Fitness Costs of Doc Expression Are Insufficient to Stabilize Its Copy Number in Drosophila melanogaster

Hsiao-Pei Yang and Sergey V. Nuzhdin

Department of Evolution and Ecology, University of California at Davis


    Abstract
 TOP
 Abstract
 Introduction
 Materials and Methods
 Results and Discussion
 Acknowledgements
 Literature Cited
 
The stable coexistence of transposable elements (TEs) with their host genome over long periods of time suggests TEs have to impose some deleterious effect upon their host fitness. Three mechanisms have been proposed to account for the deleterious effect caused by TEs: host gene interruptions by TE insertions, chromosomal rearrangements by TE-induced ectopic recombination, and costly TE expression. However, the relative importance of these mechanisms remains controversial. Here, we test specifically if TE expression accounts for the host fitness cost imposed by TE insertions. In the retrotransposon Doc, expression requires binding of the host RNA polymerase to the internal promoter. If expression of Doc elements is deleterious to their host, Doc copies with promoters would be more strongly selected against and would persist in the population for shorter periods of time compared with Docs lacking promoters. We tested this prediction using sequence-specific amplified polymorphism (SSAP) analyses. We compared the populations of these two types of Doc elements in two sets of lines of Drosophila melanogaster: selection-free isogenic lines accumulating new Doc insertions and isogenized isofemale lines sampled from a natural population. We found that (1) there is no difference in the proportion of promoter-bearing and promoter-lacking copies between sets of lines, and (2) the site occupancy distribution of promoter-bearing copies does not skew toward lower frequency compared with that of promoter-lacking copies. Thus, selection against promoter-bearing copies does not appear to be stronger than that of promoter-lacking copies. Our results show that expression is not playing a major role in stabilizing Doc copy numbers.

Key Words: Doc • retrotransposon • SSAP • gene expression • ectopic recombination


    Introduction
 TOP
 Abstract
 Introduction
 Materials and Methods
 Results and Discussion
 Acknowledgements
 Literature Cited
 
Transposable elements (TEs) are mobile DNA sequences that are found in many different organisms. Their high abundance in the genomes of many species and their capability to express, replicate, and induce recombination between nonhomologous sequences (ectopic recombination) may have a profound impact on structural changes in the host genome. In response to TE transpositions, hosts may also evolve ways to suppress TE activity (Pelisson et al. 1997). Moreover, TEs may be recruited to play functional roles, such as gene regulation, in the host genome (see citations in Kidwell and Lisch 2000; Maside, Bartolomé, and Charlesworth 2002). Knowledge about the mechanisms of TE transposition and TE domestication is important to comprehend the genome evolution of higher organisms.

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
 TOP
 Abstract
 Introduction
 Materials and Methods
 Results and Discussion
 Acknowledgements
 Literature Cited
 
Fly Populations
Two populations were studied: 2b lines and wild-type inbred (WI) lines. 2b lines were constructed from a single parental line, generating an identical genetic background except for mutations that occurred afterward. Lines were propagated at a small population size, thus, natural selection is expected to be strongly reduced in these lines. The degree of relatedness varies between 2b lines. They were kept independently for at least 100 generations. (For details of how these lines were constructed and maintained, see Pasyukova and Nuzhdin 1993; Pasyukova, Nuzhdin, and Filatov 1998). Docs are continuously transposing in these lines (Pasyukova, Nuzhdin, and Filatov 1998). Each line of the WI lines originated from a single, mated female collected from a natural population at Wolfskill orchard (Winters, California) and propagated under full-sib matings for about 40 generations. Genomes of these lines are expected to be highly homozygous. Ten 2b lines and 23 WI lines were used for this study.

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.



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FIG. 1. Schematic structure of Doc elements (the sequence is based on GenBank sequence X17551). Sequence of 350 bp in the 5' end is shown. The promoter region is shown in the box. Sequences and directions of three Doc primers are underlined with long arrows. The only MboI site in this region is marked with an arrowhead

 
The large number of simultaneously segregating bands complicated our analysis. By extending one nucleotide (A, T, C, or G) in the end of the adaptor-homologous primers, we reduced the number of bands fourfold per lane. 2b lines were assessed for primers extended by all four nucleotides. Comparable numbers of bands were detected in the WI lines with the C extended primer, providing sufficient information for data analysis. Therefore, other extended primers were not tested in the WI lines.

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
 TOP
 Abstract
 Introduction
 Materials and Methods
 Results and Discussion
 Acknowledgements
 Literature Cited
 
To infer the ratio of numbers of Docs with and without the promoter region among new inserts, we scored bands that are segregating but not fixed between 2b lines. These bands represent new copies of Doc that were inserted after the lines were constructed. The fixed bands are copies that existed in the common ancestral parental line. In WI lines, both segregating and fixed bands were scored. A copy was scored as promoter-bearing if the same band, except for length shift, was amplified with all three Doc primers. Docs amplified only with primers Doc3 or with both Doc3 and Doc2, but not Doc1, were scored as promoter-lacking copies. Examples of scoring bands from SSAP gel images are shown in figure 2.



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FIG. 2. SSAP analyses of promoter-bearing versus promoter-lacking copies of Doc elements. Samples are 10 2b lines (1 to 10) amplified with three Doc primers (Doc1 to Doc3). In the boxes are examples of the shifted band patterns produced by the same set of Doc elements amplified with three Doc primers, respectively. Bands circled are four examples of truncated copies, which were only amplified with primers Doc2 and Doc3, but not with Doc1. Three bands appear to be of the same size. They are scored as a promoter-lacking copy with site occupancy frequency equaling 0.3

 
In total, 477 inserts in 2b lines and 323 inserts in WI lines were scored. Of these, 66 inserts in 2b lines and 45 inserts in WI lines are promoter-lacking copies (table 1). The ratios of promoter-bearing versus promoter-lacking copies are not significantly different between 2b and WI lines (16.1% vs. 16.3%). Because the 2b lines were propagated under small population size and relaxed selection, we expect the ratio of the two types of copies to not be biased by selection after insertion. In contrast, inserts in WI lines have undergone natural selection. In accordance with derivations in the section Materials and Methods, the ratio of selection coefficients against promoter-lacking and promoter-bearing copies is sl/sb = 1 (the 95% confidence interval 0.89 to 1.12). The similarity of ratios in 2b and WI lines indicates that natural selection against promoter-bearing versus promoter-lacking copies was of similar magnitude, although the selection strength against promoter-bearing copies could be stronger by up to 12%.


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Table 1 Numbers of Promoter-Bearing and Promoter-Lacking Copies Scored in 2b and in W1 Lines.

 
However, it is known that Doc transpositions in 2b replicates are abnormally frequent (Pasyukova, Nuzhdin, and Filatov 1998). To avoid the complexity that they might also be abnormal in the ratio of promoter-bearing versus promoter-lacking copies, we conducted an independent analysis of the site occupancy frequencies of these two types of copies in the WI lines. If Doc expression does hurt the host, selection against promoter-bearing copies should be stronger. As a result, such copies should persist for fewer generations before being eliminated by selection, and should on average attain lower frequencies (Charlesworth and Charlesworth 1983). We did not detect a significantly smaller site occupancy frequency of the promoter-bearing copies (Mann-Whitney U test, U = 0.99). However, due to the existence of few sites with high occupancy frequencies (fig. 3), the distribution of promoter-bearing copies is significantly different from the promoter-lacking distribution (Mann-Whitney U test, U = 0.0039). Since these few high-frequency sites are likely to be sites located in low-recombination regions, such as heterochromatin, or sites closely linked to genes under selective sweeps and, thus, under completely different evolutionary trajectories compared with the rest of sites sampled, we excluded them for our further analysis. Indeed, the difference of site occupancy distribution between promoter-bearing and promoter-lacking copies became marginally significant when high-frequency sites were excluded (Mann-Whitney U test, U = 0.015). We concluded that the trend, if any, is in the direction of stronger selection against promoter-lacking copies. Overall, there is no evidence that Doc expression assists selection in removing Doc inserts.



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FIG. 3. Distribution of site occupancy frequency of promoter-bearing and promoter-lacking copies of Doc elements in the W1 lines

 
We estimated the selection coefficients, ß, of Docs based on the site occupancy distributions of two types of copies. For promoter-bearing copies, ß is 2.7 (with 95% confidence interval between 1.7 and ~3.7), and for promoter-lacking copies, ß is 3.1 (with 95% confidence interval between 2.7 and ~3.7). The selection coefficients are not significantly different between these two types of copies. They are also of the same magnitude as those of previous studies on distributions of TEs in the genomes of D. melanogaster from natural populations (Montgomery and Langley 1983; Leigh-Brown and Moss 1987; Biémont 1992; Charlesworth, Lapid, and Canada 1992). In these studies, results both from in situ hybridization to polytene salivary gland chromosomes and from fine-scale restriction mapping showed that TEs almost always occur in very low occupation frequencies, and the estimates of ß are within the range 5 to 40.

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
 TOP
 Abstract
 Introduction
 Materials and Methods
 Results and Discussion
 Acknowledgements
 Literature Cited
 
We thank Lesley Xiong for DNA extraction; Teresa Leonardo, Corbin Jones, and Dan Barbash for comments on the manuscript; and Phil Awadella and Mark Grote for help on statistics. We thank Brandon Gaut and two anonymous reviewers for helpful comments and suggestions. This work is supported by a NSF grant (#DEB-9815621) to S.V.N.


    Footnotes
 
E-mail: hpyang{at}ucdavis.edu. Back

Brandon Gaut, Associate Editor Back


    Literature Cited
 TOP
 Abstract
 Introduction
 Materials and Methods
 Results and Discussion
 Acknowledgements
 Literature Cited
 

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Accepted for publication January 7, 2003.