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.     

Mobilization of retrotransposon copia in the Drosophila melanogaster genome]

Considerable heterogeneity of retrotransposon copia sites of location on polytene chromosomes was revealed in one of the substocks of the inbred Drosophila melanogaster stock. Heterogeneity of copia sites of location was found in no other substocks analyzed. The heterogeneity was shown to be caused by copia insertions in new sites. The frequency of insertions is about 12% per haploid genome per generation. The retrotransposon excisions and somatic transpositions were not observed. The location of retrotransposons mdg1, mdg2, mdg3, mdg4, 297 and H.M.S. Beagle appeared to be stable in all the stocks analyzed. Thus, a model system allowing to study mechanisms of retrotransposon copia transpositions in D. melanogaster tissues as well as phenotypic effects of copia mobilization is described.     

Germ line transposition of the copia retrotransposon in Drosophila melanogaster is restricted to males by tissue-specific control of copia RNA levels

Germ line transposition rates of the retrotransposon copia were directly measured in males and females of an inbred Drosophila melanogaster line, 2b3, which is highly polymorphic for copia insertion sites. The elevated germ line transposition rate of copia in this line (3–8?×?10^?3 per generation per element) is confined to males, with transposition in females being undetectable under the conditions of the experiment but at most 50-fold lower than the rate for males. To determine the molecular basis of this effect, copia RNA levels were measured in whole bodies and germ lines of male and female flies of both the unstable 2b3 line and a stable line, Oregon RC-iso, which shows normal rates of copia transposition. Both male and female 2b3 flies contain much more copia RNA than flies of the stable line. However, 2b3 male germinal tissues contain much higher levels of copia RNA than the equivalent female tissues. The highest copia expression is detected in maturing primary spermatocytes. Our data show that high rates of germ line copia transposition are restricted to males by tissue-specific control of RNA levels and suggest that transposition of copia only occurs in fly tissues containing more than a relatively high threshold level of copia RNA.     

Mutation accumulation and the effect of copia insertions in Drosophila melanogaster

Repeated efforts to estimate the genomic deleterious mutation rate per generation (U) in Drosophila melanogaster have yielded inconsistent estimates ranging from 0.01 to nearly 1. We carried out a mutation-accumulation experiment with a cryopreserved control population in hopes of resolving some of the uncertainties raised by these estimates. Mutation accumulation (MA) was carried out by brother sister mating of 150 sublines derived from two inbred lines. Fitness was measured under conditions chosen to mimic the ancestral laboratory environment of these genotypes. We monitored the insertions of a transposable element, copia, that proved to accumulate at the unusually high rate of 0.24 per genome per generation in one of our MA lines. Mutational variance in fitness increased at a rate consistent with previous studies, yielding a mutational coefficient of variation greater than 3%. The performance of the cryopreserved control relative to the MA lines was inconsistent, so estimates of mutation rate by the Bateman-Mukai method are suspect. Taken at face value, these data suggest a modest decline in fitness of about 0.3% per generation. The element number of copia was a significant predictor of fitness within generations; on average, insertions caused a 0.76% loss in fitness, although the confidence limits on this estimate are wide.     

Doc and copia instability in an isogenic Drosophila melanogaster stock

A high degree of heterogeneity and an overall increase in number of insertion sites of the mobile elements Doc and copia were revealed in one substock of an isogenic Drosophila melanogaster stock, while in two other substocks the distribution of copia sites was highly homogenous, but that of Doc sites was again heterogenous. We therefore concluded that copia was unstable in one of the substocks and Doc was unstable in all. Doc instability presumably arose earlier than copia instability. Doc and copia transpositions were directly observed in experiments with one substock. An abundance of copia insertions was revealed in the X chromosome where insertions with deleterious effects are exposed to selection in hemizygous condition. The locations of many other mobile elements (mdg1, mdg2, mdg3, mdg4, 297, B104, H.M.S. Beagle, I, P, BS, FB) were found to be conserved in each substock and did not differ between them, indicating that these mobile elements were stable. This homogeneity is a strong argument against any possibility of inadvertent contamination.     

Variability of localization of retrotransposon copia ant its effect on adaptation in inbred strains of Drosophila melanogaster with the different rate of transposition

Three sublines of an inbred laboratory line of Drosophila melanogaster with the initial copia transposition rate 2 x 10(-2), 2 x 10(-3), and 5 x 10(-4) per copy per generation were reared for several dozen generations under conditions of low effective population size (by full-sib crosses or in a small mass culture of 10 females x 10 males). All six lines were tested for the transposition rate, location pattern, and copy number of copia in euchromatic genome regions and for fitness inferred from the intraspecific competition index. The copia transposition rate remained constant in both versions of the lines with an initially lower rate and decreased by an order of magnitude in both versions of the line with an initially higher rate. New copia insertions behaved as selectively neutral and were accumulated in the genome. Each new copy decreased fitness by less than 1% on average. Some of the existing unfixed insertions remained segregating after long-term inbreeding and were assumed to provide a selective advantage to heterozygotes.     

Aurora,a non-mobile retrotransposon in Drosophila melanogaster heterochromatin

A novel retrotransposon, aurora, containing 324 by long terminal repeats (LTRs) was detected in Drosophila melanogaster as a 5 kb insertion in the heterochromatic Stellate gene. This insertion causes a 5 bp duplication of the integration site. Southern analysis and in situ hybridization data show that all detectable copies of aurora are immobilized in the D. melanogaster heterochromatin. However, mobile copies of aurora were revealed in the cuchromatin of D. simulans. The element was also found in various species of the melanogaster subgroup and in the D. virilis genome.     

Evolutionary pattern of the gtwin retrotransposon in the Drosophila melanogaster subgroup

The gtwin retrotransposon was recently discovered in the Drosophila melanogaster genome and it is evolutionarily closer to gypsy endogenous retrovirus. This study has identified gtwin homologous sequences in the genome of D. simulans, D. sechellia, D. erecta and D. yakuba by performing homology searches against the public genome database of Drosophila species. The phylogenetic analyses of the gtwin env gene sequences of these species have shown some incongruities with the host species phylogeny, suggesting some horizontal transfer events for this retroelement. Moreover, we reported the existence of DNA sequences putatively encoding full-length Env proteins in the genomes of Drosophila species other than D. melanogaster. The results suggest that the gtwin element may be an infectious retrovirus able to invade the genome of new species, supporting the gtwin evolutionary picture shown in this work.     

One of the copia genes is adjacent to satellite DNA in Drosophila melanogaster.

A method for purifying sequences adjacent to satellite DNA in the heterochromatin of D. melanogaster is described. A cloned DNA segment containing part of a copia gene adjacent to 1.688 g/cm3 satellite DNA has been isolated. The copia genes compose a repeated gene family which codes for abundant cytoplasmic poly(a)-containing RNA (Young and Hogness, 1977; Finnegan et al., 1978). We have identified two major poly (A)-containing RNA species [5.2 and 2.1 kilobases (kb)] produced by the copia gene family. The cloned segment contains copia sequences homologous to the 5' end of RNA within 0.65 kb of the 1.688 satellite DNA sequences. Seven different cloned copia genes from elsewhere in the genome have also been isolated, and a 5.2 kb region present in five of the clones was identified as copia by heteroduplex analysis. In addition, three ususual copies of copia were found: a "partial" copy of the gene (3.7 kb) which has one endpoint in common with the 5.2 kb unit; a copia gene flanked on one side by a 1.6 kb sequence and on the other by the same 1.6 kb sequence in the inverted orientation; and a copia gene flanked only on one side by the same sequence.     

Wolbachia‐induced expression of kenny gene in testes affects male fertility in Drosophila melanogaster

Wolbachia are Gram‐negative endosymbionts that are known to cause embryonic lethality when infected male insects mate with uninfected females or with females carrying a different strain of Wolbachia, a situation characterized as cytoplasmic incompatibility (CI). However, the mechanism of CI is not yet fully understood, although recent studies on Drosophila melanogaster have achieved great progress. Here, we found that Wolbachia infection caused changes in the expressions of several immunity‐related genes, including significant upregulation of kenny (key), in the testes of D. melanogaster. Overexpression of key in fly testes led to a significant decrease in egg hatch rates when these flies mate with wild‐type females. Wolbachia‐infected females could rescue this embryonic lethality. Furthermore, in key overexpressing testes terminal deoxynucleotidyl transferase‐mediated dUTP‐biotin nick‐end labeling signal was significantly stronger than in the control testes, and the level of reactive oxygen species was significantly increased. Overexpression of key also resulted in alterations of some other immunity‐related gene expressions, including the downregulation of Zn72D. Knockdown of Zn72D in fly testes also led to a significant decrease in egg hatch rates. These results suggest that Wolbachia might induce the defect in male host fertility by immunity‐related pathways and thus cause an oxidative damage and cell death in male testes.