Flamenco, a Gene Controlling the Gypsy Retrovirus of Drosophila Melanogaster

Gypsy is an endogenous retrovirus of Drosophila melanogaster. It is stable and does not transpose with detectable frequencies in most Drosophila strains. However, we have characterized unstable strains, known as MG, in which it transposes at high frequency. These stocks contain more copies of gypsy than usual stocks. Transposition results in mutations in several genes such as ovo and cut. They are stable and are due to gypsy insertions. Integrations into the ovo(D1) female sterile-dominant mutation result in a null allele of the gene and occurrence of fertile females. This phenomenon, known as the ovo(D1) reversion assay, can be used to quantitate gypsy activity. We have shown that the properties of MG strains result from mutation of a host gene that we called flamenco (flam). It has a strict maternal effect on gypsy mobilization: transposition occurs at high frequency only in the germ line of the progeny of females homozygous for mutations of the gene. It is located at position 65.9 (20A1-3) on the X chromosome. The mutant allele present in MG strains is essentially recessive. Flamenco seems to control the infective properties of gypsy.     

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.     

Proviral amplification of the Gypsy endogenous retrovirus of Drosophila melanogaster involves env-independent invasion of the female germline

Gypsy is an infectious endogenous retrovirus of Drosophila melanogaster. The gypsy proviruses replicate very efficiently in the genome of the progeny of females homozygous for permissive alleles of the flamenco gene. This replicative transposition is correlated with derepression of gypsy expression, specifically in the somatic cells of the ovaries of the permissive mothers. The determinism of this amplification was studied further by making chimeric mothers containing different permissive/restrictive and somatic/germinal lineages. We show here that the derepression of active proviruses in the permissive soma is necessary and sufficient to induce proviral insertions in the progeny, even if the F1 flies derive from restrictive germ cells devoid of active proviruses. Therefore, gypsy endogenous multiplication results from the transfer of some gypsy-encoded genetic material from the soma towards the germen of the mother and its subsequent insertion into the chromosomes of the progeny. This transfer, however, is not likely to result from retroviral infection of the germline. Indeed, we also show here that the insertion of a tagged gypsy element, mutant for the env gene, occurs at high frequency, independently of the production of gypsy Env proteins by any transcomplementing helper. The possible role of the env gene for horizontal transfer to new hosts is discussed.     

A P Element Containing Suppressor of Hairy-Wing Binding Regions Has Novel Properties for Mutagenesis in Drosophila Melanogaster

P elements are widely used as insertional mutagens to tag genes, facilitating molecular cloning and analyses. We modified a P element so that it carried two copies of the suppressor of Hairy-wing [su(Hw)] binding regions isolated from the gypsy transposable element. This transposon was mobilized, and the genetic consequences of its insertion were analyzed. Gene expression can be altered by the su(Hw) protein as a result of blocking the interaction between enhancer/silencer elements and their promoter. These effects can occur over long distances and are general. Therefore, a composite transposon (SUPor-P for suppressor-P element) combines the mutagenic efficacy of the gypsy element with the controllable transposition of P elements. We show that, compared to standard P elements, this composite transposon causes an expanded repertoire of mutations and produces alleles that are suppressed by su(Hw) mutations. The large number of heterochromatic insertions obtained is unusual compared to other insertional mutagenesis procedures, indicating that the SUPor-P transposon may be useful for studying the structural and functional properties of heterochromatin.     

Precise excision of long terminal repeats of the gypsy (mdg4) retrotransposon of Drosophila melanogaster detected in Escherichia coli cells is explained by its integrase function

An Escherichia coli model system was developed to estimate the capacity of the integrase of the Drosophila melanogaster retrotransposon gypsy (mdg4) for precise excision of the long terminal repeat (LTR) and, hence, the entire gypsy. The gypsy retrotransposon was cloned in the form of a PCR fragment in the pBlueScript II KS+ (pBSLTR) vector, and the region of the second open reading frame (INT ORF2) of this element encoding integrase was cloned under the lacZ promoter in the pUC19 vector and then recloned in pACYC184 compatible with pBSLTR. The LTR was cloned in such a manner that its precise excision from the recombinant plasmid led to the restoration of the nucleotide sequence and the function of the ORF of the lacZ gene contained in the vector; therefore, it was detected by the appearance of blue colonies on a medium containing X-gal upon IPTG induction. Upon IPTG induction of E. coli XL-1 Blue cells obtained by cotransformation with plasmids pACCint and pBSLTR on an X-gal-containing medium, blue clones appeared with a frequency of 1 x 10(-3) to 1 x 10(-4), the frequency of spontaneously appearing blue colonies not exceeding 10(-9) to 10(-8). The presence of blue colonies indicated that that the integrase encoded by the INT ORF2 (pACYC 184) fragment was active. After the expression of the integrase, it recognized and excised the gypsy LTR from pBSLTR, precisely restoring the nucleotide sequence and the function of the lacZ gene, which led to the expression of the beta-galactosidase enzymatic activity. PCR analysis confirmed that the LTR was excised precisely. Thus, the resultant biplasmid model system allowed precise excisions of the gypsy LTR from the target site to be detected. Apparently, the gypsy integrase affected not only the LTR of this mobile element, but also the host genome nucleotide sequences. The system is likely to have detected only some of the events occurring in E. coli cells. Thus, the integrase of gypsy is actually capable of not only transposing this element by inserting DNA copies of the gypsy retrotransposon to chromosomes of Drosophila, but also excising them, gypsy is excised via a precise mechanism, with the original nucleotide sequence of the target site being completely restored. The obtained data demonstrate the existence of alternative ways of the transposition of retrotransposons and, possibly, retroviruses, including gypsy (mdg4).     

Retrotransposons IDEFIX and ZAM are responsible for transposition in Drosophila melanogaster MS strain mutant for the flamenco gene

Transposition activity of Drosophila melanogaster gypsy retrotransposon is controlled by the flamenco locus. Transposition activity of the gypsy, ZAM, Idefix, springer, nomad, rover, Quasimodo, 17.6, 297, and Tirant retrotransposons was investigated in isogenic SS and MS strains of D. melanogaster mutant for the flamenco gene. It has been shown that gypsy, ZAM, and Idefix have different genomic surrounding in the studied strains that evidences to their transposition in these strains.     

Evolution of gypsy endogenous retrovirus in the Drosophila obscura species group

The Ty3/gypsy family of retroelements is closely related to retroviruses, and some of their members have an open reading frame resembling the retroviral gene env. Sequences homologous to the gypsy element from Drosophila melanogaster are widely distributed among Drosophila species. In this work, we report a phylogenetic study based mainly on the analysis of the 5' region of the env gene from several species of the obscura group, and also from sequences already reported of D. melanogaster, Drosophila virilis, and Drosophila hydei. Our results indicate that the gypsy elements from species of the obscura group constitute a monophyletic group which has strongly diverged from the prototypic D. melanogaster gypsy element. Phylogenetic relationships between gypsy sequences from the obscura group are consistent with those of their hosts, indicating vertical transmission. However, D. hydei and D. virilis gypsy sequences are closely related to those of the affinis subgroup, which could be indicative of horizontal transmission.     

Molecular structure of a gypsy element of Drosophila subobscura (gypsyDs) constituting a degenerate form of insect retroviruses.

We have determined the nucleotide sequence of a 7.5 kb full-size gypsy element from Drosophila subobscura strain H-271. Comparative analyses were carried out on the sequence and molecular structure of gypsy elements of D.subobscura (gypsyDs), D.melanogaster (gypsyDm) and D.virilis (gypsyDv). The three elements show a structure that maintains a common mechanism of expression. ORF1 and ORF2 show typical motifs of gag and pol genes respectively in the three gypsy elements and could encode functional proteins necessary for intracellular expansion. In the three ORF1 proteins an arginine-rich region was found which could constitute a RNA binding motif. The main differences among the gypsy elements are found in ORF3 (env-like gene); gypsyDm encodes functional env proteins, whereas gypsyDs and gypsyDv ORF3s lack some motifs essential for functionality of this protein. On the basis of these results, while gypsyDm is the first insect retrovirus described, gypsyDs and gypsyDv could constitute degenerate forms of these retroviruses. In this context, we have found some evidence that gypsyDm could have recently infected some D.subobscura strains. Comparative analyses of divergence and phylogenetic relationships of gypsy elements indicate that the gypsy elements belonging to species of different subgenera (gypsyDs and gypsyDv) are closer than gypsy elements of species belonging to the same subgenus (gypsyDs and gypsyDm). These data are congruent with horizontal transfer of gypsy elements among different Drosophila spp.     

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.     

Elevational gradient in the cyclicity of a forest-defoliating insect

Observed changes in the cyclicity of herbivore populations along latitudinal gradients and the hypothesis that shifts in the importance of generalist versus specialist predators explain such gradients has long been a matter of intense interest. In contrast, elevational gradients in population cyclicity are largely unexplored. We quantified the cyclicity of gypsy moth populations along an elevational gradient by applying wavelet analysis to spatially referenced 31-year records (1975–2005) of defoliation. Based on geographically weighted regression and nonlinear regression, we found either a hump-shaped or plateauing relationship between elevation and the cyclicity of gypsy moth populations and a positive relationship between cyclicity and the density of the gypsy moth’s preferred host-tree species. The potential effects of elevational gradients in the density of generalist predators and preferred host-tree species on the cyclicity of gypsy moth populations were evaluated with mechanistic simulation models. The models suggested that an elevational gradient in the densities of preferred host tree species could partially explain elevational patterns of gypsy moth cyclicity. Results from a model assuming a type-III functional response of generalist predators to changes in gypsy moth density were inconsistent with the observed elevational gradient in gypsy moth cyclicity. However, a model with a more realistic type-II functional response gave results roughly consistent with the empirical findings. In contrast to classical studies on the effects of generalist predators on prey population cycles, our model with a type-II functional response predicts a unimodal relationship between generalist-predator density and the cyclicity of gypsy moth populations.     

Evolution from retrotransposons to retroviruses: origin of the env gene

In genome of Drosophila melanogaster, various families of retrotransposons with different combination of functional domens and mechanisms of transposition are present. However only retrotransposons of gypsy family are retroviruses related to errantiviruses. Other families seemingly appeared as intermediate forms of retroviruses evolution. Despite the fact that the question on origin of retroviruses remains unclear, now the hypothesis of their origin from retrotransoposons can be considered the most consistent. Infectious properties of errantiviruses are linked to the presence of the third open reading frame (the env gene). Acquisition of the env gene conversed retrotransposons into retroviruses. So, origin of this gene is of special interest. Homologues of the env gene of errantiviruses are discovered in genomes of D. melanogaster, as well as in baculoviruses and in bacteria Wolbachia pipientis, the endosymbiont of Drosophila. It was shown that homologue of the env gene come to Wolbachia genome from Drosophila genome by horizontal transfer of the gypsy group retrotransposon. Thus, Wolbachia was not a donor of the env gene for errantiviruses. Seemingly, errantiviruses captured the baculoviral homologue of the env gene (f). However origin of the f gene is not clear. At the same time the env gene homologue in D. melanogaster genome exist (Iris). It must not be ruled out that the Iris gene was the source of the env gene of errantiviruses and baculoviruses.     

The endogenous Drosophila melanogaster retrovirus gypsy can propagate in Drosophila hydei cells

The endogenous Drosophila melanogaster retrovirus gypsy (mdg4) forms virus-like particles (VLPs) which are found as extracellular particles in the medium used to culture D. melanogaster cells. The D. hydei somatic cell line DH14, which does not harbour gypsy sequences, was exposed to D. melanogaster VLPs. Subsequent PCR and Southern analysis revealed that gypsy elements had penetrated into the D. hydei cells, suggesting interspecific transmission of the retrovirus. A D. hydei cell line containing gypsy sequences was established and grown in a mixed culture together with the G418-resistant D. hydei cell line DH33, and gypsy was shown to be transmitted from cell to cell. The proportion of cells carrying gypsy increased with time. The rate of gypsy invasion of the lines DH14 and DH33 was 10(-3) and 10(-2) per cell per generation, respectively. The results demonstrate the possibility of interspecific horizontal transfer of gypsy in the form of its VLPs.     

不同地理种群二化螟Ty3/gypsy反转座子天冬氨酰蛋白酶（AP）基因序列的克隆与分析

【目的】Ty3/gypsy反转座子是广泛存在于生物体内的一类反转座子。反转座子上的天冬氨酰蛋白酶（aspartic protease, AP）基因是反转座子发生转座所需的一个重要基因。但由于该基因家族成员间变异较大,较难利用简并引物克隆得到该基因,所以对该基因家族成员的研究很少。【方法】本研究采用PCR方法克隆了二化螟Ty3/gypsy反转座子的AP基因序列,并对其序列特征和地理种群变异进行了分析。【结果】克隆获得的二化螟Chilo suppressalis （Walker）Ty3/gypsy反转座子中的AP基因具有独立的开放阅读框（open reading frame, ORF）,长528 bp,编码的蛋白含175个氨基酸残基（GenBank登录号：KF886014）。Conserved Domain Search在线工具分析显示,该蛋白中含有一个特异的Asp_protease_2保守功能域。从7个二化螟不同地理种群中共克隆获得70份AP基因拷贝。对同一基因座位上的AP序列多重比对分析,发现共存在46处碱基替换,其中碱基转换（transition）有31处,碱基颠换（transversion）有15处,70份拷贝中有69份拷贝是完整的ORF,能编码完整的蛋白。从碱基替换形式看,A→G的变异形式出现最多,有15处;其次是T→C的变异形式,有11处;其余的变异形式都很少。对比这7个不同地理种群,没有发现碱基的替换存在明显的地理区划差异。【结论】碱基的替换形式与二化螟所处的地理区域无明显相关性。本研究对于认识反转座子序列的变异特点有所帮助。