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PubMed1984

Structural organization of transposable element mdg4 from Drosophila melanogaster and a nucleotide sequence of its long terminal repeats.

A mobile dispersed genetic element, mdg4 , approximately 7.5 kilobases (kb) long has been cloned from D. melanogaster genome. Chromosomal bands have only few sites of mdg4 , but it always hybridizes to the chromocenter. The location of mdg4 varies among D. melanogaster strains. Blot hybridization shows that, in contrast to other mdg elements, mdg4 sequences are rather heterogeneous. Only few copies are full-length. A strong amplification of mdg4 has occurred during the in vitro cultivation of cells involving only one mdg4 variant. Long terminal repeats (LTRs) and flanking sequences have been sequenced in two cloned copies of transposable element mdg4 . In both cloned copies of mdg4 , LTRs have an identical nucleotide sequence 479 bp long. The mdg4 is flanked by four-base-pair direct repeats, short mismatched palindromes being present at the ends of each LTR. The termini of the mdg4 body contain an oligopurine stretch and a region partially complementary to D. melanogaster tRNA-Lys. Thus, structural organization of mdg4 LTRs is similar to that of several other mdg elements and retroviral proviruses.

Animals↗
PubMed1993

The sex determining region of Chironomus thummi is associated with highly repetitive DNA and transposable elements.

The dominant male sex determiner in chromosome III of the midge Chironomus thummi thummi is closely linked to a large cluster of tandem-repetitive DNA elements, the Cla elements, which are otherwise highly repetitive and distributed over more than 200 sites on all chromosomes. Chromosome III displays a hemizygous cluster of Cla elements in males but not in females. The chromosomal location of this hemizygous Cla element cluster is in the region of the male determiner M as localized by cytogenetic analysis. With Cla elements as hybridization probe, it was possible to clone a large part of the sex determining region. Molecular analysis of the DNA of males and females in this region displayed a number of differences between the two sexes. One striking difference is an unusual transposable element associated with the male sex determining region. The sex determining region also contains several other tandem-repetitive DNA elements in addition to the Cla elements. They are interspersed with single copy DNA. The accumulation of repetitive elements in the sex determining region interpreted as the result of a lack of recombination between the male/female heteromorphic region, although recombination in the other sections of chromosome III occurs.

Animals↗
PubMed1987

Cloned extrachromosomal circular DNA copies of the human transposable element THE-1 are related predominantly to a single type of family member.

The 2300 base-pair transposon-like human element, THE-1, has been identified in the extrachromosomal circular DNA of the established human cell line HeLa as a relatively homogeneous population of covalently closed 1900 base-pair molecules. THE-1, which has been classified tentatively as a retroviral-like transposable element (a retrotransposon), is present in the extrachromosomal circular DNA of African green monkey (BSC-1) and human lymphoblastoid (Jurkat) cell lines. The 1900 base-pair extrachromosomal elements isolated and cloned from HeLa cells (1) appear to contain only THE-1-specific nucleotide sequences, (2) are circularized versions of the linear chromosomal sequence, and (3) are related predominantly to a single, or single type of, family member.

Autoradiography↗
PubMed1998

Transmission pattern of hobo transposable element in transgenic lines of Drosophila melanogaster.

This study is an attempt to trace the fate of hobo elements in the genomes of E strains of Drosophila melanogaster that have been transfected with pHFL1, a plasmid containing an autonomous hobo. Such long-term population studies (over 105 generations) could be very useful for better understanding the population and genomic dynamics of transposable elements and their pattern of insertions. Molecular analyses of hobo elements in the transfected lines were performed using Southern blots of XhoI-digested genomic DNAs. The complete element was observed in all six injected lines. In two lines we observed, at generation 100, two deleted elements, which did not correspond to Th1 and Th2. The results obtained by the in situ method show that the number of hybridization sites increases in each line and prove that the hobo element may be amplified in an RM genome. The hobo activity does not seem to be systematically correlated with the number of hobo elements. After generation 85, the evolution of the hobo element's insertion site number depends on the injected line. In all lines, the total number of insertions remains quite small, between 0 and 11. Hobo elements are located on each of the chromosomal arms. We describe 'hotspots'-insertion sites present in all lines and in all generations. On the 3R arm, a short inversion appeared once at generation 85.

Animals↗
PubMed2025

Horizontally transferred cell-free chromatin particles function as autonomous 'satellite genomes' and vehicles for transposable elements within host cells.

Horizontal gene transfer (HGT) plays an important evolutionary role in prokaryotes, but it is less frequent in mammals. We previously reported that cell-free chromatin particles (cfChPs) - chromosomal fragments released from the billions of dying cells that circulate in human blood - are horizontally transferred to healthy cells with biological effects. However, the underlying mechanism and function of these effects remained unclear. We treated NIH3T3 mouse fibroblasts cells with cfChPs isolated from human serum and serially passaged the cells. The intracellular activities of cfChPs were analysed using chromatin fibre fluorography, cytogenetic analysis, immunofluorescence, and fluorescent in situ hybridisation. We discovered that the internalised cfChPs were almost exclusively comprised of non-coding DNA, and the disparate DNA sequences contained within them had randomly combined to form complex concatemers, some of which were multi-mega base pairs in size. The concatemers autonomously performed many functions attributable to the nuclear genome such as DNA, RNA and protein synthesis. They harboured human LINE-1 and Alu elements, with the potential to rearrange themselves within the mouse genome. Our results suggest that a cell simultaneously harbours two autonomous genome forms: one that is inherited (hereditary genome) and numerous others that are acquired (satellite genomes). The satellite genomes may have evolutionary functions given their ability to serve as vehicles for transposable elements and to generate a plethora of novel proteins. Our results also suggest that 'within-self' HGT may occur in mammals on a massive scale via the medium of cfChP concatemers that have undergone extensive and complex modifications resulting in their behaviour as 'foreign' genetic elements.

Chromatin↗
PubMed2000

Germ-line transformation of pink bollworm (Lepidoptera: gelechiidae) mediated by the piggyBac transposable element.

The pink bollworm, Pectinophora gossypiella, is a world-wide pest of cultivated cotton. In certain growing regions populations are suppressed by a sterile release strategy. Efforts to improve the sterile insect technique as well as our understanding of lepidopteran biology could benefit greatly from a germ-line transformation system. We report transformation of pink bollworm with a piggyBac transposable element carrying the enhanced green flourescent protein (EGFP) marker gene. This vector-marker system resulted in recovery of transgenics at a rate of approximately 3.5%. Integration of the transforming construct that was typical of piggyBac was demonstrated by Southern analysis and sequence determination of transposon flanks. Expression of the EGFP marker was visualized by fluorescent microscopy and Western Blot analysis. Maintenance of transformed strains indicates that the transgene segregates in a Mendelian fashion and has been stable over fourteen generations to date.

Animals↗
PubMed1980

Sequence of retrovirus provirus resembles that of bacterial transposable elements.

The nucleotide sequences of the terminal regions of an infectious integrated retrovirus cloned in the modified lambda phage cloning vector Charon 4A have been elucidated. There is a 569-base pair direct repeat at both ends of the viral DNA. The cell-virus junctions at each end consist of a 5-base pair direct repeat of cell DNA next to a 3-base pair inverted repeat of viral DNA. This structure resembles that of a transposable element and is consistent with the protovirus hypothesis that retroviruses evolved from the cell genome.

Base Sequence↗
PubMed1988

Reversion of a gypsy-induced mutation at the yellow (y) locus of Drosophila melanogaster is associated with the insertion of a newly defined transposable element.

To understand the molecular basis of the phenotype of gypsy-induced mutations, we have analyzed the structure of phenotypic revertants of the y2 allele, which is caused by the insertion of the gypsy element into the 5' region of the yellow (y) locus. Seven spontaneous revertants examined fall into two different classes. Three of these revertants arose by homologous recombination between the two gypsy long terminal repeats (LTRs), leaving behind a solo LTR. Four additional revertants contain an intact 3' LTR and half of the 5' LTR, but the central portion of gypsy has been replaced by a different 6.5-kilobase transposable element that contains a poly(A) tail. These results suggest that the mutagenic effect of the gypsy element is not due to its insertion into sequences necessary for transcription or to the distancing between the yellow promoter and remote regulatory sequences but is a consequence of idiosyncratic properties of the element itself.

Animals↗
PubMed1993

Effects of transposable elements on the expression of the forked gene of Drosophila melanogaster.

The products of the forked gene are involved in the formation and/or maintenance of a temporary fibrillar structure within the developing bristle rudiment of Drosophila melanogaster. Mutations in the forked locus alter this structure and result in aberrant development of macrochaetae, microchaetae and trichomes. The locus has been characterized at the molecular level by walking, mutant characterization and transcript analysis. Expression of the six forked transcripts is temporally restricted to mid-late pupal development. At this time, RNAs of 6.4, 5.6, 5.4, 2.5, 1.9 and 1.1 kilobases (kb) are detected by Northern analysis. The coding region of these RNAs has been found to be within a 21-kb stretch of genomic DNA. The amino terminus of the proteins encoded by the 5.4- and 5.6-kb forked transcripts contain tandem copies of ankyrin-like repeats that may play an important role in the function of forked-encoded products. The profile of forked RNA expression is altered in seven spontaneous mutations characterized during this study. Three forked mutations induced by the insertion of the gypsy retrotransposon contain a copy of this element inserted into an intron of the gene. In these mutants, the 5.6-, 5.4- and 2.5-kb forked mRNAs are truncated via recognition of the polyadenylation site in the 5' long terminal repeat of the gypsy retrotransposon. These results help explain the role of the forked gene in fly development and further our understanding of the role of transposable elements in mutagenesis.

Amino Acid Sequence↗
PubMed1988

Examination of herpes virus DNA sequences for patterns that resemble transposable elements.

Herpes viruses are associated with many types of oral tumors and can be tumorigenic in vitro. The mechanisms by which they transform cells remain unknown, but it has been suggested that they contain insertion sequences. Two algorithms were developed to search DNA sequences for the presence of patterns that characterize such elements using either selection or scoring techniques. When randomly shuffled herpes virus sequences were examined each algorithm detected many such patterns but the scoring algorithm found fewer than the selection algorithm. In the herpes virus sequences that are known to contain such patterns the computer techniques found many more than previously reported, indicating that visual inspection alone is not adequate. The frequency of these patterns suggest that although the transforming regions of herpes virus DNA could contain transposable elements experimental confirmation is necessary.

Algorithms↗
PubMed1995

Genotypic effects, maternal effects and grand-maternal effects of immobilized derivatives of the transposable element mariner.

The baseline rate of spontaneous integration of the autonomous mariner element Mos1 into the germline of Drosophila melanogaster is estimated as 16 +/- 5% (mean +/- SE) among fertile G0 flies. However, the transformation rate is reduced approximately 20-fold in Mos1 constructs with exogenous DNA in the size range 5-12 kb inserted into the SacI site. To provide alternative Mos1 helper plasmids for transformation experiments, two types of Mos1-promoter fusions were constructed: hsp-70:Mos1 and hsp26-Sgs3:Mos1. The former has the Mos1 coding region driven by the hsp70 heat-shock promoter; the latter has it driven by the basal Sgs3 promoter under the control of the hsp26 female-germline specific transcriptional regulator. When introduced into D. melanogaster by P-element-mediated germline transformation, these elements are unable to transpose or excise in the presence of autonomous Mos1-related elements (they are "marooned") because the 5' inverted repeat of Mos1 is missing. As expected, the hsp26-Sgs3:Mos1 fusions exhibit a significantly greater rate of germline excision of a target mariner element than do the hsp70:Mos1 fusions. Unexpectedly, the rate of excision of target mariner elements induced by hsp26-Sgs3:Mos1 is the same in the male germline as in the female germline. Both hsp:Mos1 fusions show strong germline expression and a maternal effect of the mariner transposase. A significant grand-maternal effect of the hsp:Mos1 fusions was also detected as a result of a maternal effect on the germline of the F1 progeny. Among flies carrying the promoter fusions inherited maternally, about three-quarters of the overall rate of germline excision derives from the direct genotypic effect and about one-quarter results from the grand-maternal effect. Despite the strong somatic expression of the hsp:Mos1 fusions, mariner transformants carrying a white+ reporter gene at the SacI site remained stable in the soma.

Animals↗
PubMed1994

The Doc transposable element in Drosophila melanogaster and Drosophila simulans: genomic distribution and transcription.

The mobile element Doc is similar in structure and coding potential to the LINE families found in various organisms. In this paper, we analyze the insertional and structural polymorphism of this element and show that it appears to have a long evolutionary history in the genome of D. melanogaster. Like the family of I elements, the Doc family seems to display three types of elements: full length elements, defective members that have recently transposed and long since immobilized members common to each D. melanogaster strain. These three classes of Doc elements seem to be present in D. simulans, a closely related species to D. melanogaster. Furthermore, we show that Doc is transcribed as a polyadenylated RNA of about 5 kb in length, presumed to be a full length RNA. This transcript is present in different tissues and at different stages of Drosophila development. These results are compared with previous records on the chromosomal distribution of LINEs or other transposable element families. Doc transcription is analyzed in an attempt to understand the link between Doc transcription and transposition.

Animals↗
PubMed1995

Characterization of a family of genes encoding a fruit-specific wound-stimulated protein of bell pepper (Capsicum annuum): identification of a new family of transposable elements.

Using a fruit-specific cDNA as a probe we isolated and sequenced the two corresponding homologous genes (Sn-1 and Sn-2) of the bell pepper (Capsicum annuum) genome. Both genes have a single intron and numerous unusual long inverted repeat sequences. The introns share 87% homology and Sn-2 contains one 450 bp additional sequence with structural features of a transposable element, which is highly repetitive in the bell pepper genome. Surprisingly, analysis in data banks showed that genes encoding the potato starch phosphorylase (EC 2.4.1.1) and patatin contain a similar element, named Alien, in their 5'-upstream region. Alien elements are characterized by a conserved 28 bp terminal inverted repeat (TIR), small size, high AT content, potential to form stable DNA secondary structures and they have probably been inserted in TA target sites. Interestingly, the TIR of the Alien elements shares high homology with sequences existing in the TIR of extrachromosomal linear pSKL DNA plasmid of Saccharomyces kluyveri. Northern blot analyses detected Sn-1 transcripts principally in the red fruit whereas no Sn-2 transcripts were detected in neither of the samples monitored. Western blot analyses detected a 16.8 kDa Sn protein principally in the ripe red fruit and wounded areas of green unripe fruit. A comparison of the deduced amino acid sequence of Sn-1 with protein sequences in data banks revealed a significant homology with proteins likely involved in the plant's disease resistance response. Analyses at the subcellular level showed that Sn-1 is localized in the membrane of vacuoles.

Amino Acid Sequence↗
PubMedDate not supplied

The MR P-type transposable elements and the genetic activities of mutagens and carcinogens in Drosophila melanogaster. I. N,N-Dimethylnitrosamine (DMN).

A technique was developed for the assay of the genetic activities of carcinogens in both the soma and germ line in the course of early larval development and to assess the extent of their modification through the introduction into the genome of the MR IInd autosome P-type transposable elements. The influence of MR on genotoxicity for a given treatment was indicated by the relative frequencies of non-MR (Cy) to MR-carrying sibs emerging within the same cultures. The viable genetic changes in the soma were classified as recombinational or mutational events on the basis of the comparative yields of mosaic sectors in females heterozygous for the markers y w sn carried in standard order (XS) or multiply inverted (XIn) X-chromosomes. The results with this technique are here described for the carcinogen DMN. In the absence of MR, the topical application of DMN induced no larval lethality up to the highest tested doses (20 mM), but raised the yields of the somatic sectors for all the test markers in the emerging females in accordance with a linear dose fit. Comparison of the XS and XIn data indicated that somatic mutagenesis by DMN entailed the induction of recombinational and mutational events in roughly equal proportions. The introduction of MR into the genome, whether patro- or matroclinously, resulted in dramatic and proportionately equivalent enhancements in the activities of DMN, both with respect to the induction of larval lethality and somatic sectoring. These activities increased exponentially with dose, following a 4th-degree polynomial course up to 10 mM, when larval lethality approached 100%. At lower dose levels, the yields of all sector types in the viable females also followed comparable polynomial curves at different heights, except for y sn in the XIn series, where sector recovery remained at the control level throughout the examined dose range. Analysis of the sector size distribution for eye and bristle mosaicism in the XS control and DMN-treated series gave statistically comparable mean values, irrespective of the presence or absence of MR, indicating that DMN alone, or in conjunction with the P elements, did not alter the timing of aberrant clone initiation. In contrast, estimates of the genetic induction events in the somatic primordia with 5 mM DMN indicated greatly increased response in the presence of MR, which was in excess of 20-fold for the mutational changes involving the sn locus.(ABSTRACT TRUNCATED AT 400 WORDS)

Animals↗
PubMed2006

Germline transformation of Aedes fluviatilis (Diptera:Culicidae) with the piggyBac transposable element.

The technique to generate transgenic mosquitoes requires adaptation for each target species because of aspects related to species biology, sensitivity to manipulation and rearing conditions. Here we tested different parameters on the microinjection procedure in order to obtain a transgenic Neotropical mosquito species. By using a transposon-based strategy we were able to successfully transform Aedes fluviatilis (Lutz), which can be used as an avian malaria model. These results demonstrate the usefulness of the piggyBac transposable element as a transformation vector for Neotropical mosquito species and opens up new research frontiers for South American mosquito vectors.

Aedes↗
PubMed1996

Germline transformation of Drosophila virilis mediated by the transposable element hobo.

A laboratory strain of Drosophila virilis was genetically transformed with a hobo vector carrying the miniwhite cassette using a helper plasmid with an hsp70-driven hobo transposase-coding sequence. The rate of transformation was 0.5% per fertile GO animal. Three transgenic insertions were cloned and characterized and found to be authentic hobo insertions. These results, together with the known widespread distribution of hobo in diverse insect species, suggest that hobo and related transposable elements may be of considerable utility in the germline transformation of insects other than D. melanogaster.

Animals↗
PubMed1980

Nucleotide sequence analysis of the long terminal repeat (LTR) of avian retroviruses: structural similarities with transposable elements.

The nucleotide sequences of the long terminal repeat (LTR) from six independently derived avian retrovirus recombinant DNA clones have been determined. The LTRs from three clones are approximately 350 bp in length and differ only in minor base insertions or substitutions. Three other clones have smaller LTRs, each with a large deletion which ranged from 89 to 161 bp. Sequence comparisons of the six LTRs indicate that there is conservation of sequences derived from the 5' terminus of viral RNA and extensive divergence of the 3'-specific sequences. The LTR sequences were obtained from clones of unintegrated viral DNA. Comparison of these LTRs with the sequence of an integrated Schmidt-Ruppin D provirus deduced previously reveals that two nucleotides present at the terminus of the LTR of the unintegrated DNA are absent in the integrated provirus. Analysis of the nucleotide sequence of the LTR from one clone, lambda RAV2-2, reveals several putative regulatory sites for the initiation and termination of transcription. There are also several structural features of the LTR which are analogous to procaryotic and eucaryotic transposable elements. These structural analogies include the presence of inverted complementary repeats at the termini of the LTR, deletions adjacent to LTR termini, and sequence homologies with transposable and other genetic elements. These observations suggest that the LTR of retroviruses function in the control of gene expression and integration.

Avian Leukosis Virus↗
PubMed1995

Effects of a transposable element insertion on alcohol dehydrogenase expression in Drosophila melanogaster.

Variation in the DNA sequence and level of alcohol dehydrogenase (Adh) gene expression in Drosophila melanogaster have been studied to determine what types of DNA polymorphisms contribute to phenotypic variation in natural populations. The Adh gene, like many others, shows a high level of variability in both DNA sequence and quantitative level of expression. A number of transposable element insertions occur in the Adh region and one of these, a copia insertion in the 5' flanking region, is associated with unusually low Adh expression. To determine whether this insertion (called R142) causes the low expression level, the insertion was excised from the cloned R142 Adh gene and the effect was assessed by P-element transformation. Removal of this insertion causes a threefold increase in the level of ADH, clearly showing that it contributes to the naturally occurring variation in expression at this locus. Removal of all but one LTR also causes a threefold increase, indicating that the mechanism is not a simple sequence disruption. Furthermore, this copia insertion, which is located between the two Adh promoters and their upstream enhancer sequences, has differential effects on the levels of proximal and distal transcripts. Finally, a test for the possible modifying effects of two suppressor loci, su(wa) and su(f), on this insertional mutation was negative, in contrast to a previous report in the literature.

Alcohol Dehydrogenase↗