About the origin of retroviruses and the co-evolution of the gypsy retrovirus with the Drosophila flamenco host gene

The gypsy element of Drosophila melanogaster is the first retrovirus identified so far in invertebrates. According to phylogenetic data, gypsy belongs to the same group as the Ty3 class of LTR-retrotransposons, which suggests that retroviruses evolved from this kind of retroelements before the radiation of vertebrates. There are other invertebrate retroelements that are also likely to be endogenous retroviruses because they share with gypsy some structural and functional retroviral-like characteristics. Gypsy is controlled by a Drosophila gene called flamenco, the restrictive alleles of which maintain the retrovirus in a repressed state. In permissive strains, functional gypsy elements transpose at high frequency and produce infective particles. Defective gypsy proviruses located in pericentromeric heterochromatin of all strains seem to be very old components of the genome of Drosophila melanogaster, which indicates that gypsy invaded this species, or an ancestor, a long time ago. At that time, Drosophila melanogaster presumably contained permissive alleles of the flamenco gene. One can imagine that the species survived to the increase of genetic load caused by the retroviral invasion because restrictive alleles of flamenco were selected. The characterization of a retrovirus in Drosophila, one of the most advanced model organisms for molecular genetics, provides us with an exceptional clue to study how a species can resist a retroviral invasion. This revised version was published online in August 2006 with corrections to the Cover Date.     

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.     

Wake up of transposable elements following Drosophila simulans worldwide colonization.

Transposable elements (TEs) make up around 10%-15% of the Drosophila melanogaster genome, but its sibling species Drosophila simulans carries only one third as many such repeat sequences. We do not, however, have an overall view of copy numbers of the various classes of TEs (long terminal repeat [LTR] retrotransposons, non-LTR retrotransposons, and transposons) in genomes of natural populations of both species. We analyzed 34 elements in individuals from various natural populations of these species. We show that D. melanogaster has higher average chromosomal insertion site numbers per genome than D. simulans for all TEs except five. The LTR retrotransposons gypsy, ZAM, and 1731 and the transposon bari-1 present similar low copy numbers in both species. The transposon hobo has a large number of insertion sites, with significantly more sites in D. simulans. High variation between populations in number of insertion sites of some elements of D. simulans suggests that these elements can invade the genome of the entire species starting from a local population. We propose that TEs in the D. simulans genome are being awakened and amplified as they had been a long time ago in D. melanogaster.     

Male reproductive behavior in Drosophila melanogaster strains with different alleles of the flamenco gene

The allelic state of gene flamenco has been determined in a number of Drosophila melanogaster strains using the ovoD test. The presence of an active copy of gypsy in these strains was detected by restriction analysis. Then male reproduction behavior was studied in the strains carrying a mutation in gene flamenco. In these experiments mating success has been experimentally estimated in groups of flies. It has been demonstrated that the presence of mutant allele flamMS decreases male mating activity irrespective of the presence or absence of mutation white. The active copy of gypsy does not affect mating activity in the absence of the mutation in gene flamenco. Individual analysis has demonstrated that that mutation flamMS results in characteristic changes in courtship: flamMS males exhibit a delay in the transition from the orientation stage to the vibration stage (the so-called vibration delay). The role of locus flamenco in the formation of male mating behavior in Drosophila is discussed.     

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.     

Distribution and conservation of mobile elements in the genus Drosophila

Essentially nothing is known of the origin, mode of transmission, and evolution of mobile elements within the genus Drosophila. To better understand the evolutionary history of these mobile elements, we examined the distribution and conservation of homologues to the P, I, gypsy, copia, and F elements in 34 Drosophila species from three subgenera. Probes specific for each element were prepared from D. melanogaster and hybridized to genomic DNA. Filters were washed under conditions of increasing stringency to estimate the similarity between D. melanogaster sequences and their homologues in other species. The I element homologues show the most limited distribution of all elements tested, being restricted to the melanogaster species group. The P elements are found in many members of the subgenus Sophophora but, with the notable exception of D. nasuta, are not found in the other two subgenera. Copia-, gypsy-, and F-element homologues are widespread in the genus, but their similarity to the D. melanogaster probe differs markedly between species. The distribution of copia and P elements and the conservation of the gypsy and P elements is inconsistent with a model that postulates a single ancient origin for each type of element followed by mating-dependent transmission. The data can be explained by horizontal transmission of mobile elements between reproductively isolated species.      

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.     

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.     

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.     

Structural and evolutionary analyses of the Ty3/gypsy group of LTR retrotransposons in the genome of Anopheles gambiae

The recent availability of the genome of Anopheles gambiae offers an extraordinary opportunity for comparative studies of the diversity of transposable elements (TEs) and their evolutionary dynamics between two related species, taking advantage of the existing information from Drosophila melanogaster. To this goal, we screened the genome of A. gambiae for elements belonging to the Ty3/gypsy group of long-terminal repeat (LTR) retrotransposons. The A. gambiae genome displays a rich diversity of LTR retrotransposons, clearly greater than D. melanogaster. We have characterized in detail 63 families, belonging to five of the nine main lineages of the Ty3/gypsy group. The Mag lineage is the most diverse and abundant, with more than 30 families. In sharp contrast with this finding, a single family belonging to this lineage has been found in D. melanogaster, here reported for the first time in the literature, most probably consisting of old inactive elements. The CsRn1 lineage is also abundant in A. gambiae but almost absent from D. melanogaster. Conversely, the Osvaldo lineage has been detected in Drosophila but not in Anopheles. Comparison of structural characteristics of different families led to the identification of several lineage-specific features such as the primer-binding site (PBS), the gag-pol translational recoding signal (TRS), which is extraordinarily diverse within the Ty3/gypsy retrotransposons of A. gambiae, or the presence/absence of specific amino acid motifs. Interestingly, some of these characteristics, although in general well conserved within lineages, may have evolved independently in particular branches of the phylogenetic tree. We also show evidence of recent activity for around 75% of the families. Nevertheless, almost all families contain a high proportion of degenerate members and solitary LTRs (solo LTRs), indicative of a lower turnover rate of retrotransposons belonging to the Ty3/gypsy group in A. gambiae than in D. melanogaster. Finally, we have detected significant overrepresentations of insertions on the X chromosome versus autosomes and of putatively active insertions on euchromatin versus heterochromatin.     

Complex evolution of gypsy in Drosophilid species

In an endeavor to contribute to the comprehension of the evolution of transposable elements (TEs) in the genome of host species, we investigated the phylogenetic relationships of sequences homologous to the retrotransposon gypsy of Drosophila melanogaster in 19 species of Drosophila, in Scaptodrosophila latifasciaeformis, and in Zaprionus indianus. This phylogenetic study was based on approximately 500 base pairs of the env gene. Our analyses showed considerable discrepancy between the phylogeny of gypsy elements and the relationship of their host species, and they allow us to infer a complex evolutionary pattern that could include ancestral polymorphism, vertical transmission, and several cases of horizontal transmission.     

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.     

Wanderings of Hobo: A Transposon in Drosophila Melanogaster and its Close Relatives

The transposon hobo is present in the genomes of Drosophila melanogaster and Drosophila simulans (and D. mauritiana and probably D. sechellia, based on Southern blots) as full-size elements and internally deleted copies. The full-size melanogaster, simulans and mauritiana hobo elements are 99.9% identical at the DNA sequence level, and internally deleted copies in these species essentially differ only in having deletions. In addition to these, hobo-related sequences are present and detectable with a hobo probe in all these species. Those in D. melanogaster are 86-94% identical to the canonical hobo, but with many indels. We have sequenced one that appears to be inserted in heterochromatin (GenBank Acc. No. AF520587). It is 87.6% identical to the canonical hobo, but quite fragmented by indels, with remnants of other transposons inserted in and near it, and clearly is defunct. Numerous similar elements are found in the sequenced D. melanogaster genome. It has recently been shown that some are fixed in the euchromatic genome, but it is probable that still more reside in heterochromatic regions not included in the D. melanogaster genome database. They are probably all relics of an earlier introduction of hobo into the ancestral species. There appear to have been a minimum of two introductions of hobo into the melanogaster subgroup, and more likely three, two ancient and one quite recent. The recent introduction of hobo was probably followed by transfers between the extant species (whether 'horizontally' or by infrequent interspecific hybridization).     

Evidence for recent horizontal transfer of gypsy-homologous LTR-retrotransposon gtwin into Drosophila erecta followed by its amplification with multiple aberrations

Long terminal repeat (LTR) retrotransposon gtwin was initially discovered in silico, and then it was isolated as gypsy-homologous sequence from Drosophila melanogaster strain, G32. The presence of ORF3 suggests, that gtwin, like gypsy, may be an endogenous retrovirus, which can leave the cell and infect another one. Therefore, in this study we decided to investigate the distribution of gtwin in different species of the melanogaster subgroup in order to find out whether gtwin can be transferred horizontally as well as vertically. Gtwin was found in all 9 species of this subgroup, hence it seems to have inhabited the host genomes for a long time. In addition, we have shown that in the Drosophila erecta genome two gtwin families are present. The first one has 93% of identity to D. melanogaster element and is likely to be a descendant of gtwin that existed in Drosophila before the divergence of the melanogaster subgroup species. The other one has >99% of identity to D. melanogaster gtwin. The most reasonable explanation is that this element has been recently horizontally transferred between D. melanogaster and D. erecta. The number and variety of gtwin copies from the "infectious" family suggest that after the horizontal transfer into D. erecta genome, gtwin underwent amplification and aberrations, leading to the rise of its diverse variants.     

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.     

Ancestral polymorphism and recent invasion of transposable elements in Drosophila species

ABSTRACT: BACKGROUND: During the evolutionary history of transposable elements, some processes, such as ancestral polymorphisms and horizontal transfer of sequences between species, can produce incongruences in phylogenies. We investigated the evolutionary history of the transposable elements Bari and 412 in the sequenced genomes of the Drosophila melanogaster group and in the sibling species D. melanogaster and D. simulans using traditional phylogenetic and network approaches. RESULTS: The maximum likelihood (ML) phylogenetic analyses revealed incongruences and unresolved relationships for both the Bari and 412 elements. The DNA transposon Bari within the D. ananassae genome is more closely related to the element of the melanogaster complex than to the sequence in D. erecta, which is inconsistent with the species phylogeny. Divergence analysis and the comparison of the rate of synonymous substitutions per synonymous site of the Bari and host gene sequences explain the incongruence as an ancestral polymorphism inherited stochastically by the derived species. Unresolved relationships were observed in the ML phylogeny of both elements involving D. melanogaster, D. simulans and D. sechellia. A network approach was used to attempt to resolve these relationships. The resulting tree suggests recent transfers of both elements between D. melanogaster and D. simulans. The divergence values of the elements between these species support this conclusion. CONCLUSIONS: We showed that an ancestral polymorphism and recent invasion of genomes due to introgression or horizontal transfer between species occurred during the evolutionary history of the Bari and 412 elements in the melanogaster group. These invasions likely occurred in Africa during the Pleistocene, before the worldwide expansion of D. melanogaster and D. simulans.