Master Copy Is Not Responsible for the High Rate of copia Transposition in Drosophila

Sarah Perdue and Sergey V. Nuzhdin2,

Section of Evolution and Ecology, University of California at Davis

The genome of Drosophila melanogaster—a model species for studying transposable element (TE) population dynamics—contains approximately 50 different families classified as transposons, long interspersed nuclear element (LINE)–type elements, and long terminal repeat (LTR)–containing retrotransposons that resemble viruses. There are, on average, 20 copies per family, with a few base pairs different between copies within a family (Charlesworth, Sniegowski, and Stephan 1994Citation ). copia and gypsy—the best studied LTR elements—are inactive in most laboratory lines. However, in several transposition-susceptible lines, they produce on the order of one new copy per genome per generation (for reviews, see Pelisson et al. 1997Citation ; Pasyukova, Nuzhdin, and Filatov 1998Citation ). Complex interactions between elements and host genes are responsible for susceptibility. In the case of gypsy elements, a copy of a permissive flamenco allele and a specific "rogue" sequence variant of gypsy are required in the maternal genome for transpositions to occur in progeny.

When flies with the permissive flamenco allele were transformed with the rogue copy of gypsy (the rogue variant was defined by the absence of the HindIII restriction site in the TE body and XbaI sites in both LTRs; Lyubomirskaya et al. 1990Citation ; Pellison et al. 1997), all new transposed copies descended from the rogue copy (Kim et al. 1994Citation ). The master-copy model, which explains accumulation of a single TE sequence variant from a family by overexpression of a specific copy (see, e.g., Smith 1993Citation ), may also be applicable to copia. Indeed, copia elements vary in the numbers of enhancer sequence motifs in the 5' LTR and the adjacent untranslated leader region. Variants differ in their abilities to drive expression when they are hooked up to reporter genes and reintroduced into flies (Matjunina, Jordan, and McDonald 1996Citation ). In Drosophila simulans, there are only weak-driving copia elements. The multiplication of strong-driving copia elements may explain the higher copia copy number in D. melanogaster (Csink and McDonald 1995Citation ).

Earlier, we described isogenic lines of D. melanogaster in which copia transposes with a frequency of 10-3–10-2 per copy per generation ("active" lines; Pasyukova and Nuzhdin 1993Citation ; Nuzhdin and Mackay 1994Citation ). For those lines, we documented a strong positive relationship between copia transposition rate (per copy) and copy number (Nuzhdin et al. 1998Citation ; Pasyukova, Nuzhdin, and Filatov 1998Citation ). This association could be explained by the accumulation of copia elements descending from only one or a few master elements. If so, when the rogue copies multiply, a larger and larger fraction of copies would become of the rogue type, increasing the overall per-copia transposition rate (Nuzhdin et al. 1998Citation ). Susceptibility for copia transposition in one of the active lines is controlled by interacting factors mapping to the regions 27B–48D on the second chromosome and 61A–65A and 97D–100A on the third chromosome (Nuzhdin et al. 1998Citation ). Similar factors turned out to be host gene alleles for copia-like Ty1 TEs in yeast (Lee et al. 1998Citation ). However, it remains possible that a master copy is one of these factors in the active Drosophila lines. To test both of these hypotheses, we directly examined with molecular tools whether different copia variants are capable of multiplication or whether, as with gypsy, a rogue element has this capacity.

LTRs and 5' untranslated regions have been sequenced for 22 D. melanogaster copia elements, and segregating single-nucleotide polymorphisms have been identified (Csink and McDonald 1995Citation ). Based on these data, we designed two pairs of primers—5'-CAATAAAAAGAGTGGTATTCTCTC-3' (primer 1.1) or T-3' (primer 1.2), and 5'-CACAGCAAAAAACGTACAAGAAGG-3' (primer 2.1) or A-3' (primer 2.2)—for which the 3' nucleotide corresponded to one of the previously identified polymorphic variants (each variant was present at least twice in a sample described by Csink and McDonald [1995Citation ]). These primers are homologous to copia LTRs and directed toward the 5' end of copia. If only one copia copy multiplies, copia elements in all new insertion sites should be of that kind. Alternatively, if we find polymorphic copia in new sites, the master-copy hypothesis will be rejected for this element.

To separately assess accumulation of copia elements homologous to different primers, we employed the technique of sequence-specific amplified polymorphism (SSAP), originally developed to study TE site heterogeneity in plants (Waugh et al. 1997Citation ). In SSAP, one ligates a ~20-bp adapter oligonucleotide to the ends of genomic DNA digested with a restriction enzyme and PCR-amplifies this DNA using a radioactively labeled TE-specific primer and a "cold" primer homologous to the adapter. Since the distance from a TE LTR to the first restriction site outside of it varies across inserts, different-sized amplification products correspond to TEs located in different chromosomal positions. copia transpositions accumulated in the replicates of the lines 2b (Pasyukova and Nuzhdin 1993Citation ) and 39 (unpublished data) for many generations (see Pletcher, Houle, and Curtsinger [1998Citation ] for the description of the latter line). If some copia sequence variant does not transpose, the set of amplification products should remain unchanged. Accumulation of a variant in new positions should cause the appearance of amplification products with new sizes (see Nuzhdin and Mackay [1994]Citation for similar logic applied to cytological positions of copia inferred by in situ hybridization with polytene chromosomes).

Genomic DNA was separately extracted from five female flies of the isogenic line Oregon R, in which copia is stable. Extractions were also made from 14 replicates of line 2b and 12 replicates of line 39. DNAs were digested with MboI. Completeness of digestion was visually checked on agarose gels. Each DNA sample was then ligated with the adapter and preamplified with the primer homologous to the adapter. Specific amplifications were made with each of the above 33P-5'-end–labeled copia-homologous primers and a cold primer homologous to the adapter. All oligonucleotides and all reaction and amplification conditions were in exact accordance with the procedure designed by Waugh et al. (1997). Bands of different sizes were resolved in 6% acrylamide gels. Signals were recorded on a Storm Phosphorimager.

The same set of bands was observed for all Oregon R flies (data are not shown). Because the 2b and 39 strains are highly inbred, all transposition accumulation lines derived from them should share a common set of initial sites (excisions of copia are rare; Pasyukova and Nuzhdin 1993Citation ). For any primer, most bands were shared between samples within a line. One stronger band was expected which corresponded to the amplification from 3' LTR toward the MboI site in the copia body. For both primers of the first pair (primers 1.1 and 1.2), many extra bands were detected (the results were very similar for 2b and 39 strains and are illustrated for the latter one in fig. 1A and B, respectively). This represents evidence for transpositions of both copia sequence variants. Note, however, that some band size changes could have come from point mutations in restriction sites as well as from small insertions or deletions (Petrov, Lozovskaya, and Hartl 1996Citation ) and from rearrangements in heterochromatin (Nurminski et al. 1994Citation ), which are frequent in Drosophila.



View larger version (148K):
[in this window]
[in a new window]
 
Fig. 1.—Plots A, B, C, and D represent products of amplification with primers 1.1, 1.2, 2.1, and 2.2, respectively. The same 12 preamplified DNA samples extracted from replicates of line 39 were used. Exposures were varied fourfold to get roughly equal band strengths in different lanes. The bands of amplification from the 3' LTR into the copia body are depicted as triangles. For illustration, several bands from new copia transpositions are shown as closed arrows, and those from copia positions fixed among replicated lines are shown as open arrows

 
Unexpectedly, the specificity of the second pair of primers was lower. The number of bands was initially too high to resolve unambiguously. We reduced the number of bands by adding selective nucleotide A to the cold primer during the specific amplification stage. This enabled us to detect copia transpositions with the second primer (2.2) but not with the first (2.1) (fig. 1D and C, respectively). Perhaps primer 2.1 recognized dead copia elements that were incapable of transposing. Incomplete DNA digestion would not explain the presence of extra bands in figure 1 , because the specific amplifications were made from the same set of preamplified DNA samples.

Here, the SSAP technique was used to prove that different sequence variants of copia transpose in laboratory lines and to rule out the hypothesis of a master copy for this element. Whether or not different copia variants (identified by Csink and McDonald 1995Citation ) transpose with different rates remains to be tested in future studies. SSAP might also be useful for obtaining multiple molecular markers highly polymorphic in natural populations. Although some SSAP bands are shared between flies due to amplification of heterochromatic TE copies fixed within species (Shevelyov 1993Citation ), most bands correspond to euchromatic sites. Those should not be shared between unrelated flies because of low frequency of TE site occupation in natural populations (Charlesworth, Sniegowski, and Stephan 1994Citation ). Conveniently, over 200 amplifications (each yielding multiple markers) could be done with DNA extracted from one fly. Inconveniently, the SSAP markers are dominant, and their positions are unknown.


    Acknowledgements
 TOP
 Acknowledgements
 literature cited
 
We thank Andy J. Flavell, in whose lab S.V.N. performed his first SSAPs. Burroughs Wellcome fund provided travel money. Charles Robin, Brad Shaffer, and Teresa Leonardo commented on the paper. Sarah Reiwitch and Katie Penkoff contributed to the establishment of the technique in the lab of S.V.N., who was supported by UC Davis start up funds and NSF grant DEB-9815621.


    Footnotes
 
Charles Aquadro, Reviewing Editor

1 Keywords: Drosophila melanogaster, transposable elements master copy molecular markers Back

2 Address for correspondence and reprints: Sergey V. Nuzhdin, Section of Evolution and Ecology, University of California at Davis, Davis, California 95616. E-mail: svnuzhdin{at}ucdavis.edu Back


    literature cited
 TOP
 Acknowledgements
 literature cited
 

    Charlesworth, B., P. Sniegowski, and W. Stephan. 1994. The evolutionary dynamics of repetitive DNA in eukaryotes. Nature 371:215–220.

    Csink, A. K., and J. F. McDonald. 1995. Analysis of copia sequence variation within and between Drosophila species. Mol. Biol. Evol. 12:83–93.[Abstract]

    Kim, A. I., N. V. Lyubomirskaya, E. S. Belyaeva, and Y. V. Ilyin. 1994. The introduction of a transpositionally active copy of retrotransposon gypsy into a stable strain of Drosophila melanogaster causes genetic instability. Mol. Gen. Genet. 242:472–477.[ISI][Medline]

    Lee, B.-S., C. P. Lichtenstein, B. Faiola, A. A. Rinckel, W. Wysock, M. J. Curcio, and D. J. Garfinkel. 1998. Posttranscriptional inhibition of Ty-1 retrotransposon by nucleotide excision repair/transcription factor TFIIH subunits Ssl2p and Rad3p. Genetics 148:1743–1761.

    Lyubomirskaya, N. V., I. R. Arkhipova, Y. V. Ilyin, and A. I. Kim. 1990. Molecular analysis of the gypsy (mdg4) retrotransposon in two Drosophila melanogaster strains differing by genetic instability. Mol. Gen. Genet. 223:305–309.[ISI][Medline]

    Matjunina, L. V., I. K. Jordan, and J. F. McDonald. 1996. Naturally occurring variation in copia expression is due to both element (cis) and host (trans) regulatory variation. Proc. Natl. Acad. Sci. USA 93:7097–7102.

    Nurminski, D. I., Y. Y. Schevelyov, S. V. Nuzhdin, and V. A. Gvozdev. 1994. Structure, molecular evolution and maintenance of copy number of extended repeated structures in the heterochromatin of Drosophila melanogaster. Chromosoma 103:277–285.

    Nuzhdin, S. V., and T. F. C. Mackay. 1994. Direct determination of retrotransposon transposition rates in Drosophila melanogaster. Genet. Res. 63:139–144.

    Nuzhdin, S. V., E. G. Pasyukova, E. A. Morozova, and A. J. Flavell. 1998. Quantitative genetic analysis of copia retrotransposon activity in inbred Drosophila melanogaster lines. Genetics 150:755–766.

    Pasyukova, E. G., and S. V. Nuzhdin. 1993. Doc and copia instability in an isogenic Drosophila melanogaster stock. Mol. Gen. Genet. 240:302–306.[ISI][Medline]

    Pasyukova, E. G., S. V. Nuzhdin, and D. A. Filatov. 1998. The relationship between the rate of transposition and transposable element copy number for copia, Doc, and roo retrotransposons. Genet. Res. 72:1–11.[ISI][Medline]

    Pelisson, A., L. Teysset, F. Chalvet, A. Kim, N. Prud'homme, C. Terzian, and A. Bucheton. 1997. About the origin of retroviruses and the co-evolution of the gypsy retrovirus with the Drosophila flamenco host gene. Genetica 100:29–37.

    Petrov, D. A., E. R. Lozovskaya, and D. L. Hartl. 1996. High intrinsic rate of DNA loss in Drosophila. Nature 384:346–349.

    Pletcher, S. D., D. Houle, and J. W. Curtsinger. 1998. Age-specific properties of spontaneous mutations affecting mortality in Drosophila melanogaster. Genetics 148:287–303.

    Shevelyov, Y. Y. 1993. Aurora, a non-mobile retrotransposon in Drosophila melanogaster heterochromatin. Mol. Gen. Genet. 239:205–209.[ISI][Medline]

    Smith, A. F. A. 1993. Identification of a new, abundant superfamily of mammalian LTR-transposons. Nucleic Acids Res. 8:1863–1872.

    Waugh, R., K. McLean, A. J. Flavell, S. R. Pearce, A. Kumar, B. B. T. Thomas, and W. Powell. 1997. Genetic distribution of Bare-1-like retrotransposable elements in the barley genome revealed by sequence-specific amplification polymorphisms (S-SAP). Mol. Gen. Genet. 253:687–694.[ISI][Medline]

Accepted for publication March 2, 2000.