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PubMed1990

Retron for the 67-base multicopy single-stranded DNA from Escherichia coli: a potential transposable element encoding both reverse transcriptase and Dam methylase functions.

The region (retron-Ec67) required for the biosynthesis of a branched-RNA-linked multicopy single-stranded DNA (msDNA-Ec67) from a clinical isolate of Escherichia coli was mapped at a position equivalent to 19 min on the K-12 chromosome. The element containing the retron consisted of a unique 34-kilobase sequence that was flanked by direct repeats of a 26-base-pair sequence found in the K-12 chromosomal DNA. This suggests that the 34-kilobase element was probably integrated into the E. coli genome by a mechanism related to transposition or phage integration. In the 34-kilobase sequence an open reading frame of 285 residues was found, which displays 44% sequence identity with the E. coli Dam methylase. Interestingly, there are three GATC sequences, the site of Dam methylation, in the promoter region of the gene for reverse transcriptase.

Amino Acid Sequence↗
PubMed1991

Reverse transcriptase encoded by a human transposable element.

L1 elements are highly repeated mammalian DNA sequences whose structure suggests dispersal by retrotransposition. A consensus L1 element encodes a protein with sequence similarity to known reverse transcriptases. The second open reading frame from the human L1 element L1.2A was expressed as a fusion protein targeted to Ty1 virus-like particles in Saccharomyces cerevisiae and shown to have reverse transcriptase activity. This activity was eliminated by a missense mutation in the highly conserved amino acid motif Y/F-X-D-D. Thus, L1 represents a potential source of the reverse transcriptase activity necessary for dispersion of the many classes of mammalian retroelements.

Base Sequence↗
PubMed1991

Trans-activation of an artificial dTam3 transposable element in transgenic tobacco plants.

In Antirrhinum majus only autonomous Tam3 transposons have been characterized. We investigated whether an artificial dTam3 element, with a deletion in the presumptive transposase coding region, can be trans-activated in tobacco by an activator Tam3 element, which was immobilized by the deletion of one inverted repeat. A phenotypic assay based on restored hygromycin resistance demonstrates that a dTam3 element harbouring a bacterial plasmid can be trans-activated with a low frequency. Molecular analysis confirms that the dTam3 element has been excised from the HPTII marker gene. Reintegration of the dTam3 element into the tobacco genome is detected only in one out of six hygromycin-resistant plants analysed. PCR analysis of empty donor sites shows that excision of the dTam3 element in tobacco results in rearrangements (deletions and additions), that have been shown to be characteristic of Tam3 excision in the original host Antirrhinum majus. This trans-activation assay allowed us to establish that, in contrast to what has been detected in Antirrhinum majus, a periodical temperature shift down to 15 degrees C does not enhance dTam3 transposition in regenerating tobacco calli.

Base Sequence↗
PubMed1991

Germline excision of the transposable element Tc1 in C. elegans.

We have examined eight germline revertants generated by the excision of Tc1 from a site within the unc-22 gene of Caenorhabditis elegans. A rich variety of rearrangements accompanied Tc1 excision at this site, including transposon 'footprints', deletions of sequences flanking the insertion site and direct nontandem duplications of flanking DNA. With only modest modification the double-strand gap repair model for transposition, recently proposed by Engles and coworkers (Cell 62: 515-525 1990), can explain even the most complex of these rearrangements. In light of this model rearrangements of the target site accompanying transposition/excision may not be the end result of imprecise excision of the element. Instead, these rearrangements may be the result of imprecise repair of the double-strand gap by the host replication and repair machinery. Sequences surrounding an insertion site influence the fidelity of gap repair by this machinery. This may lead to a number of possible resolutions of a double-strand gap as documented here for a Tc1 site in unc-22.

Animals↗
PubMed1989

Differential activity of a transposable element in Escherichia coli colonies.

In Escherichia coli colonies, patterns of differential gene expression can be visualized by the use of Mu d(lac) fusion elements. Here we report that patterned beta-galactosidase expression in colonies of strain MS1534 resulted from a novel mechanism, spatially localized replication of the Mu dII1681 element causing lacZ transposition to active expression sites. Mu dII1681 replication did not occur constitutively with a fixed probability but was dependent on the growth history of the bacterial population. The bacteria in which Mu dII1681 replication and lacZ transposition had occurred could no longer form colonies. These results lead to several interesting conclusions about cellular differentiation during colony development and the influence of bacterial growth history on gene expression and genetic change.

Blotting, Southern↗
PubMed1980

Translation and developmental regulation of RNA encoded by the eukaryotic transposable element copia.

copia-specific RNA was isolated from Drosophila melanogaster tissue culture cells by hybridization of cytoplasmic polyadenylylated RNA to copia DNA immobilized on cellulose. The purified RNA was translated in reticulocyte lysates. One major polypeptide of approximately 51,000 daltons was synthesized in addition to several others between 18,000 and 38,000 daltons. The 51,000-dalton polypeptide and several of the others are encoded by mRNAs of about 2000 nucleotides. The approximate locations on the copia element of the coding sequences for the 51,000-dalton polypeptide and several other proteins were determined by hybrid-arrested translation with copia restriction fragments. The relative abundance of copia-specific RNA was determined at various stages of the Drosophila life cycle. The level of copia-specific RNA is modulated during development of the organism, with the highest level occurring during the larval stages.

Animals↗
PubMed1993

Binding of the ubiquitous nuclear transcription factor YY1 to a cis regulatory sequence in the human LINE-1 transposable element.

The first step of the currently favored model for the mechanism of transposition of the human LINE-1 element involves the synthesis of full length LINE-1 mRNA. Previous work demonstrated that the 5'-terminal 100 base pairs of the human LINE-1 element (L1Hs) has an important role in regulating it's expression. Here we report further deletion analysis revealing the presence of a cis regulatory element overlapping the region between base pairs +12 and +18. Oligonucleotides containing this sequence form a specific complex with a nuclear protein extracted from NTera2D1 and Jurkat cells, and with recombinant YY1 produced in E. coli. The complex is competed by YY1 binding sites found in other genes, and is ablated by anti-YY1 serum. These results suggest that YY1 is involved in the regulation of L1Hs transcription and therefore transposition.

Base Sequence↗
PubMed2003

Expression of the Arabidopsis transposable element Tag1 is targeted to developing gametophytes.

The Arabidopsis transposon Tag1 undergoes late excision during vegetative and germinal development in plants containing 35S-Tag1-GUS constructs. To determine if transcriptional regulation can account for the developmental control of Tag1 excision, the transcriptional activity of Tag1 promoter-GUS fusion constructs of various lengths was examined in transgenic plants. All constructs showed expression in the reproductive organs of developing flowers but no expression in leaves. Expression was restricted to developing gametophytes in both male and female lineages. Quantitative RT-PCR analysis confirmed that Tag1 expression predominates in the reproductive organs of flower buds. These results are consistent with late germinal excision of Tag1, but they cannot explain the vegetative excision activity of Tag1 observed with 35S-Tag1-GUS constructs. To resolve this issue, Tag1 excision was reexamined using elements with no adjacent 35S promoter sequences. Tag1 excision in this context is restricted to germinal events with no detectable vegetative excision. If a 35S enhancer sequence is placed next to Tag1, vegetative excision is restored. These results indicate that the intrinsic activity of Tag1 is restricted to germinal excision due to targeted expression of the Tag1 transposase to developing gametophytes and that this activity is altered by the presence of adjacent enhancers or promoters.

Arabidopsis↗
PubMed1994

Enhanced frequency of transposition of the maize transposable element Activator following excision from T-DNA in Petunia hybrida.

Many of the systems currently employed for heterologous transposon tagging in plants rely on an excision assay to monitor transposon activity. We have used the streptomycin phosphotransferase (SPT) reporter system to assay Ac activity in Petunia hybrida. In other species, such as tobacco or Arabidopsis, excision of Ac from the SPT gene in sporogenous tissue gives rise to streptomycin-resistant seedlings in the following generation. The frequency of fully streptomycin-resistant seedlings in petunia was low (0.4%) but molecular analysis of these indicated that the actual excision frequency may be as low as 0.05%. This indicates that the SPT assay is not a reliable selection criterion for germinal excision in petunia. Extensive molecular screening for reinsertion of Ac was consistent with a low primary transposition frequency (0%-0.6%). In contrast to these findings, the progeny of confirmed germinal transpositions for three independent transformants showed frequent transposition to new sites (9.5%-17.0%). This suggests a high frequency of secondary transposition compared with primary transposition from the T-DNA. Segregation analysis indicates that the high transposition activity is closely associated with transposed copies of Ac. No evidence was found for an altered methylation state for Ac following transposition. The implications of these results for heterologous transposon tagging in petunia are discussed in the context of the reliability of excision reporter systems in general.

DNA Repair↗
PubMed2005

High-efficiency transformation of Plasmodium falciparum by the lepidopteran transposable element piggyBac.

Functional analysis of the Plasmodium falciparum genome is restricted because of the limited ability to genetically manipulate this important human pathogen. We have developed an efficient transposon-mediated insertional mutagenesis method much needed for high-throughput functional genomics of malaria parasites. A drug-selectable marker, human dihydrofolate reductase, added to the lepidopteran transposon piggyBac, transformed parasites by integration into the P. falciparum genome in the presence of a transposase-expressing helper plasmid. Multiple integrations occurred at the expected TTAA target sites throughout the genome of the parasite. We were able to transform P. falciparum with this piggyBac element at high frequencies, in the range of 10(-3), and obtain stable clones of insertional mutants in a few weeks instead of 6-12 months. Our results show that the piggyBac transposition system can be used as an efficient, random integration tool needed for large-scale, whole-genome mutagenesis of malaria parasites. The availability of such an adaptable genetic tool opens the way for much needed forward genetic approaches to study this lethal human parasite.

Animals↗
PubMed1980

The transposable element Mdg3 in Drosophila melanogaster is flanked with the perfect direct and mismatched inverted repeats.

MDg3 is a family of mobile dispersed genetic elements represented by 15 copies in the haploid genome of D. melanogaster and flanked, like other similar elements, by the regions of homology. In the present work, these regions of mdg3 have been sequenced. The existence of perfect direct repeats 268 base pairs long has been demonstrated. Inverted repeats are located on the gene distal side of them. It is possible to construct a perfect 8 b.p. palindrome or a slightly mismatched 18 b.p. palindrome. The inverted repeats are flanked by two short 5 b.p. direct repeats.

Animals↗
PubMed2001

Possible horizontal transfer of a transposable element from host to parasitoid.

Full-length mariner-like elements (MLEs) were identified from both a parasitoid wasp, Ascogaster reticulatus, and its moth host, Adoxophyes honmai. MLEs were detected in two related Tortricid moths, but not in another Ascogaster species. The MLEs of A. reticulatus and A. honmai were 97.6% identical in DNA sequence. This high similarity suggests a recent horizontal transfer, probably from the moth host to the wasp parasitoid, facilitated by the intimacy of the host-parasitoid relationship.

Amino Acid Sequence↗
PubMed1981

The nucleotide sequence and protein-coding capability of the transposable element IS5.

The nucleotide sequence of IS5, a bacterial insertion sequence, has been determined. It is 1195 bp long and contains an inverted terminal repetition of 16 bp with one mismatch. One open reading frame, spanning nearly the entire length of the element, could encode a polypeptide of 338 amino acids. Upon insertion into a DNA segment, IS5 causes a duplication of 4 bp. Based on seven examples, this site of insertion appears to be nonrandom, and the consensus target site sequence is C . T/A . A . G/A (or C/T . T . A/T . G on the opposite strand). The nucleotide sequences of IS5 insertions into the B and cim genes of bacteriophage Mu have allowed tentative identification of the protein-coding frames of B and cim.

Bacteriophage mu↗
PubMed1980

Recombination genes on the Escherichia coli sex factor specific for transposable elements.

The Escherichia coli sex factor stimulates precise excision of transposons Tn5 and Tn10 from sites either within the bacterial chromosome or within the factor itself. We have isolated two kinds of mutations that affect this activity. The ferA mutations eliminate the stimulation; the ferB mutations enhance it in the presence of FerA+. We conclude that ferA defines a sex factor gene that stimulates precise excision. The ferB mutations also specifically increase the rate of recombination between two IS3 elements on F' lac-pro (F'128) in a reaction that requires the product of recA. The stimulation of this recombination by ferB also requires an active ferA gene, which implies that the ferA gene stimulates this reaction as well as precise excision. A ferA mutation was mapped at 84.2 kilobases on the F factor, and a ferB mutation was mapped at 82.5 kilobases. The fer mutants were obtained by an approach that permits the isolation of mutants affecting precise excision.

Chromosome Mapping↗
PubMed1985

A selective screen for transposable element mobilization in Drosophila melanogaster.

A selective system is described that provides a simple and sensitive assay for the detection and analysis of induced mobile element transpositions in Drosophila melanogaster. The system will detect a single event in samples greater than 10(6) and thus provides a eucaryotic assay system for monitoring the induction of transposition by a variety of agents including, but not limited to, chemical carcinogens and toxins, ionizing radiation, and various environmental pollutants. The experimental system focuses on an X-linked rosy+ transposon and a conditional lethal system that permits the detection of a single transposition event in very large samples. The results of a pilot experiment utilizing this system are presented.

Animals↗
PubMed2006

RNAi: a defensive RNA-silencing against viruses and transposable elements.

RNA silencing is a form of nucleic-acid-based immunity, targeting viruses and genomic repeated sequences. First documented in plants and invertebrate animals, this host defence has recently been identified in mammals. RNAi is viewed as a conserved ancient mechanism protecting genomes from nucleic acid invaders. However, these tamed sequences are known to occasionally escape this host surveillance and invade the genome of their host. This response is consistent with the overall idea that parasitic sequences compete with cells to systematically counter host defences. Using examples taken from the current literature, we illustrate the dynamic move-countermove game played between these two protagonists, the host cell and its parasitic sequences, and discuss the consequences of this game on genome stability.

Animals↗
PubMed1983

Heteroduplex analysis of P-plasmid evolution: the role of insertion and deletion of transposable elements.

DNA homology of thirteen R-plasmids of group P was examined by heteroduplex analysis and Southern blotting. Ten of these plasmids showed homology for extensive regions including all genes reported as necessary for replication and conjugational transfer. The differences between these plasmids could be explained by gain or loss of DNA sequences, many of which have been shown to be transposons. Of the other three plasmids, two showed unambiguous homology with the typical P-plasmids but this homology was imperfect, implying that these plasmids are products of lines which have evolved separately for long periods. One plasmid failed to produce heteroduplexes with the reference P plasmid.

Anti-Bacterial Agents↗
PubMed1998

Pattern of chromosomal localization of the Hoppel transposable element family in the Drosophila melanogaster subgroup.

We have isolated a Hoppel-like transposon from heterochromatin of the second chromosome of Drosophila melanogaster and used a conserved DNA sequence between the different elements of this family to determine their distribution in both mitotic and polytene chromosomes. The hybridization pattern of polytene chromosomes extends throughout the entire chromocentre, as well as a substantial portion of the fourth chromosome. Analysis of different wild-type strains of D. melanogaster shows variation in euchromatic insertion sites, although most insertions are found near the chromocentre. The positions and the number of heterochromatic clusters of Hoppel on mitotic chromosomes are conserved among the several strains analysed. Accurate mapping of this element was achieved by in situ hybridization on D. melanogaster mitotic chromosomes that had previously been banded with Hoechst 33258. To evaluate the evolutionary stability of this pattern, different species were analysed by in situ hybridization and Southern blotting. We conclude that Hoppel has a conserved distribution in mitotic heterochromatin within the D. melanogaster subgroup, established around 5 million years ago. The overall conservation of heterochormatic organization supports the notion that heterochormatin does perform important structural and functional roles.

Animals↗