% for native Swedes and Finns respectively. For 2014-2015, a similar pattern was seen. The decline of TB among natives in Denmark is slower than for Sweden and Finland, and it seems Denmark has more active transmission among natives. The focused assessment on basic native TB epidemiology reveals striking differences in TB transmission among otherwise similar low-TB-incidence countries. Copyright © 2017 European Society of Clinical Microbiology and Infectious Diseases. Published by Elsevier Ltd. All rights reserved.

Validation of a pXO2-A PCR Assay To Explore Diversity among Italian Isolates of Bacillus anthracis Strains Closely Related to the Live, Attenuated Carbosap Vaccine

PubMed Central

Muscillo, M.; La Rosa, G.; Sali, M.; De Carolis, E.; Adone, R.; Ciuchini, F.; Fasanella, A.

2005-01-01

Several circulating Bacillus anthracis strains isolated in Italy and belonging to the A1.a cluster, genotype 3 (A1.a-3) are genotypically indistinguishable from Carbosap, a live attenuated vaccine strain, containing both pXO1 and pXO2 plasmids. The genotype was assessed by using eight-locus multilocus variable-number tandem repeat analysis. We describe here the use of a ninth locus able to explore variability among strains that have the same genotype. It is important to be able to genotype the wild isolate of B. anthracis strains from outbreaks of anthrax in areas where Carbosap vaccination of cattle and sheep is common practice. A total of 27 representative field strains isolated in Italy and four vaccinal strains, namely, Carbosap, Sterne, Pasteur I, and Pasteur II, were characterized by a ninth marker, called pXO2-A. Twenty-three field strains were genotype 3 and therefore identical to Carbosap. The marker was in the pXO2 plasmid and is based on the polymorphism of the already-known VX2-3 locus. Detection was obtained by PCR with fluorescence-labeled forward primers in order to produce appropriate fragments for capillary electrophoresis with an ABI 310 genetic analyzer. Genetic relationships showed heterogeneity in all of the examined samples. Interestingly, with respect to genotype 3, samples grouped into eight different subtypes, A to H, and the subtype G, had only two samples indistinguishable from Carbosap. The results of the present study confirm the validity of a hierarchical progressive protocol for discrimination among closely related isolates. PMID:16145138

Assignment of the dystonia-parkinsonism syndrome locus, DYT3, to a small region within a 1.8-Mb YAC contig of Xq13.1

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

Haberhausen, G.; Schmitt, I.; Koehler, A.

1995-09-01

A YAC contig was constructed of Xq13.1 in order to sublocalize the X-linked dystonia-parkinsonism (XDP) syndrome locus, DYT3. The contig spans a region of {approximately}1.8 Mb and includes loci DXS453/DXS348/IL2R{gamma}/GJB1/CCG1/DXS559. For the construction of the contig, nine sequence-tagged sites and four short tandem repeat polymorphisms (STRPs) were isolated. The STRPs, designated as 4704 No. 6 (DXS7113), 4704 No. 7 (DXS7114), 67601 (DXS7117), and B4Pst (DXS7119) were assigned to a region flanked by DXS348 proximally and by DXS559 distally. Their order was DXS348/4704 No. 6/4704 No. 7/67601/B4Pst/DXS559. They were applied to the analysis of allelic association and of haplotypes in 47more » not-obviously-related XDP patients and in 105 Filipino male controls. The same haplotype was found at loci 67601 (DXS7117) and B4Pst (DXS7119) in 42 of 47 patients. This percentage of common haplotypes decreased at the adjacent loci. The findings, together with the previous demonstration of DXS559 being the distal flanking marker of DYT3, assign the disease locus to a small region in Xq13.1 defined by loci 67601 (DXS7117) and B4Pst (DXS7119). The location of DYT3 was born out by the application of a newly developed likelihood method for the analysis of linkage disequilibrium. 28 refs., 1 fig., 6 tabs.« less

Polysaccharide intercellular adhesin in biofilm: structural and regulatory aspects

PubMed Central

Arciola, Carla Renata; Campoccia, Davide; Ravaioli, Stefano; Montanaro, Lucio

2015-01-01

Staphylococcus aureus and Staphylococcus epidermidis are the leading etiologic agents of implant-related infections. Biofilm formation is the main pathogenetic mechanism leading to the chronicity and irreducibility of infections. The extracellular polymeric substances of staphylococcal biofilms are the polysaccharide intercellular adhesin (PIA), extracellular-DNA, proteins, and amyloid fibrils. PIA is a poly-β(1-6)-N-acetylglucosamine (PNAG), partially deacetylated, positively charged, whose synthesis is mediated by the icaADBC locus. DNA sequences homologous to ica locus are present in many coagulase-negative staphylococcal species, among which S. lugdunensis, however, produces a biofilm prevalently consisting of proteins. The product of icaA is an N-acetylglucosaminyltransferase that synthetizes PIA oligomers from UDP-N-acetylglucosamine. The product of icaD gives optimal efficiency to IcaA. The product of icaC is involved in the externalization of the nascent polysaccharide. The product of icaB is an N-deacetylase responsible for the partial deacetylation of PIA. The expression of ica locus is affected by environmental conditions. In S. aureus and S. epidermidis ica-independent alternative mechanisms of biofilm production have been described. S. epidermidis and S. aureus undergo to a phase variation for the biofilm production that has been ascribed, in turn, to the transposition of an insertion sequence in the icaC gene or to the expansion/contraction of a tandem repeat naturally harbored within icaC. A role is played by the quorum sensing system, which negatively regulates biofilm formation, favoring the dispersal phase that disseminates bacteria to new infection sites. Interfering with the QS system is a much debated strategy to combat biofilm-related infections. In the search of vaccines against staphylococcal infections deacetylated PNAG retained on the surface of S. aureus favors opsonophagocytosis and is a potential candidate for immune-protection. PMID:25713785

Functional centromeres in Astragalus sinicus include a compact centromere-specific histone H3 and a 20-bp tandem repeat.

PubMed

Tek, Ahmet L; Kashihara, Kazunari; Murata, Minoru; Nagaki, Kiyotaka

2011-11-01

The centromere plays an essential role for proper chromosome segregation during cell division and usually harbors long arrays of tandem repeated satellite DNA sequences. Although this function is conserved among eukaryotes, the sequences of centromeric DNA repeats are variable. Most of our understanding of functional centromeres, which are defined by localization of a centromere-specific histone H3 (CENH3) protein, comes from model organisms. The components of the functional centromere in legumes are poorly known. The genus Astragalus is a member of the legumes and bears the largest numbers of species among angiosperms. Therefore, we studied the components of centromeres in Astragalus sinicus. We identified the CenH3 homolog of A. sinicus, AsCenH3 that is the most compact in size among higher eukaryotes. A CENH3-based assay revealed the functional centromeric DNA sequences from A. sinicus, called CentAs. The CentAs repeat is localized in A. sinicus centromeres, and comprises an AT-rich tandem repeat with a monomer size of 20 nucleotides.

Molecular typing of monophasic Salmonella 4,[5]:i:- strains isolated in Belgium (2008-2011).

PubMed

Boland, Cécile; Bertrand, Sophie; Mattheus, Wesley; Dierick, Katelijne; Wattiau, Pierre

2014-01-31

To assess the distribution of Salmonella 4,[5]:i:- subtypes in the Belgian food chain and compare it to the subtypes associated with human infections, a molecular assessment was initiated. Two hundred fifty-three Salmonella isolates serotyped as 4,[5]:i:- during the period 2008-2011 in Belgium and originating from animal productions, food or human clinical samples were analysed by a specific duplex PCR. One hundred ninety-four isolates (76.7%) fit the profile of a S. Typhimurium monophasic variant as defined by the European Food Safety Authority. The other isolates possessed but did not express the phase II flagellin gene (23.3%). Multiple Locus Variable Number of Tandem Repeats Analysis (MLVA) revealed many but closely related profiles in the fljB-negative S. Typhimurium monophasic variant isolates. Some MLVA types were associated with both human and animal isolates but no unique source of human contamination could be demonstrated. Copyright © 2013 Elsevier B.V. All rights reserved.

Molecular characterization of Mycobacterium tuberculosis isolates from elephants of Nepal.

PubMed

Paudel, Sarad; Mikota, Susan K; Nakajima, Chie; Gairhe, Kamal P; Maharjan, Bhagwan; Thapa, Jeewan; Poudel, Ajay; Shimozuru, Michito; Suzuki, Yasuhiko; Tsubota, Toshio

2014-05-01

Mycobacterium tuberculosis was cultured from the lung tissues of 3 captive elephants in Nepal that died with extensive lung lesions. Spoligotyping, TbD1 detection and multi-locus variable number of tandem repeat analysis (MLVA) results suggested 3 isolates belonged to a specific lineage of Indo-Oceanic clade, EAI5 SIT 138. One of the elephant isolates had a new synonymous single nucleotide polymorphism (SNP) T231C in the gyrA sequence, and the same SNP was also found in human isolates in Nepal. MLVA results and transfer history of the elephants suggested that 2 of them might be infected with M. tuberculosis from the same source. These findings indicated the source of M. tuberculosis infection of those elephants were local residents, presumably their handlers. Further investigation including detailed genotyping of elephant and human isolates is needed to clarify the infection route and eventually prevent the transmission of tuberculosis to susceptible hosts. Copyright © 2014 Elsevier Ltd. All rights reserved.

Molecular typing of isolates obtained from aborted foetuses in Brucella-free Holstein dairy cattle herd after immunisation with Brucella abortus RB51 vaccine in Egypt.

PubMed

Wareth, Gamal; Melzer, Falk; Böttcher, Denny; El-Diasty, Mohamed; El-Beskawy, Mohamed; Rasheed, Nesma; Schmoock, Gernot; Roesler, Uwe; Sprague, Lisa D; Neubauer, Heinrich

2016-12-01

Bovine brucellosis is endemic in Egypt in spite of application of surveillance and control measures. An increase of abortions was reported in a Holstein dairy cattle herd with 600 animals in Damietta governorate in Egypt after immunisation with Brucella (B.) abortus RB51 vaccine. Twenty one (10.6%) of 197 vaccinated cows aborted after 3 months. All aborted cows had been tested seronegative for brucellosis in the past 3 years. B. abortus was isolated from four foetuses. Conventional biochemical and bacteriological identification and polymerase chain reaction (PCR) confirmed two B. abortus biovar (bv.) 1 smooth and two B. abortus rough strains. None of the B. abortus isolates were identified as RB51. Genotyping analysis by multiple locus of variable number tandem repeats analysis based on 16 markers (MLVA-16) revealed two different profiles with low genetic diversity. B. abortus bv1 was introduced in the herd and caused abortions. Copyright © 2016 Elsevier B.V. All rights reserved.

Refinement of the cone-rod retinal dystrophy locus on chromosome 19q

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

Gregory, C.Y.; Evans, K.; Bhattacharya, S.S.

1994-11-01

Cone-rod dystrophy (CRD) is a severe example of an inherited retinal dystrophy: ophthalmic diseases that as a group constitute the commonest causes of blindness in children in the developed world and account for a significant proportion of visual handicap in adults. Two case reports suggested loci for CRD-causing genes on chromosomes 18q and chromosome 17q. Recently, we reported the results of a total genome search that localized an autosomal dominant form of CRD to chromosome 19q in the region 19q13.1-q13.2. Since then, using data from a short tandem repeat-polymorphism linkage map of chromosome 19 and recently developed microsatellite markers inmore » this region, we have been able to further refine the localization of the chromosome 19q CRD-causing gene. Seven new microsatellite markers were used to genotype 34 affected subjects, 22 unaffected subjects, and 15 spouses. Two-point, multipoint, and FASTMAP analyses were performed. 11 refs., 1 tab.« less

VNTR diversity in Yersinia pestis isolates from an animal challenge study reveals the potential for in vitro mutations during laboratory cultivation

USGS Publications Warehouse

Vogler, Amy J.; Nottingham, Roxanne; Busch, Joseph D.; Sahl, Jason W.; Shuey, Megan M.; Foster, Jeffrey T.; Schupp, James M.; Smith, Susan; Rocke, Tonie E.; Klein, Paul; Wagner, David M.

2016-01-01

Underlying mutation rates and other evolutionary forces shape the population structure of bacteria in nature. Although easily overlooked, similar forces are at work in the laboratory and may influence observed mutations. Here, we investigated tissue samples and Yersinia pestis isolates from a rodent laboratory challenge with strain CO92 using whole genome sequencing and multi-locus variable-number tandem repeat (VNTR) analysis (MLVA). We identified six VNTR mutations that were found to have occurred in vitro during laboratory cultivation rather than in vivo during the rodent challenge. In contrast, no single nucleotide polymorphism (SNP) mutations were observed, either in vivo or in vitro. These results were consistent with previously published mutation rates and the calculated number of Y. pestis generations that occurred during the in vitro versus the in vivo portions of the experiment. When genotyping disease outbreaks, the potential for in vitro mutations should be considered, particularly when highly variable genetic markers such as VNTRs are used.

Tuberculosis Caused by Mycobacterium africanum, United States, 2004-2013.

PubMed

Sharma, Aditya; Bloss, Emily; Heilig, Charles M; Click, Eleanor S

2016-03-01

Mycobacterium africanum is endemic to West Africa and causes tuberculosis (TB). We reviewed reported cases of TB in the United States during 2004-2013 that had lineage assigned by genotype (spoligotype and mycobacterial interspersed repetitive unit variable number tandem repeats). M. africanum caused 315 (0.4%) of 73,290 TB cases with lineage assigned by genotype. TB caused by M. africanum was associated more with persons from West Africa (adjusted odds ratio [aOR] 253.8, 95% CI 59.9-1,076.1) and US-born black persons (aOR 5.7, 95% CI 1.2-25.9) than with US-born white persons. TB caused by M. africanum did not show differences in clinical characteristics when compared with TB caused by M. tuberculosis. Clustered cases defined as >2 cases in a county with identical 24-locus mycobacterial interspersed repetitive unit genotypes, were less likely for M. africanum (aOR 0.1, 95% CI 0.1-0.4), which suggests that M. africanum is not commonly transmitted in the United States.

Genotypes of pathogenic Leptospira spp isolated from rodents in Argentina

PubMed Central

Loffler, Sylvia Grune; Pavan, Maria Elisa; Vanasco, Bibiana; Samartino, Luis; Suarez, Olga; Auteri, Carmelo; Romero, Graciela; Brihuega, Bibiana

2014-01-01

Leptospirosis is the most widespread zoonosis in the world and significant efforts have been made to determine and classify pathogenic Leptospira strains. This zoonosis is maintained in nature through chronic renal infections of carrier animals, with rodents and other small mammals serving as the most important reservoirs. Additionally, domestic animals, such as livestock and dogs, are significant sources of human infection. In this study, a multiple-locus variable-number tandem repeat analysis (MLVA) was applied to genotype 22 pathogenic Leptospira strains isolated from urban and periurban rodent populations from different regions of Argentina. Three MLVA profiles were identified in strains belonging to the species Leptospira interrogans (serovars Icterohaemorrhagiae and Canicola); one profile was observed in serovar Icterohaemorrhagiae and two MLVA profiles were observed in isolates of serovars Canicola and Portlandvere. All strains belonging to Leptospira borgpetersenii serovar Castellonis exhibited the same MLVA profile. Four different genotypes were isolated from urban populations of rodents, including both mice and rats and two different genotypes were isolated from periurban populations. PMID:24676656

The profile of repeat-associated histone lysine methylation states in the mouse epigenome

PubMed Central

Martens, Joost H A; O'Sullivan, Roderick J; Braunschweig, Ulrich; Opravil, Susanne; Radolf, Martin; Steinlein, Peter; Jenuwein, Thomas

2005-01-01

Histone lysine methylation has been shown to index silenced chromatin regions at, for example, pericentric heterochromatin or of the inactive X chromosome. Here, we examined the distribution of repressive histone lysine methylation states over the entire family of DNA repeats in the mouse genome. Using chromatin immunoprecipitation in a cluster analysis representing repetitive elements, our data demonstrate the selective enrichment of distinct H3-K9, H3-K27 and H4-K20 methylation marks across tandem repeats (e.g. major and minor satellites), DNA transposons, retrotransposons, long interspersed nucleotide elements and short interspersed nucleotide elements. Tandem repeats, but not the other repetitive elements, give rise to double-stranded (ds) RNAs that are further elevated in embryonic stem (ES) cells lacking the H3-K9-specific Suv39h histone methyltransferases. Importantly, although H3-K9 tri- and H4-K20 trimethylation appear stable at the satellite repeats, many of the other repeat-associated repressive marks vary in chromatin of differentiated ES cells or of embryonic trophoblasts and fibroblasts. Our data define a profile of repressive histone lysine methylation states for the repetitive complement of four distinct mouse epigenomes and suggest tandem repeats and dsRNA as primary triggers for more stable chromatin imprints. PMID:15678104

Two tandemly repeated telomere-associated sequences in Nicotiana plumbaginifolia.

PubMed

Chen, C M; Wang, C T; Wang, C J; Ho, C H; Kao, Y Y; Chen, C C

1997-12-01

Two tandemly repeated telomere-associated sequences, NP3R and NP4R, have been isolated from Nicotiana plumbaginifolia. The length of a repeating unit for NP3R and NP4R is 165 and 180 nucleotides respectively. The abundance of NP3R, NP4R and telomeric repeats is, respectively, 8.4 x 10(4), 6 x 10(3) and 1.5 x 10(6) copies per haploid genome of N. plumbaginifolia. Fluorescence in situ hybridization revealed that NP3R is located at the ends and/or in interstitial regions of all 10 chromosomes and NP4R on the terminal regions of three chromosomes in the haploid genome of N. plumbaginifolia. Sequence homology search revealed that not only are NP3R and NP4R homologous to HRS60 and GRS, respectively, two tandem repeats isolated from N. tabacum, but that NP3R and NP4R are also related to each other, suggesting that they originated from a common ancestral sequence. The role of these repeated sequences in chromosome healing is discussed based on the observation that two to three copies of a telomere-similar sequence were present in each repeating unit of NP3R and NP4R.

Molecular tandem repeat strategy for elucidating mechanical properties of high-strength proteins

PubMed Central

Jung, Huihun; Pena-Francesch, Abdon; Saadat, Alham; Sebastian, Aswathy; Kim, Dong Hwan; Hamilton, Reginald F.; Albert, Istvan; Allen, Benjamin D.; Demirel, Melik C.

2016-01-01

Many globular and structural proteins have repetitions in their sequences or structures. However, a clear relationship between these repeats and their contribution to the mechanical properties remains elusive. We propose a new approach for the design and production of synthetic polypeptides that comprise one or more tandem copies of a single unit with distinct amorphous and ordered regions. Our designed sequences are based on a structural protein produced in squid suction cups that has a segmented copolymer structure with amorphous and crystalline domains. We produced segmented polypeptides with varying repeat number, while keeping the lengths and compositions of the amorphous and crystalline regions fixed. We showed that mechanical properties of these synthetic proteins could be tuned by modulating their molecular weights. Specifically, the toughness and extensibility of synthetic polypeptides increase as a function of the number of tandem repeats. This result suggests that the repetitions in native squid proteins could have a genetic advantage for increased toughness and flexibility. PMID:27222581

Haplotype data for 23 Y-chromosome markers in a reference sample from Bosnia and Herzegovina.

PubMed

Kovačević, Lejla; Fatur-Cerić, Vera; Hadzic, Negra; Čakar, Jasmina; Primorac, Dragan; Marjanović, Damir

2013-06-01

To detect polymorphisms of 23 Y-chromosomal short tandem repeat (STR) loci, including 6 new loci, in a reference database of male population of Bosnia and Herzegovina, as well as to assess the importance of increasing the number of Y-STR loci utilized in forensic DNA analysis. The reference sample consisted of 100 healthy, unrelated men originating from Bosnia and Herzegovina. Sample collection using buccal swabs was performed in all geographical regions of Bosnia and Herzegovina in the period from 2010 to 2011. DNA samples were typed for 23 Y STR loci, including 6 new loci: DYS576, DYS481, DYS549, DYS533, DYS570, and DYS643, which are included in the new PowerPlex® Y 23 amplification kit. The absolute frequency of generated haplotypes was calculated and results showed that 98 samples had unique Y 23 haplotypes, and that only two samples shared the same haplotype. The most polymorphic locus was DYS418, with 14 detected alleles and the least polymorphic loci were DYS389I, DYS391, DYS437, and DYS393. This study showed that by increasing the number of highly polymorphic Y STR markers, to include those tested in our analysis, leads to a reduction of repeating haplotypes, which is very important in the application of forensic DNA analysis.

Characterization of the COL2A1 VNTR polymorphism

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

Berg, E.S.; Olaisen, B.

1993-05-01

The variable number of tandem repeat (VNTR) region 3{prime} to the collagen type II gene (COL2A1) was amplified in vitro by the polymerase chain reaction. Subsequent high-resolution gel electrophoresis showed that the five earlier reported alleles could be further subtyped. A total of 17 allelic variants with a heterozygosity of 73.0% were found in 202 unrelated Norwegians. DNA sequencing of 19 COL2A1 alleles has been performed. The internal organization of the VNTR was common for all alleles, as previously shown for a few alleles. Moreover, the polymorphism in the COL2A1 locus is mainly due to variation in the numbers ofmore » copies of two repeat units, containing 34 and 31 bp, respectively, and/or to small deletions in either of the two units. DNA sequencing of alleles with the same electrophoretic size revealed no heterogeneity such as an alternating order of the different units, a feature that might have been expected to be the result of unequal crossing-over events. The observed ordered structure of the VNTR and the possibility of single-stranded DNA from the cores in the VNTR forming hairpins and loops suggest that the COL2A1 polymorphism may have evolved mainly by replication slippage mechanisms. 23 refs., 2 figs., 3 tabs.« less

Molecular basis of length polymorphism in the human zeta-globin gene complex.

PubMed Central

Goodbourn, S E; Higgs, D R; Clegg, J B; Weatherall, D J

1983-01-01

The length polymorphism between the human zeta-globin gene and its pseudogene is caused by an allele-specific variation in the copy number of a tandemly repeating 36-base-pair sequence. This sequence is related to a tandemly repeated 14-base-pair sequence in the 5' flanking region of the human insulin gene, which is known to cause length polymorphism, and to a repetitive sequence in intervening sequence (IVS) 1 of the pseudo-zeta-globin gene. Evidence is presented that the latter is also of variable length, probably because of differences in the copy number of the tandem repeat. The homology between the three length polymorphisms may be an indication of the presence of a more widespread group of related sequences in the human genome, which might be useful for generalized linkage studies. PMID:6308667

High Fractional Occupancy of a Tandem Maf Recognition Element and Its Role in Long-Range β-Globin Gene Regulation

PubMed Central

Stees, Jared R.; Hossain, Mir A.; Sunose, Tomoki; Kudo, Yasushi; Pardo, Carolina E.; Nabilsi, Nancy H.; Darst, Russell P.; Poudyal, Rosha; Igarashi, Kazuhiko; Kladde, Michael P.

2015-01-01

Enhancers and promoters assemble protein complexes that ultimately regulate the recruitment and activity of RNA polymerases. Previous work has shown that at least some enhancers form stable protein complexes, leading to the formation of enhanceosomes. We analyzed protein-DNA interactions in the murine β-globin gene locus using the methyltransferase accessibility protocol for individual templates (MAPit). The data show that a tandem Maf recognition element (MARE) in locus control region (LCR) hypersensitive site 2 (HS2) reveals a remarkably high degree of occupancy during differentiation of mouse erythroleukemia cells. Most of the other transcription factor binding sites in LCR HS2 or in the adult β-globin gene promoter regions exhibit low fractional occupancy, suggesting highly dynamic protein-DNA interactions. Targeting of an artificial zinc finger DNA-binding domain (ZF-DBD) to the HS2 tandem MARE caused a reduction in the association of MARE-binding proteins and transcription complexes at LCR HS2 and the adult βmajor-globin gene promoter but did not affect expression of the βminor-globin gene. The data demonstrate that a stable MARE-associated footprint in LCR HS2 is important for the recruitment of transcription complexes to the adult βmajor-globin gene promoter during erythroid cell differentiation. PMID:26503787

Clustering of Tuberculosis Cases Based on Variable-Number Tandem-Repeat Typing in Relation to the Population Structure of Mycobacterium tuberculosis in the Netherlands

PubMed Central

Sloot, Rosa; Borgdorff, Martien W.; de Beer, Jessica L.; van Ingen, Jakko; Supply, Philip

2013-01-01

The population structure of 3,776 Mycobacterium tuberculosis isolates was determined using variable-number tandem-repeat (VNTR) typing. The degree of clonality was so high that a more relaxed definition of clustering cannot be applied. Among recent immigrants with non-Euro-American isolates, transmission is overestimated if based on identical VNTR patterns. PMID:23658260

RepeatsDB-lite: a web server for unit annotation of tandem repeat proteins.

PubMed

Hirsh, Layla; Paladin, Lisanna; Piovesan, Damiano; Tosatto, Silvio C E

2018-05-09

RepeatsDB-lite (http://protein.bio.unipd.it/repeatsdb-lite) is a web server for the prediction of repetitive structural elements and units in tandem repeat (TR) proteins. TRs are a widespread but poorly annotated class of non-globular proteins carrying heterogeneous functions. RepeatsDB-lite extends the prediction to all TR types and strongly improves the performance both in terms of computational time and accuracy over previous methods, with precision above 95% for solenoid structures. The algorithm exploits an improved TR unit library derived from the RepeatsDB database to perform an iterative structural search and assignment. The web interface provides tools for analyzing the evolutionary relationships between units and manually refine the prediction by changing unit positions and protein classification. An all-against-all structure-based sequence similarity matrix is calculated and visualized in real-time for every user edit. Reviewed predictions can be submitted to RepeatsDB for review and inclusion.

Tandem duplication within a Neurofibromatosis type I (NFI) gene exon in a family with features of Watson syndrome and Noonan syndrome

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

Tassabehji, M.; Strachan, T.; Colley, A.

Type 1 neurofibromatosis (NF1), Watson syndrome (WS), and Noonan syndrome (NS) show some overlap in clinical manifestations. In addition, WS has been shown to be linked to markers flanking the NF1 locus and a deletion at the NF1 locus demonstrated in a WS patient. This suggests either that WS and NF1 are allelic or the phenotypes arise from mutations in very closely linked genes. Here the authors provide evidence for the former by demonstrating a mutation in the NF1 gene in a family with features of both WS and NS. The mutation is an almost perfect in-frame tandem duplication ofmore » 42 bases in exon 28 of the NF1 gene. Unlike the mutations previously described in classical NF1, which show a preponderance of null alleles, the mutation in this family would be expected to result in a mutant neurofibromin product. 31 refs., 2 figs.« less

Marked Phenotypic Heterogeneity Associated with Expansion of a CAG Repeat Sequence at the Spinocerebellar Ataxia 3/Machado-Joseph Disease Locus

PubMed Central

Cancel, Géraldine; Abbas, Nacer; Stevanin, Giovanni; Dürr, Alexandra; Chneiweiss, Hervé; Néri, Christian; Duyckaerts, Charles; Penet, Christiane; Cann, Howard M.; Agid, Yves; Brice, Alexis

1995-01-01

The spinocerebellar ataxia 3 locus (SCA3) for type I autosomal dominant cerebellar ataxia (ADCA type I), a clinically and genetically heterogeneous group of neuro-degenerative disorders, has been mapped to chromosome 14q32.1. ADCA type I patients from families segregating SCA3 share clinical features in common with those with Machado-Joseph disease (MJD), the gene of which maps to the same region. We show here that the disease gene segregating in each of three French ADCA type I kindreds and in a French family with neuropatho-logical findings suggesting the ataxochoreic form of dentatorubropallidoluysian atrophy carries an expanded CAG repeat sequence located at the same locus as that for MJD. Analysis of the mutation in these families shows a strong negative correlation between size of the expanded CAG repeat and age at onset of clinical disease. Instability of the expanded triplet repeat was not found to be affected by sex of the parent transmitting the mutation. Evidence was found for somatic and gonadal mosaicism for alleles carrying expanded trinucleotide repeats. ImagesFigure 3Figure 5 PMID:7573040

A novel tandem repeat sequence located on human chromosome 4p: isolation and characterization.

PubMed

Kogi, M; Fukushige, S; Lefevre, C; Hadano, S; Ikeda, J E

1997-06-01

In an effort to analyze the genomic region of the distal half of human chromosome 4p, to where Huntington disease and other diseases have been mapped, we have isolated the cosmid clone (CRS447) that was likely to contain a region with specific repeat sequences. Clone CRS447 was subjected to detailed analysis, including chromosome mapping, restriction mapping, and DNA sequencing. Chromosome mapping by both a human-CHO hybrid cell panel and FISH revealed that CRS447 was predominantly located in the 4p15.1-15.3 region. CRS447 was shown to consist of tandem repeats of 4.7-kb units present on chromosome 4p. A single EcoRI unit was subcloned (pRS447), and the complete sequence was determined as 4752 nucleotides. When pRS447 was used as a probe, the number of copies of this repeat per haploid genome was estimated to be 50-70. Sequence analysis revealed that it contained two internal CA repeats and one putative ORF. Database search established that this sequence was unreported. However, two homologous STS markers were found in the database. We concluded that CRS447/pRS447 is a novel tandem repeat sequence that is mainly specific to human chromosome 4p.

A complete mitochondrial genome sequence of Asian black bear Sichuan subspecies (Ursus thibetanus mupinensis)

PubMed Central

Hou, Wan-ru; Chen, Yu; Wu, Xia; Hu, Jin-chu; Peng, Zheng-song; Yang, Jung; Tang, Zong-xiang; Zhou, Cai-Quan; Li, Yu-ming; Yang, Shi-kui; Du, Yu-jie; Kong, Ling-lu; Ren, Zheng-long; Zhang, Huai-yu; Shuai, Su-rong

2007-01-01

We obtained the complete mitochondrial genome of U.thibetanus mupinensis by DNA sequencing based on the PCR fragments of 18 primers we designed. The results indicate that the mtDNA is 16 868 bp in size, encodes 13 protein genes, 22 tRNA genes, and 2 rRNA genes, with an overall H-strand base composition of 31.2% A, 25.4% C, 15.5% G and 27.9% T. The sequence of the control region (CR) located between tRNA-Pro and tRNA-Phe is 1422 bp in size, consists of 8.43% of the whole genome, GC content is 51.9% and has a 6bp tandem repeat and two 10bp tandem repeats identified by using the Tandem Repeats Finder. U. thibetanus mupinensis mitochondrial genome shares high similarity with those of three other Ursidae: U. americanus (91.46%), U. arctos (89.25%) and U. maritimus (87.66%). PMID:17205108

Chicken microsatellite markers isolated from libraries enriched for simple tandem repeats.

PubMed

Gibbs, M; Dawson, D A; McCamley, C; Wardle, A F; Armour, J A; Burke, T

1997-12-01

The total number of microsatellite loci is considered to be at least 10-fold lower in avian species than in mammalian species. Therefore, efficient large-scale cloning of chicken microsatellites, as required for the construction of a high-resolution linkage map, is facilitated by the construction of libraries using an enrichment strategy. In this study, a plasmid library enriched for tandem repeats was constructed from chicken genomic DNA by hybridization selection. Using this technique the proportion of recombinant clones that cross-hybridized to probes containing simple tandem repeats was raised to 16%, compared with < 0.1% in a non-enriched library. Primers were designed from 121 different sequences. Polymerase chain reaction (PCR) analysis of two chicken reference pedigrees enabled 72 loci to be localized within the collaborative chicken genetic map, and at least 30 of the remaining loci have been shown to be informative in these or other crosses.

Identification of presumed ancestral DNA sequences of phaseolin in Phaseolus vulgaris.

PubMed Central

Kami, J; Velásquez, V B; Debouck, D G; Gepts, P

1995-01-01

Common bean (Phaseolus vulgaris) consists of two major geographic gene pools, one distributed in Mexico, Central America, and Colombia and the other in the southern Andes (southern Peru, Bolivia, and Argentina). Amplification and sequencing of members of the multigene family coding for phaseolin, the major seed storage protein of the common bean, provide evidence for accumulation of tandem direct repeats in both introns and exons during evolution of the multigene family in this species. The presumed ancestral phaseolin sequences, without tandem repeats, were found in recently discovered but nearly extinct wild common bean populations of Ecuador and northern Peru that are intermediate between the two major gene pools of the species based on geographical and molecular arguments. Our results illustrate the usefulness of tandem direct repeats in establishing the polarity of DNA sequence divergence and therefore in proposing phylogenies. Images Fig. 1 Fig. 3 PMID:7862642

[Molecular cloning and characterization of a novel Clonorchis sinensis antigenic protein containing tandem repeat sequences].

PubMed

Liu, Qian; Xu, Xue-Nian; Zhou, Yan; Cheng, Na; Dong, Yu-Ting; Zheng, Hua-Jun; Zhu, Yong-Qiang; Zhu, Yong-Qiang

2013-08-01

To find and clone new antigen genes from the lambda-ZAP cDNA expression library of adult Clonorchis sinensis, and determine the immunological characteristics of the recombinant proteins. The cDNA expression library of adult C. sinensis was screened by pooled sera of clonorchiasis patients. The sequences of the positive phage clones were compared with the sequences in EST database, and the full-length sequence of the gene (Cs22 gene) was obtained by RT-PCR. cDNA fragments containing 2 and 3 times tandem repeat sequences were generated by jumping PCR. The sequence encoding the mature peptide or the tandem repeat sequence was respectively cloned into the prokaryotic expression vector pET28a (+), and then transformed into E. coli Rosetta DE3 cells for expression. The recombinant proteins (rCs22-2r, rCs22-3r, rCs22M-2r, and rCs22M-3r) were purified by His-bind-resin (Ni-NTA) affinity chromatography. The immunogenicity of rCs22-2r and rCs22-3r was identified by ELISA. To evaluate the immunological diagnostic value of rCs22-2r and rCs22-3r, serum samples from 35 clonorchiasis patients, 31 healthy individuals, 15 schistosomiasis patients, 15 paragonimiasis westermani patients and 13 cysticercosis patients were examined by ELISA. To locate antigenic determinants, the pooled sera of clonorchiasis patients and healthy persons were analyzed for specific antibodies by ELISA with recombinant protein rCs22M-2r and rCs22M-3r containing the tandem repeat sequences. The full-length sequence of Cs22 antigen gene of C. sinensis was obtained. It contained 13 times tandem repeat sequences of EQQDGDEEGMGGDGGRGKEKGKVEGEDGAGEQKEQA. Bioinformatics analysis indicated that the protein (Cs22) belonged to GPI-anchored proteins family. The recombinant proteins rCs22-2r and rCs22-3r showed a certain level of immunogenicity. The positive rate by ELISA coated with the purified PrCs22-2r and PrCs22-3r for sera of clonorchiasis patients both were 45.7% (16/35), and 3.2% (1/31) for those of healthy persons. There was no cross reaction with sera of schistosomiasis and cysticercosis patients. The cross reaction with sera of paragonimiasis westermani patients was 1/15. The recombinant proteins rCs22M-2r and rCs22M-3r which only contained tandem repeats were specifically recognized by pooled sera of clonorchiasis patients. The Cs22 antigen gene of Clonorchis sinensis is obtained, and the recombinant proteins have certain diagnostic value. The antigenic determinant is located in tandem repeat sequences.

Expanded GAA repeats impair FXN gene expression and reposition the FXN locus to the nuclear lamina in single cells.

PubMed

Silva, Ana M; Brown, Jill M; Buckle, Veronica J; Wade-Martins, Richard; Lufino, Michele M P

2015-06-15

Abnormally expanded DNA repeats are associated with several neurodegenerative diseases. In Friedreich's ataxia (FRDA), expanded GAA repeats in intron 1 of the frataxin gene (FXN) reduce FXN mRNA levels in averaged cell samples through a poorly understood mechanism. By visualizing FXN expression and nuclear localization in single cells, we show that GAA-expanded repeats decrease the number of FXN mRNA molecules, slow transcription, and increase FXN localization at the nuclear lamina (NL). Restoring histone acetylation reverses NL positioning. Expanded GAA-FXN loci in FRDA patient cells show increased NL localization with increased silencing of alleles and reduced transcription from alleles positioned peripherally. We also demonstrate inefficiencies in transcription initiation and elongation from the expanded GAA-FXN locus at single-cell resolution. We suggest that repressive epigenetic modifications at the expanded GAA-FXN locus may lead to NL relocation, where further repression may occur. © The Author 2015. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oup.com.

Heme oxygenase-1 gene promoter microsatellite polymorphism is associated with progressive atherosclerosis and incident cardiovascular disease.

PubMed

Pechlaner, Raimund; Willeit, Peter; Summerer, Monika; Santer, Peter; Egger, Georg; Kronenberg, Florian; Demetz, Egon; Weiss, Günter; Tsimikas, Sotirios; Witztum, Joseph L; Willeit, Karin; Iglseder, Bernhard; Paulweber, Bernhard; Kedenko, Lyudmyla; Haun, Margot; Meisinger, Christa; Gieger, Christian; Müller-Nurasyid, Martina; Peters, Annette; Willeit, Johann; Kiechl, Stefan

2015-01-01

The enzyme heme oxygenase-1 (HO-1) exerts cytoprotective effects in response to various cellular stressors. A variable number tandem repeat polymorphism in the HO-1 gene promoter region has previously been linked to cardiovascular disease. We examined this association prospectively in the general population. Incidence of stroke, myocardial infarction, or vascular death was registered between 1995 and 2010 in 812 participants of the Bruneck Study aged 45 to 84 years (49.4% males). Carotid atherosclerosis progression was quantified by high-resolution ultrasound. HO-1 variable number tandem repeat length was determined by polymerase chain reaction. Subjects with ≥32 tandem repeats on both HO-1 alleles compared with the rest of the population (recessive trait) featured substantially increased cardiovascular disease risk (hazard ratio [95% confidence interval], 5.45 [2.39, 12.42]; P<0.0001), enhanced atherosclerosis progression (median difference in atherosclerosis score [interquartile range], 2.1 [0.8, 5.6] versus 0.0 [0.0, 2.2] mm; P=0.0012), and a trend toward higher levels of oxidized phospholipids on apolipoprotein B-100 (median oxidized phospholipids/apolipoprotein B level [interquartile range], 11364 [4160, 18330] versus 4844 [3174, 12284] relative light units; P=0.0554). Increased cardiovascular disease risk in those homozygous for ≥32 repeats was also detected in a pooled analysis of 7848 participants of the Bruneck, SAPHIR, and KORA prospective studies (hazard ratio [95% confidence interval], 3.26 [1.50, 7.33]; P=0.0043). This study found a strong association between the HO-1 variable number tandem repeat polymorphism and cardiovascular disease risk confined to subjects with a high number of repeats on both HO-1 alleles and provides evidence for accelerated atherogenesis and decreased antioxidant defense in this vascular high-risk group. © 2014 American Heart Association, Inc.

DNA replication stress restricts ribosomal DNA copy number.

PubMed

Salim, Devika; Bradford, William D; Freeland, Amy; Cady, Gillian; Wang, Jianmin; Pruitt, Steven C; Gerton, Jennifer L

2017-09-01

Ribosomal RNAs (rRNAs) in budding yeast are encoded by ~100-200 repeats of a 9.1kb sequence arranged in tandem on chromosome XII, the ribosomal DNA (rDNA) locus. Copy number of rDNA repeat units in eukaryotic cells is maintained far in excess of the requirement for ribosome biogenesis. Despite the importance of the repeats for both ribosomal and non-ribosomal functions, it is currently not known how "normal" copy number is determined or maintained. To identify essential genes involved in the maintenance of rDNA copy number, we developed a droplet digital PCR based assay to measure rDNA copy number in yeast and used it to screen a yeast conditional temperature-sensitive mutant collection of essential genes. Our screen revealed that low rDNA copy number is associated with compromised DNA replication. Further, subculturing yeast under two separate conditions of DNA replication stress selected for a contraction of the rDNA array independent of the replication fork blocking protein, Fob1. Interestingly, cells with a contracted array grew better than their counterparts with normal copy number under conditions of DNA replication stress. Our data indicate that DNA replication stresses select for a smaller rDNA array. We speculate that this liberates scarce replication factors for use by the rest of the genome, which in turn helps cells complete DNA replication and continue to propagate. Interestingly, tumors from mini chromosome maintenance 2 (MCM2)-deficient mice also show a loss of rDNA repeats. Our data suggest that a reduction in rDNA copy number may indicate a history of DNA replication stress, and that rDNA array size could serve as a diagnostic marker for replication stress. Taken together, these data begin to suggest the selective pressures that combine to yield a "normal" rDNA copy number.

Fingerprinting of Cyanobacteria Based on PCR with Primers Derived from Short and Long Tandemly Repeated Repetitive Sequences

PubMed Central

Rasmussen, Ulla; Svenning, Mette M.

1998-01-01

The presence of repeated DNA (short tandemly repeated repetitive [STRR] and long tandemly repeated repetitive [LTRR]) sequences in the genome of cyanobacteria was used to generate a fingerprint method for symbiotic and free-living isolates. Primers corresponding to the STRR and LTRR sequences were used in the PCR, resulting in a method which generate specific fingerprints for individual isolates. The method was useful both with purified DNA and with intact cyanobacterial filaments or cells as templates for the PCR. Twenty-three Nostoc isolates from a total of 35 were symbiotic isolates from the angiosperm Gunnera species, including isolates from the same Gunnera species as well as from different species. The results show a genetic similarity among isolates from different Gunnera species as well as a genetic heterogeneity among isolates from the same Gunnera species. Isolates which have been postulated to be closely related or identical revealed similar results by the PCR method, indicating that the technique is useful for clustering of even closely related strains. The method was applied to nonheterocystus cyanobacteria from which a fingerprint pattern was obtained. PMID:16349487

Identification and typing of Brucella spp. in stranded harbour porpoises (Phocoena phocoena) on the Dutch coast.

PubMed

Maio, Elisa; Begeman, Lineke; Bisselink, Yvette; van Tulden, Peter; Wiersma, Lidewij; Hiemstra, Sjoukje; Ruuls, Robin; Gröne, Andrea; Roest, Hendrik-Ido-Jan; Willemsen, Peter; van der Giessen, Joke

2014-09-17

The presence of Brucella (B.) spp. in harbour porpoises stranded between 2008 and 2011 along the Dutch coast was studied. A selection of 265 tissue samples from 112 animals was analysed using conventional and molecular methods. In total, 4.5% (5/112) of the animals corresponding with 2.3% (6/265) Brucella positive tissue samples were Brucella positive by culture and these were all confirmed by real-time polymerase chain reaction (real-time PCR) based on the insertion element 711 (IS711). In addition, two more Brucella-positive tissue samples from two animals collected in 2011 were identified using real-time PCR resulting in an overall Brucella prevalence of 6.3% (7/112 animals). Brucella spp. were obtained from lungs (n=3), pulmonary lymph node (n=3) and lungworms (n=2). Multi Locus Variable Number of Tandem Repeats (VNTR) Analysis (MLVA) typing based on the MLVA-16 showed that the Brucella isolates were B. ceti. Additional in silico Multi Locus Sequence typing (MLST) after whole genome sequencing of the 6 Brucella isolates confirmed B. ceti ST 23. According to the Brucella 2010 MLVA database, the isolated Brucella strains encountered were of five genotypes, in two distinct subclusters divided in two different time periods of harbour porpoises collection. This study is the first population based analyses for Brucella spp. infections in cetaceans stranded along the Dutch coast. Copyright © 2014 The Authors. Published by Elsevier B.V. All rights reserved.

Population genetic data and forensic parameters of 30 autosomal InDel markers in Santa Catarina State population, Southern Brazil.

PubMed

Torres, Sandra Regina Rachadel; Uehara, Clineu Julien Seki; Sutter-Latorre, Ana Frederica; de Almeida, Bibiana Sgorla; Sauerbier, Tania Streck; Muniz, Yara Costa Netto; Marrero, Andrea Rita; de Souza, Ilíada Rainha

2014-08-01

The application of DNA technology in forensic investigations has grown rapidly in the last 25 years and with an exponential increase of short tandem repeats (STRs) data, usually presented as allele frequencies, that may be later used as databases for forensic and population genetics purposes. Thereby, classes of molecular markers such as single nucleotide polymorphisms and insertions/deletions (InDels) have been presented as another option of genetic marker sets. These markers can be used in paternity cases, when mutations in STR polymorphisms are present, as well as in highly degraded DNA analysis. In the present study, the allele frequencies and heterozygosity (H) of a 30 InDel markers set were determined and the forensic efficacy was evaluated through estimation of discrimination power (DP), match probability, typical paternity index and power of paternity exclusion in 108 unrelated volunteers from the State of Santa Catarina (South Brazil). The observed H per locus showed a range between 0.370 and 0.574 (mean = 0.479). HLD128 was the locus with the highest DP (DP = 0.656). DP for all markers combined was greater than 99.9999999999646 % which provides satisfactory levels of information for forensic demands. Genetic comparisons (exact tests of population differentiation and pairwise genetic distances) revealed that the population of Santa Catarina State differs from Korea and USA Afro-American populations but is similar to the Portuguese, German, Polish, Spanish and Basque populations.

Molecular epidemiology of Bordetella pertussis in the Philippines in 2012-2014.

PubMed

Galit, Salvacion Rosario L; Otsuka, Nao; Furuse, Yuki; Almonia, Daryl Joy V; Sombrero, Lydia T; Capeding, Rosario Z; Lupisan, Socorro P; Saito, Mariko; Oshitani, Hitoshi; Hiramatsu, Yukihiro; Shibayama, Keigo; Kamachi, Kazunari

2015-06-01

The present study was designed to determine the genotypes of circulating Bordetella pertussis in the Philippines by direct molecular typing of clinical specimens. Nasopharyngeal swabs (NPSs) were collected from 50 children hospitalized with pertussis in three hospitals during 2012-2014. Multilocus variable-number tandem repeat analysis (MLVA) was performed on the DNA extracts from NPSs. B. pertussis virulence-associated allelic genes (ptxA, prn, and fim3) and the pertussis toxin promoter, ptxP, were also investigated by DNA sequence-based typing. Twenty-six DNA extracts yielded a complete MLVA profile, which were sorted into 10 MLVA types. MLVA type 34 (MT34), which is rare in Australia, Europe, Japan, and the USA, was the predominant strain (50%). Seven MTs (MT29, MT32, MT33, and MT283-286, total 42%) were single-locus variants of MT34, while two (MT141 and MT287, total 8%) were double-locus variants of MT34. All MTs had the combination of virulence-associated allelic genes, ptxP1-ptxA1-prn1-fim3A. The B. pertussis population in the Philippines comprises genetically related strains. These strains are markedly different from those found in patients from other countries where acellular pertussis vaccines are used. The differences in vaccine types between these other countries and the Philippines, where the whole-cell vaccine is still used, may select for distinct populations of B. pertussis. Copyright © 2015 The Authors. Published by Elsevier Ltd.. All rights reserved.

Predominance of Central Asian and European families among Mycobacterium tuberculosis isolates in Kashmir Valley, India.

PubMed

Bashir, Gulnaz; Wani, Tehmeena; Sharma, Pragya; Katoch, V M; Lone, Rubina; Shah, Azra; Katoch, Kiran; Kakru, D K; Chauhan, Devendra Singh

2017-10-01

As there are no data available regarding the strains of Mycobacterium tuberculosis circulating in Kashmir Valley, India, the current study aimed at describing the genetic diversity of M. tuberculosis strains in this region, by spoligotyping and 12-locus-based MIRU-VNTR typing (Mycobacterial Interspersed Repetitive Unit-Variable Number Tandem Repeat). Sputa from 207 smear positive cases with newly diagnosed pulmonary tuberculosis were subjected to culture for M. tuberculosis. Eighty-five isolates confirmed as M. tuberculosis were subjected to drug susceptibility testing and molecular typing by spoligotyping and MIRU-VNTRs. Drug susceptibility results of 72 isolates revealed 76.3% as fully sensitive while 5.5% as multidrug resistant (MDR). Spoligotyping of 85 isolates detected 42 spoligotypes with 50 isolates (58.8%) clustered into seven spoligotypes. SIT26/CAS1_Del was the major spoligotype (23, 27%) followed by SIT127/H4 (12, 14.1%); CAS lineage (37.6%) was predominant, followed by Haarlem (25.8%) and ill-defined T clade (23.5%). MIRU-VNTR analysis displayed 82 MIRU patterns from 85 strains, including 3 small clusters and 79 unique. MIRU 26 was found to be the most discriminatory locus. Kashmir Valley has CAS as the predominant lineage of M. tuberculosis similar to the rest of the Indian sub-continent, while it is peculiar in having Euro American lineages such as Haarlem and ill-defined T clade. Copyright © 2017 Tuberculosis Association of India. Published by Elsevier B.V. All rights reserved.

Tandem repeats of the 5' non-transcribed spacer of Tetrahymena rDNA function as high copy number autonomous replicons in the macronucleus but do not prevent rRNA gene dosage regulation.

PubMed Central

Pan, W J; Blackburn, E H

1995-01-01

The rRNA genes in the somatic macronucleus of Tetrahymena thermophila are normally on 21 kb linear palindromic molecules (rDNA). We examined the effect on rRNA gene dosage of transforming T.thermophila macronuclei with plasmid constructs containing a pair of tandemly repeated rDNA replication origin regions unlinked to the rRNA gene. A significant proportion of the plasmid sequences were maintained as high copy circular molecules, eventually consisting solely of tandem arrays of origin regions. As reported previously for cells transformed by a construct in which the same tandem rDNA origins were linked to the rRNA gene [Yu, G.-L. and Blackburn, E. H. (1990) Mol. Cell. Biol., 10, 2070-2080], origin sequences recombined to form linear molecules bearing several tandem repeats of the origin region, as well as rRNA genes. The total number of rDNA origin sequences eventually exceeded rRNA gene copies by approximately 20- to 40-fold and the number of circular replicons carrying only rDNA origin sequences exceeded rRNA gene copies by 2- to 3-fold. However, the rRNA gene dosage was unchanged. Hence, simply monitoring the total number of rDNA origin regions is not sufficient to regulate rRNA gene copy number. Images PMID:7784211

Transcription of highly repetitive tandemly organized DNA in amphibians and birds: A historical overview and modern concepts.

PubMed

Trofimova, Irina; Krasikova, Alla

2016-12-01

Tandemly organized highly repetitive DNA sequences are crucial structural and functional elements of eukaryotic genomes. Despite extensive evidence, satellite DNA remains an enigmatic part of the eukaryotic genome, with biological role and significance of tandem repeat transcripts remaining rather obscure. Data on tandem repeats transcription in amphibian and avian model organisms is fragmentary despite their genomes being thoroughly characterized. Review systematically covers historical and modern data on transcription of amphibian and avian satellite DNA in somatic cells and during meiosis when chromosomes acquire special lampbrush form. We highlight how transcription of tandemly repetitive DNA sequences is organized in interphase nucleus and on lampbrush chromosomes. We offer LTR-activation hypotheses of widespread satellite DNA transcription initiation during oogenesis. Recent explanations are provided for the significance of high-yield production of non-coding RNA derived from tandemly organized highly repetitive DNA. In many cases the data on the transcription of satellite DNA can be extrapolated from lampbrush chromosomes to interphase chromosomes. Lampbrush chromosomes with applied novel technical approaches such as superresolution imaging, chromosome microdissection followed by high-throughput sequencing, dynamic observation in life-like conditions provide amazing opportunities for investigation mechanisms of the satellite DNA transcription.

Transcription of highly repetitive tandemly organized DNA in amphibians and birds: A historical overview and modern concepts

PubMed Central

Krasikova, Alla

2016-01-01

ABSTRACT Tandemly organized highly repetitive DNA sequences are crucial structural and functional elements of eukaryotic genomes. Despite extensive evidence, satellite DNA remains an enigmatic part of the eukaryotic genome, with biological role and significance of tandem repeat transcripts remaining rather obscure. Data on tandem repeats transcription in amphibian and avian model organisms is fragmentary despite their genomes being thoroughly characterized. Review systematically covers historical and modern data on transcription of amphibian and avian satellite DNA in somatic cells and during meiosis when chromosomes acquire special lampbrush form. We highlight how transcription of tandemly repetitive DNA sequences is organized in interphase nucleus and on lampbrush chromosomes. We offer LTR-activation hypotheses of widespread satellite DNA transcription initiation during oogenesis. Recent explanations are provided for the significance of high-yield production of non-coding RNA derived from tandemly organized highly repetitive DNA. In many cases the data on the transcription of satellite DNA can be extrapolated from lampbrush chromosomes to interphase chromosomes. Lampbrush chromosomes with applied novel technical approaches such as superresolution imaging, chromosome microdissection followed by high-throughput sequencing, dynamic observation in life-like conditions provide amazing opportunities for investigation mechanisms of the satellite DNA transcription. PMID:27763817

Tandem Amplification of a Chromosomal Segment Harboring 5-Enolpyruvylshikimate-3-Phosphate Synthase Locus Confers Glyphosate Resistance in Kochia scoparia1[W][OPEN

PubMed Central

Jugulam, Mithila; Niehues, Kindsey; Godar, Amar S.; Koo, Dal-Hoe; Danilova, Tatiana; Friebe, Bernd; Sehgal, Sunish; Varanasi, Vijay K.; Wiersma, Andrew; Westra, Philip; Stahlman, Phillip W.; Gill, Bikram S.

2014-01-01

Recent rapid evolution and spread of resistance to the most extensively used herbicide, glyphosate, is a major threat to global crop production. Genetic mechanisms by which weeds evolve resistance to herbicides largely determine the level of resistance and the rate of evolution of resistance. In a previous study, we determined that glyphosate resistance in Kochia scoparia is due to the amplification of the 5-Enolpyruvylshikimate-3-Phosphate Synthase (EPSPS) gene, the enzyme target of glyphosate. Here, we investigated the genomic organization of the amplified EPSPS copies using fluorescence in situ hybridization (FISH) and extended DNA fiber (Fiber FISH) on K. scoparia chromosomes. In both glyphosate-resistant K. scoparia populations tested (GR1 and GR2), FISH results displayed a single and prominent hybridization site of the EPSPS gene localized on the distal end of one pair of homologous metaphase chromosomes compared with a faint hybridization site in glyphosate-susceptible samples (GS1 and GS2). Fiber FISH displayed 10 copies of the EPSPS gene (approximately 5 kb) arranged in tandem configuration approximately 40 to 70 kb apart, with one copy in an inverted orientation in GR2. In agreement with FISH results, segregation of EPSPS copies followed single-locus inheritance in GR1 population. This is the first report of tandem target gene amplification conferring field-evolved herbicide resistance in weed populations. PMID:25037215

Tissue- and age-specific DNA replication patterns at the CTG/CAG-expanded human myotonic dystrophy type 1 locus.

PubMed

Cleary, John D; Tomé, Stéphanie; López Castel, Arturo; Panigrahi, Gagan B; Foiry, Laurent; Hagerman, Katharine A; Sroka, Hana; Chitayat, David; Gourdon, Geneviève; Pearson, Christopher E

2010-09-01

Myotonic dystrophy, caused by DM1 CTG/CAG repeat expansions, shows varying instability levels between tissues and across ages within patients. We determined DNA replication profiles at the DM1 locus in patient fibroblasts and tissues from DM1 transgenic mice of various ages showing different instability. In patient cells, the repeat is flanked by two replication origins demarcated by CTCF sites, with replication diminished at the expansion. In mice, the expansion replicated from only the downstream origin (CAG as lagging template). In testes from mice of three different ages, replication toward the repeat paused at the earliest age and was relieved at later ages-coinciding with increased instability. Brain, pancreas and thymus replication varied with CpG methylation at DM1 CTCF sites. CTCF sites between progressing forks and repeats reduced replication depending on chromatin. Thus, varying replication progression may affect tissue- and age-specific repeat instability.

Integration of Lupinus angustifolius L. (narrow-leafed lupin) genome maps and comparative mapping within legumes.

PubMed

Wyrwa, Katarzyna; Książkiewicz, Michał; Szczepaniak, Anna; Susek, Karolina; Podkowiński, Jan; Naganowska, Barbara

2016-09-01

Narrow-leafed lupin (Lupinus angustifolius L.) has recently been considered a reference genome for the Lupinus genus. In the present work, genetic and cytogenetic maps of L. angustifolius were supplemented with 30 new molecular markers representing lupin genome regions, harboring genes involved in nitrogen fixation during the symbiotic interaction of legumes and soil bacteria (Rhizobiaceae). Our studies resulted in the precise localization of bacterial artificial chromosomes (BACs) carrying sequence variants for early nodulin 40, nodulin 26, nodulin 45, aspartate aminotransferase P2, asparagine synthetase, cytosolic glutamine synthetase, and phosphoenolpyruvate carboxylase. Together with previously mapped chromosomes, the integrated L. angustifolius map encompasses 73 chromosome markers, including 5S ribosomal DNA (rDNA) and 45S rDNA, and anchors 20 L. angustifolius linkage groups to corresponding chromosomes. Chromosomal identification using BAC fluorescence in situ hybridization identified two BAC clones as narrow-leafed lupin centromere-specific markers, which served as templates for preliminary studies of centromere composition within the genus. Bioinformatic analysis of these two BACs revealed that centromeric/pericentromeric regions of narrow-leafed lupin chromosomes consisted of simple sequence repeats ordered into tandem repeats containing the trinucleotide and pentanucleotide simple sequence repeats AGG and GATAC, structured into long arrays. Moreover, cross-genus microsynteny analysis revealed syntenic patterns of 31 single-locus BAC clones among several legume species. The gene and chromosome level findings provide evidence of ancient duplication events that must have occurred very early in the divergence of papilionoid lineages. This work provides a strong foundation for future comparative mapping among legumes and may facilitate understanding of mechanisms involved in shaping legume chromosomes.

Host adaptation of Chlamydia pecorum towards low virulence evident in co-evolution of the ompA, incA, and ORF663 Loci.

PubMed

Mohamad, Khalil Yousef; Kaltenboeck, Bernhard; Rahman, Kh Shamsur; Magnino, Simone; Sachse, Konrad; Rodolakis, Annie

2014-01-01

Chlamydia (C.) pecorum, an obligate intracellular bacterium, may cause severe diseases in ruminants, swine and koalas, although asymptomatic infections are the norm. Recently, we identified genetic polymorphisms in the ompA, incA and ORF663 genes that potentially differentiate between high-virulence C. pecorum isolates from diseased animals and low-virulence isolates from asymptomatic animals. Here, we expand these findings by including additional ruminant, swine, and koala strains. Coding tandem repeats (CTRs) at the incA locus encoded a variable number of repeats of APA or AGA amino acid motifs. Addition of any non-APA/AGA repeat motif, such as APEVPA, APAVPA, APE, or APAPE, associated with low virulence (P<10-4), as did a high number of amino acids in all incA CTRs (P = 0.0028). In ORF663, high numbers of 15-mer CTRs correlated with low virulence (P = 0.0001). Correction for ompA phylogram position in ORF663 and incA abolished the correlation between genetic changes and virulence, demonstrating co-evolution of ompA, incA, and ORF663 towards low virulence. Pairwise divergence of ompA, incA, and ORF663 among isolates from healthy animals was significantly higher than among strains isolated from diseased animals (P≤10-5), confirming the longer evolutionary path traversed by low-virulence strains. All three markers combined identified 43 unique strains and 4 pairs of identical strains among all 57 isolates tested, demonstrating the suitability of these markers for epidemiological investigations.

Effect of polymorphism in insulin locus and HLA on type 1 diabetes in four ethnic groups in Israel.

PubMed

Benedek, G; Brautbar, C; Vardi, P; Sharon, N; Weintrob, N; Zung, A; Israel, S

2009-01-01

This study examined a possible association of the insulin (INS) gene with type 1 diabetes (T1D) in patients and controls from four ethnic groups in Israel. We analyzed the distribution of -23HphI single nucleotide polymorphism (SNP) T/A alleles that correspond to INS variable number of tandem repeat short class I alleles (26-63 repeats) and class III alleles (141-209 repeats), respectively. The -23HphI T/T genotype was found to be positively associated with T1D in three Jewish groups (Yemenites: 93.9% patients vs 68.8% controls, P = 0.0002; Ashkenazi: 80.6% vs 50.8%, P < 10(-4); Ethiopians: 75% vs 40.5%, P = 0.002). The Yemenite healthy controls have the highest frequency of T allele from all Jewish groups studied (83.5% vs 68.8% in Ashkenazi and 64.3% in Ethiopians). The high frequency of a susceptibility allele in the Yemenites is in line with the high incidence of T1D in this population. No association was observed between T1D and the INS gene in Israeli Arabs studied (70.6% vs 66.7%). Variable incidence of T1D among different ethnicities in Israel is largely attributed to heterogeneous genetics. Human leukocyte antigen (HLA) results of our previous studies describing the susceptibility and protective haplotypes were used for combined analysis to determine possible interaction between the HLA and INS loci. Only in the Ashkenazi group such interaction was presented with statistical significance.

Complete mitochondrial genome of the whiter-spotted flower chafer, Protaetia brevitarsis (Coleoptera: Scarabaeidae).

PubMed

Kim, Min Jee; Im, Hyun Hwak; Lee, Kwang Youll; Han, Yeon Soo; Kim, Iksoo

2014-06-01

Abstract The complete nucleotide sequences of the mitochondrial genome from the whiter-spotted flower chafer, Protaetia brevitarsis (Coleoptera: Scarabaeidae), was determined. The 20,319-bp long circular genome is the longest among completely sequenced Coleoptera. As is typical in animals, the P. brevitarsis genome consisted of two ribosomal RNAs, 22 transfer RNAs, 13 protein-coding genes and one A + T-rich region. Although the size of the coding genes was typical, the non-coding A + T-rich region was 5654 bp, which is the longest in insects. The extraordinary length of this region was composed of 28,117-bp tandem repeats and 782-bp tandem repeats. These repeat sequences were encompassed by three non-repeat sequences constituting 1804 bp.

Using long ssDNA polynucleotides to amplify STRs loci in degraded DNA samples

PubMed Central

Pérez Santángelo, Agustín; Corti Bielsa, Rodrigo M.; Sala, Andrea; Ginart, Santiago; Corach, Daniel

2017-01-01

Obtaining informative short tandem repeat (STR) profiles from degraded DNA samples is a challenging task usually undermined by locus or allele dropouts and peak-high imbalances observed in capillary electrophoresis (CE) electropherograms, especially for those markers with large amplicon sizes. We hereby show that the current STR assays may be greatly improved for the detection of genetic markers in degraded DNA samples by using long single stranded DNA polynucleotides (ssDNA polynucleotides) as surrogates for PCR primers. These long primers allow a closer annealing to the repeat sequences, thereby reducing the length of the template required for the amplification in fragmented DNA samples, while at the same time rendering amplicons of larger sizes suitable for multiplex assays. We also demonstrate that the annealing of long ssDNA polynucleotides does not need to be fully complementary in the 5’ region of the primers, thus allowing for the design of practically any long primer sequence for developing new multiplex assays. Furthermore, genotyping of intact DNA samples could also benefit from utilizing long primers since their close annealing to the target STR sequences may overcome wrong profiling generated by insertions/deletions present between the STR region and the annealing site of the primers. Additionally, long ssDNA polynucleotides might be utilized in multiplex PCR assays for other types of degraded or fragmented DNA, e.g. circulating, cell-free DNA (ccfDNA). PMID:29099837

Haplotype data for 23 Y-chromosome markers in a reference sample from Bosnia and Herzegovina

PubMed Central

Kovačević, Lejla; Fatur-Cerić, Vera; Hadžić, Negra; Čakar, Jasmina; Primorac, Dragan; Marjanović, Damir

2013-01-01

Aim To detect polymorphisms of 23 Y-chromosomal short tandem repeat (STR) loci, including 6 new loci, in a reference database of male population of Bosnia and Herzegovina, as well as to assess the importance of increasing the number of Y-STR loci utilized in forensic DNA analysis. Methods The reference sample consisted of 100 healthy, unrelated men originating from Bosnia and Herzegovina. Sample collection using buccal swabs was performed in all geographical regions of Bosnia and Herzegovina in the period from 2010 to 2011. DNA samples were typed for 23 Y STR loci, including 6 new loci: DYS576, DYS481, DYS549, DYS533, DYS570, and DYS643, which are included in the new PowerPlex® Y 23 amplification kit. Results The absolute frequency of generated haplotypes was calculated and results showed that 98 samples had unique Y 23 haplotypes, and that only two samples shared the same haplotype. The most polymorphic locus was DYS418, with 14 detected alleles and the least polymorphic loci were DYS389I, DYS391, DYS437, and DYS393. Conclusion This study showed that by increasing the number of highly polymorphic Y STR markers, to include those tested in our analysis, leads to a reduction of repeating haplotypes, which is very important in the application of forensic DNA analysis. PMID:23771760

[Report of Relapse Typhoid Fever Cases from Kolkata, India: Recrudescence or Reinfection?

PubMed

Samajpati, Sriparna; Das, Surojit; Ray, Ujjwayini; Dutta, Shanta

2018-05-24

Three relapse cases were reported out of 107 hospital-attending typhoid cases within a period of 2 years (2014-2016) from Apollo Gleneagles Hospital, Kolkata, India. During the first episode of typhoid fever, 2 of the 3 cases were treated with ceftriaxone (CRO) for 7 days, and 1 was treated for 14 days. Six Salmonella Typhi (S. Typhi) isolates, obtained from the 3 patients during both typhoid episodes, were subjected to antimicrobial susceptibility testing, detection of quinolone resistance-determining region (QRDR) mutation and molecular subtyping by pulsed-field gel electrophoresis (PFGE), multiple-locus variable number tandem repeat analysis (MLVA), multilocus sequence typing (MLST), clustered regularly interspaced short palindromic repeats (CRISPR), and H58 haplotyping. Pairs of the S. Typhi strains isolated from two of the patients during the 1st and 2nd episodes were similar with respect to the antimicrobial resistance (AMR) profiles, QRDR mutations, and molecular subtypes; whereas, the S. Typhi strain pair isolated from the 3rd patient were different in their AMR profiles, QRDR mutations, and MLVA profiles. From these observations, it may be concluded that in spite of treating typhoid cases with CRO for 7-14 days, relapse of typhoid fever might occur. The article also showed the advantage of MLVA typing over PFGE, MLST, and CRISPR typing for the discrimination of strains isolated from the same patient in case of relapse of typhoid fever.

Simple Sequence Repeats in Escherichia coli: Abundance, Distribution, Composition, and Polymorphism

PubMed Central

Gur-Arie, Riva; Cohen, Cyril J.; Eitan, Yuval; Shelef, Leora; Hallerman, Eric M.; Kashi, Yechezkel

2000-01-01

Computer-based genome-wide screening of the DNA sequence of Escherichia coli strain K12 revealed tens of thousands of tandem simple sequence repeat (SSR) tracts, with motifs ranging from 1 to 6 nucleotides. SSRs were well distributed throughout the genome. Mononucleotide SSRs were over-represented in noncoding regions and under-represented in open reading frames (ORFs). Nucleotide composition of mono- and dinucleotide SSRs, both in ORFs and in noncoding regions, differed from that of the genomic region in which they occurred, with 93% of all mononucleotide SSRs proving to be of A or T. Computer-based analysis of the fine position of every SSR locus in the noncoding portion of the genome relative to downstream ORFs showed SSRs located in areas that could affect gene regulation. DNA sequences at 14 arbitrarily chosen SSR tracts were compared among E. coli strains. Polymorphisms of SSR copy number were observed at four of seven mononucleotide SSR tracts screened, with all polymorphisms occurring in noncoding regions. SSR polymorphism could prove important as a genome-wide source of variation, both for practical applications (including rapid detection, strain identification, and detection of loci affecting key phenotypes) and for evolutionary adaptation of microbes.[The sequence data described in this paper have been submitted to the GenBank data library under accession numbers AF209020–209030 and AF209508–209518.] PMID:10645951

Molecular Epidemiology of Cholera Outbreaks during the Rainy Season in Mandalay, Myanmar.

PubMed

Roobthaisong, Amonrattana; Okada, Kazuhisa; Htun, Nilar; Aung, Wah Wah; Wongboot, Warawan; Kamjumphol, Watcharaporn; Han, Aye Aye; Yi, Yi; Hamada, Shigeyuki

2017-11-01

Cholera, caused by Vibrio cholerae , remains a global threat to public health. In Myanmar, the availability of published information on the occurrence of the disease is scarce. We report here that cholera incidence in Mandalay generally exhibited a single annual peak, with an annual average of 312 patients with severe dehydration over the past 5 years (since 2011) and was closely associated with the rainy season. We analyzed cholera outbreaks, characterized 67 isolates of V. cholerae serogroup O1 in 2015 from patients from Mandalay, and compared them with 22 V. cholerae O1 isolates (12 from Mandalay and 10 from Yangon) in 2014. The isolates carried the classical cholera toxin B subunit ( ctxB ), the toxin-coregulated pilus A ( tcpA ) of Haitian type, and repeat sequence transcriptional regulator ( rstR ) of El Tor type. Two molecular typing methods, pulsed-field gel electrophoresis and multiple-locus variable-number tandem repeat analysis (MLVA), differentiated the 89 isolates into seven pulsotypes and 15 MLVA profiles. Pulsotype Y15 and one MLVA profile (11, 7, 7, 16, 7) were predominantly found in the isolates from cholera outbreaks in Mandalay, 2015. Pulsotypes Y11, Y12, and Y15 with some MLVA profiles were detected in the isolates from two remote areas, Mandalay and Yangon, with temporal changes. These data suggested that cholera spread from the seaside to the inland area in Myanmar.

Phylogenetic Characteristics of Anthrax Outbreaks in Liaoning Province, China, 2001-2015.

PubMed

Mao, Lingling; Zhang, Enmin; Wang, Zijiang; Li, Yan; Zhou, Hang; Liu, Xuesheng; Zhang, Huijuan; Cai, Hong; Liang, Xudong; Sun, Yingwei; Zhang, Zhikai; Li, Wei; Yao, Wenqing; Wei, Jianchun

2016-01-01

Anthrax is a continuous threat in China, especially in rural regions. In July 2015, an anthrax outbreak occurred in Xifeng County, Liaoning Province. A total of 10 cutaneous anthrax cases were reported, with 210 people under medical observation. In this study, the general characteristics of human anthrax outbreak occurred in Liaoning Province were described, and all cases were caused by butchering and contacting sick animal. Meanwhile, the phylogenetic relationship between outbreak-related isolates/samples of the year 2015 and previous Bacillus anthracis strains was analyzed by means of canonical single nucleotide polymorphisms (canSNP), multiple-locus variable-number tandem repeat analysis (MLVA) with 15 markers and single-nucleotide repeats (SNR) analysis. There are two canSNP subgroups found in Liaoning, A.Br.001/002 and A.Br.Ames, and a total of six MLVA 15 genotypes and five SNR genotypes were observed. The strain collected from anthrax outbreak in Xifeng County in 2015 was classified as A.Br.001/002 subgroup and identified as MLVA15-29 genotype, with same SNR profile (CL10: 17, CL12: 15, CL33: 29, and CL35: 13). So we conclude that the same clone of B.anthracis caused the anthrax outbreak in Xifeng County in 2015, and this clone is different to previous isolates. Strengthening public health education in China is one of the most important measures to prevent and control anthrax.

Tandem repeated application of organic solvents and sodium lauryl sulphate enhances cumulative skin irritation.

PubMed

Schliemann, Sibylle; Schmidt, Christina; Elsner, Peter

2014-01-01

The objective of our study was to investigate the tandem irritation potential of two organic solvents with concurrent exposure to the hydrophilic detergent irritant sodium lauryl sulphate (SLS). A tandem repeated irritation test was performed with two undiluted organic solvents, cumene (C) and octane (O), with either alternating application with SLS 0.5% or twice daily application of each irritant alone in 27 volunteers on the skin of the back. The cumulative irritation induced over 4 days was quantified using visual scoring and non-invasive bioengineering measurements (skin colour reflectance, skin hydration and transepidermal water loss). Repeated application of C/SLS and O/SLS induced more decline of stratum corneum hydration and higher degrees of clinical irritation and erythema compared to each irritant alone. Our results demonstrate a further example of additive harmful skin effects induced by particular skin irritants and indicate that exposure to organic solvents together with detergents may increase the risk of acquiring occupational contact dermatitis. © 2014 S. Karger AG, Basel.

Thermal denaturation of the BRCT tandem repeat region of human tumour suppressor gene product BRCA1.

PubMed

Pyrpassopoulos, Serapion; Ladopoulou, Angela; Vlassi, Metaxia; Papanikolau, Yannis; Vorgias, Constantinos E; Yannoukakos, Drakoulis; Nounesis, George

2005-04-01

Reduced stability of the tandem BRCT domains of human BReast CAncer 1 (BRCA1) due to missense mutations may be critical for loss of function in DNA repair and damage-induced checkpoint control. In the present thermal denaturation study of the BRCA1 BRCT region, high-precision differential scanning calorimetry (DSC) and circular dichroism (CD) spectroscopy provide evidence for the existence of a denatured state that is structurally very similar to the native. Consistency between theoretical structure-based estimates of the enthalpy (DeltaH) and heat capacity change (DeltaCp) and the calorimetric results is obtained when considering partial thermal unfolding contained in the region of the conserved hydrophobic pocket formed at the interface of the two BRCT repeats. The structural integrity of this region has been shown to be crucial for the interaction of BRCA1 with phosphorylated peptides. In addition, cancer-causing missense mutations located at the inter-BRCT-repeat interface have been linked to the destabilization of the tandem BRCT structure.

Full-length model of the human galectin-4 and insights into dynamics of inter-domain communication

NASA Astrophysics Data System (ADS)

Rustiguel, Joane K.; Soares, Ricardo O. S.; Meisburger, Steve P.; Davis, Katherine M.; Malzbender, Kristina L.; Ando, Nozomi; Dias-Baruffi, Marcelo; Nonato, Maria Cristina

2016-09-01

Galectins are proteins involved in diverse cellular contexts due to their capacity to decipher and respond to the information encoded by β-galactoside sugars. In particular, human galectin-4, normally expressed in the healthy gastrointestinal tract, displays differential expression in cancerous tissues and is considered a potential drug target for liver and lung cancer. Galectin-4 is a tandem-repeat galectin characterized by two carbohydrate recognition domains connected by a linker-peptide. Despite their relevance to cell function and pathogenesis, structural characterization of full-length tandem-repeat galectins has remained elusive. Here, we investigate galectin-4 using X-ray crystallography, small- and wide-angle X-ray scattering, molecular modelling, molecular dynamics simulations, and differential scanning fluorimetry assays and describe for the first time a structural model for human galectin-4. Our results provide insight into the structural role of the linker-peptide and shed light on the dynamic characteristics of the mechanism of carbohydrate recognition among tandem-repeat galectins.

A Lossy Compression Technique Enabling Duplication-Aware Sequence Alignment

PubMed Central

Freschi, Valerio; Bogliolo, Alessandro

2012-01-01

In spite of the recognized importance of tandem duplications in genome evolution, commonly adopted sequence comparison algorithms do not take into account complex mutation events involving more than one residue at the time, since they are not compliant with the underlying assumption of statistical independence of adjacent residues. As a consequence, the presence of tandem repeats in sequences under comparison may impair the biological significance of the resulting alignment. Although solutions have been proposed, repeat-aware sequence alignment is still considered to be an open problem and new efficient and effective methods have been advocated. The present paper describes an alternative lossy compression scheme for genomic sequences which iteratively collapses repeats of increasing length. The resulting approximate representations do not contain tandem duplications, while retaining enough information for making their comparison even more significant than the edit distance between the original sequences. This allows us to exploit traditional alignment algorithms directly on the compressed sequences. Results confirm the validity of the proposed approach for the problem of duplication-aware sequence alignment. PMID:22518086

Topological characteristics of helical repeat proteins.

PubMed

Groves, M R; Barford, D

1999-06-01

The recent elucidation of protein structures based upon repeating amino acid motifs, including the armadillo motif, the HEAT motif and tetratricopeptide repeats, reveals that they belong to the class of helical repeat proteins. These proteins share the common property of being assembled from tandem repeats of an alpha-helical structural unit, creating extended superhelical structures that are ideally suited to create a protein recognition interface.

Unrelated sequences at the 5' end of mouse LINE-1 repeated elements define two distinct subfamilies.

PubMed Central

Wincker, P; Jubier-Maurin, V; Roizès, G

1987-01-01

Some full length members of the mouse long interspersed repeated DNA family L1Md have been shown to be associated at their 5' end with a variable number of tandem repetitions, the A repeats, that have been suggested to be transcription controlling elements. We report that the other type of repeat, named F, found at the 5' end of a few L1 elements is also an integral part of full length L1 copies. Sequencing shows that the F repeats are GC rich, and organized in tandem. The L1 copies associated with either A or F repeats can be correlated with two different subsets of L1 sequences distinguished by a series of variant nucleotides specific to each and by unassociated but frequent restriction sites. These findings suggest that sequence replacement has occurred at least once in 5' of L1Md, and is related to the generation of specific subfamilies. Images PMID:3684566

Plant chromosomes from end to end: telomeres, heterochromatin and centromeres.

PubMed

Lamb, Jonathan C; Yu, Weichang; Han, Fangpu; Birchler, James A

2007-04-01

Recent evidence indicates that heterochromatin in plants is composed of heterogeneous sequences, which are usually composed of transposable elements or tandem repeat arrays. These arrays are associated with chromatin modifications that produce a closed configuration that limits transcription. Centromere sequences in plants are usually composed of tandem repeat arrays that are homogenized across the genome. Analysis of such arrays in closely related taxa suggests a rapid turnover of the repeat unit that is typical of a particular species. In addition, two lines of evidence for an epigenetic component of centromere specification have been reported, namely an example of a neocentromere formed over sequences without the typical repeat array and examples of centromere inactivation. Although the telomere repeat unit is quite prevalent in the plant kingdom, unusual repeats have been found in some families. Recently, it was demonstrated that the introduction of telomere sequences into plants cells causes truncation of the chromosomes, and that this technique can be used to produce artificial chromosome platforms.

Preliminary report for analysis of genome wide mutations from four ciprofloxacin resistant B. anthracis Sterne isolates generated by Illumina, 454 sequencing and microarrays for DHS

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

Jaing, Crystal; Vergez, Lisa; Hinckley, Aubree

2011-06-21

The objective of this project is to provide DHS a comprehensive evaluation of the current genomic technologies including genotyping, Taqman PCR, multiple locus variable tandem repeat analysis (MLVA), microarray and high-throughput DNA sequencing in the analysis of biothreat agents from complex environmental samples. As the result of a different DHS project, we have selected for and isolated a large number of ciprofloxacin resistant B. anthracis Sterne isolates. These isolates vary in the concentrations of ciprofloxacin that they can tolerate, suggesting multiple mutations in the samples. In collaboration with University of Houston, Eureka Genomics and Oak Ridge National Laboratory, we analyzedmore » the ciprofloxacin resistant B. anthracis Sterne isolates by microarray hybridization, Illumina and Roche 454 sequencing to understand the error rates and sensitivity of the different methods. The report provides an assessment of the results and a complete set of all protocols used and all data generated along with information to interpret the protocols and data sets.« less

DNA transformations of Candida tropicalis with replicating and integrative vectors.

PubMed

Sanglard, D; Fiechter, A

1992-12-01

The alkane-assimilating yeast Candida tropicalis was used as a host for DNA transformations. A stable ade2 mutant (Ha900) obtained by UV-mutagenesis was used as a recipient for different vectors carrying selectable markers. A first vector, pMK16, that was developed for the transformation of C. albicans and carries an ADE2 gene marker and a Candida autonomously replicating sequence (CARS) element promoting autonomous replication, was compatible for transforming Ha900. Two transformant types were observed: (i) pink transformants which easily lose pMK16 under non-selective growth conditions; (ii) white transformants, in which the same plasmid exhibited a higher mitotic stability. In both cases pMK16 could be rescued from these cells in Escherichia coli. A second vector, pADE2, containing the isolated C. tropicalis ADE2, gene, was used to transform Ha900. This vector integrated in the yeast genome at homologous sites of the ade2 locus. Different integration types were observed at one or both ade2 alleles in single or in tandem repeats.

Molecular epidemiological investigation of Brucella melitensis circulating in Mongolia by MLVA16.

PubMed

Kang, Sung-Il; Her, Moon; Erdenebaataar, Janchivdorj; Vanaabaatar, Batbaatar; Cho, Hyorim; Sung, So-Ra; Lee, Jin Ju; Jung, Suk Chan; Park, Yong Ho; Kim, Ji-Yeon

2017-02-01

Mongolia has a high incidence of brucellosis in human and animals due to livestock husbandry. To investigate the genetic characteristics of Mongolian B. melitensis, an MLVA (multi-locus variable-number tandem-repeat analysis)-16 assay was performed with 94 B. melitensis isolates. They were identified as B. melitensis biovar (bv.) 1 (67), 3 (10) and Rev. 1 vaccine strains (17) using a classical biotyping and multiplex PCR. In genotyping, three human isolates were grouped at 2 genotypes with sheep isolates, and it implies that B. melitensis are cross-infected between human and livestock. In the parsimony analysis, Mongolian B. melitensis isolates had high genetic similarity with Chinese strains, likely due to the geographical proximity, clustered distinctively as compared with other foreign isolates. B. melitensis Rev. 1 vaccine strains were divided into 4 genotypes with 92% similarity. In the analysis of Rev.1 strains, the risk of mutation of vaccine strain might not be overlooked. Animal quarantines should be strengthened to prevent the spread of Brucella species among adjacent countries. Copyright © 2016 Elsevier Ltd. All rights reserved.

Human U2 snRNA Genes Exhibit a Persistently Open Transcriptional State and Promoter Disassembly at Metaphase▿

PubMed Central

Pavelitz, Thomas; Bailey, Arnold D.; Elco, Christopher P.; Weiner, Alan M.

2008-01-01

In mammals, small multigene families generate spliceosomal U snRNAs that are nearly as abundant as rRNA. Using the tandemly repeated human U2 genes as a model, we show by footprinting with DNase I and permanganate that nearly all sequences between the enhancer-like distal sequence element and the initiation site are protected during interphase whereas the upstream half of the U2 snRNA coding region is exposed. We also show by chromatin immunoprecipitation that the SNAPc complex, which binds the TATA-like proximal sequence element, is removed at metaphase but remains bound under conditions that induce locus-specific metaphase fragility of the U2 genes, such as loss of CSB, BRCA1, or BRCA2 function, treatment with actinomycin D, or overexpression of the tetrameric p53 C terminus. We propose that the U2 snRNA promoter establishes a persistently open state to facilitate rapid reinitiation and perhaps also to bypass TFIIH-dependent promoter melting; this open state would then be disassembled to allow metaphase chromatin condensation. PMID:18378697

Isolation of saprophytic Leptospira spp. from a selected environmental water source of Argentina.

PubMed

Scialfa, Exequiel; Grune, Sylvia; Brihuega, Bibiana; Aguirre, Pablo; Rivero, Marina

2017-11-29

Ten Leptospira spp. strains were isolated from water samples from Nievas stream, Olavarría, Buenos Aires province (Argentina). The isolates showed the typical motility and morphology of the genus Leptospira under dark field microscopy, developing in liquid EMJH medium after eight days of incubation at 13°C and 30°C. All isolates were negative by the Multiple Locus Variable Number Tandem Repeat Analysis (MLVA). Molecular identification by 16S rRNA gene sequencing identified all isolates as nonpathogenic leptospires. Four isolates showed a genetic profile identical to that of the reference strain Leptospira biflexa serovar Patoc, and six isolates revealed sequence similarities within the 97-98% range, closely related to Leptospira yanagawae and Leptospira meyeri, respectively. Strains ScialfaASA42, ScialfaASA45, ScialfaASA44, ScialfaASA47, ScialfaASA49, ScialfaASA50 and ScialfaASA51 possibly represent a novel species of the genus Leptospira. Copyright © 2017 Asociación Argentina de Microbiología. Publicado por Elsevier España, S.L.U. All rights reserved.

Laser Capture and Single Cell Genotyping from Frozen Tissue Sections.

PubMed

Kroneis, Thomas; Ye, Jody; Gillespie, Kathleen

2016-01-01

There is an increasing requirement for genetic analysis of individual cells from tissue sections. This is particularly the case for analysis of tumor cells but is also a requirement for analysis of cells in pancreas from individuals with type 1 diabetes where there is evidence of viral infection or in the analysis of chimerism in pancreas; either post-transplant or as a result of feto-maternal cell transfer.This protocol describes a strategy to isolate cells using laser microdissection and to run a 17plex PCR to discriminate between cells of haplo-identical origin (i.e., fetal and maternal cells) in pancreas tissue but other robust DNA tests could be used. In short, snap-frozen tissues are cryo-sectioned and mounted onto membrane-coated slides. Target cells are harvested from the tissue sections by laser microdissection and pressure catapulting (LMPC) prior to DNA profiling. This is based on amplification of highly repetitive yet stably inherited loci (short tandem repeats, STR) as well as the amelogenin locus for sex determination and separation of PCR products by capillary electrophoresis.

Baluchi and Pakhtun population data of 9 X-chromosomal short tandem repeat loci.

PubMed

Israr, Muhammad; Shahid, Ahmad Ali; Rakha, Allah; Zar, Mian Sahib; Shahzad, Muhammad Saqib; Rahman, Ziaur; Husnain, Tayyab

2016-01-01

Baluchistan is the largest province of Pakistan in terms of area, constituting approximately 44% of the country's total land mass, and the smallest in terms of population, being home to less than 5% of the country's population. Khyber Pakhtunkhwa (KPK) formerly called North-West Frontier Province is located in the north-west of Pakistan having an estimated 13.4% of total population of Pakistan in which Pakhtuns are the major ethnic group. A total of 250 samples from Baluchi population and 250 samples from Pakhtun population were typed for 9 X-chromosomal STR markers: DXS101, DXS6789, DXS7132, DXS7423, DXS7424, DXS8378, GATA31E08, GATA172D05 and HPRTB along with sex typing locus, Amelogenin. A total of 59 alleles were found in Baluchi population while 61 alleles were found in Pakhtun population. This is the first study of the two populations based on these markers and the population data can be used as reference database for Baluchi and Pakhtun populations. Copyright © 2015 Elsevier Ltd and Faculty of Forensic and Legal Medicine. All rights reserved.

E. coli O157 from sheep in northeast Scotland: prevalence, concentration shed, and molecular characterization by multilocus variable tandem repeat analysis.

PubMed

Solecki, Olivia; MacRae, Marion; Strachan, Norval; Lindstedt, Bjørn-Arne; Ogden, Iain

2009-09-01

We report the prevalence, concentrations, and strain diversity of Escherichia coli O157 shed by sheep fed on root crops during a winter period in northeast Scotland. E. coli O157 was isolated on 6 farms from 14 studied during January to March 2005. The individual sheep prevalence was 5.8% and concentration excreted was <10(2) colony-forming units/g for all but one fecal sample. Verocytotoxigenic E. coli O157, determined by polymerase chain reaction and verocell assay, was recovered from 27% of samples. Four farms had sheep shedding the same strain as determined by multiple-locus variable analysis and no within-farm diversity was observed. The low numbers shed and the high levels of atoxigenic strains indicate a lower risk to human health from these animals compared to many ruminants grazing pasture during summer months. These data will be valuable for quantitative risk assessments and provide preliminary information that feeding sheep on root crops may be a practical intervention to reduce E. coli O157 infection in animals and ultimately humans.

Three novel polymorphic microsatellite markers for the glaucoma locus GLC1B by datamining tetranucleotide repeats on chromosome 2p12-q12

PubMed Central

2009-01-01

In order to identify new markers around the glaucoma locus GLC1B as a tool to refine its critical region at 2p11.2-2q11.2, we searched the critical region sequence obtained from the UCSC database for tetranucleotide (GATA)n and (GTCT)n repeats of at least 10 units in length. Three out of four potential microsatellite loci were found to be polymorphic, heterozygosity ranging from 64.56% to 79.59%. The identified markers are useful not only for GLC1B locus but also for the study of other disease loci at 2p11.2-2q11.2, a region with scarcity of microsatellite markers. PMID:21637444

Genetic profiling of Trypanosoma cruzi directly in infected tissues using nested PCR of polymorphic microsatellites.

PubMed

Valadares, Helder Magno Silva; Pimenta, Juliana Ramos; de Freitas, Jorge Marcelo; Duffy, Tomás; Bartholomeu, Daniella C; Oliveira, Riva de Paula; Chiari, Egler; Moreira, Maria da Consolação Vieira; Filho, Geraldo Brasileiro; Schijman, Alejandro Gabriel; Franco, Glória Regina; Machado, Carlos Renato; Pena, Sérgio Danilo Junho; Macedo, Andréa Mara

2008-06-01

The investigation of the importance of the genetics of Trypanosoma cruzi in determining the clinical course of Chagas disease will depend on precise characterisation of the parasites present in the tissue lesions. This can be adequately accomplished by the use of hypervariable nuclear markers such as microsatellites. However the unilocal nature of these loci and the scarcity of parasites in chronic lesions make it necessary to use high sensitivity PCR with nested primers, whose design depends on the availability of long flanking regions, a feature not hitherto available for any known T. cruzi microsatellites. Herein, making use of the extensive T. cruzi genome sequence now available and using the Tandem Repeats Finder software, it was possible to identify and characterise seven new microsatellite loci--six composed of trinucleotide (TcTAC15, TcTAT20, TcAAT8, TcATT14, TcGAG10 and TcCAA10) and one composed of tetranucleotide (TcAAAT6) motifs. All except the TcCAA10 locus were physically mapped onto distinct intergenic regions of chromosome III of the CL Brener clone contigs. The TcCAA10 locus was localised within a hypothetical protein gene in the T. cruzi genome. All microsatellites were polymorphic and useful for T. cruzi genetic variability studies. Using the TcTAC15 locus it was possible to separate the strains belonging to the T. cruzi I lineage (DTU I) from those belonging to T. cruzi II (DTU IIb), T. cruzi III (DTU IIc) and a hybrid group (DTU IId, IIe). The long flanking regions of these novel microsatellites allowed construction of nested primers and the use of full nested PCR protocols. This strategy enabled us to detect and differentiate T. cruzi strains directly in clinical specimens including heart, blood, CSF and skin tissues from patients in the acute and chronic phases of Chagas disease.

My-Forensic-Loci-queries (MyFLq) framework for analysis of forensic STR data generated by massive parallel sequencing.

PubMed

Van Neste, Christophe; Vandewoestyne, Mado; Van Criekinge, Wim; Deforce, Dieter; Van Nieuwerburgh, Filip

2014-03-01

Forensic scientists are currently investigating how to transition from capillary electrophoresis (CE) to massive parallel sequencing (MPS) for analysis of forensic DNA profiles. MPS offers several advantages over CE such as virtually unlimited multiplexy of loci, combining both short tandem repeat (STR) and single nucleotide polymorphism (SNP) loci, small amplicons without constraints of size separation, more discrimination power, deep mixture resolution and sample multiplexing. We present our bioinformatic framework My-Forensic-Loci-queries (MyFLq) for analysis of MPS forensic data. For allele calling, the framework uses a MySQL reference allele database with automatically determined regions of interest (ROIs) by a generic maximal flanking algorithm which makes it possible to use any STR or SNP forensic locus. Python scripts were designed to automatically make allele calls starting from raw MPS data. We also present a method to assess the usefulness and overall performance of a forensic locus with respect to MPS, as well as methods to estimate whether an unknown allele, which sequence is not present in the MySQL database, is in fact a new allele or a sequencing error. The MyFLq framework was applied to an Illumina MiSeq dataset of a forensic Illumina amplicon library, generated from multilocus STR polymerase chain reaction (PCR) on both single contributor samples and multiple person DNA mixtures. Although the multilocus PCR was not yet optimized for MPS in terms of amplicon length or locus selection, the results show excellent results for most loci. The results show a high signal-to-noise ratio, correct allele calls, and a low limit of detection for minor DNA contributors in mixed DNA samples. Technically, forensic MPS affords great promise for routine implementation in forensic genomics. The method is also applicable to adjacent disciplines such as molecular autopsy in legal medicine and in mitochondrial DNA research. Copyright © 2013 The Authors. Published by Elsevier Ireland Ltd.. All rights reserved.

Concordance and population studies along with stutter and peak height ratio analysis for the PowerPlex ® ESX 17 and ESI 17 Systems.

PubMed

Hill, Carolyn R; Duewer, David L; Kline, Margaret C; Sprecher, Cynthia J; McLaren, Robert S; Rabbach, Dawn R; Krenke, Benjamin E; Ensenberger, Martin G; Fulmer, Patricia M; Storts, Douglas R; Butler, John M

2011-08-01

The PowerPlex(®) ESX 17 and ESI 17 Systems for short tandem repeat (STR) amplification were developed by the Promega Corporation to meet the European Network of Forensic Science Institutes (ENFSI) and the European DNA Profiling (EDNAP) Group recommendations for increasing the number of STR loci included in the European Standard Set (ESS). The PowerPlex ESX 17 and ESI 17 Systems utilize different PCR primer combinations to co-amplify the following 17 loci: D1S1656, D2S441, D2S1338, D3S1358, D8S1179, D10S1248, D12S391, D16S539, D18S51, D19S433, D21S11, D22S1045, FGA, TH01, vWA, SE33, and the sex-typing locus amelogenin. A total of 1443 U.S. population samples were evaluated with pre-commercialization versions of both kits. Stutter and heterozygote peak height ratios have been used to characterize kit performance. Typing results have been used to estimate the match probabilities provided by the chosen loci as well as in concordance studies. Full concordance between the typing results for the two kits was observed in 99.994% (49,055 out of 49,062) STR allele calls compared. All genotyping discrepancies were confirmed by DNA sequence analysis. As a result of these comparisons, a second forward primer for the D22S1045 locus has been added to the PowerPlex ESX 17 System to address a primer binding site mutation and the D1S1656 locus reverse primer in the PowerPlex ESI 17 System was modified to eliminate an amplification-efficiency reducing primer dimer. Copyright © 2010 Elsevier Ireland Ltd. All rights reserved.

Diversification and Distribution of Ruminant Chlamydia abortus Clones Assessed by MLST and MLVA.

PubMed

Siarkou, Victoria I; Vorimore, Fabien; Vicari, Nadia; Magnino, Simone; Rodolakis, Annie; Pannekoek, Yvonne; Sachse, Konrad; Longbottom, David; Laroucau, Karine

2015-01-01

Chlamydia abortus, an obligate intracellular bacterium, is the most common infectious cause of abortion in small ruminants worldwide and has zoonotic potential. We applied multilocus sequence typing (MLST) together with multiple-locus variable-number tandem repeat analysis (MLVA) to genotype 94 ruminant C. abortus strains, field isolates and samples collected from 1950 to 2011 in diverse geographic locations, with the aim of delineating C. abortus lineages and clones. MLST revealed the previously identified sequence types (STs) ST19, ST25, ST29 and ST30, plus ST86, a recently-assigned type on the Chlamydiales MLST website and ST87, a novel type harbouring the hemN_21 allele, whereas MLVA recognized seven types (MT1 to MT7). Minimum-spanning-tree analysis suggested that all STs but one (ST30) belonged to a single clonal complex, possibly reflecting the short evolutionary timescale over which the predicted ancestor (ST19) has diversified into three single-locus variants (ST86, ST87 and ST29) and further, through ST86 diversification, into one double-locus variant (ST25). ST descendants have probably arisen through a point mutation evolution mode. Interestingly, MLVA showed that in the ST19 population there was a greater genetic diversity than in other STs, most of which exhibited the same MT over time and geographical distribution. However, the evolutionary pathways of C. abortus STs seem to be diverse across geographic distances with individual STs restricted to particular geographic locations. The ST30 singleton clone displaying geographic specificity and represented by the Greek strains LLG and POS was effectively distinguished from the clonal complex lineage, supporting the notion that possibly two separate host adaptations and hence independent bottlenecks of C. abortus have occurred through time. The combination of MLST and MLVA assays provides an additional level of C. abortus discrimination and may prove useful for the investigation and surveillance of emergent C. abortus clonal populations.

Allele frequencies of 15 STR loci in Bosnian and Herzegovinian population

PubMed Central

Pilav, Amela; Pojskić, Naris; Ahatović, Anesa; Džehverović, Mirela; Čakar, Jasmina; Marjanović, Damir

2017-01-01

Aim To determine newest the most accurate allele frequencies for 15 short tandem repeat (STR) loci in the Bosnian and Herzegovinian population, calculate statistical parameters, and compare them with the relevant data for seven neighboring populations. Methods Genomic DNA was obtained from buccal swabs of 1000 unrelated individuals from all regions of Bosnia and Herzegovina. Genotyping was performed using PowerPlex® 16 System to obtain allele frequencies for 15 polymorphic STR loci including D3S1358, TH01, D21S11, D18S51, Penta E, D5S818, D13S317, D7S820, D16S539, CSF1PO, Penta D, vWA, D8S1179, TPOX, and FGA. The calculated allele frequencies were also compared with the data from neighboring populations. Results The highest detected value of polymorphism information content (PIC) was detected at the PentaE locus, whereas the lowest value was detected at the TPOX locus. The power of discrimination (PD) values had similar distribution, with Penta E showing the highest PD of 0.9788. While D18S51 had the highest value of power of exclusion (PE), the lowest PE value was detected at the TPOX locus. Conclusion Upon comparison of Bosnian and Herzegovinian population data with those of seven neighboring populations, the highest allele frequency differentiation was noticed between Bosnian and Herzegovinian and Turkish population at 5 loci, the most informative of which was Penta E. The neighbor-joining dendrogram constructed on the basis of genetic distance showed grouping of Slovenian, Austrian, Hungarian, and Croatian populations. Bosnian and Herzegovinian population was between the mentioned cluster and Serbian population. To determine more accurate distribution of allelic frequencies and forensic parameters, our study included 1000 unrelated individuals from all regions of Bosnia and Herzegovina, and our findings demonstrated the applicability of these markers in both forensics and future population genetic studies. PMID:28613042

Associations of the interleukin-1 gene locus polymorphisms with risk to hip and knee osteoarthritis: gender and subpopulation differences.

PubMed

Kaarvatn, M H; Jotanovic, Z; Mihelic, R; Etokebe, G E; Mulac-Jericevic, B; Tijanic, T; Balen, S; Sestan, B; Dembic, Z

2013-02-01

Genetic predisposition to the complex hereditary disease like osteoarthritis (OA) of the large joints (hip and knee) includes the interleukin-1 gene (IL-1) cluster on chromosome 2. Using a case-control study with 500 OA patients (240 knee and 260 hip OA patients, all with joint replacement), we analysed frequencies of IL-1 gene cluster polymorphisms in Croatian Caucasian population. The control samples came from 531 healthy individuals including blood donors. We genotyped two single nucleotide polymorphisms in the IL-1 gene locus at IL-1A (-889, C>T, rs1800587) and IL-1B (+3594, C>T, rs1143634) and compared their frequencies between patients and controls. We predicted haplotypes by combining current data with our previous results on gene polymorphisms (IL-1B, rs16944 and the IL-1 receptor antagonist gene [IL-1RN] variable number tandem repeat [VNTR]) for the same population. Haplotype analyses revealed gender disparities and showed that women carriers of the 1-2-1-1 haplotype [IL-1A(rs1800587) - IL-1B(rs1143634) - IL-1B(rs16944) - IL-1RN(VNTR)] had sixfold lower risk to develop knee OA. However, carriers of the 1-1-1-2 haplotype of both sexes had over twofold higher predisposition to hip OA. Our results differ from some earlier studies in Caucasian subpopulations, which may be due to the fact that this is the first study to separate genders in assessing the IL-1-locus genetic risk of OA. The results suggest that inflammatory mediators like IL-1 might be implicated in the pathogenesis of primary OA in large joints and that as yet unidentified gender-specific factors exist in a Croatian Caucasian population. © 2012 The Authors. Scandinavian Journal of Immunology © 2012 Blackwell Publishing Ltd.

Allele frequencies of 15 STR loci in Bosnian and Herzegovinian population.

PubMed

Pilav, Amela; Pojskić, Naris; Ahatović, Anesa; Džehverović, Mirela; Čakar, Jasmina; Marjanović, Damir

2017-06-14

To determine newest the most accurate allele frequencies for 15 short tandem repeat (STR) loci in the Bosnian and Herzegovinian population, calculate statistical parameters, and compare them with the relevant data for seven neighboring populations. Genomic DNA was obtained from buccal swabs of 1000 unrelated individuals from all regions of Bosnia and Herzegovina. Genotyping was performed using PowerPlex® 16 System to obtain allele frequencies for 15 polymorphic STR loci including D3S1358, TH01, D21S11, D18S51, Penta E, D5S818, D13S317, D7S820, D16S539, CSF1PO, Penta D, vWA, D8S1179, TPOX, and FGA. The calculated allele frequencies were also compared with the data from neighboring populations. The highest detected value of polymorphism information content (PIC) was detected at the PentaE locus, whereas the lowest value was detected at the TPOX locus. The power of discrimination (PD) values had similar distribution, with Penta E showing the highest PD of 0.9788. While D18S51 had the highest value of power of exclusion (PE), the lowest PE value was detected at the TPOX locus. Upon comparison of Bosnian and Herzegovinian population data with those of seven neighboring populations, the highest allele frequency differentiation was noticed between Bosnian and Herzegovinian and Turkish population at 5 loci, the most informative of which was Penta E. The neighbor-joining dendrogram constructed on the basis of genetic distance showed grouping of Slovenian, Austrian, Hungarian, and Croatian populations. Bosnian and Herzegovinian population was between the mentioned cluster and Serbian population. To determine more accurate distribution of allelic frequencies and forensic parameters, our study included 1000 unrelated individuals from all regions of Bosnia and Herzegovina, and our findings demonstrated the applicability of these markers in both forensics and future population genetic studies.

The proliferation marker pKi-67 becomes masked to MIB-1 staining after expression of its tandem repeats.

PubMed

Schmidt, Mirko H H; Broll, Rainer; Bruch, Hans-Peter; Duchrow, Michael

2002-11-01

The Ki-67 antigen, pKi-67, is one of the most commonly used markers of proliferating cells. The protein can only be detected in dividing cells (G(1)-, S-, G(2)-, and M-phase) but not in quiescent cells (G(0)). The standard antibody to detect pKi-67 is MIB-1, which detects the so-called 'Ki-67 motif' FKELF in 9 of the protein's 16 tandem repeats. To investigate the function of these repeats we expressed three of them in an inducible gene expression system in HeLa cells. Surprisingly, addition of a nuclear localization sequence led to a complete absence of signal in the nuclei of MIB-1-stained cells. At the same time antibodies directed against different epitopes of pKi-67 did not fail to detect the protein. We conclude that the overexpression of the 'Ki-67 motif', which is present in the repeats, can lead to inability of MIB-1 to detect its antigen as demonstrated in adenocarcinoma tissue samples. Thereafter, in order to prevent the underestimation of Ki-67 proliferation indices in MIB-1-labeled preparations, additional antibodies (for example, MIB-21) should be used. Additionally, we could show in a mammalian two-hybrid assay that recombinant pKi-67 repeats are capable of self-associating with endogenous pKi-67. Speculating that the tandem repeats are intimately involved in its protein-protein interactions, this offers new insights in how access to these repeats is regulated by pKi-67 itself.

Medium-sized tandem repeats represent an abundant component of the Drosophila virilis genome.

PubMed

Abdurashitov, Murat A; Gonchar, Danila A; Chernukhin, Valery A; Tomilov, Victor N; Tomilova, Julia E; Schostak, Natalia G; Zatsepina, Olga G; Zelentsova, Elena S; Evgen'ev, Michael B; Degtyarev, Sergey K H

2013-11-09

Previously, we developed a simple method for carrying out a restriction enzyme analysis of eukaryotic DNA in silico, based on the known DNA sequences of the genomes. This method allows the user to calculate lengths of all DNA fragments that are formed after a whole genome is digested at the theoretical recognition sites of a given restriction enzyme. A comparison of the observed peaks in distribution diagrams with the results from DNA cleavage using several restriction enzymes performed in vitro have shown good correspondence between the theoretical and experimental data in several cases. Here, we applied this approach to the annotated genome of Drosophila virilis which is extremely rich in various repeats. Here we explored the combined approach to perform the restriction analysis of D. virilis DNA. This approach enabled to reveal three abundant medium-sized tandem repeats within the D. virilis genome. While the 225 bp repeats were revealed previously in intergenic non-transcribed spacers between ribosomal genes of D. virilis, two other families comprised of 154 bp and 172 bp repeats were not described. Tandem Repeats Finder search demonstrated that 154 bp and 172 bp units are organized in multiple clusters in the genome of D. virilis. Characteristically, only 154 bp repeats derived from Helitron transposon are transcribed. Using in silico digestion in combination with conventional restriction analysis and sequencing of repeated DNA fragments enabled us to isolate and characterize three highly abundant families of medium-sized repeats present in the D. virilis genome. These repeats comprise a significant portion of the genome and may have important roles in genome function and structural integrity. Therefore, we demonstrated an approach which makes possible to investigate in detail the gross arrangement and expression of medium-sized repeats basing on sequencing data even in the case of incompletely assembled and/or annotated genomes.

Characterization of toxin-producing cyanobacteria by using an oligonucleotide probe containing a tandemly repeated heptamer.

PubMed Central

Rouhiainen, L; Sivonen, K; Buikema, W J; Haselkorn, R

1995-01-01

Cyanobacteria produce toxins that kill animals. The two main classes of cyanobacterial toxins are cyclic peptides that cause liver damage and alkaloids that block nerve transmission. Many toxin-producing strains from Finnish lakes were brought into axenic culture, and their toxins were characterized. Restriction fragment length polymorphism analysis, probing with a short tandemly repeated DNA sequence found at many locations in the chromosome of Anabaena sp. strain PCC 7120, distinguishes hepatotoxic Anabaena isolates from neurotoxin-producing strains and from Nostoc spp. PMID:7592362

Analysis of tandem repeat units of the promoter of capsanthin/capsorubin synthase (Ccs) gene in pepper fruit.

PubMed

Tian, Shi-Lin; Li, Zheng; Li, Li; Shah, S N M; Gong, Zhen-Hui

2017-07-01

Capsanthin/capsorubin synthase ( Ccs ) gene is a key gene that regulates the synthesis of capsanthin and the development of red coloration in pepper fruits. There are three tandem repeat units in the promoter region of Ccs , but the potential effects of the number of repetitive units on the transcriptional regulation of Ccs has been unclear. In the present study, expression vectors carrying different numbers of repeat units of the Ccs promoter were constructed, and the transient expression of the β-glucuronidase ( GUS ) gene was used to detect differences in expression levels associated with the promoter fragments. These repeat fragments and the plant expression vector PBI121 containing the 35s CaMV promoter were ligated to form recombinant vectors that were transfected into Agrobacterium tumefaciens GV3101. A fluorescence spectrophotometer was used to analyze the expression associated with the various repeat units. It was concluded that the constructs containing at least one repeat were associated with GUS expression, though they did not differ from one another. This repeating unit likely plays a role in transcription and regulation of Ccs expression.

Problem Solving Appraisal of Delinquent Adolescents.

ERIC Educational Resources Information Center

Perez, Ruperto M.; And Others

The study investigated the following: (1) the relationship of problem solving appraisal to narcissistic vulnerability, locus of control, and depression; (2) the differences in problem solving appraisal, locus of control, and depression in first-time and repeat offenders; and (3) the prediction of problem solving appraisal by narcissistic…

Impact of donor mismatches at individual HLA-A, -B, -C, -DR, and -DQ loci on the development of HLA-specific antibodies in patients listed for repeat renal transplantation.

PubMed

Kosmoliaptsis, Vasilios; Gjorgjimajkoska, Olivera; Sharples, Linda D; Chaudhry, Afzal N; Chatzizacharias, Nikolaos; Peacock, Sarah; Torpey, Nicholas; Bolton, Eleanor M; Taylor, Craig J; Bradley, J Andrew

2014-11-01

We have analyzed the relationship between donor mismatches at each HLA locus and development of HLA locus-specific antibodies in patients listed for repeat transplantation. HLA antibody screening was undertaken using single-antigen beads in 131 kidney transplant recipients returning to the transplant waiting list following first graft failure. The number of HLA mismatches and the calculated reaction frequency of antibody reactivity against 10,000 consecutive deceased organ donors were determined for each HLA locus. Two-thirds of patients awaiting repeat transplantation were sensitized (calculated reaction frequency over 15%) and half were highly sensitized (calculated reaction frequency of 85% and greater). Antibody levels peaked after re-listing for repeat transplantation, were independent of graft nephrectomy and were associated with length of time on the waiting list (odds ratio 8.4) and with maintenance on dual immunosuppression (odds ratio 0.2). Sensitization was independently associated with increasing number of donor HLA mismatches (odds ratio 1.4). All mismatched HLA loci contributed to the development of HLA locus-specific antibodies (HLA-A: odds ratio 3.2, HLA-B: odds ratio 3.4, HLA-C: odds ratio 2.5, HLA-DRB1: odds ratio 3.5, HLA-DRB3/4/5: odds ratio 3.9, and HLA-DQ: odds ratio 3.0 (all significant)). Thus, the risk of allosensitization following failure of a first renal transplant increases incrementally with the number of mismatches at all HLA loci assessed. Maintenance of re-listed patients on dual immunosuppression was associated with a reduced risk of sensitization.

Statistical Enrichment of Epigenetic States Around Triplet Repeats that Can Undergo Expansions

PubMed Central

Essebier, Alexandra; Vera Wolf, Patricia; Cao, Minh Duc; Carroll, Bernard J.; Balasubramanian, Sureshkumar; Bodén, Mikael

2016-01-01

More than 30 human genetic diseases are linked to tri-nucleotide repeat expansions. There is no known mechanism that explains repeat expansions in full, but changes in the epigenetic state of the associated locus has been implicated in the disease pathology for a growing number of examples. A comprehensive comparative analysis of the genomic features associated with diverse repeat expansions has been lacking. Here, in an effort to decipher the propensity of repeats to undergo expansion and result in a disease state, we determine the genomic coordinates of tri-nucleotide repeat tracts at base pair resolution and computationally establish epigenetic profiles around them. Using three complementary statistical tests, we reveal that several epigenetic states are enriched around repeats that are associated with disease, even in cells that do not harbor expansion, relative to a carefully stratified background. Analysis of over one hundred cell types reveals that epigenetic states generally tend to vary widely between genic regions and cell types. However, there is qualified consistency in the epigenetic signatures of repeats associated with disease suggesting that changes to the chromatin and the DNA around an expanding repeat locus are likely to be similar. These epigenetic signatures may be exploited further to develop models that could explain the propensity of repeats to undergo expansions. PMID:27013954

Tracking and responding to an outbreak of tuberculosis using MIRU-VNTR genotyping and whole genome sequencing as epidemiological tools.

PubMed

Black, Anne T; Hamblion, Esther L; Buttivant, Helen; Anderson, Sarah R; Stone, Madeline; Casali, Nicola; Drobniewski, Francis; Nwoguh, Francisca; Marshall, Ben G; Booth, Linda

2017-07-05

We describe an outbreak that contributed to a near doubling of the incidence of tuberculosis in Southampton, UK. We examine the importance of 24 locus mycobacterial interspersed repetitive unit variable number tandem repeat (MIRU-VNTR) genotyping in its identification and management and the role of whole genome sequencing (WGS) in tracing the spread of the strain. Outbreak cases were defined as those diagnosed between January and December 2011 with indistinguishable 24 locus-MIRU-VNTR genotypes or, cases linked epidemiologically. A cluster questionnaire was administered by TB nurses to identify contacts and social settings. Overall, 25 patients fulfilled the case definition. No cases with this MIRU-VNTR genotype had been detected in the UK previously. Connections were found between all cases through household contacts or social venues including a football club, Internet cafe and barber's shop. Public health actions included extended contact tracing, venue screening and TB awareness-raising. The outbreak resulted in a high rate of transmission and high incidence of clinical disease among contacts. This outbreak illustrates the value of combining active case-finding with prospective MIRU-VNTR genotyping to identify settings to undertake public health action. In addition WGS revealed that the VNTR-defined cluster was a single outbreak and that active TB transmission not reactivation was responsible for this outbreak in non-UK born individuals. © The Author 2017. Published by Oxford University Press on behalf of Faculty of Public Health. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com

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

Yauk, C.L.; Quinn, J.S.

The authors used multi-locus DNA fingerprinting to examine families of herring gulls (Larus argentatus) from a genotoxically contaminated site (Hamilton Harbour) and from a pristine location (Kent Island, Bay of Fundy) to show significant differences in mutation rates between the locations. Overall the authors identified 17 mutant bands from 15 individuals of the 35 examined from Hamilton Harbour, and 7 mutant fragments from 7 individuals, of the 43 examined from Kent Island; a mutation frequency of 0.429 per nestling for Hamilton Harbour and 0.163 for Kent Island. The total number of individuals with mutant bands was significantly higher at Hamiltonmore » Harbour than at Kent Island (X{sup 2}=6.734; df = 1; P < 0.01). Ongoing analysis of other less contaminated sites also reveals lower mutation rates than those seen in Hamilton Harbour. With multi-locus DNA fingerprinting many regions of the genome can be surveyed simultaneously. The tandemly repeated arrays of nucleotides examined with DNA fingerprinting are known to have elevated rates of mutation. Furthermore, the mutations seen with DNA fingerprinting are predominantly heritable. Other biomarkers currently used in situ are not able to monitor direct and heritable DNA mutation, or measure biological endpoints that frequently result in spontaneous abortion creating difficulty in observing significantly elevated levels in viable offspring. The authors suggest that multilocus DNA fingerprinting can be used as a biomarker to identify potentially heritable risks before the onset of other types of ecological damage. This approach provides a direct measure of mutation in situ and in vivo in a vertebrate species under ambient conditions.« less

Mitochondrial FAD-glycerophosphate dehydrogenase and G-protein-coupled inwardly rectifying K+ channel: No evidence for linkage in maturity-onset diabetes of the young or NIDDM.

PubMed

Warren-Perry, M G; Stoffel, M; Saker, P J; Zhang, Y; Brown, L J; MacDonald, M J; Turner, R C

1996-05-01

Two genes that have potentially important regulatory roles in insulin secretion are both located on chromosome 2q24.1. G-protein-coupled muscarinic potassium channel (GIRK1) is an inwardly rectifying K+ channel that helps to maintain the resting potential and excitability of cells. Mitochondrial FAD-linked glycerophosphate dehydrogenase (m-GDH) catalyzes a rate-limiting step of the glycerol phosphate shuttle in pancreatic islets. Reduced m-GDH activity has been demonstrated in islets isolated from diabetic subjects compared with islets from nondiabetic control subjects and from the diabetic GK rat. To study the relationship between these candidate genes and NIDDM, we have examined a simple tandem-repeat polymorphism (STRP) close to both the KCN J3 (GIRK1) locus and the m-GDH locus. In a linkage study of three maturity-onset diabetes of the young (MODY) pedigrees, not linked to MODY1, MODY2, or MODY3, a cumulative score of - 9.6 at a recombination fraction of theta = 0 excluded linkage. In a population-association study, no linkage disequilibrium for the STRP was found between 190 unselected NIDDM patients and 60 geographically and age-matched white nondiabetic subjects (chi2 = 1.51 on 3 df, P = 0.68). Thus, mutations involving the genes for GIRK1 or FAD-glycerophosphate dehydrogenase are unlikely to cause MODY, and a common mutation in either gene is unlikely to contribute to NIDDM in whites. These data do not exclude mutations in some families or other ethnic groups.

Variation of Cats under Domestication: Genetic Assignment of Domestic Cats to Breeds and Worldwide Random Bred Populations

PubMed Central

Kurushima, J. D.; Lipinski, M. J.; Gandolfi, B.; Froenicke, L.; Grahn, J. C.; Grahn, R. A.; Lyons, L. A.

2012-01-01

Summary Both cat breeders and the lay public have interests in the origins of their pets, not only in the genetic identity of the purebred individuals, but also the historical origins of common household cats. The cat fancy is a relatively new institution with over 85% of its 40–50 breeds arising only in the past 75 years, primarily through selection on single-gene aesthetic traits. The short, yet intense cat breed history poses a significant challenge to the development of a genetic marker-based breed identification strategy. Using different breed assignment strategies and methods, 477 cats representing 29 fancy breeds were analysed with 38 short tandem repeats, 148 intergenic and five phenotypic single nucleotide polymorphisms. Results suggest the frequentist method of Paetkau (accuracy single nucleotide polymorphisms = 0.78, short tandem repeats = 0.88) surpasses the Bayesian method of Rannala and Mountain (single nucleotide polymorphisms = 0.56, short tandem repeats = 0.83) for accurate assignment of individuals to the correct breed. Additionally, a post-assignment verification step with the five phenotypic single nucleotide polymorphisms accurately identified between 0.31 and 0.58 of the mis-assigned individuals raising the sensitivity of assignment with the frequentist method to 0.89 and 0.92 single nucleotide polymorphisms and short tandem repeats respectively. This study provides a novel multi-step assignment strategy and suggests that, despite their short breed history and breed family groupings, a majority of cats can be assigned to their proper breed or population of origin, i.e. race. PMID:23171373

Dancing together and separate again: gymnosperms exhibit frequent changes of fundamental 5S and 35S rRNA gene (rDNA) organisation

PubMed Central

Garcia, S; Kovařík, A

2013-01-01

In higher eukaryotes, the 5S rRNA genes occur in tandem units and are arranged either separately (S-type arrangement) or linked to other repeated genes, in most cases to rDNA locus encoding 18S–5.8S–26S genes (L-type arrangement). Here we used Southern blot hybridisation, PCR and sequencing approaches to analyse genomic organisation of rRNA genes in all large gymnosperm groups, including Coniferales, Ginkgoales, Gnetales and Cycadales. The data are provided for 27 species (21 genera). The 5S units linked to the 35S rDNA units occur in some but not all Gnetales, Coniferales and in Ginkgo (∼30% of the species analysed), while the remaining exhibit separate organisation. The linked 5S rRNA genes may occur as single-copy insertions or as short tandems embedded in the 26S–18S rDNA intergenic spacer (IGS). The 5S transcript may be encoded by the same (Ginkgo, Ephedra) or opposite (Podocarpus) DNA strand as the 18S–5.8S–26S genes. In addition, pseudogenised 5S copies were also found in some IGS types. Both L- and S-type units have been largely homogenised across the genomes. Phylogenetic relationships based on the comparison of 5S coding sequences suggest that the 5S genes independently inserted IGS at least three times in the course of gymnosperm evolution. Frequent transpositions and rearrangements of basic units indicate relatively relaxed selection pressures imposed on genomic organisation of 5S genes in plants. PMID:23512008

Dancing together and separate again: gymnosperms exhibit frequent changes of fundamental 5S and 35S rRNA gene (rDNA) organisation.

PubMed

Garcia, S; Kovařík, A

2013-07-01

In higher eukaryotes, the 5S rRNA genes occur in tandem units and are arranged either separately (S-type arrangement) or linked to other repeated genes, in most cases to rDNA locus encoding 18S-5.8S-26S genes (L-type arrangement). Here we used Southern blot hybridisation, PCR and sequencing approaches to analyse genomic organisation of rRNA genes in all large gymnosperm groups, including Coniferales, Ginkgoales, Gnetales and Cycadales. The data are provided for 27 species (21 genera). The 5S units linked to the 35S rDNA units occur in some but not all Gnetales, Coniferales and in Ginkgo (∼30% of the species analysed), while the remaining exhibit separate organisation. The linked 5S rRNA genes may occur as single-copy insertions or as short tandems embedded in the 26S-18S rDNA intergenic spacer (IGS). The 5S transcript may be encoded by the same (Ginkgo, Ephedra) or opposite (Podocarpus) DNA strand as the 18S-5.8S-26S genes. In addition, pseudogenised 5S copies were also found in some IGS types. Both L- and S-type units have been largely homogenised across the genomes. Phylogenetic relationships based on the comparison of 5S coding sequences suggest that the 5S genes independently inserted IGS at least three times in the course of gymnosperm evolution. Frequent transpositions and rearrangements of basic units indicate relatively relaxed selection pressures imposed on genomic organisation of 5S genes in plants.

Diversity and evolution of centromere repeats in the maize genome.

PubMed

Bilinski, Paul; Distor, Kevin; Gutierrez-Lopez, Jose; Mendoza, Gabriela Mendoza; Shi, Jinghua; Dawe, R Kelly; Ross-Ibarra, Jeffrey

2015-03-01

Centromere repeats are found in most eukaryotes and play a critical role in kinetochore formation. Though centromere repeats exhibit considerable diversity both within and among species, little is understood about the mechanisms that drive centromere repeat evolution. Here, we use maize as a model to investigate how a complex history involving polyploidy, fractionation, and recent domestication has impacted the diversity of the maize centromeric repeat CentC. We first validate the existence of long tandem arrays of repeats in maize and other taxa in the genus Zea. Although we find considerable sequence diversity among CentC copies genome-wide, genetic similarity among repeats is highest within these arrays, suggesting that tandem duplications are the primary mechanism for the generation of new copies. Nonetheless, clustering analyses identify similar sequences among distant repeats, and simulations suggest that this pattern may be due to homoplasious mutation. Although the two ancestral subgenomes of maize have contributed nearly equal numbers of centromeres, our analysis shows that the majority of all CentC repeats derive from one of the parental genomes, with an even stronger bias when examining the largest assembled contiguous clusters. Finally, by comparing maize with its wild progenitor teosinte, we find that the abundance of CentC likely decreased after domestication, while the pericentromeric repeat Cent4 has drastically increased.

Laser mass spectrometry for DNA fingerprinting for forensic applications

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

Chen, C.H.; Tang, K.; Taranenko, N.I.

The application of DNA fingerprinting has become very broad in forensic analysis, patient identification, diagnostic medicine, and wildlife poaching, since every individual`s DNA structure is identical within all tissues of their body. DNA fingerprinting was initiated by the use of restriction fragment length polymorphisms (RFLP). In 1987, Nakamura et al. found that a variable number of tandem repeats (VNTR) often occurred in the alleles. The probability of different individuals having the same number of tandem repeats in several different alleles is very low. Thus, the identification of VNTR from genomic DNA became a very reliable method for identification of individuals.more » DNA fingerprinting is a reliable tool for forensic analysis. In DNA fingerprinting, knowledge of the sequence of tandem repeats and restriction endonuclease sites can provide the basis for identification. The major steps for conventional DNA fingerprinting include (1) specimen processing (2) amplification of selected DNA segments by PCR, and (3) gel electrophoresis to do the final DNA analysis. In this work we propose to use laser desorption mass spectrometry for fast DNA fingerprinting. The process and advantages are discussed.« less

Combined deficiency of MSH2 and Sμ region abolishes class switch recombination.

PubMed

Leduc, Claire; Haddad, Dania; Laviolette-Malirat, Nathalie; Nguyen Huu, Ngoc-Sa; Khamlichi, Ahmed Amine

2010-10-01

Class switch recombination (CSR) is mediated by G-rich tandem repeated sequences termed switch regions. Transcription of switch regions generates single-stranded R loops that provide substrates for activation-induced cytidine deaminase. Mice deficient in MSH2 have a mild defect in CSR and analysis of their switch junctions has led to a model in which MSH2 is more critical for switch recombination events outside than within the tandem repeats. It is also known that deletion of the whole Sμ region severely impairs but does not abrogate CSR despite the lack of detectable R loops. Here, we demonstrate that deficiency of both MSH2 and the Sμ region completely abolishes CSR and that the abrogation occurs at the genomic level. This finding further supports the crucial role of MSH2 outside the tandem repeats. It also indicates that during CSR, MSH2 has access to activation-induced cytidine deaminase targets in R-loop-deficient Iμ-Cμ sequences rarely used in CSR, suggesting an MSH2-dependent DNA processing activity at the Iμ exon that may decrease with transcription elongation across the Sμ region.

Histone and ribosomal RNA repetitive gene clusters of the boll weevil are linked in a tandem array.

PubMed

Roehrdanz, R; Heilmann, L; Senechal, P; Sears, S; Evenson, P

2010-08-01

Histones are the major protein component of chromatin structure. The histone family is made up of a quintet of proteins, four core histones (H2A, H2B, H3 & H4) and the linker histones (H1). Spacers are found between the coding regions. Among insects this quintet of genes is usually clustered and the clusters are tandemly repeated. Ribosomal DNA contains a cluster of the rRNA sequences 18S, 5.8S and 28S. The rRNA genes are separated by the spacers ITS1, ITS2 and IGS. This cluster is also tandemly repeated. We found that the ribosomal RNA repeat unit of at least two species of Anthonomine weevils, Anthonomus grandis and Anthonomus texanus (Coleoptera: Curculionidae), is interspersed with a block containing the histone gene quintet. The histone genes are situated between the rRNA 18S and 28S genes in what is known as the intergenic spacer region (IGS). The complete reiterated Anthonomus grandis histone-ribosomal sequence is 16,248 bp.

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

Zweifel,M.; Leahy, D.; Barrick, D.

Deltex is a cytosolic effector of Notch signaling thought to bind through its N-terminal domain to the Notch receptor. Here we report the structure of the Drosophila Deltex N-terminal domain, which contains two tandem WWE sequence repeats. The WWE repeats, which adopt a novel fold, are related by an approximate two-fold axis of rotation. Although the WWE repeats are structurally distinct, they interact extensively and form a deep cleft at their junction that appears well suited for ligand binding. The two repeats are thermodynamically coupled; this coupling is mediated in part by a conserved segment that is immediately C-terminal tomore » the second WWE domain. We demonstrate that although the Deltex WWE tandem is monomeric in solution, it forms a heterodimer with the ankyrin domain of the Notch receptor. These results provide structural and functional insight into how Deltex modulates Notch signaling, and how WWE modules recognize targets for ubiquitination.« less

The repeat organizer, a specialized insulator element within the intergenic spacer of the Xenopus rRNA genes.

PubMed Central

Robinett, C C; O'Connor, A; Dunaway, M

1997-01-01

We have identified a novel activity for the region of the intergenic spacer of the Xenopus laevis rRNA genes that contains the 35- and 100-bp repeats. We devised a new assay for this region by constructing DNA plasmids containing a tandem repeat of rRNA reporter genes that were separated by the 35- and 100-bp repeat region and a rRNA gene enhancer. When the 35- and 100-bp repeat region is present in its normal position and orientation at the 3' end of the rRNA reporter genes, the enhancer activates the adjacent downstream promoter but not the upstream rRNA promoter on the same plasmid. Because this element can restrict the range of an enhancer's activity in the context of tandem genes, we have named it the repeat organizer (RO). The ability to restrict enhancer action is a feature of insulator elements, but unlike previously described insulator elements the RO does not block enhancer action in a simple enhancer-blocking assay. Instead, the activity of the RO requires that it be in its normal position and orientation with respect to the other sequence elements of the rRNA genes. The enhancer-binding transcription factor xUBF also binds to the repetitive sequences of the RO in vitro, but these sequences do not activate transcription in vivo. We propose that the RO is a specialized insulator element that organizes the tandem array of rRNA genes into single-gene expression units by promoting activation of a promoter by its proximal enhancers. PMID:9111359

High Quality Maize Centromere 10 Sequence Reveals Evidence of Frequent Recombination Events

PubMed Central

Wolfgruber, Thomas K.; Nakashima, Megan M.; Schneider, Kevin L.; Sharma, Anupma; Xie, Zidian; Albert, Patrice S.; Xu, Ronghui; Bilinski, Paul; Dawe, R. Kelly; Ross-Ibarra, Jeffrey; Birchler, James A.; Presting, Gernot G.

2016-01-01

The ancestral centromeres of maize contain long stretches of the tandemly arranged CentC repeat. The abundance of tandem DNA repeats and centromeric retrotransposons (CR) has presented a significant challenge to completely assembling centromeres using traditional sequencing methods. Here, we report a nearly complete assembly of the 1.85 Mb maize centromere 10 from inbred B73 using PacBio technology and BACs from the reference genome project. The error rates estimated from overlapping BAC sequences are 7 × 10−6 and 5 × 10−5 for mismatches and indels, respectively. The number of gaps in the region covered by the reassembly was reduced from 140 in the reference genome to three. Three expressed genes are located between 92 and 477 kb from the inferred ancestral CentC cluster, which lies within the region of highest centromeric repeat density. The improved assembly increased the count of full-length CR from 5 to 55 and revealed a 22.7 kb segmental duplication that occurred approximately 121,000 years ago. Our analysis provides evidence of frequent recombination events in the form of partial retrotransposons, deletions within retrotransposons, chimeric retrotransposons, segmental duplications including higher order CentC repeats, a deleted CentC monomer, centromere-proximal inversions, and insertion of mitochondrial sequences. Double-strand DNA break (DSB) repair is the most plausible mechanism for these events and may be the major driver of centromere repeat evolution and diversity. In many cases examined here, DSB repair appears to be mediated by microhomology, suggesting that tandem repeats may have evolved to efficiently repair frequent DSBs in centromeres. PMID:27047500

A Dynamic Tandem Repeat in Monocotyledons Inferred from a Comparative Analysis of Chloroplast Genomes in Melanthiaceae.

PubMed

Do, Hoang Dang Khoa; Kim, Joo-Hwan

2017-01-01

Chloroplast genomes (cpDNA) are highly valuable resources for evolutionary studies of angiosperms, since they are highly conserved, are small in size, and play critical roles in plants. Slipped-strand mispairing (SSM) was assumed to be a mechanism for generating repeat units in cpDNA. However, research on the employment of different small repeated sequences through SSM events, which may induce the accumulation of distinct types of repeats within the same region in cpDNA, has not been documented. Here, we sequenced two chloroplast genomes from the endemic species Heloniopsis tubiflora (Korea) and Xerophyllum tenax (USA) to cover the gap between molecular data and explore "hot spots" for genomic events in Melanthiaceae. Comparative analysis of 23 complete cpDNA sequences revealed that there were different stages of deletion in the rps16 region across the Melanthiaceae. Based on the partial or complete loss of rps16 gene in cpDNA, we have firstly reported potential molecular markers for recognizing two sections ( Veratrum and Fuscoveratrum ) of Veratrum . Melathiaceae exhibits a significant change in the junction between large single copy and inverted repeat regions, ranging from trnH_GUG to a part of rps3 . Our results show an accumulation of tandem repeats in the rpl23-ycf2 regions of cpDNAs. Small conserved sequences exist and flank tandem repeats in further observation of this region across most of the examined taxa of Liliales. Therefore, we propose three scenarios in which different small repeated sequences were used during SSM events to generate newly distinct types of repeats. Occasionally, prior to the SSM process, point mutation event and double strand break repair occurred and induced the formation of initial repeat units which are indispensable in the SSM process. SSM may have likely occurred more frequently for short repeats than for long repeat sequences in tribe Parideae (Melanthiaceae, Liliales). Collectively, these findings add new evidence of dynamic results from SSM in chloroplast genomes which can be useful for further evolutionary studies in angiosperms. Additionally, genomics events in cpDNA are potential resources for mining molecular markers in Liliales.

[Molecular characteristics of Clustered Regularly Interspaced Short Palindromic Repeat in Shigella].

PubMed

Xue, Zerun; Wang, Yingfang; Duan, Guangcai; Yang, Haiyan; Xi, Yuanlin; Wang, Pengfei; Wang, Linlin; Guo, Xiangjiao

2015-08-01

To detect the molecular characteristics of Clustered Regularly Interspaced Short Palindromic Repeat (CRISPR) in Shigella and to analyze the distribution of CRISPR related to the time of isolation. Of the 52 Shigella strains, 41 were isolated from Henan, 6 from Jiangxi and 5 isolated from Beijing. Both CRISPR locus of S1, S2, S3 and S4 in Shigella were detected by polymerase chain reaction (PCR). The PCR products were sequenced and compared. The positive rates of CRISPR locus in Shigella were 33.69% (S1), 50.00% (S2), 82.69% (S3) and 73.08% (S4), respectively. Two subtypes were discovered in S1 and S3 locus. Three subtypes were discovered in S2 locus. Four different subtypes were discovered in S4 locus. The isolates from Henan strains were divided into two groups by the time of isolation. Distributions of S1 were different, before or after 2004, on Shigella. S1 could not be detected after 2004. There were no statistical differences of S2, S3 and S4 in two groups. Different CRISPR subtypes or Shigella were discovered. A significant correlation was noticed between the CRISPR S1 related to the time of isolation but not between S2, S3 or S4 on the time of isolation.

Minimal and Contributing Sequence Determinants of the cis-Acting Locus of Transfer (clt) of Streptomycete Plasmid pIJ101 Occur within an Intrinsically Curved Plasmid Region

PubMed Central

Ducote, Matthew J.; Prakash, Shubha; Pettis, Gregg S.

2000-01-01

Efficient interbacterial transfer of streptomycete plasmid pIJ101 requires the pIJ101 tra gene, as well as a cis-acting plasmid function known as clt. Here we show that the minimal pIJ101 clt locus consists of a sequence no greater than 54 bp in size that includes essential inverted-repeat and direct-repeat sequences and is located in close proximity to the 3′ end of the korB regulatory gene. Evidence that sequences extending beyond the minimal locus and into the korB open reading frame influence clt transfer function and demonstration that clt-korB sequences are intrinsically curved raise the possibility that higher-order structuring of DNA and protein within this plasmid region may be an inherent feature of efficient pIJ101 transfer. PMID:11073933

Minimal and contributing sequence determinants of the cis-acting locus of transfer (clt) of streptomycete plasmid pIJ101 occur within an intrinsically curved plasmid region.

PubMed

Ducote, M J; Prakash, S; Pettis, G S

2000-12-01

Efficient interbacterial transfer of streptomycete plasmid pIJ101 requires the pIJ101 tra gene, as well as a cis-acting plasmid function known as clt. Here we show that the minimal pIJ101 clt locus consists of a sequence no greater than 54 bp in size that includes essential inverted-repeat and direct-repeat sequences and is located in close proximity to the 3' end of the korB regulatory gene. Evidence that sequences extending beyond the minimal locus and into the korB open reading frame influence clt transfer function and demonstration that clt-korB sequences are intrinsically curved raise the possibility that higher-order structuring of DNA and protein within this plasmid region may be an inherent feature of efficient pIJ101 transfer.

Bacillus anthracis Diversity and Geographic Potential across Nigeria, Cameroon and Chad: Further Support of a Novel West African Lineage

PubMed Central

Blackburn, Jason K.; Odugbo, Moses Ode; Van Ert, Matthew; O’Shea, Bob; Mullins, Jocelyn; Perrenten, Vincent; Maho, Angaya; Hugh-Jones, Martin; Hadfield, Ted

2015-01-01

Zoonoses, diseases affecting both humans and animals, can exert tremendous pressures on human and veterinary health systems, particularly in resource limited countries. Anthrax is one such zoonosis of concern and is a disease requiring greater public health attention in Nigeria. Here we describe the genetic diversity of Bacillus anthracis in Nigeria and compare it to Chad, Cameroon and a broader global dataset based on the multiple locus variable number tandem repeat (MLVA-25) genetic typing system. Nigerian B. anthracis isolates had identical MLVA genotypes and could only be resolved by measuring highly mutable single nucleotide repeats (SNRs). The Nigerian MLVA genotype was identical or highly genetically similar to those in the neighboring countries, confirming the strains belong to this unique West African lineage. Interestingly, sequence data from a Nigerian isolate shares the anthrose deficient genotypes previously described for strains in this region, which may be associated with vaccine evasion. Strains in this study were isolated over six decades, indicating a high level of temporal strain stability regionally. Ecological niche models were used to predict the geographic distribution of the pathogen for all three countries. We describe a west-east habitat corridor through northern Nigeria extending into Chad and Cameroon. Ecological niche models and genetic results show B. anthracis to be ecologically established in Nigeria. These findings expand our understanding of the global B. anthracis population structure and can guide regional anthrax surveillance and control planning. PMID:26291625

Genetic source tracking of an anthrax outbreak in Shaanxi province, China.

PubMed

Liu, Dong-Li; Wei, Jian-Chun; Chen, Qiu-Lan; Guo, Xue-Jun; Zhang, En-Min; He, Li; Liang, Xu-Dong; Ma, Guo-Zhu; Zhou, Ti-Cao; Yin, Wen-Wu; Liu, Wei; Liu, Kai; Shi, Yi; Ji, Jian-Jun; Zhang, Hui-Juan; Ma, Lin; Zhang, Fa-Xin; Zhang, Zhi-Kai; Zhou, Hang; Yu, Hong-Jie; Kan, Biao; Xu, Jian-Guo; Liu, Feng; Li, Wei

2017-01-17

Anthrax is an acute zoonotic infectious disease caused by the bacterium known as Bacillus anthracis. From 26 July to 8 August 2015, an outbreak with 20 suspected cutaneous anthrax cases was reported in Ganquan County, Shaanxi province in China. The genetic source tracking analysis of the anthrax outbreak was performed by molecular epidemiological methods in this study. Three molecular typing methods, namely canonical single nucleotide polymorphisms (canSNP), multiple-locus variable-number tandem repeat analysis (MLVA), and single nucleotide repeat (SNR) analysis, were used to investigate the possible source of transmission and identify the genetic relationship among the strains isolated from human cases and diseased animals during the outbreak. Five strains isolated from diseased mules were clustered together with patients' isolates using canSNP typing and MLVA. The causative B. anthracis lineages in this outbreak belonged to the A.Br.001/002 canSNP subgroup and the MLVA15-31 genotype (the 31 genotype in MLVA15 scheme). Because nine isolates from another four provinces in China were clustered together with outbreak-related strains by the canSNP (A.Br.001/002 subgroup) and MLVA15 method (MLVA15-31 genotype), still another SNR analysis (CL10, CL12, CL33, and CL35) was used to source track the outbreak, and the results suggesting that these patients in the anthrax outbreak were probably infected by the same pathogen clone. It was deduced that the anthrax outbreak occurred in Shaanxi province, China in 2015 was a local occurrence.

Altered Methylation in Tandem Repeat Element and Elemental Component Levels in Inhalable Air Particles

PubMed Central

Hou, Lifang; Zhang, Xiao; Zheng, Yinan; Wang, Sheng; Dou, Chang; Guo, Liqiong; Byun, Hyang-Min; Motta, Valeria; McCracken, John; Díaz, Anaité; Kang, Choong-Min; Koutrakis, Petros; Bertazzi, Pier Alberto; Li, Jingyun; Schwartz, Joel; Baccarelli, Andrea A.

2014-01-01

Exposure to particulate matter (PM) has been associated with lung cancer risk in epidemiology investigations. Elemental components of PM have been suggested to have critical roles in PM toxicity, but the molecular mechanisms underlying their association with cancer risks remain poorly understood. DNA methylation has emerged as a promising biomarker for environmental-related diseases, including lung cancer. In this study, we evaluated the effects of PM elemental components on methylation of three tandem repeats in a highly-exposed population in Beijing, China. The Beijing Truck Driver Air Pollution Study was conducted shortly before the 2008 Beijing Olympic Games (June 15-July 27, 2008) and included 60 truck drivers and 60 office workers. On two days separated by 1-2 weeks, we measured blood DNA methylation of SATα, NBL2, D4Z4, and personal exposure to eight elemental components in PM2.5, including aluminum (Al), silicon (Si), sulfur (S), potassium (K), calcium (Ca) titanium (Ti), iron (Fe), and zinc (Zn). We estimated the associations of individual elemental component with each tandem repeat methylation in generalized estimating equations (GEE) models adjusted for PM2.5 mass and other covariates. Out of the eight examined elements, NBL2 methylation was positively associated with concentrations of Si (0.121, 95%CI: 0.030; 0.212, FDR=0.047) and Ca (0.065, 95%CI: 0.014; 0.115, FDR=0.047) in truck drivers. In office workers, SATα methylation was positively associated with concentrations of S (0.115, 95%CI: 0.034; 0.196, FDR=0.042). PM-associated differences in blood tandem-repeat methylation may help detect biological effects of the exposure and identify individuals who may eventually experience higher lung cancer risk. PMID:24273195

The production and characterization of novel heavy-chain antibodies against the tandem repeat region of MUC1 mucin.

PubMed

Rahbarizadeh, Fatemeh; Rasaee, Mohammad J; Forouzandeh, Mehdi; Allameh, Abdolamir; Sarrami, Ramin; Nasiry, Habib; Sadeghizadeh, Majid

2005-01-01

Camelidae are known to produce immunoglobulins (Igs) devoid of light chains and constant heavy-chain domains (CH1). Antigen-specific fragments of these heavy-chain IgGs (VHH) are of great interest in biotechnology applications. This paper describes the first example of successfully raised heavy-chain antibodies in Camelus dromedarius (single-humped camel) and Camelus bactrianus (two-humped camel) against a MUC1 related peptide that is found to be an important epitope expressed in cancerous tissue. Camels were immunized against a synthetic peptide corresponding to the tandem repeat region of MUC1 mucin and cancerous tissue preparation obtained from patients suffering from breast carcinoma. Three IgG subclasses with different binding properties to protein A and G were purified by affinity chromatography. Both conventional and heavy-chain IgG antibodies were produced in response to MUC1-related peptide. The elicited antibodies could react specifically with the tandem repeat region of MUC1 mucin in an enzyme linked immunosorbant assay (ELISA). Anti-peptide antibodies were purified after passing antiserum over two affinity chromatography columns. Using ELISA, immunocytochemistry and Western blotting, the interaction of purified antibodies with different antigens was evaluated. The antibodies were observed to be selectively bound to antigens namely: MUC1 peptide (tandem repeat region), human milk fat globule membrane (HMFG), deglycosylated human milk fat globule membrane (D-HMFG), homogenized cancerous breast tissue and a native MUC1 purified from ascitic fluid. Ka values of specific polyclonal antipeptide antibodies were estimated in C. dromedarius and C. bactrianus, as 7 x 10(10) M(-1) and 1.4 x 10(10) M(-1) respectively.

Tandem Repeats in Proteins: Prediction Algorithms and Biological Role.

PubMed

Pellegrini, Marco

2015-01-01

Tandem repetitions in protein sequence and structure is a fascinating subject of research which has been a focus of study since the late 1990s. In this survey, we give an overview on the multi-faceted aspects of research on protein tandem repeats (PTR for short), including prediction algorithms, databases, early classification efforts, mechanisms of PTR formation and evolution, and synthetic PTR design. We also touch on the rather open issue of the relationship between PTR and flexibility (or disorder) in proteins. Detection of PTR either from protein sequence or structure data is challenging due to inherent high (biological) signal-to-noise ratio that is a key feature of this problem. As early in silico analytic tools have been key enablers for starting this field of study, we expect that current and future algorithmic and statistical breakthroughs will have a high impact on the investigations of the biological role of PTR.

DNA replication stress restricts ribosomal DNA copy number

PubMed Central

Salim, Devika; Bradford, William D.; Freeland, Amy; Cady, Gillian; Wang, Jianmin

2017-01-01

Ribosomal RNAs (rRNAs) in budding yeast are encoded by ~100–200 repeats of a 9.1kb sequence arranged in tandem on chromosome XII, the ribosomal DNA (rDNA) locus. Copy number of rDNA repeat units in eukaryotic cells is maintained far in excess of the requirement for ribosome biogenesis. Despite the importance of the repeats for both ribosomal and non-ribosomal functions, it is currently not known how “normal” copy number is determined or maintained. To identify essential genes involved in the maintenance of rDNA copy number, we developed a droplet digital PCR based assay to measure rDNA copy number in yeast and used it to screen a yeast conditional temperature-sensitive mutant collection of essential genes. Our screen revealed that low rDNA copy number is associated with compromised DNA replication. Further, subculturing yeast under two separate conditions of DNA replication stress selected for a contraction of the rDNA array independent of the replication fork blocking protein, Fob1. Interestingly, cells with a contracted array grew better than their counterparts with normal copy number under conditions of DNA replication stress. Our data indicate that DNA replication stresses select for a smaller rDNA array. We speculate that this liberates scarce replication factors for use by the rest of the genome, which in turn helps cells complete DNA replication and continue to propagate. Interestingly, tumors from mini chromosome maintenance 2 (MCM2)-deficient mice also show a loss of rDNA repeats. Our data suggest that a reduction in rDNA copy number may indicate a history of DNA replication stress, and that rDNA array size could serve as a diagnostic marker for replication stress. Taken together, these data begin to suggest the selective pressures that combine to yield a “normal” rDNA copy number. PMID:28915237

Isolation and mapping of telomeric pentanucleotide (TAACC)n repeats of the Pacific whiteleg shrimp, Penaeus vannamei, using fluorescence in situ hybridization.

PubMed

Alcivar-Warren, Acacia; Meehan-Meola, Dawn; Wang, Yongping; Guo, Ximing; Zhou, Linghua; Xiang, Jianhai; Moss, Shaun; Arce, Steve; Warren, William; Xu, Zhenkang; Bell, Kireina

2006-01-01

To develop genetic and physical maps for shrimp, accurate information on the actual number of chromosomes and a large number of genetic markers is needed. Previous reports have shown two different chromosome numbers for the Pacific whiteleg shrimp, Penaeus vannamei, the most important penaeid shrimp species cultured in the Western hemisphere. Preliminary results obtained by direct sequencing of clones from a Sau3A-digested genomic library of P. vannamei ovary identified a large number of (TAACC/GGTTA)-containing SSRs. The objectives of this study were to (1) examine the frequency of (TAACC)n repeats in 662 P. vannamei genomic clones that were directly sequenced, and perform homology searches of these clones, (2) confirm the number of chromosomes in testis of P. vannamei, and (3) localize the TAACC repeats in P. vannamei chromosome spreads using fluorescence in situ hybridization (FISH). Results for objective 1 showed that 395 out of the 662 clones sequenced contained single or multiple SSRs with three or more repeat motifs, 199 of which contained variable tandem repeats of the pentanucleotide (TAACC/GGTTA)n, with 3 to 14 copies per sequence. The frequency of (TAACC)n repeats in P. vannamei is 4.68 kb for SSRs with five or more repeat motifs. Sequence comparisons using the BLASTN nonredundant and expressed sequence tag (EST) databases indicated that most of the TAACC-containing clones were similar to either the core pentanucleotide repeat in PVPENTREP locus (GenBank accession no. X82619) or portions of 28S rRNA. Transposable elements (transposase for Tn1000 and reverse transcriptase family members), hypothetical or unnamed protein products, and genes of known function such as 18S and 28S rRNAs, heat shock protein 70, and thrombospondin were identified in non-TAACC-containing clones. For objective 2, the meiotic chromosome number of P. vannamei was confirmed as N = 44. For objective 3, four FISH probes (P1 to P4) containing different numbers of TAACC repeats produced positive signals on telomeres of P. vannamei chromosomes. A few chromosomes had positive signals interstitially. Probe signal strength and chromosome coverage differed in the general order of P1>P2>P3>P4, which correlated with the length of TAACC repeats within the probes: 83, 66, 35, and 30 bp, respectively, suggesting that the TAACC repeats, and not the flanking sequences, produced the TAACC signals at chromosome ends and TAACC is likely the telomere sequence for P. vannamei.

Ligand binding by repeat proteins: natural and designed

PubMed Central

Grove, Tijana Z; Cortajarena, Aitziber L; Regan, Lynne

2012-01-01

Repeat proteins contain tandem arrays of small structural motifs. As a consequence of this architecture, they adopt non-globular, extended structures that present large, highly specific surfaces for ligand binding. Here we discuss recent advances toward understanding the functional role of this unique modular architecture. We showcase specific examples of natural repeat proteins interacting with diverse ligands and also present examples of designed repeat protein–ligand interactions. PMID:18602006

Exploring the repeat protein universe through computational protein design

DOE PAGES

Brunette, TJ; Parmeggiani, Fabio; Huang, Po-Ssu; ...

2015-12-16

A central question in protein evolution is the extent to which naturally occurring proteins sample the space of folded structures accessible to the polypeptide chain. Repeat proteins composed of multiple tandem copies of a modular structure unit are widespread in nature and have critical roles in molecular recognition, signalling, and other essential biological processes. Naturally occurring repeat proteins have been re-engineered for molecular recognition and modular scaffolding applications. In this paper, we use computational protein design to investigate the space of folded structures that can be generated by tandem repeating a simple helix–loop–helix–loop structural motif. Eighty-three designs with sequences unrelatedmore » to known repeat proteins were experimentally characterized. Of these, 53 are monomeric and stable at 95 °C, and 43 have solution X-ray scattering spectra consistent with the design models. Crystal structures of 15 designs spanning a broad range of curvatures are in close agreement with the design models with root mean square deviations ranging from 0.7 to 2.5 Å. Finally, our results show that existing repeat proteins occupy only a small fraction of the possible repeat protein sequence and structure space and that it is possible to design novel repeat proteins with precisely specified geometries, opening up a wide array of new possibilities for biomolecular engineering.« less

MLVA for Salmonella enterica subsp. enterica Serovar Dublin: Development of a Method Suitable for Inter-Laboratory Surveillance and Application in the Context of a Raw Milk Cheese Outbreak in France in 2012

PubMed Central

Vignaud, Marie-Léone; Cherchame, Emeline; Marault, Muriel; Chaing, Emilie; Le Hello, Simon; Michel, Valerie; Jourdan-Da Silva, Nathalie; Lailler, Renaud; Brisabois, Anne; Cadel-Six, Sabrina

2017-01-01

Salmonella enterica subspecies enterica serovar Dublin (S. Dublin) figures among the most frequently isolated Salmonella strains in humans in France. This serovar may affect production and animal health mainly in cattle herds with corresponding high economic losses. Given that the current gold standard method, pulsed-field gel electrophoresis (PFGE), provides insufficient discrimination for epidemiological investigations, we propose a standard operating procedure in this study for multiple-locus variable number tandem repeat analysis (MLVA) of S. Dublin, suitable for inter-laboratory surveillance. An in silico analysis on the genome of S. Dublin strains CT_02021853 was performed to identify appropriate microsatellite regions. Of 21 VNTR loci screened, six were selected and 401 epidemiologically unrelated and related strains, isolated from humans, food and animals were analyzed to assess performance criteria such as typeability, discriminatory power and epidemiological concordance. The MLVA scheme developed was applied to an outbreak involving Saint-Nectaire cheese for which investigations were conducted in France in 2012, making it possible to discriminate between epidemiologically related strains and sporadic case strains, while PFGE assigned only a single profile. The six loci selected were sequenced on a large set of strains to determine the sequence of the repeated units and flanking regions, and their stability was evaluated in vivo through the analysis of the strains investigated from humans, food and the farm environment during the outbreak. The six VNTR selected were found to be stable and the discriminatory power of the MLVA method developed was calculated to be 0.954 compared with that for PFGE, which was only 0.625. Twenty-four reference strains were selected from the 401 examined strains in order to represent most of the allele diversity observed for each locus. This reference set can be used to harmonize MLVA results and allow data exchange between laboratories. This original MLVA protocol could be used easily and routinely for monitoring of serovar Dublin isolates and for conducting outbreak investigations. PMID:28289408

Tandemly arranged chalcone synthase A genes contribute to the spatially regulated expression of siRNA and the natural bicolor floral phenotype in Petunia hybrida.

PubMed

Morita, Yasumasa; Saito, Ryoko; Ban, Yusuke; Tanikawa, Natsu; Kuchitsu, Kazuyuki; Ando, Toshio; Yoshikawa, Manabu; Habu, Yoshiki; Ozeki, Yoshihiro; Nakayama, Masayoshi

2012-06-01

The natural bicolor floral traits of the horticultural petunia (Petunia hybrida) cultivars Picotee and Star are caused by the spatial repression of the chalcone synthase A (CHS-A) gene, which encodes an anthocyanin biosynthetic enzyme. Here we show that Picotee and Star petunias carry the same short interfering RNA (siRNA)-producing locus, consisting of two intact CHS-A copies, PhCHS-A1 and PhCHS-A2, in a tandem head-to-tail orientation. The precursor CHS mRNAs are transcribed from the two CHS-A copies throughout the bicolored petals, but the mature CHS mRNAs are not found in the white tissues. An analysis of small RNAs revealed the accumulation of siRNAs of 21 nucleotides that originated from the exon 2 region of both CHS-A copies. This accumulation is closely correlated with the disappearance of the CHS mRNAs, indicating that the bicolor floral phenotype is caused by the spatially regulated post-transcriptional silencing of both CHS-A genes. Linkage between the tandemly arranged CHS-A allele and the bicolor floral trait indicates that the CHS-A allele is a necessary factor to confer the trait. We suppose that the spatially regulated production of siRNAs in Picotee and Star flowers is triggered by another putative regulatory locus, and that the silencing mechanism in this case may be different from other known mechanisms of post-transcriptional gene silencing in plants. A sequence analysis of wild Petunia species indicated that these tandem CHS-A genes originated from Petunia integrifolia and/or Petunia inflata, the parental species of P. hybrida, as a result of a chromosomal rearrangement rather than a gene duplication event. © 2012 The Authors. The Plant Journal © 2012 Blackwell Publishing Ltd.

[Polymorphism analysis of 20 autosomal short-tandem repeat loci in southern Chinese Han population].

PubMed

Chen, Ling; Lu, Hui-Jie; DU, Wei-An; Qiu, Ping-Ming; Liu, Chao

2016-02-20

To evaluate the value of PowerPlex ® 21 System (Promega) and study the genetic polymorphism of its 20 short-tandem repeat (STR) loci in southern Chinese Han population. We conducted genotyping experiments using PowerPlex ® 21 System on 20 autosomal STR loci (D3S1358, D1S1656, D6S1043, D13S317, Penta E, D16S539, D18S51, D2S1338, CSF1PO, Penta D, TH01, vWA, D21S11, D7S820, D5S818, TPOX, D8S1179, D12S391, D19S433 and FGA) in 2367 unrelated Chinese Han individuals living in South China. The allele frequencies and parameters commonly used in forensic science were statistically analyzed in these individuals and compared with the reported data of other populations. The PowerPlex ® 21 System had a power of discrimination (PD) ranging from 0.7839 to 0.9852 and a power of exclusion (PE) ranging from 0.2974 to 0.8099 for the 20 loci. No significant deviation from Hardy-Weinberg expectations was found for all the loci except for D5S818. This southern Chinese Han population had significant differences in the allele frequencies from 8 ethnic groups reported in China, and showed significant differences at 8 to 20 STR foci from 5 foreign populations. The allele frequency at the locus D1S1656 in this southern Chinese Han population differed significantly from those in the 5 foreign populations and from 3 reported Han populations in Beijing, Zhejiang Province and Fujian Province of China. The neighbor-joining phylogenetictree showed clustering of all the Asian populations in one branch, while the northern Italian and Argentina populations clustered in a separate branch. This southern Chinese Han population had the nearest affinity with the Yi ethnic population in Yunnan Province of China. The 20 STR loci are highly polymorphic in this southern Chinese Han population, suggesting the value of this set of STR loci in forensic personal identification, paternity testing and anthropological study.

Multiple-locus, variable number of tandem repeat analysis (MLVA) of the fish-pathogen Francisella noatunensis

PubMed Central

2011-01-01

Background Since Francisella noatunensis was first isolated from cultured Atlantic cod in 2004, it has emerged as a global fish pathogen causing disease in both warm and cold water species. Outbreaks of francisellosis occur in several important cultured fish species making a correct management of this disease a matter of major importance. Currently there are no vaccines or treatments available. A strain typing system for use in studies of F. noatunensis epizootics would be an important tool for disease management. However, the high genetic similarity within the Francisella spp. makes strain typing difficult, but such typing of the related human pathogen Francisella tullarensis has been performed successfully by targeting loci with higher genetic variation than the traditional signature sequences. These loci are known as Variable Numbers of Tandem Repeat (VNTR). The aim of this study is to identify possible useful VNTRs in the genome of F. noatunensis. Results Seven polymorphic VNTR loci were identified in the preliminary genome sequence of F. noatunensis ssp. noatunensis GM2212 isolate. These VNTR-loci were sequenced in F. noatunensis isolates collected from Atlantic cod (Gadus morhua) from Norway (n = 21), Three-line grunt (Parapristipoma trilineatum) from Japan (n = 1), Tilapia (Oreochromis spp.) from Indonesia (n = 3) and Atlantic salmon (Salmo salar) from Chile (n = 1). The Norwegian isolates presented in this study show both nine allelic profiles and clades, and that the majority of the farmed isolates belong in two clades only, while the allelic profiles from wild cod are unique. Conclusions VNTRs can be used to separate isolates belonging to both subspecies of F. noatunensis. Low allelic diversity in F. noatunensis isolates from outbreaks in cod culture compared to isolates wild cod, indicate that transmission of these isolates may be a result of human activity. The sequence based MLVA system presented in this study should provide a good starting point for further development of a genotyping system that can be used in studies of epizootics and disease management of francisellosis. PMID:21261955

spa typing for epidemiological surveillance of Staphylococcus aureus.

PubMed

Hallin, Marie; Friedrich, Alexander W; Struelens, Marc J

2009-01-01

The spa typing method is based on sequencing of the polymorphic X region of the protein A gene (spa), present in all strains of Staphylococcus aureus. The X region is constituted of a variable number of 24-bp repeats flanked by well-conserved regions. This single-locus sequence-based typing method combines a number of technical advantages, such as rapidity, reproducibility, and portability. Moreover, due to its repeat structure, the spa locus simultaneously indexes micro- and macrovariations, enabling the use of spa typing in both local and global epidemiological studies. These studies are facilitated by the establishment of standardized spa type nomenclature and Internet shared databases.

A naturally occurring, noncanonical GTP aptamer made of simple tandem repeats

PubMed Central

Curtis, Edward A; Liu, David R

2014-01-01

Recently, we used in vitro selection to identify a new class of naturally occurring GTP aptamer called the G motif. Here we report the discovery and characterization of a second class of naturally occurring GTP aptamer, the “CA motif.” The primary sequence of this aptamer is unusual in that it consists entirely of tandem repeats of CA-rich motifs as short as three nucleotides. Several active variants of the CA motif aptamer lack the ability to form consecutive Watson-Crick base pairs in any register, while others consist of repeats containing only cytidine and adenosine residues, indicating that noncanonical interactions play important roles in its structure. The circular dichroism spectrum of the CA motif aptamer is distinct from that of A-form RNA and other major classes of nucleic acid structures. Bioinformatic searches indicate that the CA motif is absent from most archaeal and bacterial genomes, but occurs in at least 70 percent of approximately 400 eukaryotic genomes examined. These searches also uncovered several phylogenetically conserved examples of the CA motif in rodent (mouse and rat) genomes. Together, these results reveal the existence of a second class of naturally occurring GTP aptamer whose sequence requirements, like that of the G motif, are not consistent with those of a canonical secondary structure. They also indicate a new and unexpected potential biochemical activity of certain naturally occurring tandem repeats. PMID:24824832

Direct repeat sequences are essential for function of the cis-acting locus of transfer (clt) of Streptomyces phaeochromogenes plasmid pJV1.

PubMed

Franco, Bernardo; González-Cerón, Gabriela; Servín-González, Luis

2003-11-01

The functionality of direct and inverted repeat sequences inside the cis acting locus of transfer (clt) of the Streptomyces plasmid pJV1 was determined by testing the effect of different deletions on plasmid transfer. The results show that the single most important element for pJV1 clt function is a series of evenly spaced 9 bp long direct repeats which match the consensus CCGCACA(C/G)(C/G), since their deletion caused a dramatic reduction in plasmid transfer. The presence of these repeats in the absence of any other clt sequences allowed plasmid transfer to occur at a frequency that was at least two orders of magnitude higher than that obtained in the complete absence of clt. A database search revealed regions with a similar organization, and in the same position, in Streptomyces plasmids pSN22 and pSLS, which have transfer proteins homologous to those of pJV1.

Length Variation in Mitochondrial DNA of the Minnow Cyprinella Spiloptera

PubMed Central

Broughton, R. E.; Dowling, T. E.

1994-01-01

Length differences in animal mitochondrial DNA (mtDNA) are common, frequently due to variation in copy number of direct tandem duplications. While such duplications appear to form without great difficulty in some taxonomic groups, they appear to be relatively short-lived, as typical duplication products are geographically restricted within species and infrequently shared among species. To better understand such length variation, we have studied a tandem and direct duplication of approximately 260 bp in the control region of the cyprinid fish, Cyprinella spiloptera. Restriction site analysis of 38 individuals was used to characterize population structure and the distribution of variation in repeat copy number. This revealed two length variants, including individuals with two or three copies of the repeat, and little geographic structure among populations. No standard length (single copy) genomes were found and heteroplasmy, a common feature of length variation in other taxa, was absent. Nucleotide sequence of tandem duplications and flanking regions localized duplication junctions in the phenylalanine tRNA and near the origin of replication. The locations of these junctions and the stability of folded repeat copies support the hypothesized importance of secondary structures in models of duplication formation. PMID:8001785

A Legionella pneumophila collagen-like protein encoded by a gene with a variable number of tandem repeats is involved in the adherence and invasion of host cells.

PubMed

Vandersmissen, Liesbeth; De Buck, Emmy; Saels, Veerle; Coil, David A; Anné, Jozef

2010-05-01

Legionella pneumophila is a Gram-negative, facultative intracellular pathogen and the causative agent of Legionnaires' disease, a severe pneumonia in humans. Analysis of the Legionella sequenced genomes revealed a gene with a variable number of tandem repeats (VNTRs), whose number varies between strains. We examined the strain distribution of this gene among a collection of 108 clinical, environmental and hot spring serotype I strains. Twelve variants were identified, but no correlation was observed between the number of repeat units and clinical and environmental strains. The encoded protein contains the C-terminal consensus motif of outer membrane proteins and has a large region of collagen-like repeats that is encoded by the VNTR region. We have therefore annotated this protein Lcl for Legionella collagen-like protein. Lcl was shown to contribute to the adherence and invasion of host cells and it was demonstrated that the number of repeat units present in lcl had an influence on these adhesion characteristics.

A silent allele in the locus D5S818 contained within the PowerPlex®21 PCR Amplification Kit.

PubMed

Chen, Ling; Tai, Yunchun; Qiu, Pingming; Du, Weian; Liu, Chao

2015-11-01

Three paternity tests cases were found with a single locus mismatch at the locus D5S818 with PowerPlex®21 PCR Amplification Kit (Promega). Forward and reverse primers were redesigned to type the samples again and to evaluate if there were alleles dropped out. The results showed the existence of a silent allele 12 in all the three families, due to a point mutation that changed cytosine to adenine at 90 nucleotides upstream from the 5' end of the AGAT repeat sequences in all the six individuals. A single locus mismatch due to a silent allele may occur in any locus using any kit. Therefore, we recommend using multiple kits to confirm the results in paternity testing cases with mismatches, especially when there is a single locus mismatch with homozygote involved. Copyright © 2015 Elsevier Ireland Ltd. All rights reserved.

Short-read, high-throughput sequencing technology for STR genotyping

PubMed Central

Bornman, Daniel M.; Hester, Mark E.; Schuetter, Jared M.; Kasoji, Manjula D.; Minard-Smith, Angela; Barden, Curt A.; Nelson, Scott C.; Godbold, Gene D.; Baker, Christine H.; Yang, Boyu; Walther, Jacquelyn E.; Tornes, Ivan E.; Yan, Pearlly S.; Rodriguez, Benjamin; Bundschuh, Ralf; Dickens, Michael L.; Young, Brian A.; Faith, Seth A.

2013-01-01

DNA-based methods for human identification principally rely upon genotyping of short tandem repeat (STR) loci. Electrophoretic-based techniques for variable-length classification of STRs are universally utilized, but are limited in that they have relatively low throughput and do not yield nucleotide sequence information. High-throughput sequencing technology may provide a more powerful instrument for human identification, but is not currently validated for forensic casework. Here, we present a systematic method to perform high-throughput genotyping analysis of the Combined DNA Index System (CODIS) STR loci using short-read (150 bp) massively parallel sequencing technology. Open source reference alignment tools were optimized to evaluate PCR-amplified STR loci using a custom designed STR genome reference. Evaluation of this approach demonstrated that the 13 CODIS STR loci and amelogenin (AMEL) locus could be accurately called from individual and mixture samples. Sensitivity analysis showed that as few as 18,500 reads, aligned to an in silico referenced genome, were required to genotype an individual (>99% confidence) for the CODIS loci. The power of this technology was further demonstrated by identification of variant alleles containing single nucleotide polymorphisms (SNPs) and the development of quantitative measurements (reads) for resolving mixed samples. PMID:25621315

Clostridium difficile infection: epidemiology, diagnosis and understanding transmission.

PubMed

Martin, Jessica S H; Monaghan, Tanya M; Wilcox, Mark H

2016-04-01

Clostridium difficile infection (CDI) continues to affect patients in hospitals and communities worldwide. The spectrum of clinical disease ranges from mild diarrhoea to toxic megacolon, colonic perforation and death. However, this bacterium might also be carried asymptomatically in the gut, potentially leading to 'silent' onward transmission. Modern technologies, such as whole-genome sequencing and multi-locus variable-number tandem-repeat analysis, are helping to track C. difficile transmission across health-care facilities, countries and continents, offering the potential to illuminate previously under-recognized sources of infection. These typing strategies have also demonstrated heterogeneity in terms of CDI incidence and strain types reflecting different stages of epidemic spread. However, comparison of CDI epidemiology, particularly between countries, is challenging due to wide-ranging approaches to sampling and testing. Diagnostic strategies for C. difficile are complicated both by the wide range of bacterial targets and tests available and the need to differentiate between toxin-producing and non-toxigenic strains. Multistep diagnostic algorithms have been recommended to improve sensitivity and specificity. In this Review, we describe the latest advances in the understanding of C. difficile epidemiology, transmission and diagnosis, and discuss the effect of these developments on the clinical management of CDI.

Drosophila Suppressor of Sable Protein [Su(s)] Promotes Degradation of Aberrant and Transposon-Derived RNAs▿

PubMed Central

Kuan, Yung-Shu; Brewer-Jensen, Paul; Bai, Wen-Li; Hunter, Cedric; Wilson, Carrie B.; Bass, Sarah; Abernethy, John; Wing, James S.; Searles, Lillie L.

2009-01-01

RNA-binding proteins act at various stages of gene expression to regulate and fine-tune patterns of mRNA accumulation. One protein in this class is Drosophila Su(s), a nuclear protein that has been previously shown to inhibit the accumulation of mutant transcripts by an unknown mechanism. Here, we have identified several additional RNAs that are downregulated by Su(s). These Su(s) targets include cryptic wild-type transcripts from the developmentally regulated Sgs4 and ng1 genes, noncoding RNAs derived from tandemly repeated αβ/αγ elements within an Hsp70 locus, and aberrant transcripts induced by Hsp70 promoter transgenes inserted at ectopic sites. We used the αβ RNAs to investigate the mechanism of Su(s) function and obtained evidence that these transcripts are degraded by the nuclear exosome and that Su(s) promotes this process. Furthermore, we showed that the RNA binding domains of Su(s) are important for this effect and mapped the sequences involved to a 267-nucleotide region of an αβ element. Taken together, these results suggest that Su(s) binds to certain nascent transcripts and stimulates their degradation by the nuclear exosome. PMID:19687295

Rapid microfluidic analysis of a Y-STR multiplex for screening of forensic samples.

PubMed

Gibson-Daw, Georgiana; Albani, Patricia; Gassmann, Marcus; McCord, Bruce

2017-02-01

In this paper, we demonstrate a rapid analysis procedure for use with a small set of rapidly mutating Y chromosomal short tandem repeat (Y-STR) loci that combines both rapid polymerase chain reaction (PCR) and microfluidic separation elements. The procedure involves a high-speed polymerase and a rapid cycling protocol to permit PCR amplification in 16 min. The resultant amplified sample is next analysed using a short 1.8-cm microfluidic electrophoresis system that permits a four-locus Y-STR genotype to be produced in 80 s. The entire procedure takes less than 25 min from sample collection to result. This paper describes the rapid amplification protocol as well as studies of the reproducibility and sensitivity of the procedure and its optimisation. The amplification process utilises a small high-speed thermocycler, microfluidic device and compact laptop, making it portable and potentially useful for rapid, inexpensive on-site genotyping. The four loci used for the multiplex were selected due to their rapid mutation rates and should proved useful in preliminary screening of samples and suspects. Overall, this technique provides a method for rapid sample screening of suspect and crime scene samples in forensic casework. Graphical abstract ᅟ.

Multiple-locus variable number of tandem repeat analysis as a tool for molecular epidemiology of botulism: The Italian experience.

PubMed

Anniballi, Fabrizio; Fillo, Silvia; Giordani, Francesco; Auricchio, Bruna; Tehran, Domenico Azarnia; di Stefano, Enrica; Mandarino, Giuseppina; De Medici, Dario; Lista, Florigio

2016-12-01

Clostridium botulinum is the bacterial agent of botulism, a rare but severe neuro-paralytic disease. Because of its high impact, in Italy botulism is monitored by an ad hoc surveillance system. The National Reference Centre for Botulism, as part of this system, collects and analyzes all demographic, epidemiologic, microbiological, and molecular data recovered during cases and/or outbreaks occurred in Italy. A panel of 312 C. botulinum strains belonging to group I were submitted to MLVA sub-typing. Strains, isolated from clinical specimens, food and environmental samples collected during the surveillance activities, were representative of all forms of botulism from all Italian regions. Through clustering analysis isolates were grouped into 12 main clusters. No regional or temporal clustering was detected, demonstrating the high heterogeneity of strains circulating in Italy. This study confirmed that MLVA is capable of sub-typing C. botulinum strains. Moreover, MLVA is effective at tracing and tracking the source of contamination and is helpful for the surveillance system in terms of planning and upgrading of procedures, activities and data collection forms. Copyright © 2016 Elsevier B.V. All rights reserved.

Genetic diversity and statistical parameters of 15 autosomal STR loci in the Pomeranian subpopulation of Espirito Santo State, Brazil.

PubMed

Silva, Beatriz Candida; de Vargas Wolfgramm, Eldamária; da Costa Aguiar, Vitor Resende; Malta, Frederico Scott Varela; de Castro, Amanda Mafia; de Souza Ferreira, Alessandro Clayton; de Paula, Flavia; Louro, Iúri Drumond

2011-06-01

Allelic frequencies and other population data analysis are reported for the 15 autosomal Short Tandem Repeats (STR) loci included in the PowerPlex(®)16 kit (CSF1PO, D13S317, D16S539, D18S51, D21S11, D3S1358, D5S818, D7S820, D8S1179, FGA, Penta D, Penta E, TPOX, TH01 and vWA) in Pomeranian's descendants from the Espirito Santo State (ES), Brazil, third largest population of Pomeranian's descendants in the world. They chose the mountain region of the state for their preferred geographic location, and they have a very peculiar lifestyle with a selective mating behavior which has maintained their characteristics as a relatively pure subpopulation. Blood samples were obtained from 82 unrelated volunteers from 11 different cities of Espirito Santo State, where there are the Pomeranian's descendants. All 15 loci analyzed showed Power of Discrimination (PD) values > 0.75. Except the TPOX locus, all analyzed loci were at Hardy-Weinberg equilibrium. This subpopulation has not yet been characterized for STR allelic frequencies used for forensic and genetic identification studies.

Molecular identification and typing of Burkholderia pseudomallei and Burkholderia mallei: when is enough enough?

PubMed

Antonov, Valery A; Tkachenko, Galina A; Altukhova, Viktoriya V; Savchenko, Sergey S; Zinchenko, Olga V; Viktorov, Dmitry V; Zamaraev, Valery S; Ilyukhin, Vladimir I; Alekseev, Vladimir V

2008-12-01

Burkholderia mallei and B. pseudomallei are highly pathogenic microorganisms for both humans and animals. Moreover, they are regarded as potential agents of bioterrorism. Thus, rapid and unequivocal detection and identification of these dangerous pathogens is critical. In the present study, we describe the use of an optimized protocol for the early diagnosis of experimental glanders and melioidosis and for the rapid differentiation and typing of Burkholderia strains. This experience with PCR-based identification methods indicates that single PCR targets (23S and 16S rRNA genes, 16S-23S intergenic region, fliC and type III secretion gene cluster) should be used with caution for identification of B. mallei and B. pseudomallei, and need to be used alongside molecular methods such as gene sequencing. Several molecular typing procedures have been used to identify genetically related B. pseudomallei and B. mallei isolates, including ribotyping, pulsed-field gel electrophoresis and multilocus sequence typing. However, these methods are time consuming and technically challenging for many laboratories. RAPD, variable amplicon typing scheme, Rep-PCR, BOX-PCR and multiple-locus variable-number tandem repeat analysis have been recommended by us for the rapid differentiation of B. mallei and B. pseudomallei strains.

Molecular methods for the detection of mutations.

PubMed

Monteiro, C; Marcelino, L A; Conde, A R; Saraiva, C; Giphart-Gassler, M; De Nooij-van Dalen, A G; Van Buuren-van Seggelen, V; Van der Keur, M; May, C A; Cole, J; Lehmann, A R; Steinsgrimsdottir, H; Beare, D; Capulas, E; Armour, J A

2000-01-01

We report the results of a collaborative study aimed at developing reliable, direct assays for mutation in human cells. The project used common lymphoblastoid cell lines, both with and without mutagen treatment, as a shared resource to validate the development of new molecular methods for the detection of low-level mutations in the presence of a large excess of normal alleles. As the "gold standard, " hprt mutation frequencies were also measured on the same samples. The methods under development included i) the restriction site mutation (RSM) assay, in which mutations lead to the destruction of a restriction site; ii) minisatellite length-change mutation, in which mutations lead to alleles containing new numbers of tandem repeat units; iii) loss of heterozygosity for HLA epitopes, in which antibodies can be used to direct selection for mutant cells; iv) multiple fluorescence-based long linker arm nucleotides assay (mf-LLA) technology, for the detection of substitutional mutations; v) detection of alterations in the TP53 locus using a (CA) array as the target for the screening; and vi) PCR analysis of lymphocytes for the presence of the BCL2 t(14:18) translocation. The relative merits of these molecular methods are discussed, and a comparison made with more "traditional" methods.

Subtyping of STEC by MLVA in Argentina.

PubMed

Bustamante, Ana V; Sanso, Andrea M; Parma, Alberto E; Lucchesi, Paula M A

2012-01-01

Shiga toxin-producing Escherichia coli (STEC) causes serious human illness such as hemolytic uremic syndrome (HUS). Argentina has the world's highest rate of this syndrome, which is the leading cause of acute renal failure among children. E. coli O157:H7 is the most common cause of HUS, but a substantial and growing proportion of this illness is caused by infection due to non-O157 strains. Multiple-locus variable-number tandem repeat analysis (MLVA) has become an established technique to subtype STEC. This review will address the use of routine STEC subtyping by MLVA in order to type this group of isolates and to get insight into the genetic diversity of native STEC. With regard to these objectives we modified and adapted two MLVA protocols, one exclusive for O157 and the other, a generic E. coli assay. A total of 202 STEC isolates, from different sources and corresponding to 20 serotypes, have been MLVA genotyped in our laboratory. In our experience, MLVA constitutes a very sensitive tool and enables us to perform an efficient STEC subtyping. The diversity found in many serotypes may be useful for future epidemiological studies of STEC clonality, applied to O157 as well as to non-O157 isolates.

In silico analysis of a disease-causing mutation in PCDH15 gene in a consanguineous Pakistani family with Usher phenotype.

PubMed

Saleha, Shamim; Ajmal, Muhammad; Jamil, Muhammad; Nasir, Muhammad; Hameed, Abdul

2016-01-01

To map Usher phenotype in a consanguineous Pakistani family and identify disease-associated mutation in a causative gene to establish phenotype-genotype correlation. A consanguineous Pakistani family in which Usher phenotype was segregating as an autosomal recessive trait was ascertained. On the basis of results of clinical investigations of affected members of this family disease was diagnosed as Usher syndrome (USH). To identify the locus responsible for the Usher phenotype in this family, genomic DNA from blood sample of each individual was genotyped using microsatellite Short Tandem Repeat (STR) markers for the known Usher syndrome loci. Then direct sequencing was performed to find out disease associated mutations in the candidate gene. By genetic linkage analysis, the USH phenotype of this family was mapped to PCDH15 locus on chromosome 10q21.1. Three different point mutations in exon 11 of PCDH15 were identified and one of them, c.1304A>C was found to be segregating with the disease phenotype in Pakistani family with Usher phenotype. This, c.1304A>C transversion mutation predicts an amino-acid substitution of aspartic acid with an alanine at residue number 435 (p.D435A) of its protein product. Moreover, in silico analysis revealed conservation of aspartic acid at position 435 and predicated this change as pathogenic. The identification of c.1304A>C pathogenic mutation in PCDH15 gene and its association with Usher syndrome in a consanguineous Pakistani family is the first example of a missense mutation of PCDH15 causing USH1 phenotype. In previous reports, it was hypothesized that severe mutations such as truncated protein of PCDH15 led to the Usher I phenotype and that missense variants are mainly responsible for non-syndromic hearing impairment.

Evaluation of MIRU-VNTR for typing of Mycobacterium bovis isolated from Sika deer in Northeast China.

PubMed

Yang, Li; Wang, Chunyu; Wang, Haijun; Meng, Qingfeng; Wang, Quankai

2015-04-11

Bovine tuberculosis has led to serious economic losses for Sika Deer producers in China. Strategies for controlling the spread of Mycobacterium bovis are often hampered by a lack of epidemiological data. Specifically, tracing infections requires the ability to trace back infections, which, in turn, requires the ability to determine isolates with a common source. This study was planned to assess the discriminatory power of each mycobacterial interspersed repetitive unit (MIRU)-variable number tandem repeats (VNTR) locus and evaluate the most appropriate combination of MIRU-VNTR loci for molecular epidemiological studies on Sika Deer in China. The discriminatory power of MIRU-VNTR typing based on 22 known loci (12 MIRUs, 2 ETRs, 4 QUBs, and 4 Mtubs) were assessed in 96 Mycobacterium bovis strains collected sequentially from Sika Deer at a slaughterhouse in northeastern China. We defined four loci (MIRU4, ETRA, QUB11b, and Mtub4) as highly discriminative, eight loci (MIRU2, MIRU23, MIRU27, MIRU31, MIRU39, MIRU40, QUB26, and Mtub21) as moderately discriminative, and three loci (MIRU16, Mtub30, and Mtub34) as poorly discriminative. The final locus showed no polymorphism between strains. MIRU-VNTR typing as a whole was highly discriminative, with an overall allelic diversity of 0.897. Of the loci tested, the four highly discriminative loci and eight moderately discriminative loci proved to be most appropriate for first line typing of M. bovis from Sika Deer, with the same resolving ability as all 22 loci (H = 0.897). MIRU-VNTR typing is quick and effective for typing bovine tuberculosis isolates from Sika Deer in China.

Streptococcus suis in invasive human infections in Poland: clonality and determinants of virulence and antimicrobial resistance.

PubMed

Bojarska, A; Molska, E; Janas, K; Skoczyńska, A; Stefaniuk, E; Hryniewicz, W; Sadowy, E

2016-06-01

The purpose of this study was to perform an analysis of Streptococcus suis human invasive isolates, collected in Poland by the National Reference Centre for Bacterial Meningitis. Isolates obtained from 21 patients during 2000-2013 were investigated by phenotypic tests, multilocus sequence typing (MLST), analysis of the TR9 locus from the multilocus variable number tandem repeat (VNTR) analysis (MLVA) scheme and pulsed-field gel electrophoresis (PFGE) of SmaI-digested DNA. Determinants of virulence and antimicrobial resistance were detected by polymerase chain reaction (PCR) and analysed by sequencing. All isolates represented sequence type 1 (ST1) and were suggested to be serotype 2. PFGE and analysis of the TR9 locus allowed the discrimination of four and 17 types, respectively. Most of the isolates were haemolysis- and DNase-positive, and around half of them formed biofilm. Genes encoding suilysin, extracellular protein factor, fibronectin-binding protein, muramidase-released protein, surface antigen one, enolase, serum opacity factor and pili were ubiquitous in the studied group, while none of the isolates carried sequences characteristic for the 89K pathogenicity island. All isolates were susceptible to penicillin, cefotaxime, imipenem, moxifloxacin, chloramphenicol, rifampicin, gentamicin, linezolid, vancomycin and daptomycin. Five isolates (24 %) were concomitantly non-susceptible to erythromycin, clindamycin and tetracycline, and harboured the tet(O) and erm(B) genes; for one isolate, lsa(E) and lnu(B) were additionally detected. Streptococcus suis isolated in Poland from human invasive infections belongs to a globally distributed clonal complex of this pathogen, enriched in virulence markers. This is the first report of the lsa(E) and lnu(B) resistance genes in S. suis.

Pretense of parentage by siblings in immigration: Polesky's paradox reconsidered.

PubMed

Wenk, Robert E; Shao, Alison

2014-02-01

Older and younger siblings occasionally attempt to impersonate parent and child to expedite immigration under US family-based visa policies. The rate with which full siblings escape detection by current relationship tests is unknown. Retrospective study of full-sibling immigrant pairs was undertaken to determine the proportion that show insufficient genetic evidence to exclude parentage. Sibship and parentage indices (SI and PI) were compared/case in unexcluded sibling cases and true parent-child cases. Alleles shared per short-tandem-repeat locus were compared in sibling and parent-child pairs. The proportion of successful parentage fraud by siblings was estimated from the parentage exclusion rate among immigrants and the proportion of sibships without genetic inconsistencies (GIs). When 11 to 25 independent loci were tested per two-sibling case to verify or refute parentage, tests failed to demonstrate any GI in 9% and PI was greater than SI in seven of 10 of these cases. Another 29% of full-sibling pairs demonstrated insufficient evidence (fewer than two GIs) to exclude parentage. Thus, 0.4% of sibling pairs could falsely claim a parent-child relationship and show no GIs. Another 1.4% could make that false claim and not present sufficient evidence to be excluded. At present, with no evidence of parentage exclusion in a full-sibling pair, the relative magnitudes of PI and SI are misleading relationship indicators because too few loci are examined and rates of sharing one and two alleles/locus vary greatly in parentage and sibling pairs. Only evidence of exclusion ascertains false parentage claims by siblings. Nevertheless, the expected rate of successful fraud is quite low. © 2013 American Association of Blood Banks.

Developmental and internal validation of a novel 13 loci STR multiplex method for Cannabis sativa DNA profiling.

PubMed

Houston, Rachel; Birck, Matthew; Hughes-Stamm, Sheree; Gangitano, David

2017-05-01

Marijuana (Cannabis sativa L.) is a plant cultivated and trafficked worldwide as a source of fiber (hemp), medicine, and intoxicant. The development of a validated method using molecular techniques such as short tandem repeats (STRs) could serve as an intelligence tool to link multiple cases by means of genetic individualization or association of cannabis samples. For this purpose, a 13 loci STR multiplex method was developed, optimized, and validated according to relevant ISFG and SWGDAM guidelines. The STR multiplex consists of 13 previously described C. sativa STR loci: ANUCS501, 9269, 4910, 5159, ANUCS305, 9043, B05, 1528, 3735, CS1, D02, C11, and H06. A sequenced allelic ladder consisting of 56 alleles was designed to accurately genotype 101 C. sativa samples from three seizures provided by a U.S. Customs and Border Protection crime lab. Using an optimal range of DNA (0.5-1.0ng), validation studies revealed well-balanced electropherograms (inter-locus balance range: 0.500-1.296), relatively balanced heterozygous peaks (mean peak height ratio of 0.83 across all loci) with minimal artifacts and stutter ratio (mean stutter of 0.021 across all loci). This multi-locus system is relatively sensitive (0.13ng of template DNA) with a combined power of discrimination of 1 in 55 million. The 13 STR panel was found to be species specific for C. sativa; however, non-specific peaks were produced with Humulus lupulus. The results of this research demonstrate the robustness and applicability of this 13 loci STR system for forensic DNA profiling of marijuana samples. Copyright © 2017 Elsevier B.V. All rights reserved.

Characterization of Genomic Island 3 and Genetic Variability of Chilean Field Strains of Brucella abortus▿

PubMed Central

Céspedes, Sandra; Salgado, Paulina; Valenzuela, Patricio; Vidal, Roberto; Oñate, Angel A.

2011-01-01

One of the capabilities developed by bacteria is the ability to gain large fragments of DNA from other bacteria or to lose portions of their own genomes. Among these exchangeable fragments are the genomic islands (GIs). Nine GIs have been identified in Brucella, and genomic island 3 (GI-3) is shared by two pathogenic species, B. melitensis and B. abortus. GI-3 encodes mostly unknown proteins. One of the aims of this study was to perform pulsed-field gel electrophoresis (PFGE) on field isolates of B. abortus from Chile to determine whether these isolates are clonally related. Furthermore, we focused on the characterization of GI-3, studying its organization and the genetic conservation of the GI-3 sequence using techniques such as tiling-path PCR (TP-PCR) and restriction fragment length polymorphism-PCR (RFLP-PCR). Our results, after PFGE was performed on 69 field isolates of B. abortus from Chile, showed that the strains were genetically homogeneous. To increase the power of genetic discrimination among these strains, we used multiple locus variable-number tandem-repeat (VNTR) analysis with 16 loci (MLVA-16). The results obtained by MLVA-16 showed that the strains of B. abortus were genetically heterogeneous and that most of them clustered according to their geographic origin. Of the genetic loci studied, panel 2B was the one describing the highest diversity in the analysis, as well as locus Bruce19 in panel 2A. In relation to the study of GI-3, our experimental analysis by TP-PCR identified and confirmed that GI-3 is present in all wild strains of B. abortus, demonstrating the high stability of gene cluster GI-3 in Chilean field strains. PMID:21543580

Lab on a chip genotyping for Brucella spp. based on 15-loci multi locus VNTR analysis.

PubMed

De Santis, Riccardo; Ciammaruconi, Andrea; Faggioni, Giovanni; D'Amelio, Raffaele; Marianelli, Cinzia; Lista, Florigio

2009-04-07

Brucellosis is an important zoonosis caused by the genus Brucella. In addition Brucella represents potential biological warfare agents due to the high contagious rates for humans and animals. Therefore, the strain typing epidemiological tool may be crucial for tracing back source of infection in outbreaks and discriminating naturally occurring outbreaks versus bioterroristic event. A Multiple Locus Variable-number tandem repeats (VNTR) Analysis (MLVA) assay based on 15 polymorphic markers was previously described. The obtained MLVA band profiles may be resolved by techniques ranging from low cost manual agarose gels to the more expensive capillary electrophoresis sequencing. In this paper a rapid, accurate and reproducible system, based on the Lab on a chip technology was set up for Brucella spp. genotyping. Seventeen DNA samples of Brucella strains isolated in Sicily, previously genotyped, and twelve DNA samples, provided by MLVA Brucella VNTR ring trial, were analyzed by MLVA-15 on Agilent 2100. The DNA fragment sizes produced by Agilent, compared with those expected, showed discrepancies; therefore, in order to assign the correct alleles to the Agilent DNA fragment sizes, a conversion table was produced. In order to validate the system twelve unknown DNA samples were analyzed by this method obtaining a full concordance with the VNTR ring trial results. In this paper we described a rapid and specific detection method for the characterization of Brucella isolates. The comparison of the MLVA typing data produced by Agilent system with the data obtained by standard sequencing or ethidium bromide slab gel electrophoresis showed a general concordance of the results. Therefore this platform represents a fair compromise among costs, speed and specificity compared to any conventional molecular typing technique.

Condensin loaded onto the replication fork barrier site in the rRNA gene repeats during S phase in a FOB1-dependent fashion to prevent contraction of a long repetitive array in Saccharomyces cerevisiae.

PubMed

Johzuka, Katsuki; Terasawa, Masahiro; Ogawa, Hideyuki; Ogawa, Tomoko; Horiuchi, Takashi

2006-03-01

An average of 200 copies of the rRNA gene (rDNA) is clustered in a long tandem array in Saccharomyces cerevisiae. FOB1 is known to be required for expansion/contraction of the repeats by stimulating recombination, thereby contributing to the maintenance of the average copy number. In Deltafob1 cells, the repeats are still maintained without any fluctuation in the copy number, suggesting that another, unknown system acts to prevent repeat contraction. Here, we show that condensin acts together with FOB1 in a functionally complemented fashion to maintain the long tandem repeats. Six condensin mutants possessing severely contracted rDNA repeats were isolated in Deltafob1 cells but not in FOB1+ cells. We also found that the condensin complex associated with the nontranscribed spacer region of rDNA with a major peak coincided with the replication fork barrier (RFB) site in a FOB1-dependent fashion. Surprisingly, condensin association with the RFB site was established during S phase and was maintained until anaphase. These results indicate that FOB1 plays a novel role in preventing repeat contraction by regulating condensin association and suggest a link between replication termination and chromosome condensation and segregation.

A Complex Structural Variation on Chromosome 27 Leads to the Ectopic Expression of HOXB8 and the Muffs and Beard Phenotype in Chickens

PubMed Central

Wang, Yanqiang; Luo, Chenglong; Liu, Ranran; Qu, Hao; Shu, Dingming; Wen, Jie; Crooijmans, Richard P. M. A.; Zhao, Yiqiang; Hu, Xiaoxiang; Li, Ning

2016-01-01

Muffs and beard (Mb) is a phenotype in chickens where groups of elongated feathers gather from both sides of the face (muffs) and below the beak (beard). It is an autosomal, incomplete dominant phenotype encoded by the Muffs and beard (Mb) locus. Here we use genome-wide association (GWA) analysis, linkage analysis, Identity-by-Descent (IBD) mapping, array-CGH, genome re-sequencing and expression analysis to show that the Mb allele causing the Mb phenotype is a derived allele where a complex structural variation (SV) on GGA27 leads to an altered expression of the gene HOXB8. This Mb allele was shown to be completely associated with the Mb phenotype in nine other independent Mb chicken breeds. The Mb allele differs from the wild-type mb allele by three duplications, one in tandem and two that are translocated to that of the tandem repeat around 1.70 Mb on GGA27. The duplications contain total seven annotated genes and their expression was tested during distinct stages of Mb morphogenesis. A continuous high ectopic expression of HOXB8 was found in the facial skin of Mb chickens, strongly suggesting that HOXB8 directs this regional feather-development. In conclusion, our results provide an interesting example of how genomic structural rearrangements alter the regulation of genes leading to novel phenotypes. Further, it again illustrates the value of utilizing derived phenotypes in domestic animals to dissect the genetic basis of developmental traits, herein providing novel insights into the likely role of HOXB8 in feather development and differentiation. PMID:27253709

Genetic variation and evolutionary stability of the FMR1 CGG repeat in six closed human populations

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

Eichler, E.E.; Nelson, D.L.

1996-07-12

In an attempt to understand the allelic diversity and mutability of the human FMR1 CGG repeat, we have analyzed the AGG substructure of this locus within six genetically-closed populations (Mbuti pygmy, Baka pygmy, R. surui, Karitiana, Mayan, and Hutterite). Most alleles (61/92 or 66%) possessed two AGG interspersions occurring with a periodicity of one AGG every nine or ten CGG repeats, indicating that this pattern is highly conserved in all human populations. Significant differences in allele distribution were observed among the populations for rare variants possessing fewer or more AGG interruptions than the canonical FMR1 CGG repeat sequence. Comparisons ofmore » expected heterozygosity of the FMR1 CGG repeat locus with 30 other microsatellite loci, demonstrated remarkably similar levels of polymorphism within each population, suggesting that most FMR1 CGG repeat alleles mutate at rates indistinguishable from other microsatellite loci. A single allele (1 out of 92) was identified with a large uninterrupted tract of pure repeats (42 pure CGG triplets). Retrospective pedigree analysis indicated that this allele had been transmitted unstably. Although such alleles mutate rapidly and likely represent evolving premutations, our analysis suggests that in spite of the estimated frequency of their occurrence, these unstable alleles do not significantly alter the expected heterozygosity of the FMR1 CGG repeat in the human population. 45 refs., 1 fig., 2 tabs.« less

ACCA phosphopeptide recognition by the BRCT repeats of BRCA1.

PubMed

Ray, Hind; Moreau, Karen; Dizin, Eva; Callebaut, Isabelle; Venezia, Nicole Dalla

2006-06-16

The tumour suppressor gene BRCA1 encodes a 220 kDa protein that participates in multiple cellular processes. The BRCA1 protein contains a tandem of two BRCT repeats at its carboxy-terminal region. The majority of disease-associated BRCA1 mutations affect this region and provide to the BRCT repeats a central role in the BRCA1 tumour suppressor function. The BRCT repeats have been shown to mediate phospho-dependant protein-protein interactions. They recognize phosphorylated peptides using a recognition groove that spans both BRCT repeats. We previously identified an interaction between the tandem of BRCA1 BRCT repeats and ACCA, which was disrupted by germ line BRCA1 mutations that affect the BRCT repeats. We recently showed that BRCA1 modulates ACCA activity through its phospho-dependent binding to ACCA. To delineate the region of ACCA that is crucial for the regulation of its activity by BRCA1, we searched for potential phosphorylation sites in the ACCA sequence that might be recognized by the BRCA1 BRCT repeats. Using sequence analysis and structure modelling, we proposed the Ser1263 residue as the most favourable candidate among six residues, for recognition by the BRCA1 BRCT repeats. Using experimental approaches, such as GST pull-down assay with Bosc cells, we clearly showed that phosphorylation of only Ser1263 was essential for the interaction of ACCA with the BRCT repeats. We finally demonstrated by immunoprecipitation of ACCA in cells, that the whole BRCA1 protein interacts with ACCA when phosphorylated on Ser1263.

A versatile palindromic amphipathic repeat coding sequence horizontally distributed among diverse bacterial and eucaryotic microbes

PubMed Central

2010-01-01

Background Intragenic tandem repeats occur throughout all domains of life and impart functional and structural variability to diverse translation products. Repeat proteins confer distinctive surface phenotypes to many unicellular organisms, including those with minimal genomes such as the wall-less bacterial monoderms, Mollicutes. One such repeat pattern in this clade is distributed in a manner suggesting its exchange by horizontal gene transfer (HGT). Expanding genome sequence databases reveal the pattern in a widening range of bacteria, and recently among eucaryotic microbes. We examined the genomic flux and consequences of the motif by determining its distribution, predicted structural features and association with membrane-targeted proteins. Results Using a refined hidden Markov model, we document a 25-residue protein sequence motif tandemly arrayed in variable-number repeats in ORFs lacking assigned functions. It appears sporadically in unicellular microbes from disparate bacterial and eucaryotic clades, representing diverse lifestyles and ecological niches that include host parasitic, marine and extreme environments. Tracts of the repeats predict a malleable configuration of recurring domains, with conserved hydrophobic residues forming an amphipathic secondary structure in which hydrophilic residues endow extensive sequence variation. Many ORFs with these domains also have membrane-targeting sequences that predict assorted topologies; others may comprise reservoirs of sequence variants. We demonstrate expressed variants among surface lipoproteins that distinguish closely related animal pathogens belonging to a subgroup of the Mollicutes. DNA sequences encoding the tandem domains display dyad symmetry. Moreover, in some taxa the domains occur in ORFs selectively associated with mobile elements. These features, a punctate phylogenetic distribution, and different patterns of dispersal in genomes of related taxa, suggest that the repeat may be disseminated by HGT and intra-genomic shuffling. Conclusions We describe novel features of PARCELs (Palindromic Amphipathic Repeat Coding ELements), a set of widely distributed repeat protein domains and coding sequences that were likely acquired through HGT by diverse unicellular microbes, further mobilized and diversified within genomes, and co-opted for expression in the membrane proteome of some taxa. Disseminated by multiple gene-centric vehicles, ORFs harboring these elements enhance accessory gene pools as part of the "mobilome" connecting genomes of various clades, in taxa sharing common niches. PMID:20626840

Prevalence, antimicrobial resistance and multiple-locus variable-number tandem-repeat analysis profiles of diarrheagenic Escherichia coli isolated from different retail foods.

PubMed

Wang, Lili; Nakamura, Hiromi; Kage-Nakadai, Eriko; Hara-Kudo, Yukiko; Nishikawa, Yoshikazu

2017-05-16

Diarrheagenic E. coli (DEC) isolates were recovered from local retail markets and the Osaka Municipal Central Wholesale Market in Japan. Retail food samples were collected for analysis in Osaka Japan from 2005 to 2008 and consisted of 32 beef, 28 pork, 20 poultry, 136 fish, 66 fruits and vegetables and 51 ready-to-eat (RTE) food samples. A total of 82 DEC strains were recovered from 64 (19%) food samples with the highest prevalence in poultry (100%, 20/20), followed by pork (54%, 15/28), beef (28%, 9/32), fruits and vegetables (12%, 8/66), fish (6.6%, 9/136) and RTE foods (5.9%, 3/51). Most of the strains belonged to E. coli possessing the enteroaggregative E. coli (EAEC) heat-stable enterotoxin 1 (EAST1) gene (EAST1EC; n=62, P<0.0001) and enteropathogenic E. coli (EPEC; n=16, P<0.01), whereas only 1 strain belonged to Shiga toxin-producing E. coli (STEC), 1 to EAEC and 2 to enterotoxigenic E. coli (ETEC) strains. Of the 82 DEC isolates, 22 O and 13H serogroups were detected, including some specific serogroups (O91, O103, O115, O119, O126, and O157) which have been associated with human diarrheal infections. Phylogenetic group A and B1 were predominant among the DEC isolates. Antimicrobial resistance to tetracycline was most common (49%), followed by nalidixic acid (28%), ampicillin (24%), sulfamethoxazole/trimethoprim (20%), and cephalothin (18%). All isolates were susceptible to aztreonam. Of the resistant strains, 44% (22/50) demonstrated resistance to >3 antimicrobial agents. Isolates resistant to >5 antimicrobials were only found in the meat samples, while isolates from the fruits and vegetables as well as RTE foods showed resistance to only 1 or 2 antimicrobial agents. Sixty one percent of EAST1EC, 56% of EPEC and all of the EAEC and ETEC were resistant to at least 1 antimicrobial agent. Multiple-locus variable-number tandem repeat analysis (MLVA) was used in this study for genotyping of DEC. The 82 isolates collected for this study showed 77 distinct MLVA profiles located among 3 branches. The Simpson's Index of Diversity (D) was 99.9% at its highest. The high diversity of these food strains would suggest their originating from a variety of sources and environments. In conclusion, retail food samples in Japan were contaminated with DEC; EAST1EC, a putative DEC, were detected at high rates in poultry, pork and beef. Isolates resistant to >3 antimicrobials were found only in raw meat and fish. Food animals may act as the reservoir for multi-resistant bacteria. Due to the finding that nearly 1/3 of EAST1EC strains were resistant to >3 antimicrobials, additional surveillance for EAST1EC should be initiated. Copyright © 2017 Elsevier B.V. All rights reserved.

Rex and a Suppressor of Rex Are Repeated Neomorphic Loci in the Drosophila Melanogaster Ribosomal DNA

PubMed Central

Rasooly, R. S.; Robbins, L. G.

1991-01-01

The Rex locus of Drosophila melanogaster induces a high frequency of mitotic exchange between two separated ribosomal DNA arrays on a single chromosome. The exchanges take place in the progeny of Rex mothers and occur very early, before the third mitotic division. A number of common laboratory stocks have also been found to carry dominant suppressors of Rex (Su(Rex)). Rex was mapped to the X centric heterochromatin, proximal to su(f), by genetic and molecular analysis of two spontaneous recombinants. Using deficiencies and duplications of the heterochromatin, both Rex and one Su(Rex) were shown to behave as neomorphs. Rex-induced exchange in a target chromosome bearing both Rex and Su(Rex) was then used to map these functions to the bb locus itself. Molecular analysis of the recombinants, using length variants of the ribosomal DNA intergenic spacer as genetic markers, mapped Su(Rex) and Rex within the bb locus and demonstrated that both are repeated elements. PMID:1936953

Complete mitochondrial genome of the Tyto longimembris (Strigiformes: Tytonidae).

PubMed

Xu, Peng; Li, Yankuo; Miao, Lujun; Xie, Guangyong; Huang, Yan

2016-07-01

The complete mitochondrial genome of Tyto longimembris has been determined in this study. It is 18,466 bp in length and consists of 13 protein-coding genes, 22 transfer RNA (tRNA) genes, 2 ribosomal RNA (rRNA) genes and a non-coding control region (D-loop). The overall base composition of the heavy strand of the T. longimembris mitochondrial genome is A: 30.1%, T: 23.5%, C: 31.8% and G: 14.6%. The structure of control region should be characterized by a region containing tandem repeats as two definitely separated clusters of tandem repeats were found. This study provided an important data set for phylogenetic and taxonomic analyses of Tyto species.

Complete mitochondrial genome of the yellowtail clownfish Amphiprion clarkii (Pisces: Perciformes, Pomacentridae).

PubMed

Tao, Yong; Li, Jian-Long; Liu, Min; Hu, Xue-Yi

2016-01-01

In this study we determined the complete mitochondrial (mt) genome of the yellowtail clownfish Amphiprion clarkii using eight consensus primer pairs with a long PCR technique. The circular mtDNA molecule was 16,976 bp in size and the overall nucleotide composition of the H-strand was 29.15% A, 26.15% T, 15.67% G and 29.03% C, with an A + T bias. The complete mitogenome contained 13 protein-coding genes, 2 rRNAs, 22 tRNAs and 1 control region (D-loop), and the gene order was typical of vertebrate mitogenomes. We determined five complete continuity tandem repeat units and one imperfect tandem repeat, all located downstream in the control region.

Juvenile myoclonic epilepsy locus in chromosome 6p21.2-p11: Linkage to convulsions and electroencephalography trait

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

Liu, A.W.; Delgado-Escueta, A.V.; Serratosa, J.M.

1995-08-01

Despite affecting 4 million Americans and 100-200 million persons worldwide, the precise molecular mechanisms of human epilepsies remain unknown. Juvenile myoclonic epilepsy (JME) is the most frequent and, hence, most important form of hereditary grand mal epilepsy. In this epilepsy, electroencephalographic (EEG) 15-30 Hz multispikes produce myoclonic and tonic-clonic convulsions beginning at 8-20 years of age. Moreover, EEG 3.5-6 Hz multispike wave complexes appear in clinically asymptomatic family members. We first studied 38 members of a four-generation LA-Belize family with classical JME but with no pyknoleptic absences. Five living members had JME; four clinically asymptomatic members had EEG multispike wavemore » complexes. Pairwise analysis tightly linked microsatellites centromeric to HLA, namely D6S272 (peak lod score [Z{sub max}]=3.564-3.560 at male-female recombination [{theta}{sub m=f}]=0-0.001) and D6S257 (Z{sub max}=3.672-3.6667 at {theta}{sub m=f}=0-0.001), spanning 7 cM, to convulsive seizures and EEG multispike wave complexes. A recombination between D6S276 and D6S273 in one affected member placed the JME locus within or below HLA. Pairwise, multipoint, and recombination analyses in this large family independently proved that a JME gene is located in chromsome 6p, centromeric to HLA. We next screened, with the same chromosome 6p21.2-p11 short tandem-repeat polymorphic markers, seven multiplex pedigrees with classic JME. When lod scores for small multiplex families are added to lod scores of the LA-Belize pedigree, Z{sub max} values for D6S294 and D6S257 are >7 ({theta}{sub m=f}=0.000). Our results prove that in chromosome 6p21.2-p11 an epilepsy locus exists whose phenotype consists of classic JME with convulsions and/or EEG rapid multispike wave complexes. 31 refs., 6 figs., 4 tabs.« less

Expanded Croatian 12 X-STR loci database with an overview of anomalous profiles.

PubMed

Mršić, Gordan; Ozretić, Petar; Crnjac, Josip; Merkaš, Siniša; Sukser, Viktorija; Račić, Ivana; Rožić, Sara; Barbarić, Lucija; Popović, Maja; Korolija, Marina

2018-05-01

In order to implement X-chromosome short tandem repeat (X-STR) typing into routine forensic practice, reference database of a given population should be established. Therefore we extended already published data with additional 397 blood samples from unrelated Croatian citizens, and analyzed the total of 995 samples (549 male and 446 female) typed by Investigator ® Argus X-12 Kit. To test genetic homogeneity of consecutively processed five historic-cultural regions covering the entire national territory, we calculated pairwise Fst genetic distances between regions based on allele and full haplotype frequencies. Since the comparison did not yield any statistically significant difference, we integrated STR profile information from all regions and used the whole data set to calculate forensic parameters. The most informative marker is DXS10135 (polymorphism information content (PIC = 0.929) and the most informative linkage group (LG) is LG1 (PIC = 0.996). We confirmed linkage disequilibrium (LD) for seven marker pairs belonging to LG2, LG3 and LG4. By including LD information, we calculated cumulative power of discrimination that amounted to 0.999999999997 in females and 0.999999005 in males. We also compared Croatia with 13 European populations based on haplotype frequencies and detected no statistically significant Fst values after Bonferroni correction in any LG. Multi-dimensional scaling plot revealed tight grouping of four Croatian regions amongst populations of southern, central and northern Europe, with the exception of northern Croatia. In this study we gave the first extensive overview of aberrant profiles encountered during Investigator ® Argus X-12 typing. We found ten profiles consistent with single locus duplication followed by tetranucleotide tract length polymorphism. Locus DXS10079 is by far the most frequently affected one, presumably mutated in eight samples. We also found four profiles consistent with X-chromosome aneuploidy (three profiles with XXX pattern and one profile with XXY pattern). In conclusion, we established integral forensic Croatian X-chromosome database, proved forensic pertinence of Investigator ® Argus X-12 Kit for the entire Croatian population and identified locus DXS10079 as a potential duplication hotspot. Copyright © 2018 Elsevier B.V. All rights reserved.

Characterization and assessment of an avian repetitive DNA sequence as an icterid phylogenetic marker.

PubMed

Quinn, J S; Guglich, E; Seutin, G; Lau, R; Marsolais, J; Parna, L; Boag, P T; White, B N

1992-02-01

The first tandemly repeated sequence examined in a passerine bird, a 431-bp PstI fragment named pMAT1, has been cloned from the genome of the brown-headed cowbird (Molothrus ater). The sequence represents about 5-10% of the genome (about 4 x 10(5) copies) and yields prominent ethidium bromide stained bands when genomic DNA cut with a variety of restriction enzymes is electrophoresed in agarose gels. A particularly striking ladder of fragments is apparent when the DNA is cut with HinfI, indicative of a tandem arrangement of the monomer. The cloned PstI monomer has been sequenced, revealing no internal repeated structure. There are sequences that hybridize with pMAT1 found in related nine-primaried oscines but not in more distantly related oscines, suboscines, or nonpasserine species. Little sequence similarity to tandemly repeated PstI cut sequences from the merlin (Falco columbarius), saurus crane (Grus antigone), or Puerto Rican parrot (Amazona vittata) or to HinfI digested sequence from the Toulouse goose (Anser anser) was detected. The isolated sequence was used as a probe to examine DNA samples of eight members of the tribe Icterini. This examination revealed phylogenetically informative characters. The repeat contains cutting sites from a number of restriction enzymes, which, if sufficiently polymorphic, would provide new phylogenetic characters. Sequences like these, conserved within a species, but variable between closely related species, may be very useful for phylogenetic studies of closely related taxa.

Characterization of the variable-number tandem repeats in vrrA from different Bacillus anthracis isolates

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

Jackson, P.J.; Walthers, E.A.; Richmond, K.L.

1997-04-01

PCR analysis of 198 Bacillus anthracis isolates revealed a variable region of DNA sequence differing in length among the isolates. Five Polymorphisms differed by the presence Of two to six copies of the 12-bp tandem repeat 5{prime}-CAATATCAACAA-3{prime}. This variable-number tandem repeat (VNTR) region is located within a larger sequence containing one complete open reading frame that encodes a putative 30-kDa protein. Length variation did not change the reading frame of the encoded protein and only changed the copy number of a 4-amino-acid sequence (QYQQ) from 2 to 6. The structure of the VNTR region suggests that these multiple repeats aremore » generated by recombination or polymerase slippage. Protein structures predicted from the reverse-translated DNA sequence suggest that any structural changes in the encoded protein are confined to the region encoded by the VNTR sequence. Copy number differences in the VNTR region were used to define five different B. anthracis alleles. Characterization of 198 isolates revealed allele frequencies of 6.1, 17.7, 59.6, 5.6, and 11.1% sequentially from shorter to longer alleles. The high degree of polymorphism in the VNTR region provides a criterion for assigning isolates to five allelic categories. There is a correlation between categories and geographic distribution. Such molecular markers can be used to monitor the epidemiology of anthrax outbreaks in domestic and native herbivore populations. 22 refs., 4 figs., 3 tabs.« less

Tandem Repeat Proteins Inspired By Squid Ring Teeth

NASA Astrophysics Data System (ADS)

Pena-Francesch, Abdon

Proteins are large biomolecules consisting of long chains of amino acids that hierarchically assemble into complex structures, and provide a variety of building blocks for biological materials. The repetition of structural building blocks is a natural evolutionary strategy for increasing the complexity and stability of protein structures. However, the relationship between amino acid sequence, structure, and material properties of protein systems remains unclear due to the lack of control over the protein sequence and the intricacies of the assembly process. In order to investigate the repetition of protein building blocks, a recently discovered protein from squids is examined as an ideal protein system. Squid ring teeth are predatory appendages located inside the suction cups that provide a strong grasp of prey, and are solely composed of a group of proteins with tandem repetition of building blocks. The objective of this thesis is the understanding of sequence, structure and property relationship in repetitive protein materials inspired in squid ring teeth for the first time. Specifically, this work focuses on squid-inspired structural proteins with tandem repeat units in their sequence (i.e., repetition of alternating building blocks) that are physically cross-linked via beta-sheet structures. The research work presented here tests the hypothesis that, in these systems, increasing the number of building blocks in the polypeptide chain decreases the protein network defects and improves the material properties. Hence, the sequence, nanostructure, and properties (thermal, mechanical, and conducting) of tandem repeat squid-inspired protein materials are examined. Spectroscopic structural analysis, advanced materials characterization, and entropic elasticity theory are combined to elucidate the structure and material properties of these repetitive proteins. This approach is applied not only to native squid proteins but also to squid-inspired synthetic polypeptides that allow for a fine control of the sequence and network morphology. The results provided in this work establish a clear dependence between the repetitive building blocks, the network morphology, and the properties of squid-inspired repetitive protein materials. Increasing the number of tandem repeat units in SRT-inspired proteins led to more effective protein networks with superior properties. Through increasing tandem repetition and optimization of network morphology, highly efficient protein materials capable of withstanding deformations up to 400% of their original length, with MPa-GPa modulus, high energy absorption (50 MJ m-3), peak proton conductivity of 3.7 mS cm-1 (at pH 7, highest reported to date for biological materials), and peak thermal conductivity of 1.4 W m-1 K -1 (which exceeds that of most polymer materials) were developed. These findings introduce new design rules in the engineering of proteins based on tandem repetition and morphology control, and provide a novel framework for tailoring and optimizing the properties of protein-based materials.

Comparative Analysis of Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) of Streptococcus thermophilus St-I and its Bacteriophage-Insensitive Mutants (BIM) Derivatives.

PubMed

Li, Wan; Bian, Xin; Evivie, Smith Etareri; Huo, Gui-Cheng

2016-09-01

The CRISPR-Cas (CRISPR together with CRISPR-associated proteins) modules are the adaptive immune system, acting as an adaptive and heritable immune system in bacteria and archaea. CRISPR-based immunity acts by integrating short virus sequences in the cell's CRISPR locus, allowing the cell to remember, recognize, and clear infections. In this study, the homology of CRISPRs sequence in BIMs (bacteriophage-insensitive mutants) of Streptococcus thermophilus St-I were analyzed. Secondary structures of the repeats and the PAMs (protospacer-associated motif) of each CRISPR locus were also predicted. Results showed that CRISPR1 has 27 repeat-spacer units, 5 of them had duplicates; CRISPR2 has one repeat-spacer unit; CRISPR3 has 28 repeat-spacer units. Only BIM1 had a new spacer acquisition in CRISPR3, while BIM2 and BIM3 had no new spacers' insertion, thus indicating that while most CRISPR1 were more active than CRISPR3, new spacer acquisition occurred just in CRSPR3 in some situations. These findings will help establish the foundation for the study of CRSPR-Cas systems in lactic acid bacteria.

Sequences spanning the leader-repeat junction mediate CRISPR adaptation to phage in Streptococcus thermophilus

PubMed Central

Wei, Yunzhou; Chesne, Megan T.; Terns, Rebecca M.; Terns, Michael P.

2015-01-01

CRISPR-Cas systems are RNA-based immune systems that protect prokaryotes from invaders such as phages and plasmids. In adaptation, the initial phase of the immune response, short foreign DNA fragments are captured and integrated into host CRISPR loci to provide heritable defense against encountered foreign nucleic acids. Each CRISPR contains a ∼100–500 bp leader element that typically includes a transcription promoter, followed by an array of captured ∼35 bp sequences (spacers) sandwiched between copies of an identical ∼35 bp direct repeat sequence. New spacers are added immediately downstream of the leader. Here, we have analyzed adaptation to phage infection in Streptococcus thermophilus at the CRISPR1 locus to identify cis-acting elements essential for the process. We show that the leader and a single repeat of the CRISPR locus are sufficient for adaptation in this system. Moreover, we identified a leader sequence element capable of stimulating adaptation at a dormant repeat. We found that sequences within 10 bp of the site of integration, in both the leader and repeat of the CRISPR, are required for the process. Our results indicate that information at the CRISPR leader-repeat junction is critical for adaptation in this Type II-A system and likely other CRISPR-Cas systems. PMID:25589547

Structure, organization, and sequence of alpha satellite DNA from human chromosome 17: evidence for evolution by unequal crossing-over and an ancestral pentamer repeat shared with the human X chromosome.

PubMed

Waye, J S; Willard, H F

1986-09-01

The centromeric regions of all human chromosomes are characterized by distinct subsets of a diverse tandemly repeated DNA family, alpha satellite. On human chromosome 17, the predominant form of alpha satellite is a 2.7-kilobase-pair higher-order repeat unit consisting of 16 alphoid monomers. We present the complete nucleotide sequence of the 16-monomer repeat, which is present in 500 to 1,000 copies per chromosome 17, as well as that of a less abundant 15-monomer repeat, also from chromosome 17. These repeat units were approximately 98% identical in sequence, differing by the exclusion of precisely 1 monomer from the 15-monomer repeat. Homologous unequal crossing-over is suggested as a probable mechanism by which the different repeat lengths on chromosome 17 were generated, and the putative site of such a recombination event is identified. The monomer organization of the chromosome 17 higher-order repeat unit is based, in part, on tandemly repeated pentamers. A similar pentameric suborganization has been previously demonstrated for alpha satellite of the human X chromosome. Despite the organizational similarities, substantial sequence divergence distinguishes these subsets. Hybridization experiments indicate that the chromosome 17 and X subsets are more similar to each other than to the subsets found on several other human chromosomes. We suggest that the chromosome 17 and X alpha satellite subsets may be related components of a larger alphoid subfamily which have evolved from a common ancestral repeat into the contemporary chromosome-specific subsets.

Genetic variation and willingness to participate in epidemiologic research: data from three studies.

PubMed

Bhatti, Parveen; Sigurdson, Alice J; Wang, Sophia S; Chen, Jinbo; Rothman, Nathaniel; Hartge, Patricia; Bergen, Andrew W; Landi, Maria Teresa

2005-10-01

The differences in common genetic polymorphism frequencies by willingness to participate in epidemiologic studies are unexplored, but the same threats to internal validity operate as for studies with nongenetic information. We analyzed single nucleotide polymorphism genotypes, haplotypes, and short tandem repeats among control groups from three studies with different recruitment designs that included early, late, and never questionnaire responders, one or more participation incentives, and blood or buccal DNA collection. Among 2,955 individuals, we compared 108 genotypes, 8 haplotypes, and 9 to 15 short tandem repeats by respondent type. Among our main comparisons, single nucleotide polymorphism genotype frequencies differed significantly (P < 0.05) between respondent groups in six instances, with 13 expected by chance alone. When comparing the odds of carrying a variant among the various response groups, 19 odds ratios were /=1.40, levels that might be notably different. Among the various respondent group comparisons, haplotype and short tandem repeat frequencies were not significantly different by willingness to participate. We observed little evidence to suggest that genotype differences underlie response characteristics in molecular epidemiologic studies, but a greater variety of genes should be examined, including those related to behavioral traits potentially associated with willingness to participate. To the extent possible, investigators should evaluate their own genetic data for bias in response categories.

Production of monoclonal antibody, PR81, recognizing the tandem repeat region of MUC1 mucin.

PubMed

Paknejad, M; Rasaee, M J; Tehrani, F Karami; Kashanian, S; Mohagheghi, M A; Omidfar, K; Bazl, M Rajabi

2003-06-01

A monoclonal antibody (MAb) was generated by immunizing BALB/c mice with homogenized breast cancerous tissues. This antibody (PR81) was found to be of IgG(1) class and subclass, containing kappa light chain. PR81 reacted with either the membrane extracts of several breast cancerous tissues or the cell surface of some MUC1 positive cell lines (MCF-7, BT-20 and T-47D) tested by enzyme immunoassay and for MCF-7 by immunofluorescence method. PR81 also reacted with two synthetic 27 and 16-amino acid peptides, TSA-P1-24 and A-P1-15, respectively, which included the core tandem repeat sequence of MUC1. However, this antibody did not react with a synthetic 14 amino acid peptide that has no similarity with tandem repeat found in MUC1. The generated antibody had good and similar affinities (2.19 x 10(8) M(-1)) toward TSA-P1-24 and A-P1-15, which are mainly shared in the hydrophilic sequence of PDTRPAP. Through Western blot analysis of homogenized breast tissues, PR81 recognized only a major band of 250 kDa. This band is stronger in malignant tissue than benign and normal tissues.

Expression of Curli by Escherichia coli O157:H7 Strains Isolated from Patients during Outbreaks Is Different from Similar Strains Isolated from Leafy Green Production Environments.

PubMed

Ravva, Subbarao V; Sarreal, Chester Z; Cooley, Michael B

2016-01-01

We previously reported that the strains of Escherichia coli O157:H7 (EcO157) that survived longer in austere soil environment lacked expression of curli, a fitness trait linked with intestinal colonization. In addition, the proportion of curli-positive variants of EcO157 decreased with repeated soil exposure. Here we evaluated 84 and 176 clinical strains from outbreaks and sporadic infections in the US, plus 211 animal fecal and environmental strains for curli expression. These shiga-toxigenic strains were from 328 different genotypes, as characterized by multi-locus variable-number tandem-repeat analysis (MLVA). More than half of the fecal strains (human and animal) and a significant proportion of environmental isolates (82%) were found to lack curli expression. EcO157 strains from several outbreaks linked with the consumption of contaminated apple juice, produce, hamburgers, steak, and beef were also found to lack curli expression. Phylogenetic analysis of fecal strains indicates curli expression is distributed throughout the population. However, a significant proportion of animal fecal isolates (84%) gave no curli expression compared to human fecal isolates (58%). In addition, analysis of environmental isolates indicated nearly exclusive clustering of curli expression to a single branch of the minimal spanning tree. This indicates that curli expression depends primarily upon the type of environmental exposure and the isolation source, although genotypic differences also contribute to clonal variation in curli. Furthermore, curli-deficient phenotype appears to be a selective trait for survival of EcO157 in agricultural environments.

Population-Scale Sequencing Data Enable Precise Estimates of Y-STR Mutation Rates

PubMed Central

Willems, Thomas; Gymrek, Melissa; Poznik, G. David; Tyler-Smith, Chris; Erlich, Yaniv

2016-01-01

Short tandem repeats (STRs) are mutation-prone loci that span nearly 1% of the human genome. Previous studies have estimated the mutation rates of highly polymorphic STRs by using capillary electrophoresis and pedigree-based designs. Although this work has provided insights into the mutational dynamics of highly mutable STRs, the mutation rates of most others remain unknown. Here, we harnessed whole-genome sequencing data to estimate the mutation rates of Y chromosome STRs (Y-STRs) with 2–6 bp repeat units that are accessible to Illumina sequencing. We genotyped 4,500 Y-STRs by using data from the 1000 Genomes Project and the Simons Genome Diversity Project. Next, we developed MUTEA, an algorithm that infers STR mutation rates from population-scale data by using a high-resolution SNP-based phylogeny. After extensive intrinsic and extrinsic validations, we harnessed MUTEA to derive mutation-rate estimates for 702 polymorphic STRs by tracing each locus over 222,000 meioses, resulting in the largest collection of Y-STR mutation rates to date. Using our estimates, we identified determinants of STR mutation rates and built a model to predict rates for STRs across the genome. These predictions indicate that the load of de novo STR mutations is at least 75 mutations per generation, rivaling the load of all other known variant types. Finally, we identified Y-STRs with potential applications in forensics and genetic genealogy, assessed the ability to differentiate between the Y chromosomes of father-son pairs, and imputed Y-STR genotypes. PMID:27126583

Genotyping of Burkholderia mallei from an outbreak of glanders in Bahrain suggests multiple introduction events.

PubMed

Scholz, Holger C; Pearson, Talima; Hornstra, Heidie; Projahn, Michaela; Terzioglu, Rahime; Wernery, Renate; Georgi, Enrico; Riehm, Julia M; Wagner, David M; Keim, Paul S; Joseph, Marina; Johnson, Bobby; Kinne, Joerg; Jose, Shanti; Hepp, Crystal M; Witte, Angela; Wernery, Ulrich

2014-09-01

Glanders, caused by the gram-negative bacterium Burkholderia mallei, is a highly infectious zoonotic disease of solipeds causing severe disease in animals and men. Although eradicated from many Western countries, it recently emerged in Asia, the Middle-East, Africa, and South America. Due to its rareness, little is known about outbreak dynamics of the disease and its epidemiology. We investigated a recent outbreak of glanders in Bahrain by applying high resolution genotyping (multiple locus variable number of tandem repeats, MLVA) and comparative whole genome sequencing to B. mallei isolated from infected horses and a camel. These results were compared to samples obtained from an outbreak in the United Arab Emirates in 2004, and further placed into a broader phylogeographic context based on previously published B. mallei data. The samples from the outbreak in Bahrain separated into two distinct clusters, suggesting a complex epidemiological background and evidence for the involvement of multiple B. mallei strains. Additionally, the samples from Bahrain were more closely related to B. mallei isolated from horses in the United Arab Emirates in 2004 than other B. mallei which is suggestive of repeated importation to the region from similar geographic sources. High-resolution genotyping and comparative whole genome analysis revealed the same phylogenetic patterns among our samples. The close relationship of the Dubai/UAE B. mallei populations to each other may be indicative of a similar geographic origin that has yet to be identified for the infecting strains. The recent emergence of glanders in combination with worldwide horse trading might pose a new risk for human infections.

Deep landscape update of dispersed and tandem repeats in the genome model of the red jungle fowl, Gallus gallus, using a series of de novo investigating tools.

PubMed

Guizard, Sébastien; Piégu, Benoît; Arensburger, Peter; Guillou, Florian; Bigot, Yves

2016-08-19

The program RepeatMasker and the database Repbase-ISB are part of the most widely used strategy for annotating repeats in animal genomes. They have been used to show that avian genomes have a lower repeat content (8-12 %) than the sequenced genomes of many vertebrate species (30-55 %). However, the efficiency of such a library-based strategies is dependent on the quality and completeness of the sequences in the database that is used. An alternative to these library based methods are methods that identify repeats de novo. These alternative methods have existed for a least a decade and may be more powerful than the library based methods. We have used an annotation strategy involving several complementary de novo tools to determine the repeat content of the model genome galGal4 (1.04 Gbp), including identifying simple sequence repeats (SSRs), tandem repeats and transposable elements (TEs). We annotated over one Gbp. of the galGal4 genome and showed that it is composed of approximately 19 % SSRs and TEs repeats. Furthermore, we estimate that the actual genome of the red jungle fowl contains about 31-35 % repeats. We find that library-based methods tend to overestimate TE diversity. These results have a major impact on the current understanding of repeats distributions throughout chromosomes in the red jungle fowl. Our results are a proof of concept of the reliability of using de novo tools to annotate repeats in large animal genomes. They have also revealed issues that will need to be resolved in order to develop gold-standard methodologies for annotating repeats in eukaryote genomes.

New paradigm in ankyrin repeats: Beyond protein-protein interaction module.

PubMed

Islam, Zeyaul; Nagampalli, Raghavendra Sashi Krishna; Fatima, Munazza Tamkeen; Ashraf, Ghulam Md

2018-04-01

Classically, ankyrin repeat (ANK) proteins are built from tandems of two or more repeats and form curved solenoid structures that are associated with protein-protein interactions. These are short, widespread structural motif of around 33 amino acids repeats in tandem, having a canonical helix-loop-helix fold, found individually or in combination with other domains. The multiplicity of structural pattern enables it to form assemblies of diverse sizes, required for their abilities to confer multiple binding and structural roles of proteins. Three-dimensional structures of these repeats determined to date reveal a degree of structural variability that translates into the considerable functional versatility of this protein superfamily. Recent work on the ANK has proposed novel structural information, especially protein-lipid, protein-sugar and protein-protein interaction. Self-assembly of these repeats was also shown to prevent the associated protein in forming filaments. In this review, we summarize the latest findings and how the new structural information has increased our understanding of the structural determinants of ANK proteins. We discussed latest findings on how these proteins participate in various interactions to diversify the ANK roles in numerous biological processes, and explored the emerging and evolving field of designer ankyrins and its framework for protein engineering emphasizing on biotechnological applications. Copyright © 2017 Elsevier B.V. All rights reserved.

Population-scale whole genome sequencing identifies 271 highly polymorphic short tandem repeats from Japanese population.

PubMed

Hirata, Satoshi; Kojima, Kaname; Misawa, Kazuharu; Gervais, Olivier; Kawai, Yosuke; Nagasaki, Masao

2018-05-01

Forensic DNA typing is widely used to identify missing persons and plays a central role in forensic profiling. DNA typing usually uses capillary electrophoresis fragment analysis of PCR amplification products to detect the length of short tandem repeat (STR) markers. Here, we analyzed whole genome data from 1,070 Japanese individuals generated using massively parallel short-read sequencing of 162 paired-end bases. We have analyzed 843,473 STR loci with two to six basepair repeat units and cataloged highly polymorphic STR loci in the Japanese population. To evaluate the performance of the cataloged STR loci, we compared 23 STR loci, widely used in forensic DNA typing, with capillary electrophoresis based STR genotyping results in the Japanese population. Seventeen loci had high correlations and high call rates. The other six loci had low call rates or low correlations due to either the limitations of short-read sequencing technology, the bioinformatics tool used, or the complexity of repeat patterns. With these analyses, we have also purified the suitable 218 STR loci with four basepair repeat units and 53 loci with five basepair repeat units both for short read sequencing and PCR based technologies, which would be candidates to the actual forensic DNA typing in Japanese population.

Variability of CAG tandem repeats in exon 1 of the androgen receptor gene is not related with dog intersexuality.

PubMed

Nowacka-Woszuk, J; Switonski, M

2010-02-01

Numerous mutations of the human androgen receptor (AR) gene cause an intersexual phenotype, called the androgen insensitivity syndrome. The intersexual phenotype is also quite often diagnosed in dogs. The aim of this study was to conduct a comparative analysis of the entire coding sequence (eight exons) of the AR gene in healthy and four intersex dogs, as well as in three other canids (the red fox, arctic fox and Chinese raccoon dog). The coding sequence of the studied species appeared to be conserved (similarity above 97%) and polymorphism was found in exon 1 only. Altogether, 2 SNPs were identified in healthy dogs, 14 in red foxes, 16 in arctic foxes and 6 were found in Chinese raccoon dogs, respectively. Moreover, a variable number of tandem repeats (CAG and CAA), encoding an array of glutamines, was also observed in this exon. The CAA codon numbers were invariable within species, but the CAG repeats were polymorphic. The highest number of the CAG and CAA repeats was found in dogs (from 40 to 42) and the observed variability was similar in intersex and healthy dogs. In the other canids the variability fell within the following ranges: 29-37 (red fox), 37-39 (arctic fox) and 29-32 (Chinese raccoon dog). In addition, a polymorphic microsatellite marker in intron 2 was found in the dog, red fox and Chinese raccoon dog. It was concluded that the polymorphism level of the AR gene in the dog was lower than in the other canids and none of the detected polymorphisms, including variability of the CAG tandem repeats, could be related with the intersexual phenotype of the studied dogs.

Accurate typing of short tandem repeats from genome-wide sequencing data and its applications.

PubMed

Fungtammasan, Arkarachai; Ananda, Guruprasad; Hile, Suzanne E; Su, Marcia Shu-Wei; Sun, Chen; Harris, Robert; Medvedev, Paul; Eckert, Kristin; Makova, Kateryna D

2015-05-01

Short tandem repeats (STRs) are implicated in dozens of human genetic diseases and contribute significantly to genome variation and instability. Yet profiling STRs from short-read sequencing data is challenging because of their high sequencing error rates. Here, we developed STR-FM, short tandem repeat profiling using flank-based mapping, a computational pipeline that can detect the full spectrum of STR alleles from short-read data, can adapt to emerging read-mapping algorithms, and can be applied to heterogeneous genetic samples (e.g., tumors, viruses, and genomes of organelles). We used STR-FM to study STR error rates and patterns in publicly available human and in-house generated ultradeep plasmid sequencing data sets. We discovered that STRs sequenced with a PCR-free protocol have up to ninefold fewer errors than those sequenced with a PCR-containing protocol. We constructed an error correction model for genotyping STRs that can distinguish heterozygous alleles containing STRs with consecutive repeat numbers. Applying our model and pipeline to Illumina sequencing data with 100-bp reads, we could confidently genotype several disease-related long trinucleotide STRs. Utilizing this pipeline, for the first time we determined the genome-wide STR germline mutation rate from a deeply sequenced human pedigree. Additionally, we built a tool that recommends minimal sequencing depth for accurate STR genotyping, depending on repeat length and sequencing read length. The required read depth increases with STR length and is lower for a PCR-free protocol. This suite of tools addresses the pressing challenges surrounding STR genotyping, and thus is of wide interest to researchers investigating disease-related STRs and STR evolution. © 2015 Fungtammasan et al.; Published by Cold Spring Harbor Laboratory Press.

Association between the dopamine D4 receptor gene exon III variable number of tandem repeats and political attitudes in female Han Chinese

PubMed Central

Ebstein, Richard P.; Monakhov, Mikhail V.; Lu, Yunfeng; Jiang, Yushi; Lai, Poh San; Chew, Soo Hong

2015-01-01

Twin and family studies suggest that political attitudes are partially determined by an individual's genotype. The dopamine D4 receptor gene (DRD4) exon III repeat region that has been extensively studied in connection with human behaviour, is a plausible candidate to contribute to individual differences in political attitudes. A first United States study provisionally identified this gene with political attitude along a liberal–conservative axis albeit contingent upon number of friends. In a large sample of 1771 Han Chinese university students in Singapore, we observed a significant main effect of association between the DRD4 exon III variable number of tandem repeats and political attitude. Subjects with two copies of the 4-repeat allele (4R/4R) were significantly more conservative. Our results provided evidence for a role of the DRD4 gene variants in contributing to individual differences in political attitude particularly in females and more generally suggested that associations between individual genes, and neurochemical pathways, contributing to traits relevant to the social sciences can be provisionally identified. PMID:26246555

Repeat-containing protein effectors of plant-associated organisms

PubMed Central

Mesarich, Carl H.; Bowen, Joanna K.; Hamiaux, Cyril; Templeton, Matthew D.

2015-01-01

Many plant-associated organisms, including microbes, nematodes, and insects, deliver effector proteins into the apoplast, vascular tissue, or cell cytoplasm of their prospective hosts. These effectors function to promote colonization, typically by altering host physiology or by modulating host immune responses. The same effectors however, can also trigger host immunity in the presence of cognate host immune receptor proteins, and thus prevent colonization. To circumvent effector-triggered immunity, or to further enhance host colonization, plant-associated organisms often rely on adaptive effector evolution. In recent years, it has become increasingly apparent that several effectors of plant-associated organisms are repeat-containing proteins (RCPs) that carry tandem or non-tandem arrays of an amino acid sequence or structural motif. In this review, we highlight the diverse roles that these repeat domains play in RCP effector function. We also draw attention to the potential role of these repeat domains in adaptive evolution with regards to RCP effector function and the evasion of effector-triggered immunity. The aim of this review is to increase the profile of RCP effectors from plant-associated organisms. PMID:26557126

Association between the dopamine D4 receptor gene exon III variable number of tandem repeats and political attitudes in female Han Chinese.

PubMed

Ebstein, Richard P; Monakhov, Mikhail V; Lu, Yunfeng; Jiang, Yushi; Lai, Poh San; Chew, Soo Hong

2015-08-22

Twin and family studies suggest that political attitudes are partially determined by an individual's genotype. The dopamine D4 receptor gene (DRD4) exon III repeat region that has been extensively studied in connection with human behaviour, is a plausible candidate to contribute to individual differences in political attitudes. A first United States study provisionally identified this gene with political attitude along a liberal-conservative axis albeit contingent upon number of friends. In a large sample of 1771 Han Chinese university students in Singapore, we observed a significant main effect of association between the DRD4 exon III variable number of tandem repeats and political attitude. Subjects with two copies of the 4-repeat allele (4R/4R) were significantly more conservative. Our results provided evidence for a role of the DRD4 gene variants in contributing to individual differences in political attitude particularly in females and more generally suggested that associations between individual genes, and neurochemical pathways, contributing to traits relevant to the social sciences can be provisionally identified. © 2015 The Author(s).

Repeat-containing protein effectors of plant-associated organisms.

PubMed

Mesarich, Carl H; Bowen, Joanna K; Hamiaux, Cyril; Templeton, Matthew D

2015-01-01

Many plant-associated organisms, including microbes, nematodes, and insects, deliver effector proteins into the apoplast, vascular tissue, or cell cytoplasm of their prospective hosts. These effectors function to promote colonization, typically by altering host physiology or by modulating host immune responses. The same effectors however, can also trigger host immunity in the presence of cognate host immune receptor proteins, and thus prevent colonization. To circumvent effector-triggered immunity, or to further enhance host colonization, plant-associated organisms often rely on adaptive effector evolution. In recent years, it has become increasingly apparent that several effectors of plant-associated organisms are repeat-containing proteins (RCPs) that carry tandem or non-tandem arrays of an amino acid sequence or structural motif. In this review, we highlight the diverse roles that these repeat domains play in RCP effector function. We also draw attention to the potential role of these repeat domains in adaptive evolution with regards to RCP effector function and the evasion of effector-triggered immunity. The aim of this review is to increase the profile of RCP effectors from plant-associated organisms.

Isolation and characterization of two overlapping cosmid clones from the 4q35 region, near the facioscapulohumeral muscular dystrophy locus

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

Deidda, G.; Grisanti, P.; Vigneti, E.

1994-09-01

The gene for facioscapulohumeral muscular dystrophy (FSHD) has been localized by linkage analysis to the 4q35 region. The most telomeric p13E-11 prove has been shown to detect 4q35 DNA rearrangements in both sporadic and familial cases of the disease. With the aim of constructing a detailed physical map of the 4q35 region and searching for the mutant gene, we used p13E-11 probe to isolate cosmid clones from a human genomic library in a pCos-EMBL 2 vector. Two positive clones were isolated, clones 3 and 5, which partially overlap and carry human genomic inserts of 42 and 45 kb, respectively. Themore » cosmids share a common region containing the p13E-11 region and a stretch of KpnI units consisting of 3.2 kb tandemly repeated sequences (about 10). The restriction maps were constructed using the following enzymes: Bam HI, BgIII, Eco RI, EcoRV, KpnI and Sfi I. Clone 3 extends 4 kb upstream of C5 and stops within the Kpn repeats. Clone 5 extends 4 kb downstream from the Kpn repeats and it presents an additional EcoRI site. Clone 5 contains a stretch of Kpn sequences of nearly 32 kb, corresponding to 10 Kpn repeats; clone 3 contains a stretch of 29 kb corresponding to 9 Kpn repeats, as determined by PFGE analysis of partial digestion of the clones. Clone 5 seems to contain the entire Eco RI region prone to rearrangements in FSHD patients. From clone 5 several subclones were obtained, from the Kpn region and from the region spanning from the last Kpn repeat to the cloning site. No single copy sequences were detected. Subclones from the 3{prime} end region contain beta-satellite or Sau3A-like sequences. In situ hybridization with the whole C5 cosmid shows hybridization signals at the tip of chromosome 4 (4q35) and chromosome 10 (10q26), in the pericentromeric region of chromosome 1 (1q12) and in the p12 region of the acrocentric chromosomes (chr. 21, 22, 13, 14, 15).« less

Ehrlichia chaffeensis Tandem Repeat Proteins and Ank200 are Type 1 Secretion System Substrates Related to the Repeats-in-Toxin Exoprotein Family

PubMed Central

Wakeel, Abdul; den Dulk-Ras, Amke; Hooykaas, Paul J. J.; McBride, Jere W.

2011-01-01

Ehrlichia chaffeensis has type 1 and 4 secretion systems (T1SS and T4SS), but the substrates have not been identified. Potential substrates include secreted tandem repeat protein (TRP) 47, TRP120, and TRP32, and the ankyrin repeat protein, Ank200, that are involved in molecular host–pathogen interactions including DNA binding and a network of protein–protein interactions with host targets associated with signaling, transcriptional regulation, vesicle trafficking, and apoptosis. In this study we report that E. chaffeensis TRP47, TRP32, TRP120, and Ank200 were not secreted in the Agrobacterium tumefaciens Cre recombinase reporter assay routinely used to identify T4SS substrates. In contrast, all TRPs and the Ank200 proteins were secreted by the Escherichia coli complemented with the hemolysin secretion system (T1SS), and secretion was reduced in a T1SS mutant (ΔTolC), demonstrating that these proteins are T1SS substrates. Moreover, T1SS secretion signals were identified in the C-terminal domains of the TRPs and Ank200, and a detailed bioinformatic analysis of E. chaffeensis TRPs and Ank200 revealed features consistent with those described in the repeats-in-toxins (RTX) family of exoproteins, including glycine- and aspartate-rich tandem repeats, homology with ATP-transporters, a non-cleavable C-terminal T1SS signal, acidic pIs, and functions consistent with other T1SS substrates. Using a heterologous E. coli T1SS, this investigation has identified the first Ehrlichia T1SS substrates supporting the conclusion that the T1SS and corresponding substrates are involved in molecular host–pathogen interactions that contribute to Ehrlichia pathobiology. Further investigation of the relationship between Ehrlichia TRPs, Ank200, and the RTX exoprotein family may lead to a greater understanding of the importance of T1SS substrates and specific functions of T1SS in the pathobiology of obligately intracellular bacteria. PMID:22919588

Comparison of taxon-specific versus general locus sets for targeted sequence capture in plant phylogenomics.

PubMed

Chau, John H; Rahfeldt, Wolfgang A; Olmstead, Richard G

2018-03-01

Targeted sequence capture can be used to efficiently gather sequence data for large numbers of loci, such as single-copy nuclear loci. Most published studies in plants have used taxon-specific locus sets developed individually for a clade using multiple genomic and transcriptomic resources. General locus sets can also be developed from loci that have been identified as single-copy and have orthologs in large clades of plants. We identify and compare a taxon-specific locus set and three general locus sets (conserved ortholog set [COSII], shared single-copy nuclear [APVO SSC] genes, and pentatricopeptide repeat [PPR] genes) for targeted sequence capture in Buddleja (Scrophulariaceae) and outgroups. We evaluate their performance in terms of assembly success, sequence variability, and resolution and support of inferred phylogenetic trees. The taxon-specific locus set had the most target loci. Assembly success was high for all locus sets in Buddleja samples. For outgroups, general locus sets had greater assembly success. Taxon-specific and PPR loci had the highest average variability. The taxon-specific data set produced the best-supported tree, but all data sets showed improved resolution over previous non-sequence capture data sets. General locus sets can be a useful source of sequence capture targets, especially if multiple genomic resources are not available for a taxon.

Complete mitochondrial genome of the Asian pencil halfbeak Hyporhamphus intermedius (Beloniformes, Hemirhamphidae).

PubMed

Song, Chao; Hu, Gengdong; Qiu, Liping; Fan, Limin; Meng, Shunlong; Chen, Jiazhang

2016-11-01

The complete mitochondrial genome of Hyporhamphus intermedius was determined to be 16,720 bp in length with (A + T) content of 56.3%, and it consists of 13 protein-coding genes, 22 tRNAs, two ribosomal RNAs, and a control region. The gene composition and the structural arrangement of the H. intermedius complete mtDNA were identical to most of the other vertebrates. Interestingly, two tandem repeat units were identified across tRNA-Pro and control region (2*41 bp), while in most of the fishes the tandem repeat units are located in the control region. The molecular data we presented here could play a useful role to study the evolutionary relationships and population genetics of Hemirhamphidae fish.

De novo generation of plant centromeres at tandem repeats.

PubMed

Teo, Chee How; Lermontova, Inna; Houben, Andreas; Mette, Michael Florian; Schubert, Ingo

2013-06-01

Artificial minichromosomes are highly desirable tools for basic research, breeding, and biotechnology purposes. We present an option to generate plant artificial minichromosomes via de novo engineering of plant centromeres in Arabidopsis thaliana by targeting kinetochore proteins to tandem repeat arrays at non-centromeric positions. We employed the bacterial lactose repressor/lactose operator system to guide derivatives of the centromeric histone H3 variant cenH3 to LacO operator sequences. Tethering of cenH3 to non-centromeric loci led to de novo assembly of kinetochore proteins and to dicentric carrier chromosomes which potentially form anaphase bridges. This approach will be further developed and may contribute to generating minichromosomes from preselected genomic regions, potentially even in a diploid background.

Genetic characterization of the UCS and Kex1 loci of Pneumocystis jirovecii.

PubMed

Esteves, F; Tavares, A; Costa, M C; Gaspar, J; Antunes, F; Matos, O

2009-02-01

Nucleotide variation in the Pneumocystis jirovecii upstream conserved sequence (UCS) and kexin-like serine protease (Kex1) loci was studied in pulmonary specimens from Portuguese HIV-positive patients. DNA was extracted and used for specific molecular sequence analysis. The number of UCS tandem repeats detected in 13 successfully sequenced isolates ranged from three (9 isolates, 69%) to four (4 isolates, 31%). A novel tandem repeat pattern and two novel polymorphisms were detected in the UCS region. For the Kex1 gene, the wild-type (24 isolates, 86%) was the most frequent sequence detected among the 28 sequenced isolates. Nevertheless, a nonsynonymous (1 isolate, 3%) and three synonymous (3 isolates, 11%) polymorphisms were detected and are described here for the first time.

High occurrence of mitochondrial heteroplasmy in nepalese indigenous sheep (Ovis aries) compared to chinese sheep.

PubMed

Gorkhali, Neena Amatya; Jiang, Lin; Shrestha, Bhola Shankar; He, Xiao-Hong; Junzhao, Qian; Han, Jian-Lin; Ma, Yue-Hui

2016-07-01

Heteroplasmy due to length polymorphism with tandem repeats in mtDNAs within individual was hardly studied in domestic animals. In the present study, we identified intra-individual length variation in the control region of mtDNAs in Nepalese sheep by molecular cloning and sequencing techniques. We observed one to four tandem repeats of a 75-bp nucleotide sequences in the mtDNA control region in 45% of the total Nepalese sheep sampled in contrast to the Chinese sheep, indicating that the heteroplasmy is specific to Nepalese sheep. The high rate of heteroplasmy in Nepalese sheep could be a resultant of the mtDNA mutation and independent segregation at intra-individual level or a strand slippage and mispairing during the replication.

Mitochondrial genome of the tomato clownfish Amphiprion frenatus (Pomacentridae, Amphiprioninae).

PubMed

Ye, Le; Hu, Jing; Wu, Kaichang; Wang, Yu; Li, Jianlong

2016-01-01

The complete mitochondrial (mt) genome of the tomato clownfish Amphiprion frenatus was obtained in this study. The circular mtDNA molecule was 16,774 bp in size and the overall nucleotide composition of the H-strand was 29.72% A, 25.81% T, 15.38% G and 29.09% C, with an A + T bias. The complete mitogenome encoded 13 protein-coding genes, 2 rRNAs, 22 tRNAs and a control region (D-loop), with the gene arrangement and translation direction basically identical to other typical vertebrate mitogenomes. The D-loop included termination associated sequence (TAS), central conserved domain (CCD) and conserved sequence block (CSB), and was composed of 6 complete continuity tandem repeat units and an imperfect tandem repeat unit.

Linkage analysis with chromosome 9 markers in hereditary essential tremor.

PubMed

Conway, D; Bain, P G; Warner, T T; Davis, M B; Findley, L J; Thompson, P D; Marsden, C D; Harding, A E

1993-07-01

Hereditary essential tremor (ET) is an autosomal dominant disorder with variable expression and reduced penetrance. A tremor indistinguishable from ET may be observed in patients with autosomal dominant idiopathic torsion dystonia (ITD), in which the disease locus has been mapped to 9q32-34 in some kindreds, tightly linked to the argininosuccinate synthetase (ASS) locus. We performed linkage analysis in 15 families with ET containing 60 definitely affected individuals, using dinucleotide repeat polymorphisms at the ASS locus and the Abelson locus (ABL). Cumulative lod scores were -19.5 for ASS and -10.8 for ABL at a recombination fraction of 0.01, and tight linkage to ASS was excluded individually in 11 of the families. These data indicate that the ET gene is not allelic to that causing ITD.

A local duplication of the Melanocortin receptor 1 locus in Astyanax

PubMed Central

Gross, Joshua B.; Weagley, James; Stahl, Bethany A.; Ma, Li; Espinasa, Luis; McGaugh, Suzanne E.

2017-01-01

In this study, we report evidence of a novel duplication of Melanocortin receptor 1 (Mc1r) in the cavefish genome. This locus was discovered following the observation of excessive allelic diversity in a ~820 bp fragment of Mc1r amplified via degenerate PCR from a natural population of Astyanax aeneus fish from Guerrero, Mexico. The cavefish genome reveals the presence of two closely related Mc1r open reading frames separated by a 1.46 kb intergenic region. One open reading frame corresponds to the previously reported Mc1r receptor, and the other open reading frame (duplicate copy) is 975 bp in length, encoding a receptor of 325 amino acids. Sequence similarity analyses position both copies in the syntenic region of the single Mc1r locus in 16 representative craniate genomes spanning bony fish (including Astyanax) to mammals, suggesting we discovered tandem duplicates of this important gene. The two Mc1r copies share ~89% sequence similarity, and, within Astyanax, are more similar to one another compared to other melanocortin family members. Future studies will inform the precise functional significance of the duplicated Mc1r locus, and if this novel copy number variant may have adaptive significance for the Astyanax lineage. PMID:28738163

Microevolution of the Direct Repeat Locus of Mycobacterium tuberculosis in a Strain Prevalent in San Francisco

PubMed Central

Aga, Roxanne S.; Fair, Elizabeth; Abernethy, Neil F.; DeRiemer, Kathryn; Paz, E. Antonio; Kawamura, L. Masae; Small, Peter M.; Kato-Maeda, Midori

2006-01-01

We describe a microevolutionary event of a prevalent strain of Mycobacterium tuberculosis that caused two outbreaks in San Francisco. During the second outbreak, a direct variable repeat was lost. We discuss the mechanisms of this change and the implications of analyzing multiple genetic loci in this context. PMID:16597893

North Carolina macular dystrophy (MCDR1) caused by a novel tandem duplication of the PRDM13 gene

PubMed Central

Sullivan, Lori S.; Wheaton, Dianna K.; Locke, Kirsten G.; Jones, Kaylie D.; Koboldt, Daniel C.; Fulton, Robert S.; Wilson, Richard K.; Blanton, Susan H.; Birch, David G.; Daiger, Stephen P.

2016-01-01

Purpose To identify the underlying cause of disease in a large family with North Carolina macular dystrophy (NCMD). Methods A large four-generation family (RFS355) with an autosomal dominant form of NCMD was ascertained. Family members underwent comprehensive visual function evaluations. Blood or saliva from six affected family members and three unaffected spouses was collected and DNA tested for linkage to the MCDR1 locus on chromosome 6q12. Three affected family members and two unaffected spouses underwent whole exome sequencing (WES) and subsequently, custom capture of the linkage region followed by next-generation sequencing (NGS). Standard PCR and dideoxy sequencing were used to further characterize the mutation. Results Of the 12 eyes examined in six affected individuals, all but two had Gass grade 3 macular degeneration features. Large central excavation of the retinal and choroid layers, referred to as a macular caldera, was seen in an age-independent manner in the grade 3 eyes. The calderas are unique to affected individuals with MCDR1. Genome-wide linkage mapping and haplotype analysis of markers from the chromosome 6q region were consistent with linkage to the MCDR1 locus. Whole exome sequencing and custom-capture NGS failed to reveal any rare coding variants segregating with the phenotype. Analysis of the custom-capture NGS sequencing data for copy number variants uncovered a tandem duplication of approximately 60 kb on chromosome 6q. This region contains two genes, CCNC and PRDM13. The duplication creates a partial copy of CCNC and a complete copy of PRDM13. The duplication was found in all affected members of the family and is not present in any unaffected members. The duplication was not seen in 200 ethnically matched normal chromosomes. Conclusions The cause of disease in the original family with MCDR1 and several others has been recently reported to be dysregulation of the PRDM13 gene, caused by either single base substitutions in a DNase 1 hypersensitive site upstream of the CCNC and PRDM13 genes or a tandem duplication of the PRDM13 gene. The duplication found in the RFS355 family is distinct from the previously reported duplication and provides additional support that dysregulation of PRDM13, not CCNC, is the cause of NCMD mapped to the MCDR1 locus. PMID:27777503

North Carolina macular dystrophy (MCDR1) caused by a novel tandem duplication of the PRDM13 gene.

PubMed

Bowne, Sara J; Sullivan, Lori S; Wheaton, Dianna K; Locke, Kirsten G; Jones, Kaylie D; Koboldt, Daniel C; Fulton, Robert S; Wilson, Richard K; Blanton, Susan H; Birch, David G; Daiger, Stephen P

2016-01-01

To identify the underlying cause of disease in a large family with North Carolina macular dystrophy (NCMD). A large four-generation family (RFS355) with an autosomal dominant form of NCMD was ascertained. Family members underwent comprehensive visual function evaluations. Blood or saliva from six affected family members and three unaffected spouses was collected and DNA tested for linkage to the MCDR1 locus on chromosome 6q12. Three affected family members and two unaffected spouses underwent whole exome sequencing (WES) and subsequently, custom capture of the linkage region followed by next-generation sequencing (NGS). Standard PCR and dideoxy sequencing were used to further characterize the mutation. Of the 12 eyes examined in six affected individuals, all but two had Gass grade 3 macular degeneration features. Large central excavation of the retinal and choroid layers, referred to as a macular caldera, was seen in an age-independent manner in the grade 3 eyes. The calderas are unique to affected individuals with MCDR1. Genome-wide linkage mapping and haplotype analysis of markers from the chromosome 6q region were consistent with linkage to the MCDR1 locus. Whole exome sequencing and custom-capture NGS failed to reveal any rare coding variants segregating with the phenotype. Analysis of the custom-capture NGS sequencing data for copy number variants uncovered a tandem duplication of approximately 60 kb on chromosome 6q. This region contains two genes, CCNC and PRDM13 . The duplication creates a partial copy of CCNC and a complete copy of PRDM13 . The duplication was found in all affected members of the family and is not present in any unaffected members. The duplication was not seen in 200 ethnically matched normal chromosomes. The cause of disease in the original family with MCDR1 and several others has been recently reported to be dysregulation of the PRDM13 gene, caused by either single base substitutions in a DNase 1 hypersensitive site upstream of the CCNC and PRDM13 genes or a tandem duplication of the PRDM13 gene. The duplication found in the RFS355 family is distinct from the previously reported duplication and provides additional support that dysregulation of PRDM13 , not CCNC , is the cause of NCMD mapped to the MCDR1 locus.

[Observation and analysis on mutation of routine STR locus].

PubMed

Li, Qiu-yang; Feng, Wei-jun; Yang, Qin-gen

2005-05-01

To observe and analyze the characteristic of mutation at STR locus. 27 mutant genes observed in 1211 paternity testing cases were checked by PAGE-silver stained and PowerPlex 16 System Kit and validated by sequencing. Mutant genes locate on 15 loci. The pattern of mutation was accord with stepwise mutation model. The mutation ratio of male-to-female was 8:1 and correlated to the age of father. Mutation rate is correlated to the geometric mean of the number of homogeneous repeats of locus. The higher the mean, the higher the mutation rate. These loci are not so appropriate for use in paternity testing.

Association study of ERβ, AR, and CYP19A1 genes and MtF transsexualism.

PubMed

Fernández, Rosa; Esteva, Isabel; Gómez-Gil, Esther; Rumbo, Teresa; Almaraz, Mari Cruz; Roda, Ester; Haro-Mora, Juan-Jesús; Guillamón, Antonio; Pásaro, Eduardo

2014-12-01

The etiology of male-to-female (MtF) transsexualism is unknown. Both genetic and neurological factors may play an important role. To investigate the possible influence of the genetic factor on the etiology of MtF transsexualism. We carried out a cytogenetic and molecular analysis in 442 MtFs and 473 healthy, age- and geographical origin-matched XY control males. The karyotype was investigated by G-banding and by high-density array in the transsexual group. The molecular analysis involved three tandem variable regions of genes estrogen receptor β (ERβ) (CA tandem repeats in intron 5), androgen receptor (AR) (CAG tandem repeats in exon 1), and CYP19A1 (TTTA tandem repeats in intron 4). The allele and genotype frequencies, after division into short and long alleles, were obtained. We investigated the association between genotype and transsexualism by performing a molecular analysis of three variable regions of genes ERβ, AR, and CYP19A1 in 915 individuals (442 MtFs and 473 control males). Most MtFs showed an unremarkable 46,XY karyotype (97.96%). No specific chromosome aberration was associated with MtF transsexualism, and prevalence of aneuploidy (2.04%) was slightly higher than in the general population. Molecular analyses showed no significant difference in allelic or genotypic distribution of the genes examined between MtFs and controls. Moreover, molecular findings presented no evidence of an association between the sex hormone-related genes (ERβ, AR, and CYP19A1) and MtF transsexualism. The study suggests that the analysis of karyotype provides limited information in these subjects. Variable regions analyzed from ERβ, AR, and CYP19A1 are not associated with MtF transsexualism. Nevertheless, this does not exclude other polymorphic regions not analyzed. © 2014 International Society for Sexual Medicine.

Sequence and Analysis of the Tomato JOINTLESS Locus1

PubMed Central

Mao, Long; Begum, Dilara; Goff, Stephen A.; Wing, Rod A.

2001-01-01

A 119-kb bacterial artificial chromosome from the JOINTLESS locus on the tomato (Lycopersicon esculentum) chromosome 11 contained 15 putative genes. Repetitive sequences in this region include one copia-like LTR retrotransposon, 13 simple sequence repeats, three copies of a novel type III foldback transposon, and four putative short DNA repeats. Database searches showed that the foldback transposon and the short DNA repeats seemed to be associated preferably with genes. The predicted tomato genes were compared with the complete Arabidopsis genome. Eleven out of 15 tomato open reading frames were found to be colinear with segments on five Arabidopsis bacterial artificial chromosome/P1-derived artificial chromosome clones. The synteny patterns, however, did not reveal duplicated segments in Arabidopsis, where over half of the genome is duplicated. Our analysis indicated that the microsynteny between the tomato and Arabidopsis genomes was still conserved at a very small scale but was complicated by the large number of gene families in the Arabidopsis genome. PMID:11457984

An annotated genetic map of loblolly pine based on microsatellite and cDNA markers

USDA-ARS?s Scientific Manuscript database

Previous loblolly pine (Pinus taeda L.) genetic linkage maps have been based on a variety of DNA polymorphisms, such as AFLPs, RAPDs, RFLPs, and ESTPs, but only a few SSRs (simple sequence repeats), also known as simple tandem repeats or microsatellites, have been mapped in P. taeda. The objective o...

Salmonella enterica serotype enteritidis in French Polynesia, South Pacific, 2008-2013.

PubMed

Le Hello, Simon; Maillard, Fiona; Mallet, Henri-Pierre; Daudens, Elise; Levy, Marc; Roy, Valérie; Branaa, Philippe; Bertrand, Sophie; Fabre, Laetitia; Weill, François-Xavier

2015-06-01

Outbreaks of Salmonella enterica serotype Enteritidis infections associated with eggs occurred in French Polynesia during 2008-2013. Molecular analysis of isolates by using clustered regularly interspaced short palindromic repeat polymorphisms and multilocus variable-number tandem-repeat analysis was performed. This subtyping made defining the epidemic strain, finding the source, and decontaminating affected poultry flocks possible.

FT Duplication Coordinates Reproductive and Vegetative Growth

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

Hsu, Chuan-Yu; Adams, Joshua P.; Kim, Hyejin

2011-01-01

Annual plants grow vegetatively at early developmental stages and then transition to the reproductive stage, followed by senescence in the same year. In contrast, after successive years of vegetative growth at early ages, woody perennial shoot meristems begin repeated transitions between vegetative and reproductive growth at sexual maturity. However, it is unknown how these repeated transitions occur without a developmental conflict between vegetative and reproductive growth. We report that functionally diverged paralogs FLOWERING LOCUS T1 (FT1) and FLOWERING LOCUS T2 (FT2), products of whole-genome duplication and homologs of Arabidopsis thaliana gene FLOWERING LOCUS T (FT), coordinate the repeated cycles ofmore » vegetative and reproductive growth in woody perennial poplar (Populus spp.). Our manipulative physiological and genetic experiments coupled with field studies, expression profiling, and network analysis reveal that reproductive onset is determined by FT1 in response to winter temperatures, whereas vegetative growth and inhibition of bud set are promoted by FT2 in response to warm temperatures and long days in the growing season. The basis for functional differentiation between FT1 and FT2 appears to be expression pattern shifts, changes in proteins, and divergence in gene regulatory networks. Thus, temporal separation of reproductive onset and vegetative growth into different seasons via FT1 and FT2 provides seasonality and demonstrates the evolution of a complex perennial adaptive trait after genome duplication.« less

Variation in the genomic locations and sequence conservation of STAR elements among staphylococcal species provides insight into DNA repeat evolution

PubMed Central

2012-01-01

Background Staphylococcus aureus Repeat (STAR) elements are a type of interspersed intergenic direct repeat. In this study the conservation and variation in these elements was explored by bioinformatic analyses of published staphylococcal genome sequences and through sequencing of specific STAR element loci from a large set of S. aureus isolates. Results Using bioinformatic analyses, we found that the STAR elements were located in different genomic loci within each staphylococcal species. There was no correlation between the number of STAR elements in each genome and the evolutionary relatedness of staphylococcal species, however higher levels of repeats were observed in both S. aureus and S. lugdunensis compared to other staphylococcal species. Unexpectedly, sequencing of the internal spacer sequences of individual repeat elements from multiple isolates showed conservation at the sequence level within deep evolutionary lineages of S. aureus. Whilst individual STAR element loci were demonstrated to expand and contract, the sequences associated with each locus were stable and distinct from one another. Conclusions The high degree of lineage and locus-specific conservation of these intergenic repeat regions suggests that STAR elements are maintained due to selective or molecular forces with some of these elements having an important role in cell physiology. The high prevalence in two of the more virulent staphylococcal species is indicative of a potential role for STAR elements in pathogenesis. PMID:23020678

An evaluation of the International Society for Animal Genetics recommended parentage and identification panel for the domestic pigeon (Columba livia domestica).

PubMed

de Groot, M; van Haeringen, W A

2017-08-01

In this study, the International Society for Animal Genetics (ISAG) recommended panel for the identification of the domestic pigeon (Columba livia domestica) is characterized based on commonly used statistical parameters. The marker panel is based on 16 short tandem repeat (STR) loci (PIGN15, PIGN10, PIGN57, PIGN26, CliμD16, CliμD19, PIGN12, CliμD17, CliμT17, PIGN04, CliμD01, CliμD11, CliμD35, CliμT02, CliμT13, CliμT43). The alleles of the 16 loci consist of a mixture of tri-, tetra-, penta- and hexameric repeat patterns. A sex determination marker was included in the multiplex for quality control. The repeat sequence of the PIGN markers was previously unpublished and therefore sequenced to reveal the sequence pattern. In total, 1421 pigeons were genotyped on 16 STR loci to generate allele frequency data for each locus. For all 16 markers combined, a PE1 (combined non-exclusion probability, first parent) of 0.9986 and PE2 (combined non-exclusion probability, second parent) of >0.9999 was observed. Comparing the alleged father and mother, a PE value of >0.9999 was observed. Two of the markers, CliμD19 and PIGN12, were found to have relatively high Hardy-Weinberg equilibrium and F(null) values. Therefore these markers may be considered to be replaced by other STRs. Another point of discussion may be to add a gender identification marker to the recommended ISAG panel. Not only can this serve as an extra identification marker, but this can also confirm the sex of a sample, because it is challenging to determine the sex based on phenotypical characteristics, especially for chicks. In conclusion, the set of 16 STR markers can be used in routine parentage verification and the identification of individuals. © 2017 Stichting International Foundation for Animal Genetics.

A multiplex PCR system for 13 RM Y-STRs with separate amplification of two different repeat motif structures in DYF403S1a.

PubMed

Lee, Eun Young; Lee, Hwan Young; Kwon, So Yeun; Oh, Yu Na; Yang, Woo Ick; Shin, Kyoung-Jin

2017-01-01

In forensic science and human genetics, Y-chromosomal short tandem repeats (Y-STRs) have been used as very useful markers. Recently, more Y-STR markers have been analyzed to enhance the resolution power in haplotype analysis, and 13 rapidly mutating (RM) Y-STRs have been suggested as revolutionary tools that can widen Y-chromosomal application from paternal lineage differentiation to male individualization. We have constructed two multiplex PCR sets for the amplification of 13 RM Y-STRs, which yield small-sized amplicons (<400bp) and a more balanced PCR efficiency with minimum PCR cycling. In particular, with the developed multiplex PCR system, we could separate three copies of DYF403S1a into two copies of DYF403S1a and one of DYF403S1b1. This is because DYF403S1b1 possesses distinguishable sequences from DYF403S1a at both the front and rear flanking regions of the repeat motif; therefore, the locus could be separately amplified using sequence-specific primers. In addition, the other copy, defined as DYF403S1b by Ballantyne et al., was renamed DYF403S1b2 because of its similar flanking region sequence to DYF403S1b1. By redefining DYF403S1 with the developed multiplex system, all genotypes of four copies could be successfully typed and more diverse haplotypes were obtained. We analyzed haplotype distributions in 705 Korean males based on four different Y-STR subsets: Yfiler, PowerPlex Y23, Yfiler Plus, and RM Y-STRs. All haplotypes obtained from RM Y-STRs were the most diverse and showed strong discriminatory power in Korean population. Copyright © 2016 Elsevier Ireland Ltd. All rights reserved.

6-mercaptopurine influences TPMT gene transcription in a TPMT gene promoter variable number of tandem repeats-dependent manner.

PubMed

Kotur, Nikola; Stankovic, Biljana; Kassela, Katerina; Georgitsi, Marianthi; Vicha, Anna; Leontari, Iliana; Dokmanovic, Lidija; Janic, Dragana; Krstovski, Nada; Klaassen, Kristel; Radmilovic, Milena; Stojiljkovic, Maja; Nikcevic, Gordana; Simeonidis, Argiris; Sivolapenko, Gregory; Pavlovic, Sonja; Patrinos, George P; Zukic, Branka

2012-02-01

TPMT activity is characterized by a trimodal distribution, namely low, intermediate and high methylator. TPMT gene promoter contains a variable number of GC-rich tandem repeats (VNTRs), namely A, B and C, ranging from three to nine repeats in length in an A(n)B(m)C architecture. We have previously shown that the VNTR architecture in the TPMT gene promoter affects TPMT gene transcription. MATERIALS, METHODS & RESULTS: Here we demonstrate, using reporter assays, that 6-mercaptopurine (6-MP) treatment results in a VNTR architecture-dependent decrease of TPMT gene transcription, mediated by the binding of newly recruited protein complexes to the TPMT gene promoter, upon 6-MP treatment. We also show that acute lymphoblastic leukemia patients undergoing 6-MP treatment display a VNTR architecture-dependent response to 6-MP. These data suggest that the TPMT gene promoter VNTR architecture can be potentially used as a pharmacogenomic marker to predict toxicity due to 6-MP treatment in acute lymphoblastic leukemia patients.

Microsatellite diversity of isolates of the parasitic nematode Haemonchus contortus.

PubMed

Otsen, M; Plas, M E; Lenstra, J A; Roos, M H; Hoekstra, R

2000-09-01

The alarming development of anthelmintic resistance in important gastrointestinal nematode parasites of man and live-stock is caused by selection for specific genotypes. In order to provide genetic tools to study the nematode populations and the consequences of anthelmintic treatment, we isolated and sequenced 59 microsatellites of the sheep and goat parasite Haemonchus contortus. These microsatellites consist typically of 2-10 tandems CA/GT repeats that are interrupted by sequences of 1-10 bp. A predominant cause of the imperfect structure of the microsatellites appeared mutations of G/C bp in the tandem repeat. About 44% of the microsatellites were associated with the HcREP1 direct repeat, and it was demonstrated that a generic HcREP1 primer could be used to amplify HcREP1-associated microsatellites. Thirty microsatellites could be typed by polymerase chain reaction (PCR) of which 27 were polymorphic. A number of these markers were used to detect genetic contamination of an experimental inbred population. The microsatellites may also contribute to the genetic mapping of drug resistance genes.

Maximal oxygen uptake is associated with allele -202 A of insulin-like growth factor binding protein-3 (IGFBP3) promoter polymorphism and (CA)n tandem repeats of insulin-like growth factor IGF1 in Caucasians from Poland.

PubMed

Gronek, Piotr; Holdys, Joanna; Kryściak, Jakub; Wieliński, Dariusz; Słomski, Ryszard

2014-01-01

Physical fitness is a trait determined by multiple genes, and its genetic basis is modified by numerous environmental factors. The present study examines the effects of the (CA)n tandem repeats polymorphism in IGFI gene and SNP Alw21I restriction site -202 A>C polymorphism in IGF1BP3 on VO2max--a physiological index of aerobic capacity of high heritability. The study sample consisted of 239 (154 male and 85 female) students of the University School of Physical Education in Poznań and athletes practicing various sports, including members of the Polish national team. An association was found between -202 A/C polymorphism of IGFBP3 gene with VO2max in men. Higher VO2max values were attained by men with CC genotype, especially male athletes practicing endurance sports and sports featuring energy metabolism of aerobic/anaerobic character. A statistically significant influence of allele 188 and genotype 188/188 of tandem repeats (CA)n polymorphism of IGF1 gene on VO2max was found in women. Also, lower values of maximal oxygen uptake were noted in individuals with allele 186 or genotype 186/186, and higher VO2max values in athletes with allele 194.

Within-Host Evolution of Burkholderia pseudomallei in Four Cases of Acute Melioidosis

PubMed Central

Limmathurotsakul, Direk; Max, Tamara L.; Sarovich, Derek S.; Vogler, Amy J.; Dale, Julia L.; Ginther, Jennifer L.; Leadem, Benjamin; Colman, Rebecca E.; Foster, Jeffrey T.; Tuanyok, Apichai; Wagner, David M.; Peacock, Sharon J.; Pearson, Talima; Keim, Paul

2010-01-01

Little is currently known about bacterial pathogen evolution and adaptation within the host during acute infection. Previous studies of Burkholderia pseudomallei, the etiologic agent of melioidosis, have shown that this opportunistic pathogen mutates rapidly both in vitro and in vivo at tandemly repeated loci, making this organism a relevant model for studying short-term evolution. In the current study, B. pseudomallei isolates cultured from multiple body sites from four Thai patients with disseminated melioidosis were subjected to fine-scale genotyping using multilocus variable-number tandem repeat analysis (MLVA). In order to understand and model the in vivo variable-number tandem repeat (VNTR) mutational process, we characterized the patterns and rates of mutations in vitro through parallel serial passage experiments of B. pseudomallei. Despite the short period of infection, substantial divergence from the putative founder genotype was observed in all four melioidosis cases. This study presents a paradigm for examining bacterial evolution over the short timescale of an acute infection. Further studies are required to determine whether the mutational process leads to phenotypic alterations that impact upon bacterial fitness in vivo. Our findings have important implications for future sampling strategies, since colonies in a single clinical sample may be genetically heterogeneous, and organisms in a culture taken late in the infective process may have undergone considerable genetic change compared with the founder inoculum. PMID:20090837

Maternal T-Cell Engraftment Interferes With Human Leukocyte Antigen Typing in Severe Combined Immunodeficiency.

PubMed

Liu, Chang; Duffy, Brian; Bednarski, Jeffrey J; Calhoun, Cecelia; Lay, Lindsay; Rundblad, Barrett; Payton, Jacqueline E; Mohanakumar, Thalachallour

2016-02-01

To report the laboratory investigation of a case of severe combined immunodeficiency (SCID) with maternal T-cell engraftment, focusing on the interference of human leukocyte antigen (HLA) typing by blood chimerism. HLA typing was performed with three different methods, including sequence-specific primer (SSP), sequence-specific oligonucleotide, and Sanger sequencing on peripheral blood leukocytes and buccal cells, from a 3-month-old boy and peripheral blood leukocytes from his parents. Short tandem repeat (STR) testing was performed in parallel. HLA typing of the patient's peripheral blood leukocytes using the SSP method demonstrated three different alleles for each of the HLA-B and HLA-C loci, with both maternal alleles present at each locus. Typing results from the patient's buccal cells showed a normal pattern of inheritance for paternal and maternal haplotypes. STR enrichment testing of the patient's CD3+ T lymphocytes and CD15+ myeloid cells confirmed maternal T-cell engraftment, while the myeloid cell profile matched the patient's buccal cells. Maternal T-cell engraftment may interfere with HLA typing in patients with SCID. Selection of the appropriate typing methods and specimens is critical for accurate HLA typing and immunologic assessment before allogeneic hematopoietic stem cell transplantation. © American Society for Clinical Pathology, 2016. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.

Optimization of ultrahigh-speed multiplex PCR for forensic analysis.

PubMed

Gibson-Daw, Georgiana; Crenshaw, Karin; McCord, Bruce

2018-01-01

In this paper, we demonstrate the design and optimization of an ultrafast PCR amplification technique, used with a seven-locus multiplex that is compatible with conventional capillary electrophoresis systems as well as newer microfluidic chip devices. The procedure involves the use of a high-speed polymerase and a rapid cycling protocol to permit multiplex PCR amplification of forensic short tandem repeat loci in 6.5 min. We describe the selection and optimization of master mix reagents such as enzyme, buffer, MgCl 2 , and dNTPs, as well as primer ratios, total volume, and cycle conditions, in order to get the best profile in the shortest time possible. Sensitivity and reproducibility studies are also described. The amplification process utilizes a small high-speed thermocycler and compact laptop, making it portable and potentially useful for rapid, inexpensive on-site genotyping. The seven loci of the multiplex were taken from conventional STR genotyping kits and selected for their size and lack of overlap. Analysis was performed using conventional capillary electrophoresis and microfluidics with fluorescent detection. Overall, this technique provides a more rapid method for rapid sample screening of suspects and victims. Graphical abstract Rapid amplification of forensic DNA using high speed thermal cycling followed by capillary or microfluidic electrophoresis.

Variation of short tandem repeats within and between species belonging to the Canidae family.

PubMed

Fredholm, M; Winterø, A K

1995-01-01

Frequency distribution and allele size in 20 canine microsatellite loci were analyzed in 33 flat-coated retrievers, 32 dachshunds, 10 red foxes, and 10 Arctic foxes. Overall, the major difference between the two dog breeds was the relative allele frequencies rather than the size ranges of alleles at the individual locus. The average heterozygosity within the two dog breeds was not significantly different. Since the average heterozygosity at several polymorphic loci is a relative measure of heterogeneity within the population, analysis of heterozygosity within microsatellite loci is suggested as a measure for the diversity of populations. Eighty percent (16 of 20) of the canine microsatellite primer pairs amplified corresponding loci in the two fox species. This reflects a very high sequence conservation within the Canidae family relative to findings in, for instance, the Muridae family. This indicates that it will be possible to utilize the well-characterized fox karyotype instead of the dog karyotype as a step towards physical mapping of the dog genome. Analysis of exclusion power and probabilities of genetic identity between unrelated animals by use of the seven most informative loci demonstrated that it will be possible to assemble a panel of microsatellite loci that is effective for parentage analysis in all breeds.

Tuberculosis Caused by Mycobacterium orygis in Dairy Cattle and Captured Monkeys in Bangladesh: a New Scenario of Tuberculosis in South Asia.

PubMed

Rahim, Z; Thapa, J; Fukushima, Y; van der Zanden, A G M; Gordon, S V; Suzuki, Y; Nakajima, C

2017-12-01

Mycobacterium orygis, commonly known as the oryx bacillus and a newly proposed Mycobacterium tuberculosis complex subspecies, was isolated from 18 cattle in a dairy farm and two captured rhesus monkeys in a zoo in Bangladesh. All the infected animals had tuberculosis lesions in their lungs, suggesting transmission and infection with M. orygis by an airborne route. The 20 isolates were analysed using a range of conventional and molecular typing methods, and RD-deletion typing and sequencing of selected genes confirmed the isolates as M. orygis. Multiple-locus variable-number tandem repeat analysis (MLVA) allowed the isolates to be divided into three clusters based on the relatedness of their MLVA profiles. The two monkey isolates shared the same MLVA pattern with 15 of the cattle isolates, whereas the remaining three cattle isolates had different patterns, even though the latter animals had been kept in the same dairy farm. The diversity observed among isolates may suggest the bacteria have been established in this area for a long period. This study along with other recent findings that report the detection of M. orygis from animals as well as humans originating from South Asia potentially indicate endemic distribution of M. orygis in South Asia. © 2016 Blackwell Verlag GmbH.

Repetitive sequences and epigenetic modification: inseparable partners play important roles in the evolution of plant sex chromosomes.

PubMed

Li, Shu-Fen; Zhang, Guo-Jun; Yuan, Jin-Hong; Deng, Chuan-Liang; Gao, Wu-Jun

2016-05-01

The present review discusses the roles of repetitive sequences played in plant sex chromosome evolution, and highlights epigenetic modification as potential mechanism of repetitive sequences involved in sex chromosome evolution. Sex determination in plants is mostly based on sex chromosomes. Classic theory proposes that sex chromosomes evolve from a specific pair of autosomes with emergence of a sex-determining gene(s). Subsequently, the newly formed sex chromosomes stop recombination in a small region around the sex-determining locus, and over time, the non-recombining region expands to almost all parts of the sex chromosomes. Accumulation of repetitive sequences, mostly transposable elements and tandem repeats, is a conspicuous feature of the non-recombining region of the Y chromosome, even in primitive one. Repetitive sequences may play multiple roles in sex chromosome evolution, such as triggering heterochromatization and causing recombination suppression, leading to structural and morphological differentiation of sex chromosomes, and promoting Y chromosome degeneration and X chromosome dosage compensation. In this article, we review the current status of this field, and based on preliminary evidence, we posit that repetitive sequences are involved in sex chromosome evolution probably via epigenetic modification, such as DNA and histone methylation, with small interfering RNAs as the mediator.

Molecular characterization by MLVA of Coxiella burnetii strains infecting dairy cows and goats of north-eastern Italy.

PubMed

Ceglie, Letizia; Guerrini, Eulalia; Rampazzo, Erika; Barberio, Antonio; Tilburg, Jeroen J H C; Hagen, Ferry; Lucchese, Laura; Zuliani, Federica; Marangon, Stefano; Natale, Alda

2015-01-01

Q fever is a worldwide zoonotic disease caused by Coxiella burnetii (C. burnetii), an obligate intracellular bacterium. In ruminants, shedding into the environment mainly occurs during parturition or abortion, but the bacterium is shed also in milk, vaginal mucus, stools and urine. In Italy few surveys have been conducted and reported seroprevalence values ranged between 10% and 60%, even if few human cases have been described. Genotyping of bacteria is crucial for enhancing diagnostic methods and for epidemiological surveillance. The objective of this study was to investigate genotypic differences of C. burnetii genotypes directly in 34 samples, collected during a 3-years survey among 11 dairy cattle and 11 goat farms in the north-eastern part of Italy using a 6-locus multiple loci variable number of tandem repeat analysis (MLVA) method. The samples analysed included 13 bulk tank milk (BTM), 6 individual milk, 11 vaginal swabs and 4 foetal spleens. MLVA-type 2 was determined as the most prevalent in cattle in this study. C. burnetii strains circulating in the studied cattle population are very similar to genotypes previously described, while genotypes from goats showed an important variability. Further investigation are needed to understand the reason of this pattern. Copyright © 2015 Institut Pasteur. Published by Elsevier Masson SAS. All rights reserved.

Salmonella Typhimurium DT193 and DT99 are present in great and blue tits in Flanders, Belgium

PubMed Central

Verbrugghe, E.; Dekeukeleire, D.; De Beelde, R.; Rouffaer, L. O.; Haesendonck, R.; Strubbe, D.; Mattheus, W.; Bertrand, S.; Pasmans, F.; Bonte, D.; Verheyen, K.; Lens, L.; Martel, A.

2017-01-01

Endemic infections with the common avian pathogen Salmonella enterica subspecies enterica serovar Typhimurium (Salmonella Typhimurium) may incur a significant cost on the host population. In this study, we determined the potential of endemic Salmonella infections to reduce the reproductive success of blue (Cyanistes caeruleus) and great (Parus major) tits by correlating eggshell infection with reproductive parameters. The fifth egg of each clutch was collected from nest boxes in 19 deciduous forest fragments. Out of the 101 sampled eggs, 7 Salmonella Typhimurium isolates were recovered. The low bacterial prevalence was reflected by a similarly low serological prevalence in the fledglings. In this study with a relatively small sample size, presence of Salmonella did not affect reproductive parameters (egg volume, clutch size, number of nestlings and number of fledglings), nor the health status of the fledglings. However, in order to clarify the impact on health and reproduction a larger number of samples have to be analyzed. Phage typing showed that the isolates belonged to the definitive phage types (DT) 193 and 99, and multi-locus variable number tandem repeat analysis (MLVA) demonstrated a high similarity among the tit isolates, but distinction to human isolates. These findings suggest the presence of passerine-adapted Salmonella strains in free-ranging tit populations with host pathogen co-existence. PMID:29112955

New paradigms for Salmonella source attribution based on microbial subtyping.

PubMed

Mughini-Gras, Lapo; Franz, Eelco; van Pelt, Wilfrid

2018-05-01

Microbial subtyping is the most common approach for Salmonella source attribution. Typically, attributions are computed using frequency-matching models like the Dutch and Danish models based on phenotyping data (serotyping, phage-typing, and antimicrobial resistance profiling). Herewith, we critically review three major paradigms facing Salmonella source attribution today: (i) the use of genotyping data, particularly Multi-Locus Variable Number of Tandem Repeats Analysis (MLVA), which is replacing traditional Salmonella phenotyping beyond serotyping; (ii) the integration of case-control data into source attribution to improve risk factor identification/characterization; (iii) the investigation of non-food sources, as attributions tend to focus on foods of animal origin only. Population genetics models or simplified MLVA schemes may provide feasible options for source attribution, although there is a strong need to explore novel modelling options as we move towards whole-genome sequencing as the standard. Classical case-control studies are enhanced by incorporating source attribution results, as individuals acquiring salmonellosis from different sources have different associated risk factors. Thus, the more such analyses are performed the better Salmonella epidemiology will be understood. Reparametrizing current models allows for inclusion of sources like reptiles, the study of which improves our understanding of Salmonella epidemiology beyond food to tackle the pathogen in a more holistic way. Copyright © 2017 Elsevier Ltd. All rights reserved.

Molecular Epidemiology of Leptospira Serogroup Pomona Infections Among Wild and Domestic Animals in Spain.

PubMed

Arent, Z J; Gilmore, C; San-Miguel Ayanz, J M; Neyra, L Quevedo; García-Peña, F J

2017-03-01

Strains of Leptospira serogroup Pomona are known to cause widespread animal infections in many parts of the world. Forty-three isolates retrieved from domestic animals and wild small mammals suggest that serogroup Pomona is epidemiologically relevant in Spain. This is supported by the high prevalence of serovar Pomona antibodies in livestock and wild animals. In this study, the strains were serologically and genetically characterized in an attempt to elucidate their epidemiology. Serological typing was based on the microscopic agglutination test but molecular typing involved species-specific polymerase chain reaction, restriction endonuclease analysis, and multiple-locus variable-number tandem repeat analysis. The study revealed that the infections are caused by two serovars, namely Pomona and Mozdok. Serovar Pomona was derived only from farm animals and may be adapted to pigs, which are recognized as the maintenance host. The results demonstrated that serovar Pomona is genetically heterogeneous and three different types were recognized. This heterogeneity was correlated with different geographical distributions of the isolates. All strains derived from small wild mammals were identified as serovar Mozdok. Some isolates of this serovar retrieved from cattle confirm that this serovar may also be the cause of infections in food-producing animals for which these wild species may be source of infection.

Evidence of Brucella strain ST27 in bottlenose dolphin (Tursiops truncatus) in Europe.

PubMed

Cvetnić, Željko; Duvnjak, Sanja; Đuras, Martina; Gomerčić, Tomislav; Reil, Irena; Zdelar-Tuk, Maja; Špičić, Silvio

2016-11-30

Marine mammal brucellosis has been known for more than 20 years, but recent work suggests it is more widespread than originally thought. Brucella (B.) pinnipedialis has been isolated from pinnipeds, while B. ceti strains have been associated with cetaceans. Here we report a Brucella strain isolated from multiple lymph nodes of one bottlenose dolphin (Tursiops truncatus) during routine examination of dolphin carcasses found in the Croatian part of the northern Adriatic Sea during the summer of 2015. Classical bacteriological biotyping, PCR-based techniques (single, multiplex, PCR-RFLP) and 16S rRNA DNA sequencing were used to identify Brucella spp. Multiple-locus variable number tandem repeat analysis of 16 loci and multilocus sequence typing of 9 loci were used for genotyping and species determination. The combination of bacteriological, molecular and genotyping techniques identified our strain as ST27, previously identified as a human pathogen. This report provides, to our knowledge, the first evidence of ST27 in the Adriatic Sea in particular and in European waters in general. The zoonotic nature of the strain and its presence in the Adriatic, which is inhabited by bottlenose dolphins, suggest that the strain may pose a significant threat to human health. Copyright © 2016 Elsevier B.V. All rights reserved.

Cell Line Data Base: structure and recent improvements towards molecular authentication of human cell lines

PubMed Central

Romano, Paolo; Manniello, Assunta; Aresu, Ottavia; Armento, Massimiliano; Cesaro, Michela; Parodi, Barbara

2009-01-01

The Cell Line Data Base (CLDB) is a well-known reference information source on human and animal cell lines including information on more than 6000 cell lines. Main biological features are coded according to controlled vocabularies derived from international lists and taxonomies. HyperCLDB (http://bioinformatics.istge.it/hypercldb/) is a hypertext version of CLDB that improves data accessibility by also allowing information retrieval through web spiders. Access to HyperCLDB is provided through indexes of biological characteristics and navigation in the hypertext is granted by many internal links. HyperCLDB also includes links to external resources. Recently, an interest was raised for a reference nomenclature for cell lines and CLDB was seen as an authoritative system. Furthermore, to overcome the cell line misidentification problem, molecular authentication methods, such as fingerprinting, single-locus short tandem repeat (STR) profile and single nucleotide polymorphisms validation, were proposed. Since this data is distributed, a reference portal on authentication of human cell lines is needed. We present here the architecture and contents of CLDB, its recent enhancements and perspectives. We also present a new related database, the Cell Line Integrated Molecular Authentication (CLIMA) database (http://bioinformatics.istge.it/clima/), that allows to link authentication data to actual cell lines. PMID:18927105

Cell Line Data Base: structure and recent improvements towards molecular authentication of human cell lines.

PubMed

Romano, Paolo; Manniello, Assunta; Aresu, Ottavia; Armento, Massimiliano; Cesaro, Michela; Parodi, Barbara

2009-01-01

The Cell Line Data Base (CLDB) is a well-known reference information source on human and animal cell lines including information on more than 6000 cell lines. Main biological features are coded according to controlled vocabularies derived from international lists and taxonomies. HyperCLDB (http://bioinformatics.istge.it/hypercldb/) is a hypertext version of CLDB that improves data accessibility by also allowing information retrieval through web spiders. Access to HyperCLDB is provided through indexes of biological characteristics and navigation in the hypertext is granted by many internal links. HyperCLDB also includes links to external resources. Recently, an interest was raised for a reference nomenclature for cell lines and CLDB was seen as an authoritative system. Furthermore, to overcome the cell line misidentification problem, molecular authentication methods, such as fingerprinting, single-locus short tandem repeat (STR) profile and single nucleotide polymorphisms validation, were proposed. Since this data is distributed, a reference portal on authentication of human cell lines is needed. We present here the architecture and contents of CLDB, its recent enhancements and perspectives. We also present a new related database, the Cell Line Integrated Molecular Authentication (CLIMA) database (http://bioinformatics.istge.it/clima/), that allows to link authentication data to actual cell lines.

Two outbreaks of diarrhoea in nurseries in Norway after farm visits, April to May 2009.

PubMed

Møller-Stray, J; Eriksen, H M; Bruheim, T; Kapperud, G; Lindstedt, B A; Skeie, Å; Sunde, M; Urdahl, A M; Øygard, B; Vold, L

2012-11-22

During a 2009 nationwide outbreak of sorbitolfermenting Escherichia coli O157 in Norway, the Norwegian Institute of Public Health was notified of diarrhoea outbreaks in two nurseries. A link to the nationwide outbreak was suspected and investigated, including retrospective cohort studies. Both nurseries had recently visited farms. Faecal specimens were obtained from symptomatic children as well as from the farm animals and tested for Campylobacter, Salmonella, Yersinia, Shigella and pathogenic E. coli, and isolates were further characterised. Nursery A had 12 symptomatic children, and we found the same strain of C. jejuni in faeces from children and lambs. Nursery B had nine symptomatic children, including one child with bloody diarrhoea carrying enterohaemorrhagic E. coli (EHEC) O26. EHEC O26 with a similar multiple-locus variable number tandem repeat analysis (MLVA)-profile was found in sheep. Five children had enteropathogenic E. coli (EPEC) O76. Animals were not tested for EPEC O76. We found no significant association between illness and risk factors for either nursery. The isolated pathogens differed from the one involved in the nationwide outbreak. In each nursery outbreak, the pathogens isolated from children matched those found in farm animals, implicating animal faeces as the source. Hygiene messages are important to prevent similar outbreaks.

Recurrence of tuberculosis in a low-incidence setting without directly observed treatment: Victoria, Australia, 2002-2014.

PubMed

Dale, K D; Globan, M; Tay, E L; Trauer, J M; Trevan, P G; Denholm, J T

2017-05-01

Victoria, Australia, is an industrialised setting with low tuberculosis (TB) incidence and universal health care. Individually tailored adherence support for self-administered daily anti-tuberculosis treatment is provided. Directly observed treatment (DOT) is very rarely used. To review the rate of recurrent TB in Victoria between 2002 and 2014. This was a retrospective cohort study. All recurrent episodes of TB were reviewed and 24-locus MIRU-VNTR (mycobacterial interspersed repetitive units-variable number of tandem repeats) molecular typing was used where possible to determine the likelihood of relapse or reinfection. Of 4766 notifications, 32 (0.7%) were recurrent episodes. Of 20 episodes that occurred in patients who had previously completed treatment, 11 were culture-positive (0.4% of 3012 culture-positive episodes): 9 were likely relapses (distinguishable at no more than one of 24 loci) and two were likely reinfections, giving a TB relapse rate among culture-positive episodes of 52.5/100 000 person-years (mean time to study end per patient of 5.7 years). The median time until relapse was 18 months (interquartile range 12-30). The low rate of relapse in our setting demonstrates that individually tailored adherence support for self-administered anti-tuberculosis treatment can achieve excellent treatment outcomes.

Methods for genotyping verotoxin-producing Escherichia coli.

PubMed

Karama, M; Gyles, C L

2010-12-01

Verotoxin-producing Escherichia coli (VTEC) is annually incriminated in more than 100,000 cases of enteric foodborne human disease and in losses amounting to $US 2.5 billion every year. A number of genotyping methods have been developed to track VTEC infections and determine diversity and evolutionary relationships among these microorganisms. These methods have facilitated monitoring and surveillance of foodborne VTEC outbreaks and early identification of outbreaks or clusters of outbreaks. Pulsed-field gel electrophoresis (PFGE) has been used extensively to track and differentiate VTEC because of its high discriminatory power, reproducibility and ease of standardization. Multiple-locus variable-number tandem-repeats analysis (MLVA) and microarrays are the latest genotyping methods that have been applied to discriminate VTEC. MLVA, a simpler and less expensive method, is proving to have a discriminatory power comparable to that of PFGE. Microarrays are successfully being applied to differentiate VTEC and make inferences on genome diversification. Novel methods that are being evaluated for subtyping VTEC include the detection of single nucleotide polymorphisms and optical mapping. This review discusses the principles, applications, advantages and disadvantages of genotyping methods that have been used to differentiate VTEC strains. These methods have been mainly used to differentiate strains of O157:H7 VTEC and to a lesser extent non-O157 VTEC. © 2009 Blackwell Verlag GmbH.

Subtyping of STEC by MLVA in Argentina

PubMed Central

Bustamante, Ana V.; Sanso, Andrea M.; Parma, Alberto E.; Lucchesi, Paula M. A.

2012-01-01

Shiga toxin-producing Escherichia coli (STEC) causes serious human illness such as hemolytic uremic syndrome (HUS). Argentina has the world’s highest rate of this syndrome, which is the leading cause of acute renal failure among children. E. coli O157:H7 is the most common cause of HUS, but a substantial and growing proportion of this illness is caused by infection due to non-O157 strains. Multiple-locus variable-number tandem repeat analysis (MLVA) has become an established technique to subtype STEC. This review will address the use of routine STEC subtyping by MLVA in order to type this group of isolates and to get insight into the genetic diversity of native STEC. With regard to these objectives we modified and adapted two MLVA protocols, one exclusive for O157 and the other, a generic E. coli assay. A total of 202 STEC isolates, from different sources and corresponding to 20 serotypes, have been MLVA genotyped in our laboratory. In our experience, MLVA constitutes a very sensitive tool and enables us to perform an efficient STEC subtyping. The diversity found in many serotypes may be useful for future epidemiological studies of STEC clonality, applied to O157 as well as to non-O157 isolates. PMID:22919698

Genotyping of Pseudomonas aeruginosa isolates from lung transplant recipients and aquatic environment-detected in-hospital transmission.

PubMed

Johansson, Ewa; Welinder-Olsson, Christina; Gilljam, Marita

2014-02-01

Lung infection with Pseudomonas aeruginosa is common in lung transplant recipients and may lead to severe complications. Bacteriological surveillance aims to detect transmission of microbes between hospital environment and patients. We sought to determine whether genotyping of P. aeruginosa isolates could improve identifications of pathways of infection. From 2004 to 2009, we performed genotyping with multiple-locus variable number of tandem repeats analysis (MLVA) and pulsed-field gel electrophoresis (PFGE) of P. aeruginosa isolates cultured from lung transplant recipients at Sahlgrenska University Hospital, Gothenburg. During a small outbreak in 2008, cultivation and genotyping of isolates from sink and drains samples from the hospital ward were performed. Pseudomona aeruginosa from 11/18 patients were genotyped to unique strains. The remaining seven patients were carriers of a P. aeruginosa strain of cluster A genotype. Pseudomona aeruginosa was isolated in 4/8 water samples, typed by MLVA also as cluster A genotype and confirmed by PFGE to be similar or identical to the isolates from four transplanted patients. In conclusion, genotyping of isolates revealed a clonal relationship between patient and water isolates, indicating in-hospital transmission of P. aeruginosa. We suggest genotyping with MLVA for rapid routine surveillance, with the PFGE method used for extended, confirmatory analyses. © 2013 APMIS. Published by John Wiley & Sons Ltd.

Selkirk Rex: Morphological and Genetic Characterization of a New Cat Breed

PubMed Central

2012-01-01

Rexoid, curly hair mutations have been selected to develop new domestic cat breeds. The Selkirk Rex is the most recently established curly-coated cat breed originating from a spontaneous mutation that was discovered in the United States in 1987. Unlike the earlier and well-established Cornish and Devon Rex breeds with curly-coat mutations, the Selkirk Rex mutation is suggested as autosomal dominant and has a different curl phenotype. This study provides a genetic analysis of the Selkirk Rex breed. An informal segregation analysis of genetically proven matings supported an autosomal, incomplete dominant expression of the curly trait in the Selkirk Rex. Homozygous curl cats can be distinguished from heterozygous cats by head and body type, as well as the presentation of the hair curl. Bayesian clustering of short tandem repeat (STR) genotypes from 31 cats that represent the future breeding stock supported the close relationship of the Selkirk Rex to the British Shorthair, Scottish Fold, Persian, and Exotic Shorthair, suggesting the Selkirk as part of the Persian breed family. The high heterozygosity of 0.630 and the low mean inbreeding coefficient of 0.057 suggest that Selkirk Rex has a diverse genetic foundation. A new locus for Selkirk autosomal dominant Rex, SADRE, is suggested for the curly trait. PMID:22837475

Efficient production of artificially designed gelatins with a Bacillus brevis system.

PubMed

Kajino, T; Takahashi, H; Hirai, M; Yamada, Y

2000-01-01

Artificially designed gelatins comprising tandemly repeated 30-amino-acid peptide units derived from human alphaI collagen were successfully produced with a Bacillus brevis system. The DNA encoding the peptide unit was synthesized by taking into consideration the codon usage of the host cells, but no clones having a tandemly repeated gene were obtained through the above-mentioned strategy. Minirepeat genes could be selected in vivo from a mixture of every possible sequence encoding an artificial gelatin by randomly ligating the mixed sequence unit and transforming it into Escherichia coli. Larger repeat genes constructed by connecting minirepeat genes obtained by in vivo selection were also stable in the expression host cells. Gelatins derived from the eight-unit and six-unit repeat genes were extracellularly produced at the level of 0.5 g/liter and easily purified by ammonium sulfate fractionation and anion-exchange chromatography. The purified artificial gelatins had the predicted N-terminal sequences and amino acid compositions and a solgel property similar to that of the native gelatin. These results suggest that the selection of a repeat unit sequence stable in an expression host is a shortcut for the efficient production of repetitive proteins and that it can conveniently be achieved by the in vivo selection method. This study revealed the possible industrial application of artificially designed repetitive proteins.

Evolution of a behavior-linked microsatellite-containing element in the 5' flanking region of the primate AVPR1A gene

PubMed Central

2008-01-01

Background The arginine vasopressin V1a receptor (V1aR) modulates social cognition and behavior in a wide variety of species. Variation in a repetitive microsatellite element in the 5' flanking region of the V1aR gene (AVPR1A) in rodents has been associated with variation in brain V1aR expression and in social behavior. In humans, the 5' flanking region of AVPR1A contains a tandem duplication of two ~350 bp, microsatellite-containing elements located approximately 3.5 kb upstream of the transcription start site. The first block, referred to as DupA, contains a polymorphic (GT)25 microsatellite; the second block, DupB, has a complex (CT)4-(TT)-(CT)8-(GT)24 polymorphic motif, known as RS3. Polymorphisms in RS3 have been associated with variation in sociobehavioral traits in humans, including autism spectrum disorders. Thus, evolution of these regions may have contributed to variation in social behavior in primates. We examined the structure of these regions in six ape, six monkey, and one prosimian species. Results Both tandem repeat blocks are present upstream of the AVPR1A coding region in five of the ape species we investigated, while monkeys have only one copy of this region. As in humans, the microsatellites within DupA and DupB are polymorphic in many primate species. Furthermore, both single (lacking DupB) and duplicated alleles (containing both DupA and DupB) are present in chimpanzee (Pan troglodytes) populations with allele frequencies of 0.795 and 0.205 for the single and duplicated alleles, respectively, based on the analysis of 47 wild-caught individuals. Finally, a phylogenetic reconstruction suggests two alternate evolutionary histories for this locus. Conclusion There is no obvious relationship between the presence of the RS3 duplication and social organization in primates. However, polymorphisms identified in some species may be useful in future genetic association studies. In particular, the presence of both single and duplicated alleles in chimpanzees provides a unique opportunity to assess the functional role of this duplication in contributing to variation in social behavior in primates. While our initial studies show no signs of directional selection on this locus in chimps, pharmacological and genetic association studies support a potential role for this region in influencing V1aR expression and social behavior. PMID:18573213

Evolution of a behavior-linked microsatellite-containing element in the 5' flanking region of the primate AVPR1A gene.

PubMed

Donaldson, Zoe R; Kondrashov, Fyodor A; Putnam, Andrea; Bai, Yaohui; Stoinski, Tara L; Hammock, Elizabeth A D; Young, Larry J

2008-06-23

The arginine vasopressin V1a receptor (V1aR) modulates social cognition and behavior in a wide variety of species. Variation in a repetitive microsatellite element in the 5' flanking region of the V1aR gene (AVPR1A) in rodents has been associated with variation in brain V1aR expression and in social behavior. In humans, the 5' flanking region of AVPR1A contains a tandem duplication of two approximately 350 bp, microsatellite-containing elements located approximately 3.5 kb upstream of the transcription start site. The first block, referred to as DupA, contains a polymorphic (GT)25 microsatellite; the second block, DupB, has a complex (CT)4-(TT)-(CT)8-(GT)24 polymorphic motif, known as RS3. Polymorphisms in RS3 have been associated with variation in sociobehavioral traits in humans, including autism spectrum disorders. Thus, evolution of these regions may have contributed to variation in social behavior in primates. We examined the structure of these regions in six ape, six monkey, and one prosimian species. Both tandem repeat blocks are present upstream of the AVPR1A coding region in five of the ape species we investigated, while monkeys have only one copy of this region. As in humans, the microsatellites within DupA and DupB are polymorphic in many primate species. Furthermore, both single (lacking DupB) and duplicated alleles (containing both DupA and DupB) are present in chimpanzee (Pan troglodytes) populations with allele frequencies of 0.795 and 0.205 for the single and duplicated alleles, respectively, based on the analysis of 47 wild-caught individuals. Finally, a phylogenetic reconstruction suggests two alternate evolutionary histories for this locus. There is no obvious relationship between the presence of the RS3 duplication and social organization in primates. However, polymorphisms identified in some species may be useful in future genetic association studies. In particular, the presence of both single and duplicated alleles in chimpanzees provides a unique opportunity to assess the functional role of this duplication in contributing to variation in social behavior in primates. While our initial studies show no signs of directional selection on this locus in chimps, pharmacological and genetic association studies support a potential role for this region in influencing V1aR expression and social behavior.

Novel variable number of tandem repeats of gibbon MAOA gene and its evolutionary significance.

PubMed

Choi, Yuri; Jung, Yi-Deun; Ayarpadikannan, Selvam; Koga, Akihiko; Imai, Hiroo; Hirai, Hirohisa; Roos, Christian; Kim, Heui-Soo

2014-08-01

Variable number of tandem repeats (VNTRs) are scattered throughout the primate genome, and genetic variation of these VNTRs have been accumulated during primate radiation. Here, we analyzed VNTRs upstream of the monoamine oxidase A (MAOA) gene in 11 different gibbon species. An abundance of truncated VNTR sequences and copy number differences were observed compared to those of human VNTR sequences. To better understand the biological role of these VNTRs, a luciferase activity assay was conducted and results indicated that selected VNTR sequences of the MAOA gene from human and three different gibbon species (Hylobates klossii, Hylobates lar, and Nomascus concolor) showed silencing ability. Together, these data could be useful for understanding the evolutionary history and functional significance of MAOA VNTR sequences in gibbon species.

[Reticulate evolution of parthenogenetic species of the Lacertidae rock lizards: inheritance of CLsat tandem repeats and anonymous RAPD markers].

PubMed

Chobanu, D; Rudykh, I A; Riabinina, N L; Grechko, V V; Kramerov, D A; Darevskiĭ, I S

2002-01-01

The genetic relatedness of several bisexual and of four unisexual "Lacerta saxicola complex" lizards was studied, using monomer sequences of the complex-specific CLsat tandem repeats and anonymous RAPD markers. Genomes of parthenospecies were shown to include different satellite monomers. The structure of each such monomer is specific for a certain pair of bisexual species. This fact might be interpreted in favor of co-dominant inheritance of these markers in bisexual species hybridogenesis. This idea is supported by the results obtained with RAPD markers; i.e., unisexual species genomes include only the loci characteristic of certain bisexual species. At the same time, in neither case parthenospecies possess specific, autoapomorphic loci that were not present in this or that bisexual species.

Length and sequence variability in mitochondrial control region of the milkfish, Chanos chanos.

PubMed

Ravago, Rachel G; Monje, Virginia D; Juinio-Meñez, Marie Antonette

2002-01-01

Extensive length variability was observed in the mitochondrial control region of the milkfish, Chanos chanos. The nucleotide sequence of the control region and flanking regions was determined. Length variability and heteroplasmy was due to the presence of varying numbers of a 41-bp tandemly repeated sequence and a 48-bp insertion/deletion (indel). The structure and organization of the milkfish control region is similar to that of other teleost fish and vertebrates. However, extensive variation in the copy number of tandem repeats (4-20 copies) and the presence of a relatively large (48-bp) indel, are apparently uncommon in teleost fish control region sequences reported to date. High sequence variability of control region peripheral domains indicates the potential utility of selected regions as markers for population-level studies.

Complex structure of knob DNA on maize chromosome 9. Retrotransposon invasion into heterochromatin.

PubMed Central

Ananiev, E V; Phillips, R L; Rines, H W

1998-01-01

The recovery of maize (Zea mays L.) chromosome addition lines of oat (Avena sativa L.) from oat x maize crosses enables us to analyze the structure and composition of specific regions, such as knobs, of individual maize chromosomes. A DNA hybridization blot panel of eight individual maize chromosome addition lines revealed that 180-bp repeats found in knobs are present in each of these maize chromosomes, but the copy number varies from approximately 100 to 25, 000. Cosmid clones with knob DNA segments were isolated from a genomic library of an oat-maize chromosome 9 addition line with the help of the 180-bp knob-associated repeated DNA sequence used as a probe. Cloned knob DNA segments revealed a complex organization in which blocks of tandemly arranged 180-bp repeating units are interrupted by insertions of other repeated DNA sequences, mostly represented by individual full size copies of retrotransposable elements. There is an obvious preference for the integration of retrotransposable elements into certain sites (hot spots) of the 180-bp repeat. Sequence microheterogeneity including point mutations and duplications was found in copies of 180-bp repeats. The 180-bp repeats within an array all had the same polarity. Restriction maps constructed for 23 cloned knob DNA fragments revealed the positions of polymorphic sites and sites of integration of insertion elements. Discovery of the interspersion of retrotransposable elements among blocks of tandem repeats in maize and some other organisms suggests that this pattern may be basic to heterochromatin organization for eukaryotes. PMID:9691055

Salmonella enterica Serotype Enteritidis in French Polynesia, South Pacific, 2008–2013

PubMed Central

Maillard, Fiona; Mallet, Henri-Pierre; Daudens, Elise; Levy, Marc; Roy, Valérie; Branaa, Philippe; Bertrand, Sophie; Fabre, Laetitia; Weill, François-Xavier

2015-01-01

Outbreaks of Salmonella enterica serotype Enteritidis infections associated with eggs occurred in French Polynesia during 2008–2013. Molecular analysis of isolates by using clustered regularly interspaced short palindromic repeat polymorphisms and multilocus variable-number tandem-repeat analysis was performed. This subtyping made defining the epidemic strain, finding the source, and decontaminating affected poultry flocks possible. PMID:25988406

Is mammalian chromosomal evolution driven by regions of genome fragility?

PubMed Central

Ruiz-Herrera, Aurora; Castresana, Jose; Robinson, Terence J

2006-01-01

Background A fundamental question in comparative genomics concerns the identification of mechanisms that underpin chromosomal change. In an attempt to shed light on the dynamics of mammalian genome evolution, we analyzed the distribution of syntenic blocks, evolutionary breakpoint regions, and evolutionary breakpoints taken from public databases available for seven eutherian species (mouse, rat, cattle, dog, pig, cat, and horse) and the chicken, and examined these for correspondence with human fragile sites and tandem repeats. Results Our results confirm previous investigations that showed the presence of chromosomal regions in the human genome that have been repeatedly used as illustrated by a high breakpoint accumulation in certain chromosomes and chromosomal bands. We show, however, that there is a striking correspondence between fragile site location, the positions of evolutionary breakpoints, and the distribution of tandem repeats throughout the human genome, which similarly reflect a non-uniform pattern of occurrence. Conclusion These observations provide further evidence that certain chromosomal regions in the human genome have been repeatedly used in the evolutionary process. As a consequence, the genome is a composite of fragile regions prone to reorganization that have been conserved in different lineages, and genomic tracts that do not exhibit the same levels of evolutionary plasticity. PMID:17156441

Development of Pineapple Microsatellite Markers and Germplasm Genetic Diversity Analysis

PubMed Central

Tong, Helin; Chen, You; Wang, Jingyi; Chen, Yeyuan; Sun, Guangming; He, Junhu; Wu, Yaoting

2013-01-01

Two methods were used to develop pineapple microsatellite markers. Genomic library-based SSR development: using selectively amplified microsatellite assay, 86 sequences were generated from pineapple genomic library. 91 (96.8%) of the 94 Simple Sequence Repeat (SSR) loci were dinucleotide repeats (39 AC/GT repeats and 52 GA/TC repeats, accounting for 42.9% and 57.1%, resp.), and the other three were mononucleotide repeats. Thirty-six pairs of SSR primers were designed; 24 of them generated clear bands of expected sizes, and 13 of them showed polymorphism. EST-based SSR development: 5659 pineapple EST sequences obtained from NCBI were analyzed; among 1397 nonredundant EST sequences, 843 were found containing 1110 SSR loci (217 of them contained more than one SSR locus). Frequency of SSRs in pineapple EST sequences is 1SSR/3.73 kb, and 44 types were found. Mononucleotide, dinucleotide, and trinucleotide repeats dominate, accounting for 95.6% in total. AG/CT and AGC/GCT were the dominant type of dinucleotide and trinucleotide repeats, accounting for 83.5% and 24.1%, respectively. Thirty pairs of primers were designed for each of randomly selected 30 sequences; 26 of them generated clear and reproducible bands, and 22 of them showed polymorphism. Eighteen pairs of primers obtained by the one or the other of the two methods above that showed polymorphism were selected to carry out germplasm genetic diversity analysis for 48 breeds of pineapple; similarity coefficients of these breeds were between 0.59 and 1.00, and they can be divided into four groups accordingly. Amplification products of five SSR markers were extracted and sequenced, corresponding repeat loci were found and locus mutations are mainly in copy number of repeats and base mutations in the flanking region. PMID:24024187

New system for multilocus variable-number tandem-repeat analysis of the enterohemorrhagic Escherichia coli strains belonging to three major serogroups: O157, O26, and O111.

PubMed

Izumiya, Hidemasa; Pei, Yingxin; Terajima, Jun; Ohnishi, Makoto; Hayashi, Tetsuya; Iyoda, Sunao; Watanabe, Haruo

2010-10-01

Enterohemorrhagic Escherichia coli (EHEC), a food- and waterborne pathogen, causes diarrhea, hemorrhagic colitis, and life-threatening HUS. MLVA is a newly developed and widely accepted genotyping tool. An MLVA system for EHEC O157 involving nine genomic loci has already been established. However, the present study revealed that the above-mentioned MLVA system cannot analyze EHEC O26 and O111 isolates-the second and third most dominant EHEC serogroups in Japan, respectively. Therefore, with several modifications to the O157 system and the use of nine additional loci, we developed an expanded MLVA system applicable to EHEC O26, O111, and O157. Our MLVA system had a relatively high resolution power for each of the three serogroups: Simpson's index of diversity was 0.991 (95% CI = 0.989-0.993), 0.988 (95% CI, 0.986-0.990), and 0.986 (95% CI, 0.979-0.993) for O26, O111, and O157, respectively. This system also detected outbreak-related isolates; the isolates collected during each of the 12 O26 and O111 outbreaks formed unique clusters, and most of the repeat copy numbers among the isolates collected during the same outbreak exhibited no or single-locus variations. These results were comparable to those of cluster analyses based on PFGE profiles. Therefore, our system can complement PFGE analysis-the current golden method. Because EHEC strains of three major serogroups can be rapidly analyzed on a single platform with our expanded MLVA system, this system could be widely used in molecular epidemiological studies of EHEC infections. © 2010 The Societies and Blackwell Publishing Asia Pty Ltd.

Dopamine Transporter Genetic Variants and Pesticides in Parkinson’s Disease

PubMed Central

Ritz, Beate R.; Manthripragada, Angelika D.; Costello, Sadie; Lincoln, Sarah J.; Farrer, Matthew J.; Cockburn, Myles; Bronstein, Jeff

2009-01-01

Background Research suggests that independent and joint effects of genetic variability in the dopamine transporter (DAT) locus and pesticides may influence Parkinson’s disease (PD) risk. Materials Methods: In 324 incident PD patients and 334 population controls from our rural California case–control study, we genotyped rs2652510, rs2550956 (for the DAT 5′ clades), and the 3′ variable number of tandem repeats (VNTR). Using geographic information system methods, we determined residential exposure to agricultural maneb and paraquat applications. We also collected occupational pesticide use data. Employing logistic regression, we calculated odds ratios (ORs) for clade diplotypes, VNTR genotype, and number of susceptibility (A clade and 9-repeat) alleles and assessed susceptibility allele–pesticide interactions. Results PD risk was increased separately in DAT A clade diplotype carriers [AA vs. BB: OR = 1.66; 95% confidence interval (CI), 1.08–2.57] and 3′ VNTR 9/9 carriers (9/9 vs. 10/10: OR = 1.8; 95% CI, 0.96–3.57), and our data suggest a gene dosing effect. Importantly, high exposure to paraquat and maneb in carriers of one susceptibility allele increased PD risk 3-fold (OR = 2.99; 95% CI, 0.88–10.2), and in carriers of two or more alleles more than 4-fold (OR = 4.53; 95% CI, 1.70–12.1). We obtained similar results for occupational pesticide measures. Discussion Using two independent pesticide measures, we a) replicated previously reported gene–environment interactions between DAT genetic variants and occupational pesticide exposure in men and b) overcame previous limitations of nonspecific pesticide measures and potential recall bias by employing state records and computer models to estimate residential pesticide exposure. Conclusion Our results suggest that DAT genetic variability and pesticide exposure interact to increase PD risk. PMID:19590691

Genotyping of Burkholderia mallei from an Outbreak of Glanders in Bahrain Suggests Multiple Introduction Events

PubMed Central

Hornstra, Heidie; Projahn, Michaela; Terzioglu, Rahime; Wernery, Renate; Georgi, Enrico; Riehm, Julia M.; Wagner, David M.; Keim, Paul S.; Joseph, Marina; Johnson, Bobby; Kinne, Joerg; Jose, Shanti; Hepp, Crystal M.; Witte, Angela; Wernery, Ulrich

2014-01-01

Background Glanders, caused by the gram-negative bacterium Burkholderia mallei, is a highly infectious zoonotic disease of solipeds causing severe disease in animals and men. Although eradicated from many Western countries, it recently emerged in Asia, the Middle-East, Africa, and South America. Due to its rareness, little is known about outbreak dynamics of the disease and its epidemiology. Methodology/Principal Findings We investigated a recent outbreak of glanders in Bahrain by applying high resolution genotyping (multiple locus variable number of tandem repeats, MLVA) and comparative whole genome sequencing to B. mallei isolated from infected horses and a camel. These results were compared to samples obtained from an outbreak in the United Arab Emirates in 2004, and further placed into a broader phylogeographic context based on previously published B. mallei data. The samples from the outbreak in Bahrain separated into two distinct clusters, suggesting a complex epidemiological background and evidence for the involvement of multiple B. mallei strains. Additionally, the samples from Bahrain were more closely related to B. mallei isolated from horses in the United Arab Emirates in 2004 than other B. mallei which is suggestive of repeated importation to the region from similar geographic sources. Conclusion/Significance High-resolution genotyping and comparative whole genome analysis revealed the same phylogenetic patterns among our samples. The close relationship of the Dubai/UAE B. mallei populations to each other may be indicative of a similar geographic origin that has yet to be identified for the infecting strains. The recent emergence of glanders in combination with worldwide horse trading might pose a new risk for human infections. PMID:25255232

Significant linkage to airway responsiveness on chromosome 12q24 in families of children with asthma in Costa Rica.

PubMed

Celedón, Juan C; Soto-Quiros, Manuel E; Avila, Lydiana; Lake, Stephen L; Liang, Catherine; Fournier, Eduardo; Spesny, Mitzi; Hersh, Craig P; Sylvia, Jody S; Hudson, Thomas J; Verner, Andrei; Klanderman, Barbara J; Freimer, Nelson B; Silverman, Edwin K; Weiss, Scott T

2007-01-01

Although asthma is a major public health problem in certain Hispanic subgroups in the United States and Latin America, only one genome scan for asthma has included Hispanic individuals. Because of small sample size, that study had limited statistical power to detect linkage to asthma and its intermediate phenotypes in Hispanic participants. To identify genomic regions that contain susceptibility genes for asthma and airway responsiveness in an isolated Hispanic population living in the Central Valley of Costa Rica, we conducted a genome-wide linkage analysis of asthma (n = 638) and airway responsiveness (n = 488) in members of eight large pedigrees of Costa Rican children with asthma. Nonparametric multipoint linkage analysis of asthma was conducted by the NPL-PAIR allele-sharing statistic, and variance component models were used for the multipoint linkage analysis of airway responsiveness as a quantitative phenotype. All linkage analyses were repeated after exclusion of the phenotypic data of former and current smokers. Chromosome 12q showed some evidence of linkage to asthma, particularly in nonsmokers (P < 0.01). Among nonsmokers, there was suggestive evidence of linkage to airway responsiveness on chromosome 12q24.31 (LOD = 2.33 at 146 cM). After genotyping 18 additional short-tandem repeat markers on chromosome 12q, there was significant evidence of linkage to airway responsiveness on chromosome 12q24.31 (LOD = 3.79 at 144 cM), with a relatively narrow 1.5-LOD unit support interval for the observed linkage peak (142-147 cM). Our results suggest that chromosome 12q24.31 contains a locus (or loci) that influence a critical intermediate phenotype of asthma (airway responsiveness) in Costa Ricans.

Polymorphisms in TS, MTHFR and ERCC1 genes as predictive markers in first-line platinum and pemetrexed therapy in NSCLC patients.

PubMed

Krawczyk, Paweł; Kucharczyk, Tomasz; Kowalski, Dariusz M; Powrózek, Tomasz; Ramlau, Rodryg; Kalinka-Warzocha, Ewa; Winiarczyk, Kinga; Knetki-Wróblewska, Magdalena; Wojas-Krawczyk, Kamila; Kałakucka, Katarzyna; Dyszkiewicz, Wojciech; Krzakowski, Maciej; Milanowski, Janusz

2014-12-01

We presented retrospective analysis of up to five polymorphisms in TS, MTHFR and ERCC1 genes as molecular predictive markers for homogeneous Caucasian, non-squamous NSCLC patients treated with pemetrexed and platinum front-line chemotherapy. The following polymorphisms in DNA isolated from 115 patients were analyzed: various number of 28-bp tandem repeats in 5'-UTR region of TS gene, single nucleotide polymorphism (SNP) within the second tandem repeat of TS gene (G>C); 6-bp deletion in 3'-UTR region of the TS (1494del6); 677C>T SNP in MTHFR; 19007C>T SNP in ERCC1. Molecular examinations' results were correlated with disease control rate, progression-free survival (PFS) and overall survival. Polymorphic tandem repeat sequence (2R, 3R) in the enhancer region of TS gene and G>C SNP within the second repeat of 3R allele seem to be important for the effectiveness of platinum and pemetrexed in first-line chemotherapy. The insignificant shortening of PFS in 3R/3R homozygotes as compared to 2R/2R and 2R/3R genotypes were observed, while it was significantly shorter in patients carrying synchronous 3R allele and G nucleotide. The combined analysis of TS VNTR and MTHFR 677C>T SNP revealed shortening of PFS in synchronous carriers of 3R allele in TS and two C alleles in MTHFR. The strongest factors increased the risk of progression were poor PS, weight loss, anemia and synchronous presence of 3R allele and G nucleotide in the second repeat of 3R allele in TS. Moreover, lack of application of second-line chemotherapy, weight loss and poor performance status and above-mentioned genotype of TS gene increased risk of early mortality. The examined polymorphisms should be accounted as molecular predictor factors for pemetrexed- and platinum-based front-line chemotherapy in non-squamous NSCLC patients.

Multilocus variable-number tandem repeat analysis for molecular typing and phylogenetic analysis of Shigella flexneri

PubMed Central

2009-01-01

Background Shigella flexneri is one of the causative agents of shigellosis, a major cause of childhood mortality in developing countries. Multilocus variable-number tandem repeat (VNTR) analysis (MLVA) is a prominent subtyping method to resolve closely related bacterial isolates for investigation of disease outbreaks and provide information for establishing phylogenetic patterns among isolates. The present study aimed to develop an MLVA method for S. flexneri and the VNTR loci identified were tested on 242 S. flexneri isolates to evaluate their variability in various serotypes. The isolates were also analyzed by pulsed-field gel electrophoresis (PFGE) to compare the discriminatory power and to evaluate the usefulness of MLVA as a tool for phylogenetic analysis of S. flexneri. Results Thirty-six VNTR loci were identified by exploring the repeat sequence loci in genomic sequences of Shigella species and by testing the loci on nine isolates of different subserotypes. The VNTR loci in different serotype groups differed greatly in their variability. The discriminatory power of an MLVA assay based on four most variable VNTR loci was higher, though not significantly, than PFGE for the total isolates, a panel of 2a isolates, which were relatively diverse, and a panel of 4a/Y isolates, which were closely-related. Phylogenetic groupings based on PFGE patterns and MLVA profiles were considerably concordant. The genetic relationships among the isolates were correlated with serotypes. The phylogenetic trees constructed using PFGE patterns and MLVA profiles presented two distinct clusters for the isolates of serotype 3 and one distinct cluster for each of the serotype groups, 1a/1b/NT, 2a/2b/X/NT, 4a/Y, and 6. Isolates that had different serotypes but had closer genetic relatedness than those with the same serotype were observed between serotype Y and subserotype 4a, serotype X and subserotype 2b, subserotype 1a and 1b, and subserotype 3a and 3b. Conclusions The 36 VNTR loci identified exhibited considerably different degrees of variability among S. flexneri serotype groups. VNTR locus could be highly variable in a serotype but invariable in others. MLVA assay based on four highly variable loci could display a comparable resolving power to PFGE in discriminating isolates. MLVA is also a prominent molecular tool for phylogenetic analysis of S. flexneri; the resulting data are beneficial to establish clear clonal patterns among different serotype groups and to discern clonal groups among isolates within the same serotype. As highly variable VNTR loci could be serotype-specific, a common MLVA protocol that consists of only a small set of loci, for example four to eight loci, and that provides high resolving power to all S. flexneri serotypes may not be obtainable. PMID:20042119

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

Rass-Rothchild, A.: Abeliovitch, D.; Kornstein, A.

The acronym LEOPARD stands for a syndromic association of Lentigines, Eletrocardiographic changes, Ocular hypertelorism, Pulmonic stenosis, Abnormal genitalia, Retardation of growth and sensorineural Deafness. Inheritance is autosomal dominant with high penetrance and variable expressivity. In 1990 Torok et al. reported on the association of LEOPARD and Marfan syndrome. In addition a clinical similarity (cardiac and cutaneous involvement) exists with the Watson syndrome (neurofibromatosis and pulmonic stenosis) which is linked to the marker D17S33 on chromosome 17. We studied possible linkage of LEOPARD syndrome to the Marfan syndrome locus on chromosome 15 (D15S1, MF13, and (TAAAA)n repeats) and to the NF-1more » locus on chromosome 17 in a family with 9 cases of LEOPARD syndrome. Close linkage between LEOPARD syndrome and both the Marfan locus on chromosome 15 and the NF-1 locus on chromosome 17 was excluded (lod score <-2.0 through {theta} = 0.1).« less

Differentiation of “Candidatus Liberibacter asiaticus” Isolates by Variable-Number Tandem-Repeat Analysis ▿

PubMed Central

Katoh, Hiroshi; Subandiyah, Siti; Tomimura, Kenta; Okuda, Mitsuru; Su, Hong-Ji; Iwanami, Toru

2011-01-01

Four highly polymorphic simple sequence repeat (SSR) loci were selected and used to differentiate 84 Japanese isolates of “Candidatus Liberibacter asiaticus.” The Nei's measure of genetic diversity values for these four SSRs ranged from 0.60 to 0.86. The four SSR loci were also highly polymorphic in four isolates from Taiwan and 12 isolates from Indonesia. PMID:21239554

Crystal structure of tandem type III fibronectin domains from Drosophila neuroglian at 2.0 A.

PubMed

Huber, A H; Wang, Y M; Bieber, A J; Bjorkman, P J

1994-04-01

We report the crystal structure of two adjacent fibronectin type III repeats from the Drosophila neural cell adhesion molecule neuroglian. Each domain consists of two antiparallel beta sheets and is folded topologically identically to single fibronectin type III domains from the extracellular matrix proteins tenascin and fibronectin. beta bulges and left-handed polyproline II helices disrupt the regular beta sheet structure of both neuroglian domains. The hydrophobic interdomain interface includes a metal-binding site, presumably involved in stabilizing the relative orientation between domains and predicted by sequence comparision to be present in the vertebrate homolog molecule L1. The neuroglian domains are related by a near perfect 2-fold screw axis along the longest molecular dimension. Using this relationship, a model for arrays of tandem fibronectin type III repeats in neuroglian and other molecules is proposed.

Phylogenic analysis and forensic genetic characterization of Chinese Uyghur group via autosomal multi STR markers.

PubMed

Jin, Xiaoye; Wei, Yuanyuan; Chen, Jiangang; Kong, Tingting; Mu, Yuling; Guo, Yuxin; Dong, Qian; Xie, Tong; Meng, Haotian; Zhang, Meng; Li, Jianfei; Li, Xiaopeng; Zhu, Bofeng

2017-09-26

We investigated the allelic frequencies and forensic descriptive parameters of 23 autosomal short tandem repeat loci in a randomly selected sample of 1218 unrelated healthy Uyghur individuals residing in the Xinjiang Uyghur Autonomous Region, northwest China. A total of 281 alleles at these loci were identified and their corresponding allelic frequencies ranged from 0.0004 to 0.5390. The combined match probability and combined probability of exclusion of all loci were 5.192 × 10 -29 and 0.9999999996594, respectively. The results of population genetic study manifested that Uyghur had close relationships with those contiguous populations, such as Xibe and Hui groups. In a word, these autosomal short tandem repeat loci were highly informative in Uyghur group and the multiplex PCR system could be used as a valuable tool for forensic caseworks and population genetic analysis.

Protein arginine methyltransferase 7 has a novel homodimer-like structure formed by tandem repeats.

PubMed

Hasegawa, Morio; Toma-Fukai, Sachiko; Kim, Jun-Dal; Fukamizu, Akiyoshi; Shimizu, Toshiyuki

2014-05-21

Protein arginine methyltransferase 7 (PRMT7) is a member of a family of enzymes that catalyze the transfer of methyl groups from S-adenosyl-l-methionine to nitrogen atoms on arginine residues. Here, we describe the crystal structure of Caenorhabditis elegans PRMT7 in complex with its reaction product S-adenosyl-L-homocysteine. The structural data indicated that PRMT7 harbors two tandem repeated PRMT core domains that form a novel homodimer-like structure. S-adenosyl-L-homocysteine bound to the N-terminal catalytic site only; the C-terminal catalytic site is occupied by a loop that inhibits cofactor binding. Mutagenesis demonstrated that only the N-terminal catalytic site of PRMT7 is responsible for cofactor binding. Copyright © 2014 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.

Performance of the SNPforID 52 SNP-plex assay in paternity testing.

PubMed

Børsting, Claus; Sanchez, Juan J; Hansen, Hanna E; Hansen, Anders J; Bruun, Hanne Q; Morling, Niels

2008-09-01

The performance of a multiplex assay with 52 autosomal single nucleotide polymorphisms (SNPs) developed for human identification was tested on 124 mother-child-father trios. The typical paternity indices (PIs) were 10(5)-10(6) for the trios and 10(3)-10(4) for the child-father duos. Using the SNP profiles from the randomly selected trios and 700 previously typed individuals, a total of 83,096 comparisons between mother, child and an unrelated man were performed. On average, 9-10 mismatches per comparison were detected. Four mismatches were genetic inconsistencies and 5-6 mismatches were opposite homozygosities. In only two of the 83,096 comparisons did an unrelated man match perfectly to a mother-child duo, and in both cases the PI of the true father was much higher than the PI of the unrelated man. The trios were also typed for 15 short tandem repeats (STRs) and seven variable number of tandem repeats (VNTRs). The typical PIs based on 15 STRs or seven VNTRs were 5-50 times higher than the typical PIs based on 52 SNPs. Six mutations in tandem repeats were detected among the randomly selected trios. In contrast, there was not found any mutations in the SNP loci. The results showed that the 52 SNP-plex assay is a very useful alternative to currently used methods in relationship testing. The usefulness of SNP markers with low mutation rates in paternity and immigration casework is discussed.

New Multilocus Variable-Number Tandem-Repeat Analysis Tool for Surveillance and Local Epidemiology of Bacterial Leaf Blight and Bacterial Leaf Streak of Rice Caused by Xanthomonas oryzae

PubMed Central

Poulin, L.; Grygiel, P.; Magne, M.; Rodriguez-R, L. M.; Forero Serna, N.; Zhao, S.; El Rafii, M.; Dao, S.; Tekete, C.; Wonni, I.; Koita, O.; Pruvost, O.; Verdier, V.; Vernière, C.

2014-01-01

Multilocus variable-number tandem-repeat analysis (MLVA) is efficient for routine typing and for investigating the genetic structures of natural microbial populations. Two distinct pathovars of Xanthomonas oryzae can cause significant crop losses in tropical and temperate rice-growing countries. Bacterial leaf streak is caused by X. oryzae pv. oryzicola, and bacterial leaf blight is caused by X. oryzae pv. oryzae. For the latter, two genetic lineages have been described in the literature. We developed a universal MLVA typing tool both for the identification of the three X. oryzae genetic lineages and for epidemiological analyses. Sixteen candidate variable-number tandem-repeat (VNTR) loci were selected according to their presence and polymorphism in 10 draft or complete genome sequences of the three X. oryzae lineages and by VNTR sequencing of a subset of loci of interest in 20 strains per lineage. The MLVA-16 scheme was then applied to 338 strains of X. oryzae representing different pathovars and geographical locations. Linkage disequilibrium between MLVA loci was calculated by index association on different scales, and the 16 loci showed linear Mantel correlation with MLSA data on 56 X. oryzae strains, suggesting that they provide a good phylogenetic signal. Furthermore, analyses of sets of strains for different lineages indicated the possibility of using the scheme for deeper epidemiological investigation on small spatial scales. PMID:25398857

"In God we trust" and other factors influencing trial of labor versus Repeat cesarean section.

PubMed

Pomeranz, Meir; Arbib, Nissim; Haddif, Limor; Reissner, Hana; Romem, Yitzhak; Biron, Tal

2018-07-01

To investigate factors influencing women's decisions to undergo trial of labor after cesarean (TOLAC) or elective repeat cesarean delivery (ERCD) based on the Multidimensional Health Locus of Control (MHLC), religious observance and family planning. Cross-sectional study of candidates for TOLAC or ERCD at two hospitals in Israel. Eligible women completed a demographic questionnaire and Form C of the MHLC scale. The study included 197 women. Those who chose TOLAC (N = 101) were more religiously observant, wanted more children and had higher Internal and Chance health locus of control. Women who chose ERCD (N = 96) were more likely to be secular and had a higher health locus of control influenced by Powerful Others, notably physicians. Women not influenced by others were more likely to choose TOLAC. A woman's choice of TOLAC or ERCD is influenced by her sense of control over her health, degree of religious observance and number of children desired. Healthcare providers can use this information to better understand, counsel and educate women regarding appropriate delivery decisions. Women who feel in control of their health, educated about delivery options and are less influenced by provider preference, might choose TOLAC; thus, reducing the rate of unnecessary ERCD.

Characterization of genic microsatellite markers derived from expressed sequence tags in Pacific abalone ( Haliotis discus hannai)

NASA Astrophysics Data System (ADS)

Li, Qi; Shu, Jing; Zhao, Cui; Liu, Shikai; Kong, Lingfeng; Zheng, Xiaodong

2010-01-01

Simple sequence repeat (SSR) markers were developed from the expressed sequence tags (ESTs) of Pacific abalone ( Haliotis discus hannai). Repeat motifs were found in 4.95% of the ESTs at a frequency of one repeat every 10.04 kb of EST sequences, after redundancy elimination. Seventeen polymorphic EST-SSRs were developed. The number of alleles per locus varied from 2-17, with an average of 6.8 alleles per locus. The expected and observed heterozygosities ranged from 0.159 to 0.928 and from 0.132 to 0.922, respectively. Twelve of the 17 loci (70.6%) were successfully amplified in H. diversicolor. Seventeen loci segregated in three families, with three showing the presence of null alleles (17.6%). The adequate level of variability and low frequency of null alleles observed in H. discus hannai, together with the high rate of transportability across Haliotis species, make this set of EST-SSR markers an important tool for comparative mapping, marker-assisted selection, and evolutionary studies, not only in the Pacific abalone, but also in related species.