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Penelope-like elements are a class of retroelement that have now been identified in >50 species belonging to at least 10 animal phyla. The Penelope element isolated from Drosophila virilis is the only transpositionally active representative of this class isolated so far. The single ORF of Penelope and its relatives contains regions homologous to a reverse transcriptase of atypical structure and to the GIY-YIG, or Uri, an endonuclease (EN) domain not previously found in retroelements. We have expressed the single ORF of Penelope in a baculovirus expression system and have shown that it encodes a polyprotein with reverse transcriptase activity that requires divalent cations (Mn2+ and Mg2+). We have also expressed and purified the EN domain in Escherichia coli and have demonstrated that it has EN activity in vitro. Mutations in the conserved residues of the EN catalytic module abolish its nicking activity, whereas the DNA-binding properties of the mutant proteins remain unaffected. Only one strand of the target sequence is cleaved, and there is a certain degree of cleavage specificity. We propose that the Penelope EN cleaves the target DNA during transposition, generating a primer for reverse transcription. Our results show that an active Uri EN has been adopted by a retrotransposon.

We report that two structurally similar transposable elements containing reverse transcriptase (RT), Penelope in Drosophila virilis and Athena in bdelloid rotifers, have proliferated as copies containing introns. The ability of Penelope-like elements (PLEs) to retain introns, their separate phylogenetic placement and their peculiar structural features make them a novel class of eukaryotic retroelements.

Analysis of the precursors of bacterial exported proteins revealed that those having bulky hydrophobic residues at position -5 have a high incidence of Pro residues at positions -6 and -4, Val at position -3, and Ser at positions -4 and -2. This led to a hypothesis that the previously observed inhibition of processing by bulky residues at position -5 can be suppressed by introduction of Pro, Ser, or Val in the corresponding nearby positions. Subsequent mutational analysis of Escherichia coli alkaline phosphatase showed that, as it was predicted, Pro on either side of bulky hydrophobic -5 Leu, Ile, or Tyr completely restores efficiency of the maturation. Introduction of Val at position -3 also partially suppresses the inhibition imposed by -5 Leu, while a Ser residue at position -4 or -2 does not restore processing. In addition, effective maturation of a mutant with Pro residues at positions from -6 throughout -4 proved that polyproline conformation of this region is permissive for processing. To understand the effects of the mutations, we modeled a peptide substrate into the active site of the signal peptidase using the known position of the beta-lactam inhibitor. The inhibitory effect of the -5 residue and its suppression by either Pro -6 or Pro -4 can be explained if we assume that Pro-containing -6 to -4 regions adopt a polyproline conformation whereas the region without Pro residues has a beta-conformation. These results permit us to specify sequence requirements at -6, -5, and -4 positions for efficient processing and to improve the prediction of yet unknown cleavage sites.

The Penelope family of retroelements was first described in species of the Drosophila virilis group. Intact elements encode a reverse transcriptase and an endonuclease of the UvrC type, which may play a role in Penelope integration. Penelope is a key element in the induction of D. virilis hybrid dysgenesis, which involves the mobilization of several unrelated families of transposable elements. We here report the successful introduction of Penelope into the germ line of Drosophila melanogaster by P element-mediated transformation with three different constructs. Penelope is actively transcribed in the D. melanogaster genome only in lines transformed with a construct containing a full-length Penelope clone. The transcript is identical to that detected in D. virilis dysgenic hybrids. Most newly transposed Penelope elements have a very complex organization. Significant proliferation of Penelope copy number occurred in some lines during the 24-month period after transformation. The absence of copy number increase with two other constructs suggests that the 5' andor 3' UTRs of Penelope are required for successful transposition in D. melanogaster. No insect retroelement has previously been reported to be actively transcribed and to increase in copy number after interspecific transformation.

Species of the virilis group of Drosophila differ by multiple inversions and chromosome fusions that probably accompanied, or led to, speciation. Drosophila virilis has the primitive karyotype for the group, and natural populations are exceptional in having no chromosomal polymorphisms. We report that the genomic locations of Penelope and Ulysses transposons are nonrandomly distributed in 12 strains of D. virilis. Furthermore, Penelope and Ulysses insertion sites in D. virilis show a statistically significant association with the breakpoints of inversions found in other species of the virilis group. Sixteen newly induced chromosomal rearrangements were isolated from the progeny of D. virilis hybrid dysgenic crosses, including 12 inversions, 2 translocations, and 2 deletions. Penelope and Ulysses were associated with the breakpoints of over half of these new rearrangements. Many rearrangement breakpoints also coincide with the chromosomal locations of Penelope and Ulysses insertions in the parental strains and with breakpoints of inversions previously established for other species of the group. Analysis of homologous sequences from D. virilis and Drosophila lummei indicated that Penelope insertion sites were closely, but not identically, located at the nucleotide sequence level. Overall, these results indicate that Penelope and Ulysses insert in a limited number of genomic locations and are consistent with the possibility that these elements play an important role in the evolution of the virilis species group.