Transposon telomeres are widely distributed in the Drosophila genus: TART elements in the virilis group

Abstract

Telomeres of most animals, plants, and unicellular eukaryotes are made up of tandem arrays of repeated DNA sequences produced by the enzyme telomerase. Drosophila melanogaster has an unusual variation on this theme; telomeres consist of tandem arrays of sequences produced by successive transpositions of two non-LTR retrotransposons, HeT-A and TART. To explore the phylogenetic distribution of these variant telomeres, we have looked for TART homologues in a distantly related Drosophila species, virilis. We have found elements that, despite many differences in nucleotide sequence, retain significant amino acid similarity to TART from D. melanogaster. These D. virilis TART elements have features that characterize TART elements in D. melanogaster: (i) they are found in tandem arrays on chromosome ends, (ii) they are not found in euchromatin, and (iii) they produce both sense and antisense transcripts, with the antisense RNA being in excess. The D. virilis TART elements have one surprising feature: both of the ORFs contain long stretches of the trinucleotide repeat CAX, encoding polyglutamine (with a few interspersed histidines). These long polyglutamine stretches are conserved in the three D. virilis elements sequenced. They do not interrupt any domains of known function in the TART proteins and are not seen in TART proteins from other species. Comparison of the D. virilis and D. melanogaster telomeres suggests that the retrotransposon mechanism of telomere maintenance may have arisen before the separation of the genus Drosophila.

Figure 1

Diagrams of TART elements in different Drosophila species drawn approximately to scale. 5′, 5′ UTR; 3′, 3′ UTR; Gag, ORF1; Pol, ORF2; AAA, the 3′oligo(A) that characterizes non-LTR retrotransposons. The TART^mel element shows an average of the sizes of the 5′ and 3′ UTRs of the three subfamilies, TART^melA, TART^melB, and TART^melC. The TART^ame is the only fragment cloned. Dotted lines represent putative flanking regions. Solid bars above and below elements correspond to probes (see Materials and Methods).

Figure 2

Diagrams of the two D. virilis phage clones, V8 and V2. Arrows above diagram identify different elements and indicate 5′ → 3′ of sense-strand. ?? indicates unidentified element. Regions in each element are marked as in Fig. 1. 3′-5′ indicates apparently complete junctions between elements. Domains in ORF2 are: E, endonuclease; RT; and X, extra domain. Black rectangles under Gag and Pol indicate high content of CAX repeats; white arrowheads mark the phage arms. Diagrams are approximately to scale.

Figure 3

Hybridization of TART^vir and TART^ame to the telomeres in polytene chromosomes. (a) D. virilis. Three telomeres from the same nucleus are shown. Each has probe hybridized to the terminal region (arrows). Note that this region is aggregated into four balls on one telomere (shown enlarged in Inset). There is no hybridization over any banded chromosome regions. (b) The free telomere of D. americana that hybridizes with TART^ame. As in D. virilis, there is no hybridization in any banded chromosome regions. (c) Chromocenter from a polytene nucleus of D. americana; two discrete regions of the heterochromatic chromocenter are labeled (arrows). [Hybridized probe detected by alkaline phosphatase activity (7). See Materials and Methods for probes.]

Figure 4

Northern hybridizations of TART^vir and TART^ame. Autoradiograph of total RNA from D. virilis and D . americana probed with the Pol coding region of the homologous TART. Both TART^vir probes detect several bands of RNA in the range of 6–9.5 kb. Sense and antisense RNAs do not migrate at exactly the same position, possibly because of differences in size or differences in conformation of the strands, which differ significantly in base composition. The exposure for the TART^vir sense-strand blot was equivalent to four times that of the antisense blot. Only the antisense blot of TART^ame is shown because we have been unable to detect sense-strand RNA for this element.

Figure 5

Phylogenetic relationships of TART, HeT-A, Doc, and jockey elements in different Drosophila species. Neighbor-joining trees for the protein sequences are shown. (The UPGMA trees yield the same relationships as do the nucleotide trees.) Bootstrap tests were performed with 500 replications and a cut-off value of 50% for the consensus tree. Numbers indicate bootstrap values of >40% in the corresponding node. Scale bar corresponds to the P value (number of differences normalized by the number of total residues). Elements are indicted by the first three letters of the species (fun, Drosophila funebris). Note that TART^yak2 groups with TART^melC as proposed (7). Gag from TART^yak was not included because all of the fragments cloned to date are 5′ truncated. GenBank accession numbers: jockey^mel ORF1, M22874; ORF2, AAA28675; jockey^fun, PIR:B38418; Doc, CAA35587.

Figure 6

Alignment of the X domain of TART^vir and TART^ame. Sequence of the TART^vir X domain is residues 1,121–1,538, and TART^ame is residues 1,146–1,518 of the Pol protein. In both cases, the sequence shown here begins just after the residue that aligns with the last residue on the TART^mel and TART^yak protein. Numbers on top indicate residues of the TART^ame protein. Residues of the same chemical property groups are shaded in black. A, TART^ame; V, TART^vir; −, gaps in alignment.