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PubMed1996

Assessment of genetic markers for species differentiation within the Mycobacterium tuberculosis complex.

It is important to correctly identify species within the Mycobacterium tuberculosis complex because of the zoonotic implications of bovine tuberculosis, especially in developing countries. We assessed the use of various genetic markers for species-specific identification of mycobacteria from the M. tuberculosis complex. A multiplex PCR designed for detection of the mtp40 and IS1081 elements was optimized and evaluated in 339 mycobacterial strains from different animal and geographic origins. The host range of the IS6110, MPB70, and 16S rRNA genes was also studied by PCR in all the strains. Finally, the usefulness of the genetic markers was compared by an immunoperoxidase test for specific identification of Mycobacterium bovis strains. The mtp40 sequence was detected in 87 of the 91 strains of M. tuberculosis and in 9 of the 11 Mycobacterium africanum strains but not in any of the M. bovis or Mycobacterium microti strains, indicating that the mtp40 element was also found in all of the M. tuberculosis complex strains isolated from seals. This organism is considered to be a true seal pathogen, but its origin is essentially unknown. The finding of the mtp40 element in the strains from seals suggests a closer relationship of these strains with a human origin than to an animal origin. The mtp40 element was not found in any other mycobacterial species included in the study. As a result of this study, we suggest that biochemical tests or alternate genetic markers are still needed to differentiate M. tuberculosis from M. africanum when these species coexist as causative agents of tuberculosis. The immunoperoxidase test worked well for the identification of M. bovis strains. We also report, for the first time, PCR amplification of the repetitive element IS6110 in an isolate of Mycobacterium ulcerans and an isolate of Mycobacterium gilvum, which emphasizes the need for further investigation of the host range of this sequence.

Animals↗

PubMed2004

Distribution of insertion sequences associated with Tn1546-like elements among Enterococcus faecium isolates from patients in Korea.

The vanA gene cluster is carried as a part of Tn1546-like elements. The genetic diversity in Tn1546-like elements has been documented previously. The differences described thus far have included the integration of insertion sequence (IS) elements IS1216V, IS1251, IS1476, and IS1542. Among these, IS1216V has been reported to be widespread in VanA enterococci of diverse geographic areas, whereas IS1542 and IS1476 have been reported only in the United Kingdom and Canada, respectively. We investigated the distribution of ISs among 20 vanA-containing Enterococcus faecium isolates from human patients in nine different university hospitals in Korea. Pulsed-field gel electrophoresis (PFGE) was performed to identify the clonality of the isolates. Moreover, PCR amplification of the internal regions of Tn1546 was performed for structural analysis of the van gene, and both DNA strands of the PCR amplicons were directly sequenced by the dideoxy termination method. The PFGE patterns revealed a high degree of clonal diversity. Structural analyses of the van gene detected IS1542 and IS1216V in the genomes of all 20 isolates, whereas it did not detect IS1476 or IS1251 in the genomes of any of the isolates. In addition, IS19 was detected in the vanS-vanH intergenic region of one isolate. These data indicate that identification of the IS within a vanA gene cluster could be a useful tool in epidemiological investigations. In addition, the distribution of ISs associated with Tn1546-like elements among the Korean isolates is therefore similar to that among European vancomycin-resistant enterococci.

Bacterial Proteins↗

PubMed2002

The 27.8-kb R-plasmid pTET3 from Corynebacterium glutamicum encodes the aminoglycoside adenyltransferase gene cassette aadA9 and the regulated tetracycline efflux system Tet 33 flanked by active copies of the widespread insertion sequence IS6100.

We determined the complete nucleotide sequence of the 27.8-kb R-plasmid pTET3 from Corynebacterium glutamicum LP-6 which encodes streptomycin, spectinomycin, and tetracycline resistance. The antibiotic resistance determinant of pTET3 comprises an intI1-like gene, which was truncated by the insertion sequence IS6100, and the novel aminoglycoside adenyltransferase gene cassette aadA9. The deduced AADA9 protein showed 61% identity and 71% similarity to AADA6 of integron In51 from Pseudomonas aeruginosa. In addition, pTET3 carries the novel repressor-regulated tetracycline resistance determinant Tet 33 which revealed amino acid sequence homology to group 1 tetracycline efflux systems. The highest level of similarity was observed to the tetracycline efflux protein TetA(Z) from the C. glutamicum plasmid pAG1 with 65% identical and 77% similar amino acids. Each antibiotic resistance region of pTET3 is flanked by identical copies of the widespread insertion sequence IS6100 initially identified in Mycobacterium fortuitum. Transposition assays with a cloned copy of IS6100 revealed that this element is transpositionally active in C. glutamicum. These data suggest a central role of IS6100 in the evolutionary history of pTET3 by mediating the cointegrative assembly of resistance gene-carrying DNA segments.

Amino Acid Sequence↗

PubMed1979

Deoxyribonucleic acid sequence homologies among bacterial insertion sequence elements and genomes of various organisms.

Plasmid and phage deoxyribonucleic acid (DNA) harboring bacterial insertion sequence (IS) elements IS1, IS2, and IS5 were characterized and used as probes to detect homologous sequences in various procaryotic and eucaryotic genomes. The hybridization method used permits the detection of sequences partially homologous to the elements. Hybridization of the IS-containing probes to each other revealed a region of limited homology between IS1 and IS2. Homologous sequences were then detected by computer analysis of the published IS1 and IS2 nucleotide sequences. The homologous sequence contains a tandemly repeated tetranucleotide sequence which resembles the repeated sequence at the hot spot for spontaneous mutations in the lacI gene (P. J. Farabaugh, U. Schmeissner, M. Hofer, and J. Miller, J. Mol. Biol. 126:847-863, 1978). Homology between the IS elements and various genomes was determined by hybridizing labeled DNA containing IS1, IS2, and IS5 sequences to Southern blots of chromosomal DNA cleaved with restriction endonucleases. IS1 and IS5 appear limited to the enteric bacteria, whereas IS2 sequences can also be detected in Pseudomonas putida, Pseudomonas aeruginosa, and Serratia marcescens. Bacteria which appear not to possess extrachromosomal elements, e.g., Caulobacter crescentus, did not show homology with any insertion sequences tested. In addition, sequences homologous to IS1, IS2, or IS5 were not detected in Saccharomyces cerevisiae, Dictyostelium discoideum, or calf thymus DNA.

Bacteria↗

PubMed1992

Detection of mycobacterial DNA in pleural fluid from patients with tuberculous pleurisy by means of the polymerase chain reaction: comparison of two protocols.

BACKGROUND: The detection of mycobacterial DNA in clinical samples on the basis of the polymerase chain reaction is a promising approach for the rapid diagnosis of tuberculous infections. No consensus exists, however, about which protocols are most sensitive, and the usefulness of this approach in the diagnosis of tuberculous effusions has been assessed in few patients. METHODS: The sensitivity of two protocols was compared for the detection of DNA from Mycobacterium tuberculosis in samples containing known amounts of mycobacterial DNA and in DNA extracted from 15 tuberculous pleural effusions. The results obtained for pleural fluid have been compared with cytological findings and with results obtained by standard microbiological techniques. RESULTS: Mycobacteria could be detected by acid fast staining in none and by culture in three of the 15 pleural fluid samples. A protocol based on the detection of the IS6110 insertion element (which could detect one mycobacterial genome/sample reproducibly) gave a positive result in nine of the 15 tuberculous effusions, though some samples were only intermittently positive (p less than 0.05 compared with culture). In contrast, a protocol based on the detection of the gene coding for the 65 kD mycobacterial antigen (which could detect mycobacterial genomes only if there were at least 10/sample) gave a positive result in three of the 15 tuberculous effusions. Pleural fluid that was always positive with the amplification procedure detecting the IS6110 sequence contained more neutrophils (30% (SD 27%)) than samples that were intermittently positive or always negative (3% (3%)); mycobacterial DNA was never detected in the four samples containing less than 1% neutrophils. CONCLUSIONS: The amplification of the IS6110 insertion element represents a rapid and sensitive means of detecting M tuberculosis in tuberculous effusions. The enrichment of cells containing mycobacteria (possibly neutrophils) before DNA extraction may be required to improve the sensitivity of this approach.

DNA Transposable Elements↗

PubMed1989

The maize En-1/Spm element transposes in potato.

The maize transposable element En-1 has been introduced into a diploid potato line via transformation with Agrobacterium tumefaciens. The element is transcriptionally active in potato. Numerous En specific RNAs are observed, including a 6 kb transcript characteristic of an active En-1 element in maize. In contrast to maize, where the 6.0 kb transcript is hardly detectable, this transcript is very abundant in the transgenic potato. Transposition of En-1 in the potato clone was analysed by Southern blot hybridization and confirmed by molecular isolation of En-1 excision and integration events. Sequence data of excision and integration sites revealed footprints and target site duplications similar to the ones described for En-1 in maize.

Blotting, Southern↗

PubMed2004

A novel IS element, ISMpa1, in Mycobacterium avium subsp. paratuberculosis.

A novel insertion element belonging to the IS110 family was identified in Mycobacterium avium subsp. paratuberculosis. The IS element, ISMpa1, is 1500 bp and has one ORF encoding a putative transposase. Three copies of ISMpa1 were identified in the M. avium subsp. paratuberculosis genome. The element had inserted into the 3' end of the highly conserved mycobacterial genes prrB and a homologue of M. tuberculosis Rv1593c, and between a putative cytochrome p450 oxygenase and a putative hydrolase. The IS element was present in all (n = 11) M. avium subsp. paratuberculosis strains but not detected in most other mycobacterial species examined, including 10 M. avium subsp. avium isolates of human, avian and porcine origin. However two porcine isolates of M. avium subsp. avium and the reference strain IWGMT49 did harbour ISMpa1. These three strains belong to a previously described subgroup of M. avium subsp. avium based on IS1245 restriction fragment length polymorphism (RFLP) pattern and serovars. All of the M. avium subsp. paratuberculosis strains examined had an identical RFLP pattern when probed with sequences corresponding to the 5' end of ISMpa1, whereas a different pattern was seen in the positive M. avium subsp. avium strains. This novel IS element might be a useful tool in strain classification of M. avium subsp. avium and also for the identification of M. avium subsp. paratuberculosis when used in combination with IS900.

DNA Transposable Elements↗

PubMed1998

Unusual insertion element polymorphisms in the promoter and terminator regions of the mucAB-like genes of R471a and R446b.

We have previously identified umu-complementing genes on two incL/M plasmids, R471a and R446b (C. Ho et al., J. Bacteriol., 175 (1993) 5411-5419). Molecular analysis of these genes revealed that they are more structurally and functionally related to mucAB from the incN plasmid pKM101 than to other members of the previously identified Umu-like family. As a consequence, we have termed these new homologs mucAB(R471a) and mucAB(R446b) respectively. Interestingly, while the location of the mucAB-like genes is essentially the same in both R471a and R446b, the regions immediately flanking the mucAB-like genes are highly polymorphic. For example, 5' to mucAB(R471a) we found an insert that appears to be a novel retroelement encoding a putative reverse transcriptase (RT). This RT is related to the reverse transcriptases encoded by group II introns but is embedded in a retron-like context. Immediately 3' to the mucAB(R471a) locus is a putative insertion element of a sparsely-dispersed class not previously reported from enteric bacteria. Both the RT and insertion element are absent in R446b. These observations suggest that the mucAB-like genes from R471a and R446b are located within regions of the R-plasmids that perhaps were once (or still are) mobile genetic elements. Such observations might help explain the distribution of umu-like genes on R-plasmids and bacterial chromosomes.

Amino Acid Sequence↗

PubMed1992

A new family of yeast nuclear pore complex proteins.

We have identified a novel family of yeast nuclear pore complex proteins. Three individual members of this family, NUP49, NUP100, and NUP116, have been isolated and then characterized by a combination of molecular genetics and immunolocalization. Employing immunoelectron and immunofluorescence microscopy on yeast cells, we found that the binding of a polyspecific monoclonal antibody recognizing this family was predominantly at the nuclear pore complexes. Furthermore, the tagging of NUP49 with a unique epitope enabled the immunolocalization of this protein to the nuclear pore complex by both fluorescence and electron microscopy. DNA sequence analysis has shown that the amino-terminal regions of NUP49, NUP100, and NUP116 share repeated "GLFG" motifs separated from each other by glutamine, asparagine, serine and threonine rich spacers. All three proteins lack a repetitive domain found in the two precisely described yeast nuclear pore complex proteins. Only NUP49 is essential for cell viability. NUP116-deficient cells grow very slowly and are temperature sensitive, whereas the lack of NUP100 has no detectable phenotype. NUP100 and NUP116 are homologous over their entire lengths. Interestingly, NUP100 and NUP116 are both flanked by a histidine tRNA gene and a transposon element suggesting that they may have arisen by gene duplication. We propose that subfamilies of pore complex proteins can be defined by their characteristic combinations of different modular domains.

Amino Acid Sequence↗

PubMed1992

Artiodactyl retroposons: association with microsatellites and use in SINEmorph detection by PCR.

During a search of polymorphic microsatellites for bovine genome mapping, we found that microsatellites often occur as tails of artiodactyl C-A retroposon elements. In this element, C (85bp) is a tRNA derivative, while A (117bp) is of unknown origin. The A element also occurs as dimer element with a connecting 27bp linker sequence comprising hexanucleotide CACTTT repeats. In 10 clones (45% of those selected deliberately for dinucleotide repeats), the microsatellite motif is associated with the C-A retroposon. In 50% of 44 database artiodactyl C-A sequences, the element also has a microsatellite tail. The microsatellite is usually a simple (CA)n repeat, but in some cases it is an apparent derivative of the linker sequence CACTTT. All but one of 33 database dimer elements have trinucleotide repeat tails (AGC)n, n = 1-9. Microsatellites, retroposons, and their truncated versions (C and/or A) often occur as clusters. We derived the consensus sequence (202bp) of the C-A element, and designed four primers for inter-SINE amplification with the aim of finding SINEmorph polymorphisms. The method is potentially powerful for rapidly producing polymorphic markers for artiodactyl genome mapping.

Animals↗

PubMed1999

New insertion sequences and a novel repeated sequence in the genome of Mycobacterium tuberculosis H37Rv.

The genome sequence of Mycobacterium tuberculosis H37Rv was found to contain 56 loci with homology to insertion sequences (ISs). As well as the previously described IS6110, IS1081, IS1547 and IS-like elements, new ISs belonging to the IS3, IS5, IS21, IS30, IS110, IS256 and ISL3 families were identified. In addition, six ISs created a grouping of their own to form a new family (the IS1535 family). Elements with similarity to ISs in other actinomycetes were identified, suggesting the movement of ISs between related genera. The location of ISs on the chromosome revealed that an approximately 600 kb region close to the origin of replication lacks ISs, pointing to the possible detrimental effect of insertions in this area. Analysis of the distribution of ISs through the tubercle strains Mycobacterium africanum, M. microti, M. bovis, M. bovis BCG Pasteur, M. tuberculosis H37Ra, M. tuberculosis CSU#93 and 29 clinical isolates revealed that only IS1532, IS1533, 1S1534, and IS1561' were absent from some of the strains tested. A novel repeated sequence, the REP13E12 family, is described that is present in seven copies on the M. tuberculosis H37Rv chromosome and which contains a probable phage attachment site. This study therefore offers an insight into the possible role of ISs and repetitive elements in the evolution of the M. tuberculosis genome, as well as identifying genetic markers that may be useful for phylogenetic and epidemiological analysis of the tubercle complex.

Base Sequence↗

PubMed1995

Negative and positive regulation of Tn10/IS10-promoted recombination by IHF: two distinguishable processes inhibit transposition off of multicopy plasmid replicons and activate chromosomal events that favor evolution of new transposons.

Tn10 is a composite transposon; inverted repeats of insertion sequence IS10 flank a tetracycline-resistance determinant. Previous work has identified several regulatory processes that modulate the interaction between Tn10 and its host. Among these, host-specified DNA adenine methylation, an IS10-encoded antisense RNA and preferential cis action of transposase are particularly important. We now find that the accessory host protein IHF and the sequences that encode the IHF-binding site in IS10 are also important regulators of the Tn10 transposition reaction in vivo and that these determinants are involved in two distinguishable regulatory processes. First, IHF and the IHF-binding site of IS10, together with other host components (e.g., HU), negatively regulate the normal intermolecular transposition process. Such negative regulation is prominent only for elements present on multicopy plasmid replicons. This multicopy plasmid-specific regulation involves effects both on the transposition reaction per se and on transposase gene expression. Second, specific interaction of IHF with its binding site stimulates transposon-promoted chromosome rearrangements but not transposition of a short Tn10-length chromosomal element. However, additional considerations predict that IHF action should favor chromosomal transposition for very long composite elements. On the basis of these and other observations we propose that, for chromosomal events, the major role of IHF is to promote the evolution of new IS10-based composite transposons.

Bacterial Proteins↗

PubMed1985

Repeated sequences similar to insertion elements clustered around the nif region of the Rhizobium japonicum genome.

Two different repeated sequences (RSs) were discovered in the Rhizobium japonicum genome: RSRj alpha is 1126 base pairs long and is repeated 12 times; RSRj beta is approximately 950 base pairs long and is repeated at least 6 times. Their arrangement in root nodule bacteroid DNA is the same as in DNA from bacteria grown in culture. Deletion analysis showed that many copies of alpha and beta are clustered around the nitrogenase genes nifDK and nifH, or, in general, they are found within a genomic region harboring genes that are nonessential for growth. One copy each of alpha and beta are located upstream of nifDK and are adjacent to each other. Neither of them, however, is involved in the expression of nifDK. Nucleotide sequence analysis of three copies of RS alpha revealed many characteristics of procaryotic insertion sequence elements: potential inverted repeats at their ends, potential target site duplication, and large open reading frames. Despite this, their genomic positions appear to be stable. One possible function of these RSs is in deletion formation probably via recombination between them.

DNA Transposable Elements↗

PubMed2004

A proposal to rename the hyperthermophile Pyrococcus woesei as Pyrococcus furiosus subsp. woesei.

Pyrococcus species are hyperthermophilic members of the order Thermococcales, with optimal growth temperatures approaching 100 degrees C. All species grow heterotrophically and produce H2 or, in the presence of elemental sulfur (S(o)), H2S. Pyrococcus woesei and P. furiosus were isolated from marine sediments at the same Vulcano Island beach site and share many morphological and physiological characteristics. We report here that the rDNA operons of these strains have identical sequences, including their intergenic spacer regions and part of the 23S rRNA. Both species grow rapidly and produce H2 in the presence of 0.1% maltose and 10-100 microM sodium tungstate in S(o)-free medium. However, P. woesei shows more extensive autolysis than P. furiosus in the stationary phase. Pyrococcus furiosus and P. woesei share three closely related families of insertion sequences (ISs). A Southern blot performed with IS probes showed extensive colinearity between the genomes of P. woesei and P. furiosus. Cloning and sequencing of ISs that were in different contexts in P. woesei and P. furiosus revealed that the napA gene in P. woesei is disrupted by a type III IS element, whereas in P. furiosus, this gene is intact. A type I IS element, closely linked to the napA gene, was observed in the same context in both P. furiosus and P. woesei genomes. Our results suggest that the IS elements are implicated in genomic rearrangements and reshuffling in these closely related strains. We propose to rename P. woesei a subspecies of P. furiosus based on their identical rDNA operon sequences, many common IS elements that are shared genomic markers, and the observation that all P. woesei nucleotide sequences deposited in GenBank to date are > 99% identical to P. furiosus sequences.

Acid Phosphatase↗

PubMed1985

Evolution of Tn21-related transposons: isolation of Tn2425, which harbours IS161.

The isolation of two multi-resistance transposons, Tn2425 and Tn1831, and their relation to Tn21 and Tn2424, is described. A 1.7 kb segment present in Tn2424 and Tn2425 was identified as an IS element by rec-independent transposition, resulting in a cointegrate structure that carries two direct repeated copies of the IS element. By the isolation of this IS element we demonstrated that transposition is one mechanism leading to sequence variations in Tn21-like structures, especially in the region between the mer operon and the sul gene.

Ampicillin↗

PubMed1997

Identification and characterization of IS1381, a new insertion sequence in Streptococcus pneumoniae.

A new insertion sequence (IS1381) was identified in the genome of Streptococcus pneumoniae R6 as an 846-bp segment containing 20-bp terminal inverted repeats and flanked by 7-bp direct repeats. The three sequenced copies of this element have two overlapping open reading frame (ORF) genes named orfA and orfB. However, significant variations between individual copies were found, suggesting that inactivating mutations have occurred in an original single ORF. Accordingly, the consensus IS1381 element derived from the comparison of the three available copies should contain a single ORF sufficient to encode a basic protein of 267 amino acids which exhibited high similarity to the putative transposases of ISL2 from Lactobacillus helveticus and of IS702 from the cyanobacterium Calothrix sp. strain PCC 7601. A minimum of five to seven copies were detected by hybridization experiments in the R6 genome. In remarkable contrast with the two previously reported pneumococcal insertion sequences, several copies of IS1381 have been detected in all of the clinical isolates tested so far. Interestingly, Streptococcus oralis NCTC 11427 (type strain), a close relative of pneumococcus, does not contain this element, but its occurrence in the type strain of Streptococcus mitis (NCTC 12261) suggests that this species has exchanged DNA with S. pneumoniae directly or through an intermediate species.

Amino Acid Sequence↗

PubMed1995

Analysis of splice donor and acceptor site function in a transposable gene trap derived from the maize element Activator.

Gene trap vectors have been used in insertional mutagenesis in animal systems to clone genes with interesting patterns of expression. These vectors are designed to allow the expression of a reporter gene when the vector inserts into a transcribed region. In this paper we examine alternative splicing events that result in the expression of a GUS reporter gene carried on a Ds element which has been designed as a gene trap vector for plants. We have developed a rapid and reliable method based on PCR to study such events. Many splice donor sites were observed in the 3' Ac border. The relative frequency of utilisation of certain splice donor and acceptor sites differed between tobacco and Arabidopsis. A higher stringency of splicing was observed in Arabidopsis.

Arabidopsis↗

PubMed1984

Complete nucleotide sequence of an unusual mobile element from trypanosoma brucei.

The complete nucleotide sequence of a mobile element from Trypanosoma brucei is presented along with the sequence of its target site, which shows that the insertion has generated a 7 base pair direct repeat. The cloned copy of the element is a dimeric structure, one end of each monomer consisting of a stretch of 14 A residues preceded by a putative trypanosome polyadenylation signal. Six base pairs of DNA of unknown origin are found in the dimer between the two copies of the element. Evidence suggests that the element is present in the genome mainly as a monomer whose sequence is conserved across several species of trypanosome. The element contains an open reading frame encoding the same 160 amino acid protein in both sequenced copies and is extensively transcribed from both strands.

Animals↗