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PubMed1998

Inversion and transposition of Tc1 transposon of C. elegans in mammalian cells.

Tc1/mariner transposons are widespread in the eukaryotes. In vitro transposition test indicated that the transposase is the only protein that is needed in transpositions. It was shown later that the reconstructed Tc1-like transposon, "sleeping beauty" in fish, and the Tc1 transposon in C. elegans jumps in human cells. This discovery indicates that the Tc1/mariner transposon may be engineered as a somatic gene therapy vector if coupled with an efficient gene delivery system. We introduced the Tc1 transposon from C. elegans into different mammalian cell lines and detected the transposition events, indicating that Tc1 transposon functions in different mammalian cells. Interestingly, a high inversion frequency of the transposon was also detected, suggesting that this type of transposon may add variations to host genome when it is horizontally transferred into a new species.

Animals↗

PubMedDate not supplied

A transposon-like element in human DNA.

Mobile genetic elements have been reported in prokaryotes, plants, yeast and Drosophila. The only transposon-like sequences reported for mammalian organisms are closely related to retroviruses, although undoubtedly other transposon families exist within the mammalian genome. Although mobile genetic elements can only be identified as such if their mobility can be demonstrated in existing populations, transposon and transposon-like elements share several common biochemical and structural features. Here we demonstrate that a repetitive human sequence has many of the diagnostic features of transposable elements. This 2.3-kilobase (kb) transposon-like element contains two flanking long terminal repeat (LTR)-like 350-base pair (bp) repetitive sequences, each of which begins with the sequence 5' TG... and ends with ...CA 3'. The transposon-like element is bounded by 5-bp direct repeats. Discrete-length polyadenylated transcripts from HeLa cells are homologous to the transposon-like element. Members of this transposon-like family are found in extrachromosomal circular DNA molecules.

Animals↗

PubMed1996

DNA sequence analyses support the role of interrupted gap repair in the origin of internal deletions of the maize transposon, MuDR.

Previous research has demonstrated that the autonomous Cy transposon can activate the excision of Mu transposons. To determine the relationship between Cy and the more recently described autonomous Mu transposon, MuDR, a Cy transposon inserted at the mutable a1 allele, a1-m5216, was isolated and cloned. DNA sequence analyses established that this Cy insertion is identical to MuDR (Mu9, GenBank accession No.: m76978.gb-pl). Therefore, Cy will henceforth be termed MuDR:Cy. Defective derivatives of MuDR:Cy were isolated that had lost their capacity to activate their own excision or the excision of a Mu7 transposon. Most of these derivatives are nonautonomous transposons because they can excise, but only in the presence of unlinked MuDR:Cy transposons. Physical mapping and DNA sequence analyses have established that six of these defective derivatives carry internal deletions. It has been proposed previously that such deletions arise via interrupted gap repair. The DNA sequences of the break points associated with all four sequenced deletions are consistent with this model. The finding that three of the excision-defective derivatives carry deletions that disrupt the coding region of the mudrA (but not the mudrB) transcript supports the view that mudrA plays a role in the excision of Mu transposons.

Alleles↗

PubMed1994

Efficient integration of artificial transposons into plasmid targets in vitro: a useful tool for DNA mapping, sequencing and genetic analysis.

We have developed efficient methods for creating artificial transposons and inserting these transposons into plasmid targets in vitro, primarily for the purpose of DNA mapping and sequencing. A novel plasmid has been engineered to convert virtually any DNA sequence, or combination of sequences, into an artificial transposon; hence, custom transposons containing any desired feature can be easily designed and constructed. Such transposons are then efficiently inserted into plasmid targets, in vitro, using the integrase activity present in yeast Ty1 virus-like particles. A single in vitro integration reaction, which resembles a simple restriction digestion in the complexity of the reaction, gives rise to thousands of recoverable insertion events within DNA target molecules; this frequency approaches one insertion per phosphodiester bond in typical plasmids. Importantly, transposon insertions are recovered from all regions of DNA inserts carried on plasmid targets, indicating that integration is a random or nearly-random process. Because of its versatility, this technology offers a generalized method of generating recombinant DNA molecules of a desired structure. We have adapted this system for DNA sequencing by developing a customized artificial transposon to insert new primer binding sites into internal regions of DNA inserts carried on cloning vectors. Transposon insertions have been generated throughout several different yeast and human DNA inserts carried on plasmids, allowing the efficient recovery of sequence information from these inserts. Our results demonstrate the overall utility of this method for both small and large-scale DNA sequencing, as well as general DNA restructuring, and indicate that it could be adapted for use with a number of additional applications including functional genetic analysis.

Base Sequence↗

PubMed1987

Transposition of Tn4551 in Bacteroides fragilis: identification and properties of a new transposon from Bacteroides spp.

Tn4551, a clindamycin resistance (Ccr) transposon from the R plasmid pBI136, was cloned onto an Escherichia coli-Bacteroides shuttle vector which could replicate normally in E. coli but was maintained unstably in Bacteroides fragilis. To aid in cloning and to ensure maintenance of Tn4551 in E. coli, a kanamycin resistance determinant (Kmr) was inserted in the transposon. The transposon-bearing shuttle vector pFD197 was transformed into B. fragilis 638, and putative insertions of Tn4551::Kmr were identified by screening for resistance to clindamycin and plasmid content. Southern hybridization analyses were used to verify integration of the transposon in the B. fragilis chromosome, and the frequency of insertion was estimated at 7.8 X 10(-5) events per generation. In 57% of the isolates tested a second integration event also occurred. This second insertion apparently involved just a single copy of the 1.2-kilobase repeat sequence which flanks the transposon. In addition, Tn4551::Kmr appeared to function as a transposon in E. coli. Evidence for this was obtained by the isolation of transposon insertions into the bacteriophage P1 genome. Finally, the transposon vector, pFD197, could be mobilized to other B. fragilis strains in which transposition was detected. Mobilization from the strain 638 background was via a conjugation like process, but occurred in the absence of known conjugative elements or other detectable plasmids. This result suggested the presence of a host-encoded transfer system in this B. fragilis strain.

Bacteroides fragilis↗

PubMed2004

Identification of candidate gammaherpesvirus 68 genes required for virus replication by signature-tagged transposon mutagenesis.

Current methods for determining the role of a given gene product in the gammaherpesvirus 68 (gammaHV68) life cycle require generation of a specific mutation by either homologous recombination in mammalian cells or bacterial artificial chromosome-mediated mutagenesis in Escherichia coli. The mutant virus is then compared to wild-type virus, and the role of the gene in the viral life cycle is deduced from its phenotype. This process is both time-consuming and labor intensive. Here we present the use of random, transposon-mediated signature-tagged mutagenesis for the identification of candidate viral genes involved in virus replication. Pools of viral mutants, each containing a random insertion of a transposon, were generated with a transposon donor library in which each transposon contains a unique sequence identifier. These pools were transfected into mammalian cells, and the ability of each mutant to replicate was assessed by comparing the presence of virus in the output pool to that present in the input pool of viral genomes. With this approach we could rapidly screen up to 96 individual mutants simultaneously. The location of the transposon insertion was determined by sequencing individual clones with a common primer specific for the transposon end. Here we present the characterization of 53 distinct viral mutants that correspond to insertions in 29 open reading frames within the gammaHV68 genome. To confirm the results of the signature-tagged mutagenesis screen, we quantitated the ability of each mutant to replicate compared to wild-type gammaHV68. From these analyses we identified 16 gammaHV68 open reading frames that, when disrupted by transposon insertions, score as essential for virus replication, and six other open reading frames whose disruption led to significant attenuation of virus replication. In addition, transposon insertion in five other gammaHV68 open reading frames did not affect virus replication. Notably, all but one of the candidate essential replication genes identified in this screen have been shown to be essential for the replication of at least one other herpesvirus.

Animals↗

PubMed2005

Transcribed Tc1-like transposons in salmonid fish.

BACKGROUND: Mobile genetic elements comprise a substantial fraction of vertebrate genomes. These genes are considered to be deleterious, and in vertebrates they are usually inactive. High throughput sequencing of salmonid fish cDNA libraries has revealed a large number of transposons, which remain transcribed despite inactivation of translation. This article reports on the structure and potential role of these genes. RESULTS: A search of EST showed the ratio of transcribed transposons in salmonid fish (i.e., 0.5% of all unique cDNA sequences) to be 2.4-32 times greater than in other vertebrate species, and 68% of these genes belonged to the Tc1-family of DNA transposons. A phylogenetic analysis of reading frames indicate repeated transposition of distantly related genes into the fish genome over protracted intervals of evolutionary time. Several copies of two new DNA transposons were cloned. These copies showed relatively little divergence (11.4% and 1.9%). The latter gene was transcribed at a high level in rainbow trout tissues, and was present in genomes of many phylogenetically remote fish species. A comparison of synonymous and non-synonymous divergence revealed remnants of divergent evolution in the younger gene, while the older gene evolved in a neutral mode. From a 1.2 MB fragment of genomic DNA, the salmonid genome contains approximately 10(5) Tc1-like sequences, the major fraction of which is not transcribed. Our microarray studies showed that transcription of rainbow trout transposons is activated by external stimuli, such as toxicity, stress and bacterial antigens. The expression profiles of Tc1-like transposons gave a strong correlation (r2 = 0.63-0.88) with a group of genes implicated in defense response, signal transduction and regulation of transcription. CONCLUSION: Salmonid genomes contain a large quantity of transcribed mobile genetic elements. Divergent or neutral evolution within genomes and lateral transmission can account for the diversity and sustained persistence of Tc1-like transposons in lower vertebrates. A small part of transposons remain transcribed and their transcription is enhanced by responses to acute conditions.

Amino Acid Sequence↗

PubMed2006

Global mapping of transposon location.

Transposable genetic elements are ubiquitous, yet their presence or absence at any given position within a genome can vary between individual cells, tissues, or strains. Transposable elements have profound impacts on host genomes by altering gene expression, assisting in genomic rearrangements, causing insertional mutations, and serving as sources of phenotypic variation. Characterizing a genome's full complement of transposons requires whole genome sequencing, precluding simple studies of the impact of transposition on interindividual variation. Here, we describe a global mapping approach for identifying transposon locations in any genome, using a combination of transposon-specific DNA extraction and microarray-based comparative hybridization analysis. We use this approach to map the repertoire of endogenous transposons in different laboratory strains of Saccharomyces cerevisiae and demonstrate that transposons are a source of extensive genomic variation. We also apply this method to mapping bacterial transposon insertion sites in a yeast genomic library. This unique whole genome view of transposon location will facilitate our exploration of transposon dynamics, as well as defining bases for individual differences and adaptive potential.

Chromosome Mapping↗

PubMed2000

Sleeping Beauty, a wide host-range transposon vector for genetic transformation in vertebrates.

Sleeping Beauty (SB), a member of the Tc1/mariner superfamily of transposable elements, is the only active DNA-based transposon system of vertebrate origin that is available for experimental manipulation. We have been using the SB element as a research tool to investigate some of the cis and trans-requirements of element mobilization, and mechanisms that regulate transposition in vertebrate species. In contrast to mariner transposons, which are regulated by overexpression inhibition, the frequency of SB transposition was found to be roughly proportional to the amount of transposase present in cells. Unlike Tc1 and mariner elements, SB contains two binding sites within each of its terminal inverted repeats, and we found that the presence of both of these sites is a strict requirement for mobilization. In addition to the size of the transposon itself, the length as well as sequence of the DNA outside the transposon have significant effects on transposition. As a general rule, the closer the transposon ends are, the more efficient transposition is from a donor molecule. We have found that SB can transform a wide range of vertebrate cells from fish to human. However, the efficiency and precision of transposition varied significantly among cell lines, suggesting potential involvement of host factors in SB transposition. A positive-negative selection assay was devised to enrich populations of cells harboring inserted transposons in their chromosomes. Using this assay, of the order of 10,000 independent transposon insertions can be generated in human cells in a single transfection experiment. Sleeping Beauty can be a powerful alternative to other vectors that are currently used for the production of transgenic animals and for human gene therapy.

Animals↗

PubMed1994

Catabolic transposons.

The structure and function of transposable elements that code for catabolic pathways involved in the biodegradation of organic compounds are reviewed. Seven of these catabolic transposons have structural features that place them in the Class I (composite) or Class II (Tn3-family) bacterial elements. One is a conjugative transposon. Another three have been found to have properties of transposable elements but have not been characterized sufficiently to assign to a known class. Structural features of the toluene (Tn4651/Tn4653) and naphthalene (Tn4655) elements that illustrate the enormous potential for acquisition, deletion and rearrangement of DNA within catabolic transposons are discussed. The recently characterized chlorobenzoate (Tn5271) and chlorobenzene (Tn5280) catabolic transposons encode different aromatic ring dioxygenases, however they both illustrate the constraints that must be overcome when recipients of catabolic transposons assemble and regulate complete metabolic pathways for environmental pollutants. The structures of the chlorobenzoate catabolic transposon Tn5271 and the related haloacetate dehalogenase catabolic element of plasmid pUO1 are compared and a hypothesis for their formation is discussed. The structures and activities of catabolic transposons of unknown class coding for the catabolism of halogenated alkanoic acids (DEH) and chlorobiphenyl (Tn4371) are also reviewed.

Biodegradation, Environmental↗

PubMed1988

Molecular structure and interrelationships of multiresistance beta-lactamase transposons.

Transposons coding for beta-lactamases OXA-3, OXA-4, OXA-5, LCR-1, and CARB-3 have been isolated and compared functionally and structurally with transposons for TEM-1, OXA-1, PSE-1, PSE-2, and PSE-4 enzymes. Each beta-lactamase gene type occurred in a unit together with resistance to other antibiotics, particularly streptomycin and sulfonamide but also chloramphenicol, mercuric ion, or gentamicin, kanamycin, and tobramycin. Restriction mapping, gene cloning, and DNA hybridization were used to compare the transposons and to localize their functional components. Although the multiresistance beta-lactamase transposons varied in size from 8 to 25 kb, the similarity of some of their restriction maps suggested a common derivation. Six of 12 transposons contained DNA segments homologous to the tnpR gene of transposon Tn21 and could complement a tnpR- Tn21 derivative. Consequently, these six transposons appear to have evolved from a common progenitor by acquisition of DNA coding for various beta-lactamases and other resistance genes.

DNA Restriction Enzymes↗

PubMed1998

High and low UV-dose responses in SOS-induction of the precise excision of transposons tn1, Tn5 and Tn10 in Escherichia coli.

UV-inducible precise excision of transposons is a specific SOS-mutagenesis process. It deals with the deletion formation which has previously been demonstrated to involve direct or inverted IS-sequences of transposons. The process was used for revisiting the targeted and untargeted SOS-mutability and its relationship to the key genes for SOS-mutagenesis: the recA, lexA and umuDC. The precise excision of transposons Tn5 and Tn10 from the chromosomal insertion sites ade128 and cyc750 is induced in Escherichia coli K-12 and B cells, wild-type for DNA-repair, both by the low doses of UV-light ranging from 0.25 J m-2 to 2.5 J m-2 and the high doses within the range 5.0-40.0 J m-2. Precise excision of these transposons induced by the range of low doses incapable to induce targeted point mutations reveals its mostly untargeted nature. This process for the transposon Tn1 is not induced by UV-light within the range of doses 0.25-2.5 J m-2 while its induction is possible by UV-fluences ranging from 5.0 to 40.0 J m-2. A dose-response of the precise excision of Tn1 is similar to that of the UV-induced reversion of trpUAA point mutation that is targeted by nature and contrasts to the UV-inducible precise excision of Tn5 and Tn10. Both types of UV-inducible precise excision, demonstrated either by Tn1 or Tn5 and Tn10, are eliminated by mutations in the lexA, recA and umuDC genes indispensable for UV-induced SOS-mutability. The palindromic structures different for the transposons Tn1, Tn5 and Tn10 are discussed to be involved and affect the targeted and untargeted precise excision of transposons induced by UV-light.

Chromosomes, Bacterial↗

PubMed2000

Efficient amplification of multiple transposon-flanking sequences.

Transposon mutagenesis is a very useful tool for gene identification in bacteria. Once the transposon mutants of interest are isolated, it is often necessary to identify the sequences that flank the transposon insertions. We devised an efficient method for specific amplification of transposon-flanking sequences that requires the sequence information of only transposon-specific sequences. The basic steps for this method consists of (1) digestion with a restriction enzyme, (2) ligation with a Y-shaped linker and (3) polymerase chain reaction amplification using a transposon-specific primer and a primer specific to the Y-shaped linker. The feasibility of this method was demonstrated with mini-Tn5 mutants of Salmonella typhimurium. We also found that this method can be used for simultaneous amplification of multiple transposon-flanking sequences.

3' Untranslated Regions↗

PubMed1998

Generalized transduction for genetic linkage analysis and transfer of transposon insertions in different Staphylococcus epidermidis strains.

Staphylococcus epidermidis phage 48 was used to efficiently transduce plasmid pTV1ts and a chromosomal Tn917 insertion M27 from S. epidermidis 13-1 to biofilm-producing clinical S. epidermidis isolates 1457, 9142, and 8400. The Tn917 insertion leading to the biofilm-negative phenotype of transposon mutant M10 was sequentially transduced to biofilm-producing S. epidermidis 1457 using S. epidermidis phage 48 and then, using the resulting biofilm-negative transductant 1457-M10 as a donor, into several unrelated biofilm-producing clinical S. epidermidis isolates using S. epidermidis phage 71. All resultant transductants displayed a completely biofilm-negative phenotype. In addition, S. epidermidis phage 71 was adapted to S. epidermidis 1457 and 8400, which allowed generalized transduction of transposon insertions in these wild-type strains. As Tn917 predominantly transposed into endogenous plasmids of all three strains used, an efficient system for chromosomal transposon mutagenesis was established by curing of S. epidermidis 1457 of a single endogenous plasmid p1457 by sodium dodecylsulfate treatment. After transduction of the resulting derivative, S. epidermidis 1457c with pTV1ts, insertion of transposon Tn917 to different sites of the chromosome of S. epidermidis 1457c was observed. Biofilm-producing S. epidermidis 1457c x pTV1ts was used to isolate a biofilm-negative transposon mutant (1457c-M3) with a chromosomal insertion apparently different from two previously isolated isogenic biofilm-negative transposon mutants, M10 and M11 (Mack, D., M. Nedelmann, A. Krokotsch, A. Schwarzkopf, J. Heesemann, and R. Laufs: Infect Immun 62 [1994] 3244-3253). S. epidermidis phage 71 was used to prove genetic linkage between transposon insertion and altered phenotype by generalized transduction. In combination with phage transduction, 1457c x pTV1ts will be a useful tool facilitating the study of bacterial determinants of the pathogenicity of S. epidermidis.

Blotting, Southern↗

PubMed2002

A collection of sequenced and mapped Ds transposon insertion sites in Arabidopsis thaliana.

Insertional mutagenesis is a powerful tool for generating knockout mutations that facilitate associating biological functions with as yet uncharacterized open reading frames (ORFs) identified by genomic sequencing or represented in EST databases. We have generated a collection of Dissociation (Ds) transposon lines with insertions on all 5 Arabidopsis chromosomes. Here we report the insertion sites in 260 independent single-transposon lines, derived from four different Ds donor sites. We amplified and determined the genomic sequence flanking each transposon, then mapped its insertion site by identity of the flanking sequences to the corresponding sequence in the Arabidopsis genome database. This constitutes the largest collection of sequence-mapped Ds insertion sites unbiased by selection against the donor site. Insertion site clusters have been identified around three of the four donor sites on chromosomes 1 and 5, as well as near the nucleolus organizers on chromosomes 2 and 4. The distribution of insertions between ORFs and intergenic sequences is roughly proportional to the ratio of genic to intergenic sequence. Within ORFs, insertions cluster near the translational start codon, although we have not detected insertion site selectivity at the nucleotide sequence level. A searchable database of insertion site sequences for the 260 transposon insertion sites is available at http://sgio2.biotec.psu.edu/sr. This and other collections of Arabidopsis lines with sequence-identified transposon insertion sites are a valuable genetic resource for functional genomics studies because the transposon location is precisely known, the transposon can be remobilized to generate revertants, and the Ds insertion can be used to initiate further local mutagenesis.

Arabidopsis↗

PubMed2001

Mobilization of a Drosophila transposon in the Caenorhabditis elegans germ line.

Transposons have been enormously useful for genetic analysis in both Drosophila and bacteria. Mutagenic insertions constitute molecular tags that are used to rapidly clone the mutated gene. Such techniques would be especially advantageous in the nematode Caenorhabditis elegans, as the entire sequence of the genome has been determined. Several different types of endogenous transposons are present in C. elegans, and these can be mobilized in mutator strains (reviewed in ref. 1). Unfortunately, use of these native transposons for regulated transposition in C. elegans is limited. First, all strains contain multiple copies of these transposons and thus new insertions do not provide unique tags. Second, mutator strains tend to activate the transposition of several classes of transposons, so that the type of transposon associated with a particular mutation is not known. Here we demonstrate that the Drosophila mariner element Mos1 can be mobilized in C. elegans. First, efficient mobilization of Mos1 is possible in somatic cells. Second, heritable insertions of the transposon can be generated in the germ line. Third, genes that have been mutated by insertion can be rapidly identified using inverse polymerase chain reaction. Fourth, these insertions can subsequently be remobilized to generate deletion and frameshift mutations by imperfect excision.

Animals↗

PubMed2000

Transposon stability and a role for conjugational transfer in adaptive mutability.

Lac(+) revertants of Escherichia coli that occur after prolonged nonlethal selection display a high frequency of transposon loss when the transposon Tn10 and the reverting lacI33 allele are linked on an F'128 episome. As many as 20% of the Lac(+) revertants are sensitive to tetracycline, about half because of transposon loss, nearly all by precise excision, and the remainder because of amplification of both the transposon and the linked lac allele. Lethality of the amplified products in the presence of tetracycline is a peculiarity of the tetA gene at high gene dosage. The selective conditions on lactose medium result in 10% transposon-free revertants, whether or not a requirement for conjugal DNA transfer is imposed. In addition, a similar fraction, about 5% of Lac(-) unreverted colonies that are products of transfer between cells experiencing nonlethal selection are also tetracycline-sensitive, and all are attributable to loss of the Tn10 transposon. These results suggest the possibility that the high frequency of transposon loss is a consequence of conjugal transfer, making this loss a marker for that transfer. We suggest that conjugal DNA transfer may be a prominent feature in the mutability process that occurs during nonlethal selection and that the subset of bacteria displaying hypermutability are those that experience such transfer.

Conjugation, Genetic↗

PubMed1992

Genetic study of the loss and restoration of Mutator transposon activity in maize: evidence against dominant-negative regulator associated with loss of activity.

The Mutator system of transposable elements is characterized by a family of transposons called Mu transposons that share common termini and are actively transposing in Robertson's Mutator (Mu) lines of maize. Mu lines lose transposition activity during propagation by either outcrossing or inbreeding. This loss of transposition activity, which can occur at non-Mendelian frequencies, is in the form of loss of forward transposition activity resulting in a decrease in the generation of new mutations, as well as the loss of mutability of Mu transposon induced mutations, and it has been correlated with hypermethylation of the Mu elements. Previous studies have concluded that restoration of Mutator transposon activity by crossing inactive lines back to active lines is incomplete or transient, and depends upon the sex of the inactive parent. Further, it has been proposed that the inactive system is dominant to the active system, with the dominance possibly mediated through a negative regulatory factor that is preferentially transmitted through the female. In this study, we have examined the frequencies of loss and restoration of Mu transposon activity using a Mu line carrying an insertion in the bronze 1 locus. We find that transmission of Mu transposon activity to non-Mu plants can occur at high rates through males and females, but individual cases of decreased transmission through the male were observed. We also find that in crosses between inactive-Mu and active-Mu plants, reactivation was efficient as well as heritable, regardless of the sex of the inactive parent. Similar results were obtained whether the inactivation occurred in an outcross or a self. In all cases examined, loss of Mu transposon activity was correlated with hypermethylation of Mu elements, and reactivation was correlated with their demethylation. Our results indicate that an inactive Mu system does not exhibit dominance over an active Mu system. We conclude that contrary to current models, inactivation and its maintenance is not obligatorily associated with a dominant negative regulatory factor whether nuclear or cytoplasmic, and we propose a revised model to account for these and other observations.

Alleles↗