Sex and the Spread of Retrotramposon Ty 3 in Experimental Populations of Saccharomyces cerevisiae

Mobile genetic elements may be molecular parasites that reduce the fitness of individuals that bear them by causing predominantly deleterious mutations, but increase in frequency when rare because transposition increases their rates of transmission to the progeny of crosses between infected and uninfected individuals. If this is true, then the initial spread of a mobile element requires sex. We tested this prediction using the yeast retrotransposon Ty3 and a strain of Saccharomyces cerevisiae lacking Ty3. We infected replicate isogenic sexual and asexual populations with a galactose-inducible Ty3 element at an initial frequency of 1%. In two of six asexual populations, active Ty3 elements increased in frequency to 38 and 86%, due to the spread in each population of a competitively superior mutant carrying a new Ty3 insertion. Ty3 frequencies increased above 80% in all sexual populations in which transposition was induced in haplophase or in diplophase. Ty3 did not increase in frequency when active during both haplophase and diplophase, apparently because of selective sweeps during adaptation to galactose. Repressed Ty3 elements spread in sexual populations, by increasing sexual fitness. These results indicate that active Ty3 elements are more likely to become established in sexual populations than in asexual populations. M OBILE genetic elements of various types are widespread and abundant in eukaryotic genomes. They have long been hypothesized to be parasitic, spreading because their replication within genomes and dispersal among chromosomes increases their rates of transmission (DOOLITTLE and SAPIENZA 1980; ORGEL and CRICK 1980). Because a mobile element may be transmitted to most of the offspring of crosses between individuals with and without the element, it can spread despite reducing fitness to as little as one-half of the fitness of uninfected individuals (HICKEY 1982). Mobile elements reduce fitness in two ways. First, their insertion into new locations causes mutations, altering either gene products or their patterns of regulation or expression. Second, some elements induce ectopic recombination and chromosomal rearrangements. Mobile elements can produce adaptive mutations and can increase variability in quantitative and metabolic traits (WILKE and ADAMS 1992; CLARK et al. 1995), thus accelerating the response of experimental populations to artificial selection (WCKAY 1985; TORKAMANZEHI et al. 1992). However, the mobile elements responsible for this variation were already present in most or all of the individuals in the experimental populations. Because only a small fraction of the mutations they produce are beneficial, mobile elements must be abundant to produce adaptive variation and usually reCorresponding author: Clifford Zeyl, Department of Biology, McCill University, 1205 Avenue Docteur Penfield, Montreal, Quebec, Canada H3A 1B1. E-mail: [email protected] Genetics 143 1567-1577 (August, 1996) duce the mean fitness of individuals (CHARLESWORTH 1987). For example, transposition by Tyl reduced the mean fitness and increased the variance in fitness of 60 genotypes tested by WILKE and ADAMS (1992). Only one of the 60 mutants had higher fitness, as estimated by density in stationary growth phase, than did the control. Therefore, the effects of established elements on whole populations do not explain their increase in frequency from initial rarity. If mobile elements reduce individual fitness, their spread should occur only in sexual, outcrossing populations, because in clonal or inbreeding populations, biased transmission and the invasion of uninfected lineages cannot occur, and elements that reduce the fitness of a clonal lineage will be eliminated (CAVALIER-SMITH 1980; HICKEY 1982). The chromosomal distributions of mobile elements in natural populations of Drosophila support the view that they produce few if any beneficial mutations: particular insertions are almost always very rare, indicating that they confer no selective advantage (CHARLES WORTH et al. 1994). In many cases, what is known of the molecular biology of mobile elements is consistent with sexual parasitism (ZEYL and BELL 1996). For example, the germ-line specificity of transposition by some elements is an attribute that would be expected of a sexual parasite of metazoans. Although the invasion of populations of D. melanogaster by P elements has been experimentally reproduced (KIDWELL et al. 1981; GOOD et al. 1989), the obligate association of sex with reproduction in D. melanogaster precludes the demonstration that this spread depends on sex. The most direct test 1568 C. Zeyl, G. Bell and D. M. Green of molecular parasitism would be to introduce a mobile element at a low frequency into experimental populations of a facultatively sexual eukaryote, with the expectation that the element would spread in sexual populations but decline in asexual populations. FUTCHER et al. (1988) used precisely such an experiment to demonstrate the parasitic nature of the 2p plasmid of Saccham myces cerevisiae. This plasmid encodes no vegetative functions and reduces fitness by 1%, but invades experimental populations provided that they are sexual and outcrossing. S. cerevisiae is an ideal test organism for the hypothesis that the spread of mobile elements requires sex, because, in addition to being a facultatively sexual and genetically tractable eukaryote with a very short generation time, it hosts four families of retrotransposons. Retrotransposons are elements several kilobases in length, bounded by direct repeats several hundred base pairs long. They are transcribed and encode structural proteins and enzymes that reverse-transcribe the RNA transcripts and integrate the resulting DNA copies into the chromosomes. The molecular biology of the yeast retrotransposons Tyl and Ty3 is especially well characterized and has been reviewed by BOEKE and SANDMEYER (1991). We chose Ty3 as a test element because BILANCHONE et al. (1993) have constructed a yeast strain with no Ty3 elements by experimentally deleting three endogenous copies from a preexisting strain. Moreover, HANSEN et al. (1988) and KIRCHNER et al. (1992) have constructed galactose-inducible Ty3 elements by splicing the GALl-IO upstream activating sequence into the 5' copy of the long terminal repeat, immediately upstream of the site where transcription of wild-type Ty3 elements begins. Transposed copies of this element therefore lack the GALI-losequence, but the galactoseinducible source element permits both the experimental induction of transposition to elevated rates and the repression of Ty3 in control populations cultured on glucose (HANSEN et al. 1988; MENEES and SANDMEYER 1994; KINSEY and SANDMEYER 1995). We used this galactose-inducible retrotransposon and yeast strains lacking Ty3 to test the hypothesis that Ty3 spread would occur only in sexual populations in which transposition was induced. MATERIALS AND METHODS Strains and plasmids: Yeast strains and plasmids are described in Table l. Those used to construct the initial experimental populations were a generous gift from S. SANDMEYER, and additional plasmids used as genetic markers in subsequent tests of relative fitness were constructed from the original plasmids or obtained from H. BUSSEY. Plasmids were maintained in Eschm'chia coli strain DHlOa following transformation by electroporation. To construct isogenic yeast strains each carrying single, chromosomally integrated copies of Ty3 and a selectable marker, yVBll0 was transformed with the large EcoRI fragment of plasmid pEGTy3-1, carrying Ty3 and URA3, and strains yVBl14 and yVBl15 were transformed with the large EcoRI fragment of pJK31lAC, carrying Ty3 and TRPl, using the lithium acetate method as described by GO LEMIS et al. (1994). The Ty3free strains and these transformed derivatives were used to construct the initial experimental populations, as described below. Additional strains, used in subsequent tests for an effect of Ty3 on mating and sporulation, were constructed by transforming yVB110 and yVBll4 with the appropriate restriction fragments of 2p"based plasmids carrying TRPl, URA3 or HZS3 markers, or with the entire plasmids. All media were prepared as described by GOLEMIS et al. (1994), and in addition to standard components included 20 mg/mL uracil and 40 mg/mL tryptophan, except for selective plates used to score genotype frequencies. Construction of base populations and experimental procedure: Four base populations were established: haploid mating type a (MATa), haploid mating type a (MATa), diploid MATa/a, and sexual (both mating types present in equal frequencies). Each base population comprised 99% genotypes lacking Ty3 and 1% isogenic transformants each carrying a single integrated copy of Ty3 and URA3 or TRPl. To construct these base populations, the constituent genotypes were grown overnight in 5 mL liquid W D at 30" with agitation, and mixtures were prepared using ODsoo measurements to determine the appropriate volumes of each genotype. From each mixture, diluted aliquots of equal volumes were thinspread on WD and on SC -Ura or SC -Trp plates to confirm that the Ty3bearing genotypes comprised 1 2 0.2% of each population. These four base populations and the experimental lines established from them are outlined in Figure 1. The MATa, MATa, and diploid mixtures were each used to establish two replicate asexual populations in which transposition was induced by growth on 2% galactose (WGal medium), and two replicate asexual lines in which transposition was repressed by growth on 2% glucose (YF'D medium). For these asexual lines the experimental cycle consisted of culture for 3 days in 5 mL liquid media at 30" with agitation at 250 rpm, spreading 40 pl aliquots on agar plates of the same media, and 3 days' growth at room temperature on the plates, followed by 6 days' refrigeration while the sexual lines completed their cycle. From the sexual base population, three replicates each of four treatments were established: transposition repressed by growth on glucose, transposition induced throughout the cycle by growth on galactose, transposition induced during haplophase, and transposition induced during diplophase. In each sexual cycle, mated cultures were presporulated overnight at 30" in 250-mL Erlenmeyer flasks containing 25 mL YPA (1% yeast extract, 2% peptone, 2% potassium acetate) shaken at 150 rpm. The cultures were then pelleted and resuspended in 25 mL sporulation medium and transferred to new 250 mL flasks. Sporulating cultures were shaken at 350 rpm at room temperature for 5 days. Unsporulated cells were killed by digesting the cultures overnight in 100 pg/mL zymolyase lOOT and 0.2% 0-mercaptoethanol, as described by GoLEMIS et al. (1994). To prevent the inbreeding that results from matings between sibling spores that remain associated (FUTCHER et al. 1988), suspensions of the spores in 5 mL 0.25% Triton-X were shaken at 300 rpm for 30 min with 2 mL of glass beads (450-600 pm; Sigma) to separate sibling spores. Aliquots of 200 p1 from each culture were spread on either WD (for the lines in which transposition was repressed, and those in which it was induced in diplophase) or WGal (for lines in which transposition was induced in haplophase or throughout the cycle). After growth on these plates at room temperature for 2 days, the cells covering about a quarter of the plate were thoroughly mixed with a sterile loop to maxTy3 Spread in Yeast Populations