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 289 records · Page 16Linked to original sources

PubMed1994

Localization and characterization of the gene encoding release factor RF3 in Escherichia coli.

Two protein release factors (RFs) showing codon specificity, RF1 and RF2, are known to be required for polypeptide chain termination in Escherichia coli. A third protein component has also been described that stimulates termination in vitro, but it has remained uncertain whether this protein, RF3, participates in termination in vivo or is essential to cell growth. We report (i) the purification and N-terminal sequencing of RF3; (ii) the isolation of transposon insertion mutants similar to miaD, a suppressor of a leaky UAA mutation affecting the gene miaA, leading to enhanced nonsense suppression; (iii) the localization of the affected gene on the physical map of the chromosome; and (iv) the cloning and sequencing of the wild-type gene, providing proof that it encodes the factor RF3. We designate the gene prfC. Two transposon insertions were shown to interrupt the coding sequence of prfC, at codons 287 and 426. The enhanced nonsense suppression in the insertion mutants shows that the product participates in termination in vivo. The isolation of such mutants strongly suggests that the gene product is not essential to cell viability, though cell growth is affected. RF3 is a protein with a molecular weight of 59,460 containing 528 amino acids and displays much similarity to elongation factor EF-G, a GTP binding protein necessary for ribosomal translocation, and other GTP binding proteins known or thought to interact with the ribosome.

Amino Acid Sequence↗

PubMed2005

Amplification of genomic sequences flanking T-DNA insertions by thermal asymmetric interlaced polymerase chain reaction.

Thermal asymmetric interlaced polymerase chain reaction (TAIL-PCR) is an efficient tool for the recovery of DNA fragments adjacent to known sequences. A protocol is presented for the amplification of genomic sequences flanking DNA (T-DNA or transposon) insertions using the TAIL-PCR method. The amplified products are suitable as templates for direct sequencing, or for cloning in vectors. Examples are given in use of the protocol for T-DNA tagging in rice using the pCAMBIA binary vectors.

Base Sequence↗

PubMed1998

A methylated Neurospora 5S rRNA pseudogene contains a transposable element inactivated by repeat-induced point mutation.

In an analysis of 22 of the roughly 100 dispersed 5S rRNA genes in Neurospora crassa, a methylated 5S rRNA pseudogene, Psi63, was identified. We characterized the Psi63 region to better understand the control and function of DNA methylation. The 120-bp 5S rRNA-like region of Psi63 is interrupted by a 1.9-kb insertion that has characteristics of sequences that have been modified by repeat-induced point mutation (RIP). We found sequences related to this insertion in wild-type strains of N. crassa and other Neurospora species. Most showed evidence of RIP; but one, isolated from the N. crassa host of Psi63, showed no evidence of RIP. A deletion from near the center of this sequence apparently rendered it incapable of participating in RIP with the related full-length copies. The Psi63 insertion and the related sequences have features of transposons and are related to the Fot1 class of fungal transposable elements. Apparently Psi63 was generated by insertion of a previously unrecognized Neurospora transposable element into a 5S rRNA gene, followed by RIP. We name the resulting inactivated Neurospora transposon PuntRIP1 and the related sequence showing no evidence of RIP, but harboring a deletion that presumably rendered it defective for transposition, dPunt.

Amino Acid Sequence↗

PubMed1999

Genetic organization of the O7-specific lipopolysaccharide biosynthesis cluster of Escherichia coli VW187 (O7:K1).

In previous studies the authors cloned and characterized the DNA sequence of the regions at both ends of the O7-specific lipopolysaccharide (LPS) biosynthesis cluster of Escherichia coli VW187 (O7:K1), and identified the biosynthetic genes for dTDP-rhamnose and GDP-mannose, as well as one of the candidate glycosyltransferases. In this work the complete DNA sequence of a 6.9 kb intervening region is presented. Seven new ORFs were identified. All the functions required for the synthesis and transfer of the O7 LPS were assigned on the basis of complementation experiments of transposon insertion mutants, and amino acid sequence homology to proteins involved in LPS synthesis of other bacteria. Of the seven ORFs, two encoded membrane proteins that were homologous to the O-antigen translocase (Wzx) and polymerase (Wxy), two were involved in the biosynthesis of dTDP-N-acetylviosamine, and the remaining three showed homologies to sugar transferases. The O antigen chain length regulator gene wzz was also identified in the vicinity of the O7 polysaccharide cluster. O7-specific DNA primers were designed and tested for serotyping of O7 E. coli strains.

Amino Acid Sequence↗

PubMed1982

Regulation of white locus expression: the structure of mutant alleles at the white locus of Drosophila melanogaster.

We have analyzed the structures of 19 mutant alleles at the white locus of Drosophila melanogaster. Thirteen of the mutant alleles in our selected sample arose spontaneously, and of these, seven are associated with insertions of non-white-region DNA sequence elements. Several lines of evidence strongly suggest that these insertions are responsible for their associated mutant alleles, and further suggest that most or all of these insertions are transposons. Moreover, the white locus DNA sequences can be divided into two nonoverlapping domains on the basis of the properties of the two domains as mutational targets. One of these domains behaves, in this regard, in the manner expected of functional coding sequences, whereas the other does not. We propose a model for the nature and function of the presumptive noncoding white locus genetic elements. The two domains of the white locus defined by our studies are approximately coextensive with the functionally distinct subintervals of the locus defined by previous genetic analysis. Lastly, our results strongly suggest that the dominant, mutable wDZL allele results from the insertion of a transposon outside of, but near, the white locus. This putative transposon apparently carries genetic elements that act at a distance to repress expression of the white locus.

Alleles↗

PubMed1994

par-2, a gene required for blastomere asymmetry in Caenorhabditis elegans, encodes zinc-finger and ATP-binding motifs.

The par-2 gene of Caenorhabditis elegans functions in early embryogenesis to ensure an asymmetric first cleavage and the segregation of cytoplasmic factors. Both processes appear to be required to generate daughter blastomeres with distinct developmental potential. We isolated an allele of par-2 by using a screen for maternal-effect lethal mutations in a strain known for its high frequency of transposition events. A transposable element was found to be linked to this allele. Sequences flanking the site of transposon insertion were cloned and found to rescue the par-2 mutant phenotype. DNA in the par-2 region hybridized to a 2.3-kb germ-line-enriched mRNA. The cDNA corresponding to this germ-line-enriched message was cloned, sequenced, and used to identify the molecular lesions associated with three par-2 alleles. Sequence analysis of the par-2 cDNA revealed that the predicted protein contained two distinct motifs found in other known proteins: an ATP-binding site and a cysteine-rich motif which identifies the par-2 gene product as a member of a growing class of putative zinc-binding proteins.

Adenosine Triphosphate↗

PubMed1999

Development of a transposon mutagenesis system for Mycobacterium avium subsp. paratuberculosis.

Mycobacterium avium subspecies paratuberculosis, a slow-growing Mycobacterium, is the causative agent of Johne's disease. Although M. paratuberculosis is difficult to manipulate genetically, our laboratory has recently demonstrated the ability to introduce DNA into these bacteria by transformation and phage infection. In the current study we develop the first transposon mutagenesis system for M. paratuberculosis using the conditionally replicating mycobacteriophage phAE94 to introduce the mycobacterial transposon Tn5367. Southern blotting and sequence analysis demonstrated that the transposon insertion sites are distributed relatively randomly throughout the M. paratuberculosis genome. We constructed a comprehensive bank of 5620 insertion mutants using this transposon. The transposition frequency obtained using this delivery system was 1.0 x 10(-6) transposition events per recipient cell. Auxotrophic mutants were observed in this library at a frequency of 0.3%.

Base Sequence↗

PubMed2002

Influence of Tn917 insertion on transcription of the icaADBC operon in six biofilm-negative transposon mutants of Staphylococcus epidermidis.

Insertion of Tn917 into the icaADBC operon determines a biofilm-negative phenotype in biofilm-producing Staphylococcus epidermidis due to the inactivation of the genes responsible for the synthesis of the polysaccharide intercellular adhesin. We previously characterized six isogenic biofilm-negative transposon mutants of S. epidermidis 1457 with Tn917 insertions in either icaA or icaC. Northern blot analysis using ica- and Tn917-specific probes revealed that ica sequences located upstream and downstream of the transposon insertion site were still transcribed in five mutants in which Tn917 was inserted in the same transcriptional orientation. Outward-directed transcription initiating from within the transposon resulted in the complete expression of individual ica genes. Our results indicate that not only the inactivation of the entire operon but the isolated interruption of individual icaA and icaC genes led to a biofilm-negative phenotype in S. epidermidis. Tn917 mutagenesis may also result in the activation of genes located downstream of the insertion site.

Amino Acid Sequence↗

PubMed1999

An efficient DNA sequencing strategy based on the bacteriophage mu in vitro DNA transposition reaction.

A highly efficient DNA sequencing strategy was developed on the basis of the bacteriophage Mu in vitro DNA transposition reaction. In the reaction, an artificial transposon with a chloramphenicol acetyltransferase (cat) gene as a selectable marker integrated into the target plasmid DNA containing a 10.3-kb mouse genomic insert to be sequenced. Bacterial clones carrying plasmids with the transposon insertions in different positions were produced by transforming transposition reaction products into Escherichia coli cells that were then selected on appropriate selection plates. Plasmids from individual clones were isolated and used as templates for DNA sequencing, each with two primers specific for the transposon sequence but reading the sequence into opposite directions, thus creating a minicontig. By combining the information from overlapping minicontigs, the sequence of the entire 10,288-bp region of mouse genome including six exons of mouse Kcc2 gene was obtained. The results indicated that the described methodology is extremely well suited for DNA sequencing projects in which considerable sequence information is on demand. In addition, massive DNA sequencing projects, including those of full genomes, are expected to benefit substantially from the Mu strategy.

Amino Acid Sequence↗

PubMed1994

Lipooligosaccharide biosynthesis in pathogenic Neisseria. Cloning, identification, and characterization of the phosphoglucomutase gene.

The lipooligosaccharide (LOS) of pathogenic Neisseria is an important factor in disease pathogenesis. Little is known about the genes involved in neisserial LOS biosynthesis. To elucidate specific LOS biosynthetic genes, we screened a Tn916 library that was constructed in Neisseria meningitidis strain NMB. This strain expresses a single LOS that has an molecular mass of 4.5 kDa and binds monoclonal antibody (mAb) 3F11. This library was screened using a mAb panel that recognizes structural differences in neisserial LOS oligosaccharides. A stable LOS mutant of strain NMB was identified which we designated NMB-R6. This mutant expressed an LOS with an molecular mass of approximately 3.1-3.2 kDa and did not bind mAb 3F11. Genomic DNA from this mutant transformed N. meningitidis strain NMB to the tetracycline resistant NMB-R6 phenotype greater than 10(-4)/recipient/micrograms of DNA. In addition, we transformed Neisseria gonorrhoeae strain 1291 (LOS phenotype molecular mass 4.5 kDa, mAb 3F11+) to the NMB-R6 LOS phenotype with N. meningitidis NMB-R6 genomic DNA. Analysis of N. gonorrhoeae strain 1291-R6 LOS by mass spectroscopy showed that the LOS oligosaccharide structure is GlcNAc-->Hep2phosphoethanolamine-->2-keto-3-deoxymannooctuloson ic acid (where Hep is heptose). Sequence analysis showed that the transposon is inserted into the 3' end of a gene that has homology to the human phosphoglucomutase (PGM) gene. Sequence comparison indicated that the putative PGM gene in N. gonorrhoeae 1291 and N. meningitidis NMB had 92% identity at the DNA level. PGM and glucokinase activity was present in cell free extracts of N. meningitidis NMB and N. gonorrhoeae strain 1291. N. meningitidis NMB-R6 and N. gonorrhoeae strain 1291-R6 had no detectable PGM activity, whereas glucokinase activity was similar to the wild type strains. PGM activity can be reconstituted in N. meningitidis strain NMB-R6 by transformation with the cloned PGM gene. SDS-polyacrylamide gel electrophoresis demonstrated that NMB-R6 transformed with the PGM gene expressed the 3F11+, 4.5-kDa LOS of the parent NMB strain. The inability of N. meningitidis NMB-R6 and N. gonorrhoeae strain 1291-R6 to convert glucose 6-phosphate to glucose 1-phosphate results in the truncated LOS phenotype expressed by these mutants.

Amino Acid Sequence↗

PubMed1987

Tn2501, a component of the lactose transposon Tn951, is an example of a new category of class II transposable elements.

Tn2501 is a cryptic class II transposon found as part of the lactose transposon Tn951. Insertional inactivation and nucleotide sequence analysis of Tn2501 allowed us (i) to localize the transposase (tnpA) and the resolvase (tnpR) genes as well as the resolution site (res) of Tn2501 and (ii) to compare Tn2501 with other well-known elements of the two subgroups of class II transposons (Tn3, gamma delta, Tn951, IS101; and Tn21, Tn501, Tn1721). The genetic organization of Tn2501 is similar to that of Tn3 with divergent transcription of the tnpA and tnpR genes away from the intervening res site. The tnpR gene of Tn2501 shows weak homology with that of Tn3 and even less with those of Tn21 and Tn501. However, the tnpA gene and the inverted repeat sequences of Tn2501 present more homology with those of Tn21 and Tn501 than with those of Tn3. Complementation studies showed that TnpA- mutants of Tn2501 can be complemented, at a low frequency, by the Tn21 transposase. None of the tested transposons complemented TnpR- mutants of Tn2501.

Amino Acid Sequence↗

PubMed2005

EU-OSTID: a collection of transposon insertional mutants for functional genomics in rice.

A collection of 1373 unique flanking sequence tags (FSTs), generated from Ac/Ds and Ac transposon lines for reverse genetics studies, were produced in japonica and indica rice, respectively. The Ds and Ac FSTs together with the original T-DNAs were assigned a position in the rice genome sequence represented as assembled pseudomolecules, and found to be distributed evenly over the entire rice genome with a distinct bias for predicted gene-rich regions. The bias of the Ds and Ac transposon inserts for genes was exemplified by the presence of 59% of the inserts in genes annotated on the rice chromosomes and 41% present in genes transcribed as disclosed by their homology to cDNA clones. In a screen for inserts in a set of 75 well annotated transcription factors, including homeobox-containing genes, we found six Ac/Ds inserts. This high frequency of Ds and Ac inserts in genes suggests that saturated knockout mutagenesis in rice using this strategy will be efficient and possible with a lower number of inserts than expected. These FSTs and the corresponding plant lines are publicly available through OrygenesDB database and from the EU consortium members.

Binding Sites↗

PubMed1995

An rpoE-like locus controls outer membrane protein synthesis and growth at cold temperatures and high pressures in the deep-sea bacterium Photobacterium sp. strain SS9.

Many deep-sea bacteria have evolved specialized adaptations for life at cold temperatures and high pressures. A locus required for both psychro- and baro-adaptation in the psychrophilic, moderate barophile, Photobacterium species strain SS9 was identified among SS9 transposon mutants. DNA sequence analysis of this locus identified four complete open reading frames (ORFs), which appear to comprise an operon, and a fifth incomplete ORF. All transposon insertions isolated are in ORF3. Extensive sequence similarity exists between the translation products of ORFs 1-3 and a collection of gene products proposed to include alternative RNA polymerase sigma factors and modifiers of sigma-factor activity involved in extracytoplasmic sensing and regulation. Based on the similarity between ORF1 and Escherichia coli rpoE, we have tentatively designated this locus the rpoE locus. SS9 rpoE locus ORF3 insertion mutants showed altered abundances of numerous outer membrane proteins and were both baro- and psychro-sensitive. ORF3 mutant revertants that displayed enhanced high-pressure growth also displayed concomitant enhanced low-temperature growth. Most of these revertants possessed DNA rearrangements at the site of the transposon insertion, further demonstrating the importance of the rpoE locus to high-pressure and cold-temperature growth. Complementation analyses indicated that ORF3 functions in OMP synthesis regulation while ORF4 is required for baro- and psychro-adaptation.

Bacterial Outer Membrane Proteins↗

PubMed1985

Transposon-mediated mutagenesis of a baculovirus.

Spontaneous mutants of insect nuclear polyhedrosis viruses of Autographa californica (AcMNPV) and Galleria mellonella (GmMNPV) were analyzed. These mutants produce few polyhedra in infected cells and have insertions of host cell DNA. All the insertions mapped to two adjacent sites in the genome. The junctions between two host insertions and the viral DNA were sequenced. One of the insertions contained a perfect 7-bp inverted terminal repeat, and had caused a direct duplication of 4 bp of viral DNA at the point of insertion. Therefore, this insertion sequence has properties of a transposon of the host cell Trichoplusia ni.

Animals↗

PubMed1996

A transposon insertion in the Arabidopsis SSR16 gene causes an embryo-defective lethal mutation.

The SSR16 gene of Arabidopsis has been identified as a gene encoding a ribosomal protein S16 homolog through analysis of a transposon insertion mutation. The insertion mutation is lethal, arresting embryonic development at approximately the transition from the globular to the heart stage of embryonic development. Co-segregation of the mutant phenotype with the transposon-borne drug-resistance marker and loss of the inserted transposon concomitant with phenotypic reversion provided evidence that the transposon had caused the mutation. Sequences flanking the insertion site were amplified from DNA of viable heterozygotes by thermal asymmetric interlaced (TAIL) PCR. The amplified fragment flanking the 3' end of the inserted element was sequenced and found to be identical to an Arabidopsis expressed sequence tag (EST). The EST, in turn, contained a coding sequence homologous to the ribosomal protein S16 (RPS16) of bacteria such as Escherichia coli, Bacillus subtilis and Salmonella typhimurium, as well as Neurospora crassa mitochondria and higher plant plastids. Thus the gene identified by the embryo-defective lethal insertion mutation encodes an RPS16 homolog and has been designated the SSR16 gene.

Amino Acid Sequence↗

PubMed1994

The nitrogen-regulated Bacillus subtilis nrgAB operon encodes a membrane protein and a protein highly similar to the Escherichia coli glnB-encoded PII protein.

Expression of beta-galactosidase encoded by the nrg-29::Tn917-lacZ insertion increases 4,000-fold during nitrogen-limited growth (M.R. Atkinson and S. H. Fisher, J. Bacteriol. 173:23-27, 1991). The chromosomal DNA adjacent to the nrg-29::Tn917-lacZ insertion was cloned and sequenced. Analysis of the resulting nucleotide sequence revealed that the Tn917-lacZ transposon was inserted into the first gene of a dicistronic operon, nrgAB. The nrgA gene encodes a 43-kDa hydrophobic protein that is likely to be an integral membrane protein. The nrgB gene encodes a 13-kDa protein that has significant sequence similarity with the Escherichia coli glnB-encoded PII protein. Primer extension analysis revealed that the nrgAB operon is transcribed from a single promoter. The nucleotide sequence of this promoter has significant similarity with the -10 region, but not the -35 region, of the consensus sequence for Bacillus subtilis sigma A-dependent promoters.

Amino Acid Sequence↗

PubMed1999

Streptococcus mutans ffh, a gene encoding a homologue of the 54 kDa subunit of the signal recognition particle, is involved in resistance to acid stress.

The ability of Streptococcus mutans, a bacterial pathogen associated with dental caries, to tolerate rapid drops in plaque pH (acidurance), is considered an important virulence factor. To study this trait, Tn917 mutants of S. mutans strain JH1005 which display acid sensitivity have been isolated and partially characterized. In this paper, the characterization of one of these mutants, AS17, is reported. Preliminary sequence analysis revealed that the transposon insertion in AS17 occurred in the intergenic region of a two-gene locus which has been named sat for secretion and acid tolerance. This locus displays a high degree of homology to the ylxM-ffh operon of Bacillus subtilis. The sat+ locus was cloned by complementation of a conditional Escherichia coli ffh mutant with an S. mutans genomic library. Sequencing of the complementing clone identified the intact ylxM and ffh genes as well as a partial ORF with homology to the proUlopuAC gene of B. subtilis which encodes the binding protein of the ProU/OpuA osmoregulated glycine betaine transport system. RNA dot blot experiments indicated steady-state levels of ffh mRNA in the mutant that were approximately eightfold lower compared to parental levels. This suggests a partial polar effect of the sat-1::Tn917 mutation on ffh expression. Upon acid shock (pH 5), wild-type ffh mRNA levels were found to increase approximately four- to eightfold compared to unstressed (pH 7.5) levels. Mutant levels remained unaltered under the same conditions. Experiments designed to investigate the origins of the acid-sensitivity of the mutant revealed a lack of an acid-adaptive/tolerance response. Assays of proton-extruding ATPase (H+/ATPase) specific activity measured with purified membranes derived from acid-shocked AS17 showed twofold lower levels compared to the parent strain. Also, AS17 was found to be unable to ferment sorbitol although it was able to grow in glucose and a variety of other sugar substrates. These findings suggest that Ffh may be involved in the maintenance of a functional membrane protein composition during adaptation of S. mutans to changing environmental conditions.

Bacterial Proteins↗

PubMed2006

Genetic analysis of a multiresistant strain of Pseudomonas aeruginosa producing PER-1 beta-lactamase.

A multiresistant strain of Pseudomonas aeruginosa, PA2345, belonging to serotype O:1, was isolated at the Teaching Hospital of Besançon, France. Resistance to beta-lactams, including third-generation cephalosporins, depended upon a chromosomally-located composite transposon carrying the bla(PER-1) gene encoding extended-spectrum beta-lactamase PER-1. PA2345 was unrelated genotypically to two previous PER-1-producing isolates of P. aeruginosa. Sequence analysis of the transposon in PA2345 revealed the presence of two insertion sequences (ISPa23 and ISPa24) with very different predicted transposases (TnpA1, TnpA2), which were both bordered by closely related 16-bp inverted repeats. High resistance of PA2345 to aminoglycosides was caused, in part, by a chromosomal class-I integron containing gene cassettes aadB, encoding an ANT(2'') enzyme, and aadA11, encoding a new ANT(3'') enzyme with 281 amino-acids that conferred elevated resistance to streptomycin and spectinomycin. Stable overproduction of efflux system MexXY contributed to resistance to amikacin, while mutations in the quinolone resistance-determining regions of gyrA and parC accounted for the high resistance of PA2345 to fluoroquinolones. The study indicates that multidrug resistance in P. aeruginosa might arise from sequential acquisition of a variety of mechanisms provided by both horizontal gene transfers and mutations in chromosomal genes.

Drug Resistance, Multiple, Bacterial↗