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.     

Comparative analysis of the localization and mobility of retrotransposons in sibling species Drosophila simulans and Drosophila melanogaster]

The distribution of four retrotransposon families (MDG1, MDG3, MDG4 and copia) on polytene chromosomes of different (from 9 to 15) Drosophila simulans strains is studied. The mean number of MDG1 and copia euchromatic hybridization sites (3 sites for each element) is drastically decreased in D. simulans in comparison with D. melanogaster (24 and 18 sites respectively). The mean number of MDG3 sites of hybridization is 5 in D. simulans against 12 in D. melanogaster. As for MDG4 both species have on the average about 2-3 euchromatic sites. The majority of MDG1 and copia and about a half of MDG3 euchromatic copies are localized in restricted number of sites (hot spots) on D. simulans polytene chromosomes. In D. melanogaster these elements are scattered along the chromosomes though there are some hot spots too. It appears that euchromatic copies of MDG1 and copia are considerably less mobile in D. simulans in contrast to D. melanogaster. Some common hot spots of retrotransposon localization in D. simulans and D. melanogaster were earlier described as intercalary heterochromatin regions in D. melanogaster. The level of interstrain variability of MDG4 hybridization sites is comparable in both species. Comparative blot-analysis of adult and larval salivary gland DNA shows that MDG1 and copia are situated mainly in euchromatic regions of D. melanogaster chromosomes. In D. simulans genome they are located mainly in heterochromatic regions underreplicated in salivary gland polytene chromosomes. There are interspecies differences in the distribution of retrotransposons in beta-heterochromatic chromosome regions.     

Maintenance of the copy number of retrotransposon MDG3 in the Drosophila melanogaster genome]

The genomes of laboratory stocks and natural population of Drosophila melanogaster contain 8-12 copies of retrotransposon MDG3 detected by in situ hybridization. Construction of genotypes with decreased MDG3 copy number using X-chromosome and chromosome 3 free of MDG3 copies results in appearance of hybrid genomes carrying up to 7-10 copies, instead of 2-4 copies expected. New MDG3 copies are detected in different genome regions, including the 42B hot spot of their location. The chromosomes, where new clusters of MDG3 were observed, carry conserved "parental pattern" of MDG1 arrangement. The data obtained suggest the existence of genomic mechanism for maintenance of retrotransposon copy number on a definite level.     

Mobile dispersed genetic element MDG1 of Drosophila melanogaster: nucleotide sequence of long terminal repeats.

Long terminal repeats (LTRs) of two members of mdg1 family were sequenced. In the both cases, they are represented by perfect direct repeats 442 and 444 bp in length. Sixteen nucleotides in the LTRs of two different mdg1 elements are different. Each LTR contains slightly mismatched 16-nucleotide inverted repeats located at the ends of the LTR. Six base pairs closest to the termini of LTR form perfect inverted repeats. On the gene-distal sides of LTRs, short 4-nucleotide direct repeats are located, probably representing the duplication of a target DNA sequence arising from insertion of mdg. They are different in the two cases analyzed. Just as the other analyzed eukaryotic transposable elements, mdg1 starts with TGT and ends with ACA. Within the both strands of LTR, the sequences similar to Hogness box (a putative signal for RNA initiation, or a selector) and AATAAA blocks (putative polyadenylation signals) are present. The LTR of mdg1 contains many short direct and inverted repetitive sequences. These include a 10-nucleotide sequence forming a perfect direct repeat with the first ten nucleotides of the LTR. A region of LTR about 70 bp long is represented by simple repetitive sequences (TAT).     

Mobile genetic element MDG4 (gypsy) in Drosophila melanogaster. Features of structure and regulation of transposition]

Distribution of two structural functional variants of the MDG4 (gypsy) mobile genetic element was examined in 44 strains of Drosophila melanogaster. The results obtained suggest that less transpositionally active MDG4 variant is more ancient component of the Drosophila genome. Using Southern blotting, five strains characterized by increased copy number of MDG4 with significant prevalence of the active variant over the less active one were selected for further analysis. Genetic analysis of these strains led to the suggestion that some of them carry factors that mobilize MDG4 independently from the cellular flamenco gene known to be responsible for transposition of this element. Other strains probably contained a suppressor of the flam- mutant allele causing active transpositions of the MDG4. Thus, the material for studying poorly examined relationships between the retrovirus and the host cell genome was obtained.     

HeT-A telomere-specific retrotransposons in the centric heterochromatin of Drosophila melanogaster chromosome 3

We have isolated two yeast artificial chromosome (YAC) clones from Drosophila melanogaster that contain a small amount of dodeca satellite (a satellite DNA located in the centromeric region of chromosome 3) and sequences homologous to the telomeric retrotransposon HeT-A. Using these YACs as probes for fluorescence in situ hybridization to mitotic chromosomes, we have localized these HeT-A elements to the centric heterochromatin of chromosome 3, at region h55. The possible origin of these telomeric elements in a centromeric position is discussed. Received: 30 July 1999 / Accepted: 19 September 1999     

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.     

Evolution of errantiviruses of Drosophila melanogaster. Strategy 2: from retroviruses to retrotransposons

Drosophila melanogaster retrotransposons of the gypsy group are considered to be potential errantiviruses. Their infectivity is caused by the functional activity of the third open reading frame (ORF3) encoding the Env protein, which was probably captured from baculoviruses. Mobile genetic elements (MGEs) of the gypsy group can be conventionally divided into three subgroups: with three ORFs, with a defective ORF3, and without the ORF3. To establish the patterns of evolution of gypsy retrotransposons in D. melanogaster, the members of the three subgroups were examined. Structural analysis of retrotransposons opus and rover, which carry a defective ORF3, as well as retrotransposons Burdock, McClintock, qbert, and HMS-Beagle, which lack the ORF3, suggests that the evolution of these MGEs followed the pattern of loosing the ORF3. At the same time, an MGE of the same subgroup, Transpac, may be an ancestral form, which had acquired the env gene and gave rise to the first errantiviruses. The capture of the ORF3 by retrotransposons provided their conversion to a fundamentally new state. However, the ORF3 in the genome is not subjected to strong selective pressure, because it is not essential for intragenomic transpositions. Because of this, the process of its gradual loss seems quite natural.     

Heterochromatic regions in different Drosophila melanogaster stocks contain similar arrangements of moderate repeats with inserted copia-like elements (MDG1)

Seven out of twenty 30–50 kb genome fragments with an MDG1 copia-like element cloned in cosmids were found to carry homologous sequences which belong to a new family of non-mobile heterochromatic moderate repeats (the HMR family). These repeats along with the MDG1 copies inserted in them are under-replicated in polytene chromosomes. Such repeats may also be located in the intercalary heterochromatin site 12E of the X chromosome. Chromosomal heterochromatic regions are enriched with one of the two main genomic variants of MDG1, MDG1^het, identifiable by EcoRI restriction. From Southern DNA blot analysis the number of MDG1^het copies and their sites within the heterochromatin are invariant in all the stocks examined, while there is not a single MDG1 site along the polytene chromosomes shared by all the stocks in question.     

Mechanisms regulating the copy numbers of six LTR retrotransposons in the genome of Drosophila melanogaster

There has been debate over the mechanisms that control the copy number of transposable elements in the genome of Drosophila melanogaster. Target sites in D. melanogaster populations are occupied at low frequencies, suggesting that there is some form of selection acting against transposable elements. Three main theories have been proposed to explain how selection acts against transposable elements: insertions of a copy of a transposable element are selected against; chromosomal rearrangements caused by ectopic exchange between element copies are selected against; or the process of transposition itself is selected against. The three theories give different predictions for the pattern of transposable element insertions in the chromosomes of D. melanogaster. We analysed the abundance of six LTR (long terminal repeat) retrotransposons on the X and fourth chromosomes of multiple strains of D. melanogaster, which we compare with the predictions of each theory. The data suggest that no one theory can account for the insertion patterns of all six retrotransposons. Comparing our results with earlier work using these transposable element families, we find a significant correlation between studies in the particular model of copy number regulation supported by the proportion of elements on the X for the different transposable element families. This suggests that different retrotransposon families are regulated by different mechanisms.     

Interlineage distribution and characteristics of the structure of two subfamilies of Drosophila melanogaster MDG4 (gypsy) retrotransposon

The distribution of two variants of MDG4 (gypsy) was analyzed in several Drosophila melanogaster strains. Southern blot hybridization revealed the inactive variant of MDG4 in all strains examined and active MDG4 only in some of them. Most of the strains harboring the active MDG4 variant were recently isolated from natural populations. It is of interest that the active MDG4 prevailed over the inactive one only in strains carrying the mutant flamenco gene. Several lines were analyzed in more detail. The number of MDG4 sites on salivary-gland polytene chromosomes was established via in situ hybridization, and MDG4 was tested for transposition using the ovoD test.     

Regulatory elements of copia retrotransposons, controlling the level of its expression in Drosophila melanogaster testes

Expression of the lacZ reporter gene under the control of five deletion derivatives of the copia regulatory region including the 5' long terminal repeat (LTR) and the 5' untranslated region (UTR) was assayed in the testes of transgenic Drosophila melanogaster males (larvae and imago). The full-length copia regulatory region (LTR + UTR) ensured expression of the reporter gene in testes of both larvae and adult males. Deletion of UTR or 3' end of LTR increased lacZ expression in the testes, whereas deletion of the 5' end of LTR increased it. This indicated that a positive regulator of copia expression is at the 5' end of LTR and that negative regulators are at the 3' end of LTR and in UTR. The effects of the fragments of the copia regulatory region on reporter gene expression in the testes in vivo did not completely coincide with the effects observed earlier in cultured cells. We suggest that this difference is due to different regulation of expression of the fusion constructs integrated into chromatin as compared to their transient expression.