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PubMed2006

Evaluation and selection of tandem repeat loci for a Brucella MLVA typing assay.

BACKGROUND: The classification of Brucella into species and biovars relies on phenotypic characteristics and sometimes raises difficulties in the interpretation of the results due to an absence of standardization of the typing reagents. In addition, the resolution of this biotyping is moderate and requires the manipulation of the living agent. More efficient DNA-based methods are needed, and this work explores the suitability of multiple locus variable number tandem repeats analysis (MLVA) for both typing and species identification. RESULTS: Eighty tandem repeat loci predicted to be polymorphic by genome sequence analysis of three available Brucella genome sequences were tested for polymorphism by genotyping 21 Brucella strains (18 reference strains representing the six 'classical' species and all biovars as well as 3 marine mammal strains currently recognized as members of two new species). The MLVA data efficiently cluster the strains as expected according to their species and biovar. For practical use, a subset of 15 loci preserving this clustering was selected and applied to the typing of 236 isolates. Using this MLVA-15 assay, the clusters generated correspond to the classical biotyping scheme of Brucella spp. The 15 markers have been divided into two groups, one comprising 8 user-friendly minisatellite markers with a good species identification capability (panel 1) and another complementary group of 7 microsatellite markers with higher discriminatory power (panel 2). CONCLUSION: The MLVA-15 assay can be applied to large collections of Brucella strains with automated or manual procedures, and can be proposed as a complement, or even a substitute, of classical biotyping methods. This is facilitated by the fact that MLVA is based on non-infectious material (DNA) whereas the biotyping procedure itself requires the manipulation of the living agent. The data produced can be queried on a dedicated MLVA web service site.

Animals↗
PubMed2000

Genome screen for QTLs contributing to normal variation in bone mineral density and osteoporosis.

A major determinant of the risk for osteoporosis is peak bone mineral density (BMD), which is largely determined by genetic factors. We recently reported linkage of peak BMD in a large sample of healthy sister pairs to chromosome 11q12-13. To identify additional loci underlying normal variations in peak BMD, we conducted an autosomal genome screen in 429 Caucasian sister pairs. Multipoint LOD scores were computed for BMD at four skeletal sites. Chromosomal regions with LOD scores above 1.85 were further pursued in an expanded sample of 595 sister pairs (464 Caucasians and 131 African-Americans). The highest LOD score attained in the expanded sample was 3.86 at chromosome 1q21-23 with lumbar spine BMD. Chromosome 5q33-35 gave a LOD score of 2.23 with femoral neck BMD. At chromosome 6p11-12, the 464 Caucasian pairs achieved a LOD score of 2.13 with lumbar spine BMD. Markers within the 11q12-13 region continued to support linkage to femoral neck BMD, although the peak LOD score was decreased to 2.16 in the sample of 595 sibling pairs. Our study is the largest genome screen to date for genes underlying variations in peak BMD and represents an important step toward identifying genes contributing to osteoporosis in the general population.

Adult↗
PubMed1992

Pax genes, mutants and molecular function.

The paired domain is a conserved DNA binding motif which was first found in Drosophila segmentation gene products. This paired domain is encoded by a well conserved, paired box DNA sequence, also detected in the genomes of other species. The mouse paired box-containing genes are referred to as Pax genes and are expressed in a distinct spatiotemporal pattern during embryogenesis. Pax proteins are able to bind to specific DNA sequences and modulate transcriptional activity. Interestingly, three different Pax genes have already been shown to correspond to some mouse and human mutants, emphasizing their role as developmental control genes.

Animals↗
PubMed2006

Clinical expression of C282Y homozygous HFE haemochromatosis at 14 years of age.

A 14-year-old boy who presented with debilitating lethargy was shown to have an elevated serum ferritin of 572 microg/L and a C282Y homozygous HFE genotype. Liver iron concentration was measured non-invasively by magnetic resonance imaging, which revealed a liver iron concentration of 59 micromol/g dry weight (children's reference range < 14). The early phenotypic expression was further investigated by screening genomic DNA for the presence of co-inherited mutations in genes responsible for non-HFE haemochromatosis. Coding regions and splice sites in genes encoding hepcidin and haemojuvelin were sequenced and previously described mutations in ferroportin 1 and transferrin receptor 2 genes were screened. Although no mutations were found, the most likely cause for the early expression is the presence of novel mutations or gene(s).

Adult↗
PubMed2001

A transgenic approach to studying imprinted genes: modified BACs and PACs.

The advantages of using large genomic clones in the analysis of imprinted genes is described in Chapter 4 with particular reference to yeast artificial chromosomes (YACs). These contain on average 500-600 kb of DNA but can be much larger (>1 Mb). YACS are propagated in yeast and are therefore amenable to genetic modification by homologous recombination, and there are now many examples of their use to generate transgenic mice. This chapter describes a relatively new strategy for using large genomic clones that relies on Escherichia coli-based systems.

Animals↗
PubMedDate not supplied

[Genomic and antigenic analysis of strains of influenza A virus (H1N1) isolated in 1982-1983: approaches to the choice of the optimal vaccine strain].

Features of the genome and antigenic specificity of hemagglutinin of some influenza A (H1N1) virus strains circulating in the epidemic period of 1982-1983 were studied comparatively. Analysis of the genome of the isolates under study in comparison with that of the reference A/England/333/80 strain and with each other has established changes not only in the genes coding for hemagglutinin and neuraminidase but also most of the genes coding for unglycolysed proteins. The antigenic specificity of hemagglutinin of the isolates under study examined with rat antisera and monoclonal antibodies was found to be quite dissimilar. Hemagglutinin of the A/Dunedin/27/83 strain induced antibodies capable of reacting predominantly with the homologous strain whereas antibodies to hemagglutinin of the A/Leningrad/16/16/82 and A/Chile/1/83 strains had a wide spectrum of antigenic specificity and neutralized well the hemagglutinin of different variants of influenza virus of H1N1 serotype circulating at that time. Among the 1982-1983 isolates studied, the A/Leningrad/16/16/82 strain was selected which, by its hemagglutinin properties, is optimal for preparation of inactivated vaccine, as was confirmed by the study of an experimental batch of such vaccine in volunteers.

Animals↗
PubMed1989

Molecular cloning of DNA from a bovine herpesvirus 1 strain isolated in Hungary.

Molecular cloning of the HindIII fragments of bovine herpesvirus 1 (BHV-1) strain HB144, isolated from infectious bovine rhinotracheitis (IBR) in Hungary, and of an infectious pustular vulvovaginitis (IPV) reference strain (K22) is reported. So far 52% of the IBR viral genome and 28% of the IPV viral genome have been cloned. The analysis of differences between the strains is currently in progress.

Animals↗
PubMed2004

Amplification of Echoviruses genomic regions by different RT-PCR protocols--a comparative study.

In the present report, the results of a comparative study in the detection of all Echoviruses reference strains as well as of 38 clinical isolates are presented. Using RT-PCR with already published primer pairs (UG(52)-UC(53), 292-222, 012-011 and EUG2a, 2b, 2c-EUC2) from the 5'UTR, the VP1 region as well as a long genomic fragment including the VP1 3' end, the entire coding sequence of 2A, 2B, and the 5' moiety of the 2C-coding region amplification was effective with all reference and clinical Echovirus isolates with primer pair UG(52)-UC(53) while with 292-222 and 012-011 were amplified 27/28 reference Echovirus strains and all clinical isolates. As far as EUG2a,2b,2c-EUC2 is concerned, the RT-PCR gave a positive result for 26/28 reference Echovirus strains and 34/38 clinical isolates. The sequence analysis of a large part of the 5'UTR has revealed that there is no correlation between 5'UTR identity and the currently recognized human enterovirus species. It has been suggested that part of VP1 coding sequence would correlate well with serotype since a number of important neutralization epitopes, as well as receptor recognition sequences, lie within the VP1 coding sequence. Therefore, UG(52)-UC(53) and 292-222 primer pairs seem to be the most appropriate for Echovirus detection and, moreover, UG(52)-UC(53) is useful for the classification of enteroviruses into genetic clusters (sub-groups) while 292-222 for the identification of enteroviruses by amplicon sequencing.

DNA Primers↗
PubMed2006

Streptomyces turgidiscabies and Streptomyces reticuliscabiei: one genomic species, two pathogenic groups.

Three strains of Streptomyces reticuliscabiei and two strains of Streptomyces turgidiscabies were analysed, together with reference and type strains of other Streptomyces species, for phenotypic traits, DNA-DNA relatedness, comparison of 16S rRNA gene sequences and presence of necrotic protein gene (nec1) homologues in order to clarify their phylogenetic relationships. A numerical analysis of phenotypic characteristics showed that S. reticuliscabiei and S. turgidiscabies belong to the same cluster and share almost all morphological and biochemical traits that are important in the identification of Streptomyces species. DNA-DNA hybridization and phylogenetic comparisons of 16S rRNA gene sequences confirmed that the two species are genomically closely related. In contrast, pathological data showed that S. turgidiscabies and S. reticuliscabiei cause two distinct diseases. Gene homologues of nec1 were detected in S. turgidiscabies and other common scab species (Streptomyces scabiei, Streptomyces europaeiscabiei and Streptomyces stelliscabiei), but not in S. reticuliscabiei. To avoid confusion between agents causing separate diseases, it is proposed that the existing distinct species names are retained: S. turgidiscabies involved in common scab and S. reticuliscabiei involved in netted scab.

Bacterial Proteins↗
PubMed1998

Mapping the genome of Eimeria tenella: use of a yeast artificial chromosome library as the basis for a physical map.

Efforts to derive a physical map of the genome of Eimeria tenella are being made to complement genetic mapping and to assist in the production of an integrated map. A large insert library of DNA from the Houghton (H) reference strain of E. tenella has been constructed in yeast artificial chromosomes (YACs). The library contains a tenfold E. tenella genome equivalent consisting of 3177 clones arrayed in 96-well microtitre plates and gridded on nylon membranes at 1728 clones/86 cm2. Size fractionation of a random sample of 185 YACs revealed an average insert size of approximately 170 kb. Hybridisation of four E. tenella single-copy probes to the gridded colony filters produced around four or more positive clones. A probe representing the whole of the 1.2-Mb chromosome 2, excised from a preparative gel following pulsed-field gel electrophoresis, revealed between 80 and 100 positive clones.

Animals↗
PubMed2006

Genetic diversity of the Q fever agent, Coxiella burnetii, assessed by microarray-based whole-genome comparisons.

Coxiella burnetii, a gram-negative obligate intracellular bacterium, causes human Q fever and is considered a potential agent of bioterrorism. Distinct genomic groups of C. burnetii are revealed by restriction fragment-length polymorphisms (RFLP). Here we comprehensively define the genetic diversity of C. burnetii by hybridizing the genomes of 20 RFLP-grouped and four ungrouped isolates from disparate sources to a high-density custom Affymetrix GeneChip containing all open reading frames (ORFs) of the Nine Mile phase I (NMI) reference isolate. We confirmed the relatedness of RFLP-grouped isolates and showed that two ungrouped isolates represent distinct genomic groups. Isolates contained up to 20 genomic polymorphisms consisting of 1 to 18 ORFs each. These were mostly complete ORF deletions, although partial deletions, point mutations, and insertions were also identified. A total of 139 chromosomal and plasmid ORFs were polymorphic among all C. burnetii isolates, representing ca. 7% of the NMI coding capacity. Approximately 67% of all deleted ORFs were hypothetical, while 9% were annotated in NMI as nonfunctional (e.g., frameshifted). The remaining deleted ORFs were associated with diverse cellular functions. The only deletions associated with isogenic NMI variants of attenuated virulence were previously described large deletions containing genes involved in lipopolysaccharide (LPS) biosynthesis, suggesting that these polymorphisms alone are responsible for the lower virulence of these variants. Interestingly, a variant of the Australia QD isolate producing truncated LPS had no detectable deletions, indicating LPS truncation can occur via small genetic changes. Our results provide new insight into the genetic diversity and virulence potential of Coxiella species.

Chromosomes, Bacterial↗
PubMed2005

Canine DNA subjected to whole genome amplification is suitable for a wide range of molecular applications.

Molecular and genetic studies of canine disease phenotypes can be limited by the amount of DNA available for analysis. New methods have been developed to amplify the genomic DNA of a species producing large quantities of DNA from small starting amounts. Whole genome amplification (WGA) of DNA is now being used in human studies, although this technique has not been applied extensively in veterinary research. We evaluated WGA of canine DNA for suitability in a range of molecular tests. DNA from 93 canine blood extracted and 18 buccal swab samples was subjected to WGA using the GenomiPhi kit (Amersham). Genomic DNA was compared with WGA product using a range of techniques, including reference strand-mediated conformation analysis, denaturing high-performance liquid chromatography analysis, microsatellite genotyping, direct DNA sequencing, and single nucleotide polymorphism allelic discrimination. All samples amplified well, giving an average yield of 3 mug of DNA from 2.5 ng of starting material. Extremely high levels of experimental reproducibility and concordance were observed between source and WGA DNA samples for all analyses used: greater than 95% for blood extracted DNA and greater than 80% for buccal swab DNA. These studies clearly demonstrate the usefulness of WGA of canine DNA as a means of increasing DNA quantities for canine studies. This technique will have major implications for future veterinary research.

Animals↗
PubMed2026

Segzoo: a turnkey system that summarizes genome annotations.

MOTIVATION: Segmentation and automated genome annotation (SAGA) techniques, such as Segway and ChromHMM, assign labels to every part of the genome, identifying similar patterns across multiple genomic input signals. Inferring biological meaning in these patterns remains challenging. Doing so requires a time-consuming process of manually downloading reference data, running multiple analysis methods, and interpreting many individual results. RESULTS: To simplify these tasks, we developed the turnkey system Segzoo. As input, Segzoo only requires a genome annotation file in browser extensible data (BED) format. It automatically downloads the rest of the data required for comparisons. Segzoo performs analyses using these data and summarizes results in a single visualization. AVAILABILITY AND IMPLEMENTATION: The source code for Python &#x2265; 3.7 on Linux is freely available for download at https://github.com/hoffmangroup/segzoo under the GNU General Public License (GPL) version 2. Segzoo is also available in the Bioconda package segzoo: https://anaconda.org/bioconda/segzoo. We have deposited in Zenodo the version of the Segzoo source which produced the results in this article (https://doi.org/10.5281/zenodo.10988775), other code and data used to produce the results (https://doi.org/10.5281/zenodo.10477083), and the results (https://doi.org/10.5281/zenodo.10477106).

Software↗
PubMed1995

Random subcloning.

Random subcloning strategies are commonly employed for analyzing pieces of DNA that are too large for direct analysis. Such strategies are applicable to gene finding, physical mapping, and DNA sequencing. Random subcloning refers to the generation of many small, directly analyzable fragments of DNA that represent random fragments of a larger whole, such as a genome. Following analysis of these fragments, a map or sequence of the original target may be reconstructed. Mathematical modeling is useful in planning such strategies and in providing a reference for their evaluation, both during execution and following completion. The statistical theory necessary for constructing these models has been developed independently over the last century. This paper brings this theory together into a statistical model for random subcloning strategies. This mathematical model retains its utility even at high subclone redundancies, which are necessary for project completion. The discussion here centers on shotgun sequencing, a random subcloning strategy envisioned as the method of choice for sequencing the human genome.

Chromosome Mapping↗
PubMed2003

A new circulating recombinant form, CRF15_01B, reinforces the linkage between IDU and heterosexual epidemics in Thailand.

HIV-1 subtype B and CRF01_AE have been in circulation in Thailand and Southeast Asia for more than a decade. Initially separated by risk group, the two strains are increasingly intermixed, and two recombinant strains of essentially reciprocal structure have been recently reported. Here we identify additional CRF_01B recombinants and provide the evidence that HIV-1 strains now pass freely between the two high-risk populations. HIV isolates that showed discordance between CRF01_AE and subtype B in multi-region genotyping assays were selected for the study. They were drawn from 3 different cohorts in Thailand representing different risk behaviors and demographic characteristics: a drug user cohort in the north, a family planning clinic attendee cohort in the southeast, and a cohort study of the mucosal virology and immunology of HIV-1 infection in Thailand. The DNA from these isolates was PCR amplified to recover the full HIV-1 genome and subjected to sequencing and phylogenetic analysis. We establish that one particular CRF_01B recombinant, with the external envelope of subtype B and the rest of the genome from CRF01_AE, is circulating widely in Thailand. Termed CRF15_01B (also referred to as CRF15), the strain was primarily heterosexually transmitted, although injecting drug use (IDU) also played a role. In aggregate data from the studies, CRF15 constituted 1.7% of all HIV-1 infections (95% confidence interval 0.5-4.4%) and was dispersed widely in the country. The previously separate heterosexual and IDU epidemics have apparently been bridged by a new CRF. The entry of CRF15 into the mainstream of the epidemic signals new complexity in the long stable molecular picture in Thailand. These recombinants must be considered in ongoing or projected efficacy evaluations of HIV-1 vaccines and antiviral therapies.

Adult↗
PubMed1990

A GGCAGG motif in minisatellites affecting their germline instability.

Mouse and human genomes contain hypervariable DNA regions consisting of tandem repeats of a short sequence referred to as minisatellites. This variation is thought to arise through processes such as unequal crossover or replication slippage. A mo-1 minisatellite probe comprising a 14-base pair repeat sequence reveals many polymorphic fragments even in DNA of BALB/c sublines. Oligonucleotide probes with single base substitution in the mo-1 have been synthesized and used for assessing sequence involved in generation of polymorphisms. The results indicate that the loci containing mo-1 homologues with mutation in the GGCAGG sequence are monomorphic despite the other mutants showing polymorphism. Reciprocally, locus-specific polymorphic clones, Pc-1 and Pc-2, have been isolated with hybridization to mo-1, and both are shown to contain repeated sequence comprising the GGCAGG sequence. They reveal high mutation rates of 8.8% and 3.3% per gamete, respectively. These results strongly suggest that the motif contributes to the germline instability of minisatellites.

Animals↗