Department of Evolution and Ecology, University of California at Davis
![]() |
Abstract |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Key Words: Doc retrotransposon SSAP gene expression ectopic recombination
![]() |
Introduction |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
The stable existence of active TEs in the host genome over a long period of time has been a puzzle to many biologists. One plausible hypothesis is that TEs survive by increasing copy numbers by transposition, and natural selection purges them because of lower fitness of hosts with heavier loads of TEs (Charlesworth, Sniegowski, and Stephan 1994). The deleterious effect of TEs on host fitness can come directly from the disruption of host genes due to TE insertions (Finnegan 1992), or it may come from increasing the probability for ectopic recombination to occur due to increased copy numbers of TEs (Goldberg et al. 1983). A third possibility is that the deleterious effect may come from TE expression required for transposition. Abundant retrotransposon transcripts may be resource consuming for the host and, thus, may have negative effects upon host fitness (Nuzhdin, Pasyukova, and Mackay 1996). Further, transposase activity may cause chromosome breaks and decrease host fitness (Brookfield and Badge 1997). The first two mechanisms have been tested in multiple studies (reviewed by Charlesworth, Sniegowski, and Stephan 1994; Biémont et al. 1997; Charlesworth, Langley, and Sniegowski 1997), but the third mechanism has never been tested.
Transcriptional regulation has been well-characterized in several LINE type elements, including Doc and I. Expression of LINEs requires RNA polymerase to bind to the promoter region in the 5' end. The Doc promoter is located within the first 60 bp from the 5' end (Contursi, Minchiotti, and Di Nocera 1995; Minchiotti, Contursi, and Di Nocera 1997). Copies of Doc cDNA synthesized by reverse transcriptase are frequently truncated at the 5' end. After inserting into the host genome, the 5' truncated copies may no longer transpose, as they lack the promoter region needed for RNA polymerase to bind (Luan et al. 1993). All new inserts are therefore replicating from transposition of full-length copies with promoters, but not all new inserts will have promoters.
If TE expression harms the host, we would expect Docs with promoters to persist in natural population for fewer numbers of generations than Docs without promoters. Therefore, we predict that (1) the ratio of promoter-bearing versus promoter-lacking copies is lower for elements segregating in natural populations than among new Doc inserts observed in the lab, and (2) Doc copies lacking a promoter are more likely to drift to higher frequency in natural populations than promoter-bearing copies. We test these hypotheses specifically by comparing genome-wide copy numbers and distributions of these two types of Docs in two sets of lines of Drosophila melanogaster. One set is isogenic lines that have been propagated under small population size and relaxation of selection (Pasyukova and Nuzhdin 1993; Pasyukova, Nuzhdin, and Filatov 1998), and the other set is isofemale lines collected from a natural population. Our results suggested that expression of Docs does not significantly impact host fitness.
![]() |
Materials and Methods |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
SSAP Analyses
Genomic DNA from 20 to 30 female flies of each line was phenol-chloroform extracted, ethanol precipitated, and diluted to a final concentration of about 0.5µg/µl. After overnight digestion with MboI, the restricted genomic DNA fragments were ethanol precipitated and ligated to MboI adaptors with DNA ligase (Promega). About 0.05 ng of the ligation product was used for PCR preamplification with MboI primer (5'-GATGAGTCCTGAGGATC-3') and Taq DNA polymerase (Promega). The program for PCR reaction starts with 94°C for 5 min, followed by 30 cycles of 94°C (30 s), 60°C (30 s), and 70°C (1 min), and it ends with 70°C for 7 min. The product was then selectively PCR amplified with the MboI primer and one of the three P33-labeled Doc primers complementary to the 5' end of Doc and designed to amplify the 5' end of Doc and surrounding host DNA. The primer sequences relative to the transcription initiation and the promoter region of Doc elements is shown in figure 1. Note that each primer is about 100 bp apart and the promoter is located between primers Doc1 and Doc2. The thermal program for selective PCR was 94°C for 5 min, 14 cycles of denaturing at 94°C (30 s), and annealing at temperatures starting at 65°C and descending with 1.4°C every two cycles (30 s), followed by 23 cycles of 94°C (30 s) and 56°C (30 s) and terminated with1 cycle at 72°C (10 min). Sequences of the three Doc primers are Doc1 (5'-AAAGAAACACGTCTCCACCC-3'), Doc2 (5'-ATTACTATAGTCAATAATTGTTAGTTG-3'), Doc3 (5'-AGAGCCGGCGCTCGTCTTGTT-3'). The PCR products were denatured at 70°C and separated on 6% acrylamide gels under 1800 V, 100 W, and 50 mAmp for 3.5 h. Each amplification yielded multiple bands corresponding to the distances between the Doc primer binding site and the Mbo1 recognition site closest to the 5' end of DocI, which are different between Doc inserts. After the gel was transferred, dried, and exposed to a screen for a few days, bands were visualized by Phosphorimaging on the Storm 8600 imager.
|
Statistical Analyses
Chi-square tests were performed to verify whether the proportions of promoter-bearing versus promoter-lacking copies are different between 2b and WI lines. To infer the relative strengths of selection on promoter-bearing versus promoter-lacking copies and to calculate the selection boundaries, we did the following calculations. nb(unselected) and nl(unselected) are the numbers of promoter-bearing and promoter-lacking copies found among new Doc inserts in 2b replicates, and nb(selected) and nl(selected) are the numbers of copies detected in flies extracted from nature. Suppose qb (ql) is the frequency of the presence of promoter-bearing (or promoter-lacking) Doc insert per unit of DNA length (e.g., per cytological subdivision) in natural population, ub (ul) is the rate of transposition per unit of DNA, sb (sl) is the average strength of selection against an insert, and h is their average recessivity. Assuming TE-caused mutations, h > 0 (see Fry and Nuzhdin 2003; the following conclusions will need only a trivial modification if h = 0), under mutation-selection balance, the equilibrium frequency of occupancy per DNA unit is qb = ub/hsb for full copies and ql = ul/hsl for promoter-lacking copies. Then, nb(selected)/nl(selected) = qb/ql = (ub/sb)/(ul/sl). The ratio of transposition rates is equal to the ratio of accumulated unselected copies: ub/ul = nb(unselected)/nl (unselected). It follows that sl/sb = (nb(selected) nl (unselected))/(nl (selected) nb(unselected)). Given the number of detected unselected and selected promoter-lacking and promoter-bearing copies, how different could natural selection on them be? nb(unselected), nl(unselected), nb(selected), and nl(selected) are Poisson distributed (Charlesworth and Charlesworth 1983), and their upper and lower 95% confidence limits are straightforward to calculate. Combining the boundaries giving the smallest and the largest sl/sb estimates, we calculate the confidence interval of this ratio.
The Mann-Whitney U test was used to test whether the distributions of site occupancy frequencies differ between promoter-bearing verses promoter-lacking copies. More specifically, we tested whether the promoter-bearing copies have significantly smaller occupancy frequencies than the promoter-lacking copies due to stronger selection against the former.
The selection coefficient of Doc insertions was estimated following method B in appendix of Biémont et al. (1994). Briefly, assuming ß distribution of Doc frequencies, under an infinite site model, the stationary probability distribution of element frequencies at individual sites is proportional to x-1(1-x)ß-1, where ß equals to 4Nes (Ne is the effective population size and s is the selection coefficient against Docs) (Charlesworth and Charlesworth 1983). Transforming the probability density function into a discrete distribution, the expected numbers of sites with i chromosomes occupied out of all chromosomes sampled j(j = 23 in our WI samples) is (j/i)(1-i/j)ß-1. We estimated ß with the minimum chi-square method comparing the expected site occupancy distribution under the discrete distribution with the observed one.
![]() |
Results and Discussion |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
|
|
|
The reported Doc elements in the Drosophila genome database (release 3, Kaminker et al. 2002) consist of 28 sequences in the euchromatic regions. Among them, eight copies (30%) lack the 5' end, and three copies (10.7% out of total) of these eight copies have 5' truncations located within first 300 bp of Doc. This is consistent with our whole genome results, which suggest 16.1% of Docs are truncated within the first 300 bp of the 5' end. The rest of the truncated copies have their truncation points far downstream from the promoter region. Assuming these proportions represent general features of the fly genome, our method would only allow us to detect three-eighths of all the 5' truncated copies in the genome. This fact, however, does not affect our conclusion, as our experiment was designed explicitly to test the hypothesis of deleterious effect of TE expression. Moreover, the dynamics of short Doc copies with the 5' truncation points far away from the promoter region may also be affected by reduced probability of ectopic recombination due to shorter sequence homology. Thus, our analyses, focused only on long copies with or without promoter regions, can avoid the complexity caused by differences in the rate of ectopic recombination.
To test whether ectopic recombination biased our comparisons, we compared strength of selection on two groups of promoter-lacking Docs that are different in size: those that were amplified both with Doc2 and Doc3 primers (D2+3) and those that were amplified with Doc3 primers only (D3). The estimated selection coefficients of these two types of copies based on their distributions of site occupancy frequencies were not significantly different (data not shown). The test lacks power because of our small sample sizes of both types of copies and the small differences in length (100 bp) between them. In contrast to our results, Petrov et al. (personal communication) focused on the differences in the distributions of site occupancy frequency among several TE families and found that families with longer sequence length are more strongly selected against than families with shorter sequence length, providing evidence supporting ectopic recombination. Furthermore, Bartolomé and Charlesworth (2002) presented evidence for the deleterious effect of ectopic recombination based on a genome-wide survey. They found TE abundance to be strongly associated with local recombination rates but not gene densities.
Curiously, our results also show that copies at a few sites reached high frequencies. It would be interesting to check if these copies are located in regions of high recombination, which would suggest that they are very likely to either be sites that have undergone selective sweeps or linked to sites under gene selective sweeps but not driven by genetic drift. Although TE insertions are considered mostly deleterious, some evidence suggests that a few cases of TE insertions can be beneficial to the host (for review see Kidwell and Lisch 2000; Maside, Bartolomé, and Charlesworth 2002; Schlenke and Begun, personal communication). In these cases, the TE insertions are usually located in the upstream regulatory regions of a gene, and their beneficial effect is mediated through regulating the expression levels of the adjacent gene. It would be interesting to further investigate these high-frequency Doc copies and their relationship with neighboring genes.
In conclusion, Doc expression does not impose a detectable effect on the host fitness in this study, assuming expression is aptly represented by the presence and absence of promoter sequence. The force opposing the spread of TEs is most likely due to gene disruptions by TE insertions and from ectopic recombination induced by sequence homology among TEs.
![]() |
Acknowledgements |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
![]() |
Footnotes |
---|
Brandon Gaut, Associate Editor
![]() |
Literature Cited |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Bartolomé, C., and B. Charlesworth. 2002. On the abundance and distribution of transposable elements in the genome of Drosophila melanogaster. Mol. Biol. Evol. 19:926-937.
Biémont, C. 1992. Population genetics of transposable DNA elements: a Drosophila point of view. Genetica 86:67-84.[ISI][Medline]
Biémont, C., F. Lemeunier, M. P. Garcia Guerreiro, J. F. Brookfield, C. Gautier, S. Aulard, and E. G. Pasyukova. 1994. Population dynamics of the copia, mdg1, mdg3, gypsy, and P transposable elements in a natural population of Drosophila melanogaster. Genet. Res. 63:197-212.[ISI][Medline]
Biémont, C., A. Tsitrone, C. Vieira, and C. Hoogland. 1997. Transposable element distribution in Drosophila. Genetics 147:1997-9.
Brookfield, J. F., and R. M. Badge. 1997. Population genetics models of transposable elements. Genetica 100:281-294.[CrossRef][ISI][Medline]
Charlesworth, B., and D. Charlesworth. 1983. The population dynamics of transposable elements. Genet. Res. 42:1-27.[ISI]
Charlesworth, B., C. H. Langley, and P. D. Sniegowski. 1997. Transposable element distributions in Drosophila. Genetics 147:1993-5.
Charlesworth, B., A. Lapid, and D. Canada. 1992. The distribution of transposable elements within and between chromosomes in a population of Drosophila melanogaster. I. Element frequencies and distribution. Genet. Res. 60:103-14.[ISI][Medline]
Charlesworth, B., P. Sniegowski, and W. Stephan. 1994. The evolutionary dynamics of repetitive DNA in eukaryotes. Nature 371:215-220.[CrossRef][ISI][Medline]
Contursi, C., G. Minchiotti, and P. P. Di Nocera. 1995. Identification of sequences which regulate the expression of Drosophila melanogaster Doc elements. J. Biol. Chem. 70:26570-26576.[CrossRef]
Finnegan, D. J. 1992. Transposable elements. Pp. 10961107 in D. L. Lindsley and G. Zimm, eds. The genome of Drosophila melanogaster. Academic Press, New York.
Fry, J. D., and S. V. Nuzhdin. 2003. Dominance of mutations affecting viability in Drosophila. Genetics (in press).
Goldberg, M. L., J.-Y. Sheen, W. J. Gehrubg, and M. M. Green. 1983. Unequal crossing-over associated with asymmetrical synapses between nomadic elements in the Drosophila melanogaster genome. Proc. Natl. Acad. Sci. USA 80:5017-5021.[Abstract]
Kaminker, J., C. Bergman, and B. Kronmiller, et al. (12 co-authors). 2002. The transposable elements of the Drosophila melanogaster euchromatin: a genomics perspective. Genome Biol. 3:1-20.
Kidwell, M. G., and D. R. Lisch. 2000. Transposable elements and host genome evolution. Trends Ecol. Evol. 15:95-99.[CrossRef][ISI][Medline]
Leigh-Brown, A. J., and J. E. Moss. 1987. Transposition of the I element and copia in a natural population of Drosophila melanogaster. Genet. Res. 49:121-128.[ISI]
Luan, D. D., M. H. Korman, J. L. Jackubczak, and T. H. Eickbush. 1993. Reverse transcription of R2Bm is primed by a nick at the chromosomal target site: a mechanism for non-LTR retrotransposition. Cell 72:595-605.[ISI][Medline]
Maside, X., C. Bartolomé, and B. Charlesworth. 2002. S-element insertions are associated with the evolution of the Hsp70 genes in Drosophila melanogaster. Curr. Biol. 12:1686-1691.[CrossRef][ISI][Medline]
Montgomery, E. A., and C. H. Langley. 1983. Transposable elements in Mendelian population II. Distribution of three copia-like elements in a natural population of Drosophila melanogaster. Genetics 104:473-483.
Minchiotti, G., C. Contursi, and P. P. Di Nocera. 1997. Multiple downstream promoter modules regulate the transcription of the Drosophila melanogaster I, Doc and F elements. J. Mol. Biol. 267:37-46.[CrossRef][ISI][Medline]
Nuzhdin, S. V., E. G. Pasyukova, and T. F. C. Mackay. 1996. Positive association between copia transposition rate and copy number in Drosophila melanogaster. Proc. R. Soc. Lond. B Biol. Sci. 263:823-831.[ISI][Medline]
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. 72:79-91.[CrossRef][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.[CrossRef][ISI][Medline]