SEARCH · PubMed Health

Results for “transposon insertion sequencing”

Explore indexed PubMed citations for clinical trials, systematic reviews and public health research. Read source abstracts and follow each citation to its original PubMed record.

Quote a phrase for an exact phrase match. Source license links do not imply unrestricted reuse.

At least 775 records · Page 43Linked to original sources

PubMed1996

Characterization of cpsF and its product CMP-N-acetylneuraminic acid synthetase, a group B streptococcal enzyme that can function in K1 capsular polysaccharide biosynthesis in Escherichia coli.

Group B Streptococcus (GBS) is the foremost cause of neonatal sepsis and meningitis in the United States. A major virulence factor for GBS is its capsular polysaccharide, a high molecular weight polymer of branched oligosaccharide subunits. N-acetylneuraminic acid (Neu5Ac or sialic acid), at the end of the polysaccharide side chains, is critical to the virulence function of the capsular polysaccharide. Neu5Ac must be activated by CMP-Neu5Ac synthetase before it is incorporated into the polymer. We showed previously that a transposon mutant of a serotype III GBS strain which had no detectable capsular Neu5Ac was deficient in CMP-Neu5Ac-synthetase activity (Wessels et al., 1992). In this paper, we report the identification and characterization of cpsF, a gene interrupted by transposon insertion in the previously described Neu5Ac-deficient mutant. The predicted amino acid sequence of the cpsF gene product shares 57% similarity and 37% identity with CMP-Neu5Ac synthase encoded by the Escherichia coli K1 gene, neuA. The enzymatic function of the protein encoded by cpsF was established by cloning the gene E. coli under the control of the T7 polymerase/promoter. Lysates of E. coli in which the cpsF gene product was expressed, catalysed the condensation of CTP with Neu5Ac to form CMP-Neu5Ac. In addition, when CMP-Neu5Ac synthetase-deficient mutant of E. coli K1 was transformed with cpsF, K1 antigen expression was restored. We concluded that cpsF encodes CMP-Neu5Ac synthetase in type III GBS, and that the GBS enzyme can function in the capsule-synthesis of a heterologous bacterial species.

Antigens, Bacterial↗

PubMed1993

Cloning by insertional mutagenesis of a cDNA encoding Caenorhabditis elegans kinesin heavy chain.

An additional genetic locus in Caenorhabditis elegans, unc-116, was identified in a screen for mutations resulting in defective locomotion. unc-116 was cloned by use of a transposon insertion mutant and the physical and genetic map of the genome. The cDNA sequence predicts an 815-amino acid protein. Based upon sequence comparison and secondary structure predictions, unc-116 encodes all three domains of the kinesin heavy chain: the motor, stalk, and tail. While the motor and tail domains have a high degree of identity to the equivalent domains of cloned kinesin heavy chains, the rodII domain of the stalk is significantly shorter than those previously reported and is not predicted to form a coiled-coil alpha-helix. Analysis of mutational defects in two C. elegans genes encoding anterograde motor molecules, unc-116 and unc-104, should provide insight into the in vivo functions of these members of the kinesin heavy chain superfamily.

Amino Acid Sequence↗

PubMed1982

Genetic transformation of Drosophila with transposable element vectors.

Exogenous DNA sequences were introduced into the Drosophila germ line. A rosy transposon (ry1), constructed by inserting a chromosomal DNA fragment containing the wild-type rosy gene into a P transposable element, transformed germ line cells in 20 to 50 percent of the injected rosy mutant embryos. Transformants contained one or two copies of chromosomally integrated, intact ry1 that were stably inherited in subsequent generations. These transformed flies had wild-type eye color indicating that the visible genetic defect in the host strain could be fully and permanently corrected by the transferred gene. To demonstrate the generality of this approach, a DNA segment that does not confer a recognizable phenotype on recipients was also transferred into germ line chromosomes.

Animals↗

PubMed1985

Structural and functional analyses of the fosfomycin resistance transposon Tn2921.

The fosfomycin resistance transposon Tn2921 is flanked by directly repeated sequences homologous to the Tn10-related insertion sequence IS10. The nonrepeated DNA sequences of Tn2921 can be deleted without affecting the transposition ability of the element, showing that at least one of the direct repeats is an active insertion sequence. Transposition of Tn2921 seems to occur through direct transposition, since cointegrates have not been observed. The evolutionary relatedness of Tn2921 and IS10 is discussed.

Anti-Bacterial Agents↗

PubMed1991

The spo0K locus of Bacillus subtilis is homologous to the oligopeptide permease locus and is required for sporulation and competence.

Spore formation in Bacillus subtilis is a dramatic response to environmental signals that is controlled in part by a two-component regulatory system composed of a histidine protein kinase (SpoIIJ) and a transcriptional regulator (Spo0A). The spo0K locus plays an important but undefined role in the initiation of sporulation and in the development of genetic competence. spoIIJ spo0K double mutants had a more severe defect in sporulation than either single mutant. Overproduction of the spoIIJ gene product resulted in the suppression of the sporulation defect, but not the competence defect, caused by mutations in the spo0K locus. On the basis of the phenotype of the spoIIJ spo0K double mutant and the effect of overproduction of the spoIIJ gene product, a transposon insertion in the spo0K locus was isolated. The spo0K locus was cloned and sequenced. spo0K proved to be an operon of five genes that is homologous to the oligopeptide permease (opp) operon of Salmonella typhimurium and related to a large family of membrane transport systems. The requirement for the transport system encoded by spo0K in the development of competence was somewhat different than its requirement in the system encoded by spo0K in the development of competence was somewhat different than its requirement in the initiation of sporulation. Disruption of the last open reading frame in the spo0K operon caused a defect in competence but had little or no effect on sporulation. We hypothesize that the transport system encoded by spo0K may have a role in sensing extracellular peptide factors that we have shown are required for efficient sporulation and perhaps in sensing similar factors that may be necessary for genetic competence.

Amino Acid Sequence↗

PubMed1999

Random transposition by Tn916 in Desulfitobacterium dehalogenans allows for isolation and characterization of halorespiration-deficient mutants.

To allow for the molecular analysis of halorespiration by the strictly anaerobic gram-positive bacterium Desulfitobacterium dehalogenans, halorespiration-deficient mutants were selected and characterized following insertional mutagenesis by the conjugative transposon Tn916. To facilitate rapid screening of transconjugants, a highly efficient method for the growth of single colonies on solidified medium has been developed. A streptomycin-resistant mutant of D. dehalogenans was isolated and mated with Enterococcus faecalis JH2-2 carrying Tn916. Insertion of one or two copies of Tn916 into the chromosome of D. dehalogenans was observed. From a total of 2,500 transconjugants, 24 halorespiration-deficient mutants were selected based upon their inability to use 3-chloro-4-hydroxyphenylacetic acid as an electron acceptor. Physiological characterization led to the definition of three phenotypic classes of mutants that differed in their ability to use the additional terminal electron acceptors nitrate and fumarate. The activities of hydrogenase and formate dehydrogenase were determined, and the transposon insertion sites in selected mutants representing the different classes were analyzed on the sequence level following amplification by inverse PCR. The results of the molecular characterization as well as the pleiotropic phenotypes of most mutants indicate that genes coding for common elements shared by the different respiratory chains present in the versatile D. dehalogenans have been disrupted.

Bacteria, Anaerobic↗

PubMed1987

[Comparative analysis of the structure of incompatibility plasmids N, P and W].

Evolutionary relationships of the IncN plasmid R15 and other broad host range plasmids (IncN plasmids N3 and R46, IncP plasmids RP4 and R906, IncW plasmids Sa and R388) were studied by Southern blot hybridization technique. The IncN plasmids were shown to harbour homologous determinants for replication and conjugation. No homology was found between the rep and tra genes in R15 and in the IncW and IncP plasmids, respectively. The second rep region of the N3 plasmid is distinctive from the corresponding determinants in the IncN plasmids. Homology was demonstrated for the plasmid genes that mediate restriction and modification in R15 and N3, mercury resistance in R15 and R906, sulfanilamide resistance in R15, N3, R46, Sa, R388, and R906, streptomycin resistance in R15, R46 and Sa. The latter genes are different from the R906 SmR gene. In addition to the three known mobile elements in the plasmid R15, the fourth one (IS46) that is a part of the transposon Tn2353 was identified in this study. Besides, the third copy of this insertion sequence was found in the N3 plasmid.

Base Sequence↗

PubMed2005

Transposition of ISHp608, member of an unusual family of bacterial insertion sequences.

ISHp608 from Helicobacter pylori is active in Escherichia coli and represents a recently recognised group of insertion sequences. Its transposase and organisation suggest that it transposes using a different mechanism to that of other known transposons. The IS was shown to excise as a circular form, which is accompanied by the formation of a resealed donor plasmid backbone. We also demonstrate that TnpA, which is less than half the length of other transposases, is responsible for this and for ISHp608 transposition. Transposition was shown to be site specific: both insertion and transposon excision require a conserved target, 5'TTAC. Deletion analysis suggested that potential secondary structures at the left and right ends are important for transposition. In vitro TnpA bound both ends, showed a strong preference for a specific single-stranded DNA and introduced a single-strand break on the same strand at each end. Although many of the characteristics of ISHp608 appear similar to rolling-circle transposons, there are differences suggesting that, overall, transposition occurs by a different mechanism. The results have permitted the formulation of several related models.

Amino Acid Sequence↗

PubMed1997

Organization and regulation of the D-xylose operons in Escherichia coli K-12: XylR acts as a transcriptional activator.

The metabolism of D-xylose in Escherichia coli K-12 is known to be mediated by the xylAB gene. However, the nearby xylFGHR genes were found by genome sequencing and predicted to be responsible for transport and regulation for xylose based on their sequence similarities to other functionally related genes. Here, we investigated transcriptional organization and functions of the xyl genes. An analysis with random transposon insertions revealed that the xyl genes are organized into two major transcriptional units, xylAB and xylFGHR, governed by the promoters PA and PF, respectively. However, there is an additional weak promoter, PR, which is specific for xylR. Sites of transcription initiation were determined by primer extension analysis. When studied with operon fusions to lacZ, the PA and PF promoters were activated by D-xylose and repressed by glucose. In contrast, the PR promoter was not regulated by these sugars. A mutation in xylR completely abolished expression from the PA and PF promoters, causing a defect in both growth and transport. Binding of XylR to the xyl promoter was enhanced by the presence of D-xylose, suggesting that transcription was positively regulated by XylR. In vivo footprinting analysis revealed that XylR binds to at least two DNA regions, IA and IF, each with a direct repeat. It is very likely that XylR interacts with IA and IF as a dimer. The presumed binding sites are located just upstream of the promoter consensus sequences (-35), while IA is additionally flanked by a cyclic AMP receptor protein-binding site on the other side. The proposed structure of xyl promoters is consistent with the regulation of xyl gene expression and with phenotypes of transposon insertions obtained in the promoter regions.

Bacterial Proteins↗

PubMed1982

Isolation of a Caulobacter gene cluster specifying flagellum production by using nonmotile Tn5 insertion mutants.

Caulobacter crescentus assembles a single polar flagellum from protein components synthesized at a specific time in the cell cycle. Of the 26 genes required for flagellum production, at least 4 of them-flaY, E, F, and G-map together in a single cluster. We have isolated DNA from this region of the chromosome by using a nonmotile mutant with a Tn5 insertion into flaE. C. crescentus DNA carrying the Tn5-flaE region and adjacent sequences was cloned into pBR325 and selected by transposon-encoded kanamycin resistance. The resulting plasmid was used as a probe to isolate the flaE region from a wild-type gene bank and to determine the chromosomal location of several deletion and insertion mutations within the flaY/E/F/G cluster. At least three promotors and three major transcripts were shown to originate from the cloned gene cluster. The role of these genes in flagellar biogenesis was examined by immunoprecipitation of mutant cell extracts with antiflagellin antibody. Deletions extending rightward into this gene cluster eliminated one of the two flagellin proteins normally synthesized by C. crescentus. Mutations mapping to the left permitted synthesis of both normal flagellins but at significantly decreased levels. These results suggest that the leftward end of this cluster contains a region that may function in a regulatory capacity whereas the rightward end may contain sequences overlapping a flagellin structural gene.

Journal Article↗

PubMed1990

Structural and functional characterization of tnpI, a recombinase locus in Tn21 and related beta-lactamase transposons.

A novel discrete mobile DNA element from Tn21 from the plasmid R100.1 is described, and its mobilization function was confirmed experimentally. In addition, the element behaves as a recombinase-active locus (tnpI) which facilitates insertions of antibiotic resistance genes as modules or cassettes at defined hot spots or integration sites. A similar tnpI sequence was detected by DNA hybridization in a series of beta-lactamase transposons and plasmids and localized on their physical maps. The genetic function of the locus cloned from Tn21 into pACYC184 was tested for conduction and integration into the plasmids R388 and pOX38Km, and the results suggested recombinase-integrase activity and recA independence. DNA sequence analysis of the tnpI locus revealed no inverted or direct terminal repeats or transposition features of class I and class II transposons. The coding capacity revealed three putative open reading frames encoding 131, 134, and 337 amino acids. Orf3 encoded a putative polypeptide product of 337 amino acids that shared highly significant identity with the carboxyl region of integrase proteins. A comparison and an alignment of the tnpI locus from Tn21 and its flanking sequences identified similar sequences in plasmids and in transposons. The alignment revealed discrete nucleotide changes in these tnpI-like loci and a conserved 3' and 5' GTTA/G hot spot as a duplicated target site. Our data confirm the remarkable ubiquity of tnpI associated with antibiotic resistance genes. We present a model of transposon modular evolution into more complex multiresistant units via tnpI and site-specific insertions, deletions, and DNA rearrangements at this locus.

Amino Acid Sequence↗

PubMed2001

Transposons Tn1696 and Tn21 and their integrons In4 and In2 have independent origins.

The first 13.6 kb of the mercury and multidrug resistance transposon Tn1696, which includes the class 1 integron In4, has been sequenced. In4 is 8.33 kb long and contains the 5'-conserved segment (5'-CS) and 2.24 kb of the 3'-conserved segment (3'-CS) flanking four integrated cassettes. The 3'-CS region is followed by one full copy and an adjacent partial copy of the insertion sequence IS6100 flanked, in inverse orientation, by two short segments (123 and 152 bp) from the outer right-hand end of class 1 integrons. This structure is representative of a distinct group of class 1 integrons that differs from In2, found in Tn21, and other related class 1 integrons. In4 does not include transposition genes but is bounded by characteristic 25-bp inverted repeats and flanked by a direct duplication of 5 bp of the target sequence, indicating that it was inserted by a transpositional mechanism. In4 lies between the resII and resI sites of a backbone mercury resistance transposon which is >99.5% identical to Tn5036. Although Tn21 and Tn1696 are both classified as members of the Tn21 subfamily of the Tn3 transposon family, the backbone mercury resistance transposons are only 79 to 96% identical. Tn21 also contains a region of about 0.7 kb not found in Tn1696. The integrons In2 and In4 carrying the antibiotic resistance genes have been inserted at different locations into distinct ancestral mercury resistance transposons. Thus, Tn21 and Tn1696 have independent histories and origins. Other transposons (Tn1403 and Tn1412) that include a class 1 integron also have independent origins. In all except Tn21, the integron is located within the res region of the backbone transposon.

Anti-Infective Agents, Local↗

PubMed1993

Transpositional activity of IS986 in Mycobacterium smegmatis.

The insertion sequence IS986 (also known as IS6110) has been widely used for typing Mycobacterium tuberculosis isolates, due to the extensive multiple polymorphism shown using this probe. Although this polymorphism is presumed to be due to the mobility of IS986, transposition of this element has not previously been demonstrated in the laboratory. Using artificial composite transposons constructed in a vector unable to replicate in mycobacteria, we have succeeded in demonstrating the mobility of IS986 in M. smegmatis, apparently through cointegrate formation and transposition. The apparently random nature of IS986 insertions in M. tuberculosis is advantageous for transposon mutagenesis, although its efficient use will require more effective delivery systems.

Base Sequence↗

PubMed1999

Cloning and nucleotide sequence determination of the entire mec DNA of pre-methicillin-resistant Staphylococcus aureus N315.

In methicillin-resistant Staphylococcus aureus, the methicillin resistance gene mecA is localized within a large chromosomal region which is absent in the methicillin-susceptible S. aureus chromosome. The region, designated mec DNA, is speculated to have originated from the genome of another bacterial species and become integrated into the chromosome of the S. aureus cell in the past. We report here cloning and determination of the structure of the entire mec DNA sequence from a Japanese S. aureus strain, N315. The mec DNA was found to be 51,669 bp long, including terminal inverted repeats of 27 bp and a characteristic pair of direct repeat sequences of 15 bp each: one is situated in the right extremity of mec DNA, and the other is situated outside the mec DNA and abuts the left boundary of mec DNA. The integration site of mec DNA was found to be located in an open reading frame (ORF) of unknown function, designated orfX. Clusters of antibiotic resistance genes were noted in mec DNA carried by transposon Tn554 and an integrated copy of plasmid pUB110. Both the transposon and plasmid were integrated in the proximity of the mecA gene, the latter being flanked by a pair of insertion sequence IS431 elements. Many ORFs other than those encoding antibiotic resistance were considered nonfunctional because of the acquired mutations or partial deletions found in the ORFs. Two ORFs potentially encoding novel site-specific recombinases were found in mec DNA. However, there was no ORF that might encode mec DNA-specific transposase or integrase proteins, indicating that the mec DNA is not a transposon or a bacteriophage in nature.

Amino Acid Sequence↗

PubMed1999

Mutation in GDP-fucose synthesis genes of Sinorhizobium fredii alters Nod factors and significantly decreases competitiveness to nodulate soybeans.

We mutagenized Sinorhizobium fredii HH103-1 with Tn5-B20 and screened about 2,000 colonies for increased beta-galactosidase activity in the presence of the flavonoid naringenin. One mutant, designated SVQ287, produces lipochitooligosaccharide Nod factors (LCOs) that differ from those of the parental strain. The nonreducing N-acetylglucosamine residues of all of the LCOs of mutant SVQ287 lack fucose and 2-O-methylfucose substituents. In addition, SVQ287 synthesizes an LCO with an unusually long, C20:1 fatty acyl side chain. The transposon insertion of mutant SVQ287 lies within a 1.1-kb HindIII fragment. This and an adjacent 2.4-kb HindIII fragment were sequenced. The sequence contains the 3' end of noeK, nodZ, and noeL (the gene interrupted by Tn5-B20), and the 5' end of nolK, all in the same orientation. Although each of these genes has a similarly oriented counterpart on the symbiosis plasmid of the broad-host-range Rhizobium sp. strain NGR234, there are significant differences in the noeK/nodZ intergenic region. Based on amino acid sequence homology, noeL encodes GDP-D-mannose dehydratase, an enzyme involved in the synthesis of GDP-L-fucose, and nolK encodes a NAD-dependent nucleotide sugar epimerase/dehydrogenase. We show that expression of the noeL gene is under the control of NodD1 in S. fredii and is most probably mediated by the nod box that precedes nodZ. Transposon insertion into neoL has two impacts on symbiosis with Williams soybean: nodulation rate is reduced slightly and competitiveness for nodulation is decreased significantly. Mutant SVQ287 retains its ability to form nitrogen-fixing nodules on other legumes, but final nodule number is attenuated on Cajanus cajan.

Amino Acid Sequence↗

PubMed2006

Gene disruption in Cryptococcus neoformans and Cryptococcus gattii by in vitro transposition.

Cryptococcus neoformans and Cryptococcus gattii are basidiomycetous fungi that infect immunocompromised and immunocompetent people. We developed an insertional mutagenesis strategy for these species based on in vitro transposition and we tested the method by disrupting the URA5 gene in a strain of C. neoformans and the CAP10 gene in three strains of C. gattii. We targeted plasmid DNA containing the URA5 gene or plasmid DNA containing the CAP10 gene from genomic libraries from the shotgun sequencing project for the C. gatti strain WM276. In the latter case, the availability of the end sequences of the clones from the assembled genomic sequence allows rapid selection of target genes for disruption. Modified transposons containing the nourseothricin (NAT) or neomycin (Neo) resistance cassettes were randomly inserted into the target DNA by in vitro transposition. The disrupted genes were used for biolistic transformation and homologous integration was subsequently confirmed by PCR and Southern blot analysis. These results demonstrate that the emerging genomic resources, combined with in vitro transposition into plasmid DNAs from shotgun sequencing libraries or cloned PCR products, will facilitate high-throughput genetic analysis in Cryptococcus species.

Biolistics↗

PubMed1987

[Transfer of "artificial transposons" constructed on the basis of insertion element IS1].

Terminal inverted repeats of the insertion element IS1 were synthesized chemically and plasmids containing these sequences flanking kanamycin-resistance gene in different combinations were constructed. Further incorporation of a whole-sized copy of the IS1 into such plasmids caused in some cases the autonomous transfer of Km-resistance from plasmid to bacteriophage lambda DNA. The transposition of the Km-resistance gene was only observed in those cases when the gene was enclosed between IS1 copy and one of the terminal repeats. The data obtained are discussed with regard to the evolution of bacterial transposons.

Bacteriophage lambda↗

PubMed1986

Evidence that white-blood is a novel type of temperature-sensitive mutation resulting from temperature-dependent effects of a transposon insertion on formation of white transcripts.

We report results indicating that the temperature-sensitive white-blood (wbl) mutation has a novel molecular basis, involving the formation of RNA transcripts of the affected gene rather than the behavior of the polypeptide product. First, we show that the temperature-sensitive mutant phenotype of wbl correlates with (and presumably results from) a temperature-dependent effect on levels of the 2.6-kb polyadenylated white transcript. Second, DNA sequence analysis and other studies show that wbl is associated with the insertion into the second white intron of a previously uncharacterized retrotransposon (designated the blood transposon). We discuss the potential origins of the novel temperature-sensitive molecular phenotype of wbl and the prospects for exploiting wbl to engineer temperature-sensitive mutations in other genes.

Alleles↗