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PubMed2006

Common regions e.g. orf513 and antibiotic resistance: IS91-like elements evolving complex class 1 integrons.

The ability of bacteria to procure, sometimes rearrange, and evince acquired DNA continues to impress us-even more so if this genetic plasticity involves the sequestering of antibiotic resistance genes. The acquisition of genes in bacteria is often facilitated by transposons, integrons and archetype insertion elements. Recently however, a new element, 'orf513', has been increasingly associated with class 1 integrons. Moreover, these 'complex' class 1 integrons can potentially mediate resistance to chloramphenicol, trimethoprim, aminoglycosides and tetracycline and may carry a range of beta-lactamase genes as well as the qnrA gene. Elements such as 'orf513' demonstrate IS91-like characteristics and will mobilize adjacent DNA via a process called rolling circle replication, and thus we have renamed them 'insertion sequence CRs' (ISCRs) to appropriately reflect their structure-function properties. In this article, we provide a brief description of these new and clinically important mobile elements, and how they are able to mobilize antibiotic resistance genes.

Anti-Bacterial Agents↗

PubMed1982

Ribosomal RNA genes of Trypanosoma brucei. Cloning of a rRNA gene containing a mobile element.

An ordered restriction map of the ribosomal RNA genes of Trypanosoma brucei brucei is presented. Bgl II fragments of T.b.brucei genomic DNA were cloned into pAT 153, and the clones containing rDNA identified. Restriction maps were established and the sense strands identified. One clone was shown by heteroduplex mapping to contain a 1.1 kb inserted sequence which was demonstrated to be widely distributed throughout the genomes of members of the subgenus Trypanozoon. However, in two other subgenera of Trypanosoma, Nannomonas and Schizotrypanum, the sequence is far less abundant. Analysis of the genomic DNA from two serodemes of T.b.brucei showed that the sequence was present in the rRNA of only one of them, implying that the sequence is a mobile element and that its appearance in rDNA is a comparitively recent occurrence.

Animals↗

PubMed1996

Gene structure and differential regulation of the Rhizobium-induced peroxidase gene rip1.

Primary expression of the Rhizobium meliloti-induced peroxidase gene rip1 occurs prior to nodule morphogenesis, specifically at the site of impending rhizobial infection (D. Cook, D. Dreyer, D. Bonnet, M. Howell, E. Nony, K. VandenBosch [1995] Plant Cell 7: 43-55). We examined the distribution and structure of rip1 transcript throughout nodule development. We determined that expression of rip1 in root tips is correlated with the competence of this zone for symbiotic association, whereas after rhizobial infection rip1 transcript is specifically associated with the zone of nodule development, including nascent nodule primordia. rip1 transcripts are characterized by multiple polyadenylation sites distributed within 200 to 400 bp of the translation stop site, and a single major transcription initiation site in close proximity to the rip1 open reading frame. Thus, rip1 expression is likely to be mediated through effects on a single transcription unit. Immediately 5' of the rip1 transcription unit DNA sequence analysis identified a 377-bp DNA element containing extensive repeat structure that is widely distributed in the Medicago truncatula genome.

Base Sequence↗

PubMed1992

A Coxiella burnetti repeated DNA element resembling a bacterial insertion sequence.

A DNA fragment located on the 3' side of the Coxiella burnetii htpAB operon was determined by Southern blotting to exist in approximately 19 copies in the Nine Mile I genome. The DNA sequences of this htpAB-associated repetitive element and two other independent copies were analyzed to determine the size and nature of the element. The three copies of the element were 1,450, 1,452, and 1,458 bp long, with less than 2% divergence among the three sequences. Several features characteristic of bacterial insertion sequences were discovered. These included a single significant open reading frame that would encode a 367-amino-acid polypeptide which was predicted to be highly basic, to have a DNA-binding helix-turn-helix motif, to have a leucine zipper motif, and to have homology to polypeptides found in several other bacterial insertion sequences. Identical 7-bp inverted repeats were found at the ends of all three copies of the element. However, duplications generated by many bacterial mobile elements in the recipient DNA during insertion events did not flank the inverted repeats of any of the three C. burnetii elements examined. A second pair of inverted repeats that flanked the open reading frame was also found in all three copies of the element. Most of the divergence among the three copies of the element occurred in the region between the two inverted repeat sequences in the 3' end of the element. Despite the sequence changes, all three copies of the element have retained significant dyad symmetry in this region.

Amino Acid Sequence↗

PubMed1993

Identification of an insertion sequence-like element in a subspecies of Mycobacterium kansasii.

Analysis of a genomic DNA clone library of a strain from the genetic subspecies of Mycobacterium kansasii determined the existence of a repetitive insertion sequence-like element. The element is 947 bp long and is present in a minimum of 1 to 11 copies per genome. Similar to insertion sequences, it contains a 3-bp (TAG) direct repeat at its extremities and a transcription promoter-like sequence. In addition, for one of the clones sequenced, a potential cointegrate formation, a characteristic frequently observed with insertion sequences, was revealed. This insertion sequence does not contain short inverted repeats near the ends or a large open reading frame to code for a transposase enzyme. Its host range is restricted to a previously described genetic subspecies of M. kansasii and is not present in typical M. kansasii or other mycobacterial species. When used as a probe for Southern blot hybridization, significant heterogeneity between different isolates of the M. kansasii subspecies was observed. This repeated element will be useful in further studies on the characterization, diagnosis, and epidemiology of M. kansasii.

Base Sequence↗

PubMed1989

Mechanisms of genetic variability in Halobacterium halobium: the purple membrane and gas vesicle mutations.

Several phenotypic variants of Halobacterium halobium arise spontaneously at extremely high frequencies (up to 1%) and are readily identified by inspection of bacterial colonies. Two mutant types, those lacking the buoyant gas vesicles or the photosynthetic purple membrane, have been studied in detail by phenotypic and molecular genetic analysis. In the wild-type NRC-1 strain, the bop gene, encoding the purple membrane protein bacterio-opsin, is found on the bacterial chromosome, while the gas vesicle protein genes, gvpA and gvpC, are present on pNRC100, a multicopy plasmid of approximately 150 kilobase pairs. The gvpA and gvpC genes are on a single transcription unit, while the major bop mRNA is monocistronic. Essentially all of the purple membrane deficient mutants contain insertion sequence (IS) elements into or upstream of the bop gene. Two elements, ISH 1 and ISH 2, account for most (80-90%) of the purple membrane mutations, but at least three other elements, ISH 23, ISH 26, and ISH S1, have also been implicated. The gas vesicle mutants are more heterogeneous, with many displaying partial phenotypes. Three major classes of gas vesicle mutants are distinguishable: class I and class III mutants are the result of large deletions in pNCR100; however, while class I mutants are partially gas vesicle deficient and contain a correspondingly reduced number of gvpAC operon copies, class III mutants contain no detectable copies of the gas vesicle genes and are essentially completely gas vesicle deficient. Class II mutants, like the purple membrane mutants, contain IS elements into or upstream of the gas vesicle genes.(ABSTRACT TRUNCATED AT 250 WORDS)

Archaeal Proteins↗

PubMed1984

[Molecular genetic studies of the DNA promotor regions in Bacillus thuringiensis subsp. galleriae 69-6. II. Increased level of gene expression resulting from IS1 element integration].

The 1.45 kb promoter containing HindIII fragment of Bacillus thuringiensis DNA promotes the expression of the tet gene of recombinant pPBT9 plasmid in Escherichia coli cells. Spontaneous mutants of this plasmid were isolated and analysed. They are responsible for an increase in the level of tetracycline resistance. This 3-fold increase resulted from integration of IS1 element into the bacillar promoter containing HindIII fragment, which led to formation of a mutant pPBT9::IS1 plasmid. The IS sequence integrated was defined as an IS1 element of the E. coli HB101 chromosomal DNA. The integration site of IS1 was localized.

Bacillus thuringiensis↗

PubMedDate not supplied

[Creation and analysis of Brassica napus + Arabidopsis thaliana somatic hybrids possessing maize Spm/dSpm heterologous transposable system].

Functionally asymmetric somatic hybrids possessing heterologous transposable element Spm/dSpm were obtained following intertribal somatic hybridization between Brassica napus and transgenic Arabidopsis thaliana. Mobile genetic elements actively transposed in the hybrid genomes. Complete elimination of A. thaliana genome was not observed.

Arabidopsis↗

PubMed2002

Human L1 element target-primed reverse transcription in vitro.

L1 elements are ubiquitous human transposons that replicate via an RNA intermediate. We have reconstituted the initial stages of L1 element transposition in vitro. The reaction requires only the L1 ORF2 protein, L1 3' RNA, a target DNA and appropriate buffer components. We detect branched molecules consisting of junctions between transposon 3' end cDNA and the target DNA, resulting from priming at a nick in the target DNA. 5' junctions of transposon cDNA and target DNA are also observed. The nicking and reverse transcription steps in the reaction can be uncoupled, as priming at pre-existing nicks and even double-strand breaks can occur. We find evidence for specific positioning of the L1 RNA with the ORF2 protein, probably mediated in part by the polyadenosine portion of L1 RNA. Polyguanosine, similar to a conserved region of the L1 3' UTR, potently inhibits L1 endonuclease (L1 EN) activity. L1 EN activity is also repressed in the context of the full-length ORF2 protein, but it and a second cryptic nuclease activity are released by ORF2p proteolysis. Additionally, heterologous RNA species such as Alu element RNA and L1 transcripts with 3' extensions are substrates for the reaction.

Base Sequence↗

PubMed1987

Detection and characterization of IS256, an insertion sequence in Staphylococcus aureus.

Resistance to the aminoglycosides gentamicin (Gmr), tobramycin (Tmr) and kanamycin (Kmr) in strains of Staphylococcus aureus isolated in Australia is mediated by the transposon Tn4001. The 1.35 kb inverted repeat of this transposon exhibits many of the characteristics of an insertion sequence and has consequently been designated IS256. Tandem duplication of IS256 contiguous with Tn4001 results in an increase in the level of GmrTmrKmr, thereby implying that the element possesses strong promoter sequences. Both contiguous and independent insertions of IS256 into the staphylococcal chromosome have been observed, the latter suggesting that the element may play a role in molecular rearrangements of the genome.

Chromosome Mapping↗

PubMed1990

The Bacillus subtilis gene for the development transcription factor sigma K is generated by excision of a dispensable DNA element containing a sporulation recombinase gene.

The structural gene (sigK) for the mother-cell RNA polymerase sigma-factor sigma K in Bacillus subtilis is a composite of two truncated genes, named spoIVCB and spoIIIC, which are brought together by site-specific recombination during sporulation. We now show that the recombination event is compartmentalized in that the mother cell, but not the forespore chromosome, undergoes rearrangement. We also show that spoIIIC (encoding the carboxy-terminal portion of sigma K) lies approximately 42 kb downstream of spoIVCB (encoding the amino-terminal portion) and that the joining of the truncated coding sequences is a reciprocal recombination event in which intervening DNA is deleted from the chromosome as a circle. The rearrangement is governed by the product of a gene named spoIVCA located in the excised DNA, as demonstrated by the observations (1) that the product of spoIVCA, but not the product of any other stage-IV sporulation gene tested, is required for the rearrangement, and (2) that the presence of a cloned copy of the rearranged sigK gene in the chromosome bypasses the requirement for the spoIVCA gene product in sporulation. Because cells engineered to contain an intact copy of sigK sporulate normally, we conclude that the sigK rearrangement is not essential for the control of gene expression during sporulation, and we infer the existence of an additional mechanism for restricting sigma K-directed transcription to the mother-cell chamber of the sporangium. Finally, the construction of a strain deleted for the entire sigK intervening sequence shows that the 42-kb element contains no genes essential for viability.

Bacillus subtilis↗

PubMed1998

A reliable amplification technique for the characterization of genomic DNA sequences flanking insertion sequences.

A simple and efficient ligation-mediated PCR (LMPCR) is described for amplifying DNA adjacent to known sequences. The method uses one primer specific for the known sequence and a second specific for a synthetic linker ligated to restricted genomic DNA. Perkin-Elmer AmpliTaq Gold polymerase is used to minimize non-specific primer annealing and amplification. This LMPCR method was successfully applied to isolate DNA sequences flanking mobile elements present in mycobacterial mutants generated by transposon mutagenesis.

Cloning, Molecular↗

PubMed1995

DNA rearrangements in Euplotes crassus coincide with discrete periods of DNA replication during the polytene chromosome stage of macronuclear development.

Macronuclear development in Euplotes crassus begins with polytenization of micronuclear chromosomes and is accompanied by highly precise excision of DNA sequences known as internal eliminated sequences and transposon-like elements (Tecs). Quantitation of radiolabeled-precursor incorporation into DNA indicates that DNA synthesis during formation of polytene chromosomes is not continuous and occurs during two distinct periods. We demonstrate that the timing of Tec excision coincides with these replication periods and that excision can occur during both periods even at a single locus. We also show that Tec and internal eliminated sequence excisions are coincident in the second replication period, thus providing further evidence for similarity in their excision mechanism. Inhibition of DNA synthesis with hydroxyurea diminishes Tec element excision, indicating that replication is an important aspect of the excision process.

Animals↗

PubMed1999

A new Ac-like transposon of Arabidopsis is associated with a deletion of the RPS5 disease resistance gene.

The RPS5 and RFL1 disease resistance genes of Arabidopsis ecotype Col-0 are oriented in tandem and are separated by 1.4 kb. The Ler-0 ecotype contains RFL1, but lacks RPS5. Sequence analysis of the RPS5 deletion region in Ler-0 revealed the presence of an Ac-like transposable element, which we have designated Tag2. Southern hybridization analysis of six Arabidopsis ecotypes revealed 4-11 Tag2-homologous sequences in each, indicating that this element is ubiquitous in Arabidopsis and has been active in recent evolutionary time. The Tag2 insertion adjacent to RFL1 was unique to the Ler-0 ecotype, however, and was not present in two other ecotypes that lack RPS5. DNA sequence from the latter ecotypes lacked a transposon footprint, suggesting that insertion of Tag2 occurred after the initial deletion of RPS5. The deletion breakpoint contained a 192-bp insertion that displayed hallmarks of a nonhomologous DNA end-joining event. We conclude that loss of RPS5 was caused by a double-strand break and subsequent repair, and cannot be attributed to unequal crossing over between resistance gene homologs.

Amino Acid Sequence↗

PubMed1981

The sequence of IS4.

IS-elements are devoid of easily recognizable transacting functions and exert their visible effects in the position cis only (recent reviews Calos and Miller 1980; Starlinger 1980). It has been a matter of debate, whether these elements encode functions for their own transposition. In the case of the E. coli IS-elements this could not easily be determined by genetic methods, because most of these elements are present in several copies (Saedler and Heiss 1973; Deonier et al. 1979). In the case of the IS-elements flanking transposons, evidence has recently been brought forward that these carry the transposition specificity (Rothstein et al. 1980; Kleckner 1980; Grindley 1981). IS4 is present in one copy only in several E. coli K12 strains and should, therefore, be suitable for genetic and physiological studies (Chadwell et al. 1979). It has been cloned from several sites on the E. coli chromosome in pBR322 (Klaer and Starlinger 1980). Here we report the DNA sequence of IS4 which contains an open reading frame for 442 amino acids, and of the junctions of this element with surrounding DNA at three different sites in the E. coli chromosome.

Base Sequence↗

PubMed1980

Cloning of Lac+ BamHI fragment into transposon Tn3 and transposition of the Tn3[lac] element.

By use of recombinant DNA techniques, we have inserted the lac+ operon into a transposon (Tn3). We constructed the recombinant in such a way that the essential step in assaying for transposition consisted of screening for bacteria with a thermostable Lac+ phenotype. Our results showed that transposition of the Tn3[lac+] element occurred and that its frequency was derepressed compared to frequencies reported by others for wild-type Tn3 transposition.

Cloning, Molecular↗

PubMed1998

Oncoviral DNAs induce transposition of endogenous mobile elements in the genome of Drosophila melanogaster.

Previously, we have shown that particles of Rous sarcoma virus or cloned fragments of RSV cDNA as well as DNA of oncogenic simian adenovirus Sa7, injected into the polar plasm of early Drosophila melanogaster embryos, were able to induce, with high frequency, unstable visible mutations in different groups of genetic loci. The genetic instability of the recovered mutations, i.e., their ability to revert to normal state or to generate new mutant alleles at the affected locus, was manifest in mutant lines through several generations. The molecular analysis undertaken in this study of the yellow-scute loci region which is highly sensitive to the microinjected Sa7 DNA, and of the white locus, that frequently mutates under the influence of RSV cDNA, clearly shows that the induced mutations and reversions are accompanied by insertion/excision of endogenous mobile elements. This conclusion is confirmed by in situ hybridization experiments which demonstrate that the adenovirus DNA is able to change, though with different efficiency, the chromosomal localization of certain Drosophila retrotransposons. These results partially elucidate the molecular mechanism of the genetic instability in D. melanogaster induced by microinjection of oncoviruses into early embryos, implying that is results from mobilization of endogenous transposons which play the role of insertional elements directly causing unstable mutations.

Adenoviruses, Simian↗

PubMed1990

SLACS retrotransposon from Trypanosoma brucei gambiense is similar to mammalian LINEs.

We have characterized a retrotransposon in Trypanosoma brucei gambiense uniquely associated with the spliced-leader (SL) RNA gene cluster (Spliced Leader Associated Conserved Sequence, SLACS). There are nine copies of SLACS and DNA sequence analysis of one shows the hallmarks of Line-1 like elements. SLACS has generated a 49 bp target DNA duplication at its insertion site and its 3'-end is preceded by a poly(A) stretch. Two putative open reading frames (ORFs) span 75% of the element. ORF1 has CysHis motif associated with the retroviral gag polypeptide while ORF2 shows homology with reverse transcriptase sequences. Its 5'-end contains a repeated segment of a 185 bp that varies in copy number in different SLACS insertions. Retrotransposon-like sequences inserted into the SL-RNA genes occur in several hemoflagellates. These elements may represent a related family which has maintained its target site specificity.

Amino Acid Sequence↗