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PubMed2000

Tca5, a Ty5-like retrotransposon from Candida albicans.

This report describes the identification and characterization of a retrotransposon, termed Tca5, from the pathogenic yeast Candida albicans. Tca5 has identical 685 bp LTRs flanking 4218 bp of internal sequence within which lies a single long ORF. Immediately internal to the left LTR is a primer binding site complementary to an internal portion of the initiator methionine tRNA and upstream of the right LTR is a polypurine tract. The ORF predicts a protein containing all the conserved motifs characteristic of Gag, protease, integrase, reverse transcriptase and RNaseH. Genomic Southern blots probed with Tca5 sequences show that it is a low copy number element and is present at different loci in different strains. This, together with the apparently intact structure of Tca5, suggests that it has transposed very recently. Potentially full-length Tca5 transcripts were detected in some strains raising the possibility that some copies of Tca5 may still be active. Phylogenetic analyses and other sequence comparisons suggest that Tca5 is most closely related to the Ty5 element of Saccharomyces cerevisiae and S. paradoxus. The nucleotide sequence of Tca5 has been submitted to GenBank under Accession No. AF093417.

Amino Acid Sequence↗

PubMed1985

Long interspersed sequences in mammalian DNA. Properties of newly identified specimens.

The genomes of primates and of rodents contain numerous long interspersed sequences or LINEs, which are mutually homologous and show characteristics of inserted reverse transcripts or retroposons. Here I report the identification of five new specimens in published DNA sequences, including the first two examples from the rat. These specimens demonstrate the generality of certain sequence arrangements seen in LINEs, viz.: 5' truncation; internal inversion with deletion; clustering with other retroposons; and evolutionary divergence at the 3' end. The 3' segments show a patchwork pattern of homology suggestive of frequent sequence exchanges between multiple subfamilies.

Animals↗

PubMed1998

Host proteins can stimulate Tn7 transposition: a novel role for the ribosomal protein L29 and the acyl carrier protein.

The bacterial transposon Tn7 is distinguished by its ability to insert at a high frequency into a specific site in the Escherichia coli chromosome called attTn7. Tn7 insertion into attTn7 requires four Tn7-encoded transposition proteins: TnsA, TnsB, TnsC and TnsD. The selection of attTn7 is determined by TnsD, a sequence-specific DNA-binding protein. TnsD binds attTn7 and interacts with TnsABC, the core transposition machinery, which facilitates the insertion of Tn7 into attTn7. In this work, we report the identification of two host proteins, the ribosomal protein L29 and the acyl carrier protein (ACP), which together stimulate the binding of TnsD to attTn7. The combination of L29 and ACP also stimulates Tn7 transposition in vitro. Interestingly, mutations in L29 drastically decrease Tn7 transposition in vivo, and this effect of L29 on Tn7 transposition is specific for TnsABC+D reactions.

Acyl Carrier Protein↗

PubMed2006

Identification and differentiation of European and African/Australian strains of Mycoplasma mycoides subspecies mycoides small-colony type using polymerase chain reaction analysis.

Mycoplasma mycoides subspecies mycoides small-colony type (M. m. m. SC) is the cause of the economically important contagious bovine pleuropneumonia. Isolates from Africa and Australia have previously been documented to have a fragment of approximately 8.84 kb, which is absent in European strains. A set of polymerase chain reaction (PCR) primers over this region was designed to identify M. m. m. SC isolates and separate European strains from those of Africa/Australia. Specificity of the PCR assay was achieved through the positioning of an oligonucleotide within the insertion sequence IS1296, upstream of this deletion, which then was paired with a reverse primer, upstream of the deletion, within the 8.84 kb-deleted region or downstream of the deletion, generating fragments of 1.1 kb (all M. m. m. SC strains), 1.4 kb (African/Australian strains only) and 1.3 kb (European strains only), respectively. Identification and differentiation was specific for DNA from M. m. m. SC with no amplification of DNA from other cluster members or closely related species. The PCR products did not require differentiation by use of a restriction endonuclease, and have potential for use in detection of this organism in clinical samples.

Africa↗

PubMed2004

Rapid identification and mapping of insertion sequences in Escherichia coli genomes using vectorette PCR.

BACKGROUND: Insertion sequences (IS) are small DNA segments capable of transposing within and between prokaryotic genomes, often causing insertional mutations and chromosomal rearrangements. Although several methods are available for locating ISs in microbial genomes, they are either labor-intensive or inefficient. Here, we use vectorette PCR to identify and map the genomic positions of the eight insertion sequences (IS1, 2, 3, 4, 5, 30, 150, and 186) found in E. coli strain CGSC6300, a close relative of MG1655 whose genome has been sequenced. RESULTS: Genomic DNA from strain CGSC6300 was digested with a four-base cutter Rsa I and the resulting restriction fragments ligated onto vectorette units. Using IS-specific primers directed outward from the extreme ends of each IS and a vectorette primer, flanking DNA fragments were amplified from all but one of the 37 IS elements identified in the genomic sequence of MG1655. Purification and sequencing of the PCR products confirmed that they are IS-associated flanking DNA fragments corresponding to the known IS locations in the MG1655 genome. Seven additional insertions were found in strain CGSC6300 indicating that very closely related isolates of the same laboratory strain (the K12 isolate) may differ in their IS complement. Two other E. coli K12 derivatives, TD2 and TD10, were also analyzed by vectorette PCR. They share 36 of the MG1655 IS sites as well as having 16 and 18 additional insertions, respectively. CONCLUSION: This study shows that vectorette PCR is a swift, efficient, reliable method for typing microbial strains and identifying and mapping IS insertion sites present in microbial genomes. Unlike Southern hybridization and inverse PCR, our approach involves only one genomic digest and one ligation step. Vectorette PCR is then used to simultaneously amplify all IS elements of a given type, making it a rapid and sensitive means to survey IS elements in genomes. The ability to rapidly identify the IS complements of microbial genomes should facilitate subtyping closely related pathogens during disease outbreaks.

Bacterial Typing Techniques↗

PubMed1992

Control of transcription of Drosophila retrotransposons.

Studies of transcriptional control sequences responsible for regulated and basal-level RNA synthesis from promoters of Drosophila melanogaster retrotransposons reveal novel aspects of gene regulation and lead to identification of trans-acting factors that can be involved in RNA polymerase II transcription not only of retrotransposons, but of many other cellular genes. Comparisons between promoters of retrotransposons and some other Drosophila genes demonstrate that there is a greater variety in basal promoter structure than previously thought and that many promoters may contain essential sequences downstream from the RNA start site.

Animals↗

PubMed1997

Identification and characterization of IS1476, an insertion sequence-like element that disrupts VanY function in a vancomycin-resistant Enterococcus faecium strain.

The vanY gene of vancomycin-resistant enterococci encodes a D,D-carboxypeptidase. By using a PCR detection strategy, a VanA Enterococcus faecium clinical isolate was found to have an insertion sequence (IS)-like element designated IS1476 in vanY. The activity of the VanY D,D-carboxypeptidase in this isolate was decreased in a fluorometric fluoraldehyde o-phthalaldehyde assay with diacetyl-L-Lys-D-Ala-D-Ala as the substrate. This, to our knowledge, is the first report of an IS-like element in a vancomycin resistance gene.

Anti-Bacterial Agents↗

PubMed1993

Diversity of chromosomal genetic elements and gene identification in antibiotic-resistant strains of Streptococcus pneumoniae and Streptococcus bovis.

Antibiotic-resistant Streptococcus pneumoniae (26 strains) and Streptococcus bovis (28 strains), devoid of R plasmids, were examined for DNA-DNA homology to Tn916 and Tn3701. Tn916-like structures were found in 17 S. pneumoniae and 21 S. bovis strains. Tn916-modified structures were present in 6 S. pneumoniae and 2 S. bovis strains. Two strains of each species carried elements having a Tn3701-like composite structure. All these elements were chromosome-borne. No chromosomal elements were detected in 1 S. pneumoniae and 3 S. bovis strains.

Chromosomes, Bacterial↗

PubMed2005

Identification of Actinobacillus suis genes essential for the colonization of the upper respiratory tract of swine.

Actinobacillus suis has emerged as an important opportunistic pathogen of high-health-status swine. A colonization challenge method was developed, and using PCR-based signature-tagged transposon mutagenesis, 13 genes belonging to 9 different functional classes were identified that were necessary for A. suis colonization of the upper respiratory tract of swine.

Actinobacillus Infections↗

PubMed2004

Fluorescence resonance energy transfer (FRET) based molecular detection of a genetically modified PCB degrader in soil.

Genetic analysis of the location of a mini-Tn5 promoted insertion of the LB400 bph operon in the rhizosphere coloniser Pseudomonas fluorescens F113rifPCB, allowed the development of a specific PCR detection system based on the unique DNA sequence at this insertion site. Real time PCR using both SYBR green chemistry and Fluorescence Resonance Energy Transfer probes allowed the precise identification of the recombinant strain and its quantitative detection in soil microcosms over a (bacteria/g) range of five orders of magnitude. This new assay can detect the genetically modified microorganism from soil in less than 90 min and at levels below the detection limits of standard PCR or cultivable counts on selective media.

Benzothiazoles↗

PubMed2000

Evaluation of the accuracy and reproducibility of a practical PCR panel assay for rapid detection and differentiation of Mycobacterium avium subspecies.

The Mycobacterium avium subspecies (MAs) include the closely related MAs avium and MAs paratuberculosis. This study was conducted to evaluate the performance of a PCR panel assay as a diagnostic tool to detect and differentiate MAs avium and MAs paratuberculosis infection. Specific oligonucleotides primers derived from the 16 S rRNA (MAs) sequence, insertion elements IS 901 (MAs avium), IS 1245 Mycobacterium avium complex (MAC), IS 900 (MAs paratuberculosis), and the hspX (MAs paratuberculosis) gene sequences were synthesized and used in preassembled PCR reaction mixtures. These five primer sets made up the PCR panel assay. To determine the accuracy of the PCR panel assay for MAs avium and MAs paratuberculosis strain detection and differentiation, lysates of mycobacterial DNA from 120 (n=120) strains were tested with the PCR panel assay by one laboratory (#1). The PCR panel assay specifically detected and differentiated 91/91 (100%) of MAs avium and MAs paratuberculosis strains tested in this study. The PCR panel assay also specifically differentiated all MAs avium and MAs paratuberculosis strains from all but one (M. intracellulare, serovar 23) of the other mycobacterial strains tested. To confirm the accuracy and evaluate the reproducibility of the PCR panel assay, samples were numbered and given to a different laboratory (#2) as 'unknowns' for identification by the PCR panel assay. In this study, the overall accuracy for strain identification using the PCR panel assay was 99.2% (119/120). The reproducibility of the PCR panel assay when comparing data from laboratory #1 with laboratory #2 was found to be 100% (120/120). These results indicate that this 'easy-to-use', rapid PCR method can accurately and reliably detect and differentiate closely related MAs avium and MAs paratuberculosis from each other and from other mycobacterial species. The PCR panel assay can also differentiate mixed cultures of MAs. The simplicity of this PCR method could be beneficial to laboratories that test for members of MA.

Animals↗

PubMed1988

Safe, live Vibrio cholerae vaccines?

Mutants of Vibrio cholerae defective in intestinal colonization have been constructed. Characterization of these mutants has led to the identification of a gene cluster involved in the assembly of a pilus colonization factor called TCP. The tcp operon has been cloned and strains of V. cholerae have been constructed that overproduce this pilus and the B subunit of cholera toxin. Together these studies may contribute to the eventual construction of efficient live and killed, oral cholera vaccines.

Administration, Oral↗

PubMed2000

Development of a genetically modified bacteriophage for use in tracing sources of pollution.

Bacteriophage are frequently used as biotracers to identify the source of water pollutants. Genetic manipulation of bacteriophage M13mp18 has been used to enhance this technique by creating a library in which each recombinant bacteriophage genome contains a unique identification sequence. Techniques that identify a recombinant bacteriophage by the presence of the identification sequence, including polymerase chain reaction, restriction site polymorphism and plaque hybridization, have been developed. Recombinant bacteriophage can be used to test a large number of suspected sources simultaneously. The identification sequence also eliminates confusion with natural bacteriophage present in water samples. The performance of the modified bacteriophage and the techniques were assessed in simulated field trials on a restricted site carried out under a consent for environmental release of a genetically modified organism. The techniques were also field tested at sites in northwest England using wild-type M13 bacteriophage.

Bacteriophage M13↗

PubMed2006

Characterization of a Brucella sp. strain as a marine-mammal type despite isolation from a patient with spinal osteomyelitis in New Zealand.

Naturally acquired infection of humans with a marine mammal-associated Brucella sp. has only been reported once previously in a study describing infections of two patients from Peru. We report the isolation and characterization of a strain of Brucella from a New Zealand patient that appears most closely related to strains previously identified from marine mammals. The isolate was preliminarily identified as Brucella suis using conventional bacteriological tests in our laboratory. However, the results profile was not an exact match, and the isolate was forwarded to four international reference laboratories for further identification. The reference laboratories identified the isolate as either B. suis or B. melitensis by traditional bacteriological methods in three laboratories and by a molecular test in the fourth laboratory. Molecular characterization by PCR, PCR-restriction fragment length polymorphism, and DNA sequencing of the bp26 gene; IS711; the omp genes omp25, omp31, omp2a, and omp2b; IRS-PCR fragments I, III, and IV; and five housekeeping gene fragments was conducted to resolve the discrepant identification of the isolate. The isolate was identified to be closely related to a Brucella sp. originating from a United States bottlenose dolphin (Tursiops truncatus) and common seals (Phoca vitulina).

Animals↗

PubMed1994

Identification of spliced RNA species of Drosophila melanogaster gypsy retrotransposon. New evidence for retroviral nature of the gypsy element.

We have identified a novel RNA species of Drosophila melanogaster gypsy retrotransposon that is ca. 2 kb in length and corresponds to the third open reading frame (ORF3) of the gypsy element. This RNA is generated by splicing of the primary gypsy transcript, as is the case for retroviral env gene expression. Therefore, the striking resemblance between gypsy and retroviruses has now been extended by this study to the expression strategies of these retroelements. The primary structure of spliced RNA was determined, and its analysis shows that both gypsy subfamilies (6K and 7K) apparently are able to encode functionally active ORF3 translation products.

Animals↗

PubMed2001

The diversity of retrotransposons in the yeast Cryptococcus neoformans.

We have undertaken an analysis of the retrotransposons in the medically important basidiomycetous fungus Cryptococcus neoformans. Using the data generated by a C. neoformans genome sequencing project at the Stanford Genome Technology Center, 15 distinct families of LTR retrotransposons and several families of non-LTR retrotransposons were identified. Members of at least seven families have transposed recently and are probably still active. For several families, only partial elements could be identified and these are quite diverse in sequence, suggesting that they are ancient components of the C. neoformans genome. Most C. neoformans elements are not closely related to previously identified fungal retrotransposons, suggesting that the diversity of fungal retrotransposons has been only sparsely sampled to date. C. neoformans has fewer distinct retrotransposon families than Candida albicans (37 or more), in particular fewer families represented solely by ancient and inactive elements, but it has considerably more families than either Saccharomyces cerevisiae (five) or Schizosaccharomyces pombe (two). The findings suggest that elimination of retrotransposons is faster in C. neoformans than in C. albicans, but perhaps not as rapid as in S. cerevisiae or Sz. pombe. The identification of the retrotransposons of C. neoformans should assist in the molecular characterization of this important pathogen, and also further our understanding of the role played by retroelements in genome evolution.

Amino Acid Sequence↗

PubMed1984

The identification and cloning of genes encoding haloaromatic catabolic enzymes and the construction of hybrid pathways for substrate mineralization.

This paper reviews the genetic basis of haloaromatic biodegradation by bacteria, with a focus on the genetic analysis of Alcaligenes eutrophus JMP134, an organism which can utilize 3-chlorobenzoate, 2,4-dichlorophenoxyacetate (2,4-D) and related compounds as sole carbon and energy sources, and Pseudomonas sp. B13, a chlorobenzoate degrader. The involvement of transmissible plasmids pJP4 and pWR1, isolated from strains JMP134 and B13, respectively, in chloroaromatic mineralization has been examined, and restriction fragments of both plasmids have been cloned on the broad host range plasmid vector pKT231. Transposon Tn5 mutagenesis of these and other soil isolates enriched in and purified from mixed cultures utilizing 2,4,5-trichlorophenoxyacetate (2,4,5-T) as sole carbon and energy source, has been carried out using a "suicide" transposon donor, pLG221 (Co1Ibdrd-1::Tn5). Mapping of Tn5 insertions in mutants which accumulate pathway intermediates has facilitated the identification and cloning of genes encoding chlorocatechol 1,2-dioxygenase, and other key enzymes in haloaromatic catabolism. There are good prospects for the genetic construction of hybrid haloaromatic catabolic pathways by combining genes encoding broad specificity enzymes, capable of transforming halogenated analogues of their natural substrates, with genes for halocatechol degradation.

2,4-Dichlorophenoxyacetic Acid↗

PubMed2003

Identification of a new hobo element in the cabbage moth, Mamestra brassicae (Lepidoptera).

A complete hobo-like element, called Mbhobo, was identified in the cabbage moth, Mamestra brassicae. This element has a high sequence similarity to the HFL1 hobo element of Drosophila melanogaster. Amplification of Mbhobo termini indicated that transposition occurred into a 5'-GTGGGTAC-3' target sequence that was duplicated upon insertion. This target site conforms to the consensus sequence established for the insertion sites of insect hAT elements. Mbhobo has a single 1935 bp long ORF with significant homology to the D. melanogaster HFL1 hobo transposase. FISH experiments evidenced Mbhobo clusters located in heterochromatic regions of Z and W sex chromosomes and in heterochromatic areas of chromosome pair 10.

Amino Acid Sequence↗