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Results for “transposon insertion sequencing”

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PubMed1984

Functional analysis of the white gene of Drosophila by P-factor-mediated transformation.

A 12-kb DNA segment spanning the white (w) locus of Drosophila has been inserted into a P-transposon vector and used for P-factor-mediated germ-line transformation. Several red-eyed transformants were recovered which complement the white mutant phenotype. Analysis of the eye pigments and the interaction with the zeste mutation indicates that the w gene inserted at several new chromosomal sites is expressed normally. The tissue-specific accumulation of w transcripts, as studied by in situ hybridization to tissue sections, is the same in transformant and wild-type larvae. This indicates that all the genetic information specified by the w locus is contained within this 12-kb segment of DNA. By secondary mobilization it was shown that the w sequences have been inserted as a functional P(w) transposon which is capable of further transposition.

Journal Article↗

PubMed1994

Tn5401 disruption of the spo0F gene, identified by direct chromosomal sequencing, results in CryIIIA overproduction in Bacillus thuringiensis.

The Bacillus thuringiensis spo0F gene was identified by chromosomal DNA sequencing of sporulation mutants derived from a B. thuringiensis transposon insertion library. A spo0F defect in B. thuringiensis, which was suppressed by multicopy hknA or kinA, resulted in the overproduction of the CryIIIA insecticidal crystal protein.

Amino Acid Sequence↗

PubMed2001

Genomic analysis of the erythromycin resistance element Tn5398 from Clostridium difficile.

Clostridium difficile is a nosocomial pathogen that causes a range of chronic intestinal diseases, usually as a result of antimicrobial therapy. Macrolide-lincosamide-streptogramin B (MLS) resistance in C. difficile is encoded by the Erm B resistance determinant, which is thought to be located on a conjugative transposon, Tn5398. The 9630 bp Tn5398 element has been cloned and completely sequenced and its insertion site determined. Analysis of the resultant data reveals that Tn5398 is not a classical conjugative transposon but appears to be a mobilizable non-conjugative element. It does not carry any transposase or site-specific recombinase genes, nor any genes likely to be involved in conjugation. Furthermore, using PCR analysis it has been shown that isolates of C. difficile obtained from different geographical locations exhibit heterogeneity in the genetic arrangement of both Tn5398 and their Erm B determinants. These results indicate that genetic exchange and recombination between these determinants occurs in the clinical and natural environment.

Anti-Bacterial Agents↗

PubMed1996

DNA sequence analyses support the role of interrupted gap repair in the origin of internal deletions of the maize transposon, MuDR.

Previous research has demonstrated that the autonomous Cy transposon can activate the excision of Mu transposons. To determine the relationship between Cy and the more recently described autonomous Mu transposon, MuDR, a Cy transposon inserted at the mutable a1 allele, a1-m5216, was isolated and cloned. DNA sequence analyses established that this Cy insertion is identical to MuDR (Mu9, GenBank accession No.: m76978.gb-pl). Therefore, Cy will henceforth be termed MuDR:Cy. Defective derivatives of MuDR:Cy were isolated that had lost their capacity to activate their own excision or the excision of a Mu7 transposon. Most of these derivatives are nonautonomous transposons because they can excise, but only in the presence of unlinked MuDR:Cy transposons. Physical mapping and DNA sequence analyses have established that six of these defective derivatives carry internal deletions. It has been proposed previously that such deletions arise via interrupted gap repair. The DNA sequences of the break points associated with all four sequenced deletions are consistent with this model. The finding that three of the excision-defective derivatives carry deletions that disrupt the coding region of the mudrA (but not the mudrB) transcript supports the view that mudrA plays a role in the excision of Mu transposons.

Alleles↗

PubMed1987

Identification and nucleotide sequence of the regions of Autographa californica nuclear polyhedrosis virus genome carrying insertion elements derived from Spodoptera frugiperda.

A morphology mutant of the baculovirus Autographa californica nuclear polyhedrosis virus called M5 was previously shown to synthesize two size classes of viral DNA, one of which had a deletion of 42% of the genome. It was hypothesized that the presence of the smaller M5 circular DNA resulted from the specific deletion of the region located between two sites carrying short DNA insertions. These sites have now been identified by DNA-DNA hybridization using cloned EcoRI fragments of M5 DNA. One cloned M5 EcoRI fragment was found to correspond to the deletion junction fragment where sequences from the insertion site in the 2.6-map unit region were covalently linked to sequences from the insertion site in the 46-map unit region. The 2.6- and 46-map unit regions of Wt AcMNPV DNA were sequenced. Potential long open reading frames which would be disrupted by the M5 inserts were detected. Nucleotide sequence analysis of the same regions of M5 DNA revealed the presence of almost identical inserts of 290 bp. The primary sequence of the inserts revealed characteristics similar to the termini of transposons. Hybridization studies suggested that the inserts were derived from repetitive elements of the Spodoptera frugiperda host cell genome.

Animals↗

PubMed2000

DNA sequencing with transposons.

The use of transposons offers the possibility of a directed approach to DNA sequencing, where a target DNA up to about 6kb in length can be sequenced quickly and with minimal redundancy. Transposons are mobile DNA elements which can be inserted in a reasonably random fashion into the target DNA. An important part of this process is the location of the transposon insertions (known as mapping) and the selection of a sensible subset of transposons to use as priming sites for sequencing reactions. This paper presents a probabilistic method of scoring selected subsets of transposons and a graph-theoretic algorithm for selection of a subset of maximal score.

Algorithms↗

PubMed1994

Genetic analysis of Escherichia coli O9 rfb: identification and DNA sequence of phosphomannomutase and GDP-mannose pyrophosphorylase genes.

Subcloning, transposon insertion, and deletion analysis revealed that the Escherichia coli O9 rfb region is about 12 kb in size. The region encodes at least seven polypeptides of 89, 74, 55, 50, 44, 41 and 39.5 kDa. Southern hybridization analysis of rfb regions of E. coli O8 and O9, and Klebsiella O3 and O5 serotypes (all of these O polysaccharides are mannose homopolymers and the structures of the repeating unit of E. coli O9 and Klebsiella O3 are identical) showed that a central region specific for E. coli O9 and Klebsiella O3 is flanked by two regions common to all four. Complementation experiments using strains with known defects and specific tests for the enzymic activity showed that the 50 and 55 kDa polypeptides, encoded by the common region, are phosphomannomutase (PMM) and GDP-mannose pyrophosphorylase (GMP), respectively. Nucleotide sequencing of the region revealed the presence of two genes, rfbK and rfbM, analogous to the corresponding genes of Salmonella typhimurium. In E. coli O9, rfbK and rfbM encode proteins of 460 amino acids (50,809 Da) and 471 amino acids (52,789 Da). The amino acid sequence of GMP was conserved in RfbMs of E. coli O7 and Salmonella groups B, C1 and C2, CpsB of S. typhimurium, AlgA of Pseudomonas aeruginosa, and XanB of Xanthomonas campestris. The phylogenetic trees of PMM and GMP were different in topology and in the evolutionary distances from ancestors.

Amino Acid Sequence↗

PubMed2005

A transposon-based activation-tagging population in Arabidopsis thaliana (TAMARA) and its application in the identification of dominant developmental and metabolic mutations.

A population of 9471 stable activation-tagged lines was generated by transposable element mediated activation tagging mutagenesis in Arabidopsis (TAMARA) using the maize En/Spm transposon system. Based on DNA gel blot and flanking sequence analysis, this population contains approximately 6000 independent transposon insertions. A greenhouse-based screen identified six dominant or semi-dominant activation tagged mutants with obvious developmental alterations, among these a new pistillata mutant allele. In addition, a subset of 1500 lines was screened by a HPLC based high-throughput method for dominant activation tagged mutants with enhanced contents of phenolic compounds. One dominant activation tagged mutant (hpc1-1D) was isolated showing accumulation of a particular compound due to the upregulation of an R2R3-MYB transcription factor.

Amino Acid Sequence↗

PubMed1998

Exochelin genes in Mycobacterium smegmatis: identification of an ABC transporter and two non-ribosomal peptide synthetase genes.

Many strains of mycobacteria produce two ferric chelating substances that are termed exochelin (an excreted product) and mycobactin (a cell-associated product). These agents may function as iron acquisition siderophores. To examine the genetics of the iron acquisition system in mycobacteria, ultraviolet (UV) and transposon (Tn611) mutagenesis techniques were used to generate exochelin-deficient mutants of Mycobacterium smegmatis strains ATCC 607 and LR222 respectively. Mutants were identified on CAS siderophore detection agar plates. Comparisons of the amounts of CAS-reactive material excreted by the possible mutant strains with that of the wild-type strains verified that seven UV mutant strains and two confirmed transposition mutant strains were deficient in exochelin production. Cell-associated mycobactin production in the mutants appeared to be normal. From the two transposon mutants, the mutated gene regions were cloned and identified by colony hybridization with an IS6100 probe, and the DNA regions flanking the transposon insertion sites were then used as probes to clone the wild-type loci from M. smegmatis LR222 genomic DNA. Complementation assays showed that an 8 kb PstI fragment and a 4.8 kb PstI/SacI subclone of this fragment complemented one transposon mutant (LUN2) and one UV mutant (R92). A 10.1 kb SacI fragment restored exochelin production to the other transposon mutant (LUN1). The nucleotide sequence of the 15.3 kb DNA region that spanned the two transposon insertion sites overlapped the 5' region of the previously reported exochelin biosynthetic gene fxbA and contained three open reading frames that were transcribed in the opposite orientation to fxbA. The corresponding genes were designated exiT, fxbB and fxbC. The deduced amino acid sequence of ExiT suggested that it was a member of the ABC transporter superfamily, while FxbB and FxbC displayed significant homology with many enzymes (including pristinamycin I synthetase) that catalyse non-ribosomal peptide synthesis. We propose that the peptide backbone of the siderophore exochelin is synthesized in part by enzymes resembling non-ribosomal peptide synthetases and that the ABC transporter ExiT is responsible for exochelin excretion.

ATP-Binding Cassette Transporters↗

PubMed1986

Molecular cloning of suppressor of sable, a Drosophila melanogaster transposon-mediated suppressor.

A hybrid dysgenesis-induced allele [su(s)w20] associated with a P-element insertion was used to clone sequences from the su(s) region of Drosophila melanogaster by means of the transposon-tagging technique. Cloned sequences were used to probe restriction enzyme-digested DNAs from 22 other su(s) mutations. None of three X-ray-induced or six ethyl methanesulfonate-induced su(s) mutations possessed detectable variation. Seven spontaneous, four hybrid dysgenesis-induced, and two DNA transformation-induced mutations were associated with insertions within 2.0 kilobases (kb) of the su(s)w20 P-element insertion site. When the region of DNA that included the mutational insertions was used to probe poly(A)+ RNAs, a 5-kb message was detected in wild-type RNA that was present in greatly reduced amounts in two su(s) mutations. By using strand-specific probes, the direction of transcription of the 5-kb message was determined. The mutational insertions lie in DNA sequences near the 5' end of the 5-kb message. Three of the seven spontaneous su(s) mutations are associated with gypsy insertions, but they are not suppressible by su(Hw).

Animals↗

PubMed1985

Evidence for related virulence sequences in plasmids of Salmonella dublin and Salmonella typhimurium.

Transposon-insertion mutants were prepared from virulent field isolates of Salmonella dublin and Salmonella typhimurium. Detailed restriction-enzyme mapping of the single sites of TnA insertion in two mutants (M51 and M173) of S. dublin that showed diminished virulence in a mouse assay indicated that these sites were about 5 kbp apart on the approximately 70 kbp plasmid harboured by the isolate. A Tn10-insertion mutant (M242) of S. typhimurium that showed diminished virulence was also identified. A single copy of Tn10 was inserted into the approximately 90 kbp plasmid harboured by this isolate. Hybridization studies indicated that homology existed between the region encompassing the sites of TnA insertion in M51 and M173 and that encompassing the site of Tn10 insertion in M242. Restriction mapping indicated that the two regions were very similar and could even be identical and, if so, the Tn10 insertion in M242 could be mapped to a point 1.5 kbp from the TnA insertion in M51 and 6.5 kbp from that in M173. It appeared that the maximal extent of the putative similarity/identity was between 13 and 23 kbp. It is proposed that this stretch of high homology could represent a virulence sequence that has been conserved during the evolutionary divergence of the two Salmonella serotypes.

Animals↗

PubMed1995

Simultaneous identification of bacterial virulence genes by negative selection.

An insertional mutagenesis system that uses transposons carrying unique DNA sequence tags was developed for the isolation of bacterial virulence genes. The tags from a mixed population of bacterial mutants representing the inoculum and bacteria recovered from infected hosts were detected by amplification, radiolabeling, and hybridization analysis. When applied to a murine model of typhoid fever caused by Salmonella typhimurium, mutants with attenuated virulence were revealed by use of tags that were present in the inoculum but not in bacteria recovered from infected mice. This approach resulted in the identification of new virulence genes, some of which are related to, but functionally distinct from, the inv/spa family of S. typhimurium.

Animals↗

PubMed2003

Insertion sequence ISEcp1B is involved in expression and mobilization of a bla(CTX-M) beta-lactamase gene.

The genetic structures (ca. 10-kb DNA fragment) surrounding the plasmid-borne extended-spectrum beta-lactamase bla(CTX-M-19) gene in a Klebsiella pneumoniae clinical isolate were determined. This beta-lactamase gene was part of a 4,797-bp transposon inserted inside orf1 of Tn1721. Inside this transposon, bla(CTX-M-19) was bracketed upstream and downstream by insertion sequences ISE cp1B and IS903D, respectively, and further downstream by a truncated gene encoding an outer membrane protein for iron transport. The single-copy ISEcp1B element was probably involved alone in the mobilization process that led to a 5-bp duplication at the target site of the transposed fragment. This mobilization event probably involved one inverted repeat of ISE cp1B and a second sequence farther away, resembling its second inverted repeat. Additionally, ISEcp1B provided -35 and -10 promoter sequences, contributing to the high-level expression of the bla(CTX-M-19) gene. Southern blot analysis failed to identify a reservoir of ISEcp1-like sequences among a series of gram-negative and gram-positive bacterial species usually found in the skin and intestinal human floras. The ability of ISEcp1-like elements to mobilize and to promote the expression of beta-lactamase genes may explain, in part, the current spread of CTX-M-type enzymes worldwide.

Anti-Bacterial Agents↗

PubMed1999

The chlorocatechol-catabolic transposon Tn5707 of Alcaligenes eutrophus NH9, carrying a gene cluster highly homologous to that in the 1,2,4-trichlorobenzene-degrading bacterium Pseudomonas sp. strain P51, confers the ability to grow on 3-chlorobenzoate.

Alcaligenes eutrophus (Ralstonia eutropha) NH9, isolated in Japan, utilizes 3-chlorobenzoate as its sole source of carbon and energy. Sequencing of the relevant region of plasmid pENH91 from strain NH9 revealed that the genes for the catabolic enzymes were homologous to the genes of the modified ortho-cleavage pathway. The genes from strain NH9 (cbnR-ABCD) showed the highest homology (89 to 100% identity at the nucleotide level) to the tcbR-CDEF genes on plasmid pP51 of the 1,2,4-trichlorobenzene-degrading bacterium Pseudomonas sp. strain P51, which was isolated in The Netherlands. The structure of the operon, including the lengths of open reading frames and intervening sequences, was completely conserved between the cbn and tcb genes. Most nucleotide substitutions were localized within and proximal to the cbnB (tcbD) gene. The difference in the chloroaromatics that the two strains could use as growth substrates seemed to be due to differences in enzymes that convert substrates to chlorocatechols. The restriction map of plasmid pENH91 was clearly different from that of pP51 except in the regions that contained the cbnR-ABCD and tcbR-CDEF genes, respectively, suggesting that the chlorocatechol gene clusters might have been transferred as units. Two homologous sequences, present as direct repeats in both flanking regions of the cbnR-ABCD genes on pENH91, were found to be identical insertion sequences (ISs), designated IS1600, which formed a composite transposon designated Tn5707. Although the tcbR-CDEF genes were not associated with similar ISs, a DNA fragment homologous to IS1600 was cloned from the chromosome of strain P51. The sequence of the fragment suggested that it might be a remnant of an IS. The two sequences, together with IS1326 and nmoT, formed a distinct cluster on a phylogenetic tree of the IS21 family. The diversity of the sources of these IS or IS-like elements suggests the prevalence of ISs of this type.

Amino Acid Sequence↗

PubMed1987

Transposition of Tn4551 in Bacteroides fragilis: identification and properties of a new transposon from Bacteroides spp.

Tn4551, a clindamycin resistance (Ccr) transposon from the R plasmid pBI136, was cloned onto an Escherichia coli-Bacteroides shuttle vector which could replicate normally in E. coli but was maintained unstably in Bacteroides fragilis. To aid in cloning and to ensure maintenance of Tn4551 in E. coli, a kanamycin resistance determinant (Kmr) was inserted in the transposon. The transposon-bearing shuttle vector pFD197 was transformed into B. fragilis 638, and putative insertions of Tn4551::Kmr were identified by screening for resistance to clindamycin and plasmid content. Southern hybridization analyses were used to verify integration of the transposon in the B. fragilis chromosome, and the frequency of insertion was estimated at 7.8 X 10(-5) events per generation. In 57% of the isolates tested a second integration event also occurred. This second insertion apparently involved just a single copy of the 1.2-kilobase repeat sequence which flanks the transposon. In addition, Tn4551::Kmr appeared to function as a transposon in E. coli. Evidence for this was obtained by the isolation of transposon insertions into the bacteriophage P1 genome. Finally, the transposon vector, pFD197, could be mobilized to other B. fragilis strains in which transposition was detected. Mobilization from the strain 638 background was via a conjugation like process, but occurred in the absence of known conjugative elements or other detectable plasmids. This result suggested the presence of a host-encoded transfer system in this B. fragilis strain.

Bacteroides fragilis↗

PubMed2002

Integration of a transposon into the Gli3 gene in the Pdn mouse.

The phenotype of the genetic polydactyly/ arhinencephaly mouse (Pdn/Pdn) is similar to Greig cephalopolysyndactyly syndrome (GCPS), whose responsible gene is GLI3. Suppression of Gli3 gene expression has been observed in the Pdn/Pdn and integration of retrotransposon in Gli3 gene in the Pdn mouse has been reported. Thus, the responsible gene for Pdn/Pdn is thought to be Gli3, but the site of mutation within the gene has not been demarcated. In the present study, we demonstrated that 5442 bp of early retrotransposon was inserted into intron 3 of Gli3 gene in the Pdn mouse (Gli3 (Pdn) ). This transposon had almost the same sequence as MMY17106 (EMBL). It had 317-bp long terminal repeat at both ends followed by the identical 6-bp target duplication sequence, GAGACT. Forward and reverse PCR primers were constructed in intron 3 near the insertion point, and a forward primer in the transposon was also constructed. These primers allowed us to discriminate +/+, Pdn/+ and Pdn/Pdn embryos by the PCR products. Morphological determination of the genotypes in the Pdn mouse embryos is impossible before day 12 of gestation. Quick discrimination method of genotypes developed in the present study allows us to investigate the early dysmorphogenetic mechanisms in the brain and limbs in the Pdn/Pdn embryos. Then, the dysmorphogenetic mechanisms in the Pdn/Pdn may be extrapolated to those in GCPS.

Animals↗

PubMed1991

IS231A from Bacillus thuringiensis is functional in Escherichia coli: transposition and insertion specificity.

A kanamycin resistance gene was introduced within the insertion sequence IS231A from Bacillus thuringiensis, and transposition of the element was demonstrated in Escherichia coli. DNA sequencing at the target sites showed that IS231A transposition results in direct repeats of variable lengths (10, 11, and 12 bp). These target sequences resemble the terminal inverted repeats of the transposon Tn4430, which are the preferred natural insertion sites of IS231 in B. thuringiensis.

Bacillus thuringiensis↗

PubMed1994

Characterization of a repetitive element detected by NheI in the genomes of Salmo species.

The genomes of the two species in the genus Salmo (Atlantic salmon, S. salar; brown trout, S. trutta) contain a 380-bp repetitive element that is flanked by the recognition sequence of the restriction enzyme NheI. These elements, which comprise approximately 1.2% of the salmon genome, do not exist in long tandem arrays as is typical of satellite DNA. A comparison of the sequences of 16 salmon and 7 trout elements revealed that members of this family of repetitive DNA are closely related to one another (over 95% identity). Subfamily structure exists and there is evidence that members of the same subfamilies are found in both Salmo species. A search of the GenBank database indicated that sequences homologous to the NheI repeat are located within a 1424-bp segment inserted immediately downstream of the 5' end of a Tc1 transposon-like sequence isolated from Atlantic salmon (A.R. Radice, B. Bugaj, D.H. Fitch, and S.W. Emmons, unpublished data; GenBank accession No. L12206).

Animals↗