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Results for “transposon insertion sequencing”

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PubMed1997

Evolution of the rice Xa21 disease resistance gene family.

The rice disease resistance gene Xa21, encoding a receptor-like kinase, is a member of a multigene family. Sequence analysis of seven family members revealed two distinct classes of genes. One member from each class encodes a receptor kinase-like open reading frame. The other five members encode truncated open reading frames of the predicted receptor kinase. A highly conserved 233-bp sequence (HC) was also identified among the seven family members. Recombination at the HC region between family members apparently resulted in the precise swapping of promoter regions. Large sequence duplications were generated by a presumed unequal crossover event in intergenic regions. Insertions of transposon-like sequences truncated two of the predicted open reading frames. A model for amplification and diversification of the Xa21 gene family is presented.

Amino Acid Sequence↗

PubMed1992

A system of transposon mutagenesis for bacteriophage T4.

We have developed a system of transposon mutagenesis for bacteriophage T4. The transposon is a plasmid derivative of Tn5 which contains the essential T4 gene 24, permitting a direct selection for transposition events into a gene 24-deleted phage. The transposition occurred at a frequency of only 10(-7) per progeny phage, even though a dam- host was used to increase transposition frequency. Phage strains with a transposon insert were distinguished from most pseudorevertants of the gene 24 deletion by plaque hybridization using a transposon-specific probe. Mapping analysis showed that the transposon inserts into a large number of sites in the T4 genome, probably with a preference for certain regions. The transposon insertions in four strains were analysed by DNA sequencing using primers that hybridize to each end of the transposon and read out into the T4 genome. In each case, a 9 bp T4 target sequence had been duplicated and the insertions had occurred exactly at the IS50 ends of the transposon, demonstrating that bona fide transposition had occurred. Finally, the transposon insert strains were screened on the TabG Escherichia coli strain, which inhibits the growth of T4 motA mutants, and a motA transposon insert strain was found.

Base Sequence↗

PubMed2002

Cointegrate resolution following transposition of Tn1792 in Streptomyces avermitilis facilitates analysis of transposon-tagged genes.

The insertion sequence IS6100, belonging to the IS6 family, normally forms a cointegrate as an end product of transposition. The IS6100-based minitransposon, Tn1792, has been developed as a genetic tool to mutagenise antibiotic-producing Streptomyces. Here, we describe resolution of Tn1792 cointegrates in Streptomyces avermitilis that can facilitate both the initial isolation of Tn1792 insertion mutants and also the subsequent rescue of Tn1792-tagged sequences. This is the first reported example of cointegrate resolution for an IS6-type transposable element. As a result of mutagenesis, several putative genes involved in morphological development and antibiotic production have been isolated.

Anti-Bacterial Agents↗

PubMed1994

Transposon mutagenesis of coryneform bacteria.

The Corynebacterium glutamicum insertion sequence IS31831 was used to construct two artificial transposons: Tn31831 and miniTn31831. The transposition vectors were based on a gram-negative replication origin and do not replicate in coryneform bacteria. Strain Brevibacterium flavum MJ233C was mutagenized by miniTn31831 at an efficiency of 4.3 x 10(4) mutants per microgram DNA. Transposon insertions occurred at different locations on the chromosome and produced a variety of mutants. Auxotrophs could be recovered at a frequency of approximately 0.2%. Transposition of IS31831 derivatives led not only to simple insertion, but also to cointegrate formation (5%). No multiple insertions were observed. Chromosomal loci of B. flavum corresponding to auxotrophic and pigmentation mutants could be rescued in Escherichia coli, demonstrating that these transposable elements are useful genetic tools for studying the biology of coryneform bacteria.

Base Sequence↗

PubMed2005

Cloning of a CACTA transposon-like insertion in intron I of tomato invertase Lin5 gene and identification of transposase-like sequences of Solanaceae species.

Very few CACTA transposon-like sequences have been described in Solanaceae species. Sequence information has been restricted to partial transposase (TPase)-like fragments, and no target gene of CACTA-like transposon insertion has been described in tomato to date. In this manuscript, we report on a CACTA transposon-like insertion in intron I of tomato (Lycopersicon esculentum) invertase gene Lin5 and TPase-like sequences of several Solanaceae species. Consensus primers deduced from the TPase region of the tomato CACTA transposon-like element allowed the amplification of similar sequences from various Solanaceae species of different subfamilies including Solaneae (Solanum tuberosum), Cestreae (Nicotiana tabacum) and Datureae (Datura stramonium). This demonstrates the ubiquitous presence of CACTA-like elements in Solanaceae genomes. The obtained partial sequences are highly conserved, and allow further detection and detailed analysis of CACTA-like transposons throughout Solanaceae species. CACTA-like transposon sequences make possible the evaluation of their use for genome analysis, functional studies of genes and the evolutionary relationships between plant species.

Amino Acid Sequence↗

PubMed1988

Derived amino acid sequence and identification of active site residues of Escherichia coli beta-hydroxydecanoyl thioester dehydrase.

The nucleotide sequence of the fabA gene encoding beta-hydroxydecanoyl thioester dehydrase, a key enzyme of the unsaturated fatty acid synthesis pathway of Escherichia coli, has been determined by the dideoxynucleotide sequencing technique. Most of the sequence was obtained by sequencing intragenic insertions of the transposon, Tn1000, isolated in vivo. A synthetic primer complementary to a portion of the inverted repeat sequences at the ends of the transposon was used to prime DNA synthesis into the flanking fabA sequences. The gene is composed of 516 nucleotides (171 amino acid residues) encoding a protein with a molecular weight of 18,800. Approximately half of the derived amino acid sequence was confirmed by automated Edman sequencing of peptides obtained by cyanogen bromide cleavage. The active site histidine residue (His-70) has been identified by analysis of the peptides labeled by reaction with 14C-labeled 3-decynoyl-N-acetylcysteamine, a specific mechanism-activated inhibitor. A cysteine residue (Cys-69) adjacent to the active site histidine may play the role in catalysis previously assigned to a tyrosine residue. We also report a simplified purification process for the dehydrase beginning with extracts of a brain which greatly overproduces the enzyme.

Amino Acid Sequence↗

PubMed1978

The transposon Tn9 generates a 9 bp repeated sequence during integration.

We performed a genetic and sequencing analysis of insertions of the transposon Tn9 into the lac operon of E. coli. Genetic mapping of 70 insertions into lacl and Z shows that starting from the same point on the chromosome, Tn9 goes to at least 50 different points in these two genes. Although there are preferred regions for insertion, these consist of multiple integration points within a small area, as demonstrated by pairwise crosses and restriction mapping. Sequence analysis of three Tn9 insertions reveals that Tn9 integration is associated with a direct repeat of 9 base pairs (bp) of host sequence. We show that these extra 9 nucleotide pairs are generated upon insertion and not brought in with the element.

Base Sequence↗

PubMed1997

Tn916 transposition in Haemophilus influenzae Rd: preferential insertion into noncoding DNA.

The availability of completely sequenced genomes has created an opportunity for high throughput mutational studies. Using the conjugative transposon Tn916, a pilot project was initiated to determine the efficiency of gene disruption in the first completely sequenced bacterium, Haemophilus influenzae Rd strain KW20. DNA was isolated from Tn916-mutagenized cells, and the point of transposon insertion was determined by inverse PCR, DNA sequencing, and mapping to the wild-type genome sequence. Analysis of the insertion sites at the nucleotide level demonstrated a biased pattern of insertion into regions rich in stretches of A's and T's. Although Tn916 integrated at multiple dispersed positions throughout the chromosome, 9 of 10 insertion events occurred in noncoding, intergenic DNA. It was determined that the intergenic DNA was over 5% more A + T-rich than that of protein coding sequences. This suggests that A + T-rich sequences similar to the Tn916 insertion site would be more likely to reside in the intergenic DNA. In an effort to identify other likely sites for transposon integration, a hidden Markov model of the consensus target insertion site was derived from the Tn916-H. influenzae junction fragments and searched against the entire genome. Eighty percent of the 30 highest-scoring predicted Tn916 target sites were from intergenic, nonprotein-coding regions of the genome. These data support the hypothesis that Tn916 has a marked preference for insertion into noncoding DNA for H. influenzae, suggesting that this mobile element has evolved to minimize disruption of host cell function on integration.

Base Sequence↗

PubMed1994

Efficient integration of artificial transposons into plasmid targets in vitro: a useful tool for DNA mapping, sequencing and genetic analysis.

We have developed efficient methods for creating artificial transposons and inserting these transposons into plasmid targets in vitro, primarily for the purpose of DNA mapping and sequencing. A novel plasmid has been engineered to convert virtually any DNA sequence, or combination of sequences, into an artificial transposon; hence, custom transposons containing any desired feature can be easily designed and constructed. Such transposons are then efficiently inserted into plasmid targets, in vitro, using the integrase activity present in yeast Ty1 virus-like particles. A single in vitro integration reaction, which resembles a simple restriction digestion in the complexity of the reaction, gives rise to thousands of recoverable insertion events within DNA target molecules; this frequency approaches one insertion per phosphodiester bond in typical plasmids. Importantly, transposon insertions are recovered from all regions of DNA inserts carried on plasmid targets, indicating that integration is a random or nearly-random process. Because of its versatility, this technology offers a generalized method of generating recombinant DNA molecules of a desired structure. We have adapted this system for DNA sequencing by developing a customized artificial transposon to insert new primer binding sites into internal regions of DNA inserts carried on cloning vectors. Transposon insertions have been generated throughout several different yeast and human DNA inserts carried on plasmids, allowing the efficient recovery of sequence information from these inserts. Our results demonstrate the overall utility of this method for both small and large-scale DNA sequencing, as well as general DNA restructuring, and indicate that it could be adapted for use with a number of additional applications including functional genetic analysis.

Base Sequence↗

PubMed2005

Identification of novel non-autonomous CemaT transposable elements and evidence of their mobility within the C. elegans genome.

We describe here two new transposable elements, CemaT4 and CemaT5, that were identified within the sequenced genome of Caenorhabditis elegans using homology based searches. Five variants of CemaT4 were found, all non-autonomous and sharing 26 bp inverted terminal repeats (ITRs) and segments (152-367 bp) of sequence with similarity to the CemaT1 transposon of C. elegans. Sixteen copies of a short, 30 bp repetitive sequence, comprised entirely of an inverted repeat of the first 15 bp of CemaT4's ITR, were also found, each flanked by TA dinucleotide duplications, which are hallmarks of target site duplications of mariner-Tc transposon transpositions. The CemaT5 transposable element had no similarity to maT elements, except for sharing identical ITR sequences with CemaT3. We provide evidence that CemaT5 and CemaT3 are capable of excising from the C. elegans genome, despite neither transposon being capable of encoding a functional transposase enzyme. Presumably, these two transposons are cross-mobilised by an autonomous transposon that recognises their shared ITRs. The excisions of these and other non-autonomous elements may provide opportunities for abortive gap repair to create internal deletions and/or insert novel sequence within these transposons. The influence of non-autonomous element mobility and structural diversity on genome variation is discussed.

Animals↗

PubMed1988

Activation of silent genes by transposons Tn5 and Tn10.

The presence of transposons Tn10 or Tn5 in the genome increases the frequency with which a silent (promoter-less) gene (hisD) is mutationally activated. The activation frequency is increased 5-25-fold by Tn10 and 30-90-fold by Tn5. Activation of the hisD gene is achieved by transposition of the entire transposon or one of its flanking insertion sequences to a region just upstream of the silent gene, between a Rho-dependent termination site in the adjacent hisG gene and the hisD gene. For both Tn5 and Tn10 the component insertion sequences were found to transpose much more frequently than the entire composite element. Transposons Tn5 and Tn10 have previously been shown to carry promoters which direct transcripts into sequences adjacent to their insertion sites.

Alcohol Oxidoreductases↗

PubMed1999

Genetic and functional characterization of the alpAB gene locus essential for the adhesion of Helicobacter pylori to human gastric tissue.

In this study, we isolated and characterized a chromosomal locus of Helicobacter pylori previously identified by transposon shuttle mutagenesis as being involved in the adhesion of the pathogen to gastric epithelial cells. Two closely homologous genes were identified, designated as alpA and alpB, encoding outer membrane (OM) proteins of 518 amino acids each. They are members of the outer membrane protein supergene family identified in the H. pylori 26695 complete genome sequence. AlpA carries a functional lipoprotein signal sequence. AlpB carries a putative standard N-terminal signal sequence and shows a strong amino-acid sequence identity to AlpA. Transposon insertion mutagenesis, immunoblotting and primer extension studies indicate that both genes are organized in an operon, but no obvious consensus promoter sequence was found upstream of the transcriptional start site. The C-terminal portion of both proteins is predicted to form a porin-like beta-barrel in the outer membrane, consisting of 14 transmembrane amphipathic beta-strands. Adhesion experiments with defined isogenic mutants indicate that both proteins are necessary for specific adherence of H. pylori to human gastric tissue. The pattern of AlpAB-dependent adherence of H. pylori to the gastric epithelial surface shows a clear difference to the BabA2-mediated adherence to Lewis, suggesting that a different receptor is involved.

Algorithms↗

PubMed2001

Identification of a new plasmid-encoded sec-dependent bacteriocin produced by Listeria innocua 743.

Listeria innocua 743 produces an inhibitory activity demonstrating broad-spectrum inhibition of Listeria monocytogenes isolates. Gel-electrophoretic analysis of culture supernatants indicated that two inhibitors with different molecular weights were produced by this strain. Insertion of Tn917 into a 2.9 Kb plasmid (pHC743) generated mutants with either an impaired ability or a loss in ability to produce one of the inhibitors. Sequence analysis of the transposon insertion regions revealed the presence of two continuous open reading frames, the first encoding a new pediocin-like bacteriocin (lisA) and the second encoding a protein homologous with genes involved in immunity toward other bacteriocins (lisB). Translation of the bacteriocin gene (lisA) initiates from a noncanonical start codon and encodes a 71-amino-acid prebacteriocin which lacked the double glycine leader peptidase processing site common in other type II bacteriocins. Alignment of the sequence with the processed N termini of related bacteriocins suggests that the mature bacteriocin consists of 43 amino acids, with a predicted molecular mass of 4,484 Da. Mutants containing insertions into lisA were sensitive to the inhibitor, indicating that lisAB forms a single operon and that lisB represents the immunity protein. Cloning of an amplicon containing the lisAB operon into Escherichia coli resulted in expression and export of the bacteriocin. This finding confirms that the phenotype is dependent on the structural and immunity gene only and that export of this bacteriocin is sec dependent. This is the first confirmation of bacteriocin production in a Listeria spp., and it is of interest that this bacteriocin is closely related to the pediocin family of bacteriocins produced by lactic acid bacteria.

Amino Acid Sequence↗

PubMed2026

Gene Contribution of Streptococcus dysgalactiae Subspecies equisimilis, an Emerging Pathogen, to Experimental Primate Necrotizing Myositis.

Streptococcus dysgalactiae subspecies equisimilis (SDSE) is an emerging human pathogen closely related to group A Streptococcus. However, its genetic requirements for survival and growth in different conditions and for causing invasive infections remain poorly understood. To address this gap, transposon-directed insertion-site sequencing was used to identify genes contributing to fitness in experimental necrotizing myositis in nonhuman primates. Using two SDSE stG62647 human clinical isolates, MGCS36044 and MGCS36089, highly saturated transposon mutant libraries were generated and analyzed following in vitro growth and in vivo infection in eight nonhuman primates. A total of 398 essential genes were identified to be shared by both strains during growth in vitro and in vivo, and 17 and 7 conditionally essential genes required only in vitro or only in vivo, respectively. Additionally, 117 and 110 genes in MGCS36044 and MGCS36089, respectively, were found to be associated with fitness during necrotizing myositis. Transposon insertions in 34 MGCS36044 genes conferred increased fitness, whereas mutation of 83 genes conferred decreased fitness. Similarly, in MGCS36089, mutations in 38 and 72 genes conferred increased or decreased fitness, respectively. Importantly, both strains shared 46 fitness-associated genes, including an enrichment of transporter genes, highlighting nutrient acquisition as a dominant requirement during infection. The results provide critical information for guiding future translational efforts to develop preventive and therapeutic strategies against human SDSE infections.

Animals↗

PubMed1981

Tn9 and IS1 inserts in a ribosomal ribonucleic acid operon of Escherichia coli are incompletely polar.

Transcription is known to be coupled to translation in many or all bacterial operons which code for proteins. In these operons, nonsense codons which prevent normal translation often result in premature termination of transcription (polarity). However, efficient transcription of ribosomal ribonucleic acid operons (rrn operons) occurs, although rrn transcripts are not translated. It therefore seemed possible that insertion sequences and transposable elements which are polar in protein-coding operons might not be polar in rrn operons. Previously, it has been shown (E. A. Morgan, Cell 21:257-265, 1980) that Tn10 is incompletely polar in the rrnX operon. Here we show that the transposon Tn9 and the insertion sequence IS1 also incompletely polar in rrnX. In normal cells expression of sequences distal to the insertions can be detected by genetic methods. In ultraviolet-irradiated cells expression of distal sequences is about 80% of that observed in uninterrupted rrnX operons. These observations provide evidence that ribonucleic acid polymerase molecules beginning at rrnX promoters can read through Tn9 and IS1 and that, at least in ultraviolet-irradiated cells, read-through is very efficient.

Bacteriophage lambda↗

PubMed2006

TraA is required for megaplasmid conjugation in Rhodococcus erythropolis AN12.

Pulsed-field gel electrophoresis (PFGE) revealed three previously uncharacterized megaplasmids in the genome of Rhodococcus erythropolis AN12. These megaplasmids, pREA400, pREA250, and pREA100, are approximately 400, 250, and 100kb, respectively, based on their migration in pulsed-field gels. Genetic screening of an AN12 transposon insertion library showed that two megaplasmids, pREA400, and pREA250, are conjugative. Mobilization frequencies of these AN12 megaplasmids to recipient R. erythropolis SQ1 were determined to be approximately 7x10(-4) and 5x10(-4) events per recipient cell, respectively. It is known for other bacterial systems that a relaxase encoded by the traA gene is required to initiate DNA transfer during plasmid conjugation. Sequences adjacent to the transposon insertion in megaplasmid pREA400 revealed a putative traA-like open reading frame. A targeted gene disruption method was developed to generate a traA mutation in AN12, which allowed us to address the role of the traA gene product for Rhodococcus megaplasmid conjugation. We found that the AN12 traA mutant is no longer capable of transferring the pREA400 megaplasmid to SQ1. Furthermore, we confirmed that the conjugation defect was specifically due to the disruption of the traA gene, as pREA400 megaplasmid conjugation defect is restored with a complementing copy of the traA gene.

Amino Acid Sequence↗

PubMed1995

The sequence of 24.3 kb from chromosome X reveals five complete open reading frames, all of which correspond to new genes, and a tandem insertion of a Ty1 transposon.

We have determined the complete nucleotide sequence of a 24.3 kb segment from chromosome X carried by the cosmid pEJ103. The sequence encodes five complete open reading frames (ORFs), none of which correspond to previously described genes; however, four of these ORFs display interesting similarities with sequences present in the databanks. The sequence also contains a tandem insertion of a Ty1 element. An investigation of the Ty1 polymorphism in other strains has revealed that the original insertion occurred within an ORF. Finally, the structure of the Ty1 repeat suggests a mechanism by which it may have been generated.

Amino Acid Sequence↗

PubMed1998

Identification of csrR/csrS, a genetic locus that regulates hyaluronic acid capsule synthesis in group A Streptococcus.

The hyaluronic acid capsule of group A Streptococcus (GAS) is an important virulence factor, but little is known about mechanisms that regulate capsule expression. Transposon Tn916 mutagenesis of the poorly encapsulated M-type 3 GAS strain DLS003 produced a transconjugant that exhibited a mucoid colony morphology, reflecting increased hyaluronic acid capsule production. Analysis of chromosomal DNA sequence immediately downstream of the transposon insertion identified two open reading frames, designated csrR and csrS, which exhibited sequence similarity to bacterial two-component regulatory systems. We constructed an in-frame deletion mutation within csrR, which encodes the putative response component. Replacement of the native csrR gene in the DLS003 chromosome with the mutant allele resulted in a sixfold increase in capsule production and a corresponding increase in transcription of the has operon, which contains the essential genes for hyaluronic acid synthesis. Increased capsule production by the csrR mutant strain was associated with enhanced resistance to complement-mediated opsonophagocytic killing in vitro and with a 500-fold increase in virulence in mice. These results establish CsrR as a negative regulator of hyaluronic acid capsule synthesis and suggest that it is part of a two-component regulatory system that influences capsule expression and virulence.

Amino Acid Sequence↗