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PubMed2004

Tn5 as an insect gene vector.

The purpose of this study was to explore alternatives to insect-derived transposable elements as insect gene vectors with the intention of improving existing insect transgenesis methods. The mobility properties of the bacterial transposon, Tn5, were tested in mosquitoes using a transient transposable element mobility assay and by attempting to create transgenic insects. Tn5 synaptic complexes were assembled in vitro in the absence of Mg(2+) and co-injected with a target plasmid into developing yellow fever mosquito, Aedes aegypti, embryos. Target plasmids recovered from embryos a day later were screened for the presence of Tn5. Recombinants (transposition events) were found at a frequency of 1.2 x 10(-3). Some transposition events did not appear to be associated with canonical 9 bp direct duplications at the site of insertion and also were associated with either deletions or rearrangements. A Tn5 element containing the brain-specific transgene, 3 x P3DsRed, was assembled into synaptic complexes in vitro and injected into pre-blastoderm embryos of Ae. aegypti. Of the approximately 900 embryos surviving injection and developing into adults, two produced transgenic progeny. Both transgenic events involved the co-integrations of approximately five elements resulting in nested and tandem arrayed Tn5::3 x P3DsRed elements. This study extends the known host range of Tn5 to insects and makes available to insect biologists and others another eukaryotic genome-manipulation tool. The hyperactivity of synaptic complexes may be responsible for the unusual clustering of elements and managing this aspect of the element's behavior will be important in future applications of this technology to insects.

Aedes↗
PubMed1990

Identification of a potential RNA intermediate for transposition of the LINE-like element I factor in Drosophila melanogaster.

The I factor, a transposable element related to mammalian LINEs, controls the I-R system of hybrid dysgenesis in Drosophila melanogaster. It transposes at high frequency in the germ-line of the female progeny of crosses between females of the reactive class of strains and males of the inducer class. The structure and DNA sequence of the I factor suggest that it transposes by reverse transcription of an RNA intermediate. Northern blot and S1 mapping experiments show that a full-length RNA of the I factor is synthesized specifically in the conditions of which I factors transpose. This RNA has all characteristics of a transposition intermediate. It is only found in the ovaries of dysgenic females suggesting that I factor activity is restricted to this tissue because of regulation at the level of the initiation of transcription or RNA stability.

Animals↗
PubMed1994

Mariner: its prospects as a DNA vector for the genetic manipulation of medically important insects.

Originally described in Drosophila mauritiana, the mariner transposable element has very recently been identified in 63 other insect species, representing ten insect orders, and including the malaria-vector mosquito, Anopheles gambiae. Ann Warren and Julian Crampton here discuss how transposable elements can be exploited as valuable research tools for the molecular characterization of genomes and as DNA vectors for genome manipulation and the 'creation' of transgenic organisms.

Journal Article↗
PubMed1997

Expression of intracisternal A-particle retrotransposons in primary tumors of oncogene-expressing transgenic mice.

Intracisternal A-Particle (IAP) sequences are endogenous retrovirus-like mobile elements, present at 1000 copies in the mouse genome. These elements transpose in a replicative manner via an RNA intermediate and its reverse transcription, and their transposition should therefore be tightly controlled by their transcription level. The in vivo pattern of expression of these potentially mutagenic elements had previously been analysed in normal mice, and we have now investigated their expression in transgenic mice carrying different oncogenes (e.g. c-myc, v-Ha-ras, SV40 T-antigen) under tissue-specific promoters and disclosing tumors within the brain, the mammary or salivary glands, or the lymphoid organs. Northern blot analysis of IAP expression within the resulting tumors demonstrates a lack of significant and/or systematic effect of v-Ha-ras and SV40 T-antigen expression, but a systematic IAP induction in the myc-induced tumors. In this case, however, analysis of double transgenic mice obtained by crossing the tumor-prone mice with previously described transgenic mice carrying IAP reporter genes did not provide any evidence for induction of the IAP transgenes, therefore strongly suggesting that c-myc expression had an effect on only a limited number of IAP sequences--most probably depending on their position and/or methylation state. These results strengthen the importance of in vivo studies for a correct appraisal of complex biological processes, and moderate previous conclusions derived from in vitro analyses on the general activation of IAPs by oncogenes and on the role of these transposable elements in tumorigenesis.

Animals↗
PubMed1989

Mobility of the maize suppressor-mutator element in transgenic tobacco cells.

Maize Suppressor-mutator (Spm) transposable elements have been introduced into tobacco cells and a visual assay for Spm activity has been developed using a bacterial beta-glucuronidase gene. The Spm element is mobile in tobacco and can trans-activate excision of a transposition-defective Spm (dSpm) element either from a different site on the same transforming Ti plasmid or from a second plasmid. An Spm element expressed from the stronger cauliflower mosaic virus 35S promoter trans-activates transposition of a dSpm element earlier after its introduction into tobacco cells than an element expressed from its own promoter.

Base Sequence↗
PubMed1996

Reduced germline mobility of a mariner vector containing exogenous DNA: effect of size or site?

Germline mobilization of the transposable element mariner is severely inhibited by the insertion of a 4.5- to 11.9-kb fragment of exogenous DNA into a unique SacI site approximately in the middle of the 1286-bp element. In the presence of transposase driven by the germline-specific hsp26-sgs3 promoter, mobilization of the MlwB construct (containing a 11.9-kb insertion) is detected at low frequency. Analysis of a mobilized MlwB element indicated that mobilization is mediated by the mariner transposase. However, transposed MlwB elements are also defective in germline mobilization. Rare, transposase-induced germline excision events were also recovered for such vectors. The estimated rate of excision is < 0.1% per chromosome per generation. Excision appears to be accompanied by gap repair if a suitable template is available. The data imply that the reduced mobility of mariner vectors with exogenous DNA in the SacI site results from disruption of sequences necessary for efficient mobilization. The relative stability may be a valuable property in the uses of mariner-like elements in genetic engineering of insects of economic importance.

Animals↗
PubMed1992

I elements and the Drosophila genome.

LINEs are a large class of transposable elements in eukaryotes. They transpose by reverse transcription of an RNA intermediate. I elements of Drosophila melanogaster belong to this class and are responsible for the I-R system of hybrid dysgenesis. Many results indicate that at the beginning of the century natural populations of this species were devoid of active I elements and that they were invaded by functional I elements in the last decades. Many Drosophila species contain both defective and active I elements. It seems that the latter were lost in Drosophila melanogaster before its spread throughout the world, and that the recent invasion results from the spread of functional elements originating either from another species by horizontal transfer or from an isolated population of the same species. These data are discussed, as well as their significance in evolutionary processes.

Amino Acid Sequence↗
PubMed1989

Extrachromosomal circular DNAs in Drosophila melanogaster: comparison between embryos and Kc0% cells.

We established the size distribution of extrachromosomal covalently closed circular DNA molecules from embryos of various Drosophila melanogaster strains and from Kc0% tissue culture cells. In embryos, more than 80% of the circular DNA molecules are smaller than 2.5 kb and all the distributions show a peak of molecules of between 200 and 400 bp. The Kc0% cell distribution differs mainly from that of embryos in that 48% of the molecules have a size between 4 and 8 kb. Correlating with this, circular molecules homologous to copia, 412 and 297 were detected only in Kc0% cells. The three tandemly repeated families containing the 5S genes, the histone genes and the 240 bp repeat of the ribosomal DNA intergenic spacer, which had previously been identified in circular DNAs from embryos, were also found in cultured cells. A fourth tandemly repeated family corresponding to the 1.688 g/cm3 satellite DNA was detected, both in embryos and Kc0% cells. It consists of circular multimeric molecules containing multiple copies of the 359 bp repeated unit. No circular DNA molecules homologous to the actin genes, the type I ribosomal DNA insertion, or the F and I transposable elements were found in embryos or Kc0% cells. Thus it appears that the extrachromosomal circular DNA molecules from embryos and from tissue culture cells differ mainly in the presence of circular copies of the copia-like transposable elements.

Animals↗
PubMed1995

IS1500, an IS3-like element from Leptospira interrogans.

Copies of an insertion-sequence (IS)-like element were isolated from two closely related serovars of Leptospira interrogans sensu stricto. Nucleotide sequence analysis of the 1236 bp element showed a characteristic IS structure with terminal imperfect inverted repeats (IRs) flanking a 1159 bp central region. This element was designated IS1500. Four open reading frames (orfA-orfD) were found in the central 'unique' region of IS1500. Similarities were detected between ORFA and ORFB and the putative transposases from members of the IS3 family of transposable elements. IS1500 or IS1500-like sequences were also detected in all other pathogenic leptospiral serovars, but not in the saprophytic species L. biflexa. Differences in IS1500 copy numbers in members of the same species suggest that this element can transpose. Physical mapping of IS1500 insertions in L. interrogans serovars icterohaemorrhagiae and pomona showed insertions were only on the large chromosomal replicon. The location of some IS1500 insertions coincides with regions of the genome that have undergone large rearrangements.

Amino Acid Sequence↗
PubMed1992

Distribution and structure of cloned P elements from the Drosophila melanogaster P strain pi 2.

P transposable elements of Drosophila melanogaster cloned from the strong P strain pi 2 have been analysed. The structures and chromosomal locations of 26 of the 30-50 elements estimated to be present in pi 2 have been determined. At one location two elements are inserted 100 base pairs (bp) apart, and in a second location two elements are only separated by the 8 bp duplicated upon P-element insertion. In addition to 2.9 kilobase-pair (kbp) elements, elements with 14 different internal deletions from 1.3 to 2.3 kbp in size have been isolated. There are 7 copies of the 2.9 kbp element, 2 copies each of 5 internally deleted elements and a single copy of 9 internally deleted elements. One of the elements found twice is the KP element, which may play a role in the regulation of hybrid dysgenesis in strains which contain many copies of this element. Apart from internal deletions the elements are extremely homogeneous in DNA sequence, with only 2 single base polymorphisms detected twice each in over 16 kbp of P-element sequence. Although transpositions are infrequent in an inbred P cytotype strain such as pi 2, the distribution of these cloned elements indicates that when the genomic library was made, the strain was polymorphic with respect to element location. The distribution and structures of the element are discussed with respect to models for regulation of P-element transposition.

Animals↗
PubMed2004

Shuffling of Sulfolobus genomes by autonomous and non-autonomous mobile elements.

Each of the sequenced Sulfolobus genomes contains large numbers of putatively mobile elements, both IS elements (insertion sequence elements) and MITEs (miniature inverted-repeat transposable elements). There are 344 in the 3.0 Mb genome of Sulfolobus solfataricus P2 and 95 in the 2.7 Mb genome of Sulfolobus tokodaii. In the former they constitute more than 10% of the genome. Experimental data suggest that transposition of IS elements occurs frequently. Moreover, the gene order between the two organisms differs greatly, indicating that multiple rearrangements have occurred. This has also led to considerable speculation as to how the cells are viable. Recently, a third Sulfolobus genome was completed which contains no IS elements or MITEs. This enabled us to compare the gene orders of the three genomes and provide evidence for mobile element-induced rearrangements of sections of the genomes.

DNA Transposable Elements↗
PubMed1995

Identification of a mariner element from the tsetse fly, Glossina palpalis palpalis.

In the present study, the polymerase chain reaction was used initially to demonstrate the presence of mariner sequences in seven species/subspecies of tsetse flies. DNA hybridization experiments show mariner sequences to be dispersed within the tsetse genome and that there are large variations in copy numbers among the various taxa. A genomic library was used to isolate and characterize a full-length mariner element from G. p. palpalis. The results indicate that this element is 1257 bp in length, flanked by two 32 bp inverted repeats differing at only one position, and belongs to the mellifera subfamily. The nucleotide sequence that is translated into a reading frame of 337 amino acids requires the introduction of two frame shifts and one stop codon to maximize sequence homology with a mariner element from Drosophila erecta. Based on this evidence, we conclude that the G. p. palpalis mariner element clearly represents a non-functional transposable element and that the protein product is not an active transposase.

Amino Acid Sequence↗
PubMed2000

Impact of multiple insertions of two retroelements, ZAM and Idefix at an euchromatic locus.

Transposable elements represent a large fraction of eukaryotic genomes and they are thought to play an important role in chromatin structure. ZAM and Idefix are two LTR-retrotransposons from Drosophila melanogaster very similar in structure to vertebrate retroviruses. In all the strains where their distribution has been studied, ZAM appears to be present exclusively in the intercalary heterochromatin while Idefix copies are mainly found in the centromeric heterochromatin with very few copies in euchromatin. Their distribution varies in a specific strain called RevI in which the mobilization of ZAM and Idefix is highly induced. In this strain, 15 copies of ZAM and 30 copies of Idefix are found on the chromosomal arms in addition to their usual distribution. Amongst the loci where new copies are detected, a hotspot for their insertion has been detected at the white locus where up to four elements occurred within a 3-kb fragment at the 5' end of this gene. This property of ZAM and Idefix to accumulate at a defined site provides an interesting paradigm to bring insight into the effect exerted by multiple insertions of transposable elements at an euchromatic locus.

Animals↗
PubMed2006

[Length polymorphism of integrated copies of R1 and R2 retrotransposons in the German cockroach (Blattella germanica) as a potential marker for population and phylogenetic studies].

Using polymerase chain reaction technique with primers flanking target sites of retrotransposons R1 and R2, integrated copies of these transposable elements were amplified in various cockroach species (Blattodea). It was shown that each species has a unique pattern of "5'-undertranscripts" with the definite set of amplified fragments of different lengths. Intraspecies polymorphism was revealed in analysis of German cockroach specimens obtained upon individual mating. This is the first report providing results of identifying, cloning, and sequencing extended fragments (5'-truncated copies) of Blatella germanica R1 and R2 retrotransposons. It may be assumed that patterns of 5'-truncated copies of R1 and R2 elements can be used as markers in population and phylogenetic studies. Moreover, cloned and sequenced fragments will be employed in our further studies for screening of the German cockroach genomic library in order to detect full-length copies in this class transposable elements.

Animals↗
PubMed1998

Characterization of Soymar1, a mariner element in soybean.

Mariner elements, a family of DNA-mediated transposable elements with short, inverted terminal repeats, have been reported in a wide variety of arthropods, as well as planarians, nematodes, and humans. No such element has been reported in a plant. Here we report a mariner element in the plant soybean (Glycine max (L.) Merr.). Although this sequence belongs to the mariner family, it is clearly distinct from previously reported mariner-like elements, as well as from the Tc1 transposon family. Novel aspects of its sequence could be useful as a starting point to identify mariner-like elements in new organisms, and it may prove useful in creating a transformation vector for plants.

Amino Acid Sequence↗
PubMed1992

Unusual features of the retroid element PAT from the nematode Panagrellus redivivus.

The PAT retroid transposable elements differ from other retroids in that they have a 'split direct repeat' structure, i.e., and internal 300bp sequence is found repeated, about one half at each element extremity. A very abundant transcript of about 900 nt, the start of which maps to the preferentially deleted portion of PAT elements, is detected on total Panagrellus redivius RNA bearing Northern blots. A potentially corresponding ORF encodes a protein of 265 residues having a carboxy terminal Cystein motif, believed to be exclusively characteristic of the GAG protein in retoid elements. A much fainter, 1800nt long transcript, is also detected on Northern blots and maps slightly downstream of the first ORF. The predicted protein sequence of this region bears motifs typical of reverse transcriptase and RNaseH, as found in the Pol genes of retroid elements. Peptide motif similarities are greatest with the DIRS-1 element derived from Dictyostelium discoideum. The possibility of using PAT elements as transposon tagging system for Caenorhabditis elegans is discussed.

Amino Acid Sequence↗
PubMed1993

Characterization of an IS-like element from Mycobacterium tuberculosis.

A DNA sequence, present in members of the Mycobacterium tuberculosis complex, has been identified and characterized. The distribution of this DNA sequence among mycobacterial species was analysed by DNA hybridization and PCR experiments. As the sequence was detected only in bacteria belonging to the M. tuberculosis complex, it may be useful for the rapid discrimination of mycobacteria. Interestingly, the sequence has some characteristics of an insertion element (IS) and codes for a hypothetical protein with significant homologies to proteins encoded by several IS elements of other organisms, namely IS427 and IS869 from Agrobacterium tumefaciens, IS402 from Pseudomonas cepacia, Tn4811 from Streptomyces lividans and ISRm4 from Rhizobium meliloti. Together, these elements form a previously unrecognized family of transposable elements. This finding suggests the possibility of horizontal gene transfer between pathogenic mycobacteria and other organisms including Gram-negative plant-pathogenic bacteria.

Amino Acid Sequence↗
PubMed2002

Length distribution of long interspersed nucleotide elements (LINEs) and processed pseudogenes of human endogenous retroviruses: implications for retrotransposition and pseudogene detection.

Deciphering the human genome includes reliable identification and structural characterization of individual retrotransposon elements. The most active group of autonomous transposable elements, the long interspersed nuclear elements (LINE), transpose themselves as well as other RNAs, including those of human endogenous retroviruses (HERV). During this transposition, however, the LINE-encoded reverse transcriptase (RT) often abortively dissociates from the RNA template, leaving a prematurely terminated, 5' truncated copy. We have analyzed the length distributions of LINEs and of processed pseudogenes derived from HERV-W. As expected, we have found that the majority of 5' truncated LINEs and HERV-W processed pseudogenes show a prevalence of very short elements terminated close to the 3' end. On the other hand, the number of complete elements is far above the expectation. The characteristic distribution in both cases indicates two important conclusions: (i) dissociation of LINE RT from the template cannot be fully explained by low processivity of RT modelled as a stochastic, Poisson-type process. (ii) Currently cited numbers of pseudogenes within the human genome are underestimated, since a large percentage of pseudogenes are terminated in the 3' untranslated region and remain undetectable in translated homology searches of protein databases against the human genome.

Endogenous Retroviruses↗