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Results for “transposon insertion sequencing”

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PubMed1987

[Transfer of "artificial transposons" constructed on the basis of insertion element IS1].

Terminal inverted repeats of the insertion element IS1 were synthesized chemically and plasmids containing these sequences flanking kanamycin-resistance gene in different combinations were constructed. Further incorporation of a whole-sized copy of the IS1 into such plasmids caused in some cases the autonomous transfer of Km-resistance from plasmid to bacteriophage lambda DNA. The transposition of the Km-resistance gene was only observed in those cases when the gene was enclosed between IS1 copy and one of the terminal repeats. The data obtained are discussed with regard to the evolution of bacterial transposons.

Bacteriophage lambda↗

PubMed1986

Evidence that white-blood is a novel type of temperature-sensitive mutation resulting from temperature-dependent effects of a transposon insertion on formation of white transcripts.

We report results indicating that the temperature-sensitive white-blood (wbl) mutation has a novel molecular basis, involving the formation of RNA transcripts of the affected gene rather than the behavior of the polypeptide product. First, we show that the temperature-sensitive mutant phenotype of wbl correlates with (and presumably results from) a temperature-dependent effect on levels of the 2.6-kb polyadenylated white transcript. Second, DNA sequence analysis and other studies show that wbl is associated with the insertion into the second white intron of a previously uncharacterized retrotransposon (designated the blood transposon). We discuss the potential origins of the novel temperature-sensitive molecular phenotype of wbl and the prospects for exploiting wbl to engineer temperature-sensitive mutations in other genes.

Alleles↗

PubMed1983

Genetic transposition and insertional mutagenesis in Bacillus subtilis with Streptococcus faecalis transposon Tn917.

The Streptococcus faecalis transposon Tn917 was introduced into Bacillus subtilis by transformation of competent cells with the plasmid pAM alpha 1::Tn917 and was tested for transposition activity by selection for insertions into the temperate phage SP beta. Insertions were obtained at a frequency indicating relatively efficient movement of the element, and Southern hybridization analysis of a particular insertion confirmed it to be the result of a genuine transposition event. A restriction fragment from pAM alpha 1::Tn917 containing the transposon sequences was ligated into a temperature-sensitive plasmid (pBD95), and transpositions into the B. subtilis chromosome were selected by requiring the transposon drug resistance to be maintained at temperatures nonpermissive for plasmid replication. Insertions have been recovered at many chromosomal sites, including ones that produced auxotrophy of different kinds and ones that produced various different sporulation-defective phenotypes, indicating good prospects for the use of Tn917 as a tool for insertional mutagenesis in B. subtilis.

Bacillus subtilis↗

PubMed1984

Generation of a Tn5 promoter probe and its use in the study of gene expression in Caulobacter crescentus.

A promoter probe, Tn5-VB32, was constructed and placed in a P group R plasmid containing bacteriophage Mu sequences, allowing transfer of the transposon to bacteria such as Caulobacter, Rhizobium, and Agrobacterium without retention of the plasmid. The probe carries an altered Tn5 transposon that allows detection of chromosomal promoter regions by virtue of acquired kanamycin resistance. A fragment of DNA containing the neomycin phosphotransferase II (NPT II) gene from Tn5, lacking its promoter region but retaining its translation initiation signal, was inserted into a Tn5 derivative that lacked the entire NPT II gene and a large portion of the IS50L sequence while retaining its ability to transpose. This Tn5 derivative also contained the intact tetracycline resistance-encoding region of the transposon Tn10. Transposition of the Tn5-VB32 promoter probe into the Caulobacter crescentus chromosome generated auxotrophic and motility mutants and Southern blot analysis of DNA from these mutants showed Tn5-VB32 sequences in random-sized chromosomal restriction fragments. Transcriptional regulation by exogenous cysteine of NPT II gene expression was demonstrated in a cysteine auxotroph generated by Tn5-VB32 insertional inactivation. NPT II synthesis, measured by agar plate assays of kanamycin resistance and by immunoprecipitation of the NPT II protein, was repressed in the presence of cysteine and derepressed in its absence. Several fla- mutants were also isolated by Tn5-VB32 mutagenesis and shown to confer kanamycin resistance. Insertions within temporally regulated genes, such as those involved in flagellar biosynthesis and chemotaxis functions, can now be used directly to monitor transcriptional regulation from Caulobacter promoter sequences.

Bacteria↗

PubMed2001

The minimum internal and external sequence requirements for transposition of the eukaryotic transformation vector piggyBac.

The piggyBac element from Trichoplusia ni is recognized as a useful vector for transgenesis of a wide variety of species. This transposable element is 2472 bp in length, and has a complex repeat configuration consisting of an internal repeat (IR), spacer, and terminal repeat (TR) at both ends, and a single ORF encoding the transposase. Excision assays performed in microinjected T. ni embryos using plasmids deleted for progressively larger portions of the piggyBac internal sequence reveal that the 5' and 3' IR, spacer, and TR configuration is sufficient for precise excision of piggyBac when transposase is provided in trans. Interplasmid transposition assays using plasmids carrying varying lengths of intervening sequence between the piggyBac termini in T. ni demonstrate that a minimum of 55 bp of intervening sequence is required for optimal transposition, while lengths less than 40 bp result in a dramatic decrease in transposition frequency. These results suggest that the piggyBac transposase may bind both termini simultaneously before cleavage can occur, and/or that the formation of a transposition complex requires DNA bending between the two termini. Based on these results we constructed a 702-bp cartridge with minimal piggyBac 5' and 3' terminal regions separated by an intervening sequence of optimal length. Interplasmid transposition assays demonstrate that the minimal terminal configuration is sufficient to mediate transposition, and also verify that simply inserting this cartridge into an existing plasmid converts that plasmid into a non-autonomous piggyBac transposon. We also constructed a minimal piggyBac vector, pXL-Bac, that contains an internal multiple cloning site sequence between the minimal terminal regions. These vectors should greatly facilitate the utilization of the piggyBac transposon in a wide range of hosts.

Animals↗

PubMed2005

Gene duplication and exon shuffling by helitron-like transposons generate intraspecies diversity in maize.

We report a whole-genome comparison of gene content in allelic BAC contigs from two maize inbred lines. Genic content polymorphisms involve as many as 10,000 sequences and are mainly generated by DNA insertions. The termini of eight of the nine genic insertions that we analyzed shared the structural hallmarks of helitron rolling-circle transposons. DNA segments defined by helitron termini contained multiple gene-derived fragments and had a structure typical of nonautonomous helitron-like transposons. Closely related insertions were found in multiple genomic locations. Some of these produced transcripts containing segments of different genes, supporting the idea that these transposition events have a role in exon shuffling and the evolution of new proteins. We identified putative autonomous helitron elements and found evidence for their transcription. Helitrons in maize seem to continually produce new nonautonomous elements responsible for the duplicative insertion of gene segments into new locations and for the unprecedented genic diversity. The maize genome is in constant flux, as transposable elements continue to change both the genic and nongenic fractions of the genome, profoundly affecting genetic diversity.

Base Sequence↗

PubMed2002

Construction of mini-Tn4001tet and its use in Mycoplasma gallisepticum.

The Mollicutes are a group of cell-wall-less bacteria and are important plant and animal pathogens. Progress toward analyzing their pathogenic mechanisms has been hampered by the few available genetic tools. Of the two transposons shown to function in mycoplasmas, only Tn4001 is readily amenable to modification and development. One disadvantage of using Tn4001 in mycoplasmas has been independent insertion of the insertion sequence, IS256, probably as a result of inadequate control of the transposase expression in mycoplasmas. In this study, we describe the construction of a mini-Tn4001 containing the tetM antibiotic resistance gene from Tn916. The transposase gene was placed outside the inverted repeats to lower the frequency of independent transposition events. Transposition of mini-Tn4001tet in Mycoplasma gallisepticum occurred at a frequency of 1-8 x 10(-6), a frequency similar to that of the parent transposon. Insertions of mini-Tn4001tet were random and only single insertions were observed. Several unique restriction sites between the inverted repeat sequences provide for further development of mini-Tn4001.

Amino Acid Sequence↗

PubMed1996

Control of hemA expression in Rhodobacter sphaeroides 2.4.1: regulation through alterations in the cellular redox state.

Rhodobacter sphaeroides 2.4.1 has the ability to synthesize a variety of tetrapyrroles, reflecting the metabolic versatility of this organism and making it capable of aerobic, anaerobic, photosynthetic, and diazotrophic growth. The hemA and hemT genes encode isozymes that catalyze the formation of 5-aminolevulinic acid, the first step in the biosynthesis of all tetrapyrroles present in R. sphaeroides 2.4.1. As part of our studies of the regulation and expression of these genes, we developed a genetic selection that uses transposon mutagenesis to identify loci affecting the aerobic expression of the hemA gene. In developing this selection, we found that sequences constituting an open reading frame immediately upstream of hemA positively affect hemA transcription. Using a transposon-based selection for increased hemA expression in the absence of the upstream open reading frame, we isolated three independent mutants. We have determined that the transposon insertions in these strains map to three different loci located on chromosome 1. One of the transposition sites mapped in the vicinity of the recently identified R. sphaeroides 2.4.1 homolog of the anaerobic regulatory gene fnr. By marker rescue and DNA sequence analysis, we found that the transposition site was located between the first two genes of the cco operon in R. sphaeroides 2.4.1, which encodes a cytochrome c terminal oxidase. Examination of the phenotype of the mutant strain revealed that, in addition to increased aerobic expression of hemA, the transposition event also conferred an oxygen-insensitive development of the photosynthetic membranes. We propose that the insertion of the transposon in cells grown in the presence of high oxygen levels has led to the generation of a cellular redox state resembling either reduced oxygen or anaerobiosis, thereby resulting in increased expression of hemA, as well as the accumulation of spectral complex formation. Several models are presented to explain these findings.

Aerobiosis↗

PubMed1986

Cloning of chromosomal DNA encoding the F41 adhesin of enterotoxigenic Escherichia coli and genetic homology between adhesins F41 and K88.

The genetic determinant for production of the adhesive antigen F41 was isolated from a porcine enterotoxigenic Escherichia coli strain by cosmid cloning. The cloned DNA included sequences homologous to those of hybridization probes prepared from the K88 adhesive antigen operon. Transposon insertions which inactivated F41 production mapped to the same region of DNA showing homology with the K88 genes, demonstrating the genetic relatedness of F41 and K88. Hybridization of a K88 gene probe to plasmid and total DNA from the porcine E. coli isolate from which the F41 gene was cloned indicated that F41 is chromosomally encoded by this strain. This observation was extended to other F41-producing animal isolates. A large number of animal E. coli isolates were examined with K88, F41, and K99 gene probes and for mannose-resistant hemagglutination of human group O erythrocytes and K88 and F41 antigen production. All K88 and F41 antigen producers possessed genetic homology with the K88 and F41 gene probes. Most, but not all, F41-producing strains possessed homology to the K99 gene probe, reflecting the previously observed association of F41 and K99 antigen production. In the strains examined, homology with the K99 gene probe was plasmid associated, whereas homology with the F41 gene probe was chromosomal. The K88 antigen-producing strains showed no homology with the K99 probe. A number of strains possessed homology with the K88 and F41 gene probes and were mannose-resistant hemagglutination positive, but did not produce K88 or F41 antigens. This suggests that there are adhesins among animal isolates of E. coli which are genetically related to but antigenically distinct from K88 and F41.

Adhesins, Escherichia coli↗

PubMed2004

Requirement of IS911 replication before integration defines a new bacterial transposition pathway.

Movement of transposable elements is often accompanied by replication to ensure their proliferation. Replication is associated with both major classes of transposition mechanisms: cut-and-paste and cointegrate formation (paste-and-copy). Cut-and-paste transposition is often activated by replication of the transposon, while in cointegrate formation replication completes integration. We describe a novel transposition mechanism used by insertion sequence IS911, which we call copy-and-paste. IS911 transposes using a circular intermediate (circle), which then integrates into a target. We demonstrate that this is derived from a branched intermediate (figure-eight) in which both ends are joined by a single-strand bridge after a first-strand transfer. In vivo labelling experiments show that the process of circle formation is replicative. The results indicate that the replication pathway not only produces circles from figure-eight but also regenerates the transposon donor plasmid. To confirm the replicative mechanism, we have also used the Escherichia coli terminators (terC) which, when bound by the Tus protein, inhibit replication forks in a polarised manner. Finally, we demonstrate that the primase DnaG is essential, implicating a host-specific replication pathway.

DNA Primase↗

PubMed1995

Structure and expression of clustered P element homologues in Drosophila subobscura and Drosophila guanche.

Sequence relationships and functional aspects were analysed in the P element homologues of Drosophila subobscura (Ds) and D. guanche (Dg). In both species, the P homologues are clustered at a single genomic position. They lack the characteristic terminal structures of actively transposing P elements, but they have the coding capacity for a 66-kDa 'repressor-like' protein. Two different types of cluster units (G-type and A-type) can be distinguished. The A-type unit, which is present in multiple copies, is transcribed in adult flies. In contrast, the G-type unit has a much lower copy number and is apparently not expressed. In Dg, the isolated G-type sequence carries a 420-bp insertion in the promoter region, which is probably responsible for inactivation. Sequence comparisons of different cluster units show that differentiation of the two types precedes the lineage split of these species. Substitution rates of the deduced proteins reveal two distinct subregions: high variability at the N terminus and strong sequence conservation in the rest of the protein. The variable region contains motifs characteristic of DNA-binding proteins. Adaptive diversification of the cluster units towards specific binding properties might be a plausible explanation for variability in the N-termini. Both unit types have lost the weak promoter region characteristic of P transposons. In the A-type unit, a new promoter has been formed which is apparently composed of parts of insertion sequences derived from two different mobile elements.(ABSTRACT TRUNCATED AT 250 WORDS)

Amino Acid Sequence↗

PubMed2006

Discovery of Bacillus thuringiensis virulence genes using signature-tagged mutagenesis in an insect model of septicaemia.

Transposon Tn917 was used to identify Bacillus thuringiensis genes required for virulence and survival in a Manduca sexta (tobacco hornworm) septicaemia model. Uniquely tagged transposons, n = 72, were constructed and used to generate 1152 insertion mutants. Sixteen pools of 72 mutants were screened in the infection model, and 12 virulence-attenuated mutants were unable to survive the infection. Analysis of the mutated DNA sequences implicated an arsR family transcriptional regulator, a histone-like DNA-binding protein, a transposon, and several sequences of unknown function in B. thuringiensis pathogenesis.

Animals↗

PubMed2004

Isolation of carotenoid-deficient mutant from alkylated dibenzothiophene desulfurizing nocardioform bacteria, Gordonia sp. TM414.

The dibenzothiophene-desulfurizing nocardioform bacteria, Gordonia sp. TM414, was isolated from oil-contaminated soil. To avoid coloration of the oil layer after the desulfurization reaction, which could decrease the quality of the oil, two colorless knock-out mutants, TPc and TPd, were isolated by using a broad-host-range transposon complex. Genomic sequence analysis revealed that the same gene was disrupted in these mutants and that the transposon-inserted gene was assigned as the gene for phytoene desaturase, crt I. The crt I mutants also showed desulfurization activity comparable to that of the parent strain in a model-oil/aqueous bi-phasic reaction, suggesting that the carotenoid production is not responsible for the bi-phasic desulfurization reaction that requires hydrophobic substrate incorporation from the organic phase.

Amino Acid Sequence↗

PubMed1993

A versatile system for detecting transposition in Arabidopsis.

The maize transposable element Activator (Ac) has been shown to be active in a number of dicots, including Arabidopsis thaliana, whose small genome and short generation time have favored its wide adoption as a model organism for molecular genetic approaches to plant physiology and development. Using the Ac element and several bacterial and plant marker genes, we have devised a versatile system for identifying plants in which a transposon has excised and reinserted elsewhere in the genome. The transposons have been designed to facilitate the identification of insertions downstream of promoters and in the vicinity of enhancers by the inclusion of a beta-glucuronidase (GUS) gene either lacking a promoter or having a minimal promoter sequence. The system permits the transposon and the source of transposase to be maintained either stably in separate plants or in the same plant. Plants in which transposition is occurring can be identified by the frequent somatic activation of the GUS gene. The herbicide chlorsulfuron is used as a selective agent to identify progeny plants in which the transposon has excised from its original insertion site within a chlorsulfuron-resistant acetolactate synthase gene. Additional selectable markers permit the identification of plants containing a transposed element, but lacking transposase. Here we describe our initial characterization of the system and demonstrate its reliability and efficiency in identifying plants with transposed elements.

Acetolactate Synthase↗

PubMed1995

Conjugative transposition.

Conjugative transposons are important determinants of antibiotic resistance, especially in gram-positive bacteria. They are remarkably promiscuous and can conjugate between bacteria belonging to different species and genera. Transposon-promoted conjugation may be similar to F plasmid-promoted conjugation, as it appears that only one strand of the transposon DNA is transferred from donor to recipient. The recent determination of the entire nucleotide sequence of Tn916 allowed us to make specific predictions about the possible function of different open reading frames and the position of a (hypothetical) origin of transfer. The mechanism of recombination during conjugative transposition differs from that of other transposons, as shown by the absence of a duplication of the target sequence upon integration. The current model for recombination postulates that staggered double-stranded cleavages occur at each end of the transposon. One DNA strand is cut six bases from the end of the transposon, and the other strand is cut immediately adjacent to the end. The ends of the excised transposon are then ligated to form a circular intermediate with a six-base heteroduplex. Staggered cleavages of the circular intermediate and the target DNA allow the transposon to insert into the target, where it is flanked by heteroduplex regions that are resolved by replication. All hosts examined contain preferential target sites: these are not specific sequences but apparently consist of bent DNA. The site-specific recombinases encoded by conjugative transposons belong to the integrase family. Like phage lambda integrase, the integrase of Tn916 has two DNA-binding domains that recognize different sequences, one within the ends of the element and one that includes target DNA. The affinity of Tn916 integrase for target sites correlates with the frequency of integration into a particular site. The similarity between conjugative transposons and phage lambda is striking and suggests that both use the same mechanism of recombination. In lambda, however, recombining sites must be homologous. Homology may be necessary because of branch migration, which is thought to occur during recombination. In conjugative transposition, the recombining sites are nearly always different, and therefore branch migration probably does not occur. This review presents a speculative model for the alignment of the ends of Tn916 during excision that was adapted from one recently proposed for lambda.

Bacteria↗

PubMed1993

Insertion and deletion mutations in the repA4 region of the IncFII plasmid NR1 cause unstable inheritance.

Mutants of IncFII plasmid NR1 that have transposons inserted in the repA4 open reading frame (ORF) are not inherited stably. The repA4 ORF is located immediately downstream from the replication origin (ori). The repA4 coding region contains inverted-repeat sequences that are homologous to the terC inverted repeats located in the replication terminus of the Escherichia coli chromosome. The site of initiation of leading-strand synthesis for replication of NR1 is also located in repA4 near its 3' end. Transposon insertions between ori and the right-hand terC repeat resulted in plasmid instability, whereas transposon insertions farther downstream did not. Derivatives that contained a 35-bp frameshift insertion in the repA4 ORF were all stable, even when the frameshift was located very near the 5' end of the coding region. This finding indicates that repA4 does not specify a protein product that is essential for plasmid stability. Examination of mutants having a nest of deletions with endpoints in or near repA4 indicated that the 3' end of the repA4 coding region and the site of leading-strand initiation could be deleted without appreciable effect on plasmid stability. Deletion of the pemI and pemK genes, located farther downstream from repA4 and reported to affect plasmid stability, also had no detectable effect. In contrast, mutants from which the right-hand terC repeat, or both right- and left-hand repeats, had been deleted were unstable. None of the insertion or deletion mutations in or near repA4 affected plasmid copy number. Alteration of the terC repeats by site-directed mutagenesis had little effect on plasmid stability. Plasmid stability was not affected by a fus mutation known to inactivate the termination function. Therefore, it appears that the overall integrity of the repA4 region is more important for stable maintenance of plasmid NR1 than are any of the individual known features found in this region.

Amino Acid Sequence↗

PubMed2002

Efficient recovery of centric heterochromatin P-element insertions in Drosophila melanogaster.

Approximately one-third of the human and Drosophila melanogaster genomes are heterochromatic, yet we know very little about the structure and function of this enigmatic component of eukaryotic genomes. To facilitate molecular and cytological analysis of heterochromatin we introduced a yellow(+) (y(+))-marked P element into centric heterochromatin by screening for variegated phenotypes, that is, mosaic gene inactivation. We recovered >110 P insertions with variegated yellow expression from approximately 3500 total mobilization events. FISH analysis of 71 of these insertions showed that 69 (97%) were in the centric heterochromatin, rather than telomeres or euchromatin. High-resolution banding analysis showed a wide but nonuniform distribution of insertions within centric heterochromatin; variegated insertions were predominantly recovered near regions of satellite DNA. We successfully used inverse PCR to clone and sequence the flanking DNA for approximately 63% of the insertions. BLAST analysis of the flanks demonstrated that either most of the variegated insertions could not be placed on the genomic scaffold, and thus may be inserted within novel DNA sequence, or that the flanking DNA hit multiple sites on the scaffold, due to insertions within different transposons. Taken together these data suggest that screening for yellow variegation is a very efficient method for recovering centric insertions and that a large-scale screen for variegated yellow P insertions will provide important tools for detailed analysis of centric heterochromatin structure and function.

Animals↗

PubMed1997

Insertion mutagenesis as a tool in the modification of protein function. Extended substrate specificity conferred by pentapeptide insertions in the omega-loop of TEM-1 beta-lactamase.

The TEM-1 beta-lactamase enzyme efficiently hydrolyzes beta-lactam antibiotics such as ampicillin but cleaves third generation cephalosporin antibiotics poorly. Variant beta-lactamases that conferred elevated levels of resistance to the cephalosporin ceftazidime were identified in a set of beta-lactamase derivatives previously generated by pentapeptide scanning mutagenesis in which a variable 5-amino acid cassette was introduced randomly in the target protein. This mutagenesis procedure was also modified to allow the direct selection of variant beta-lactamases with pentapeptide insertions that conferred extended substrate specificities. All insertions associated with enhanced resistance to ceftazidime were targetted to the 19-amino acid Omega-loop region, which forms part of the catalytic pocket of the beta-lactamase enzyme. However, pentapeptide insertions in the C- and N-terminal halves of this region had different effects on the ability of the enzyme to hydrolyze ampicillin in vivo. Larger insertions that increased the length of the Omega-loop by up to 2-fold also retained catalytic activity toward ampicillin and/or ceftazidime in vivo. In accord with previous substitution mutation studies, these results emphasize the extreme flexibility of the Omega-loop with regards the primary structure requirements for ceftazidime hydrolysis by beta-lactamase. The potential of pentapeptide scanning mutagenesis in mimicking evolution events that result from the insertion and excision of transposons in nature is discussed.

Amino Acid Sequence↗