Search PubMed⌕ Search

SEARCH · Search PubMed

Results for “Transposons”

Search indexed PubMed citations on genomics, clinical trials, systematic reviews and public health. Explore titles, authors and supplied subject terms, then open the PubMed record.

Quote a phrase for an exact phrase match. Source license links do not imply unrestricted reuse.

At least 127 records · Page 7Linked to original sources
PubMed1989

New derivatives of transposon Tn5 suitable for mobilization of replicons, generation of operon fusions and induction of genes in gram-negative bacteria.

Three types of new variants of the broad-host-range transposon Tn5 are described. (i) Tn5-mob derivatives with the new selective resistance (R) markers GmR, SpR and TcR facilitate the efficient mobilization of replicons within a wide range of Gram-negative bacteria. (ii) Promoter probe transposons carry the promoterless reporter genes lacZ, nptII, or luc, and NmR, GmR or TcR as selective markers. These transposons can be used to generate transcriptional fusions upon insertion, thus facilitating accurate determinations of gene expression. (iii) Tn5-P-out derivatives carry the npt- or tac-promoter reading out from the transposon, and TcR, NmR or GmR genes. These variants allow the constitutive expression of downstream genes. The new Tn5 variants are available on mobilizable Escherichia coli vectors suitable as suicidal carriers for transposon mutagenesis of non-E. coli recipients and some on a phage lambda mutant to be used for transposon mutagenesis in E. coli.

Base Sequence↗
PubMed1991

Transposon vectors for stable chromosomal integration of cloned genes in rhizosphere bacteria.

A series of Tn5-based transposon-cloning vectors, in which many unique restriction sites lie within the transposon, have been constructed. These transposon vectors can be delivered, by conjugation, using a delivery vehicle containing a pBR322 replicon and the mobilization genes of plasmid RK2. In Pseudomonas sp., this delivery vehicle acts as a suicide plasmid, permitting transposition to the chromosome to be detected. To facilitate cloning into the transposon vector, the delivery vehicle has been simplified so that the useful cloning sites in the transposon are not duplicated. As a model system for the transposition of cloned genes, the xylE (coding for catechol-2,3-dioxygenase) has been transposed to a variety of Pseudomonads. The transposon vectors should be useful when a stable single copy of a cloned gene is desired. They should be particularly advantageous for the genetic engineering of soil bacteria for environmental studies (agriculture or pollution control) where the stability of the engineered strains, in the absence of continuous antibiotic selection, may be important.

Blotting, Southern↗
PubMedDate not supplied

Transposition in prokaryotes: transposon Tn501.

Bacteria contain a large number of transposable elements that can be categorized in four major groups according to their mechanisms of transposition. These are: class I: insertion sequences (IS) and compound transposons (with IS sequences at their termini) which usually require only one protein for transposition to occur (e.g. Tn10); class II: complex transposons and insertion sequences with short inverted repeats in which transposition is replicative and requires two gene products (e.g. Tn3); class III: transposable bacteriophage (e.g. Mu). The fourth group consists of the transposons and IS of variable mechanism, which do not fall into the above classes (e.g. Tn7). We have studied the mechanism of transposition of Tn501 and Tn21, closely-related class II mercury-resistance transposons, which transpose via a cointegrate intermediate. By using genetic methods, we have shown that the region of the 989 amino acid transposase between amino acids 57 and 186 determines the specificity for recognition of the 38-bp terminal inverted repeats of the transposon in normal transposition and for replicon fusion catalysed by a single transposon terminus. The Tn501 transposase has been over-expressed and is functional in vivo, raising the frequency of transposition approximately 10(4)-fold.

Amino Acid Sequence↗
PubMed2005

Cloning of a CACTA transposon-like insertion in intron I of tomato invertase Lin5 gene and identification of transposase-like sequences of Solanaceae species.

Very few CACTA transposon-like sequences have been described in Solanaceae species. Sequence information has been restricted to partial transposase (TPase)-like fragments, and no target gene of CACTA-like transposon insertion has been described in tomato to date. In this manuscript, we report on a CACTA transposon-like insertion in intron I of tomato (Lycopersicon esculentum) invertase gene Lin5 and TPase-like sequences of several Solanaceae species. Consensus primers deduced from the TPase region of the tomato CACTA transposon-like element allowed the amplification of similar sequences from various Solanaceae species of different subfamilies including Solaneae (Solanum tuberosum), Cestreae (Nicotiana tabacum) and Datureae (Datura stramonium). This demonstrates the ubiquitous presence of CACTA-like elements in Solanaceae genomes. The obtained partial sequences are highly conserved, and allow further detection and detailed analysis of CACTA-like transposons throughout Solanaceae species. CACTA-like transposon sequences make possible the evaluation of their use for genome analysis, functional studies of genes and the evolutionary relationships between plant species.

Amino Acid Sequence↗
PubMed2005

Sleeping beauty transposon-mediated gene therapy for prolonged expression.

The Sleeping Beauty (SB) transposon system represents a new vector for non-viral gene transfer that melds advantages of viruses and other forms of naked DNA transfer. The transposon itself is comprised of two inverted terminal repeats of about 340 base pairs each. The SB system directs precise transfer of specific constructs from a donor plasmid into a mammalian chromosome. The excision of the transposon from a donor plasmid and integration into a chromosomal site is mediated by Sleeping Beauty transposase, which can be delivered to cells vita its gene or its mRNA. As a result of its integration in chromosomes, and its lack of viral sequences that are often detected by poorly understood cellular defense mechanisms, a gene in a chromosomally integrated transposon can be expressed over the lifetime of a cell. SB transposons integrate nearly randomly into chromosomes at TA-dinucleotide base pairs although the sequences flanking the TAs can influence the probability of integration at a given site. Although random integration of vectors into human genomes is often thought to raise significant safety issues, evidence to date does not indicate that random insertions of SB transposons represent risks that are equal to those of viral vectors. Here we review the activities of the SB system in mice used as a model for human gene therapy, methods of delivery of the SB system, and its efficacy in ameliorating disorders that model human disease.

Animals↗
PubMed2003

A genome-wide screen identifies 27 genes involved in transposon silencing in C. elegans.

Transposon jumps are a major cause of genome instability. In the C. elegans strain Bristol N2, transposons are active in somatic cells, but they are silenced in the germline, presumably to protect the germline from mutations. Interestingly, the transposon-silencing mechanism shares factors with the RNAi machinery. To better understand the mechanism of transposon silencing, we performed a genome-wide RNAi screen for genes that, when silenced, cause transposition of Tc1 in the C. elegans germline. We identified 27 such genes, among which are mut-16, a mutator that was previously found but not identified at the molecular level, ppw-2, a member of the argonaute family, and several factors that indicate a role for chromatin structure in the regulation of transposition. Some of the newly identified genes are also required for cosuppression and therefore represent the shared components of the two pathways. Since most of the newly identified genes have clear homologs in other species, and since transposons are found from protozoa to human, it seems likely that they also protect other genomes against transposon activity in the germline.

Animals↗
PubMed2002

Genetic analyses of gene function and pathogenesis of murine cytomegalovirus by transposon-mediated mutagenesis.

Murine cytomegalovirus (MCMV) has a linear genome of 230 kb and encodes more than 170 genes, many of which have not been extensively studied for their functions in pathogenesis in vivo. A Tn3-based transposon was constructed and used to generate MCMV mutants by disrupting viral gene targets. The functions of the mutated genes were investigated by studying the viral mutants in cultured cells and in immunocompetent Balb/c and immunodeficient SCID mice. A pool of MCMV mutants that contained the transposon sequence randomly inserted at the viral genome was generated. Studies of several mutants (e.g. a viral mutant with the transposon inserted at open reading frame m09) in cultured cells and in mice indicate that the presence of the transposon sequence per se in the viral genome does not significantly affect viral growth in vitro and in vivo. Moreover, the genome structures of the viral mutants, including the transposon insertion regions, were stable during replication in cultured cells and in animals. Several viral mutants (e.g. a viral mutant with the transposon at M27) that are attenuated in growth and virulence in animals were identified. These results suggest that the genes mutated in these viral mutants may be important for viral virulence and pathogenesis. The Tn3-based system may be a useful tool for the systematic construction of CMV mutants and for studies of CMV gene functions in viral replication in vitro and in pathogenesis in vivo.

Animals↗
PubMed2003

Gene transfer into genomes of human cells by the sleeping beauty transposon system.

The Sleeping Beauty (SB) transposon system, derived from teleost fish sequences, is extremely effective at delivering DNA to vertebrate genomes, including those of humans. We have examined several parameters of the SB system to improve it as a potential, nonviral vector for gene therapy. Our investigation centered on three features: the carrying capacity of the transposon for efficient integration into chromosomes of HeLa cells, the effects of overexpression of the SB transposase gene on transposition rates, and improvements in the activity of SB transposase to increase insertion rates of transgenes into cellular chromosomes. We found that SB transposons of about 6 kb retained 50% of the maximal efficiency of transposition, which is sufficient to deliver 70-80% of identified human cDNAs with appropriate transcriptional regulatory sequences. Overexpression inhibition studies revealed that there are optimal ratios of SB transposase to transposon for maximal rates of transposition, suggesting that conditions of delivery of the two-part transposon system are important for the best gene-transfer efficiencies. We further refined the SB transposase to incorporate several amino acid substitutions, the result of which led to an improved transposase called SB11. With SB11 we are able to achieve transposition rates that are about 100-fold above those achieved with plasmids that insert into chromosomes by random recombination. With the recently described improvements to the transposon itself, the SB system appears to be a potential gene-transfer tool for human gene therapy.

Blotting, Western↗
PubMed2003

Helper-Independent Sleeping Beauty transposon-transposase vectors for efficient nonviral gene delivery and persistent gene expression in vivo.

Transposon-based vectors represent promising new tools for chromosomal transgene insertion and establishment of persistent gene expression in vivo. Here, we report the development of helper-independent transposon-transposase (HITT) vectors, which contain on single plasmids (i) a Sleeping Beauty (SB) transposon containing the transgene and (ii) a SB transposase expression cassette. To obtain an optimal level of transposase expression from HITT vectors, we determined the relative strength of a panel of different promoters in mouse liver and used these promoters to drive transposase expression from injected HITT vectors carrying a human alpha(1)-antitrypsin (hAAT) expression cassette flanked by transposon inverted repeats. By correlating promoter strength with stabilized serum hAAT levels, a narrow expression window supporting high-level transposition in the liver was defined. Peak levels of long-term gene expression were obtained with promoters 30- to 40-fold less active than CMV in mouse liver, whereas reduced stable levels of hAAT were detected with both weaker and stronger promoters. Injected HITT vectors induced transposase-dependent insertion of transposon DNA into the genome of at least 5-6% of transfected hepatocytes, generating levels of persistent hAAT expression that were 2- to 4-fold higher than with an optimized two-plasmid approach. In addition, we show that HITT vectors carrying a human factor IX (hFIX)-containing transposon support (i) long-term hFIX expression in normal mice and (ii) partial phenotypic correction in a mouse model of hemophilia B. SB-based HITT vectors represent a major advance in the establishment of persistent transgene expression from nonviral gene delivery systems and should prove useful for gene transfer to tissues or cell types in which transfection efficiencies are low.

Animals↗
PubMed2004

Activity of the En/Spm-like transposons in meiosis as a base for chromosome repatterning in a small, isolated, peripheral population of Aegilops speltoides Tausch.

Chromosomal repatterning is considered to be one of the main mechanisms for plant genome evolution. Here, we report the first cytogenetic evidence for the involvement of En/Spm transposons in ongoing chromosomal repatterning leading to the rise of new fertile genomic forms in a small, isolated, peripheral plant population. Cytogenetical screening of original individual plants of Aegilops speltoides Tausch. with different phenotypes revealed a wide spectrum of chromosomal abnormalities including extra chromosomes, chromosomal rearrangements, and variability in chromosomal position/number of 45S and 5S rDNA sites. Cytogenetic analysis of the dynamics of En/Spm transposons in meiosis indicates that: (i) this type of transposon is active during male gametogenesis; (ii) separately or in conjunction with rDNA they form clusters in the hot spots of large chromosomal rearrangements; (iii) appearance of at least part of the mobile rDNA sites in genome of Ae. speltoides are connected with meiotic activity of En/Spm transposons. PCR screening for the site-selected transposon insertions confirm the presence of combined fragments that consist partly of the sequence of En/Spm transposon and partly of 5S rDNA sequence.

Chromosomes↗
PubMedDate not supplied

A retrovirus-like strategy for expression of a fusion protein encoded by yeast transposon Ty1.

Eukaryotic transposons such as the Ty element of yeast or the copia-like sequences of Drosophila show structural and functional similarities to both prokaryotic transposons and retroviral proviruses, but the prokaryotic transposons and retroviral proviruses use markedly different expression strategies which yield products having entirely different functions. To determine the phylogenetic relationship between eukaryotic transposons, prokaryotic transposons and retroviruses, we have sought to identify and characterize the proteins encoded by the yeast Ty element and to describe the strategies used to express these proteins. We show here that the yeast transposon produces a fusion protein by a specific frameshifting event that fuses two out-of-phase open reading frames (ORFs). The process is remarkably similar to that used by retroviruses such as Rous sarcoma virus (RSV) to produce Pr180gag-pol.

Amino Acid Sequence↗
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↗
PubMed2001

Regulated transposition of a fish transposon in the mouse germ line.

Tc1/mariner elements are able to transpose in species other than the host from which they were isolated. As potential vectors for insertional mutagenesis and transgenesis of the mouse, these cut-and-paste transposons were tested for their ability to transpose in the mouse germ line. First, the levels of activity of several Tc1/mariner elements in mammalian cells were compared; the reconstructed fish transposon Sleeping Beauty (SB) was found to be an order of magnitude more efficient than the other tested transposons. SB then was introduced into the mouse germ line as a two-component system: one transgene for the expression of the transposase in the male germ line and a second transgene carrying a modified transposon. In 20% of the progeny of double transgenic male mice the transposon had jumped from the original chromosomal position into another locus. Analysis of the integration sites shows that these jumps indeed occurred through the action of SB transposase, and that SB has a strong preference for intrachromosomal transposition. Analysis of the excision sites suggests that double-strand breaks in haploid spermatids are repaired via nonhomologous end joining. The SB system may be a powerful tool for transposon mutagenesis of the mouse germ line.

3T3 Cells↗
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↗
PubMed2006

Role of horizontal transfer of the transposon Tn1546 in the nosocomial spread of vanA vancomycin-resistant enterococci at a tertiary care hospital in Korea.

OBJECTIVE: To investigate the epidemiologic characteristics of vancomycin-resistant enterococci (VRE) infection. DESIGN: An epidemiologic description by means of chromosomal DNA fingerprinting and transposon typing. SETTING: A 2,200-bed tertiary care hospital in Korea. PATIENTS: First VRE isolates were obtained from patients hospitalized from April 1997 to December 2001. INTERVENTIONS: The van genotypes of isolates were identified by means of multiplex polymerase chain reaction (PCR). The macrorestriction patterns of chromosomal DNA were determined by pulsed-field gel electrophoresis (PFGE). The transposon Tn1546 was typed by means of 2 sets of long PCR restriction fragment-length polymorphism analysis, which were ClaI restriction of a 10.4-kb region from orf1 to vanZ and DdeI restriction of a 4.4-kb region from vanR to vanX. RESULTS: VRE isolates were recovered from 215 patients. All were vanA genotype. PFGE analysis of the 215 isolates showed 172 types, including 21 clusters composed of 64 isolates and 151 types of as many isolates. Each type was composed of 2-10 isolates; the isolates within each PFGE cluster were detected within a 10-month period and mostly shared a transposon type. Transposon typing classified 169 strains into 15 types and 158 strains belonged to 4 major transposon clusters. Each of these 4 transposon clusters was isolated from patients treated in 5-22 different wards during a 31-52 month period and consisted of 9-80 PFGE types. Each of the other 11 types were found in only one strain. CONCLUSIONS: Our findings suggest that the horizontal transfer of Tn1546 has a major role in the nosocomial spread of vanA VRE. Clonal spread of VRE seemed to contribute to short-term dissemination in limited areas.

Cross Infection↗
PubMed2004

Harbinger transposons and an ancient HARBI1 gene derived from a transposase.

In this study we report main properties of Harbinger DNA transposons identified in protists, plants, insects, worms, and vertebrates. This is the first superfamily of eukaryotic DNA transposons where all autonomous transposons, even those that are hosted by species from different kingdoms, encode two proteins: a superfamily-specific transposase and a DNA-binding protein characterized by the presence of the conserved SANT/myb/trihelix motif. The last motif is known to be important for the DNA binding by different transcription regulators. Therefore, we suggest that this protein is necessary for coordinated expression of the Harbinger transposase. Although mammalian genomes are free of recognizable remnants of Harbingers, we identified a widely expressed HARBI1 gene encoding a 350-aa protein entirely derived from a Harbinger transposase some 450-500 million years ago. The HARBI1 proteins are conserved in humans, rats, mice, cows, pigs, chickens, frogs, and various bony fish, as well as other extremely important proteins, including RAG1 and RAG2. Conserved motifs detected in the Harbinger transposases are also well preserved in the HARBI1 proteins. Therefore, the HARBI1 proteins are expected to be nucleases important for functioning of bony vertebrates. We also found that the protein most similar to HARBI1 is encoded by an autonomous Harbinger 3_DR transposon that was transpositionally active in the zebrafish genome a few million years ago. Nonautonomous transposons derived from Harbinger3_DR are characterized by a striking preference for a 17-bp target site never seen previously in any other DNA transposon. Based on this observation, we suggest that the hypothetical HARBI1 nucleases are also characterized by a strong DNA-target specificity.

Amino Acid Motifs↗
PubMed1987

A Tn10-lacZ-kanR-URA3 gene fusion transposon for insertion mutagenesis and fusion analysis of yeast and bacterial genes.

We describe here a new variant of transposon Tn10 especially adapted for transposon analysis of cloned yeast genes; it can equally well be used for analysis of prokaryotic genes. We have applied this element to analysis of the LEU2, RAD50, and CDC48 genes of Saccharomyces cerevisiae. This transposon, nicknamed mini-Tn10-LUK, contains a lacZ gene without efficient transcription or translation start signals, an intact URA3 gene, and a kanR determinant. The lacZ gene can be activated by appropriate insertion of the element into an actively expressed gene. Other yeast genes can easily be substituted for URA3 in the available constructs. The mini-Tn10-LUK system has several important advantages. Transposition events occur in Escherichia coli at high frequency and into many different sites in yeast DNA. It is easy to obtain enough insertions to sensitively define the functional limits of a gene. Transposon insertions can be obtained in a single step by standard transposon procedures and can be screened immediately for phenotype either in yeast or in E. coli. The LacZ phenotypes of the insertion mutations provide a good circumstantial indication of the orientation of the target gene. Under favorable circumstances, usable lacZ protein fusions are created. Transposon insertion mutations obtained by this method directly facilitate additional genetic, functional, physical and DNA sequence analysis of the gene or region of interest.

Base Sequence↗
PubMed1997

Mobilization of a Minos transposon in Drosophila melanogaster chromosomes and chromatid repair by heteroduplex formation.

Transposase-mediated mobilization of the element Minos has been studied in the Drosophila melanogaster genome. Excision and transposition of a nonautonomous Minos transposon in the presence of a Minos transposase gene was detected with a dominant eye color marker carried by the transposon. Frequencies of excision in somatic tissues and in the germ line were higher in flies heterozygous for the transposon than in homozygotes or hemizygotes. Transposition of a X chromosome-linked insertion of Minos into new autosomal sites occurred in 1-12% of males expressing transposase, suggesting that this system is usable for gene tagging and enhancer trapping in Drosophila. Sequence analysis of PCR-amplified donor sites after excision showed precise restoration of the original target sequence in approximately 75% of events in heterozygotes and the presence of footprints or partially deleted elements in the remaining events. Most footprints consisted of the four terminal bases of the transposon, flanked by the TA target duplication. Sequencing of a chromosomal donor site that was directly cloned after excision showed a characteristic two-base mismatch heteroduplex in the center of the 6-bp footprint. Circular extrachromosomal forms of the transposon, presumably representing excised Minos elements, could be detected only in the presence of transposase. A model for chromatid repair after Minos excision is discussed in which staggered cuts are first produced at the ends of the inverted repeats, the broken chromatid ends are joined, and the resulting heteroduplex is subsequently repaired. The model also suggests a simple mechanism for the production of the target site duplication and for regeneration of the transposon ends during reintegration.

Animals↗