Retrotransposon MDG4 and its role in genetic instability of a mutator strain of Drosophila melanogaster

This article summarizes the results of a ten-year study of genetic instability of a mutator strain of Drosophila melanogaster caused by transposition of the gypsy retrotransposon. The results of other authors working with an analogous system are analyzed. Possible mechanisms are suggested for the interaction of gypsy with the cell gene flamenco that participates in transposition control of this mobile element.     

Transposition of mobile elements gypsy (mdg4) and hobo in germ-line and somatic cells of a genetically unstable mutator strain of Drosophila melanogaster.

Summary Using the in situ hybridization technique, we have analysed the distribution of mobile elements in the X chromosomes of male offspring of individual mutator strain (MS) males crossed to attached-X females. The experiments demonstrate varying cytological localization of the mobile elements gypsy (mdg4) and hobo among different individuals. The other mobile elements investigated (mdgl, mdg3, 412, 297, copia, 17.6, Doc, H.M.S. Beagle, Springer, FB) display no changes in insertion sites. Such an experiment is equivalent to analysis of separate gametes of an MS individual. Thus, the ability of gypsy and hobo to transpose in germ-line cells is demonstrated directly. Transpositions occur at premeiotic stages of germ cell development, since they appear in clusters. Analysis of gypsy and hobo transposition events shows that they occur independently. The same experiment demonstrates that gypsy localization varies significantly between different salivary gland cells of an MS individual. Two types of gypsy hybridization sites can be distinguished: permanent sites, common to all cells, and additional ones varying between neighbouring salivary gland cells. These additional sites indicate gypsy transposition in somatic cells of the MS. Transposition of the hobo element in somatic cells has also been observed.     

Transpositions of mobile elements mdg4 (gypsy) and hobo in somatic and germ cells of a genetically unstable mutator strain of Drosophila melanogaster]

Analysis of distribution of the several families of mobile genetic elements has been performed. The analysis dealt with the X chromosomes of male progeny from the crosses of individual males of Mutator strain (MS) with attached-X females. The experimental results demonstrated different localization of the elements gypsy and hobo in the salivary gland squashes of different males-brothers. Location of other elements under study--mdg1, 412, mdg3, copia, 297, 17.6, Beagle, BS, Doc, FB, Springer--was invariant in all larvae. The analysis is equal to the study of transposition events at the level of gametes. Thus, doubtless, the capability of gypsy and hobo to transpose in germ cells of the MS individuals has been detected. Mobilization of the elements occurs at premiotic stages of gametes' development, as indicated by appearance of the clusters of transpositions. In the process of studies on coincidence of gypsy and hobo transposition acts, independent character of the elements' movement has been revealed. It has been detected in the same experiment that the distribution of the gypsy copies in different cells of the same salivary gland varies strongly. All hybridization sites were divided into two groups: "constant" sites common for all cells and "additional" ones, whose locations did not coincide in neighbouring cells of salivary gland. The existence of additional sites is major evidence of gypsy transpositions in somatic cells of MS. Transposition events have been as well discovered for hobo in somatic cells.     

Autonomous transposition of gypsy mobile elements and genetic instability in Drosophila melanogaster

Summary The laboratory imitator strain (MS) of Drosophila melanogaster is characterized by an elevated frequency of spontaneous mutation (10^–3–10^–4). Mutations occur in both sexes at premeiotic stages of germ cell development. The increased mutability is a characteristic feature of MS itself, since it appears in the absence of outcrossing. Most of the mutations arising in this strain are unstable: reversions to wild type, high frequency mutation to new mutant states and replicating instability were observed. We have investigated the localization of the transposable genetic elements mdg1, 412, mdg3, gypsy (mdg4), copia and P in the X chromosomes of the MS and in the mutant lines y, ct, sbt derived from it by in situ hybridization. The P element was not found in any of these strains. The distributions of mdg1, 412, mdg3 and copia were identical in the X chromosomes of the MS and its derivatives. However, the sites of hybridization with gypsy differ in the various lines tested. In the polytene chromosomes of MS animals significant variation in location and number of copies of the gypsy element was demonstrated between different larvae; copy numbers as high as 30–40 were observed. These results suggest autonomous transposition of gypsy in the MS genome while several other mobile elements remain stable.     

Participation of mobile element hobo in transposition events in the long-term instability system of Drosophila melanogaster]

The lines with an active hobo elements as well as those without any hobo fragments were hybridized with the y2sc1waG line. This resulted in the appearance of a number of mutations at the white, miniature, and some other loci. The authors analysed, in which way the hobo transposable elements take part in mutagenesis in these crosses. Most of the white mutants obtained were analysed and transpositions of hobo and Stalker elements were demonstrated. Both independent and simultaneous transpositions were found. It was shown by means of the Southern blot analysis that additional hobo or Stalker insertion into or close to the parental unknown waG insertion resulted in mutant white phenotype's shift toward both extreme and partial reversion. Possible participation in mutagenesis of other mobile elements is also under debate.     

Site-specific instability in Drosophila melanogaster: evidence for transposition of destabilizing element

In this study, we show that at least one lethal mutation at the 3F-4A region of the X chromosome can generate an array of chromosome rearrangements, all with one chromosome break in the 3F-4A region. The mutation at 3F-4A (secondary mutation) was detected in an X chromosome carrying a reverse mutation of an unstable lethal mutation, which was mapped in the 6F1-2 doublet (primary mutation). The primary lethal mutation at 6F1-2 had occurred in an unstable chromosome (Uc) described previously (LIM 1979). Prior to reversion, the fF1-2 doublet was normal and stable, as was the 3F-4A region in the X chromosome carrying the primary lethal mutation. The disappearance of the instability having a set of genetic properties at one region (6F1-2) accompanied by its appearance elsewhere in the chromosome (3F-4A) implies that a transposition of the destabilizing element took place. The mutant at 3F-4A and other secondary mutants exhibited all but one (reinversion of an inversion to the normal sequence) of the eight properties of the primary lethal mutations. These observations support the view that a transposable destabilizing element is responsible for the hypermutability observed in the unstable chromosome and its derivatives.     

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.     

Structural instability of 297 element in Drosophila melanogaster

297 element Southern pattern modifications previously detected in mutation accumulation lines of Drosophila melanogaster were further investigated by in situ hybridisation, Southern blotting with different combinations of genomic digest-probe, and PCR. Only one out of the nine pattern modifications studied could be interpreted as an excision and was detectable by in situ hybridisation to polytene chromosomes. Results were consistent with most pattern modifications being small rearrangements within the body of the element. In agreement with the existence of spontaneous rearrangements of this kind is the observation that many genomic copies of element 297 are defective and these are not limited to heterochromatin. These findings have important implications for the models of transposable element (TE) number regulation as well as for the study of genome evolution. This revised version was published online in July 2006 with corrections to the Cover Date.     

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.     

Interaction of mobile elements P and mdg3 in Drosophila melanogaster: genetic aspects

It was found earlier that two unstable sn mutants isolated from natural populations are connected with insertion of mobile element mdg3 into the 7D1-2 region where singed gene (1-21.0) is localised. From two original sn mutants, a series of unstable sn alleles, both mutant and normal for phenotype, was extracted. Then we studied, how they change the mutation rate in germinal and somatic cells of different hybrids with pi 2 stock having P cytotype and active P elements in the chromosomes. Addition of P chromosomes, independently of the background of cytoplasm, proved to reduce the sn instability. The level of sn mutability was decreased with increasing the dose of P chromosomes. It is suggested that mutation events are caused by transposition of mdg3 and that both mdg3 and P elements compete for the same cellular factor, capable of activation of transposition process.