Transposition bursts in analysis of repeated mutations in unstable strains of Drosophila melanogaster

The phenomenon of transposition memory was earlier demonstrated for the cut locus and mdg4. This work has been aimed at finding out, in what way the transposition memory can be realized. An unstable stock cmMR17ctMRpN17 was analysed which had high frequency of double cm+ct+ reversions and cmMRctMRpN repeated mutations. A series of five such transpositions could be followed. The ctMRpN17 mutation is a result of insertion at the cut locus mdg4 with the jockey element inserted within it. As seen from in situ hybridization analysis, transitions to the normal phenotype correlate, as a rule, with the excision of mdg4 and the jockey from the cut locus. Analysis of distribution of mdg1, mdg2, mdg3 and jockey on the X-chromosome of unstable revertants and repeated mutants indicated that not only transpositions of mdg4 and jockey, but also those of all mobile elements tested occur. So, we propose that the transposition memory in our genetic system is manifested in the process of transposition bursts.     

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.     

A novel transposition system in Drosophila melanogaster depending on the Stalker mobile genetic element.

Crosses between the Drosophila melanogaster y2sc1waG strain or some of its derivatives and the FM4 strain yielded insertional mutagenesis with a frequency of 10(-3)-10(-4). The system differs in several respects from the known cases of hybrid dysgenesis: (i) it does not depend on the direction of a cross; (ii) destabilization continues for a long time after initial crosses; (iii) mutations may occur at different stages of development. The mutation in the yellow locus has been cloned and found to depend on insertion into the coding region of the gene of a novel mobile genetic element designated as Stalker. The sequencing of Stalker termini reveals 405 bp direct repeats (LTRs) and a target 3 bp duplication, as well as some other sequences typical of retrovirus-like retrotransposons. The number of Stalker copies per genome and chromosomal localization vary among D. melanogaster strains. Before crosses, the location of Stalker on chromosomes is fairly stable in a particular strain but thereafter numerous changes in Stalker distribution take place. Most novel substrains are internally heterogenous which is indicative of the continuing Stalker transposition. Other mobile elements tested do not move. Possibly, only Stalker is mobilized in the system. Many known and novel mutations have been obtained. Comparison of their genetic localization with Stalker distribution suggests that the majority of them have been induced by the Stalker insertion.     

Genetic instability and transposition of mobile element mdg4 in a mutator strain of Drosophila melanogaster

Laboratory mutator strain of Drosophila melanogaster is characterized by increased (up to 10(-3)-10(-4) frequency of spontaneous mutability. Mutations appear in premeiotic stages of gametes development. The majority of mutations were unstable (high frequencies of reversions, appearance of new mutations at the same and other loci, replicating instability). Localization of mobile elements mdg1, mdg2, mdg3, mdg4, copia and P element in X chromosomes of mutator individuals and its mutations y, ct, sbt was studied by hybridization in situ. In all strains P element was absent. The distribution of mdg1, mdg2, mdg3 and copia was identical in mutator strains and its derivatives, but distribution of mdg4 was different. The essential heterogeneity in localization of mdg4 and increased (up to 30-40) copy number in the mutator strain individuals was observed. The ability of single element mdg4 to autonomous transpositions was thus shown.     

Mobile dispersed genetic elements in eukaryotes and their possible relationship to carcinogenesis

During last three years, the mobile dispersed genetic elements (mdg) were isolated from the genome of Drosophila melanogaster, yeasts and mammals. According to a number of their properties, mdg elements are quite similar to endogenous pro-retroviruses. It is known that in many cases oncogeneity of retroviruses depends on the incorporation of the certain host genes (potential oncogenes) into the viral genome. We suggest that in some cases mdg elements could entrap the potential oncogenes in the course of transposition. As a result, oncogenes become uncontrollable by host regulatory systems and may induce cell transformation. Another possible mechanism underlying switch off of the gene responsible for differentiation control may be mdg transposition to a region in close vicinity of the gene. As transposition of mdg elements seems to occur rather often, they may be regarded as one of the most important factors of genome rearrangements leading to cell transformation.     

The nature of unstable insertion mutations and reversions in the locus cut of Drosophila melanogaster: molecular mechanism of transposition memory

The segment of the locus cut containing the mobile genetic element mdg4 (gypsy) insertions which induce unstable ct^MR2 and ct^MRpN10 mutations has been cloned. Both mutations depend on the insertion of mdg4 into the same sequence, which coincides with that in ct⁶ allele. The ct^MRpN10 mutation differs from ct^MR2 by additional insertion of a novel mobile element jockey into mdg4. Jockey is 2.8 kb long, represented by ˜2–100 copies per genome, very homogeneous and lacks long terminal repeats (LTRs). The excision of mdg4 takes place in stable ct⁺ reversions. On the other hand, a complete single LTR is retained in the case of unstable ct reversions characterized by a high level of reverse directed transpositions of mdg4 into the locus cut. The LTR serves as a guide for reinsertion of mdg4 itself or mdg4 with jockey into the same site of the genome. A possible mechanism of transposition memory (homologous recombination with extrachromosomal circular DNA) is discussed.     

Superunstable systems in Drosophila melanogaster: analysis of mutations in the ocelliless locus

Six super-unstable mutations were obtained after crossing the M-cytotype strain of Drosophila melanogaster, where transpositions of the Stalker mobile element occur, with the pi 2 strain. One of these mutations appeared at the ocelliless locus. Detailed genetic analysis of this mutation and its numerous super-unstable derivatives was performed. Eleven allelic classes with different expression of the oc phenotype were isolated. The main features of super-instability at the ocelliless locus have been described and found to be common for all super-unstable systems obtained in our experiments. The role of the P element in the induction of super-instability is discussed.     

Evidence for horizontal transfer of the LTR retrotransposon mdg3, which lacks an env gene

Horizontal (interspecific) transfer is regarded as a possible strategy for the propagation of transposable elements through evolutionary time. To date, however, conclusive evidence that transposable elements are capable of horizontal transfer from one species to another has been limited to class II or DNA-type elements. We tested the possibility of such transfer for several Drosophila melanogaster LTR retrotransposons of the gypsy group in an experiment in which D. melanogaster and D. virilis somatic cell lines were used as donor and recipient cells, respectively. This approach was chosen in light of the high levels of LTR retrotransposon amplification and expression observed in cultured D. melanogaster cells. In the course of the experiment, parallel analysis for mdg1, mdg3, 17.6, 297, 412 and B104/roo retrotransposons was performed to detect their presence in the genome of recipient cells. Only the mdg3 retrotransposon, which lacks an env gene, was found to be transmitted into recipient cells. This model, based on the use of cultured cells, is a promising system for further investigating the mechanisms of LTR retrotransposon transfer.     

Mobile dispersed genetic element MDG1 of Drosophila melanogaster: structural organization.

The whole-length mobile dispersed genetic element mdg1 has been cloned from D. melanogaster genome. It contains DNA fragments described earlier as Dm225 and Dm234, Mdg1 is 7.2 kb long and framed with two direct repeats of 300-400 base pairs each. Mdg1 family is represented by about 25 copies in the genome of flies and by 200 copies in the genome of cultured cell line 67J25D. Virtually all the copies in the genome of D. melanogaster have the same restriction map. Oligo(dA)-oligo(dT) regions were found within mdg1.     

Transpositional bursts and chromosome rearrangements in unstable lines of Drosophila

The phenomenon of transpositional bursts-massive simultaneous transpositions of mobile elements belonging to different structural classes and accompanied by multiple mutagenesis were earlier described. Although the mechanisms of this phenomenon are still unclear, it is obvious now that it embraces total genome and includes not only transpositions of different mobile elements but also recombination processes--homologous recombination for LTR's and gene conversion. It is shown in this work that transpositional bursts may be accompanied by appearance of grass chromosomal rearrangements. The chain of closely related mutations which is characterized, as well as pedigrees described earlier, by coordinated mutational transitions and multiple transpositions of mdg1, mdg2 and retrotransposon jokey was analyzed. Spontaneous appearance of mutations in the loci yellow, white (deficiency for 462 kb) and cut (insertion of mdg4, together with jokey) correlates with appearance of inversion In(I), 14A-20B, and the reversions of these mutations to the wild type (y+w+ct+) or to other alleles (ctMR2--insertion of mdg4 without jokey) are accompanied by reversions of inversion. The relationship of all lines analyzed in this work as well as the lines from other pedigrees was proved using analysis of polymorphic restriction sites at the scute and cut loci (5 probes were used). All "y w ctpN"--type mutants are shown to have insertion of about 7 kb at the scute locus which causes no alteration of phenotype. This once again proves multiple and coordinated character of changes taking place during hybrid dysgenesis.     

The Drosophila mobile element jockey is a LINE element and contains coding sequences homologous to retroviral proteins

A detailed investigation of Drosophila melanogaster mobile dispersed repetitive element jockey is performed. Its structural features resemble those of LINE elements. Sequencing of the complete jockey 5020 bp in length revealed two long open reading frames ORF1 and ORF2 overlapping with a frameshift-1. Judging by amino acid homologies, ORF1 encodes a nucleic acid binding protein, characteristic of replication competent retroviruses; the 3' part of ORF2 encodes an RNA-dependent DNA polymerase which has an amino acid sequence, similar to recently published sequences of LINE elements of Drosophila, Trypanosoma and mammals. This fact demonstrates their evolutionary relationship. Sequencing of several deleted copies of jockey revealed the absence of the major part of ORF2, though the rest of the element, including the ends, is highly conservative.