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Results for “transposon sequencing”

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At least 145 records · Page 8Linked to original sources
PubMed1984

A mechanism that couples DNA transposition to cell differentiation (hypothesis).

A hypothesis underlying the significance of cytoplasmic events in the process of DNA transposition is proposed. To appear at a new chromosomal site, the transposon sequence is assumed to be first transcribed and then, in a form of the RNA copy, transported to the cytoplasm. A translation-dependent selection of RNA transcripts for reverse transcription is then made and the resulting single-stranded DNA copies return to the nucleus. The free copy may integrate within a new, transcriptionally inactive region of the chromosomal DNA. As a net result, cell differentiation advances in a manner coupled to the message utilization. The hypothesis finds support in many observations, particularly those made for plant cells.

Cell Differentiation↗
PubMed1983

Investigation of nosocomial infections by plasmid analysis.

Molecular biological techniques, including agarose gel electrophoresis, restriction endonuclease analysis and DNA-DNA hybridization (Southern blotting and heteroduplex analysis) are being applied very successfully to the investigation of nosocomial infections. 'Plasmid fingerprinting' by electrophoresis, with or without restriction endonuclease analysis, can be used to identify epidemic strains of bacteria and 'epidemic plasmids' which have spread through several different bacterial species. This technique is rapid and inexpensive, and can be applied to drug-sensitive as well as resistant strains. This approach is especially useful for investigation of organisms for which no standard typing system is available. DNA-DNA hybridization techniques can be used to study the evolution of plasmids in the hospital environment, and to demonstrate the presence and spread of translocatable DNA sequences (transposons) carrying drug resistance determinants from plasmid to plasmid within a bacterial cell.

Cross Infection↗
PubMedDate not supplied

DNA hybridization studies of a nucleotide sequence homologous to transposon Tn1545 in the "Minnesota" strain of multiresistant Streptococcus pneumoniae isolated in 1977.

Nucleotide sequences homologous to the transposon-specific region and the tetM determinant mediating tetracycline resistance of the multiresistance transposon Tn1545, originating in Streptococcus pneumoniae, were detected by hybridization in the first multiresistant pneumococcus reported in the United States. The sequences were localized to a common 25-kb EcoRI restriction fragment of the chromosome.

Autoradiography↗
PubMed2004

Transposon Express, a software application to report the identity of insertions obtained by comprehensive transposon mutagenesis of sequenced genomes: analysis of the preference for in vitro Tn5 transposition into GC-rich DNA.

Comprehensive mutant libraries can be readily constructed by transposon mutagenesis. To systematically mutagenise the genome of the Gram-positive bacterium Streptomyces coelicolor A3(2), we have employed high-throughput shuttle transposon mutagenesis of a cosmid library prepared in Escherichia coli. The location of transposon insertions is determined using automated procedures for cosmid isolation and DNA sequencing. However, a major bottleneck was the subsequent analysis of DNA sequence files. To overcome this limitation, a software application, Transposon Express, was written to allow the rapid location of transposon insertions in a sequenced genome (available at http://www.swan.ac.uk/genetics/dyson/InstallTE). Transposon Express determines the identity both of a disrupted open reading frame (ORF), and the short target site duplication created by transposition. Transposon Express also reports the orientation of the transposon and can therefore predict transcriptional coupling between an upstream promoter and a promoter-less reporter gene carried by the transposon. Analysis of a large dataset of independent insertions created using a Tn5-based transposon revealed an insertional preference for GC-rich streptomycete DNA compared to E.coli vector DNA. In addition to demonstrating the value of Transposon Express as a generic tool supporting genome-wide transposon mutagenesis programs, these data provide insight into target site selection by Tn5.

Computational Biology↗
PubMed2004

Post-integration stabilization of a transposon vector by terminal sequence deletion in Drosophila melanogaster.

Germline transformation systems for nearly 20 insect species have been derived from transposable elements, allowing the development of transgenic insects for basic and applied studies. These systems use a defective nonautonomous vector that results in stable vector integrations after the disappearance of transiently provided transposase helper plasmid, which is essential to maintain true breeding lines and consistent transgene expression that would otherwise be lost after vector remobilization. The risk of remobilization by an unintended transposase source has so far not been a concern for laboratory studies, but the prospective use of millions of transgenic insects in biocontrol programs will likely increase the risk, therefore making this a critical issue for the ecological safety of field release programs. Here we describe an efficient method that deletes a terminal repeat sequence of a transposon vector after genomic integration. This procedure prevents transposase-mediated remobilization of the other terminal sequence and associated genes, ensuring their genomic stability.

Animals↗
PubMed1991

A transposon-related palindromic repetitive sequence from C. elegans.

A family of transposon-like sequences in the C. elegans genome is described. This family, termed the Tc6 family, consists mostly of conserved, 1.6 kb elements. Four Tc6 elements or partial elements have been cloned and the DNA sequences of three were determined. One appears to be a complete element of 1603 nucleotides, consisting of a palindrome of 765 nucleotides, with a central, non-palindromic region of 73 nucleotides. Another has an identical structure except for an internal deletion. A third is a partial element terminating at a probable internal restriction site used for cloning. A fourth clone contained portions of the Tc6 sequence juxtaposed to non-Tc6 sequences. All C. elegans strains examined contain 20-30 Tc6 elements. The ends of Tc6 elements are conserved and have sequence similarity to the ends of C. elegans transposons Tc1 and Tc3. The ends of Tc6 elements also have sequence similarity to the heptamer portion of the immunoglobulin and T-cell receptor recombination signal sequence, raising the possibility of wide phylogenetic conservation of the recombination mechanism. Tc6 elements also share sequence motifs with plant-pathogenic viroid RNA's, possibly indicative of a Tc6 RNA replicative phase.

Animals↗
PubMed1994

Deletion and transposon mutagenesis and sequence analysis of the pRO1600 OriR region found in the broad-host-range plasmids of the pQF series.

The nucleotide sequence of the replicative origin (OriR) region of the small cryptic broad-host-range plasmid, pRO1600, which forms the basis of a number of useful cloning vectors has been determined. In addition it has been subjected to Tn5 mutagenesis, deletion analysis, and subcloning in order to define the regions essential for replication in Pseudomonas aeruginosa. The sequence (1894 bp) contains a fragment derived from transposon Tn1. The OriR region is structurally related to other replication (Rep) protein-dependent origins in that it has an A-T-rich region upstream of four 17-bp direct repeats (iterons) which presumably function in initiator protein binding. The sequence also contains a DNA-A-binding site and an open reading frame which could encode a basic (pI 10.6) 25,343-Da Rep protein with homology to RepA from the Neisseria gonorrhoeae beta-lactamase plasmid pFA3. The possible evolutionary origin of this plasmid in P. aeruginosa (RP1) is discussed.

Amino Acid Sequence↗
PubMed1985

A rapid procedure for DNA sequencing using transposon-promoted deletions in Escherichia coli.

A simple procedure has been developed for sequencing long fragments of DNA. The fragment (which can be several kb in length) is cloned in pAA3.7X, and subdivided into many overlapping segments by Tn9-promoted deletions. The deletions are isolated by positive selection for galactose resistance. A rapid plasmid preparation from several hundred galactose-resistant colonies is fractionated by agarose gel electrophoresis to pick a series of deletions terminating at approx. 200-bp intervals across the entire length of the fragment. Selected plasmids are purified by rapid alkaline extraction, and used directly for supercoil sequencing with a primer derived from IS1. Sequences of adjacent deletions contain overlaps which are used to connect individual sequences to give the complete sequence.

Base Sequence↗
PubMed1987

Characterization of the terminal sequences flanking the transposon that carries the Escherichia coli enterotoxin STII gene.

The Escherichia coli enterotoxin STII gene is flanked by two repeat sequences, approx. 600 bp each and 8 kb apart. This 9-kb DNA fragment has been shown to transpose as a unit and is thus considered a transposon. It is presently designated as Tn4521. In this study, the two terminal sequences of Tn4521 cloned in pPS1 were localized, isolated, and characterized. The two terminal sequences were found to be composed of IS2 sequences and were in an inverted repeat orientation. However, neither repeat contained a complete IS2. The LTR contained bp 1-722, whereas the RTR contained bp 17-536 and 969-1327, all three of the IS2 sequence.

Bacterial Toxins↗
PubMed1988

Mutational analysis of insertion sequence 50 (IS50) and transposon 5 (Tn5) ends.

Insertion sequence 50 (IS50) transposition utilizes a 19-base-pair "outside" end and a 19-base-pair "inside" end in inverted orientation relative to each other, whereas transposon 5 (Tn5) transposition utilizes two inverted outside ends. The frequency of transposition events that involve an inside end is regulated 1000-fold by the host dam methylase system. The end sequence requirements for transposition and its regulation by dam methylase were analyzed in Escherichia coli by generating random single base pair mutations in either an IS50 inside end or outside end placed in inverted orientation with respect to an unmutagenized outside end. The mutations were then isolated, assayed for transposition phenotype, and sequenced. Mutations were isolated at 15 of the 19 sites in the outside end. All of these mutations except those at position 4 decreased transposition. Mutations at position 4 (which is the only nonidentical base pair in a region of homology between the outside and inside ends) had no effect on transposition. Mutations were isolated at 11 of the 19 sites in the inside end. All of these mutations, including one at position 4, decreased transposition in dam- cells. Mutations at position 10 (within a dam recognition sequence) and 2 (not within a dam recognition sequence) reduced the magnitude of dam regulation. A mutation within a dam recognition sequence adjacent to the required 19 base pairs of the inside end did not reduce the magnitude of dam regulation.

Bacterial Proteins↗
PubMed1997

Delta sequence of Ty1 transposon can initiate transcription of the distal part of the URA2 gene complex in Saccharomyces cerevisiae.

Expression of a silent aspartate transcarbamylase (ATCase) domain can occur by insertion of a Tyl retrotransposon within the coding sequence of a mutated ura2 allele. This unusual type of Ty-mediated gene activation is possible as the URA2 gene product is a multifunctional protein containing the carbamoyl phosphate synthetase (CPSase), the ATCase and a cryptic dihydroorotase (DHOase) domain. The region in which transcription of the corresponding allele is initiated was determined by RT-PCR experiments. Expression is initiated by a sequence located in the delta element of the Tyl and not by a sequence of the URA2 gene itself. This situation differs with the Ty-mediated gene activation described thus far, in which the transposon substitutes only the 5' regulatory sequences and in which the normal transcription start point is used. The corresponding protein carries both the DHOase-like domain and the ATCase domain, suggesting that the DHOase-like domain is at least involved in the architecture of the protein and necessary to render the ATCase domain functional.

Alleles↗
PubMed1990

A single oligonucleotide can be used to rapidly isolate DNA sequences flanking a transposon Tn5 insertion by the polymerase chain reaction.

We have developed a strategy to rapidly construct DNA hybridization probes for the isolation of genes disrupted by transposon Tn5 insertions. A single oligonucleotide complementary to and extending outward from the ends of the inverted repeat of Tn5 was used to prime DNA synthesis in the polymerase chain reaction. The amplified product consisted of DNA sequences adjacent to both ends of the transposon insertion. The general feasibility of the approach was tested by amplifying pBR322 sequences from a derivative of pBR322 containing a Tn5 insertion. To amplify genomic DNA sequences flanking a Tn5 insertion in the chromosome of a Pseudomonas syringae strain, circular substrates were generated by ligating EcoRI-digested genomic DNA. Tn5 was contained intact within one such circular molecule, as the transposon does not contain sites for cleavage by EcoRI. The amplified product (approximately 2.5 kb) was used as a DNA hybridization probe to isolate the homologous fragment from a cosmid library of wild-type Pseudomonas syringae genomic DNA. This approach may be applied to the efficient isolation of sequences flanking any Tn5 insertion.

Base Sequence↗
PubMed1996

Typing of Staphylococcus aureus by PCR for DNA sequences flanked by transposon Tn916 target region and ribosomal binding site.

The continuous intra- and interhospital spread of multiresistant Staphylococcus aureus demands a rapid molecular typing system. This study describes the fingerprinting of S. aureus by PCR amplification of DNA sequences flanked by the target site for transposon Tn916 and the ribosomal binding site and neighboring nucleotides (target 916-Shine-Dalgarno PCR [tar 916-shida PCR]). Both starting points for PCR are known to be randomly distributed on the S. aureus chromosome. By use of SmaI-macrorestriction patterns as the reference method it was shown that this PCR genotyping discriminates among strains of the major clonal groups of the species S. aureus (strains with phage patterns 29, +, 94, 96, and 95 as well as group II and group III patterns) and identifies the six epidemic methicillin-resistant S. aureus strains prevalent in German hospitals. All of the investigated strains including methicillin-sensitive. S. aureus were typeable. Tar 916-shida patterns are stable during the dissemination of epidemic methicillin-resistant S. aureus among different hospitals.

Bacterial Typing Techniques↗
PubMed1997

Rapid amplification of uncharacterized transposon-tagged DNA sequences from genomic DNA.

Although the entire DNA sequence of the yeast genome has been determined, the functions of nearly a third of the identified genes are unknown. Recently, we described a collection of mutants, each with a transposon-tagged disruption in an essential gene in Saccharomyces cerevisiae. Identification of these essential genes and characterization of their mutant phenotypes should help assign functions to these thousands of novel genes, and since each mutation in our collection is physically marked by the uniform, unique DNA sequence of the transposable element, it should be possible to use the polymerase chain reaction (PCR) to amplify the DNA adjacent to the transposon. However, existing PCR methods include steps that make their use on a large scale cumbersome. In this report, we describe a semi-random, two-step PCR protocol, ST-PCR. This method is simpler and more specific than current methods, requiring only genomic DNA and two pairs of PCR primers, and involving two successive PCR reactions. Using this method, we have rapidly and easily identified the essential genes identified by several of our mutants.

Cloning, Molecular↗
PubMed1994

Hypertransposing derivatives of the streptomycete insertion sequence IS493.

Transposons derived from the Streptomyces lividans insertion sequence IS493 are useful for the genetic analysis and manipulation of a number of Streptomyces spp. Tn5099-10, an IS493 derivative that contains a spontaneous deletion terminating in the left inverted repeat (IR-L), transposed at a 1000-fold higher frequency in Streptomyces griseofuscus, and at a tenfold higher frequency in Streptomyces fradiae, than the IS493 derivatives, Tn5096 and Tn5099. The IR-L from Tn5099-10 was used to construct a cassette which hypertransposes from plasmids containing the transposon genes, ORFA and ORFB, outside of the inverted repeats. The target sequences of two Tn5099-10 insertions conformed to the consensus target sequence of the other IS493 derivatives, gNCaNTgNNy (where lower-case letters indicate that other nt have been observed at this position and N is any nt). Transposition mutant libraries of S. griseofuscus and S. fradiae can be easily prepared in broth culture by using the hypertransposing elements and a temperature-sensitive delivery plasmid.

Base Sequence↗
PubMed1983

Early differential tissue expression of transposon-like repetitive DNA sequences of the mouse.

Another family of long moderately repetitive and dispersed sequences has been identified in the mouse genome. These sequences have a transposon-like structure. A 6-kilobase RNA transcript is detected in undifferentiated embryonal carcinoma cell lines but not in any of the differentiated cell types tested. By R-loop formation, the RNA is colinear with a DNA fragment from a randomly selected genomic clone.

Animals↗