The gypsy element of Drosophila differs from most LTR retrotransposons in containing a third open reading frame that resembles retroviral env genes. The protein encoded by ORF3 is glycosylated and processed, like all retroviral envelope proteins. The protein is expressed at high levels in fly strains in which gypsy elements are active. In these strains the protein is found primarily in viral particles. When larvae of fly strains in which gypsy is normally inactive are exposed to sucrose gradient fractions containing these particles, a high level of gypsy insertion activity is observed in their progeny. Thus, gypsy has the expected properties of an insect retrovirus. [Key Words: Drosophila; mutagenesis; transposable element; insect retrovirus] Received May 6, 1994; revised version accepted July 11, 1994.The gypsy element of Drosophila melanogaster has been classified traditionally as a long terminal repeat (LTR) retrotransposon; however, it is one of a small group of LTR retrotransposons from insects that are unusual in that they contain three open reading frames (ORFs). In these retroelements, the first two ORFs correspond to retroviral gag and poi, whereas ORF3 is of unknown function but corresponds in size and genomic location to retroviral env (Fig. 1). All elements with three ORFs described so far are from insects, either Drosophila or the lepidopteran Trichoplusia ni. Recent results have shown that in two elements encoding an ORF3, gypsy and tom, a subgenomic mRNA similar in structure to retroviral env mRNAs can be found (P61isson et al. 1994;Tanda et al. 1994). In the case of gypsy, this transcript is observed only in certain strains in which gypsy transpositional activity is high (P41isson et al. 1994). The primary sequences of the encoded ORF3 proteins of these elements are quite variable and show no obvious similarity to retroviral Env proteins. However, retroviral Env proteins are themselves very variable in primary sequence. Like retroviral Env proteins, the proteins encoded by "retrotransposon" ORF3s contain a putative transmembrane domain near their carboxyl terminus, multiple putative N-glycosylation sites, and putative protease cleavage sites (resembling the cleavage sites in a variety of retroviral Env proteins) at conserved positions (Fig. 1 }. These features of gypsy and the other ORF3-containing insect retrotransposons have prompted the suggestion that these elements may represent endogenous insect retro- viruses (Boeke 1988;Boeke and Corces 1989;Coffin 1993).To address the question of whether gypsy represents a virus or a transposon more directly, we have turned to the use of Drosophila strains that show genetic instabilities associated with high-frequency insertion by gypsy elements. These strains are characterized by a large number of full-length gypsy elements in the euchromatin, appearance of gypsy insertion mutations at high frequency, and the presence of both large amounts of gypsy full-length RNA and spliced ORF3 mRNA (P61isson et al. 1994; N. Prud'homme, M. Gans, M. Masson, C. Ter...

Retroviruses and related genetic elements generate terminally redundant RNA products by differential polyadenylylation within a long terminal repeat. Expression of the white-apricot (wa) allele ofDrosophila melanogaster, which carries an insertion of the 5.1-kilobase retrovirus-like transposable element copia in a small intron, is influenced by signals within copia. By using this indicator, we have isolated a 518-base-pair deletion, 312 base pairs upstream of the copia polyadenylylation site, that is phenotypically like much larger deletions and eliminates RNA species polyadenylylated in copia. This requirement of distant upstream sequences for copia polyadenylylation has implications for the expression of many genetic elements bearing long terminal repeats.TV white-apricot (wa) allele of Drosophila melanogaster .-rs an apricot eye color rather than the dark red of wild ty_ This mutation is caused by disruption of the 74-basep-(bp) second white intron by the transposable element copia (1-4). Consequently, the level of normally spliced [2.6 kilobase (kb)] mRNA is greatly reduced and a number of aberrant RNAs (5.7 kb, 3.0 kb, and 1.25 kb) that are polyadenylylated within copia are observed (5-8). Interest in Wa derives from the existence of genetic modifiers that act in trans to alter its expression. At least one ofthese, suppressorof-white-apricot [su(wa)], is a regulator of RNA splicing (9, 10). Here we describe waRE714 , a partial revertant of Wa with nearly wild-type eye color that is unresponsive to most extragenic modifiers of Wa. Molecular analysis of waRE714 reveals a 518-bp deletion of internal copia sequences 312 bp upstream of the site of copia polyadenylylation and a complete absence of the aberrant RNAs polyadenylylated at that site. Thus, unexpectedly distant upstream sequences appear to be required for copia polyadenylylation. We suggest that the copia element in Wa allows polyadenylylation to compete with splicing of the second white intron and that the deletion found in WaRE714 relieves this competition, resulting in higher steady-state levels of both a processing intermediate and the mRNA. MATERIALS AND METHODSEthyl Methanesulfonate (EMS) Mutagenesis. An EMS mutagenic screen for dominant modifiers of white-apricot was conducted. wa-iso2,3 males were fed EMS as described by Ashburner (11) using a solution of 10 mM Tris-HCI (pH 7.5), 1% sucrose, and 25 mM EMS. These males were mated to females ofthe same strain, and 24,000 progeny were screened for altered eye coloration. The partial revertant WaRE714 was one of 16 eye color mutations observed among 24,000 F1 progeny. (13)] were determined from the sequence (14) of mpMK27, a Cla I-Hpa II M13 subclone of a PCR-amplified product from WaRE714 genomic DNA. The PCR was conducted using oligomer JW1 at copia nt 4040, ACATATGGA'ITTTAACTACTGCAGTAA, and oligomer UCB5 at white nt -141 (4), CGGAATTCATC-CCGGATGGCGATAC.Fly Genotypes. Alleles are generally described in Lindsley and Grell (15) with exceptions specified in the text. Oregon R is a wild-type...

No abstract

The gypsy retroelement of Drosophila moves at high frequency in the germ line of the progeny of females carrying a mutation in the flamenco (flam) gene. This high rate of de novo insertion correlates with elevated accumulation of full-length gypsy RNA in the ovaries of these females, as well as the presence of an env-specific RNA. We have prepared monoclonal antibodies against the gypsy Pol and Env products and found that these proteins are expressed in the ovaries of flam females and processed in the manner characteristic of vertebrate retroviruses. The Pol proteins are expressed in both follicle and nurse cells, but they do not accumulate at detectable levels in the oocyte. The Env proteins are expressed exclusively in the follicle cells starting at stage 9 of oogenesis, where they accumulate in the secretory apparatus of the endoplasmic reticulum. They then migrate to the inner side of the cytoplasmic membrane where they assemble into viral particles. These particles can be observed in the perivitelline space starting at stage 10 by immunoelectron microscopy using anti-Env antibodies. We propose a model to explain flamenco-mediated induction of gypsy mobilization that involves the synthesis of gypsy viral particles in the follicle cells, from where they leave and infect the oocyte, thus explaining gypsy insertion into the germ line of the subsequent generation.