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Results for “Transposon insertion library”

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At least 199 records · Page 11Linked to original sources

PubMed1995

Rhizobium leguminosarum nodulation gene (nod) expression is lowered by an allele-specific mutation in the dicarboxylate transport gene dctB.

To identify host genes that might influence nod (nodulation) gene expression in Rhizobium leguminosarum, a nodC-phoA reporter plasmid (carrying nodD) was introduced into a chemically mutagenized population of a R. leguminosarum strain lacking a symbiotic plasmid. The transconjugants were screened for expression of alkaline phosphatase (PhoA) on plates containing hesperetin, an inducer of nod genes, and a mutant with reduced expression was identified. When the nodC-phoA plasmid was cured from the mutant and the symbiotic plasmid pRL1Jl introduced, the mutant formed nodules, but symbiotic nitrogen fixation was less than 20% of normal. When the nodC-phoA allele was introduced on pRL1Jl a low level of nod gene induction was found. The reduced nodC expression appeared to be caused by a decrease in expression of the regulatory gene nodD, since expression of a nodD-lacZ fusion was also lower in the mutant than in the control. These mutant phenotypes and the low nitrogen fixation were complemented with a plasmid (plJ1848) from a R. leguminosarum cosmid library. DNA hybridization confirmed that plJ1848 was not from the symbiotic plasmid and showed that a DNA insertion was present in the mutant. The complementing region of plJ1848 was defined by transposon mutagenesis; DNA sequencing revealed that it carried the dicarboxylic acid transport (dct) genes. However, the mutant grew well with succinate as sole C-source. Genetic analysis revealed that the mutant appeared to contain IS50 in the regulatory gene dctB and that this mutation caused the reduction in nod gene expression. The effect was allele-specific since other mutations in dctB did not influence nod gene expression. Surprisingly, the mutant had a constitutive high level of succinate transport, indicating that the mutation caused unregulated expression of dctA the structural gene for dicarboxylic acid transport. This in some way appears to have lowered the expression of nodD, indicating that the nodD promoter may be influenced by the metabolic status of the cells or by expression of dctD in the absence of dctB.

Alkaline Phosphatase↗

PubMed1997

Role of the citrate pathway in glutamate biosynthesis by Streptococcus mutans.

In work previously reported (J. A. Gutierrez, P. J. Crowley, D. P. Brown, J. D. Hillman, P. Youngman, and A. S. Bleiweis, J. Bacteriol. 178:4166-4175, 1996), a Tn917 transposon-generated mutant of Streptococcus mutans JH1005 unable to synthesize glutamate anaerobically was isolated and the insertion point of the transposon was determined to be in the icd gene encoding isocitrate dehydrogenase (ICDH). The intact icd gene of S. mutans has now been isolated from an S. mutans genomic plasmid library by complementation of an icd mutation in Escherichia coli host strain EB106. Genetic analysis of the complementing plasmid pJG400 revealed an open reading frame (ORF) of 1,182 nucleotides which encoded an enzyme of 393 amino acids with a predicted molecular mass of 43 kDa. The nucleotide sequence contained regions of high (60 to 72%) homology with icd genes from three other bacterial species. Immediately 5' of the icd gene, we discovered an ORF of 1,119 nucleotides in length, designated citZ, encoding a homolog of known citrate synthase genes from other bacteria. This ORF encoded a predicted protein of 372 amino acids with a molecular mass of 43 kDa. Furthermore, plasmid pJG400 was also able to complement a citrate synthase (gltA) mutation of E. coli W620. The enzyme activities of both ICDH, found to be NAD+ dependent, and citrate synthase were measured in cell extracts of wild-type S. mutans and E. coli mutants harboring plasmid pJG400. The region 5' from the citZ gene also revealed a partial ORF encoding 264 carboxy-terminal amino acids of a putative aconitase gene. The genetic and biochemical evidence indicates that S. mutans possesses the enzymes required to convert acetyl coenzyme A and oxalacetate to alpha-ketoglutarate, which is necessary for the synthesis of glutamic acid. Indeed, S. mutans JH1005 was shown to assimilate ammonia as a sole source of nitrogen in minimal medium devoid of organic nitrogen sources.

Aconitate Hydratase↗

PubMed2001

Identification of genes required for growth under ethanol stress using transposon mutagenesis in Saccharomyces cerevisiae.

The yeast Saccharomyces cerevisiae exhibits high ethanol tolerance compared with other microorganisms. The mechanism of ethanol tolerance in yeast is thought to be regulated by many genes. To identify some of these genes, we screened for ethanol-sensitive S. cerevisiae strains among a collection of mutants obtained using transposon mutagenesis. Five ethanol-sensitive (ets) mutants were isolated from approximately 7,000 mutants created by transforming yeast cells with a transposon (mTn-lacZ/LEU2)-mutagenized genomic library. Although these mutants grew normally in a rich medium, they could not grow in the same medium containing 6% ethanol. Sequence analysis of the ets mutants revealed that the transposon was inserted in the coding regions of BEM2, PAT1, ROM2, VPS34 and ADA2. We constructed deletion mutants for these genes by a PCR-directed disruption method and confirmed that the disruptants, like the ets mutants, were ethanol sensitive. Thus, these five genes are indeed required for growth under ethanol stress. These mutants were also more sensitive than normal cells to Calcofluor white, a drug that affects cell wall architecture, and Zymolyase, a yeast lytic enzyme containing mainly beta-1,3- glucanase, indicating that the integrity of the cell wall plays an important role in ethanol tolerance in S. cerevisiae.

Base Sequence↗

PubMed1999

Identification of a virulence gene cluster of Mycobacterium tuberculosis by signature-tagged transposon mutagenesis.

Tuberculosis remains the greatest cause of death worldwide due to a single pathogen. In order to identify the genes required for the pathogenicity of Mycobacterium tuberculosis, a functional genomic approach was developed. A library of signature-tagged transposon mutants of this bacterium was constructed and screened for those affected in their multiplication within the lungs of mice. From 1927 mutants tested, 16 were attenuated for their virulence. The insertions harboured by the selected mutants were mapped on the M. tuberculosis genome and most of the mutated loci appeared to be involved in lipid metabolism or transport across the membrane. Four independent mutations identified a cluster of virulence genes located on a 50 kb chromosomal region. These genes might be involved in the production of phthiocerol and phenolphthiocerol derivatives, a group of molecules restricted to eight mycobacterial species, seven of them being either strict or opportunistic pathogens. The interaction of five mutant strains with mouse bone marrow macrophages was investigated. These five mutants were still able to multiply in this cell type. However, in three cases, there was a growth defect in comparison with the wild-type strain. The other two strains exhibited no clear difference from the virulent strain, MT103, in this model. This study, which is the first global research of virulence factors of M. tuberculosis, opens the way to a better understanding of the molecules that are key players in the interaction of this pathogen with its host.

Animals↗

PubMed2006

Genomic analysis of insertion behavior and target specificity of mini-Tn7 and Tn3 transposons in Saccharomyces cerevisiae.

Transposons are widely employed as tools for gene disruption. Ideally, they should display unbiased insertion behavior, and incorporate readily into any genomic DNA to which they are exposed. However, many transposons preferentially insert at specific nucleotide sequences. It is unclear to what extent such bias affects their usefulness as mutagenesis tools. Here, we examine insertion site specificity and global insertion behavior of two mini-transposons previously used for large-scale gene disruption in Saccharomyces cerevisiae: Tn3 and Tn7. Using an expanded set of insertion data, we confirm that Tn3 displays marked preference for the AT-rich 5 bp consensus site TA[A/T]TA, whereas Tn7 displays negligible target site preference. On a genome level, both transposons display marked non-uniform insertion behavior: certain sites are targeted far more often than expected, and both distributions depart drastically from Poisson. Thus, to compare their insertion behavior on a genome level, we developed a windowed Kolmogorov-Smirnov (K-S) test to analyze transposon insertion distributions in sequence windows of various sizes. We find that when scored in large windows (>300 bp), both Tn3 and Tn7 distributions appear uniform, whereas in smaller windows, Tn7 appears uniform while Tn3 does not. Thus, both transposons are effective tools for gene disruption, but Tn7 does so with less duplication and a more uniform distribution, better approximating the behavior of the ideal transposon.

Base Sequence↗

PubMed1990

Location and organization of the dimethylphenol catabolic genes of Pseudomonas CF600.

The gene organization of the phenol catabolic pathway of Pseudomonas CF600 has been investigated. This strain can grow on phenol and some methylated phenols by virtue of an inducible phenol hydroxylase and metacleavage pathway enzymes. The genes coding for these enzymes are located on pVI150, an IncP-2 degradative mega plasmid of this strain. Twenty-three kilobases of contiguous DNA were isolated from lambda libraries constructed from strains harbouring wild type and Tn5 insertion mutants of pVI150. A 19.9 kb region of this DNA has been identified which encodes all the catabolic genes of the pathway. Using transposon mutagenesis, polypeptide analysis and expression of subfragments of DNA, the genes encoding the first four enzymatic steps of the pathway have been individually mapped and found to lie adjacent to each other. The order of these genes is the same as that for isofunctional genes of TOL plasmid pWWO and plasmid NAH7.

Bacterial Outer Membrane Proteins↗

PubMed1990

Spore colour in Streptomyces coelicolor A3(2) involves the developmentally regulated synthesis of a compound biosynthetically related to polyketide antibiotics.

Streptomyces coelicolor produces spores whose development of a grey colour requires the activity of the whiE locus. The cloned whiE locus was identified after mobilization into a whiE mutant of a library of S. coelicolor DNA inserted into a transmissible plasmid vector. The whiE region of the cloned DNA was localized both by subcloning and by mutagenesis of the cloned DNA with the Streptomyces transposon Tn4560. Nucleotide sequencing of this region revealed seven open reading frames, of which six show homology at the level of deduced gene products with genes involved in the synthesis of polyketide antibiotics. A previously described S. coelicolor DNA segment encoding biosynthesis of a brown pigment (Horinouchi and Beppu, 1985) corresponds to the cloned whiE DNA. It is proposed that whiE is normally expressed only in the aerial hyphae, where the biosynthetic product is responsible for spore colour.

Amino Acid Sequence↗

PubMed2006

The carnegie protein trap library: a versatile tool for Drosophila developmental studies.

Metazoan physiology depends on intricate patterns of gene expression that remain poorly known. Using transposon mutagenesis in Drosophila, we constructed a library of 7404 protein trap and enhancer trap lines, the Carnegie collection, to facilitate gene expression mapping at single-cell resolution. By sequencing the genomic insertion sites, determining splicing patterns downstream of the enhanced green fluorescent protein (EGFP) exon, and analyzing expression patterns in the ovary and salivary gland, we found that 600-900 different genes are trapped in our collection. A core set of 244 lines trapped different identifiable protein isoforms, while insertions likely to act as GFP-enhancer traps were found in 256 additional genes. At least 8 novel genes were also identified. Our results demonstrate that the Carnegie collection will be useful as a discovery tool in diverse areas of cell and developmental biology and suggest new strategies for greatly increasing the coverage of the Drosophila proteome with protein trap insertions.

Animals↗

PubMed2003

Functional genetic identification of PRP1, an ABC transporter superfamily member conferring pentamidine resistance in Leishmania major.

Pentamidine (PEN) is a second-line agent in the treatment of leishmaniasis whose mode of action and resistance is not well understood. Here, we used a genetic strategy to search for loci able to mediate PEN resistance (PENr) when overexpressed in Leishmania major. A shuttle cosmid library containing genomic DNA inserts was transfected into wild-type promastigotes and screened for PEN-resistant transfectants. Two different cosmids identifying the same locus were found, which differed from other known Leishmania drug resistance genes. The PENr gene was mapped by deletion and transposon mutagenesis to an open reading frame (ORF) belonging to the P-glycoprotein (PGP)/MRP ATP-binding cassette (ABC) transporter superfamily that we named pentamidine resistance protein 1 (PRP1). The predicted PRP1 protein encodes 1,807 amino acids with the typical dimeric structure involving 10 transmembrane domains and two nucleotide-binding domains (NBDs). PRP1-mediated PENr could be reversed by verapamil and PRP1 overexpressors showed cross-resistance to trivalent antimony but not to pentavalent antimony (glucantime). Although the degree of PENr was modest (1.7- to 3.7-fold), this may be significant in clinical drug resistance given the marginal efficacy of PEN against Leishmania.

ATP-Binding Cassette Transporters↗

PubMed2003

Construction and utility of 10-kb libraries for efficient clone-gap closure for rice genome sequencing.

Rice is an important crop and a model system for monocot genomics, and is a target for whole genome sequencing by the International Rice Genome Sequencing Project (IRGSP). The IRGSP is using a clone by clone approach to sequence rice based on minimum tiles of BAC or PAC clones. For chromosomes 10 and 3 we are using an integrated physical map based on two fingerprinted and end-sequenced BAC libraries to identifying a minimum tiling path of clones. In this study we constructed and tested two rice genomic libraries with an average insert size of 10 kb (10-kb library) to support the gap closure and finishing phases of the rice genome sequencing project. The HaeIII library contains 166,752 clones covering approximately 4.6x rice genome equivalents with an average insert size of 10.5 kb. The Sau3AI library contains 138,960 clones covering 4.2x genome equivalents with an average insert size of 11.6 kb. Both libraries were gridded in duplicate onto 11 high-density filters in a 5 x 5 pattern to facilitate screening by hybridization. The libraries contain an unbiased coverage of the rice genome with less than 5% contamination by clones containing organelle DNA or no insert. An efficient method was developed, consisting of pooled overgo hybridization, the selection of 10-kb gap spanning clones using end sequences, transposon sequencing and utilization of in silico draft sequence, to close relatively small gaps between sequenced BAC clones. Using this method we were able to close a majority of the gaps (up to approximately 50 kb) identified during the finishing phase of chromosome-10 sequencing. This method represents a useful way to close clone gaps and thus to complete the entire rice genome.

Chromosomes, Artificial, Bacterial↗

PubMed1999

Molecular characterization of the maize Rp1-D rust resistance haplotype and its mutants.

The Rp1-D gene for resistance to maize common rust (Puccinia sorghi) is a member of a complex locus (haplotype) composed of Rp1-D and approximately eight other gene homologs. The identity of Rp1-D was demonstrated by using two independent gene-tagging approaches with the transposons Mutator and Dissociation. PIC20, a disease resistance (R) gene analog probe previously mapped to the rp1 locus, detected insertion of Dissociation in an Rp1-D mutation and excision in three revertants. Independent libraries probed with the PIC20 or Mutator probes resulted in isolation of the same gene sequence. Rp1-D belongs to the nucleotide binding site, leucine-rich repeat class of R genes. However, unlike the rust resistance genes M and L6 from flax, the maize Rp1-D gene does not encode an N-terminal domain with similarity to the signal transduction domains of the Drosophila Toll protein and mammalian interleukin-1 receptor. Although the abundance of transcripts of genes from the rp1 complex changed with leaf age, there was no evidence of any change due to inoculation with avirulent or virulent rust biotypes. A set of 27 Rp1-D mutants displayed at least nine different deletions of Rp1-D gene family members that were consistent with unequal crossing-over events. One mutation (Rp1-D*-24) resulted in deletion of all but one gene family member. Other unique deletions were observed in the disease lesion mimic Rp1-D*-21 and the partially susceptible mutant Rp1-D*-5. Different rp1 specificities have distinct DNA fingerprints (haplotypes). Analysis of recombinants between rp1 specificities indicated that recombination had occurred within the rp1 gene complex. Similar analyses indicated that the rust R genes at the rp5 locus, 2 centimorgans distal to rp1, are not closely related to Rp1-D.

Amino Acid Sequence↗

PubMed2001

Tn10 insertional mutations of Bacillus subtilis that block the biosynthesis of bacilysin.

Transposon mutagenesis was employed to isolate the gene(s) related with the biosynthesis of dipeptide antibiotic in Bacillus subtilis PY79 (a prototrophic derivative of the standard 168 strain). The blocked mutants were phenotypically selected from the transposon library by bioassay and the complete loss of biosynthetic ability was verified through ESI-mass spectrometry analysis. Four different bacilysin nonproducer mutants (Bac(-)::Tn10(ori-spc)) were isolated from the transposon library. The genes involved in bacilysin biosynthesis were identified as thyA (thymidilate synthetase), ybgG (unknown; similar to homocysteine methyl transferase) and oppA (oligopeptide permease), respectively. The other blocked gene was yvgW (unknown; similar to heavy metal-transporting ATPase); however, backcross studies did not verify its involvement in bacilysin biosynthesis. This gene, on the other hand, appeared to be necessary for efficient sporulation and transformation. Opp involvement was significant as it suggested that bacilysin biosynthesis is under or a component of the quorum sensing pathway which has been shown to be responsible for the establishment of sporulation, competence development and onset of surfactin biosynthesis. For verification, it was necessary to check the involvement of peptide pheromones (PhrA or PhrC) internalized by the Opp system and response regulator ComA as the essential components of this global control. phrA, phrC and comA deleted mutants of PY79 were thus constructed and the latter two genes were shown to be essential for bacilysin biosynthesis.

ATP-Binding Cassette Transporters↗

PubMed1982

The construction of cosmid libraries which can be used to transform eukaryotic cells.

Cosmid vectors have been developed which carry selective markers for growth in bacteria (beta lactamase gene) and animal cells (the Herpes Simplex virus thymidine kinase gene, the transposon Tn-5 aminoglycosyl 3' phosphotransferase gene and the E. coli guanine phosphoribosyltransferase gene). The design of the cosmids allows the exchange of the eukaryotic markers in recombinant cosmids. Human and mouse cosmid libraries containing DNA inserts of about 40kb have been generated by an improved method. Several clones from the human beta-globin locus were isolated. These cosmids transform mouse L cells at high efficiency in both circular and linear form. The newly introduced genes are expressed accurately in L cells.

Animals↗

PubMed2002

Identification of motility and autoagglutination Campylobacter jejuni mutants by random transposon mutagenesis.

Campylobacter jejuni has been identified as the leading cause of acute bacterial diarrhea in the United States, yet compared with other enteric pathogens, considerably less is understood concerning the virulence factors of this human pathogen. A random in vivo transposon mutagenesis system was recently developed for the purpose of creating a library of C. jejuni transformants. A total of 1,065 C. jejuni transposon mutants were screened for their ability to swarm on motility agar plates and autoagglutinate in liquid cultures; 28 mutants were subsequently identified. The transposon insertion sites were obtained by using random-primed PCR, and the putative genes responsible for these phenotypes were identified. Of these mutants, all 28 were found to have diminished motility (0 to 86% that of the control). Seventeen motility mutants had insertions in genes with strong homology to functionally known motility and chemotaxis genes; however, 11 insertions were in genes of unknown function. Twenty motility mutants were unable to autoagglutinate, suggesting that the expression of flagella is correlated with autoagglutination (AAG). However, four mutants expressed wild-type levels of surface FlaA, as indicated by Western blot analysis, yet were unable to autoagglutinate (Cj1318, Cj1333, Cj1340c, and Cj1062). These results suggest that FlaA is necessary but not sufficient to mediate the AAG phenotype. Furthermore, two of the four AAG mutants (Cj1333 and Cj1062) were unable to invade INT-407 intestinal epithelial cells, as determined by a gentamicin treatment assay. These data identify novel genes important for motility, chemotaxis, and AAG and demonstrate their potential role in virulence.

Agglutination↗

PubMed2001

Selection of several classes of mimosine-degradation-defective Tn3Hogus-insertion mutants of Rhizobium sp. strain TAL1145 on the basis of mimosine-inducible GUS activity.

Rhizobium sp. strain TAL1145 that nodulates Leucaena leucocephala degrades mimosine, a toxin produced by this tree legume. A cosmid clone, pUHR263, containing approximately 25 kb cloned DNA was isolated by plating Escherichia coli cells containing the cosmid clone library of TAL1145 on a minimal medium in which 3-hydroxy-4-pyridone (HP), a degradation product of mimosine, was used as the source of nitrogen. Cosmid pUHR263 was mutagenized by random insertions of Tn3Hogus, a transposon that makes transcriptional gus fusions when it is inserted in a gene in the correct orientation. Various pUHR263::Tn3Hogus derivatives that showed mimosine-inducible or mimosine-repressible GUS activities when transferred to the Rhizobium sp. strain TAL1145 were selected. Mutants of TAL1145 were constructed by transferring these Tn3Hogus insertions into the TAL1145 chromosome through double-homologous recombination. These mutants were classified into five classes on the basis of defects in mimosine degradation. The growth of these mutants was inhibited to different extents by mimosine applied to the growth medium. Mimosine forms a red-colored Fe-mimosine complex when FeCI3 is added to the medium. The inhibitory effect of Fe-mimosine on growth of the mutants was much less than that of mimosine.

Bacterial Proteins↗

PubMed1986

In vivo repackaging of recombinant cosmid molecules for analyses of Salmonella typhimurium, Streptococcus mutans, and mycobacterial genomic libraries.

Strains of Escherichia coli K-12 were constructed that permitted the amplification of in vitro-packaged recombinant cosmid-transducing particles by in vivo repackaging of recombinant cosmid molecules. Thermal induction of these thermoinducible, excision-defective lysogens containing recombinant cosmid molecules yielded high titers of packaged recombinant cosmids and low levels of PFU. These strains were used to amplify packaged recombinant cosmid libraries of Mycobacterium leprae, Mycobacterium vaccae, Salmonella typhimurium, and Streptococcus mutans DNA. Contiguous and noncontiguous libraries were compared for the successful identification of cloned genes. Construction of noncontiguous libraries allowed the dissociation of desired genes from genes that were deleterious to the survival of a cosmid recombinant and permitted selection for unlinked traits that resulted in a selected phenotype. In vivo repackaging of recombinant cosmids permitted amplification of the original in vitro-packaged collection of transducing particles, storage of cosmid libraries as phage lysates, facilitation of complementation screening, expression analysis of repackaged recombinant cosmids after UV-irradiated cells were infected, in situ enzyme or immunological screening, and facilitation of recovery of recombinant cosmid molecules containing transposon inserts.

ATP-Dependent Proteases↗

PubMed2005

Triplet nucleotide removal at random positions in a target gene: the tolerance of TEM-1 beta-lactamase to an amino acid deletion.

The deletion of amino acids is one of the evolutionary mechanisms by which nature adapts the function of proteins. A simple method has been developed that mimics this event in vitro by introducing a deletion of exactly three nucleotides at random positions in a target gene. The method involved the engineering of the mini-Mu transposon to introduce a recognition sequence for the restriction enzyme MlyI. The new transposon, MuDel, was capable of efficient insertion into a target DNA sequence. To determine the efficacy of the method, the bla gene that encodes the TEM-1 beta-lactamase was used as the target and a small library containing 22 different sequence variants was created. Of these 22 variants, 8 were identified that conferred resistance to ampicillin on Escherichia coli. Each of the TEM-1 variants possessed a distinct ampicillin minimum inhibitory concentration, ranging from 500 to >10,000 microg/ml. Sequence analysis revealed that active TEM-1 variants contained deletions not just in loops but also helices, and included regions known to be involved in catalysis, antibiotic resistance and inhibitor binding. This new technology is transferable to most genes, permitting an extensive analysis of deletion mutations on protein function.

Amino Acid Sequence↗

PubMed2002

Construction of a novel transposon mutagenesis system useful in the isolation of Streptococcus parasanguis mutants defective in Fap1 glycosylation.

Streptococcus parasanguis, a primary colonizer of the tooth surface, has long, peritrichous fimbriae. A fimbria-associated protein, Fap1, is identified as an adhesin of S. parasanguis FW213. The mature Fap1 protein is glycosylated, and the glycosylation is required for fimbria biogenesis and bacterial adhesion. Little is known about the mechanism of Fap1 glycosylation due to the lack of identifiable mutants. A novel transposon mutagenesis system was established and used to generate a mutant library. Screening of the library with a monoclonal antibody specific for a glycan epitope of Fap1 yielded six mutants with decreased expression levels of surface-associated glycosylated Fap1 protein. Southern blot analyses revealed that three of the mutants had the transposon inserted in the fap1 locus, whereas the other three mutants had insertions in other genes. Among the latter three mutants, two expressed Fap1 polypeptides on which no glycosylation was detected by glycan-specific antibodies; the other mutant expressed a partially glycosylated Fap1 polypeptide. These data suggest that three mutants were isolated with defects in genes implicated in Fap1 glycosylation.

DNA Transposable Elements↗