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PubMed1992

Ty insertions upstream and downstream of native DUR1,2 promoter elements generate different patterns of DUR1,2 expression in Saccharomyces cerevisiae.

Expression of allantoin pathway genes is subject to induction and nitrogen catabolite repression. Two classes of cis-dominant mutations (DUR80 and DUR1,2-Oh) result in overproduction of DUR1,2 mRNA. In DUR80 mutants, DUR1,2 expression remained inducible, nitrogen catabolite repression sensitive, and unresponsive to cell ploidy, i.e., overproduction was superimposed on normal gene regulation. DUR1,2-Oh mutations, in contrast, generated a pattern of DUR1,2 expression similar to that often reported when a Ty element inserts upstream of a gene, the ROAM phenotype. We analyzed four independent DUR80 and DUR1,2-Oh alleles. The DUR1,2-Oh mutation was, as expected, a Ty insertion at -445 3' of the native DUR1,2 upstream activation sequences (UASs). All three DUR80 alleles were also Ty insertions between -644 and -653 immediately 5' of the native DUR1,2 USASs. We suggest that the difference in DUR1,2-Oh and DUR80 phenotypes depends on whether the native cis-acting elements and transcription factors associated with them can operate. If they can, enhancement of normally regulated DUR1,2 expression is observed. This is a novel phenotype for Ty insertions. If the native DUR1,2 cis-acting elements are not present, the case when Ty insertion occurs 3' of them, a ROAM phenotype is generated. Nitrogen-regulated upstream activation sequence (UASNTR)-homologous sequences present in the Ty delta elements rather than cis-acting elements required for Ty transcription are the most likely candidates to serve as the cis-acting elements mediating the DUR80 phenotype.

Base Sequence↗
PubMed2001

The genome of the Queensland fruit fly Bactrocera tryoni contains multiple representatives of the mariner family of transposable elements.

Representatives of five distinct types of transposable elements of the mariner family were detected in the genomes of the Queensland fruit fly Bactrocera tryoni and its sibling species Bactrocera neohumeralis by phylogenetic analysis of transposase gene fragments. Three mariner types were also found in an additional tephritid, Bactrocera jarvisi. Using genomic library screening and inverse PCR, full-length elements representing the mellifera subfamily (B. tryoni.mar1) and the irritans subfamily (B. tryoni.mar2) were isolated from the B. tryoni genome. Nucleotide consensus sequences for each type were derived from multiple defective copies. Predicted transposase sequences share approximately 23% amino acid identity. B. tryoni.mar1 elements have an estimated copy number of about 900 in the B. tryoni genome, whereas B. tryoni.mar2 element types appear to be present in low copy number.

Amino Acid Sequence↗
PubMed1985

IS10 transposition is regulated by DNA adenine methylation.

We show that dam- mutants are a major class of E. coli mutants with increased IS10 activity. IS10 has two dam methylation sites, one within the transposase promoter and one within the inner terminus where transposase presumably binds. Absence of methylation results in increased activity of both promoter and terminus, and completely accounts for increased transposition in dam- strains. Transposition of Tn903 and Tn5 are also increased in dam- strains, probably for analogous reasons. Transposition is also increased when IS10 is hemimethylated. One hemimethylated species is much more active than the other and is estimated to be at least 1000 times more active than a fully methylated element. Evidence is presented that the promoter and inner terminus of IS10 are coordinately activated in a dam-dependent fashion, presumably because they are hemimethylated at the same time. Thus, in dam+ strains, IS10 will transpose preferentially when DNA is hemimethylated. We suggest specifically that IS10 transposition may preferentially occur immediately after passage of a chromosomal replication fork.

Adenine↗
PubMed2002

The clostridial mobilisable transposons.

Mobilisable transposons are transposable genetic elements that also encode mobilisation functions but are not in themselves conjugative. They rely on coresident conjugative elements to facilitate their transfer to recipient cells. Clostridial mobilisable transposons include Tn4451 and Tn4452 from Clostridium perfringens, and Tn4453a and Tn4453b from Clostridium difficile, all of which are closely related, and Tn5398 from C. difficile. The Tn4451 group of elements encodes resistance to chloramphenicol and is unusual in that transposition is dependent upon a large resolvase protein rather than a more conventional transposase or integrase. This group of elements also encodes the mobilisation protein TnpZ that, by acting at the RS(A) or oriT site located on the transposon, and in the presence of a coresident conjugative element, promotes the movement of the nonreplicating circular intermediate and of plasmids on which the transposon resides. The erythromycin resistance element Tn5398 is unique in that it encodes no readily identifiable transposition or mobilisation proteins. However, the element is still capable of intraspecific transfer between C. difficile isolates, by an unknown mechanism. The detailed analysis of these mobilisable clostridial elements provides evidence that the evolution and dissemination of antibiotic resistance genes is a complex process that may involve the interaction of genetic elements with very different properties.

Clostridium↗
PubMed1993

Effects of gene dosage and sequence modification on the frequency and timing of transposition of the maize element Activator (Ac) in tobacco.

The effect of Ac copy number on the frequency and timing of germinal transposition in tobacco was investigated using the streptomycin phosphotransferase gene (SPT) as an excision marker. The activity of one and two copies of the element was compared by selecting heterozygous and homozygous progeny of transformants carrying single SPT::Ac inserts. It was observed that increasing gene copy not only increases the transposition frequency, but also occasionally alters the timing of transposition such that earlier events are obtained. The result is that some homozygous plants generate multiple streptomycin resistant progeny carrying the same transposed Ac (trAc) element. We have also investigated the effect of modification of the sequence in the region around 82 bp downstream of the polyadenylation site and 177 bp from the 3' end of the element on germinal excision frequencies. Alteration of three bases to create a Bgl II site at this location caused a minor decrease in germinal excision events, but insertion of four bases to create a Cla I site caused a 10-fold decrease in the transposition activity of the Ac element.

Agrobacterium tumefaciens↗
PubMed1993

Reverse transcription of R2Bm RNA is primed by a nick at the chromosomal target site: a mechanism for non-LTR retrotransposition.

R2 is a non-LTR retrotransposable element that inserts at a specific site in the 28S rRNA genes of most insects. We have expressed the open reading frame of the R2 element from Bombyx mori, R2Bm, in E. coli and shown that it encodes both sequence-specific endonuclease and reverse transcriptase activities. The R2 protein makes a specific nick in one of the DNA strands at the insertion site and uses the 3' hydroxyl group exposed by this nick to prime reverse transcription of its RNA transcript. After reverse transcription, cleavage of the second DNA strand occurs. A similar mechanism of insertion may be used by other non-LTR retrotransposable elements as well as short interspersed nucleotide elements.

Animals↗
PubMed2003

Three new insertion sequence elements ISLdl2, ISLdl3, and ISLdl4 in Lactobacillus delbrueckii: isolation, molecular characterization, and potential use for strain identification.

A group of new insertion sequence (IS) elements, ISLdl2, ISLdl3, and ISLdl4, from Lactobacillus delbrueckii subsp. lactis ATCC 15808 was isolated, characterized, and used for strain identification together with ISLdl1, recently characterized as an L. delbrueckii IS element belonging to the ISL3 family. ISLdl2 was 1367 bp in size and had a 24 bp IR and an 8 bp DR. The single ORF of ISLdl2 encoded a protein of 392 aa similar to transposases of the IS256 family. ISLdl3 had a single ORF encoding a protein of 343 aa similar to transposases of the IS30 family. Finally, ISLdl4 had a single ORF encoding a protein of 406 aa and displayed homology to the transposases of the IS110 family. ISLdl4 was only slight different from ISL4 (Accession No. AY040213). ISLdl1, ISLdl2, and ISLdl4 were present in all of the 10 L. delbrueckii subsp. lactis and subsp. delbrueckii strains tested, as well as in three of the 11 L. delbrueckii subsp. bulgaricus strains tested. ISLdl3 was present only in four closely related strains of L. delbrueckii subsp. lactis. These IS elements were not observed in Lactobacillus rhamnosus, Lactobacillus acidophilus, Lactobacillus helveticus, or Lactobacillus plantarum. A cluster of IS elements, ISLdl1, ISLdl2, ISLdl3, ISLdl4, and ISL6, was observed in L. delbrueckii subsp. lactis strain ATCC 15808. Within this cluster, ISLdl4 was inserted into ISLdl1 between the left IR and the start codon of ORF455, encoding a putative transposase. Most of the integration sites of the IS elements were strain-specific. We have observed that IS elements can migrate from one strain to another as integral parts of bacterial DNA by using phage LL-H as a vehicle. We demonstrate for the first time that inverse PCR and vectorette PCR methods with primers based on sequences of the IS elements could be used for identification of L. delbrueckii strains.

Bacteriophages↗
PubMed2000

Mutator-like elements in Arabidopsis thaliana. Structure, diversity and evolution.

While genome-wide surveys of abundance and diversity of mobile elements have been conducted for some class I transposable element families, little is known about the nature of class II transposable elements on this scale. In this report, we present the results from analysis of the sequence and structural diversity of Mutator-like elements (MULEs) in the genome of Arabidopsis thaliana (Columbia). Sequence similarity searches and subsequent characterization suggest that MULEs exhibit extreme structure, sequence, and size heterogeneity. Multiple alignments at the nucleotide and amino acid levels reveal conserved, potentially transposition-related sequence motifs. While many MULEs share common structural features to Mu elements in maize, some groups lack characteristic long terminal inverted repeats. High sequence similarity and phylogenetic analyses based on nucleotide sequence alignments indicate that many of these elements with diverse structural features may remain transpositionally competent and that multiple MULE lineages may have been evolving independently over long time scales. Finally, there is evidence that MULEs are capable of the acquisition of host DNA segments, which may have implications for adaptive evolution, both at the element and host levels.

Amino Acid Motifs↗
PubMedDate not supplied

Role of an upstream regulatory element in leucine repression of the Saccharomyces cerevisiae leu2 gene.

The expression of a number of eukaryotic genes has been shown to involve at least two sequences located upstream of the actual transcription unit: one of these sequences, centred on a widely conserved TATAAT sequence, is thought to be involved in determining the precise site of initiation of transcription; the other has a gene-specific sequence, can function at a variable distance upstream of the initiation site, and is involved in the regulation of transcription. By constructing beta-galactosidase gene fusions, to facilitate measuring gene expression in vivo, we have now defined a cis-acting regulatory element of the Saccharomyces cerevisiae leu2 gene. This element is located within a 280 base pair (bp) fragment which occurs 125 bp upstream of the leu2 translation initiation codon and which contains a short G + C-rich palindromic sequence. A fragment of the Escherichia coli transposable element Tn9 which contains a similar palindromic sequence can functionally replace the natural leu2 regulatory element. Our results are contrary to previous speculations that the leu2 gene is regulated by an attenuation mechanism.

3-Isopropylmalate Dehydrogenase↗
PubMed2001

A novel transposon tagging element for obtaining gain-of-function mutants based on a self-stabilizing Ac derivative.

A novel tagging system AcREH, designed for obtaining gain-of-function mutations, was prepared on the basis of a self-stabilizing Ac transposon derivative. The transposable element, DsAT, was constructed in a way that it can activate transcription of neighboring genes by two 35S promoters and/or by four tandem repeats of the enhancer fragment of this promoter. DsAT revealed somatic excision in the first generation of the tobacco transformants. The element exhibited germinal excision to the next generation, as demonstrated by PCR and Southern hybridization analysis. In spite of the structure of the element, which may inhibit the expression of the transposase gene, the frequency of germinal excision was comparable to or higher than those so far reported, suggesting the applicability of the element for gene tagging.

Base Sequence↗
PubMed1993

Functional dissection of two promoters that control sense and antisense transcription of Drosophila melanogaster F elements.

Drosophila melanogaster F elements are members of the super-family of LINEs, mobile repeated DNA sequences that lack LTRs and propagate by the reverse transcription of unit-length RNA intermediates. The F 5' end region harbours two promoters (F(in) and F(out)) that transcribe in a convergent manner. Each promoter has been functionally dissected by assaying in D. melanogaster cultured cells templates carrying base substitutions and/or deletions across the +1 to +245 region of the element F12. F(in), that likely controls the synthesis of gene products and transposition intermediates, is internal to the transcribed region. Two elements play a major role in F-sense transcription. The proximal element spans the interval +6 to +14 and includes a major RNA start site. Heterologous DNA featuring a nearly identical purine-pyrimidine sequence can functionally replace the initiator-like module only when properly spaced from downstream F sequences. The distal element is within the interval +18 to +46 and may correspond to a motif (AGACGTTT, +34 to +41) conserved in other Drosophila LINEs. F(out) is a TATA-less promoter that directs transcription predominantly from three nearby sites (a to c). F(out) expression is influenced by multiple elements located upstream of residue -68 relative to site a as +1 within a region (alpha) shown to stimulate the D. melanogaster hsp70 promoter in an orientation and position-independent fashion. Changes within the -43 to +24 interval may suppress or stimulate transcription from sites a and c. Initiation from a site approximately 30 nucleotides upstream of site a is enhanced by alterations of the interval -43 to -5. The expression of the two F promoters, determined by the interaction of the transcriptional machinery with distinct DNA sequences, is influenced by a common element within the alpha region.

Animals↗
PubMed2001

Analysis of a Bacteroides conjugative transposon using a novel "targeted capture" model system.

Large conjugative transposons (CTn's) are widespread among Bacteroides spp. and they are responsible for the high rates of Bacteroides tetracycline resistance, which is mediated by the tetQ gene. These elements are self-transmissible and conjugation can be induced up to 1000-fold by the addition of tetracycline to cultures prior to mating. In addition to self-transfer, the Bacteroides CTn's, such as CTn341, are able to mobilize unlinked genetic elements such as plasmids and mobilizable transposons in a tetracycline-inducible manner. To study the molecular properties of these unique elements, a vector was designed to capture CTn's for analysis in heterologous hosts. This plasmid, pFD670, consisted of the low-copy vector pWSK29, the RK2 oriT, an ermF gene, and a tetQ gene fragment containing the N-terminus and promoter. The vector was transferred into Bacteroides recipients containing CTn341 where it integrated into the tetQ gene by homologous recombination. This integrated construct then was transferred back into an Escherichia coli host where it replicated as a plasmid, pFD699, about 56 kb in size. Further analysis showed that pFD699 could be transferred into Bacteroides hosts where it displayed the same tetracycline-inducible properties as the native CTn341. The captured element appeared to utilize a circular intermediate in both transfer and transposition, and integration into the chromosome seemed to be random. Hybridization studies with a range of Bacteroides CTn's encoding tetracycline resistance revealed a great deal of homology between most of the CTn's but there was much variation seen in the restriction patterns of these elements, suggesting great diversity among this group.

Bacteroides↗
PubMed1980

Nucleotide sequences of integrated Moloney sarcoma provirus long terminal repeats and their host and viral junctions.

Integrated Moloney murine sarcoma provirus (MSV) has direct terminal repeat sequences (TRS). We determined the nucleotide sequence of both 588-base-pair TRS elements and the adjacent host and viral junctions of an integrated MSV cloned in bacteriophage lambda. Sequences were identified corresponding to the tRNAPro primer binding site in genomic RNA and the reverse-transcribed minus strong stop DNA. Each 588-base-pair repeat contains putative sites for promoting RNA synthesis and RNA polyadenylylation. The first and last 11 nucleotides of the TRS are inverted with respect to each other, and the same four-nucleotide host sequence is found bracketing integrated MSV. Some similarities of TRS and prokaryotic insertion sequence elements are discussed.

Animals↗
PubMed1987

Type I-like intervening sequences are found in the rDNA of the nematode Ascaris lumbricoides.

The intervening sequences in the large ribosomal RNA gene of Ascaris lumbricoides var. suum show many similarities to the type I insertions, previously found only in some insect species. They include structural features, but also a presumed transcriptional inactivity in vivo: No transcript of the rDNA intervening sequence in A. lumbricoides could be detected in Northern and dot blot hybridizations. However, the primary structure of the Pol I promoter region is well conserved in interrupted and uninterrupted genes. Moreover, genes with an intervening sequence are correctly initiated in a whole-cell in vitro extract from Ascaris oogonia. Hence, the presence of the intervening sequence alone does not seem to account for a transcriptional inhibition in rRNA genes. As with the type I insertions of insect rDNA, some copies of the A. lumbricoides intervening sequence are also present in locations outside the rDNA cluster. About 50% of the extraribosomal copies are found in a repetitive sequence of the genome, and additional copies are inserted in unique sequences. These striking analogies to type I insertions are discussed, and lead to the conclusion that the two phenomena are undoubtedly related. This is the first report proving the presence of a type I-like insertion element outside of the class Insecta.

Animals↗
PubMed1995

Transfer of the IncJ plasmid R391 to recombination deficient Escherichia coli K12: evidence that R391 behaves as a conjugal transposon.

A study of the IncJ plasmid R391 confirmed a low frequency of transfer between recombination proficient (recA+) Escherichia coli (10(-5) donor -1). Reanalysis of its transfer to recombination deficient (recA) E. coli revealed an equivalent transfer frequency to and from all mutants tested. Extrachromosomal DNA could not be detected in either recA+ or recA transconjugants, while R391 proved refractory to curing in both backgrounds implying a high degree of stability. The integration of R391 into a specific region of the chromosome was demonstrated by its transfer as part of the exogenote mobilised from the transfer origins of Hfr strains BW6165 and JC158. Transfer of R391 coupled to recA independent chromosomal integration has significant implications as to the nature and classification of the element. We propose that R391 behaves like a conjugal transposon.

Conjugation, Genetic↗
PubMed1990

The 987P gene cluster in enterotoxigenic Escherichia coli contains an STpa transposon that activates 987P expression.

The genetic determinant for the production of 987P fimbriae has been cloned into pBR322. Analysis of frequently occurring deletions in the resultant recombinant plasmid, pPK180, revealed that the 987P gene cluster contains a transposon that encodes the synthesis of heat-stable enterotoxin STpa and is flanked by inverted repeats of IS1. Hybridization experiments with STpa- and 987P-specific probes demonstrated that a variety of STpa+ 987P+ wild-type Escherichia coli strains contained contiguous STpa-987P DNA, most likely on their chromosome. Transcription of the 987P gene cluster appeared to be activated by the adjacent IS1 element.

Base Sequence↗
PubMed1999

Construction of plasmid vectors bearing a NotI-expression cassette based on the lac promoter.

We have constructed two plasmid vectors for cloning and expression of DNA fragments controlled by the lac promoter as a NotI-expression cassette. Whereas plasmid pSJ33 allows mobilization of the expression cassette into a wide variety of Gram-negative bacteria by RP4-mediated conjugation, the low-copy-number plasmid pSJP18Not facilitates cloning and expression in Escherichia coli when high gene dosage may be detrimental. In addition to their suitable cloning features (e.g. multiple cloning site, lacZ alpha fragment, compatible with ColE1-derived vectors), these plasmids are particularly useful as auxiliary vectors for cloning of the expression cassettes at the NotI site of mini-transposon elements [1, 2] and their eventual stable insertion into the host chromosome.

Cloning, Molecular↗