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PubMed1994

TUBIS, a fossilized retroposon in the tubulin gene cluster of Trypanosoma brucei.

The genome of Trypanosoma brucei contains many copies of TRS/ingi, a retroposon like element of about 5 kb length. One vestigial member of this family, TUBIS, is found in the tubulin gene cluster. The element has sequence homologies to reverse transcriptase. The similarity to TRS/ingi is lost after 4 kb, resulting in a 3' truncated element. The sequence following downstream shows similarities to 5' flanking regions of VSG genes.

Amino Acid Sequence↗

PubMed1986

Structure of a conjugative element in Streptococcus pneumoniae.

We have cloned and mapped a 69-kilobase (kb) region of the chromosome of Streptococcus pneumoniae DP1322, which carries the conjugative omega (cat-tet) insertion from S. pneumoniae BM6001. This element proved to be 65.5 kb in size. Location of the junctions was facilitated by cloning a preferred target region from the wild-type strain Rx1 recipient genome. This target site was preferred by both the BM6001 element and the cat-erm-tet element from Streptococcus agalactiae B109. Within the BM6001 element cat and tet were separated by 30 kb, and cat was flanked by two copies of a sequence that was also present in the recipient strain Rx1 DNA. Another sequence at least 2.4 kb in size was found inside the BM6001 element and at two places in the Rx1 genome. Its role is unknown. The ends of the BM6001 element appear to be the same as those of the B109 element, both as seen after transfer to S. pneumoniae and as mapped by others in pDP5 after transposition in Streptococcus faecalis. We see no homology between the ends of the BM6001 element and find no evidence suggesting that it ever circularizes.

Cloning, Molecular↗

PubMed1988

Simple assay for quantitation of Tn5 inversion events in Escherichia coli and use of the assay in determination of plasmid copy number.

In this report, we describe a simple method for measuring the frequency of sequence inversion in the transposable element Tn5 as a result of recombination across its duplicated IS50 elements. The structure of Tn5 was manipulated so that the neomycin phosphotransferase gene of the transposon would be expressed only if a sequence inversion event occurred. This highly sensitive assay also served as the basis for a novel means of estimating plasmid copy number.

Chromosome Inversion↗

PubMed1996

A promoter identified in the 3' end of the Ac transposon can be activated by cis-acting elements in transgenic Arabidopsis lines.

In experiments directed to develop a promoter trap strategy in Arabidopsis, using a Ds chimaeric element containing a promoterless beta-glucuronidase (GUS) gene, we identified a promoter in the 3' end region of the Ac transposable element. The promoter initiates most of the transcripts at coordinate 4250 in the Ac sequence and is oriented towards the internal part of the element. When fused to a promoterless GUS gene, the promoter allows transient expression in Arabidopsis leaves. After stable integration into the Arabidpsis genome, no GUS activity was observed in most of the transformed lines analysed. Only two of them exhibited different tissue-specific GUS expression. When a CaMV 35S promoter was introduced into the transformation vector, downstream to the reporter gene, a high level of GUS activity was observed in all the transformants. These results strongly suggest that the promoter is not normally expressed at a significant level in Arabidopsis transformed lines except when activated by neighbouring cis-acting enhancer elements. This opens an interesting possibility for using this promoter to develop 'enhancer trap' strategies in Arabidopsis. Since only one Ac transcript, initiating in the 5' end region of the element has been reported to date in maize, the putative biological function of the promoter remains an open question.

Animals↗

PubMed1988

Insertional mutagenesis of the Drosophila genome with single P elements.

A versatile genetic method for identifying and cloning Drosophila melanogaster genes affecting any recognizable phenotype is described. Strains are constructed in which the insertion of a single P transposable element has caused a new mutation, greatly simplifying the genetic and molecular analysis of the affected gene. Mutagenesis is initiated by crossing two strains, each of which contains a specially designed P element. One element (jumpstarter), encoding P element transposase, efficiently mobilizes the second nonautonomous transposon (mutator), whose structure facilitates selection and cloning of new insertion mutations. Random mutator transpositions are captured in individual stocks that no longer contain jumpstarter, where they remain stable. This method was used to construct 1300 single P element insertion stocks which were then screened for recessive mutations. A library of single-element insertion strains will allow the structure and function of Drosophila genes to be readily correlated, and should have many other applications in Drosophila molecular genetics.

Animals↗

PubMed1990

Tn552, a novel transposable element from Staphylococcus aureus.

Tn552, one of several closely related beta-lactamase-encoding transposons from Staphylococcus aureus, has a novel set of putative transposition functions. Each is homologous with a well-characterized function from a different type of mobile genetic element. Thus, Tn552 encodes: (i) resL-binL, a co-integrate resolution system homologous with those of Tn3 family elements; (ii) p480, a potential transposase significantly homologous with the DNA integrases of eukaryotic retroviruses and retrotransposons; and (iii) p271, a potential ATP-binding protein that shows homology with the B protein of phage Mu. The 3' terminal nucleotides of Tn552 (CA), adjacent to which p480 might cleave, are the same as those of retroviruses, retrotransposons and phage Mu. The presumptive resolvase (BinL) is very closely related to BinR, which was identified as a DNA invertase and is now shown to resolve an artificial co-integrate in vivo. Furthermore, the structure of the derivative of Tn552 found in the staphylococcal plasmid pI258 can be explained by a BinL (or BinR)-mediated site-specific deletion ('resolution') event. Thus, pI258 contains only the right-hand half of Tn552, which encodes the beta-lactamase and two regulatory proteins. The latter are homologous with the beta-lactamase gene repressor and co-inducer of Bacillus licheniformis. Interestingly, the order of the regulatory genes is reversed in S. aureus compared with Bacillus licheniformis.

Amino Acid Sequence↗

PubMed2003

Chloramphenicol resistance transposable element TnSs1 of Streptococcus suis, a transposon flanked by IS6-family elements.

A new transposon, designated TnSs1, which contains a chloramphenicol acetyltransferase gene flanked by direct repeats of an IS6-family element was found in a field isolate of Streptococcus suis. Polymerase chain reaction and hybridization analyses indicated that another field isolate carried the same transposon in a different location on the chromosome. A transposition assay done with a thermosensitive suicide vector showed that, among the seven TnSs1 mutants tested in this study, six formed a cointegrate between the S. suis genome and the vector with the generation of the third copy of the insertion sequence element, and one harbored one copy of TnSs1 on the chromosome as a result of a subsequent resolution step. On transposition, TnSs1 duplicated an 8-bp sequence at the target site.

Chloramphenicol Resistance↗

PubMed1988

Overproduction of the protein encoded by the maize transposable element Ac in insect cells by a baculovirus vector.

The polypeptide encoded in the Activator (Ac) element of Zea mays L. has been expressed in Spodoptera frugiperda insect cells using plasmids which carry the strong polyhedrin promoter of the baculovirus Autographa californica nuclear polyhedrosis virus (AcNPV). Recombinant AcNPVs with the Ac-cDNA integrated and under the control of the viral polyhedrin promoter have been isolated and their genomes have been partly characterized as to the location of the foreign DNA insert. Upon infection of S. frugiperda cells with the recombinant AcNPV, maize Ac element specific messenger RNAs, as well as a newly synthesized polypeptide with an apparent molecular weight of about 116 kDa, have been detected in extracts of recombinant infected cells. This polypeptide is absent from extracts of wild-type infected cells expressing the polyhedrin polypeptide which can be recognized by the presence of nuclear inclusion bodies. Recombinant infected cells lack this protein. The Ac specific polypeptide is detected by antisera, which have been raised against fusion proteins containing Ac sequences synthesized in Escherichia coli, both in immunoprecipitation and in Western blotting experiments. The Ac specific protein is a nuclear phosphoprotein and represents about 1%-2% of the newly synthesized protein.

Animals↗

PubMed1982

Ty1 and delta elements occur adjacent to several tRNA genes in yeast.

A comparative analysis of a number of yeast DNA-pBR322 recombinant plasmids carrying repetitive sequence elements has revealed that Ty1 or delta elements occur in the vicinity of several tRNA genes. Four examples have been characterized in detail: three glutamate tRNA genes and a serine tRNA gene. The tRNAGlu3 genes occupy different chromosomal locations; two of these genes are found adjacent to Ty1 elements, and the third is found adjacent to an independent delta element. A delta unit is also found adjacent to a tRNASer2 gene. Next to one of the tRNAGlu3 genes, the delta element is joined to a truncated sigma element. Junctions between different delta units were characterized by the sequence analysis of two DNA segments that carry no tRNA genes.

Base Sequence↗

PubMed1995

The maize transposable element Ac is mobile in the legume Lotus japonicus.

To evaluate the prospects for transposon mutagenesis in the autogamous diploid legume Lotus japonicus, the behaviour of the maize transposable element Ac was analysed in the progeny of 38 independent transgenic plants. The conditions for monitoring donor site excision using histochemical localization of beta-glucuronidase activity or the alternative spectinomycin resistance assay were established, and used to follow Ac mobility through two generations. Somatic excision was monitored as variegated cotyledons in the T2 generation and germinal excision events were scored in segregating T3 families as complete beta-glucuronidase-mediated staining of cotyledons or as a fully green spectinomycin-resistant phenotype. Using these assays an average germinal excision frequency of 12% was estimated in the T3 offspring from variegated plants. The fidelity of the excision assays was ascertained by comparing the frequency of germinal excision to the frequency of Ac reinsertion at new positions of the genome. Transposition of Ac in 42% of the plants and detection of the characteristic Ac insertion/excision footprints suggests that insertion mutagenesis with the autonomous maize Activator element is feasible in Lotus japonicus. Parameters influencing Ac behaviour, such as dosage, position effects and modification of the element itself, were also investigated comparing homozygous and hemizygous plants from the same family and by analysing different transformants.

Base Sequence↗

PubMed1995

Characterization of a family of genes encoding a fruit-specific wound-stimulated protein of bell pepper (Capsicum annuum): identification of a new family of transposable elements.

Using a fruit-specific cDNA as a probe we isolated and sequenced the two corresponding homologous genes (Sn-1 and Sn-2) of the bell pepper (Capsicum annuum) genome. Both genes have a single intron and numerous unusual long inverted repeat sequences. The introns share 87% homology and Sn-2 contains one 450 bp additional sequence with structural features of a transposable element, which is highly repetitive in the bell pepper genome. Surprisingly, analysis in data banks showed that genes encoding the potato starch phosphorylase (EC 2.4.1.1) and patatin contain a similar element, named Alien, in their 5'-upstream region. Alien elements are characterized by a conserved 28 bp terminal inverted repeat (TIR), small size, high AT content, potential to form stable DNA secondary structures and they have probably been inserted in TA target sites. Interestingly, the TIR of the Alien elements shares high homology with sequences existing in the TIR of extrachromosomal linear pSKL DNA plasmid of Saccharomyces kluyveri. Northern blot analyses detected Sn-1 transcripts principally in the red fruit whereas no Sn-2 transcripts were detected in neither of the samples monitored. Western blot analyses detected a 16.8 kDa Sn protein principally in the ripe red fruit and wounded areas of green unripe fruit. A comparison of the deduced amino acid sequence of Sn-1 with protein sequences in data banks revealed a significant homology with proteins likely involved in the plant's disease resistance response. Analyses at the subcellular level showed that Sn-1 is localized in the membrane of vacuoles.

Amino Acid Sequence↗

PubMed1996

Characterization of a mosaic ISS1 element and evidence for the recent horizontal transfer of two different types of ISS1 between Streptococcus thermophilus and Lactococcus lactis.

A 12-kb region of the Streptococcus thermophilus CNRZ368 chromosome was found to contain two copies of IS981 (one complete and one truncated) and three copies of ISS1 (two complete, ISS1SA and ISS1SC, and one truncated, delta ISS1SB). Comparison of the nucleotide sequences of these ISS1 elements with those of previously identified iso-ISS1 elements from Lactococcus lactis and the Enterococcus genus indicated that the ISS1 group is divided into three distinct subgroups which we have named alpha, beta and gamma. Nucleotide sequences of elements belonging to the same subgroup share more than 97% identity whereas sequences of elements from different groups share only 75-85% identity. Sequence analysis of ISS1SA and delta ISS1SB showed that they are members of the alpha group. We found that ISS1SC from S. themophilus CNRZ368, an ISS1 from L. lactis IL964 and IS946 from L. lactis TEK1 resulted from recombinations between alpha and beta elements. In addition, ISS1W from L. lactis Wg2 resulted from a recombination event between a gamma element and an ISS1 belonging to an unidentified subgroup. ISS1 sequences belonging to the alpha and beta subgroups were found in both S. thermophilus and L. lactis and gamma sequences were found in both the Enterococcus genus and L. lactis. The quasi-identity of some ISS1 elements in S. thermophilus and L. lactis and the distribution of alpha and beta elements suggest that horizontal transfer of ISS1 elements recently took place from L. lactis to S. thermophilus, two lactic acid bacteria used in the manufacture of cheeses. Since the presence of IS981 in S. thermophilus CNRZ368 also probably resulted from a horizontal transfer from L. lactis [Guédon et al. (1995) Mol. Microbiol. 16, 69-78], the 12-kb region bearing IS981 and ISS1 elements could be due to the integration of a lactococcal DNA fragment into the chromosome.

Base Sequence↗

PubMed1998

Target specificity of insertion element IS30.

The Escherichia coli resident mobile element IS30 has pronounced target specificity. Upon transposition, the element frequently inserts exactly into the same position of a preferred target sequence. Insertion sites in phages, plasmids and in the genome of E. coli are characterized by an exceptionally long palindromic consensus sequence that provides strong specificity for IS30 insertions, despite a relatively high level of degeneracy. This 24-bp-long region alone determines the attractiveness of the target DNA and the exact position of IS30 insertion. The divergence of a target site from the consensus and the occurrence of 'non-permitted' bases in certain positions influence the target activity. Differences in attractiveness are emphasized if two targets are present in the same replicon, as was demonstrated by quantitative analysis. In a system of competitive targets, the oligonucleotide sequence representing the consensus of genomic IS30 insertion sites proved to be the most efficient target. Having compared the known insertion sites, we suppose that IS30-like target specificity, which may represent an alternative strategy in target selection among mobile elements, is characteristic of the insertion sequences IS3, IS6 and IS21, too.

Binding Sites↗

PubMed2004

Variation in the Shiga toxin region of 20th-century epidemic and endemic Shigella dysenteriae 1 strains.

The Shiga toxin (Stx) region of Shigella dysenteriae 1 lies on a defective prophage homologous to lambdoid bacteriophages in Stx-producing Escherichia coli. S. dysenteriae 1 strains obtained in locations throughout the world over the course of the past 60 years were assessed for variations in the Stx region by use of polymerase chain reaction and sequence analysis. The defective prophage was present in all strains examined, suggesting that all S. dysenteriae 1 isolates derive from a clone that resulted from a single phage-integration event. All western-hemisphere strains have an additional iso-IS1 insertion element upstream of stxAB, implying that there has been minimal exchange of strains between hemispheres in recent decades.

DNA Transposable Elements↗

PubMed1997

Sequence analysis of the 36-kb region between gntZ and trnY genes of Bacillus subtilis genome.

Within the framework of an international Bacillus subtilis genome sequencing project, we have determined a 36-kb sequence covering the region between the gntZ and trnY genes. In addition to five genes sequenced and characterized previously, 27 putative protein coding sequences (open reading frame; ORF) were identified. A homology search for the newly identified ORFs revealed that six of them had similarities to known proteins. It is notable that new ORFs belonging to response-regulator aspartate phosphatase (Rap) and its regulator (Phr) families, and response regulator and sensory kinase families of two-component signal transduction systems have been identified. Furthermore, we found that some 180-bp non-coding sequence, that might be an remnant of an ancient IS element, is preserved in at least five loci of the B. subtilis genome.

Bacillus subtilis↗

PubMed1996

Epigenetic control of a transposon-inactivated gene in Neurospora is dependent on DNA methylation.

An unstable allele of the Neurospora am (GDH) gene resulting from integration of the retrotransposon Tad3-2 into 5' noncoding sequences was found in previous work. We report that reversion to Am+ depends on DNA methylation within and upstream of Tad. Levels of methylation were correlated with the proportion of Am+ conidia, whether the cultures were derived from Am- or Am+ isolates. Reversion to Am+ did not occur when conidia were plated on 5-azacytidine, which reduces DNA methylation. The mutation dim-2, which appears to abolish DNA methylation, also prevented reversion to Am+. The native am allele, in a strain that lacked Tad elements, was replaced with am::Tad3-2 or with a deletion derivative that prevents transposition of Tad. Transformants of both classes showed instability comparable with that of the original isolates, which contain multiple Tad elements. Deletion of the upstream enhancer-like sequences, URSam alpha and beta, did not prevent the instability of am::Tad3-2. The results suggest that am expression is dependent on DNA methylation but not on proliferation or transposition of the Tad element and that the instability does not require the upstream sequences of am.

Alleles↗

PubMed2006

The presence of a conjugative Gram-positive Tn2009 in Gram-negative commensal bacteria.

OBJECTIVES: To determine whether mef(A)-msr(D) and tet(M) genes are linked in representative Gram-negative isolates and/or transferred together during conjugation. To molecularly characterize the Acinetobacter junii element and compare the structure and sequence with the non-conjugative Streptococcus pneumoniae Tn2009 element. METHODS: PCR assays, DNA-DNA hybridization and sequencing of PCR products were used. Nucleotide sequences were determined at the integration site of the mef(A) element into Tn916 and upstream and downstream flanking regions of the element. RESULTS: A total of 10 mef(A)-msr(D)- and tet(M)-positive isolates carried conjugative element(s). The A. junii Tn2009 element was indistinguishable from S. pneumoniae Tn2009. The region upstream of the A. junii Tn2009 contained an orf that was 89-91% identical to an S. pneumoniae spr1206 gene found upstream of the streptococcal Tn2009. In the A. junii, the spr1206 gene was separated by 67 bp from the end of the Tn2009, while 29 bp were found separating spr1206 from the streptococcal Tn2009. The 1201 bp downstream A. junii sequences included 913 unique sequences. CONCLUSIONS: A total of 10 different Gram-negative genera were found to carry the tet(M) genes, including the first description in three genera (Citrobacter, Proteus and Stenotrophomonas). All isolates were able to transfer the genes into > or =1 recipient with macrolide selection. Over 3000 bp were sequenced on each side of the insertion mef junction region in the A. junii and were indistinguishable from the streptococcal Tn2009. The A. junii Tn2009 element was flanked by an S. pneumoniae gene upstream and a unique sequence downstream, suggesting that the A. junii Tn2009 could be part of a larger element.

Acinetobacter↗

PubMed2006

New variants of the tet(M) gene in Clostridium difficile clinical isolates harbouring Tn916-like elements.

OBJECTIVES: To detect Tn916-like elements in Clostridium difficile clinical isolates from different time periods and to analyse the genetic structure of these elements, in particular the tet(M) region. METHODS: Ninety C. difficile clinical isolates were examined by PCR assays for tet(M) and int, which are markers for the Tn916 family of elements. Positive isolates were typed by PCR-ribotyping, and tetracycline MIC values were evaluated by Etest. The genetic organization of the Tn916 elements was investigated by PCR mapping and hybridization assays. The tet(M) region of eight selected C. difficile isolates was sequenced. RESULTS: Nineteen isolates were tet(M)/int positive and the majority (12/19) belonged to PCR-ribotype R, previously found to be predominant in clinical strains of more recent isolation. Eleven isolates were tetracycline resistant, three inducibly resistant and five susceptible. Fifty-eight per cent of the C. difficile isolates harboured one Tn916 element and 42% harboured two. Most isolates showed elements with a genetic organization very similar to that of Enterococcus faecalis DS16 Tn916. Sequence analysis highlighted variations in the leader peptide and six tet(M) variants were identified, five of which have never been described before. CONCLUSIONS: C. difficile isolates harbouring Tn916-like elements have mainly been isolated since 1997, suggesting a recent circulation of these elements among C. difficile strains in Italian hospitals. Molecular analysis of these Tn916-like elements showed that they may have different genetic structures and carry new tet(M) alleles.

Bacterial Proteins↗