Penelope, a new family of transposable elements and its possible role in hybrid dysgenesis in Drosophila virilis

Abstract

A hybrid dysgenesis syndrome occurs in Drosophila virilis when males from an established laboratory strain are crossed to females obtained from the wild, causing the simultaneous mobilization of several different transposable elements. The insertion sequence responsible for the mutant phenotype of a dysgenic yellow allele has been characterized and named Penelope. In situ hybridization and Southern analyses reveal the presence of more than 30 copies of this element in the P-like parental strain, whereas Penelope is absent in all M-like strains tested. Penelope contains one 2.5-kb-long ORF that could encode products with homology to integrase and reverse transcriptase. Northern analysis and whole-mount in situ hybridization show strong induction of a 2.6-kb RNA in the ovaries of dysgenic females that is expressed at very low levels in the parental strains or in the progeny from the reciprocal cross. Injection of Penelope-containing plasmids into preblastoderm embryos of an M-like strain results in mutant progeny caused by insertion of Ulysses and perhaps other transposons, suggesting that Penelope expression might be responsible for the observed dysgenesis syndrome and the simultaneous mobilization of other transposable elements.

Figure 1

Southern blot analysis of genomic DNA from wild-type and mutant strains. (A) Ten micrograms of total DNA from strain 9 (lane 1), strain 160 (lane 2), and the y^d mutant strain (lane 3) was digested with HindIII and subjected to Southern blot analysis using the SalI–EcoRI fragment of a D. melanogaster yellow-containing clone (16) as a probe. (B) Genomic DNA from various D. virilis strains and mutants isolated from the progeny of dysgenic crosses was digested with XhoI and probed with the XhoI fragment of Penelope. Lanes: 1, strain 160; 2, strain 2; 3, strain 9; 4, white mutant; 5, sn²⁵ mutation; 6, revertant of sn²⁵ mutation; 7, y^d mutation. The arrow indicates the position of the 2.8-kb band. (C) Genomic DNA of various D. virilis strains was digested with BamHI and subjected to Southern blot analysis. Lanes: 1, strain 160; 2, Pasadena strain; 3, Krasnodar strain. The arrow indicates the position of a prominent 2.7-kb band present in all strains containing Penelope. (D) DNA from strain 9 (lane 2) and two independent droop mutants (lanes 1 and 3) obtained from the progeny of embryos injected with Penelope-containing clones were digested with EcoRI and HindIII and hybridized with a probe containing an internal SalI–BamHI fragment of the Ulysses element. The arrow indicates an additional restriction fragment seen in both droop mutant strains.

Figure 2

In situ hybridization of Penelope and Ulysses elements to D. virilis polytene chromosomes. (A) Hybridization of a yellow-containing clone to chromosomes of the M-like strain 9. The arrowhead indicates the only labeled site corresponding to the yellow locus in the D. virilis X-chromosome. (B) Hybridization of the yellow-containing clone to chromosomes of the P-like strain 160; multiple sites are seen due to hybridization of Penelope. (C) Hybridization of the SalI–BamHI fragment of Ulysses with the proximal end of chromosome X of D. virilis strain 9. (D) Hybridization of Ulysses with polytene chromosomes from a droop mutant obtained in the progeny of injected embryos of strain 9; the arrowhead indicates the appearance of an additional site of hybridization. (E) Hybridization of Penelope (clone p1) with strain 9 chromosomes; the only site of hybridization in the 49B section of chromosome 4 is indicated by the arrowhead and is due to the presence of flanking sequences in the clone. (F) Hybridization of the same clone with the chromosomes of strain D8, an unstable strain displaying a Delta phenotype obtained in the progeny of embryos injected with the Penelope element (see Table 1). An additional site of hybridization in chromosome 5 indicated by an arrow resulted from the insertion of Penelope sequences.

Figure 3

Schematic representation of the structure of different Penelope copies isolated from various genomic libraries. (A) Penelope from strain 160 containing terminal repeats in direct orientation and a complete ORF (p6). (B) Penelope element inserted in the yellow locus in the dysgenic y^d allele (clone py2). (C) Penelope from strain 160 with two repeats in inverse orientation (p17). (D) Copy of Penelope isolated from strain sn²⁵ obtained from a dysgenic cross; this copy contains two tandemly arranged central cores carrying two terminal repeats in direct orientation and one in inverted orientation (clone p1). The dotted line represents the genomic tandem organization. (E) Penelope from a dysgenic mutant strain with part of the core region in antisense orientation (clone psn25-4).

Figure 4

Sequence alignments of retroviral and Penelope-encoded proteins. (A) Sequence alignment of representative integrases of various retroelements with the putative integrase of Penelope. Amino acids identical or chemically similar to those of the putative Penelope protein are shown as gray boxes. Residues that form part of the Zn finger motif have been indicated by a + symbol, whereas amino acids in the DD35E motif are indicated by ∗. (B) Sequence alignment of RTs of various retroelements and the RT of Penelope. Dotted lines separate conserved blocks of RTs described by Xiong and Eickbush (21). Identical or chemically conserved residues are indicated as shaded boxes. Chemically similar amino acids are grouped as follows: A, S, T, P, and G; N, D, E, and Q; H, R, and K; M, L, I, and V; F, Y, and W (22).

Figure 5

Analysis of Penelope expression in parental strains and reciprocal hybrids. (A) Transfer analysis of RNA isolated from parental strains and dysgenic hybrids using the ³²P-labeled XhoI fragment of Penelope as a probe. Lanes: 1, dysgenic females; 2, dysgenic males; 3, females from the reciprocal cross; 4, males from the reciprocal cross; 5, strain 160 flies; 6, strain 9 flies. The position of the 2.6-kb transcript induced in dysgenic hybrids is indicated by the arrow. (B) Five micrograms of poly(A)⁺-containing RNA isolated either from ovaries or from fly carcasses lacking ovaries was subjected to Northern blot analysis as described above. Lanes: 1, carcasses of females from the reciprocal cross without ovaries; 2, carcasses of dysgenic females without ovaries; 3, ovaries isolated from females of the reciprocal cross; 4, ovaries isolated from dysgenic females. The position corresponding to the 2.6-kb transcript induced in the ovaries is indicated by the arrow. (C) The same blot was rehybridized with a Drosophila actin gene and is shown as a marker for the amount of RNA. The position of the actin RNA is indicated by the arrow.