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Results for “transposon insertion sequencing”

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At least 433 records · Page 24Linked to original sources
PubMed1996

Site-selected insertional mutagenesis of tomato with maize Ac and Ds elements.

Site-selected insertion (SSI) is a PCR-based technique which uses primers located within the transposon and a target gene for detection of transposon insertions into cloned genes. We screened tomato plants bearing single or multiple copies of maize Ac or Ds transposable elements for somatic insertions at one close-range target and two long-range targets. Eight close-range Ds insertions near the right border of the T-DNA were recovered. Sequence analysis showed a precise junction between the transposon and the target for all insertions. Two insertions in separate plants occurred at the same site, but others appeared dispersed in the region of the right T-DNA border with no target specificity. However, insertions showed a preference for one orientation of the transposon. Use of plants with multiple Ac (HiAc) or Ds (HiDs) elements allowed detection of somatic insertions at two single-copy genes, PG (polygalacturonase) and DFR (dihydroflavonol 4-reductase). Certain HiDs plants showed much higher rates of insertion into PG than others. Insertions in PG and DFR were found throughout the gene regions monitored and, with the exception of one insertion in PG, the junctions between transposon and target were exact. SSI analysis of progeny from the HiDs parents revealed that in some cases the tendency to incur high levels of somatic insertions in PG was inherited. Inheritance of this character is an indication that SSI could be used to direct a search for germinal PG insertions in tomato.

Alcohol Oxidoreductases↗
PubMed2003

Identification of Actinobacillus pleuropneumoniae genes important for survival during infection in its natural host.

Actinobacillus pleuropneumoniae is a strict respiratory tract pathogen of swine and is the causative agent of porcine pleuropneumonia. We have used signature-tagged mutagenesis (STM) to identify genes required for survival of the organism within the pig. A total of 2,064 signature-tagged Tn10 transposon mutants were assembled into pools of 48 each, and used to inoculate pigs by the endotracheal route. Out of 105 mutants that were consistently attenuated in vivo, only 11 mutants showed a >2-fold reduction in growth in vitro compared to the wild type, whereas 8 of 14 mutants tested showed significant levels of attenuation in pig as evidenced from competitive index experiments. Inverse PCR was used to generate DNA sequence of the chromosomal domains flanking each transposon insertion. Only one sibling pair of mutants was identified, but three apparent transposon insertion hot spots were found--an anticipated consequence of the use of a Tn10-based system. Transposon insertions were found within 55 different loci, and similarity (BLAST) searching identified possible analogues or homologues for all but four of these. Matches included proteins putatively involved in metabolism and transport of various nutrients or unknown substances, in stress responses, in gene regulation, and in the production of cell surface components. Ten of the sequences have homology with genes involved in lipopolysaccharide and capsule production. The results highlight the importance of genes involved in energy metabolism, nutrient uptake and stress responses for the survival of A. pleuropneumoniae in its natural host: the pig.

ATP-Binding Cassette Transporters↗
PubMed2002

The identification of five genetic loci of Francisella novicida associated with intracellular growth.

Five transposon mutants of Francisella novicida were isolated that are compromised in their ability to grow in mouse macrophages in vitro. Sequence analysis of the DNA flanking the transposon insertions identified the genes that were interrupted in these mutants. One of the inactivated loci corresponds to the Francisella tularensis gene that encodes a 23-kDa protein that is the most prominently induced protein following macrophage infection. Another insertion was localised to approximately 2 kb upstream of the gene encoding the 23-kDa protein. By analysis of the incomplete Francisella genome sequence it was surmised that these two insertions disrupt different portions of a putative operon that encodes four proteins, none of which have discernible functions. Three other interrupted loci associated with poor intramacrophage growth showed similarity at the deduced amino acid level to alanine racemase, the ClpB heat-shock protease, and the purine biosynthetic enzyme, glutamine phosphoribosylpyrophosphate amidotransferases.

Animals↗
PubMed2004

Identification of Mycobacterium marinum virulence genes using signature-tagged mutagenesis and the goldfish model of mycobacterial pathogenesis.

Mycobacterium marinum, a causative agent of fish tuberculosis, is one of the most closely related Mycobacterium species (outside the M. tuberculosis complex) to M. tuberculosis, the etiologic agent of human tuberculosis. Signature-tagged mutagenesis was used to identify genes of M. marinum required for in vivo survival in a goldfish model of mycobacterial pathogenesis. Screening the first 1008 M. marinum mutants led to the identification of 40 putative virulence mutants. DNA sequence analysis of these 40 mutants identified transposon insertions in 35 unique loci. Twenty-eight out of 33 (85%) loci encoding putative virulence genes have homologous genes in M. tuberculosis.

Animals↗
PubMed1993

Cloning, expression and sequencing of Helicobacter felis urease genes.

Urease genes from Helicobacter felis were cloned and expressed in Escherichia coli cells. A genomic bank of Sau3A-digested H. felis chromosomal DNA was created using a cosmid vector. Cosmid clones were screened for urease activity following subculture on a nitrogen-limiting medium. Subcloning of DNA from an urease-positive cosmid clone led to the construction of pILL205 (9.5 kb) which conferred a urease activity of 1.2 +/- 0.5 mumole urea min-1 mg-1 bacterial protein to E. coli HB101 bacteria grown on a nitrogen-limiting medium. Random mutagenesis using a MiniTn3-Km transposable element permitted the identification of three DNA regions on pILL205 which were necessary for the expression of an urease-positive phenotype in E. coli clones. To localize the putative structural genes of H. felis on pILL205, extracts of clones harbouring the mutated copies of the plasmid were analysed by Western blotting with anti-H. felis rabbit serum. One mutant clone did not synthesize the putative UreB subunit of H. felis urease and it was postulated that the transposable element had disrupted the corresponding structural gene. By sequencing the DNA region adjacent to the transposon insertion site two open reading frames, designated ureA and ureB, were identified. The polypeptides encoded by these genes had calculated molecular masses of 26,074 and 61,663 Da, respectively, and shared 73.5% and 88.2% identity with the corresponding gene products of Helicobacter pylori urease.

Amino Acid Sequence↗
PubMed1984

Replicative and conservative transpositional recombination of insertion sequences.

We have presented the results of experiments with IS903- and IS10- derived transposons that have led us to the following conclusions: The predominant mechanism of transpositional recombination of these IS elements is a donor-suicide process that results intermolecularly in a simple IS insertion. This process presumably involves little or no replication of the IS. Intramolecular transposition by this process normally results in nonviable products. However, in the particular situation where the transpositional target lies within the transposon, viable products are obtained; these are deletions and deletion-inversions. Deletions between an IS and a target lying outside the element (the conventional "adjacent deletion") occur by a fully replicative process analogous to the formation of cointegrate molecules in intermolecular transposition. The ability of an IS to promote adjacent deletions correlates closely with its ability to fuse replicons into a cointegrate. Before transposition can occur, a complex of the transposase and both IS ends is probably formed. Requirement for such a pretranspositional complex is suggested by the effect on transpositional frequency of changing the distance between the ends. Our results do not support any of the asymmetrical models for transposition. They are, however, compatible with a modified version of the symmetric model proposed by Shapiro (1979). It is interesting to note the similarity between the structures generated by intramolecular simple transposition of an inverse transposon and the circular structures apparently formed by retroviral and copia autointegrative transposition. Shoemaker et al. (1981a,b) and Flavell and Ish-Horowicz (1983) have characterized circular molecules from retrovirally infected cells and Drosophila tissue-culture cells, respectively. The structures of some of the circular molecules resemble deletions and deletion-inversions (Fig. 3B). To our knowledge, a circular species containing two long terminal repeats (LTRs) and an adjacent deletion, which we predict could only occur by a fully replicative process given the similarity in geometry of an LTR to an IS, have not been found. It would appear, then, that the molecule containing two LTRs acts as an inverse transposon, integrating into itself. Shoemaker et al. (1981b) and Flavell and Ish-Horowicz (1983) have also suggested that these products arise from molecules containing two LTRs. We suggest that the two inside LTR ends interact in a conservative, intramolecular, simple transpositionlike event.

DNA Replication↗
PubMed1995

Inducible transposition in Streptomyces lividans of insertion sequence IS6100 from Mycobacterium fortuitum.

Transposition of IS6100, originally isolated as part of the compound transposon Tn610 from Mycobacterium fortuitum, was tested in the related actinomycete Streptomyces lividans. Cointegrate formation was observed, as expected for this IS6-related element, and involved apparent random integration of the temperature-sensitive vector carrying IS6100 and concomitant duplication of the insertion sequence. This establishes that a single copy of the insertion sequence can promote transposition and is a precedent for the functioning of a heterologous transposable element in Streptomyces. Transposition could be induced 100-fold by external transcription emanating from a copy of the thiostrepton-inducible promoter ptipA located outwith the insertion sequence and resulting in overexpression of the transposase gene. Thus, in contrast to other prokaryotic transposable elements, IS6100 appears to have no effective means of protecting itself from external activation.

Base Sequence↗
PubMed1999

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

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

Amino Acid Sequence↗
PubMed1992

Hyperhemolytic phenomena associated with insertions of Tn916 into the hemolysin determinant of Enterococcus faecalis plasmid pAD1.

Members of the Tn916 family of conjugative transposons are able to insert themselves into Enterococcus faecalis hemolysin/bacteriocin plasmid pAD1 (and related elements) in such a way as to generate hyperexpression of the hemolysin/bacteriocin. To examine this phenomenon in more detail, E. faecalis (pAD1::Tn916) derivatives defective or altered in hemolysin expression were isolated and characterized with respect to production of the L (lytic) or A (activator) component (also known as CylA) and the specific location of the transposon. The mutants fell into five classes. Class 1 strains were nonhemolytic, and the related insertions mapped in a location known to affect expression of the L component. The other four classes varied from an inability to express hemolysin (class 2) to different degrees of hyperhemolytic expression (classes 3 to 5); the insertions in these classes mapped in a similar place within cylA, near the 3' end of the determinant. A previous study provided evidence that CylA is also necessary for bacteriocin immunity; however, these insertions did not destroy this function. (A Tn917 insertion in the 5' half of the determinant eliminates immunity.) In mutant classes 3 to 5, the presence of tetracycline enhanced hemolysin expression. In late-exponential-phase broth cultures, hemolysin could not be detected in supernatants of classes 2 to 5, in contrast to a wild-type control strain; however, different amounts of the L component could be detected, with the lowest in class 2 and greater-than-normal amounts in classes 3 to 5. Although nucleotide sequencing showed that the Tn916 insertions in classes 2 to 5 were at identical sites, the transposon junction sequences differed in some cases. The data indicated that cylA translation into the transposon would result in different truncation sites, and these differences were probably related to phenotype differences.

Amino Acid Sequence↗
PubMed1995

Specificity of insertion of Tn1545 transposon family in Lactococcus lactis subsp. lactis.

A collection of Lactococcus lactis subsp. lactis strains carrying a derivative of Tn1545 inserted in the chromosome was generated. Some 34 insertions were cloned in Escherichia coli and the flanking DNA sequences determined. Insertions are distributed non randomly and several hot spots were observed. The hot spots do not have sequence features which would distinguish them from other insertion sites, suggesting that they may have special structural properties. Insertions in open reading frames occurred at a far lower frequency than expected for random transposition and were most often located near potential terminator and promoter sequences. Different mutations in the extremities of the transposon do not affect the specificity of insertion. We suggest that target specificity is mainly due to the properties of the intergrase itself.

Amino Acid Sequence↗
PubMed2004

Activity of the En/Spm-like transposons in meiosis as a base for chromosome repatterning in a small, isolated, peripheral population of Aegilops speltoides Tausch.

Chromosomal repatterning is considered to be one of the main mechanisms for plant genome evolution. Here, we report the first cytogenetic evidence for the involvement of En/Spm transposons in ongoing chromosomal repatterning leading to the rise of new fertile genomic forms in a small, isolated, peripheral plant population. Cytogenetical screening of original individual plants of Aegilops speltoides Tausch. with different phenotypes revealed a wide spectrum of chromosomal abnormalities including extra chromosomes, chromosomal rearrangements, and variability in chromosomal position/number of 45S and 5S rDNA sites. Cytogenetic analysis of the dynamics of En/Spm transposons in meiosis indicates that: (i) this type of transposon is active during male gametogenesis; (ii) separately or in conjunction with rDNA they form clusters in the hot spots of large chromosomal rearrangements; (iii) appearance of at least part of the mobile rDNA sites in genome of Ae. speltoides are connected with meiotic activity of En/Spm transposons. PCR screening for the site-selected transposon insertions confirm the presence of combined fragments that consist partly of the sequence of En/Spm transposon and partly of 5S rDNA sequence.

Chromosomes↗
PubMed1997

Identification and characterization of a K88- and CS31A-like operon of a rabbit enteropathogenic Escherichia coli strain which encodes fimbriae involved in the colonization of rabbit intestine.

Initiation of attaching-effacing lesions, which characterize infections with rabbit enteropathogenic Escherichia coli (REPEC), requires bacteria to adhere to the intestinal epithelium. This adherence is reflected in vitro by the affinity of these E. coli strains for various types of eukaryotic cells. TnphoA mutants of REPEC 83/39 (O15:H-) which had lost the ability to adhere to HEp-2 epithelial cells, guinea pig ileal brush borders, and mouse erythrocytes were generated. DNA sequencing of the region surrounding the inactivating transposon insertions within a 95-kb plasmid, designated pRAP for REPEC adherence plasmid, revealed extensive homology between that region and the structural genes of enterotoxigenic E. coli operons encoding the K88 and CS31A fimbrial adhesins and the genes for the afr2 adhesin from REPEC B10 (O103:H2). Seven genes of the ral operon (for REPEC adherence locus), including three putative minor fimbrial subunit genes (ralC, ralF, and ralH), a major fimbrial subunit gene (ralG), a gene of unknown function (ralI), and genes for two fimbrial subunit chaperones (ralD and ralE), were sequenced. When inoculated perorally into weanling rabbits, a mutant with a TnphoA insertion in the ralE gene showed a 10-fold reduction in colonizing ability, with only 1 of 10 rabbits excreting bacteria compared to all 5 of those infected with the wild-type parent strain (P = 0.002). The severity of the diarrheal illness caused by the mutant strain was also reduced. Western blotting of surface protein extracts of strain 83/39 with hyperimmune anti-83/39 antiserum, adsorbed with the ralE mutant, revealed a 32-kDa protein which was absent from protein extracts of two nonadherent mutants. The adsorbed antiserum also bound to the surface of strain 83/39 but not to nonadherent mutants, as detected by immunogold labeling. These results indicate that the ral operon of REPEC 83/39 contains genes necessary for the biosynthesis of fine fimbriae which are responsible for in vitro adherence of the bacteria and play a role in their colonization of, and hence virulence for, rabbits. The putative major fimbrial subunit is a protein with an observed molecular size of approximately 32 kDa which, when assembled, appears to form a capsule of fimbriae surrounding the bacterium similar to that described for CS31A.

Amino Acid Sequence↗
PubMed2001

gmhX, a novel gene required for the incorporation of L-glycero-D-manno-heptose into lipooligosaccharide in Neisseria meningitidis.

Lipooligosaccharide (LOS) is a critical virulence factor of Neisseria meningitidis. A Tn916 insertion mutant, designated 469, was found to exhibit a markedly truncated LOS of 2.9 kDa when compared by Tricine/SDS-PAGE to the parental LOS (4.6 kDa). Electrospray mass spectrometry analysis of 469 LOS revealed that it consisted of the deep rough, heptose-deficient structure, Kdo(2)-lipid A. Sequencing of chromosomal DNA flanking the Tn916 insertion in mutant 469 revealed that the transposon had inserted into an ORF predicted to encode a 187 aa protein with sequence homology to the histidinol-phosphate phosphatase domain of Escherichia coli HisB and to a family of genes of unknown function. The gene, designated gmhX, is part of a polycistronic operon (ice-2) containing two other genes, nlaB and orfC. nlaB encodes a lysophosphatidic-acid acyltransferase and orfC is predicted to encode a N-acetyltransferase. Specific polar and non-polar gmhX mutations in the parental strain, NMB, exhibited the truncated LOS structure of mutant 469, and repair of gmhX mutants by homologous recombination with the wild-type gmhX restored the LOS parental phenotype. GmhX mutants demonstrated increased sensitivity to polymyxin B. GmhX mutants and other Kdo(2)-lipid A mutants also demonstrated increased sensitivity to killing by normal human serum but were not as sensitive as inner-core mutants containing heptose. In the genomes of Helicobacter pylori and Synechocystis, gmhX homologues are associated with heptose biosynthesis genes; however, in N. meningitidis, gmhX was found in a location distinct from that of gmhA, rfaD, rfaE, aut and rfaC. GmhX is a novel enzyme required for the incorporation of L-glycero-D-manno-heptose into meningococcal LOS, and is a candidate for the 2-D-glycero-manno-heptose phosphatase of the heptose biosynthesis pathway.

Bacterial Proteins↗
PubMed1998

A novel plasmid pIJB1 possessing a putative 2,4-dichlorophenoxyacetate degradative transposon Tn5530 in Burkholderia cepacia strain 2a.

A 102-kb plasmid, pIJB1, was isolated from Burkholderia cepacia strain 2a, which is able to use 2,4-dichlorophenoxyacetate (2,4-D) as a sole carbon source, and a physical map of the plasmid has been established. It was observed that spontaneous loss of a 37-kb fragment of the plasmid after growth in nonselective medium occurred, generating a plasmid of diminished size, pIJB2. The deletion event is concomitant with the loss of the 2,4-D dissimilatory phenotype, indicating that at least some of the 2,4-D degradative genes are on the missing fragment. The missing fragment is flanked by two identical 4.3-kb insertion sequences (IS) and shows a typical composite transposon structure of 41-kb in size, designated Tn5530. The mutant plasmid pIJB2 possesses a single copy of the IS element.

2,4-Dichlorophenoxyacetic Acid↗
PubMed1992

NotI genomic cleavage map of Escherichia coli K-12 strain MG1655.

Several approaches were used to construct a complete NotI restriction enzyme cleavage map of the genome of Escherichia coli MG1655. The approaches included use of transposable element insertions that created auxotrophic mutations and introduced a NotI site into the genome, hybridization of NotI fragments to the ordered lambda library constructed by Kohara et al. (BioTechniques 10:474-477, 1991), Southern blotting of NotI digests with cloned genes as probes, and analysis of the known E. coli DNA sequence for NotI sites. In all, 22 NotI cleavage sites were mapped along with 26 transposon insertions. These sites were localized to clones in the lambda library and, when possible, sequenced genes. The map was compared with that of strain EMG2, a wild-type E. coli K-12 strain, and several differences were found, including a region of about 600 kb with an altered restriction pattern and an additional fragment in MG1655. Comparison of MG1655 with other strains revealed minor differences but indicated that this map was representative of that for many commonly used E. coli K-12 strains.

Base Sequence↗
PubMed2001

Screening insertion libraries for mutations in many genes simultaneously using DNA microarrays.

We describe a method to screen pools of DNA from multiple transposon lines for insertions in many genes simultaneously. We use thermal asymmetric interlaced-PCR, a hemispecific PCR amplification protocol that combines nested, insertion-specific primers with degenerate primers, to amplify DNA flanking the transposons. In reconstruction experiments with previously characterized Arabidopsis lines carrying insertions of the maize Dissociation (Ds) transposon, we show that fluorescently labeled, transposon-flanking fragments overlapping ORFs hybridize to cognate expressed sequence tags (ESTs) on a DNA microarray. We further show that insertions can be detected in DNA pools from as many as 100 plants representing different transposon lines and that all of the tested, transposon-disrupted genes whose flanking fragments can be amplified individually also can be detected when amplified from the pool. The ability of a transposon-flanking fragment to hybridize declines rapidly with decreasing homology to the spotted DNA fragment, so that only ESTs with >90% homology to the transposon-disrupted gene exhibit significant cross-hybridization. Because thermal asymmetric interlaced-PCR fragments tend to be short, use of the present method favors recovery of insertions in and near genes. We apply the technique to screening pools of new Ds lines using cDNA microarrays containing ESTs for approximately 1,000 stress-induced and -repressed Arabidopsis genes.

DNA Transposable Elements↗
PubMed1996

Cloning, sequencing, and characterization of a gene (narT) encoding a transport protein involved in dissimilatory nitrate reduction in Staphylococcus carnosus.

A Tn917 mutant of Staphylococcus carnosus TM300, nrIII, was isolated and characterized. Mutant nrIII did not take up nitrate or accumulate nitrite when grown in B-medium supplemented with up to 10 mM nitrate under anoxic conditions; however, it displayed wild-type levels of benzyl Delta viologen-linked nitrate reductase activity. Cultivated in B-medium with nitrate under oxic conditions, mutant nrIII accumulated fivefold less nitrite than the wild-type. The mutation in S. carnosus nrIII could be complemented with a 2-kb chromosomal EcoRI-HpaI fragment from the wild-type. The gene affected by transposon insertion in mutant nrIII was cloned and sequenced. Analysis of the deduced amino acid sequence revealed that this gene, designated narT, encodes a highly hydrophobic 42-kDa transmembrane protein of 388 amino acids and shows similarities to transport proteins that play a role in nitrate import or nitrite export. The inability of nrIII to take up nitrate under anoxic conditions and its ability to take up and accumulate nitrite in the presence of benzyl viologen, a nitrate ionophore, under the same conditions suggest that NarT represents a transport protein required for nitrate uptake under anoxic conditions in S. carnosus.

Amino Acid Sequence↗