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At least 865 records · Page 48Linked to original sources
PubMed2000

Species identification of Mycobacterium avium complex isolates by a variety of molecular techniques.

Organisms in the Mycobacterium avium complex (MAC; M. avium, M. intracellulare, and "nonspecific or X" MAC) are emerging pathogens among individual organisms of which significant genetic variability is displayed. The objective of the present study was to evaluate various molecular methods for the rapid and definitive identification of MAC species. Isolates were obtained from both human immunodeficiency virus (HIV)-positive patients and HIV-negative patients with and without known predisposing conditions. The isolates were initially hybridized with nucleic acid probes complementary to the rRNA of the respective mycobacterial species (AccuProbe Culture Confirmation kits for M. avium, M. intracellulare, and MAC species; Gen-Probe). Isolates were also examined by PCR and in some cases by Southern blot hybridization for the insertion element IS1245. Two other techniques included a PCR assay that amplifies the mig gene, a putative virulence factor for MAC, and hsp65 gene amplification and sequencing. This study led to the following observations. Eighty-five percent of the isolates from HIV-positive patients were M. avium and 86% of the isolates from HIV-negative patients were M. intracellulare. Fifteen of the M. avium isolates did not contain IS1245 and 7% of the M. intracellulare isolates were found to carry IS1245. All of the M. avium strains were mig positive, and all of the M. intracellulare strains were mig negative.

AIDS-Related Opportunistic Infections↗
PubMed2003

Accessory proteins functioning selectively and pleiotropically in the biosynthesis of [NiFe] hydrogenases in Thiocapsa roseopersicina.

There are at least two membrane-bound (HynSL and HupSL) and one soluble (HoxEFUYH) [NiFe] hydrogenases in Thiocapsa roseopersicina BBS, a purple sulfur photosynthetic bacterium. Genes coding for accessory proteins that participate in the biosynthesis and maturation of hydrogenases seem to be scattered along the chromosome. Transposon-based mutagenesis was used to locate the hydrogenase accessory genes. Molecular analysis of strains showing mutant phenotypes led to the identification of hupK (hoxV ), hypC1, hypC2, hypD, hypE, and hynD genes. The roles of hynD, hupK and the two hypC genes were investigated in detail. The putative HynD was found to be a hydrogenase-specific endoprotease type protein, participating in the maturation of the HynSL enzyme. HupK plays an important role in the formation of the functionally active membrane-bound [NiFe] hydrogenases, but not in the biosynthesis of the soluble enzyme. In-frame deletion mutagenesis showed that HypC proteins were not specific for the maturation of either hydrogenase enzyme. The lack of either HypC protein drastically reduced the activity of every hydrogenase. Hence both HypCs might participate in the maturation of [NiFe] hydrogenases. Homologous complementation with the appropriate genes substantiated the physiological roles of the corresponding gene products in the H2 metabolism of T. roseopersicina.

Bacterial Proteins↗
PubMed2005

Large-scale screen highlights the importance of capsule for virulence in the zoonotic pathogen Streptococcus iniae.

Zoonotic pathogens have the unique ability to cross the species barrier, causing disease in both humans and specific animal hosts. Streptococcus iniae is a zoonotic pathogen of both fish and humans, and the clinical presentations of S. iniae infections in fish and humans are very similar to those caused by various human-specific streptococcal pathogens. Virulence mechanisms required for infection by this pathogen of either host have yet to be determined. Using the previously reported zebrafish infectious disease model, we performed a large-scale screening to determine genes required for systemic infection. Screening 1,128 signature-tagged transposon mutants through the zebrafish model allowed identification of 41 potential mutants that were unable to survive within the host environment. Greater than 50% of the mutants that could be identified through homology searches were highly homologous to genes found in other human-specific streptococcal pathogens, while 32% were found to have no homology to any sequences found in the databases, suggesting as yet unknown gram-positive bacterial virulence factors. A large percentage of the insertions were found to be located in several putative capsule synthesis genes, an important virulence component for other systemic pathogens. Density gradient assays demonstrated that several of these putative capsule mutants have dissimilar buoyant densities, suggesting different levels of capsule synthesis. Putative capsule mutants were also less resistant to phagocytosis in whole-blood assays than wild-type S. iniae. Our initial large-scale characterization of S. iniae virulence highlights the importance of the capsule for successful infection.

Animals↗
PubMed1990

Characterization of a transpositionally active Ty3 element and identification of the Ty3 integrase protein.

Ty3 is a Saccharomyces cerevisiae retrotransposon associated with tRNA genes. Two Ty3 elements have been cloned and characterized. The complete nucleotide sequence for one element, Ty3-2, was reported previously (L. J. Hansen, D. L. Chalker, and S. B. Sandmeyer, Mol. Cell. Biol. 9:5245-5256, 1988). However, this element is incapable of autonomous transposition. The complete DNA sequence of a transpositionally competent Ty3 element, Ty3-1, is presented here. Its sequence translates into two overlapping open reading frames, TYA3-1 and TYB3-1, which encode proteins with homology to the proteins specified by the retroviral gag and pol genes, respectively. Comparison of the Ty3-1 nucleotide sequence to Ty3-2 suggests that the TYB3-2 open reading frame of Ty3-2 is truncated by the deletion of a single nucleotide, which causes a frameshift mutation. Restoration of the reading frame with insertion of a single adenine by site-directed mutagenesis converted Ty3-2 into a transpositionally active element, Ty3-2(+ A). Western blot analysis with antibodies made against synthetic peptides identified integrase (IN) proteins in viruslike particle preparations from cells expressing Ty3 elements. Cells expressing Ty3-1 and Ty3-2 (+A) produce antibody-reactive proteins with approximate molecular masses of 61 and 58 kilodaltons (kDa), while cells expressing Ty3-2 produce reactive proteins of approximately 52 and 49 kDa. Together, these data show that the 61- or 58-kDa protein, or both, provides the integrase function of Ty3.

Amino Acid Sequence↗
PubMed2005

[Mutation mechanisms and their consequences].

The identification of mutations leading to human genetic diseases has grown into an intensive research field during the last few years. Through novel DNA analysis progress, it is now possible to determine the mutational spectrum for a given genetic disease and international databases are now available online. Genetic diagnosis of hereditary diseases has become an essential tool in genetic counselling and prenatal diagnosis. The knowledge of the deleterious mutation type and the molecular associated mechanism is fundamental in order to devise the optimal molecular diagnosis strategy. This review aims to present the various mutation categories involved in genetic diseases (single base-pair substitutions, small deletions or insertions, dynamic mutations, gross DNA lesions...) and to summarize our current knowledge about the main molecular mechanisms responsible for these mutations. Their deleterious consequences on gene expression, including transcription and transcript maturation, and protein loss or gain of function are also discussed in this review.

Chromosome Inversion↗
PubMed2002

Identification of genes induced by rapid intraoperative tissue expansion in mouse skin.

A method of rapid skin stretching, i.e. hemispherical load cycling with an inflated subcutaneous silicone balloon (Rapid Intraoperative Tissue Expansion or RITE), permits the surgeon to rapidly elongate skin and create a flap of greater length for reconstructive plastic surgery. We have previously developed an experimental mouse model to evaluate RITE, and have shown that rapid stretching prevents ischemia and significantly reduces necrosis. Although the advantages of RITE have been demonstrated both clinically and experimentally, the cellular and molecular mechanisms underlying these benefits were unknown. In the study reported here, we used differential display reverse transcription polymerase chain reaction to identify genes that are specifically induced by RITE. Among four differential gene fragments, the expression of one was confirmed by Northern blot hybridization. The cDNA fragment was extended and the resultant sequence analyzed to reveal induction of truncated long interspersed nucleotide element 1 (LINE-1 or L1). Truncated L1 elements are located inside introns of many genes and among these genes myotubularin and insulin I are known to regulate cell growth. Northern hybridization using specific cDNA probes for myotubularin and insulin I demonstrated that it also was induced by RITE. This is the first reported study to show that L1, myotubularin and insulin I are responsive to rapid hemispherical and not rapid linear stretch.

Amino Acid Sequence↗
PubMed1994

Mucopolysaccharidosis type I: identification of 8 novel mutations and determination of the frequency of the two common alpha-L-iduronidase mutations (W402X and Q70X) among European patients.

A group of 46 European patients with mucopolysaccharidosis type I (MPS I) was screened for mutations of the alpha-L-iduronidase gene. The 2 common nonsense mutations, W402X and Q70X, were identified in, respectively, 37% and 35% of mutant alleles. Considerable differences were seen in the frequency of these 2 mutations in patients from North Europe (Norway and Finland) and other European countries (mainly The Netherlands and Germany). In Scandinavia, W402X and Q70X account for 17% and 62% of the MPS I alleles, respectively, while in other European countries W402X is about 2.5 times more frequent (48%) than Q70X (19%). Eight novel mutations are described including 4 missense mutations, 1 nonsense mutation, 1 insertion of 2 base pairs, and 2 deletions of 1 and 12 base pairs.

Alleles↗
PubMed1991

987P fimbrial gene identification and protein characterization by T7 RNA polymerase-induced transcription and TnphoA mutagenesis.

The 987P fimbrial gene cluster has been previously cloned as a 12 kb fragment from prototype strain 987. Gene products encoded by the whole clone were analysed by utilizing an in vivo system based on the induction of transcription by T7 RNA polymerase. The sensitivity of this technique permitted us to identify new proteins involved in 987P fimbriation. In total, eight proteins were detected, their genes (fasA to fasH) were mapped and their orientation of transcription determined. Several of the gene products demonstrated typical properties of exported proteins. Precursor and processed forms could be correlated after inhibiting protein transport with ethanol. The detection of enzymatically active fusion proteins after TnphoA (Tn5IS50L::phoA) mutagenesis supported and complemented these results. One protein encoded by the 12kb fragment was found not to be related to fimbriation but rather the product of the STla gene, identified as a component of a Tn1681-like transposon.

Adhesins, Escherichia coli↗
PubMed2006

Identification of low-temperature-regulated genes in the fire blight pathogen Erwinia amylovora.

Genes involved in pathogenicity of several plant pathogens were shown to be induced at relatively cold temperatures. Loci from the fire blight pathogen Erwinia amylovora (Burrill) induced at 18 degrees C were identified using the miniTn5 transposon that contains the promoterless reporter gene gusA coding for beta-glucuronidase (GUS). Certain mutants (2.7%) expressed GUS predominantly at 18 degrees C on minimal medium plates, indicating that the transposon had been inserted downstream of a putatively thermoregulated promoter. Those mutants were further screened with a quantitative GUS fluorometric assay. A total of 21 mutants were selected: 19 mutants had a transposon insertion in temperature-dependent genetic loci, with a 2.2- to 6.3-fold induction of gusA gene expression at 18 degrees C, and two mutants with impaired growth at 18 degrees C. Some of these genetic loci encoded (i) proteins implicated in flagella biosynthesis, biotin biosynthesis, multi-drug efflux, and type II secretion protein, and (ii) proteins of unknown function.

Bacterial Proteins↗
PubMed2001

Identification and characterization of Tn4656, a novel class II transposon carrying a set of toluene-degrading genes from TOL plasmid pWW53.

It has been reported that the toluene-degrading (xyl) genes from Pseudomonas putida plasmid pWW53 are able to translocate to broad-host-range drug resistance plasmid RP4, and pWW53-4 is one of the smallest RP4 derivatives (H. Keil, S. Keil, R. W. Pickup, and P. A. Williams, J. Bacteriol. 164:887-895, 1985). Our investigation of pWW53-4 in this study demonstrated that such a translocated region that is 39 kb long is a transposon. This mobile element, Tn4656, was classified as a class II transposon since its transposition occurred by a two-step process: transposase (TnpA)-mediated formation of the cointegrate and resolvase (TnpR)-mediated site-specific resolution of the cointegrate at the two copies of the res site. The Tn4656 TnpA and TnpR functions encoded in the rightmost 4-kb region were found to be exchangeable with those specified by other Tn1721-related class II transposons, including another toluene transposon, Tn4653. Sequence analysis of the transposition-related genes and sites of Tn4656 also supported the hypothesis that this transposon is closely related to the Tn1721-related transposons. The lower transposition frequency of Tn4656 has been suggested to be due to the unique nucleotide sequence of one of the terminal 39-bp inverted repeats.

Bacterial Proteins↗
PubMed2000

GUP1 and its close homologue GUP2, encoding multimembrane-spanning proteins involved in active glycerol uptake in Saccharomyces cerevisiae.

Many yeast species can utilize glycerol, both as a sole carbon source and as an osmolyte. In Saccharomyces cerevisiae, physiological studies have previously shown the presence of an active uptake system driven by electrogenic proton symport. We have used transposon mutagenesis to isolate mutants affected in the transport of glycerol into the cell. Here we present the identification of YGL084c, encoding a multimembrane-spanning protein, as being essential for proton symport of glycerol into S. cerevisiae. The gene is named GUP1 (glycerol uptake) and, for growth on glycerol, is important as a carbon and energy source. In addition, in strains deficient in glycerol production it also provides osmotic protection by the addition of glycerol. Another open reading frame (ORF), YPL189w, presenting a high degree of homology to YGL084c, similarly appears to be involved in active glycerol uptake in salt-containing glucose-based media in strains deficient in glycerol production. Analogously, this gene is named GUP2. To our knowledge, this is the first report on a gene product involved in active transport of glycerol in yeasts. Mutations with the same phenotypes occurred in two other ORFs of previously unknown function, YDL074c and YPL180w.

Amino Acid Sequence↗
PubMed1999

Direct sequencing of bacterial and P1 artificial chromosome-nested deletions for identifying position-specific single-nucleotide polymorphisms.

A loxP-transposon retrofitting strategy for generating large nested deletions from one end of the insert DNA in bacterial artificial chromosomes and P1 artificial chromosomes was described recently [Chatterjee, P. K. & Coren, J. S. (1997) Nucleic Acids Res. 25, 2205-2212]. In this report, we combine this procedure with direct sequencing of nested-deletion templates by using primers located in the transposon end to illustrate its value for position-specific single-nucleotide polymorphism (SNP) discovery from chosen regions of large insert clones. A simple ampicillin sensitivity screen was developed to facilitate identification and recovery of deletion clones free of transduced transposon plasmid. This directed approach requires minimal DNA sequencing, and no in vitro subclone library generation; positionally oriented SNPs are a consequence of the method. The procedure is used to discover new SNPs as well as physically map those identified from random subcloned libraries or sequence databases. The deletion templates, positioned SNPs, and markers are also used to orient large insert clones into a contig. The deletion clone can serve as a ready resource for future functional genomic studies because each carries a mammalian cell-specific antibiotic resistance gene from the transposon. Furthermore, the technique should be especially applicable to the analysis of genomes for which a full genome sequence or radiation hybrid cell lines are unavailable.

Base Sequence↗
PubMed2005

Regulation of the sphingoid long-chain base kinase Lcb4p by ergosterol and heme: studies in phytosphingosine-resistant mutants.

Sphingoid long-chain base 1-phosphates (LCBPs) are widely conserved, bioactive lipid molecules. In yeast, LCBPs are known to be involved in several cellular responses such as heat shock resistance and Ca(2+) mobilization, although their target molecules and signaling pathways remain unclear. To identify genes involved in LCBP signaling and in regulation of LCBP synthesis, we performed transposon mutagenesis in cells lacking the LCBP lyase DPL1 and LCBP phosphatase LCB3 genes and screened for phytosphingosine-resistant clones. Further isolation and identification revealed eight genes (PBP1, HEM14, UFD4, HMG1, TPS1, KES1, WHI2, and ERG5), in addition to the previously characterized LCB4 and PDR5 genes, that are involved in phytosphingosine resistance. Of these eight, four are ergosterol-related genes (HEM14, HMG1, KES1, and ERG5). We also demonstrated that protein expression of the long-chain base kinase Lcb4p was reduced in Deltahem14 and Deltahmg1 cells, likely as a consequence of decreased activity of the heme-dependent transcription factor Hap1p. In addition, phosphorylation of Lcb4p was decreased in all the ergosterol-related mutants isolated and other ergosterol mutants constructed (Deltaerg2, Deltaerg3, and Deltaerg6). Finally, plasma membrane localization of Lcb4p was found to be reduced in Deltaerg6 cells. These results suggest that changes in sterol composition affect the phosphorylation of Lcb4p because of the altered localization. The other genes isolated (PBP1, UFD4, TPS1, and WHI2) may be involved in LCBP signaling.

Biomarkers↗
PubMed1998

Identification of mutations in the human PATCHED gene in sporadic basal cell carcinomas and in patients with the basal cell nevus syndrome.

Mutations in PATCHED (PTC), the human homolog of the Drosophila patched gene, have been identified in most exons of the gene in patients with the basal cell nevus syndrome and in sporadic basal cell carcinomas. We have screened the 23 PTC exons for mutations using single strand conformation polymorphism analysis of DNA from 86 basal cell nevus syndrome probands, 26 sporadic basal cell carcinomas, and seven basal cell nevus syndrome-associated basal cell carcinomas. This screen identified mutations located in eight exons in 13 of the basal cell nevus syndrome patients and in three of the tumors. The most common mutations were frameshifts resulting in premature chain termination. These results provide further evidence for the crucial role of PTC as a tumor suppressor in human keratinocytes.

Amino Acid Substitution↗
PubMed1995

Identification of chromosomal mobile element conferring high-level vancomycin resistance in Enterococcus faecium.

A clinical isolate of Enterococcus faecium that contains a chromosomally encoded vanA gene cluster, Tn1546::IS1251, transferred vancomycin resistance to the plasmid-free strain Enterococcus faecalis JH2-2 during filter matings. Hybridization of a vanHAXY probe to SmaI restriction-digested genomic DNA separated by pulsed-field gel electrophoresis showed that the vanA gene cluster was located on a 40-kb fragment in the original donor strain and on fragments of different sizes (150 to 450 kb) in the transconjugants. No hybridization to vanA gene cluster probes was obtained with plasmid DNA preparations from the donor or transconjugants. These results suggested that in each case, the van genes had integrated into the recipient chromosome. The transconjugants in turn could act as donors of vancomycin resistance, and resistance was transferable to a Rec- recipient. The results of restriction analyses and DNA hybridizations of genomic DNA from the donor and transconjugants were consistent with the transfer of a mobile element that includes the 12.3-kb Tn1546::IS1251 gene cluster and at least 13 kb of additional DNA. This element has been tentatively designated Tn5482. DNA sequence analysis of a fragment predicted to contain the left end of Tn5482 revealed two insertion sequence-like elements: IS1216V and an apparently truncated IS3-like element. Restriction mapping and DNA hybridization patterns of the van gene clusters of three additional clinical isolates from New York City showed an element similar to Tn5482. Transfer of Tn5482 and related elements may be involved in dissemination of vancomycin resistance.

Amino Acid Sequence↗
PubMedDate not supplied

Genetic basis of trimethoprim and O/129 resistance in Vibrio cholerae.

Because of its important consequences on prophylaxis and therapy of cholera and on bacterial identification, we have studied the genetic basis of cross-resistance to trimethoprim and O/129 of strains of Vibrio cholerae O1 independently isolated in Africa. Two classes of bacteria were found. In the first class, the strains were also resistant to ampicillin and kanamycin and to high levels of streptomycin by synthesis of a 3"- or 6-aminoglycoside phosphotransferase. The strains hybridized weakly with a Tn7 probe and all the resistance characters were transferable en bloc to Escherichia coli. The second class included strains which, in addition to trimethoprim and O/129, were resistant to moderate levels of streptomycin and spectinomycin by production of a 3",9-aminoglycoside-aminocyclitol adenylyltransferase. The resistance characters were not self-transferable to E. coli and the host strain hybridized strongly with Tn7. It therefore appears, that both plasmids and transposons are responsible for the dissemination of resistance to trimethoprim and O/129 in Vibrio.

Anti-Bacterial Agents↗
PubMed2002

A PCR test for detecting Escherichia coli O157:H7 based on the identification of the small inserted locus (SILO157).

AIMS: This paper provides identification of a DNA sequence derived from Shiga toxin-producing Escherichia coli (STEC) O157:H7 and information on its utilization for detecting STEC O157 by PCR. METHODS AND RESULTS: Random Amplified Polymorphic DNA and DNA library were used to identify in STEC O157:H7 (strain EDL 933) a 2634-bp Small Inserted Locus, designated SILO157. Analysis of 211 bacterial strains showed that the PCR assays amplifying the SILO157 region could be used to detect STEC O157 with a good specificity. CONCLUSIONS: Characterization of a novel locus in STEC O157 is attractive since the serotype O157:H7 of STEC is still by far the most important serotype associated with more serious diseases. This island encodes putative proteins and especially one that is predicted to be an outer membrane protein designated IHP1. SIGNIFICANCE AND IMPACT OF THE STUDY: Further investigations could now be developed to appreciate the role of the SILO157 in pathogenicity.

Bacterial Outer Membrane Proteins↗
PubMed1999

Identification of Yersinia enterocolitica genes affecting survival in an animal host using signature-tagged transposon mutagenesis.

Pathogenic Yersinia species are associated with both localized and systemic infections in mammalian hosts. In this study, signature-tagged transposon mutagenesis was used to identify Yersinia enterocolitica genes required for survival in a mouse model of infection. Approximately 2000 transposon insertion mutants were screened for attenuation. This led to the identification of 55 mutants defective for survival in the animal host, as judged by their ability to compete with the wild-type strain in mixed infections. A total of 28 mutants had transposon insertions in the virulence plasmid, validating the screen. Two of the plasmid mutants with severe virulence defects had insertions in an uncharacterized region. Several of the chromosomal insertions were in a gene cluster involved in O-antigen biosynthesis. Other chromosomal insertions identified genes not previously demonstrated as being required for in vivo survival of Y. enterocolitica. These include genes involved in the synthesis of outer membrane components, stress response and nutrient acquisition. One severely attenuated mutant had an insertion in a homologue of the pspC gene (phage shock protein C) of Escherichia coli. The phage shock protein operon has no known biochemical or physiological function in E. coli, but is apparently essential for the survival of Y. enterocolitica during infection.

Animals↗