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Kimberley J. Dej, Tatiana Gerasimova, Victor G. Corces, Jef D. The Drosophila retroelement gypsy has a of unusual features including an unusual LTR terminal sequence and an apparent target sequence preference. The ovo locus is a known hotspot for gypsy insertion. Insertions were found at multiple sites within the ovo locus. However, this target sequence is at best necessary but not sufficient to specify a hotspot, as there were several other sequences conforming to this consensus in the ovo locus that were not hit.

The indicate that gypsy may have a higher degree of target specificity than most infectious LTR retroelements. The gypsy LTR retroelement of Drosophila melanogaster is of great interest because it represents a genetically tractable yryryr twitter that is closely related to the non-infectious LTR retrotransposons but is infectious like retroviruses 12.

Like retroviruses, gypsy encodes genes equivalent to gagpol and envwhereas most retrotransposons encode only gag and pol. Moreover, the envelope protein is encoded by a subgenomic mRNA, just as is the case in retroviruses 3. The latter feature is shared with another Drosophila retroelement, Tom 4. Although this aspect of gypsy's biology has attracted considerable attention, relatively little is known about how gypsylike all retrotransposons and retroviruses, inserts its DNA into the host genome during the essential integration phase of its life cycle.

Like these other elements, gypsy encodes an integrase IN protein that presumably mediates the integration reaction. Unlike the other elements, gypsy has unusual terminal sequences, and may have sequence specificity for insertion. LTR retroelement life cycles can be divided into a few discrete stages, reviewed in 5. These are: i the gene expression stage, when element RNA s and proteins are produced, ii the assembly stage, when virion or virus-like particles are put together and matured via a series of endoproteolytic cleavages, iii the reverse transcription stage, when element DNA is produced from the genomic RNA in the particle and iv the integration stage, during which the newly synthesized element DNA is integrated at a novel site in the host yryryr twitter.

An absolutely critical aspect of the integration stage is the identification of target sequences in host DNA. Craigie 6 pointed out that non-infectious elements are bound to the genome of the host organism they inhabit, and the progeny thereof. In contrast, the infectious retroviruses are relatively free of this constraint because of their ability to move horizontally to other members of the host population. Indeed, recent studies of both avian and mammalian retroviral integration suggest that only modest specificity of integration relative to host DNA targets is observed 78.

In contrast, recent studies of the integration specificity of the non-infectious elements Ty1, Ty3 and Ty5 of yeast have borne this prediction out rather spectacularly. Interestingly Ty1 and Ty3, which are extremely distantly related elements 13target these regions by what appear to be distinct mechanisms 14and so tRNA targeting appears to be an example of convergent evolution for these two elements. Because the gypsy element lies at the phylogenetic interface between the retrotransposons and retroviruses, analysis of its integration specificity is of special interest.

The gypsy element is unusual among the LTR retroelements in another way. All retroviruses and nearly all LTR retrotransposons share the terminal sequences TG…CA usually embedded within a somewhat longer imperfect, inverted repeat sequence; Fig. However gypsy is one of a very small of elements, thus far known only from insects, that have non-TG…CA terminal sequences. Interestingly, and perhaps not coincidentally, all of these elements share the property of having three reading frames corresponding to retroviral gag, pol and env.

All of these elements are pd to be infectious, although this has only been demonstrated, thus far, for gypsy. Retroelement LTR termini. Interestingly, gypsy sequences from different Drosophila species do not completely conserve the terminal dinucleotide, suggesting that gypsy's IN may have less stringent terminal sequence requirements than those of the other LTR retroelements.

Alternatively, the INs of different gypsy species could recognize the individual termini with high specificity. The gypsy element can insert into many different loci in genomic DNA. Based on the sequencing of a relatively yryryr twitter of genomic gypsy elements it was concluded that gypsy was highly sequence-specific in its insertion into genomic DNA; in nearly every example, the target sequence TATATA or TACATA 17—19 was duplicated upon insertion.

A recent study in which three gypsy insertions into the ovo gene were sequenced 2 suggested that other target sequences could also be recognized, and suggested a relaxation of the proposed recognition sequence to YRYRYR. The gypsy element moves at high frequency during crosses of flamenco strains to other strains The ovo locus, required yryryr twitter differentiation of the female germ line, codes for two OVO protein isoforms that differ in their N-termini 22 We have used the ovoD1 reversion assay system to generate a larger collection of gypsy insertions, and report here the insertion sites of a total of 26 new insertion mutations within the ovo locus.

OvoD1 revertants were isolated by two methods, described in Figure 2. The two methods are genetic crosses Fig. The genetic crosses involved three steps. First, females of one of three strains, listed in Table 1were crossed by y v f mal flam 1 males F 0. Second, progeny females were crossed to ovo D1 v males F 1 ; the F 2 female progeny were then scored for fertility ovoD1 reversion. The high frequency of reversion observed Table 1 indicates that the three strains from which the females were derived must bear flamenco mutations.

In the feeding experiments, gypsy particles were first isolated from females of the y v f mal flam strain; these were fed to SS strain larvae as described ly 2. Genomic DNA was prepared from 50 flies for each insertion stock by using a potassium acetate quickprep.

This was spun down at 14 r. The supernatant was transferred to a fresh 1. Isolation of ovo D1 revertants caused by gypsy insertions diagram of two methods, crossing and feeding. A Method 1: genetic cross. A genetic cross between females of a donor strain y w a4 ct 6 sn w flam 2 and ovo D1 males was carried out as outlined in the figure. Stocks of ovoDR revertant lines were then generated. Fed SS females were mated with male ovo D1 flies and fertile female progeny were identified and studied further. PCR products were run on 0. The 18 kb ovo clone D1B2NR was used as a probe This clone covers the 7.

PCR products that hybridized with the ovo DNA were then isolated from the gel by electroelution and sequenced directly. Reactions were done using the Taq Extender PCR additive Stratagene which was used to generate up to an 8 kb product in control experiments using cloned DNA and vector primers. Insertions of gypsy into ovo D1 were obtained in either of two ways, by crossing as described 21 or by feeding of gypsy virus particles as described by Song et al.

The remaining non- gypsy revertants were not characterized further. The progeny of revertant female flies were studied for whether or not they bore gypsy elements within the ovo locus by a series of PCR amplification experiments using one primer reading outward from one of the gypsy ends and one primer chosen from among a set of primers spanning the ovo locus Fig. Finally, selected PCR products were chosen for DNA sequence analysis, and the DNA sequencing provided final proof that the observed products indeed corresponded to gypsy insertions into the ovo locus.

The features included are the major transcription start site TSS; vertical arrowmajor and minor splice donor sites SD; overline for the first intron two other extremely minor splice donors are not shownthe splice acceptor site in intron 1 SA; overlinethe position of the new ATG formed by the ovo D1 mutation 24yryryr twitter arrow pointing upward and a single gypsy insertion site A, underline.

Also indicated are seven sites of gypsy insertion uncovered during this study sites B-H ; the inferred target site duplications are underlined. The diagram indicates the position of sites A-H within the ovo locus; insertions are symbolized by triangles, exons by boxes and the ORF by shading.

The arrows below the line indicate the approximate positions of the yryryr twitter used. A total of 26 PCR products corresponding to gypsy-ovo junction fragments were directly sequenced or cloned and then sequenced and the sequences of the junction between the end of the gypsy element and ovo were tabulated Fig. Remarkably, all of the new insertion sites clustered within a bp region, and seven different target sites, defined by their inferred 6 bp target site duplication, were observed; four of these target sites consisted of either a single insertion or insertions in a single orientation and the other three included insertions in both yryryr twitter.

All of the insertions lie within the boundaries of the other class of transcripts, the ovo a transcript. The insertion sites obtained by crossing and feeding methods were very similar to each other, and also agree well with the positions of ly restriction mapped insertions 1 It is expected that insertion of gypsy at these positions in ovo will lead to premature termination of transcription within the gypsy sequences. The ovo locus contains two transcription start sites at positions and bp The transcript initiating at bp codes for the OVO-A isoform, a amino acid protein believed to be expressed late in oogenesis The OVO-B isoform is believed to be expressed early in oogenesis Several dominant female sterile alleles yryryr twitter ovosuch as ovo D1result from point mutations that create new in-frame translation initiator codons upstream of the OVO-B initiator site and result in translation of a new protein with an N-terminal extension 24 We hypothesize that insertion of gypsy at these positions will lead to the premature termination of ovo transcription within the gypsy sequences.

It is not known whether gypsy inserted in the opposite orientation will result in truncation of the ovo mRNA, but we note that there are four copies of the sequence AATAAA, a sequence known to specify polyadenylation, on the negative strand of the gypsy sequence.

We believe these gypsy insertions may represent null yryryr twitter or at least alleles that ificantly reduce ovo D1 expression because they phenotypically completely reverse the dominant female-sterile phenotype of the starting ovo D1 mutation. However, if the consensus is relaxed to a slightly more degenerate one consisting of alternating pyrimidine Y and purine R residues, that is, a YRYRYR consensus target sequence, it is apparent that most of the ovo insertions conform. Interestingly, all of the single mismatches in our studies were in the 5th or 6th position of the target site duplication.

The alternating purines and pyrimidines are a typical feature of DNA sequences able to adopt the Z conformation 27raising the possibility that gypsy recognizes this DNA structural feature. Alternatively, other sequence variations, such as kinks have been associated with YR steps in DNA sequences 28and these could conceivably be recognized by the gypsy integration machinery. Site C, our biggest hotspot, lies within a stretch of 11 alternating pyrimidines and purines and yryryr twitter F, G and H lie within stretches of In particular, there is a remarkable sequence of 39 consecutive alternating pyrimidines and purines nt — that was not hit in our study, although a single insertion in this region has been reported ly as a mutation that reverts ovoD1 23 In any case, it is very clear that gypsy sequences integrate very non-randomly in response to the selection for ovo D1 revertants.

We thank Maryellen Kurkulos for helpful discussions and Sun Song for doing some of the feeding experiments. Google Scholar. Google Preview.

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A hotspot for the Drosophila gypsy retroelement in the ovo locus.