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Results for “transposon insertion sequencing”

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PubMed2000

Nucleotide sequence analysis of hypervariable junctions of Haemophilus influenzae pilus gene clusters.

Haemophilus influenzae pili are surface structures that promote attachment to human epithelial cells. The five genes that encode pili, hifABCDE, are found inserted in genomes either between pmbA and hpt (hif-1) or between purE and pepN (hif-2). We determined the sequence between the ends of the pilus clusters and bordering genes in a number of H. influenzae strains. The junctions of the hif-1 cluster (limited to biogroup aegyptius isolates) are structurally simple. In contrast, hif-2 junctions are highly diverse, complex assemblies of conserved intergenic sequences (including genes hicA and hicB) with evidence of frequent recombination. Variation at hif-2 junctions seems to be tied to multiple copies of a 23-bp Haemophilus intergenic dyad sequence. The hif-1 cluster appears to have originated in biogroup aegyptius strains from invasion of the hpt-pmbA region by a DNA template containing the hif-2 genes with termini in the hairpin loop of flanking intergenic dyad sequences. The pilus gene clusters are an interesting model of a mobile "pathogenicity island" not associated with a phage, transposon, or insertion element.

Base Sequence↗

PubMed1998

Molecular screening for P-element insertions in a large genomic region of Drosophila melanogaster using polymerase chain reaction mediated by the vectorette.

As an alternative to existing methods for the detection of new insertions during a transposon mutagenesis, we adapted the method of vectorette ligation to genomic restriction fragments followed by PCR to obtain genomic sequences flanking the transposon. By combining flies containing a defined genomic transposon with an excess of flies containing unrelated insertion sites, we demonstrate the specificity and sensitivity of the procedure in the detection of integration events. This method was applied in a transposon-tagging screen for BJ1, the Drosophila homolog of the vertebrate gene Regulator of Chromosome Condensation (RCCI). Genetic mobilization of a single genomic P element was used to generate preferentially new local insertions from which integrations into a genomic region surrounding the BJ1 gene were screened. Flies harboring new insertions were phenotypically selected on the basis of the zeste1-dependent transvection of white. We detected a single transposition to a 13-kb region close to the BJ1 gene among 6650 progeny that were analyzed. Southern analysis of the homozygous line confirmed the integration 3 kb downstream of BJ1.

Animals↗

PubMed2004

Strain-specific genomic regions of Ruminococcus flavefaciens FD-1 as revealed by combinatorial random-phase genome sequencing and suppressive subtractive hybridization.

Two closely related strains of the Gram-positive, cellulolytic ruminal bacterium Ruminococcus flavefaciens were compared at the genomic level by suppressive subtractive hybridization. The two strains investigated in this study differ by 1.94% in their respective 16S rDNA genes. Three hundred and eighty-four PCR-amplified products were cloned and then screened for their strain identity by dot blot hybridization. Based on redundancy percentages of the clones sequenced, 9.5% of the genome of the R. flavefaciens FD-1 strain is not present in the JM1 strain. The majority of identities of individual cloned subtracted products (642 bp average length) bore no relation to deposited sequences in GenBank (42% of the subtracted library), whereas of those with putative assigned functions 7% are loosely associated with fibre-degradation, 6% with insertion elements, transposons and phage-like ORFs, 5% with cell membrane associated proteins and 3% with signal transduction. Subtracted sequences were then supplemented with the draft (2 x coverage) genome sequence of R. flavefaciens FD-1 to indicate potential regions of rearrangement within the genome, including a novel insertion sequence.

Base Sequence↗

PubMed1989

Listeriolysin O is essential for virulence of Listeria monocytogenes: direct evidence obtained by gene complementation.

The role of listeriolysin O in the intracellular multiplication of Listeria monocytogenes and, therefore, its pathogenicity was questioned through a genetic complementation study. A nonhemolytic mutant was generated by inserting a single copy of transposon Tn917 in the bacterial chromosome. This insertion was localized by DNA sequence analysis in hlyA, the gene coding for listeriolysin O. As was another mutant that we previously characterized, this mutant was avirulent in the mouse. It was transformed with a plasmid carrying only hlyA, able to replicate in L. monocytogenes, and stably maintained in vitro and in vivo. The complemented strain displayed a hemolytic phenotype identical to that of the wild-type strain and was fully virulent, therefore attributing a crucial role to listeriolysin O in virulence and excluding the hypothesis of a polar effect of the transposon insertion on genes adjacent to hlyA and possibly involved in virulence.

Animals↗

PubMed2004

The Rice PIPELINE: a unification tool for plant functional genomics.

The Rice Genome Research Project in Japan performs genome sequencing and comprehensive expression profiling, constructs genetic and physical maps, collects full-length cDNAs and generates mutant lines, all aimed at improving the breeding of the rice plant as a food source. The National Institute of Agrobiological Sciences in Tsukuba, Japan, has accumulated numerous rice biological resources and has already successfully produced a high-quality genome sequence, a high-density genetic map with 3000 markers, 30,000 full-length cDNAs, over 700 expression profiles with a 9000 cDNA microarray and 15,000 flanking sequences with Tos17 insertions in about 3765 mutant lines from about 50,000 transposon insertion lines. These resources are available in the public domain. A new unification tool for functional genomics, called Rice PIPELINE, has also been developed for the dynamic collection and compilation of genomics data (genome sequences, full-length cDNAs, gene expression profiles, mutant lines, cis elements) from various databases. The mission of Rice PIPELINE is to provide a unique scientific resource that pools publicly available rice genomic data for search by clone sequence, clone name, GenBank accession number, or keyword. The web-based form of Rice PIPELINE is available at http://cdna01.dna.affrc.go.jp/PIPE/.

Computational Biology↗

PubMed1986

Identification of Tn4430, a transposon of Bacillus thuringiensis functional in Escherichia coli.

The mobile genetic element Tn4430, originating from the gram-positive bacterium, Bacillus thuringiensis, and previously described as the Th-sequence, is the first transposon isolated from the genus Bacillus. In the present work a gene (APH-III) conferring resistance to kanamycin was inserted into this 4.2 kb transposon. Transposition experiments showed that Tn4430 omega APH-III could transpose in the gram-negative host Escherichia coli when its insertion functions were supplied by an intact copy of Tn4430. By transposing Tn4430 omega APH-III directly onto pBR322, it was possible to determine the nucleotide sequence of the terminal inverted repeats of Tn4430 and of the target DNA site. Identical 38 bp in inverted orientation are situated at each end of the transposon and there is a direct duplication of 5 bp at the insertion site. Thus, it is clear that Tn4430 is closely related to the transposons belonging to the Tn3 family (class II elements).

Bacillus thuringiensis↗

PubMed1988

Isolation and characterization of methyl viologen-sensitive mutants of Escherichia coli K-12.

Escherichia coli mutants sensitive to methyl viologen (MV), an active oxygen propagator, were isolated. Among them, the new genes mvrA and mvrB were mapped at 7 and 28 min on the E. coli linkage map, respectively. MV toxicity was exerted only in the presence of oxygen and was suppressed by the radical scavenger uric acid but not by the hydroxyl radical scavenger mannitol. The mvr mutants were sensitive only to MV and had a normal repair capacity for the MV-damaged DNA. From these results, these mutants were assumed to be related to the elimination of MV-specific toxic species. Gene mvrA was cloned into vector pBR322 and its sequence was determined. The mvrA gene, which was predicted to range in size from 600 to 900 base pairs (bp) by transposon Tn1000 insertion analysis, was identified to be 807 bp, with an approximately 60-bp promoter sequence carrying consensus sequences for the -35 region, the -10 region, and a ribosome-binding site. The MvrA protein deduced from the DNA sequence was 29.7 kilodaltons, which was in good agreement with the 29 kilodaltons of the MvrA protein identified by sodium dodecyl sulfate-polyacrylamide gel electrophoresis after a maxicell labeling experiment.

Amino Acid Sequence↗

PubMed2006

[Progress of the PCR amplification techniques for chromosome walking].

Various PCR-based methods are available for chromosome walking from a known sequence to an unknown region. It is promising in genome-related research for the following experiments: promoter cloning, obtaining non-conservative parts of genes in new species, identification of T-DNA or transposon insertion sites and filling in gaps or unknown chromosome regions in genome sequencing. These methods consisted of three types: inverse PCR, ligation-mediated PCR and specific primer PCR. In this review, we illustrated and compared the current techniques.

Animals↗

PubMed2006

Functional characterization of IS1999, an IS4 family element involved in mobilization and expression of beta-lactam resistance genes.

IS1999 and a point mutant derivative, IS1999.2, have been described inserted upstream of emerging antibiotic resistance genes bla(VEB-1) and bla(OXA-48). 5' Rapid amplification of cDNA ends experiments revealed that expression of these beta-lactamase genes was driven by the outward-directed promoter, P(out), located in the IS1999 elements. These findings led us to study IS1999-mediated gene mobilization. Thus, the transposition properties of IS1999 and of IS1999-based composite transposons, made of two copies of IS1999 in different orientations, were investigated. IS1999 or IS1999-based composite transposons were capable of transposing onto the conjugative plasmid pOX38-Gen. Sequence analysis of the insertion sites revealed that IS1999 inserted preferentially into DNA targets containing the consensus sequence NGCNNNGCN. Transposition was more efficient when at least one left inverted repeat end was located at an outside end of the transposon. The transposition frequency of IS1999.2 was 10-fold lower than that of IS1999, and transposition frequencies of the putative natural transposon, Tn1999, were below detection limits of our transposition assay. This reduced transposition frequency of IS1999.2-based elements may result from a lower transcription of the transposase gene, as revealed by reverse transcription-PCR analyses.

Amino Acid Sequence↗

PubMed1993

The importance of starch biosynthesis in the wrinkled seed shape character of peas studied by Mendel.

The wrinkled-seed mutant (rr) of pea (Pisum sativum L.) arose through mutation of the gene encoding starch-branching enzyme isoform I (SBE1) by insertion of a transposon-like element into the coding sequence. Two isoforms of starch-branching enzyme have been documented in the developing pea embryo. The second isoform, SBEII, is expressed towards the later stages of embryo development while SBEI is expressed highly in the early stages. Due to mutation of SBEI the total amount of starch and the proportion of amylopectin, a branched starch polymer, are greatly reduced in the wrinkled (rr) line as compared to that in the wild-type, round (RR) line. Consequently, the level of sucrose in the rr line is nearly two fold that of the RR line. Increased sucrose concentration in the developing embryos of this mutant line causes increased uptake of water and thereby increases the cell size and fresh weight. During seed maturation in these mutant seeds a greater loss of water occurs. As a result, the wrinkled seed phenotype develops. Besides this morphological variation, the mutation also causes changes in the amount of lipid and of one storage protein, legumin. This review article discusses the role of the SBEI enzyme in causing such metabolic changes in the developing embryos with the implication that metabolism can play a central role in plant development.

1,4-alpha-Glucan Branching Enzyme↗

PubMed2003

The putative sensor histidine kinase CKI1 is involved in female gametophyte development in Arabidopsis.

Embryo sac formation is a fundamental step in sexual reproduction in plants. However, the key players involved in the development of the female gametophyte remain elusive. We present data indicating that a two-component sensor histidine kinase, CKI1, originally implicated in cytokinin perception, is required for completion of megagametogenesis in Arabidopsis. We isolated a loss-of-function mutation in CKI1 resulting from an insertion of the En-1 transposon into the CKI1 coding sequence. Genetic analysis revealed that the mutant allele, cki1-i, could not be transmitted through the female germ line. Confocal laser scanning microscopy identified a block in megagametogenesis, characterized by the abortion of the central vacuole in mutant embryo sacs, and degradation of the developing female gametophyte after completion of all mitotic divisions. The recovery of two independent stable alleles and one revertant wild-type allele resulting from En-1 excision confirmed unambiguously the causal link between the cki1-i mutation and the abnormal phenotype. In situ localization of CKI1 mRNA and histochemical analysis of stable transformants harboring the uidA gene under the control of CKI1 promoter revealed that expression of CKI1 starts at the very beginning of female gametophyte development, and continues until fertilization. This suggests that the developing embryo sac may remain sensitive to signals recognized by CKI1 throughout megagametogenesis. Furthermore, expression of the paternally transmitted CKI1 was detected early after fertilization. The results indicate a role for a two-component signaling system during female gametophyte development, and provide the first evidence that gametophytic expression of a sensor-like molecule is essential for specific processes during megagametogenesis.

Amino Acid Sequence↗

PubMed1984

Nucleotide sequence of the transposable element IS15.

We have determined the complete nucleotide sequence of the right (R) copy of the insertion sequence IS15 which flanks, in direct orientation, the composite transposon Tn1525. IS15-R, which is capable of independent transposition, is 1648 bp long and has short (14 bp) perfect inverted repeats at its termini. Analysis of the nucleotide sequence indicates that IS15-R results from the transposition, in direct orientation, of a smaller (820 bp long) IS, designated IS15-delta, into itself. This integration event is accompanied by the duplication of 8 bp in the target DNA. IS15-delta possesses two large overlapping open reading frames (ORF) located on opposite strands. Because of this particular structure, IS15 possesses four large ORFs which, due to the integration event, exhibit some differences with those of the parental IS15-delta.

Base Sequence↗

PubMed2007

Tools for the genetic analysis of Mycoplasma.

The mycoplasmas have attracted much scientific attention since they contain the smallest known genomes of any independently viable bacterial species. However, the detailed genetic analysis of these bacteria has lagged behind the well-analyzed bacterial model organisms for a long time. This is due to the use of the UGA codon for tryptophan rather than as stop codon, which had often prevented the expression of full-length Mycoplasma proteins in heterologous hosts. Additionally, insufficient efficiency of homologous recombination prevented the targeted disruption of genes in some species such as M. pneumoniae whereas homologous recombination is operative in other mycoplasmas. Only recently, efficient screening systems for the use of transposon-based mutagenesis have been developed to circumvent this problem and to allow the targeted isolation of desired transposon insertion mutants. With the availability of several Mycoplasma genome sequences, artificial plasmids based on the chromosomal origin of replication were constructed that can now be used for complementation studies and for the stable introduction of foreign genetic material. In this review, we give an overview on recent developments in Mycoplasma genetics that facilitate the genetic manipulation of these interesting organisms.

Codon↗

PubMed1996

Identification of sigma S-dependent genes associated with the stationary-phase acid-resistance phenotype of Shigella flexneri.

Shigella flexneri grown to stationary phase has the ability to survive for several hours at pH 2.5. This acid resistance, which may contribute to the low infective dose associated with shigellosis, is dependent upon the expression of the stationary-phase-specific sigma factor sigma S. Using random TnphoA and TnlacZ mutagenesis we isolated five acid-sensitive mutants of S. flexneri, which had lost their ability to survive at pH 2.5 for 2 h in vitro. Each transposon insertion with flanking S. flexneri DNA was cloned and sequenced. Database searches indicated that two TnlacZ mutants had an insertion within the hdeA gene, which is the first gene in the hdeAB operon. Acid resistance was restored in one of these mutants by a plasmid carrying the entire hdeAB operon. Further sequence analysis from the remaining TnlacZ and two TnphoA mutants demonstrated that they all had insertions within a previously unidentified open reading frame (ORF), which is directly downstream from the gadB gene. This putative ORF encodes a protein that has homology to a number of inner membrane amino acid antiporters. A 1.8 kb polymerase chain reaction (PCR) product containing this gene was cloned, which was able to restore acid resistance in each mutant. These fusions were induced during entry into late exponential phase and were positively regulated by RpoS. We confirmed that the expression of the acid-resistance phenotype in acidified minimal media was dependent upon the supplementation of glutamic acid and that this glutamate-dependent system was RpoS regulated. Southern hybridization revealed that both the gadC and hdeAB loci are absent in Salmonella. An rpoS deletion mutant of S. flexneri was also constructed to confirm the important role played by this gene in acid resistance. This rpoS- derivative was extremely acid sensitive. Two-dimensional gel electrophoresis of this mutant revealed that it no longer expressed 27 proteins in late log phase that were present in its isogenic parent. These data indicate that the expression of acid resistance in S. flexneri may be multifactorial and involve proteins located at different subcellular locations.

Acids↗

PubMed1991

IS10 promotes adjacent deletions at low frequency.

Some transposable elements move by a replicative mechanism involving cointegrate formation. Intramolecular cointegration can generate a product called an "adjacent deletion" in which a contiguous chromosomal segment adjacent to the transposon is deleted while the element responsible remains intact. Insertion sequence IS10 is thought to transpose by a nonreplicative mechanism. In the simplest models, nonreplicative transposition cannot give rise to an adjacent deletion because an intrinsic feature of such transposition is excision of the IS element from the donor location. We report here that IS10 can generate adjacent deletions, but at a frequency which is approximately 1/30th the frequency of transposition for the same element. We suggest that these deletions might arise either by nonreplicative transposition events that involve two IS10 elements located on sister chromosomes or by aberrant nonreplicative events involving cleavage and ligation at only one end of the element.

Blotting, Southern↗

PubMed1991

IS900-promoted stable integration of a foreign gene into mycobacteria.

An artificial mycobacterial transposon was constructed by placing two copies of the insertion sequence IS900 flanking a kanamycin resistance gene into a non-(mycobacterial) replicating vector. Constructs were introduced into mycobacteria by electroporation and transposition events conferring kanamycin resistance were selected. Integration of IS900 into several genomic sites was analysed by Southern blotting and shown to involve both simple insertions and cointegrate formation, suggesting that IS900 can transpose by a replicative mechanism. Kanamycin resistance of IS900-integrated transformants was shown to be stable in the absence of selection.

Blotting, Southern↗

PubMed2000

Complete nucleotide sequence of ubiquitous plasmid pEA29 from Erwinia amylovora strain Ea88: gene organization and intraspecies variation.

The complete sequence of plasmid pEA29 from Erwinia amylovora strain Ea88 consists of 28,185 bp with a 50.2% G+C content. As deletions and insertions were detected in other derivatives of pEA29, its size actually varied from 27.6 to 34.9 kb. Thirteen open reading frames that encoded predicted proteins with similarities to known proteins from other bacteria were identified along with two open reading frames related to hypothetical proteins found in GenBank and six open reading frames with no similarities to existing GenBank entries. Predicted products of open reading frames with similarity to the thiamine biosynthetic genes thiO, thiG, and thiF; a betT gene coding for choline transport; an msrA gene for the enzyme methionine sulfoxide reductase; a putative methyl-accepting chemotaxis gene; an aldehyde dehydrogenase gene; an hns DNA binding gene; a LysR-type transcriptional regulator; and parA and parB partitioning genes were identified. A putative iteron-containing theta-type origin of replication with an AT-rich region and a gene for a RepA protein was identified. PstI and KpnI restriction patterns for pEA29 isolated from tree fruit strains of E. amylovora were homogenous and different from those for pEA29 isolated from Rubus (raspberry) strains. All Rubus derivatives of pEA29 contained a point mutation that eliminated a PstI site and a 1,264-bp region that replaced 1, 890 bp of sequence found in pEA29 from strain Ea88. This change eliminated a second PstI site and increased the length of a KpnI fragment. An insertion sequence, ISEam1, was detected in one Rubus strain, and transposon Tn5393 was detected in three apple strains in two separate locations on the plasmid. Plasmid-cured strains exhibited reduced virulence and modified colony morphology on minimal medium without thiamine, indicating that some of the genes in pEA29 play a role in the physiology or metabolism of E. amylovora.

Base Sequence↗

PubMed2000

Identification of three essential regulatory gene loci governing expression of Staphylococcus epidermidis polysaccharide intercellular adhesin and biofilm formation.

The formation of adherent multilayered biofilms embedded into a glycocalyx represents an essential factor in the pathogenesis of Staphylococcus epidermidis biomaterial-related infections. Using biofilm-producing S. epidermidis 1457 and transposon Tn917 carried on plasmid pTV1ts, we isolated nine isogenic biofilm-negative transposon mutants. Transduction by S. epidermidis phage 71 was used to prove the genetic linkage of transposon insertions and altered phenotypes. Mapping of the different transposon insertions by Southern hybridization and pulsed-field gel electrophoresis indicated that these were inserted in four unlinked genetic loci. According to their phenotypes, including quantitative differences in biofilm production in different growth media, in the amount of the polysaccharide intercellular adhesin (PIA) produced, in the hemagglutination titers, and in the altered colony morphology, the mutants could be separated into four phenotypic classes corresponding with the genetic classes. Synthesis of PIA was not detectable with class I and II mutants, whereas the amount of PIA produced reflected the residual degree of biofilm production of class III and IV mutants in different growth media. Chromosomal DNA flanking the transposon insertions of five class I mutants was cloned and sequenced, and the insertions were mapped to different locations of icaADBC, representing the synthetic genes for PIA. Expression of icaADBC from a xylose-dependent promoter in the different isogenic mutant classes reconstituted biofilm production in all mutants. In a Northern blot analysis no icaADBC-specific transcripts were observed in RNA isolated from mutants of classes II, III, and IV. Apparently, in addition to icaADBC, three other gene loci have a direct or indirect regulatory influence on expression of the synthetic genes for PIA on the level of transcription.

Adhesins, Bacterial↗