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Results for “transposon insertion sequencing”

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At least 649 records · Page 36Linked to original sources
PubMed1989

Trimethoprim resistance transposon Tn4003 from Staphylococcus aureus encodes genes for a dihydrofolate reductase and thymidylate synthetase flanked by three copies of IS257.

Trimethoprim resistance mediated by the Staphylococcus aureus multi-resistance plasmid pSK1 is encoded by a structure with characteristics of a composite transposon which we have designated Tn4003. Nucleotide sequence analysis of Tn4003 revealed it to be 4717 bp in length and to contain three copies of the insertion element IS257 (789-790 bp), the outside two of which are flanked by directly repeated 8-bp target sequences. IS257 has imperfect terminal inverted repeats of 27-28 bp and encodes for a putative transposase with two potential alpha-helix-turn-alpha-helix DNA recognition motifs. IS257 shares sequence similarities with members of the IS15 family of insertion sequences from Gram-negative bacteria and with ISS1 from Streptococcus lactis. The central region of the transposon contains the dfrA gene that specifies the S1 dihydrofolate reductase (DHFR) responsible for trimethoprim resistance. The S1 enzyme shows sequence homology with type I and V trimethoprim-resistant DHFRs from Gram-negative bacteria and with chromosomally encoded DHFRs from Gram-positive and Gram-negative bacteria. 5' to dfrA is a thymidylate synthetase gene, designated thyE.

Amino Acid Sequence↗
PubMed1979

DNA sequence analysis of the transposon Tn3: three genes and three sites involved in transposition of Tn3.

The complete nucleotide sequence of the transposon Tn3 and of 20 mutations which affect its transposition are reported. The mutations, generated in vitro by random insertion of synthetic restriction sites, proved to contain small duplications or deletions immediately adjacent to the new restriction site. By determining the phenotype and DNA sequence of these mutations we were able to generate an overlapping phenotypic and nucleotide map. This 4957 bp transposon encodes three polypeptides which account for all but 350 bp of its total coding capacity. These proteins are the transposase, a high molecular weight polypeptide (1015 amino acids) encoded by the tnpA gene; the Tn3-specific repressor, a low molecular weight polypeptide (185 amino acids) encoded by the tnpR gene; and the 286 amino acid beta-lactamase. The 38 bp inverted repeats flanking Tn3 appear to be absolutely required in cis for Tn3 to transpose. Genetic data suggest that Tn3 contains a third site (Gill et al., 1978), designated IRS (internal resolution site), whose absence results in the insertion of two complete copies of Tn3 as direct repeats into the recipient DNA. We suggest that these direct repeats of complete copies of Tn3 are intermediates in transposition, and that the IRS site is required for recombination and subsequent segregation of the direct repeats to leave a single copy of Tn3 (Gill et al., 1978). A 23 nucleotide sequence within the amino terminus of the transposase which shares strong sequence homology with the inverted repeat may be the internal resolution site.

Base Sequence↗
PubMed2000

Inactivation of gltB abolishes expression of the assimilatory nitrate reductase gene (nasB) in Pseudomonas putida KT2442.

By using mini-Tn5 transposon mutagenesis, random transcriptional fusions of promoterless bacterial luciferase, luxAB, to genes of Pseudomonas putida KT2442 were generated. Insertion mutants that responded to ammonium deficiency by induction of bioluminescence were selected. The mutant that responded most strongly was genetically analyzed and is demonstrated to bear the transposon within the assimilatory nitrate reductase gene (nasB) of P. putida KT2442. Genetic evidence as well as sequence analyses of the DNA regions flanking nasB suggest that the genes required for nitrate assimilation are not clustered. We isolated three second-site mutants in which induction of nasB expression was completely abolished under nitrogen-limiting conditions. Nucleotide sequence analysis of the chromosomal junctions revealed that in all three mutants the secondary transposon had inserted at different sites in the gltB gene of P. putida KT2442 encoding the major subunit of the glutamate synthase. A detailed physiological characterization of the gltB mutants revealed that they are unable to utilize a number of potential nitrogen sources, are defective in the ability to express nitrogen starvation proteins, display an aberrant cell morphology under nitrogen-limiting conditions, and are impaired in the capacity to survive prolonged nitrogen starvation periods.

Amino Acid Sequence↗
PubMed1999

Mobile catabolic genes in bacteria.

The recent findings of various mobile catabolic genes have provided some insight into the evolution of microbial degradation systems for xenobiotic compounds. The catabolic genes undergo marked genetic rearrangements due to their presence on transposons or association with mobile genetic elements. Bacterial catabolic transposons fall into three defined structural classes. Class I elements include catabolic genes flanked by two copies of insertion sequences. Class II elements carry short terminal inverted repeats and transpose by the replicative mode in which transposase and resolvase are involved. Conjugative catabolic transposons represent the third class of mobile genetic elements. They carry all the genes required for excision, conjugal transfer to a new host, and integration. This review focuses on the structures, functions and roles of the recently characterized catabolic transposons in bacteria. Also described are the mobile catabolic elements that share structural similarity with the pathogenicity and symbiosis islands.

Journal Article↗
PubMed2006

Exploring strategies for protein trapping in Drosophila.

The use of fluorescent protein tags has had a huge impact on cell biological studies in virtually every experimental system. Incorporation of coding sequence for fluorescent proteins such as green fluorescent protein (GFP) into genes at their endogenous chromosomal position is especially useful for generating GFP-fusion proteins that provide accurate cellular and subcellular expression data. We tested modifications of a transposon-based protein trap screening procedure in Drosophila to optimize the rate of recovering useful protein traps and their analysis. Transposons carrying the GFP-coding sequence flanked by splice acceptor and donor sequences were mobilized, and new insertions that resulted in production of GFP were captured using an automated embryo sorter. Individual stocks were established, GFP expression was analyzed during oogenesis, and insertion sites were determined by sequencing genomic DNA flanking the insertions. The resulting collection includes lines with protein traps in which GFP was spliced into mRNAs and embedded within endogenous proteins or enhancer traps in which GFP expression depended on splicing into transposon-derived RNA. We report a total of 335 genes associated with protein or enhancer traps and a web-accessible database for viewing molecular information and expression data for these genes.

Animals↗
PubMed1999

Transposition of the autonomous Fot1 element in the filamentous fungus Fusarium oxysporum.

Autonomous mobility of different copies of the Fot1 element was determined for several strains of the fungal plant pathogen Fusarium oxysporum to develop a transposon tagging system. Two Fot1 copies inserted into the third intron of the nitrate reductase structural gene (niaD) were separately introduced into two genetic backgrounds devoid of endogenous Fot1 elements. Mobility of these copies was observed through a phenotypic assay for excision based on the restoration of nitrate reductase activity. Inactivation of the Fot1 transposase open reading frame (frameshift, deletion, or disruption) prevented excision in strains free of Fot1 elements. Molecular analysis of the Nia+ revertant strains showed that the Fot1 element reintegrated frequently into new genomic sites after excision and that it can transpose from the introduced niaD gene into a different chromosome. Sequence analysis of several Fot1 excision sites revealed the so-called footprint left by this transposable element. Three reinserted Fot1 elements were cloned and the DNA sequences flanking the transposon were determined using inverse polymerase chain reaction. In all cases, the transposon was inserted into a TA dinucleotide and created the characteristic TA target site duplication. The availability of autonomous Fot1 copies will now permit the development of an efficient two-component transposon tagging system comprising a trans-activator element supplying transposase and a cis-responsive marked element.

Base Sequence↗
PubMed2003

Alternative interactions between the Tn7 transposase and the Tn7 target DNA binding protein regulate target immunity and transposition.

The Tn7 transposon avoids inserting into a target DNA that contains a pre-existing copy of Tn7. This phenomenon, known as 'target immunity', is established when TnsB, a Tn7 transposase subunit, binds to Tn7 sequences in the target DNA and mediates displacement of TnsC, a critical transposase activator, from the DNA. Paradoxically, TnsB-TnsC interactions are also required to promote transposon insertion. We have probed Tn7 target immunity by isolating TnsB mutants that mediate more frequent insertions into a potentially immune target DNA because they fail to provoke dissociation of TnsC from the DNA. We show that a single region of TnsB mediates the TnsB-TnsC interaction that underlies both target immunity and transposition, but that TnsA, the other transposase subunit, channels the TnsB-TnsC interaction toward transposition.

Amino Acid Sequence↗
PubMed1989

Nucleotide sequence of the transcriptional control region of the osmotically regulated proU operon of Salmonella typhimurium and identification of the 5' endpoint of the proU mRNA.

Southern blot analysis of 15 proU transposon insertions in Salmonella typhimurium indicated that this operon is at least 3 kilobase pairs in length. The nucleotide sequence of 1.5-kilobase-pair fragment that contains the transcriptional control region of the proU operon and the coding sequences specifying 290 amino acids of the first structural gene of the operon was determined. The predicted amino acid sequence of the product of this gene shows extensive similarity to the HisP, MalK, and other proteins that are inner membrane-associated components of binding protein-dependent transport systems. S1 mapping and primer extension analysis of the proU mRNAs revealed several species with different 5' ends. Two of these endpoints are sufficiently close to sequences that have weak similarities to the consensus -35 and -10 promoter sequences that they are likely to define two transcription start sites. However, we cannot rule out the possibility that some or all of the 5' endpoints detected arose as a result of the degradation of a longer mRNA. The expression of proU-lacZ operon fusions located on plasmids was normal in S. typhimurium regardless of the plasmid copy number. The sequences mediating normal, osmoregulated expression of the proU operon were shown by subcloning to be contained on an 815-base-pair fragment. A 350-base-pair subclone of this fragment placed onto a lacZ expression vector directed a high-level constitutive expression of beta-galactosidase, suggesting that there is a site for negative regulation in the proU transcriptional control region which has been deleted in the construction of this plasmid.

Amino Acid Sequence↗
PubMed2006

Rapid identification of non-essential genes for in vitro replication of Marek's disease virus by random transposon mutagenesis.

Marek's disease virus (MDV) is a highly oncogenic alphaherpesvirus that induces rapid-onset T-cell lymphomas in poultry. The MDV genome encodes more than 100 genes. However, the role of many of these genes in virus replication is not known. The construction of an infectious bacterial artificial chromosome (BAC) clone of the highly oncogenic RB-1B strain of MDV has been described previously. Virus reconstituted from the BAC clone induced rapid-onset lymphomas in chickens very similar to the wildtype viruses. In this paper, the construction of a high-density random transposon-insertion mutant library of the RB-1B BAC clone using a high throughput in vitro transposon mutagenesis technique is described. Furthermore a PCR screening method, using primers specific for the transposon sequence and the MDV gene(s) of interest, was developed for the rapid identification of specific insertion mutants. The application of the screening method to identify some of the non-essential genes for MDV replication in vitro is described.

Chromosomes, Artificial, Bacterial↗
PubMed1989

Nucleotide sequences from the colicin E5, E6 and E9 operons: presence of a degenerate transposon-like structure in the ColE9-J plasmid.

The nucleotide sequences of 1288 bp of plasmid ColE5-099, 1609 bp of ColE6-CT14 and 2099 bp of ColE9-J were determined. These sequences encompass the structural genes for the C-terminal receptor-binding and nuclease domains of colicins E5, E6 and E9, their cis- or trans-acting immunity proteins and four lysis proteins including an atypical one of non-lipoprotein nature (Lys) present in the ColE9-J plasmid. The ColE6 gene organisation, in the order col-imm-E8imm-lys, is identical to that found in the previously described double-immunity gene system of ColE3-CA38 (an RNase producer). The corresponding genes in the two plasmids are 87%-94% homologous. In ColE9-J, the genes are organised as col-imm-lys-E5imm-lys. The E9 col-imm gene pair is homologous to the colicin E2-P9 type (a DNase producer). Downstream from E9imm is an E5imm (designated E5imm[E9]) which is trans-acting. Neither the predicted structures of E5Imm[E9] nor the cis-acting Imm resident in the ColE5-099 plasmid which differs by a single amino acid shows any resemblance to other immunity structures which have been sequenced. Furthermore, the E5col sequences differ from those predicted previously for other colicins except for the conserved btuB-specified receptor-binding domain. A novel 205 nucleotide long insertion sequence is found in the ColE9-J plasmid. This insertion sequence, which we named ISE9, has features reminiscent of the degenerate transposon IS101 previously found in plasmid pSC101. One effect of ISE9 is the presence of the atypical lysis gene, lys.(ABSTRACT TRUNCATED AT 250 WORDS)

Amino Acid Sequence↗
PubMed1997

Use of a promoterless lacZ gene insertion to investigate chitinase gene expression in the marine bacterium Pseudoalteromonas sp. strain S9.

Sequence data for genes encoding 16S rRNA indicated that the marine strain previously named Pseudomonas sp. strain S9 would be better identified as a Pseudoalteromonas sp. By use of transposon mutagenesis, a chitinase-negative mutant of S9 with a lacZ reporter gene insertion was isolated. Part of the interrupted gene was cloned and sequenced. The deduced amino acid sequence had homology to sequences of bacterial chitinases. Expression of the chitinase gene promoter was quantified by measuring the lacZ reporter gene product, beta-galactosidase, beta-Galactosidase production was induced 10-fold by N-acetylglucosamine and 3-fold by chitin in minimal medium. Repression of beta-galactosidase synthesis was observed in rich medium either with or without chitin but was not observed in minimal medium containing glucose. The chitinase gene promoter was induced by starvation and higher-than-ambient levels of carbon dioxide but not by cadmium ion, heat or cold shock, or UV exposure.

Acetylglucosamine↗
PubMed1990

Analysis of protein localization by use of gene fusions with complementary properties.

This report describes a new transposon designed to facilitate the combined use of beta-galactosidase and alkaline phosphatase gene fusions in the analysis of protein localization. The transposon, called TnlacZ, is a Tn5 derivative that permits the generation of gene fusions encoding hybrid proteins carrying beta-galactosidase at their C termini. In tests with plasmids, TnlacZ insertions that led to high cellular beta-galactosidase activity were restricted to sequences encoding either cytoplasmic proteins or cytoplasmic segments of a membrane protein. The fusion characteristics of TnlacZ are thus complementary to those of TnphoA, a transposon able to generate alkaline phosphatase fusions whose high-activity insertion sites generally correspond to periplasmic sequences. The structure of TnlacZ allows the conversion of a TnlacZ fusion into the corresponding TnphoA fusion (and vice versa) through recombination or in vitro manipulation in a process called fusion switching. Fusion switching was used to generate the following two types of fusions with unusual properties: a low-specific-activity beta-galactosidase-alkaline phosphatase gene fusion and two toxic periplasmic-domain serine chemoreceptor-beta-galactosidase gene fusions. The generation of both beta-galactosidase and alkaline phosphatase fusions at exactly the same site in a protein permits a comparison of the two enzyme activities in evaluating the subcellular location of the site, such as in studies of membrane protein topology. In addition, fusion switching makes it possible to generate gene fusions whose properties should facilitate the isolation of mutants defective in the export or membrane anchoring of different cell envelope proteins.

Alkaline Phosphatase↗
PubMed2003

Mobilization of a transposon in the rice genome.

Rice (Oryza sativa L.) is an important crop worldwide and, with the availability of the draft sequence, a useful model for analysing the genome structure of grasses. To practice efficient rice breeding through genetic engineering techniques, it is important to identify the economically important genes in this crop. The use of mobile transposons as gene tags in intact plants is a powerful tool for functional analysis because transposon insertions often inactivate genes. Here we identify an active rice transposon named miniature Ping (mPing) through analysis of the mutability of a slender mutation of the glume-the seed structure that encloses and determines the shape of the grain. The mPing transposon is inserted in the slender glume (slg) mutant allele but not in the wild-type allele. Search of the O. sativa variety Nipponbare genome identified 34 sequences with high nucleotide similarity to mPing, indicating that mPing constitutes a family of transposon elements. Excision of mPing from slg plants results in reversion to a wild-type phenotype. The mobility of the transposon mPing in intact rice plants represents a useful alternative tool for the functional analysis of rice genes.

Alleles↗
PubMed1988

Evidence for natural gene transfer from gram-positive cocci to Escherichia coli.

High-level resistance to macrolide-lincosamide-streptogramin type B (MLS) antibiotics in Escherichia coli BM2570 is due to the presence on the conjugative plasmid pIP1527 of the MLS resistance determinant ermBC, which is almost identical to the erm genes previously described in plasmid pAM77 from Streptococcus sanguis (ermAM) and in transposon Tn917 from Enterococcus faecalis (ermB). This gene and its regulatory region are located downstream from the insertion sequence IS1. The 23S rRNA methylase encoded by pIP1527 differs by three and six amino acids from those encoded by Tn917 and pAM77, respectively. Unlike the streptococcal elements which confer the inducible MLS phenotype, the ermBC gene is expressed constitutively in E. coli and Bacillus subtilis, due to several mutations in the regulatory region. Transcription of the ermBC gene starts from three different sites following three overlapping promoters which function in both E. coli and B. subtilis. Promoters P2 and P3 are located within the region homologous to pAM77 and Tn917, and P1 is a hybrid promoter constituted by -35 and -10 sequences located at the end of IS15 and in the streptococcal region, respectively. These results constitute evidence for the recent in vivo transfer from Streptococcus spp. to E. coli. This transfer could have been mediated by transposons such as Tn917 or Tn1545 from Streptococcus pneumoniae, which also bears an MLS determinant that is homologous to ermB. We speculate that the insertion sequences IS15 and IS1 could have played a role in the expression and dissemination of ermBC, which has been found in numerous strains of enterobacteria.

Amino Acid Sequence↗
PubMed2001

Disruption of a gene encoding a putative gamma-butyrolactone-binding protein in Streptomyces tendae affects nikkomycin production.

A 2.6-kb BamHI fragment from the genome of the wild-type, nikkomycin-producing strain of Streptomyces tendae ATCC 31160 was cloned and sequenced. This 2.6-kb BamHI fragment corresponds to the DNA site where transposon Tn4560 had inserted to create a nikkomycin-nonproducing mutant. A possible ORF of 660 nucleotides was found in this 2.6-kb BamHI fragment, in which the third base of each codon was either G or C in 92% of the codons. The deduced amino acid sequence coded by this ORF (TarA, tendae autoregulator receptor) shows strong homology with several Gamma-butyrolactone-binding proteins that negatively regulate antibiotic production in other streptomycetes and have a helix-turn-helix DNA-binding motif. A portion (179 nucleotides) of tarA that encodes the helix-turn-helix motif was replaced with ermE, and wild-type S. tendae was transformed with this construct borne in pDH5, a gene-disruption vector. Southern hybridization indicated that ermE had inserted in the 2.6-kb BamHI region in one isolate that is erythromycin resistant. Northern hybridization indicated that tarA disruption significantly increased the amount of disrupted-tarA mRNA. This suggests that TarA negatively regulates its own synthesis. Nikkomycin production by the tarA disruptant was delayed but reached the wild-type level after longer incubation in production medium.

4-Butyrolactone↗
PubMed1990

Mobile element insertions causing mutations in the Drosophila suppressor of sable locus occur in DNase I hypersensitive subregions of 5'-transcribed nontranslated sequences.

The locations of 16 mobile element insertions causing mutations at the Drosophila suppressor of sable [su(s)] locus were determined by restriction mapping and DNA sequencing of the junction sites. The transposons causing the mutations are: P element (5 alleles), gypsy (3 alleles), 17.6, HMS Beagle, springer, Delta 88, prygun, Stalker, and a new mobile element which was named roamer (2 alleles). Four P element insertions occur in 5' nontranslated leader sequences, while the fifth P element and all 11 non-P elements inserted into the 2053 nucleotide, 5'-most intron that is spliced from the 5' nontranslated leader approximately 100 nucleotides upstream of the translation start. Fifteen of the 16 mobile elements inserted within a approximately 1900 nucleotide region that contains seven 100-200-nucleotide long DNase I-hypersensitive subregions that alternate with DNase I-resistant intervals of similar lengths. The locations of these 15 insertion sites correlate well with the roughly estimated locations of five of the DNase I-hypersensitive subregions. These findings suggest that the features of chromatin structure that accompany gene activation may also make the DNA susceptible to insertion of mobile elements.

Animals↗