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Results for “transposon insertion sequencing”

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PubMed1982

A symmetrical six-base-pair target site sequence determines Tn10 insertion specificity.

Transposon Tn10 inserts at many sites in the bacterial chromosome, but preferentially inserts at particular hotspots. We believe we have identified the target DNA signal responsible for this specificity. We have determined the DNA sequences of 11 Tn10 insertion sites and identified a particular 6 base pair (bp) symmetrical consensus sequence (GCTNAGC) common to those sites. The sequences at some sites differ from the consensus sequence but only in limited and well defined ways. The sequences at some sites differ from the consensus sequence than do sequences at other sites, and the consensus sequence and closely related sequences are generally absent from potential target regions where Tn10 is known not to insert. Other aspects of the target DNA can significantly influence the efficiency with which a particular target site sequence is used. The 6 bp consensus sequence is symmetrically located within the 9 bp target DNA sequence that is cleaved and duplicated during Tn10 insertion. This juxtaposition of recognition and cleavage sites plus the symmetry of the perfect consensus sequence suggest that the target DNA may be both recognized and cleaved by the symmetrically disposed subunits of a single protein, as suggested for type II restriction endonucleases. There is plausible homology between the consensus sequence and the very ends of Tn10, compatible with recognition of transposon ends and target DNA by the same protein. The sequences of actual insertion sites deviate from the perfect consensus sequence in a way which suggests that the 6 bp specificity determinant may be recognized through protein-DNA contacts along the major groove of the DNA double helix.

Base Sequence↗

PubMed1991

Chlorobenzoate catabolic transposon Tn5271 is a composite class I element with flanking class II insertion sequences.

The structure of a transposon specifying the biodegradation of chlorobenzoate contaminants is described. Tn5271 is a 17-kilobase (kb) transposon that resides in the plasmid or chromosome of Alcaligenes sp. strain BR60 and allows this organism to grow on 3- and 4-chlorobenzoate. The transposon is flanked by a directly repeated sequence of 3201 base pairs (bp), which in turn is flanked by 110-bp inverted repeats. The 3.2-kb repeated sequence, designated IS1071, exists in multiple copies in the genome of Alcaligenes sp. strain BR60 and is involved in recombination of the catabolic genes into the chromosome of this strain. Sequence analysis revealed that the inverted repeat of IS1071 and the derived amino acid sequence of the single open reading frame within IS1071 are related to the inverted repeats and transposase (TnpA) proteins of the class II (Tn3 family) transposable elements. The absence of a resolvase gene within IS1071 suggests that this element is capable of determining the first step in class II transposition only. This was confirmed by observations on the IS1071-dependent formation of stable cointegrates in a recombination-deficient Escherichia coli. These results support an evolutionary scheme in which the class II transposable elements descended from simple insertion sequences.

Alcaligenes↗

PubMed1987

Identification of the structural gene encoding the SH-activated hemolysin of Listeria monocytogenes: listeriolysin O is homologous to streptolysin O and pneumolysin.

By immunoblotting with an antiserum raised against purified listeriolysin O, we have detected the presence of a truncated protein of 52 kilodaltons in culture supernatants of a Tn1545-induced nonhemolytic mutant of Listeria monocytogenes (J.L. Gaillard, P. Berche, and P. Sansonetti, Infect. Immun. 52:50-55, 1986). The region of insertion of the transposon has been cloned and sequenced. The transposon had inserted in an open reading frame the listeriolysin O gene. The deduced amino acid sequence of this open reading frame revealed that listeriolysin O is homologous to streptolysin O and pneumolysin, although homologies were not detectable at the DNA level.

Bacterial Proteins↗

PubMed1988

Nucleotide sequence of insertion sequence IS3411, which flanks the citrate utilization determinant of transposon Tn3411.

The nucleotide sequences of insertion sequences IS3411L (left) and IS3411R (right), present as direct terminal repeats in the citrate utilization of citrate utilization transposon Tn3411, and of IS3411 (generated by intramolecular recombination between IS3411L and IS3411R) were determined. The three IS3411 elements (IS3411R, IS3411L, and IS3411) were 1,309 base pairs long and identical in DNA sequence. IS3411 had 27-base-pair terminal inverted repeats with three bases mismatched and one long open reading frame (240 amino acids) that was proposed to be a transposase. Three polypeptides of 29,000, 27,000, and about 10,000 molecular weight, determined by IS3411, were identified in minicells. Since Tn3411 generates a 3-base-pair repeat upon integration, the nucleotide sequences of IS3411 were compared with those of IS3.

Amino Acid Sequence↗

PubMed2006

Remarkable site specificity of local transposition into the Hsp70 promoter of Drosophila melanogaster.

Heat-shock genes have numerous features that ought to predispose them to insertional mutagenesis via transposition. To elucidate the evolvability of heat-shock genes via transposition, we have exploited a local transposition technique and Drosophila melanogaster strains with EPgy2 insertions near the Hsp70 gene cluster at 87A7 to produce numerous novel EPgy2 insertions into these Hsp70 genes. More than 50% of 45 independent insertions were made into two adjacent nucleotides in the proximal promoter at positions -96 and -97, and no insertions were into a coding or 3'-flanking sequence. All inserted transposons were in inverse orientation to the starting transposon. The frequent insertion into nucleotides -96 and -97 is consistent with the DNase hypersensitivity, absence of nucleosomes, flanking GAGA-factor-binding sites, and nucleotide sequence of this region. These experimental insertions recapitulated many of the phenotypes of natural transposition into Hsp70: reduced mRNA expression, less Hsp70 protein, and decreased inducible thermotolerance. The results suggest that the distinctive features of heat-shock promoters, which underlie the massive and rapid expression of heat-shock genes upon heat shock, also are a source of evolutionary variation on which natural selection can act.

Animals↗

PubMed2004

Merlin, a new superfamily of DNA transposons identified in diverse animal genomes and related to bacterial IS1016 insertion sequences.

Several new families of DNA transposons were identified by computer-assisted searches in a wide range of animal species that includes nematodes, flat worms, mosquitoes, sea squirt, zebrafish, and humans. Many of these elements have coding capacity for transposases, which are related to each other and to those encoded by the IS1016 group of bacterial insertion sequences. Although these transposases display a motif similar to the DDE motif found in many transposases and integrases, they cannot be directly allied to any of the previously described eukaryotic transposases. Other common features of the new eukaryotic and bacterial transposons include similarities in their terminal inverted repeats and 8-bp or 9-bp target-site duplications. Together, these data indicate that these elements belong to a new superfamily of DNA transposons, called Merlin/IS1016, which is common in many eubacterial and animal genomes. We also present evidence that these transposons have been recently active in several animal species. This evidence is particularly strong in the parasitic blood fluke Schistosoma mansoni, in which Merlin is also the first described DNA transposon family.

Animals↗

PubMed2000

Analysis of a clonal selection event during transposon-mediated nested-deletion formation in rare BAC and PAC clones.

Nested deletions from one end of the genomic DNA in bacterial artificial chromosomes (BACs) and P1 artificial chromosomes (PACs) are readily generated by inserting a loxP site-containing Tn10 minitransposon into the recombinant clone and transducing with P1 phage. Although the size of clones in the deletion series is largely random, in about 5% of BACs and PACs the distribution appears skewed to a certain length, and in rare cases (<1%) is definitely skewed to a particular size. Here we investigate this relatively rare phenomenon and validate that sequence-specific transposon insertions are not the cause of such skewed nested-deletion libraries. Instead, a detailed analysis of our experiments with a BAC clone demonstrating this unusual feature indicates that deletions of a certain size arise from clonal expansion of a transposon insertion as a result of transient derepression of the transposase gene prior to IPTG induction. Transposition itself shows no bias to any particular region of insert DNA in the clone. We suggest a simple modification to the procedure for generating nested-deletions that allows all BACs and PACs to produce nested-deletions of random size. These findings should provide additional insight into the causes of site selectivity in genomic clones with other inducible transposon systems.

Chromosomes, Artificial↗

PubMed1986

Shuttle mutagenesis: a method of transposon mutagenesis for Saccharomyces cerevisiae.

We have extended the method of transposon mutagenesis to the eukaryote, Saccharomyces cerevisiae. A bacterial transposon containing a selectable yeast gene can be transposed into a cloned fragment of yeast DNA in Escherichia coli, and the transposon insertion can be returned to the yeast genome by homologous recombination. Initially, the cloned yeast DNA fragment to be mutagenized was transformed into an E. coli strain containing an F factor derivative carrying the transposable element. The culture was grown to allow transposition and cointegrate formation and, upon conjugation, recipients were selected that contained yeast sequences with transposon insertions. The yeast DNA was removed from the vector by restriction endonuclease digestion, and the transposon insertion was transformed into yeast. The procedure required a minimum number of manipulations, and each transconjugant colony contained an independent insertion. We describe 12 transposon Tn3 derivatives for this procedure as well as several cloning vectors to facilitate the method.

Cloning, Molecular↗

PubMed1995

Four genes, two ends, and a res region are involved in transposition of Tn5053: a paradigm for a novel family of transposons carrying either a mer operon or an integron.

The complete nucleotide sequence of an 8447 bp-long mercury-resistance transposon (Tn5053) has been determined. Tn5053 is composed of two modules: (i) the mercury-resistance module and (ii) the transposition module. The mercury-resistance module carries a mer operon, merRTPFAD, and appears to be a single-ended relic of a transposon closely related to the classical mercury-resistance transposons Tn21 and Tn501. The transposition module of Tn5053 is bounded by 25 bp terminal inverted repeats and contains four genes involved in transposition, i.e. tniA, tniB, tniQ, and tniR. Transposition of Tn5053 occurs via cointegrate formation mediated by the products of the tniABQ genes, followed by site-specific cointegrate resolution. This is catalysed by the product of the tniR gene at the res region, which is located upstream of tniR. The same pathway of transposition is used by Tn402 (Tn5090) which carries the integron of R751. Transposition genes of Tn5053 and Tn402 are interchangeable. Sequence analysis suggests that Tn5053 and Tn402 are representatives of a new family of transposable elements, which fall into a recently recognized super-family of transposons including retroviruses, insertion sequences of the IS3 family, and transposons Tn552 and Tn7. We suggest that the tni genes were involved in the dissemination of integrons.

Amino Acid Sequence↗

PubMed2005

Phylogenetic analysis of the DNA polymerase gene of a novel alphaherpesvirus isolated from an Indian Gyps vulture.

The DNA polymerase gene of a novel herpesvirus, vulture herpesvirus (VHV), isolated from an Indian Gyps vulture was completely sequenced using primer walking and transposon insertion strategies. DNA sequencing analysis revealed a single open reading frame (ORF) of 3660 nucleotides (53% G-C content) able to encode 1219 amino acids. Identification was based on a nucleotide sequence identity of approximately 50% to other herpesvirus sequences found in Genbank. Nine motifs were identified that are conserved amongst all known herpesviruses and are found within the 3'-5' exonuclease and DNA binding functional domains of the DNA polymerase enzyme. Phylogenetic analysis using Clustal W with neighbour-joining revealed VHV to group within the subfamily Alphaherpesvirinae, more closely related to the avian herpesviruses than to those of other species. Partial sequence data also revealed VHV to contain other genes fundamental to the structure and replication of all herpesvirus genomes. A Real Time PCR Taqman assay specific for the VHV DNA polymerase gene was designed to detect the presence of VHV genomic material in post mortem tissue samples from diseased birds. Positive tissues included the spleen, rectum, thymus, kidney and brain. A herpesvirus specific to vultures may pose a threat to the management of captive breeding programs being established to assist the survival of wild populations of Gyps vultures.

Alphaherpesvirinae↗

PubMed1998

A cluster of genes involved in polysaccharide biosynthesis from Enterococcus faecalis OG1RF.

Our previous work identified a cosmid clone containing a 43-kb insert from Enterococcus faecalis OG1RF that produced a nonprotein antigen in Escherichia coli. In the present work, we studied this clone in detail. Periodate treatment of lysates of the clone confirmed that the antigen was carbohydrate in nature. Analysis of DNA sequences and transposon insertion mutants suggested that the insert contained a multicistronic gene cluster. Database comparison showed that the cluster contained genes similar to genes involved in the biosynthesis of dTDP-rhamnose, glycosyltransferases, and ABC transporters involved in the export of sugar polymers from both gram-positive and gram-negative organisms. Insertions in several genes within the cluster abolished the immunoreactivity of the clone. This is the first report on a gene cluster of E. faecalis involved in the biosynthesis of an antigenic polysaccharide.

Antigens, Bacterial↗

PubMed2003

IS Rm31, a new insertion sequence of the IS 66 family in Sinorhizobium meliloti.

Sinorhizobium meliloti natural populations show a high level of genetic polymorphism possibly due to the presence of mobile genetic elements such as insertion sequences (IS), transposons, and bacterial mobile introns. The analysis of the DNA sequence polymorphism of the nod region of S. meliloti p SymA megaplasmid in an Italian isolate led to the discovery of a new insertion sequence, IS Rm31. IS Rm31 is 2,803 bp long and has 22-bp-long terminal inverted repeat sequences, 8-bp direct repeat sequences generated by transposition, and three ORFs (A, B, C) coding for proteins of 124, 115, and 541 amino acids, respectively. ORF A and ORF C are significantly similar to members of the transposase family. Amino acid and nucleotide sequences indicate that IS Rm31 is a member of the IS 66 family. IS Rm31 sequences were found in 30.5% of the Italian strains analyzed, and were also present in several collection strains of the Rhizobiaceae family, including S. meliloti strain 1021. Alignment of targets sites in the genome of strains carrying IS Rm31 suggested that IS Rm31 inserts randomly into S. meliloti genomes. Moreover, analysis of IS Rm31 insertion sites revealed DNA sequences not present in the recently sequenced S. meliloti strain 1021 genome. In fact, IS Rm31 was in some cases linked to DNA fragments homologous to sequences found in other rhizobia species.

Base Composition↗

PubMed2000

Transposon insertions in the Flavobacterium johnsoniae ftsX gene disrupt gliding motility and cell division.

Flavobacterium johnsoniae is a gram-negative bacterium that exhibits gliding motility. To determine the mechanism of flavobacterial gliding motility, we isolated 33 nongliding mutants by Tn4351 mutagenesis. Seventeen of these mutants exhibited filamentous cell morphology. The region of DNA surrounding the transposon insertion in the filamentous mutant CJ101-207 was cloned and sequenced. The transposon was inserted in a gene that was similar to Escherichia coli ftsX. Two of the remaining 16 filamentous mutants also carried insertions in ftsX. Introduction of the wild-type F. johnsoniae ftsX gene restored motility and normal cell morphology to each of the three ftsX mutants. CJ101-207 appears to be blocked at a late stage of cell division, since the filaments produced cross walls but cells failed to separate. In E. coli, FtsX is thought to function with FtsE in translocating proteins involved in potassium transport, and perhaps proteins involved in cell division, into the cytoplasmic membrane. Mutations in F. johnsoniae ftsX may prevent translocation of proteins involved in cell division and proteins involved in gliding motility into the cytoplasmic membrane, thus resulting in defects in both processes. Alternatively, the loss of gliding motility may be an indirect result of the defect in cell division. The inability to complete cell division may alter the cell architecture and disrupt gliding motility by preventing the synthesis, assembly, or functioning of the motility apparatus.

Amino Acid Sequence↗

PubMed2003

Comprehensive transposon mutant library of Pseudomonas aeruginosa.

We have developed technologies for creating saturating libraries of sequence-defined transposon insertion mutants in which each strain is maintained. Phenotypic analysis of such libraries should provide a virtually complete identification of nonessential genes required for any process for which a suitable screen can be devised. The approach was applied to Pseudomonas aeruginosa, an opportunistic pathogen with a 6.3-Mbp genome. The library that was generated consists of 30,100 sequence-defined mutants, corresponding to an average of five insertions per gene. About 12% of the predicted genes of this organism lacked insertions; many of these genes are likely to be essential for growth on rich media. Based on statistical analyses and bioinformatic comparison to known essential genes in E. coli, we estimate that the actual number of essential genes is 300-400. Screening the collection for strains defective in two defined multigenic processes (twitching motility and prototrophic growth) identified mutants corresponding to nearly all genes expected from earlier studies. Thus, phenotypic analysis of the collection may produce essentially complete lists of genes required for diverse biological activities. The transposons used to generate the mutant collection have added features that should facilitate downstream studies of gene expression, protein localization, epistasis, and chromosome engineering.

Escherichia coli↗

PubMed2005

piggyBac internal sequences are necessary for efficient transformation of target genomes.

A previously reported piggyBac minimal sequence cartridge, which is capable of efficient transposition in embryo interplasmid transposition assays, failed to produce transformants at a significant frequency in Drosophila melanogaster compared with full-length or less extensive internal deletion constructs. We have re-examined the importance of these internal domain (ID) sequences for germline transformation using a PCR strategy that effectively adds increasing lengths of ID sequences to each terminus. A series of these piggyBac ID synthetic deletion plasmids containing the 3xP3-ECFP marker gene are compared for germline transformation of D. melanogaster. Our analyses identify a minimal sequence configuration that is sufficient for movement of piggyBac vectored sequences from plasmids into the insect genome. Southern hybridizations confirm the presence of the piggyBac transposon sequences, and insertion site analyses confirm these integrations target TTAA sites. The results verify that ID sequences adjacent to the 5' and 3' terminal repeat domains are crucial for effective germline transformation with piggyBac even though they are not required for excision or interplasmid transposition. Using this information we reconstructed an inverted repeat cartridge, ITR1.1k, and a minimal piggyBac transposon vector, pXL-BacII-ECFP, each of which contains these identified ID sequences in addition to the terminal repeat configuration previously described as essential for mobility. We confirm in independent experiments that these new minimal constructs yield transformation frequencies similar to the control piggyBac vector. Sequencing analyses of our constructs verify the position and the source of a point mutation within the 3' internal repeat sequence of our vectors that has no apparent effect on transformation efficiency.

Animals↗

PubMed1995

Meiotic recombination break points resolve at high rates at the 5' end of a maize coding sequence.

Sequence analysis of recombination break points has defined a 377-bp recombination hot spot within the anthocyanin 1 (a1) gene. One-fifth of all recombination events that occurred within the 140-kb a1-shrunken 2 interval resolved within this 377-bp hot spot. In yeast, meiotic double-strand breaks in chromosomal DNA are thought to initiate recombination and are generally located 5' of coding regions, near transcription promoter sequences. Because the a1 recombination hot spot is located within the 5' transcribed region of the a1 gene, the sites at which recombination events initiate and resolve appear to be different, but both appear to be regulated in relation to transcribed sequences. Although transposon insertions are known to suppress recombination and alter the ratio of crossovers to apparent gene conversions, the Mutator 1 transposon insertion in the a1-mum2 allele does not alter the sites at which recombination events resolve.

Alleles↗

PubMed2001

Genetic organization of the region encoding regulation, biosynthesis, and transport of rhizobactin 1021, a siderophore produced by Sinorhizobium meliloti.

Eight genes have been identified that function in the regulation, biosynthesis, and transport of rhizobactin 1021, a hydroxamate siderophore produced under iron stress by Sinorhizobium meliloti. The genes were sequenced, and transposon insertion mutants were constructed for phenotypic analysis. Six of the genes, named rhbABCDEF, function in the biosynthesis of the siderophore and were shown to constitute an operon that is repressed under iron-replete conditions. Another gene in the cluster, named rhtA, encodes the outer membrane receptor protein for rhizobactin 1021. It was shown to be regulated by iron and to encode a product having 61% similarity to IutA, the outer membrane receptor for aerobactin. Transcription of both the rhbABCDEF operon and the rhtA gene was found to be positively regulated by the product of the eighth gene in the cluster, named rhrA, which has characteristics of an AraC-type transcriptional activator. The six genes in the rhbABCDEF operon have interesting gene junctions with short base overlaps existing between the genes. Similarities between the protein products of the biosynthesis genes and other proteins suggest that rhizobactin 1021 is synthesized by the formation of a novel siderophore precursor, 1,3-diaminopropane, which is then modified and attached to citrate in steps resembling those of the aerobactin biosynthetic pathway. The cluster of genes is located on the pSyma megaplasmid of S. meliloti 2011. Reverse transcription-PCR with RNA isolated from mature alfalfa nodules yielded no products for rhbF or rhtA at a time when the nifH gene was strongly expressed, indicating that siderophore biosynthesis and transport genes are not strongly expressed when nitrogenase is being formed in root nodules. Mutants having transposon insertions in the biosynthesis or transport genes induced effective nitrogen-fixing nodules on alfalfa plants.

Amino Acid Sequence↗

PubMed2025

SegMantX: A Novel Tool for Detecting DNA Duplications Uncovers Prevalent Duplications in Plasmids.

Segmental duplications play an important role in genome evolution via their contribution to copy-number variation, gene-family diversification, and the emergence of novel functions. The detection of segmental duplications is challenging due to heterogeneous amelioration of sequence similarity among duplicates, which hinders the reconstruction of continuous sequence alignment. Here we introduce SegMantX, a novel approach for the identification of diverged segmental duplications in prokaryote genomes using local alignment chaining. In this approach, local alignments resulting from a preliminary sequence similarity search (e.g. BLASTn) are chained into continuous segments. Evaluating the performance of SegMantX using simulated sequences shows that the tool can detect diverged duplications beyond the sensitivity limits of standard alignment-based methods. Applying SegMantX to 6,784 enterobacterial plasmids, we find that 65% plasmids contain duplicated regions and gene duplications, most of which correspond either to dispersed, noncoding regions or duplicated mobile genetic elements (MGEs; e.g. transposons and insertion sequences). Furthermore, we demonstrate the applicability of SegMantX for the identification of diverged gene transfers between replicons and plasmid hybridization events. Our findings highlight MGEs as drivers of segmental duplications in plasmid evolution, leading to the amplification of their cargo genes, including antibiotic resistance genes. SegMantX provides a powerful framework for reconstructing diverged segmental duplications and other alignment problems.

Plasmids↗