The potential adaptive significance of transposable elements (TEs) to the host genomes in which they reside is a topic that has been hotly debated by molecular evolutionists for more than two decades. While the biological importance of TEs seemed self-evident to those scientists involved in their initial discovery [e.g., 12], the subsequent realization that TEs could be maintained in populations even while imparting slight selective disadvantage to their hosts [e.g., 345] drew into question the presumption of adaptive significance. However, even if TEs can be maintained in populations on a day-to-day basis without providing selective advantage, it does not preclude the possibility that the insertion of TEs in or near genes may, in some instances, be of adaptive advantage.

If TE insertion variants have contributed to adaptive gene evolution, such variants might be expected to be in high frequency or fixed in populations and species. Initial surveys of natural populations of Drosophila melanogaster showing that TE insertion alleles are in uniformly low frequency seemed to negate the adaptive hypothesis 6. However, the sporadic discovery of degenerate TEs or TE fragments as critical components of functional genes in both plants and animals was sufficient to keep the adaptive hypothesis alive throughout the pre-genomic era 7891011.

The current availability of the complete or nearly complete sequence of select genomes representing a variety of species is providing an unprecedented opportunity to examine the frequency and distribution of TEs in eukaryotic genomes. The results have been dramatic. TEs not only comprise a significant fraction of nearly all eukaryotic genomes thus far sequenced, they have been found to be components of the regulatory and/or coding regions of a surprisingly large number of genes [e.g., 12]. For example, a recent genomic analysis of 13,799 human genes revealed that approximately 4% harbored retrotransposon sequences within protein-coding regions 13. Similar results have been recently reported for the nematode Caenorhabditis elegans14. Here we analyze the polymorphism of two LTR retrotransposon / host gene associations across geographically widespread D. melanogaster populations and a representative population of the D. melanogaster sibling species, Drosophila mauritiana.

We have initiated a genomic analysis of LTR retrotransposons present in the Drosophila melanogaster genome [e.g., 15]. Of particular interest is identification of genes harboring TEs and determining if these insertion alleles are in high frequency or fixed among natural populations as would be expected from the adaptive hypothesis. We report here the results of an analysis of two LTR retrotransposon-containing genes located on the second chromosome of the sequenced D. melanogaster y; cn bw sp strain. These two genes present an interesting contrast in that one of them, Chitinase 3 (Cht3), is located within constitutive heterochromatin (Genbank accession: AE002743) while the other, cathD, is located in a euchromatic region of the chromosome (Genbank accession: AE003839). Our findings demonstrate that while the euchromatic cathD insertion variant was not detected in any of the natural populations examined, the insertion variant present in the heterochromatic Cht3 gene was found to be apparently fixed throughout the species. These results are consistent with the view that the presence of TEs in constitutive heterochromatin may have relevance to the expression of heterochromatic genes [e.g., 1617].

Genomic analysis of the sequenced y; cn bw sp strain of Drosophila melanogaster identified a full-length Burdock LTR retrotransposon located just 3' to the cathD gene and a 359bp LTR fragment (complete LTR is 659 bp) of an Antonia LTR retrotransposon 15 located within an intron of the Cht3 gene (Figure 1). A set of PCR primers were designed to amplify regions of both genes and retrotransposon sequences. Appropriate pairs of gene and element primers were used to detect the presence or absence of the respective retrotransposon inserts associated with each gene in strains representing 12 geographically dispersed populations of D. melanogaster. The results presented in Figure 2 and Table 1 demonstrate that while the Burdock insertion located just 3' to cathD gene is not present in any of 12 strains representing a geographically diverse sampling of natural populations, the Antonia LTR fragment located in the intron of the heterochromatic Cht3 gene is fixed in all 12 strains tested.

It is formally possible that the presence of the Antonia LTR within the Cht3 intron was the result of a chance fixation event prior to the expansion of D. melanogaster around the world. Thus, to further test the adaptive hypothesis we compared the level of sequence divergence within the LTR and its flanking intronic sequence between the two sibling species Drosophila melanogaster and Drosophila mauritiana. If the LTR-containing intron is under stabilizing selection, a lower than neutral rate of substitution would be expected. A total of 685 bp of the Cht3 intron was sequenced. This region spans 264 bp of the 359 bp Antonia LTR fragment. The sequence of this region in a D. melanogaster (Dimonika, Africa) and D. mauritiana (Mauritius, Africa) strain was aligned with the homologous region in the sequenced D. melanogaster y; cn bw sp strain (Figure 3). The two melanogaster strains were 100% identical. The melanogaster sequences were found to be only 1.3% (9 substitutions/685 nucleotide sites) diverged from that of D. mauritiana. This value is significantly less than half of the expected 4.3 % (± 2.7) divergence based on the Drosophila neutral substitution rate of 0.016 (± 0.005) substitutions/site/million year 18 over the estimated 2.7 million years separating the two species 19.

To directly compare the substitution rate for the Cht3 intron with that of another Drosophila gene intron, we randomly selected intron 1 of the Drosophila alcohol dehydrogenase (Adh) gene. Adh is a widely studied Drosophila gene and it has been sequenced in several Drosophila species including D. melanogaster, accession X60793 20 and D. mauritiana, accession M19264 21. The sequence divergence between D. melanogaster and D. mauritiana in the Adh intron 1 (7.9%, Figure 4), is higher than that for the LTR containing Cht3 intron (1.3%). These results strongly suggest that conservative selection has been operating on the LTR containing intron associated with the Drosophila Cht3 gene over the past 2.7 million years.

For many years, constitutive heterochromatin was considered to be of little or no functional significance 22. This view seemed to be supported by early molecular studies showing that heterochromatin consists almost exclusively of highly repeated and middle repetitive DNA [e.g., 2324]. The middle repetitive fraction was viewed as the descendent of once active TEs that had the misfortune of inserting into transcriptionally inert heterochromatin at some point in their evolutionary history [e.g., 620]. The view of heterochromatin as a genetic wasteland gradually changed with the mapping of a number of functionally important Drosophila genes to constitutive heterochromatin [e.g., 2425262728293031]. Reexamination of Drosophila constitutive heterochromatin revealed that long stretches of highly repetitive DNA are interrupted by "islands" of retrotransposon sequences [e.g., 3233]. Drosophila genes in heterochromatin are typically associated with these islands of retrotransposons 231343536. It has been suggested that transposable elements inserted into heterochromatin may locally alter chromatin structure [e.g., 16]. Our results suggest that in at least some instances, the association of heterochromatic genes with transposable element sequences may be of adaptive significance.

The results presented here are consistent with the hypothesis that a 359 bp fragment of the Antonia retrotransposon located within the intron of the heterochromatic Drosophila melanogaster Cht3 gene may be of adaptive evolutionary significance. Further genomic and molecular analyses will be required to assess the general importance of LTR retrotransposon sequences to the evolution of heterochromatic gene structure and function.