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PubMed1998

Variability in the distribution and composition of insertion sequence-like elements in strains of Mycoplasma fermentans.

Mycoplasma fermentans has been reported to be pathogenic for man. All fourteen strains tested contain an insertion sequence-like element (ISLE) which may be present in multiple copies. To determine whether ISLE copies are similarly distributed in different strains of M. fermentans, restriction enzyme digest fragments of genomic DNA from 14 isolates, from a variety of sources, were separated by electrophoresis, blotted and hybridized to a biotin labelled polymerase chain reaction (PCR) amplified fragment of ISLE. A range of patterns was observed suggesting that the element has a tendency to undergo rearrangement within the genome. Analysis of ISLE sequences revealed inter- and intra-strain polymorphisms.

Base Sequence↗

PubMed2000

cis and trans factors affecting Mos1 mariner evolution and transposition in vitro, and its potential for functional genomics.

Mos1 and other mariner / Tc1 transposons move horizon-tally during evolution, and when transplanted into heterologous species can transpose in organisms ranging from prokaryotes to protozoans and vertebrates. To further develop the Drosophila Mos1 mariner system as a genetic tool and to probe mechanisms affecting the regulation of transposition activity, we developed an in vitro system for Mos1 transposition using purified transposase and selectable Mos1 derivatives. Transposition frequencies of nearly 10(-3)/target DNA molecule were obtained, and insertions occurred at TA dinucleotides with little other sequence specificity. Mos1 elements containing only the 28 bp terminal inverted repeats were inactive in vitro, while elements containing a few additional internal bases were fully active, establishing the minimal cis -acting requirements for transposition. With increasing transposase the transposition frequency increased to a plateau value, in contrast to the predictions of the protein over-expression inhibition model and to that found recently with a reconstructed Himar1 transposase. This difference between the 'natural' Mos1 and 'reconstructed' Himar1 transposases suggests an evolutionary path for down-regulation of mariner transposition following its introduction into a naïve population. The establishment of the cis and trans requirements for optimal mariner transposition in vitro provides key data for the creation of vectors for in vitro mutagenesis, and will facilitate the development of in vivo systems for mariner transposition.

Animals↗

PubMed1994

Insertion sequence-related genetic variation in resting Escherichia coli K-12.

Bacterial subclones recovered from an old stab culture of Escherichia coli K-12 revealed a high degree of genetic diversity, which occurred in spite of a very reduced rate of propagation during storage. This conclusion is based on a pronounced restriction fragment length polymorphism (RFLP) detected upon hybridization with internal fragments of eight resident insertion sequences (IS). Genetic diversity was dependent on the IS considered and, in many cases, a clear consequence of IS transposition. IS5 was particularly active in the generation of variation. All subclones in which IS30 had been active testify to a burst of IS30 transposition. This was correlated with a loss of prototrophy and a reduced growth on rich media. A pedigree of the entire clone could be drawn from the RFLP patterns of the subclones. Out of 118 subclones analyzed, 68 different patterns were found but the putative ancestral population had disappeared. A few patterns were each represented by several subclones displaying improved fitness. These results offer insights into the role of IS elements in the plasticity of the E. coli genome, and they further document that enzyme-mediated DNA rearrangements do occur in resting bacterial cultures.

Biological Evolution↗

PubMed2000

Genetic linkage of the vanB2 gene cluster to Tn5382 in vancomycin-resistant enterococci and characterization of two novel insertion sequences.

VanB-type vancomycin resistance is encoded by the vanB gene cluster, which disseminates by horizontal gene transfer and clonal spread of vancomycin-resistant enterococci (VRE). Genetic linkage of the vanB gene cluster to transposon Tn5382 and the insertion sequences IS16 and IS256-like has previously been shown. In this study linkage of defined vanB gene cluster subtypes to these elements was examined. All the vanB2 subtype strains studied (n=14) revealed co-hybridization of vanB and Tn5382, whereas the strains of vanB1 (n=8) and vanB3 (n=1) subtypes were Tn5382 negative. Conjugative cotransfer of the vanB2 gene cluster and Tn5382 was demonstrated for two strains. DNA sequencing of the vanX(B)-ORFC region in vanB2 strains confirmed that the vanB2 gene cluster is an integral part of Tn5382. No general pattern of linkage was observed with regard to IS16 and IS256-like. Two novel insertion sequences were identified in specific vanB2 subtype strains. (i) A 1611 bp element (ISEnfa110) was detected in the left flank of Tn5382. Its insertion site, lack of terminal inverted and direct repeats, and two conserved motifs in its putative transposase all conform to the conventions of the IS110 family. (ii) A 787 bp element (ISEnfa200) was detected in the vanS(B)-vanY(B) intergenic region. Its ORF encoded a putative protein with 60-70% identity to transposases of the IS200 family. No further copies of ISEnfa110 were found by colony hybridization of 181 enterococcal isolates, whereas ISEnfa200 was found in four additional vanB2 strains from the USA. The five strains had identical ISEnfa200 element insertion sites, and Tn5382 was located downstream from a pbp5 gene conferring high-level ampicillin resistance. These isolates showed related PFGE patterns, suggesting possible clonal spread of a VRE strain harbouring a Tn5382-vanB2-ISEnfa200 element linked to a pbp5 gene conferring ampicillin resistance.

Animals↗

PubMed2003

New insertion sequence elements in the upstream region of cfiA in imipenem-resistant Bacteroides fragilis strains.

The 747-bp cfiA gene, which encodes a metallo-beta-lactamase, and the regions flanking cfiA in six imipenem-resistant and four imipenem-susceptible Bacteroides fragilis strains isolated in Japan were analyzed by PCR and DNA sequencing. The nucleotide sequences of the cfiA genes (designated cfiA(1) to cfiA(10)) of all 10 strains tested varied from that of the standard cfiA gene from B. fragilis TAL2480. However, putative proteins encoded by the cfiA variants contained conserved amino acid residues important for zinc binding and hairpin loop formation, suggesting that cfiA variants have the capability of producing metallo-beta-lactamases with full catalytic activities. PCR assay indicated that six metallo-beta-lactamase-producing, imipenem-resistant strains had an insertion mutation in the region immediately upstream of cfiA. Nucleotide sequencing of the PCR-amplified fragments along with the upstream region of cfiA revealed that there were five new kinds of insertion sequence (IS) elements (designated IS612, IS613, IS614, IS615, and IS616, with a size range of 1,594 to 1,691 bp), of which only IS616 was found to be almost identical to IS1188, one of the IS elements previously identified in the upstream region of cfiA. These elements had target site duplications of 4 or 5 bp in length, terminal inverted repeats (14, 15, or 17 bp in size), and a large open reading frame encoding a putative transposase which is required for the transcription of IS elements. Each element was inserted such that the transcriptional direction of the transposase was opposite to that of cfiA. A computer-aided homology search revealed that, based on the homology of their putative transposases, the sizes of their terminal inverted repeat sequences, and their target site duplications, IS612, IS613, IS614, and IS615 belong to the IS4 family, which includes IS942, previously found in some drug-resistant B. fragilis strains, but that IS616 belongs to the IS1380 family. All the IS elements appear to have putative promoter motif sequences (the -7 region's TAnnTTTG motif and the -33 region's TTG or TG) in their end regions, suggesting that the IS elements provide a promoter for the transcription of cfiA upon insertion. These data provide additional proof that various IS elements may exist to provide a promoter to express the cfiA gene.

Amino Acid Sequence↗

PubMed1988

Site-specific insertion of biologically functional adventitious genes into the Streptomyces lividans chromosome.

We report that transformation of Streptomyces lividans with cloned DNA of the SLP1 genetic element results in integration of the element at the same chromosomal locus (attB) normally occupied by SLP1 in its original host, Streptomyces coelicolor, and in S. lividans that has received SLP1 by mating. We constructed SLP1 derivatives that can integrate foreign DNA at the attB site and used these to introduce adventitious DNA sequences into the S. lividans chromosome. We also identified three regions of SLP1 essential for its integration and demonstrated that integration of the SLP1 element does not require expression of functions necessary for stable maintenance or transfer of extrachromosomal forms of SLP1.

Cloning, Molecular↗

PubMed1994

Transposition of IS117, the 2.5 kb Streptomyces coelicolor A3(2) 'minicircle': roles of open reading frames and origin of tandem insertions.

IS117 is a 2527 bp transposable element from Streptomyces coelicolor A3(2) with a circular transposition intermediate. Disruption of ORF1 of IS117, presumed to encode a transposase, abolished transposition. Deletion or mutation of ORF2 and ORF3, which overlap each other on opposite strands of IS117, caused a c. 20-fold reduction in integration frequency of the circular form of IS117 into the Streptomyces lividans chromosome or into the preferred chromosomal target site cloned on a plasmid in transformation experiments. In contrast, inactivation of ORF2/3 did not significantly influence transposition of IS117 derivatives from an already integrated state in the chromosome to the preferred target site cloned on a plasmid. ORF2 mutants apparently excised readily from the S. lividans chromosome, whereas excision of integrated wild-type IS117 derivatives to yield the unoccupied site was not detected; presumably, therefore, the circular transposition intermediate normally arises replicatively. Attempts to promote integration of a plasmid carrying the attachment site of IS117 by providing the ORF1 product in trans were unsuccessful. Most transformation of S. lividans with circular IS117 derivatives yielded tandem chromosomal insertions, which arose by co-transformation rather than dimerization of a monomeric insert. Typically, two to three transforming elements gave a transformed strain, suggesting a local concentration of transposase as a limit on integration.

Base Sequence↗

PubMed2004

BAC end sequences and a physical map reveal transposable element content and clustering patterns in the genome of Magnaporthe grisea.

Transposable elements (TEs) are viewed as major contributors to the evolution of fungal genomes. Genomic resources such as BAC libraries are an underutilized resource for studying genome-wide TE distribution. Using the BAC end sequences and physical map that are available for the rice blast fungus, Magnaporthe grisea, we describe a likelihood ratio test designed to identify clustering of TEs in the genome. A significant variation in the distribution of three TEs, MAGGY, MGL, and Pot2 was observed among the fingerprint contigs of the physical map. We utilized a draft sequence of M. grisea chromosome 7 to validate our results and found a similar pattern of clustering. By examining individual BAC end sequences, we found evidence for 11 unique integrations of MAGGY or MGL into Pot2 but no evidence for the reciprocal integration of Pot2 into another TE. This suggests that: (a) the presence of Pot2 in the genome predates that of the other TEs, (b) Pot2 was less transpositionally active than other TEs, or (c) that MAGGY and MGL have integration site preference for Pot2. High transition/transversion mutation ratios as well as bias in transition site context was observed in MAGGY and MGL elements, but not in Pot2 elements. These features are consistent with the effects of a Repeat-Induced Point (RIP) mutation-like process occurring in MAGGY and MGL elements. This study illustrates the general utility of a physical map and BAC end sequences for the study of genome-wide repetitive DNA content and organization.

Chromosomes, Artificial, Bacterial↗

PubMed1984

Evolutionary relationship between Tn21-like elements and pBP201, a plasmid from Klebsiella pneumoniae mediating resistance to gentamicin and eight other drugs.

We have characterized pBP201 one of the plasmids from a collection of 46 strains producing adenylyltransferase ANT(2") (Schmidt 1984). It confers resistance to sulphonamides and produces aminoglycoside adenylyltransferases AAD(3") and ANT(2") and beta-lactamase TEM-1. Plasmid pBP201 has a size of 24.8 kilobases (kb) and contains TnA and a Tn21-related element, Tn4000 delta, with deletions in mer and the termini and a substitution at tnpR. In complementation assays with transposition-deficient mutants of Tn21 the element in pBP201 appears to be TnpA+ but TnpR-. It represents a naturally occurring defective transposon. The sequence organization of pBP201 has been compared with that of Tn21-related elements such as Tn2410, Tn2603, Tn2424, Tn1696, and Tn4000. In these transposons the integration sites of resistance genes cat, bla, aacA, aacC or aadB have been identified at two preferential locations; these are at the termini of the streptomycin resistance gene aadA. Two additional sites have been localized in the Tn21 backbone to the right of the mer operon and at res (internal resolution site) and are probably involved in the evolution of these elements. Based on these results a model for the possible genealogy of class II transposons is presented.

Aminoglycosides↗

PubMed1990

Isolation of DNA insertion elements from Rhizobium meliloti which are able to promote transcription of adjacent genes.

In order to select insertion sequences able to promote transcription of flanking genes (ISp elements), three mobilizable RSF1010 derived vectors were constructed. Using promoterless antibiotic resistance genes, ISp elements ranging from 0.75 to 2.9 kb were isolated from Escherichia coli and Rhizobium meliloti. Restriction and hybridization experiments revealed that identical ISp elements could be isolated from different R. meliloti strains and that one of these is similar to an insertion sequence found previously in R. meliloti 2011.

Cloning, Molecular↗

PubMed1991

High fidelity developmental excision of Tec1 transposons and internal eliminated sequences in Euplotes crassus.

Following the sexual phase of its life cycle, the hypotrichous ciliate Euplotes crassus transforms a copy of its chromosomal micronucleus into a transcriptionally active macronucleus containing short, linear, gene-sized DNA molecules. Tens of thousands of DNA breakage and joining, or splicing, events occur during macronuclear development. The DNA removed by such events includes transposon-like elements, referred to as Tec1 elements, as well as segments of unique sequence DNA, termed internal eliminated sequences (IESs). Both types of elements are bounded by short direct repeats. In the current study, a polymerase chain reaction (PCR) and DNA sequencing strategy has been used to examine the fidelity of excision of two Tec1 elements and three IESs. In all cases, the vast majority of excision events were found to be precise, with one copy of the terminal direct repeats retained at the empty site in the macronuclear DNA molecule. These results, in combination with previous studies that have characterized the excised DNA elements, indicate that the two products of excision (the free element and the macronuclear DNA molecule) share DNA sequences. This suggests that excision events are initiated by staggered cuts in the chromosomal DNA.

Animals↗

PubMed1993

Ribosome assembly in three strains of Escherichia coli with mutations in the rpmB,G operon.

The rpmB,G operon of Escherichia coli codes for the synthesis of ribosomal proteins L28 and L33. In one mutant strain (TP28), these two proteins are made at about half their normal rates, ribosome assembly is greatly perturbed and precursor particles accumulate. The mutation in strain TP28 is in a Shine-Dalgarno sequence in the leader region of the rpmB,G messenger RNA. Another mutant, strain AM108, makes neither protein because it has the mobile element IS1 inserted into the rpmB coding sequence. Surprisingly, ribosome assembly in this strain is virtually normal with respect to growth rate. Strain AM90, which fails to make protein L33, has the element IS3 inserted into rpmG and also shows no major defects in ribosome assembly.

Amino Acid Sequence↗

PubMed2006

An active DNA transposon nDart causing leaf variegation and mutable dwarfism and its related elements in rice.

While characterized mutable alleles caused by DNA transposons have been abundant in maize since the discovery of Dissociation conferring variegation by Barbara McClintock, only a few mutable alleles have been described in rice even though the rice genome contains various transposons. Here, we show that a spontaneous mutable virescent allele, pyl-v, is caused by the disruption of the nuclear-coded essential chloroplast protease gene, OsClpP5, due to insertion of a 607-bp non-autonomous DNA transposon, non-autonomous DNA-based active rice transposon one (nDart1), belonging to the hAT superfamily. The transposition of nDart1 can be induced by crossing with a line containing an autonomous element, aDart, and stabilized by segregating out of aDart. We also identified a novel mutable dwarf allele thl-m caused by an insertion of nDart1. The japonica cultivar Nipponbare carries no aDart, although it contains epigenetically silenced Dart element(s), which can be activated by 5-azacytidine. Nipponbare bears four subgroups of about 3.6-kb Dart-like sequences, three of which contain potential transposase genes, and around 3.6-kb elements without an apparent transposase gene, as well as three subgroups of about 0.6-kb nDart1-related elements that are all internal deletions of the Dart-like sequences. Both nDart1 and 3.6-kb Dart-like elements were also present in indica varieties 93-11 and Kasalath. nDart1 appears to be the most active mutagen among nDart1-related elements contributing to generating natural variations. A candidate for an autonomous element, aDart, and a possible application of nDart1 for transposon tagging are discussed.

Alleles↗

PubMed1999

Distribution of insertion sequence-like element IS1272 and its position relative to methicillin resistance genes in clinically important Staphylococci.

The distribution of insertion sequence-like element IS1272 was analyzed for clinical isolates of Staphylococcus aureus, Staphylococcus epidermidis, and Staphylococcus haemolyticus. In each of the staphylococcal species, IS1272 was detected in both methicillin-resistant (MR) and methicillin-susceptible strains of different genetic types. In MR isolates, IS1272 was generally located downstream of the truncated mecR1 gene (DeltamecR1), with an identical junction sequence occurring between DeltamecR1 and IS1272, although insertion of an additional gene sequence in the junction sequence was detected in one S. epidermidis isolate. These findings suggest that the mec element with the rearranged form of mecR1 (DeltamecR1-IS1272) has been transmitted to multiple clones of staphylococci.

Bacterial Proteins↗

PubMed2000

Effects of combination of different -10 hexamers and downstream sequences on stationary-phase-specific sigma factor sigma(S)-dependent transcription in Pseudomonas putida.

The main sigma factor activating gene expression, necessary in stationary phase and under stress conditions, is sigma(S). In contrast to other minor sigma factors, RNA polymerase holoenzyme containing sigma(S) (Esigma(S)) recognizes a number of promoters which are also recognized by that containing sigma(70) (Esigma(70)). We have previously shown that transposon Tn4652 can activate silent genes in starving Pseudomonas putida cells by creating fusion promoters during transposition. The sequence of the fusion promoters is similar to the sigma(70)-specific promoter consensus. The -10 hexameric sequence and the sequence downstream from the -10 element differ among these promoters. We found that transcription from the fusion promoters is stationary phase specific. Based on in vivo experiments carried out with wild-type and rpoS-deficient mutant P. putida, the effect of sigma(S) on transcription from the fusion promoters was established only in some of these promoters. The importance of the sequence of the -10 hexamer has been pointed out in several published papers, but there is no information about whether the sequences downstream from the -10 element can affect sigma(S)-dependent transcription. Combination of the -10 hexameric sequences and downstream sequences of different fusion promoters revealed that sigma(S)-specific transcription from these promoters is not determined by the -10 hexameric sequence only. The results obtained in this study indicate that the sequence of the -10 element influences sigma(S)-specific transcription in concert with the sequence downstream from the -10 box.

Artificial Gene Fusion↗

PubMed2005

Development of a nested PCR method targeting a unique multicopy element, ISMap02, for detection of Mycobacterium avium subsp. paratuberculosis in fecal samples.

This study describes the development of a nested PCR assay that uses a unique element (ISMap02) for Mycobacterium avium subsp. paratuberculosis that is present at six copies within the genome. In addition, the sensitivity of the assay with this element was compared to the sensitivity of detection of the IS900 element in both conventional and real-time PCR assays. The specificity of the ISMap02 element was evaluated by PCR of the DNA extracted from isolates of M. avium subsp. paratuberculosis and M. avium subsp. avium, as well as DNA from M. fortuitum, M. scofulaceum, M. phlei, M. smegmatis, and M. gordonae. Only M. avium subsp. paratuberculosis DNA was detectable after amplification with the ISMap02 primers. The sensitivity of detection for the ISMap02 element in either a conventional or a real-time PCR format was less than 100 fg DNA or 10(2) CFU/ml in serial titration curves with pure bacteria. These results were comparable to those obtained for the IS900 element. Experimental spiking of a negative fecal sample followed by M. avium subsp. paratuberculosis DNA extraction resulted in detection thresholds of 10(2) CFU/g for the IS900 element and 10(3) CFU/g for the ISMap02 element by using a real-time PCR format, but this sensitivity dropped 10-fold for both elements in a conventional PCR format. Analyses of fecal samples obtained from naturally infected animals demonstrated a sensitivity for the detection of M. avium subsp. paratuberculosis DNA by use of the ISMap02 element similar to that achieved by use of the IS900 element when it was used in a conventional PCR format. The real-time PCR format improved the levels of detection of both elements, but not to a significant degree. In conclusion, the ISMap02 element provides a very sensitive and specific alternative as a diagnostic reagent for use in PCR assays for the detection of paratuberculosis.

Animals↗

PubMed1987

[Induction of unstable mutations in Drosophila melanogaster by microinjection of DNA from oncogenic viruses into the embryo polar plasma. II. Analysis of the role of injected DNA].

We have demonstrated that the mutagenic effect of oncovirus DNA injected into Drosophila embryos is of two-type locus specificity: the spectrum of mutations induced by the retroviral cDNA (RSV) changed in different recipient stocks and those induced by the adenoviral DNA (Sa7) did not differ in the stocks studied. The Sa7 DNA and the cDNA of RSV induce mutations in different groups of loci. Transpositions of the copia element were found in mutant lines obtained in both cases.

Adenoviridae↗

PubMed2003

Rapid inactivation of the maize transposable element En/Spm in Medicago truncatula.

Transposable elements have been widely used as mutagens in many organisms. Among them, the maize transposable element En/Spm has been shown to transpose efficiently in several plant species including the model plant Arabidopsis, where it has been used for large-scale mutagenesis. To determine whether we could use this transposon as a mutagen in the model legume plant Medicago truncatula, we tested the activity of the autonomous element, as well as two defective elements, in this plant, and in Arabidopsis as a positive control. In agreement with previous reports, we observed efficient excision of the autonomous En/Spm element in A. thaliana. This element was also active in M. truncatula, but the transposition activity was low and was apparently restricted to the tissue culture step necessary for the production of transgenic plants. The activity of one of the defective transposable elements, dSpm, was very low in A. thaliana and even lower in M. truncatula. The use of different sources of transposases suggested that this defect in transposition was associated with the dSpm element itself. Transposition of the other defective element, I6078, was also detected in M. truncatula, but, as observed with the autonomous element, transposition events were very rare and occurred during tissue culture. These results suggest that the En/Spm element is rapidly inactivated in the regenerated plants and their progeny, and therefore is not suitable for routine insertion mutagenesis in M. truncatula.

Amino Acid Sequence↗