MOLECULAR AND CELLULAR BIOLOGY, Apr. 1992, p. 1613-1620

Vol. 12, No. 4

0270-7306/92/041613-08$02.00/0 Copyright C) 1992, American Society for Microbiology

Involvement of cDNA in Homologous Recombination between Ty Elements in Saccharomyces cerevisiae CATHY MELAMED,t YAEL NEVO, AND MARTIN KUPIEC* Department of Molecular Microbiology and Biotechnology, Tel Aviv University, Ramat Aviv 69978, Israel Received 25 November 1991/Accepted 28 January 1992

Strains carrying a marked Ty element (TyUra) in the LYS2 locus were transformed with plasmids bearing a differently marked Tyl element (TylNeo) under the control of the GAL promoter. When these strains were grown in glucose, a low level of gene conversion events involving TyUra was detected. Upon growth on galactose an increase in the rate of gene conversion was seen. This homologous recombination is not the consequence of increased levels of transposition. When an intron-containing fragment was inserted into TylNeo, some of the convertants had the intron removed, implying an RNA intermediate. Mutations that affect reverse transcriptase or reverse transcription of TylNeo greatly reduce the induction of recombination in galactose. Thus, Ty cDNA is involved in homologous gene conversion with chromosomal copies ofTy elements. Our results have implications about the way families of repeated sequences retain homogeneity throughout evolution.

Repetitive sequences are present in the genomes of all of the eukaryotic organisms studied to date. They are scattered through the genome, sometimes in very large numbers. Although most of these sequences have no known function, they represent a potential source of genomic instability, since recombination between dispersed repeats can lead to chromosomal rearrangements, such as translocations, inversions, and deletions. Ectopic recombination (recombination between sequences that share homology but are located at different positions in the genome) occurs readily between artificial repeats in the yeast Saccharomyces cerevisiae in both vegetative growth (26, 30, 35) and meiosis (17, 18, 22). Homologous recombination can be reciprocal or nonreciprocal (gene conversion). When gene conversion events are selected for, approximately half of the cases (17 to 64% in meiosis, 10 to 55% in mitosis) show an associated exchange of flanking markers (11, 14). When a conversion event involves sequences located on nonhomologous chromosomes, the associated crossover will produce a reciprocal translocation. Translocations were indeed recovered at the expected frequency when selection was made for ectopic gene conversion events (18, 19, 22). Several families of naturally occurring repeats exist in the yeast genome. One of the most prominent, representing -1 to 2% of the genome, is the Ty element family. These elements are present in 30 to 40 copies per haploid genome and are related structurally and functionally to retroviruses (for reviews, see references 2 and 31). The similarities are a consequence of the fact that both retroviruses and Tys use reverse transcription of an end-to-end transcript as the means of transposition (4). Ty elements direct the synthesis of viruslike particles (VLPs) (15, 25), and the mechanism of transposition has been shown to resemble that of retroviruses, except that the entire cycle is intracellular. The RNA is reverse transcribed to cDNA by Ty-encoded proteins present in the VLPs. As VLPs are rather big, it is possible that the particles do not enter the nucleus. Instead, a cDNA

intermediate

may

be injected and then integrated into the

yeast genome with the help of a Ty integrase, creating a 5-bp duplication (72). Ty elements transpose at low frequencies (10-' to 10- per locus per cell division [2]). The two main

classes of Ty elements, Tyl and Ty2, have a similar overall structure and share extensive homology, differing in two regions that show sequence divergence (2, 31). When ectopic meiotic recombination was measured between Ty elements, the frequency of gene conversion was shown to be low (about 10-6). In contrast to the -50% association seen with artificial repeats, almost no reciprocal exchange was detected in hundreds of conversion events (19, 20). Reciprocal translocations associated with mitotic recombination are detected, but at very low frequencies (23). Since almost no translocations are detected associated with Ty conversion, there is no evidence for physical contact between chromosomes. In the present study we investigate the possibility that diffusible molecules (RNA or cDNA) are involved in homologous recombination between Ty elements. We report that cDNA participates in gene conversion events involving marked Ty elements.

MATERIALS AND METHODS

Media, growth conditions, and general procedures. Yeast cells were grown vegetatively at 32°C in either SD-Leu medium (0.67% yeast nitrogen base-2% glucose plus nutrients and amino acids, except leucine) or SGal-Leu medium (0.67% yeast nitrogen base-2% galactose plus nutrients and amino acids, except leucine) (34). Leucine was added (40 ,ug/ml) for strains without plasmids. Ura- colonies were selected on SD-Complete plates containing 5-fluoro-orotic acid (5-FOA) (5). CAN medium is SD-Complete without arginine, plus 40 ,ug of canavanine sulfate per ml. YPD contains 1% yeast extract, 2% Bacto Peptone, and 2% glucose. Solid media had 1.5% Bacto Agar added. Standard molecular biology procedures such as cloning, restriction enzyme analysis, and Southern blot analysis were done as described in reference 24. Yeast molecular biology (transformations and DNA preparations, etc.) were done as described in reference 34. Yeast strains. Yeast strains used in the present study are listed in Table 1. MK67 was derived from MK39 (19) and

* Corresponding author. Electronic mail address: MARTIN@ TAUNOS.BITNET. t Present address: Department of Organic Chemistry, Weizmann Institute of Science, Rehovot 76100, Israel.

1613

1614

MELAMED ET AL.

MOL. CELL. BIOL.

TABLE 1. Strains and plasmids used in the present study or relevant marker(s) - - 1- I Genotype I. ---

Strain or plasmid

Strains MK67 MK87 MK104

---

--- --

..MA4Ta ura3-Nco- his3-11,15 leu2-3,112 trpl-Xba- canl-101 lys2::Ty2Ura ..M....AL4Ta ura3-Nco- his3-11,15 leu2-3,112 trpl-Xba- canl-101 lys2::Ty2Ura ..MA4Ta ura3-Nco- his3-11,15 leu2-2, 113 trpl-Xba- canl-101 lys2::TylUra

Plasmids

pM48 ....... pM52 ....... pM97 ....... pM102 ....... pM109 .......

21Lm LEU2 Gal-TylNeo 2pm LEU2 Gal-TylNeo+RP51 2pm LEU2 CANM Gal-TylNeo As pM97, reverse transcriptase negative (Asp 718 fill-in) As pM97, primer binding site mutated

carries a Ty2Ura inserted at the LYS2 locus in chromosome II. Since it grew very poorly on galactose, it was crossed to W303-1B (32) to create MK87. MK104 was created by first transforming MK87 to Lys' with pRE194, thus creating MK89, and then transforming MK89 to Ura+ Lys- with pM115 digested with BamHI and XbaI. All of the chromosomal configurations were confirmed by Southern blot analysis. Plasmid constructions. Plasmids used in this study are listed in Table 1. pM48 was constructed by replacing the URA3-containing BamHI-ApaI fragment of pGTyNeo (6) with the BamHI-HpaI fragment of YIp33 carrying the LEU2 gene. pM97 was created by inserting a BamHI fragment carrying most of the CANI gene (1) in the BamHI site of pM48. pM52 was created by inserting a RP51-containing BglII-AvaII fragment from pHZ18 (37) (obtained from M. Rosbash) in the SmaI site of the neo insertion. pM102 was created by filling in with the Klenow fragment of DNA polymerase I and ligation after partial cutting with Asp 718 (an isoschizomer of KpnI). The filling in creates a SnaBI site in place of the Asp 718 site. pM109 was created by replacing a SnaBI fragment of pM97 with the same fragment from plasmid pCK7 (6a) (a generous gift from Jef Boeke). This fragment contains 5-bp substitutions in the primer binding site without affecting the TYA open reading frame. Another plasmid used, pRE194, is identical to pDP6 (13) and contains an XbaI-HindIII fragment of the LYS2 gene. Two other plasmids were also used as LYS2 probes: YIp333, carrying a bigger PstI-EcoRI fragment (from R. Rothstein), and pM43 (19), carrying an overlapping PvuII fragment. pGH54 contains a 1-kb insert carrying the neomycin resistance gene from Tn9O3 (a generous gift from J. Boeke). pM21 carries the 1.15-kb URA3 gene in pUC19 (19). pM115 was constructed in several steps. First, GalTylUra was constructed by inserting the URA3 1.15-kb BamHI fragment from pM21 in the 3' BglII site of pGalTy (Ty1H3 [4]). Then the GalTylUra was inserted as a BamHI fragment into the unique BglII site of pRE194, thus creating pM106. pM115 is identical to pM106, except that the 5' long terminal repeat (LTR) was reconstructed by replacing the XbaI-ClaI GAL-containing fragment with the XbaI-ClaI fragment carrying an intact 5' LTR derived from pGN821 (from J. Boeke). Finally, a BamHI fragment containing the whole TylUra was inserted in the BglII site of pRE194. Measurement of Ty recombination. Twelve to 36 small independent cultures were grown to the late logarithmic phase on SD-Leu or SGal-Leu medium, washed, and resuspended in water. Aliquots were plated on 5-FOA plates or were diluted and plated on YPD. Colonies were scored after 3 days. Recombination rates were calculated by the method of the median (21).

The YPD plates were routinely replica plated onto SD-Leu plates to score the presence of the plasmids. Typically,

about 50 and 30% of the colonies were Leu+ after growth on SD-Leu and SGal-Leu medium, respectively. One independent Ura+ Leu- colony from each culture was transferred to a master plate and replica plated onto YPD plates containing G418 (Geneticin; Sigma Co.) at a final concentration of 200 ,ug/ml, to assay for transposition proficiency (6). Independent Ura- colonies were picked from 5-FOA plates, streaked on YPD or CAN plates and replica plated onto SD medium lacking either uracil (to confirm the Uraphenotype) or leucine (to look for colonies that have lost the plasmid). Ura- Leu- colonies were picked to a master plate and replica plated onto G418-containing YPD plates. DNA was prepared from G418-resistant and G418-sensitive colonies and subjected to Southern blot analysis. LYS2, URA3, neo, and RP51 sequences were used as probes. DNA sequencing. Two Ura- G418-resistant colonies derived from MK67/pM52 grown on SGal-Leu and carrying intronless TylNeos at LYS2 were chosen for cloning and sequencing. The TylNeos were cloned by eviction (39) after transformation with pRE194 to Lys'. Appropriate fragments were subcloned into M13mpl8 and M13mpl9 and sequenced with the Sequenase 2.0 kit (United States Biochemical) as recommended by the supplier. RESULTS Experimental design. Strains MK67 and MK87 are Ura+ because of the presence of a single functional copy of the URA3 gene located inside a Ty2 element designated Ty2Ura (19). Ty2Ura is inserted at the LYS2 locus, in chromosome II. Ura- derivatives can be selected by plating on 5-FOA media (5). Previous experiments have shown that most of these Ura- derivatives (80 to 90%) are created by ectopic gene conversion between Ty2Ura and unmarked Ty elements. As a consequence of this nonreciprocal recombination, part of the information in Ty2Ura (carrying the URA3 gene) is replaced by information of unmarked Tys, resulting in a Ura- cell. A smaller proportion of these Ura- cells are created by mutations in the functional UR43 of the TyUra or by gene conversion with the ura3-Nco- allele at the URA3 locus (19). We transformed strains MK67 and MK87 either with plasmid pM48, a high-copy-number plasmid that carries a Tyl element under the control of the GALI promoter, or with pM97, a similar plasmid that carries also the CANM gene (allowing selection against the plasmid). The Ty elements in these plasmids carry the bacterial neo gene at a position in the Tyl identical to the position of the URA3 gene in the chromosomal Ty2Ura (6). Under inducing conditions (galac-

Ty cDNA IS INVOLVED IN HOMOLOGOUS RECOMBINATION

VOL. 12, 1992

TyUra

1615

TABLE 2. Rate of appearance of Ura- colonies

p

Strain

MK67

TyNeo

MK67/pM48 MK67/pM52 MK87

MK87/pM97 MK87/pM102 MK87/pM109

Rate of appearance (10-7) on:

Induction (od

(fold)

Glucose

Galactose

2.41 ± 0.37 2.07 ± 0.45 2.31 ± 0.47

2.22 ± 0.54 6.08 ± 0.28 7.29 ± 0.98

0.92 2.94 3.16

± + + ±

1.09 10.44 0.81 0.88

3.03 3.66 3.58 3.89

± ± ± ±

0.63 0.56 0.52 0.61

3.31 38.20 2.89 3.41

0.66 5.29 0.56 0.50

:"a

Select Ura-, Lose plasmid

TyNeo FIG. 1. General strategy. pM48, a plasmid carrying a GalTyl Neo, was introduced into strains with TyUra inserted in the LYS2 locus. After growth in glucose or galactose, Ura- cells were selected for and after plasmid curing were checked for the presence of a TylNeo at LYS2 in place of the TyUra. Open triangles, Ty LTRs; 1, URA3 sequences; W, LYS2 open square, GAL promoter; sequences; _, neo sequences; C, LEU2 sequences.

tose-containing medium), the marked Ty is highly expressed and the marked Ty transposes at high rates (4, 6). The transposition efficiency is dependent on the temperature (28, 29). Our experiments were carried out at 32°C, a temperature at which the rate of transposition is rather low (see below). MK67/pM48 and MK87/97 were grown at 32°C in galactose- or glucose-based media (inducing and noninducing conditions, respectively), and Ura- derivatives were selected on 5-FOA plates (Fig. 1). As a control, the strains without a plasmid were grown under similar conditions. Results are shown in Table 2. A similar rate of events that give rise to Ura- colonies was seen for these strains grown in glucose or galactose and for MK67/pM48 and MK87/pM97 in glucose. When the strains carrying the plasmids were grown on galactose-containing medium, however, a 3- to 10-fold increase in Ura- colonies was seen (Table 2). Independent colonies were isolated for each strain and after plasmid curing were subjected to replica plating to G418containing plates and to Southern blot analysis with LYS2 sequences as a probe. MK67 and MK87 alone and the strains carrying plasmids, when grown in glucose-containing medium, showed the same results previously seen: most of the Ura- colonies were created by gene conversion between Ty2Ura and unmarked Tys (19, 20) (Fig. 2A). No homology to TylNeo was found in 40 Ura- colonies of each strain. In

contrast, 7 of 21 and 11 of 14 Ura- colonies of galactosegrown MK67/pM48 and MK87/pM97, respectively, showed in Southern blot analysis the pattern expected if the Ty2Ura in the chromosome has been replaced by a TylNeo (Fig. 2B). Thus, growth of the strains carrying GAL-promoted Ty elements caused an increase in the level of homologous recombination involving chromosomal Tys, as measured at the Ty2Ura locus. Many of these events involve TylNeo information. The actual increase in the level of Ty2UraTylNeo recombination cannot be accurately measured, since we have not found such an event in glucose-grown cells (0 of 80 colonies), but can be estimated as at least 40-fold for MK67/pM48 and 300-fold for MK89/pM97. It should be noticed that although a low level of transposition of the TylNeo occurred in our cultures, the LYS2 probe does not detect transposition events, since only the Ty element located at the original position of the Ty2Ura can be detected. In all of the cases, the fragment sizes obtained showed that the position of the Ty element inside the LYS2 gene was not changed but the Ty2Ura was replaced by TylNeo sequences. We have never detected a transposition event within the Ty2Ura. These results were confirmed by rehybridizing the Southern blots with neo or URA3 sequences; in all of the cases in which a conversion event had occurred, the neo probe hybridized to bands previously shown to hybridize with the LYS2 sequences, and only chromosome V sequences were detected with the URA3

probe. We will address the issue of transposition in these experiments further below. Experiments with TylUra. The marked TyUra used in the above-described experiments was a Ty2, whereas the TyNeo carried in the plasmid belongs to the Tyl class. Although both classes share extensive homology, they are not completely identical (2, 31). In order to assess the homology requirements of this type of recombination, we constructed a new strain, MK104, isogenic to MK87 but carrying TylUra (TyH3 [3]) inserted at the LYS2 locus instead of the Ty2Ura carried by MK87. Strain MK104 showed a basal level of Ty-Ty recombination much higher than the one observed for MK67 and MK87 (Table 3). Southern blot analysis showed that only 1 of 33 Ura- colonies grown on glucose and 1 of 35 Ura- colonies grown on galactose were the product of a Ty-Ty interaction; the rest had been created by an event that replaced the whole TylUra by a solo LTR element. Solo LTRs can result from (i) an intrachromosomal crossover involving the two LTRs of the marked Ty element, (ii) a gene conversion event in which the whole Ty was replaced by a single LTR, or (iii) an unequal exchange involving sister chromatids (32). If we calculate the basal level of interactions between the

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marked Ty and other full-length Ty elements in this strain (1/34 x 32.3 x 10-7 = 0.95 10-7), we observe that it is similar to the one seen in MK67 and MK87. When MK104/pM97 was grown on glucose, the same basal level of Ura- colonies was seen, but upon growth of the strain on galactose, a threefold increase in the rate of appearance of Ura- colonies was seen. Southern analysis showed that 20 of 20 colonies analyzed from MK104/pM97 grown on glucose showed solo LTRs at LYS2, whereas only 9 of 32 colonies isolated after growth on galactose belonged to this category. Four of the 32 showed the pattern of a conversion event with unmarked Tys, and 19 of 32 were the result of a conversion event in which TylUra was replaced by TylNeo. Thus, upon growth on galactose, there is an increase in the level of recombination between TylUra and other Tys (23/32 x 113.6 x 10-7 = 81.65 x 10-7, an 86-fold increase). In most of these events (83%) TylUra has been replaced by TylNeo. Again, the actual increase in TylUra-TylNeo interactions cannot be accurately calculated, since no such event was detected among 40 colonies grown in glucose, but can be estimated as at least 70-fold. Mechanism of recombination. What is the origin of the x

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TylNeo information now present at LYS2? Three main possibilities must be considered. (i) Chromosomal TyUra interacts with TylNeo on the plasmid, and a gene conversion event introduces TylNeo information in place of TyUra. This plasmid-chromosome interaction must be dependent on the presence of galactose, since no TylNeos were seen among the Ura- colonies selected after growth on glucose. This dependence could be indicative of a role of transcription in the recombination process: sequences that are heavily transcribed could be in a chromatin configuration that allows them to act as donors of information in a conversion event. (ii) Another possible origin for the TylNeo information is the Ty transcript: in galactose-containing medium the Ty on the plasmid is heavily transcribed, resulting in an increase in the level of Ty mRNA and possibly of Ty cDNA as well (4, 15). In this model either of these molecules (RNA transcript or cDNA) is engaged in homologous recombination with the chromosomal copy of TyUra. (iii) A third possibility is that the increased transcription of Ty on galactose causes an increased level of Ty transposition. As a result, many copies of the TylNeo are inserted at different places in the genome, and then any one of them serves as a donor of information in a subsequent genomic Ty-genomic Ty gene conversion event. This model predicts that every Ura- cell in which TyUra was replaced by TABLE 3. Rate of appearance of Ura- colonies Strain

MK104 MK104/pM97 MK104/pM52 MK104/pM102 MK104/pM109

Rate of appearance

Glucose 33.5 ± 7.4 37.9 ± 5.7 35.6 ± 9.3 42.7 ± 3.5 35.6 ± 4.4

(10-') on: Galactose

31.10 113.60 109.52 13.00 12.02

± 2.8 ± 19.0 ± 13.5 ± 1.9 + 2.8

Induction

(fold)

(od

0.93 3.0 3.08 0.30 0.34

VOL. 12, 1992

Ty cDNA IS INVOLVED IN HOMOLOGOUS RECOMBINATION

TylNeo would show at least one additional copy of TylNeo integrated in the genome. It is also possible in theory to have a transposition event in G2 of the cell cycle, followed by an ectopic conversion event and then segregation of the TylNeos to different daughter cells. We do not think that this mechanism is very probable, since it involves several concerted events and in at least half of the cases we would expect both TylNeos to segregate to the same daughter cell. As shown below, this was found not to be the case: most of the G418-resistant colonies had no additional copies of neo in their genome. We have performed our experiments at 32°C, a temperature at which the level of transposition was minimal, and we could still detect recombination events. The level of transposition after growth on galactose was measured (by Southern blot hybridization with a neo probe after plasmid loss): Ura- cells showed the same level of transposition as Ura+ cells, and the distribution of events was similar in Ura- cells converted by TylNeos or in Uracells converted by unmarked Tys. The number of neohybridizing bands (apart from the one at LYS2) was measured in galactose-grown MK67/pM48: 6 of 21 Ura- colonies showed one band, and 1 of 21 showed two bands (average number of transposition, 38%). Ten of 36 randomly picked Ura+ colonies showed a single neo-hybridizing band (28%). As expected, no hybridizing band was seen among colonies grown on glucose (4). Similar results were obtained for MK87. In total, 16 of 18 Neor Ura- colonies in which Ty2Ura was replaced by TylNeo did not show extra neohybridizing bands. For MK104/pM97 grown in galactose, 3 of 24 Ura- and 4 of 27 Ura+ colonies showed one neo-hybridizing band outside the LYS2 locus. Thus, most (17 of 19) of the colonies in which TylUra was replaced by TylNeo didn't show extra bands that hybridized with the neo probe. We conclude that conversion of TyUra by TylNeo is not correlated with high levels of transposition and that most of the conversion events do not occur after transposition. These results rule out the third explanation. A transcription product is involved in recombination. In order to distinguish between the two possibilities left, namely, (i) transcription-stimulated recombination of the plasmid with the chromosome and (ii) gene conversion of the Ty RNA or cDNA with the chromosome, we designed the following experiment (Fig. 3). MK67 and MK104 were transformed with a new plasmid, called pM52, that is identical to pM48, except that it carries a piece of the RP51 gene inserted into the neo gene inside the inducible Tyl. The RP51 gene codes for a ribosomal protein and contains a small intron (37). We reasoned that while in plasmid-to-chromosome conversion the intron will always be coconverted with the neo gene, in RNA or cDNA-tochromosome conversion the intron may be spliced out sometimes from the RNA intermediate. Thus, if we find cases in which TyUra has been replaced by an intronless TylNeo, we will have a demonstration that there is an RNA intermediate in the flow of information between the plasmid and TyUra. Independent Ura- colonies were isolated after growth on glucose or galactose, and after plasmid curing, they were subjected to Southern blot analysis with a neo probe. No TylNeo was observed among 20 Ura- colonies isolated after growth of MK67/pM52 on glucose. Of 62 galactose-grown colonies analyzed, however, 24 (39%) had TylNeo replacing Ty2Ura. Southern analysis with different restriction enzymes showed that 9 of these TylNeos lacked the intron and

1617

TyUra

{_~~~~~~~~~~~~2RPSI +Intron

Select Ura-, Lose plasmid

TyNeow/o Intron or

TyNeo + Intron

FIG. 3. Experiment that distinguishes conversion by plasmid from conversion by transcription products (RNA or cDNA). pM52 is identical to pM48, except for the insertion of an intron-containing fragment from RP51 S. Conversion events in which plasmid sequences acted as donors of information should retain the intron. If there is an RNA step before recombination, intronless TyNeos should be found among the Ura- colonies. Symbols are as in Fig. 1.

15 retained it (Fig. 4). These results were confirmed by using a DNA probe internal to the RP51 intron (Fig. 5). Two of the intronless TylNeos were cloned from the chromosome, and the RP51 insertion was sequenced (see Materials and Methods). The sequence confirmed the absence of the intron sequences and the presence of a precise splicing junction as predicted (37) (data not shown). We also performed the same experiments with MK104/ pM52. When this strain was grown in glucose, no TylNeo replacing TylUra was seen in 12 Ura- colonies analyzed by Southern blot analysis. Growth in galactose caused a threefold increase in the number of Ura- colonies (Table 3). Of 27 Ura- colonies that grew in galactose, 19 had TylNeo at LYS2. Of these, 13 had the intron removed, 5 still had the intact intron, and 1 showed a deletion of the intron and part of the neo gene. The intronless TyNeos provide positive evidence for the involvement of either RNA or cDNA in homologous recombination. Since not all of the TylNeos had the intron removed, we checked the TylNeos created by transposition events in these strains. Five transposition events were detected in 22 Ura- colonies (5 of 22, 22.7%); of these, four had the intron removed and one did not. Two transposition

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iounmarked

events were detected among 12 randomly picked Ura+ colonies (16.7%): one of them retained the initron, and the other did not. Thus, intron-containing arnd intronless TylNeos are detected in these strains in both transposition and recombination, suggesting that transposiltion and gene conversion involve the same intermediate(s). 'The detection of TylNeo convertants containing the intro n and of the

partially deleted TylNeo is best explained by low splicing efficiency of the unusual TylNeo-Rp5l transcript. The Uracolonies carrying TylNeo containing the intron could be due to conversion events involving unspliced RNA molecules or their cDNA derivatives. In a transposition system using intron-marked GalTyHis, Curcio and Garfinkel (7) report a 12.5% efficiency of splicing. We cannot, however, completely rule out the possibility that transcription stimulates direct plasmid-to-chromosome gene conversions. Ty cDNA is involved in homologous recombination. The experiments described above provide positive evidence for the involvement of a diffusible molecule in ectopic recombination between Tys. Is this molecule RNA or DNA? In order to distinguish between these two possibilities we repeated the above-described experiments with plasmids pM102 and pM109, derivatives of pM97. In the first plasmid, a frameshift mutation was introduced in the TYB open reading frame, and thus the Ty cannot encode for reverse transcriptase activity but can be properly transcribed. We reasoned that if cDNA is the molecule that participates in homologous recombination with genomic Tys, this mutation should reduce the induction seen in galactose. On the other hand, if RNA is directly involved in recombination, we shouldn't see any effect. Plasmid pM109 carries five base substitutions in the

primer binding site of the Ty (6a). Thus, although it carries an intact reverse transcriptase, the Ty is unable to initiate the reverse transcription. Again, if cDNA is the molecule involved, we expect a reduction in the rate of Ura- colonies obtained on galactose. Tables 2 and 3 show the results obtained with both plasmids in strains MK87 and MK104: no induction of recombination involving the TylUra was seen in strains MK87 and MK104 (in fact, an actual decrease in the rate of appearance of Ura- colonies was seen). As expected, no transposition events were observed among 12 randomly picked Ura+ colonies from each strain grown on galactose. Since mutations which eliminate reverse transcription dramatically reduce the induction on galactose, we conclude that homologous recombination between Tys can be mediated by a cDNA molecule.

Ty cDNA IS INVOLVED IN HOMOLOGOUS RECOMBINATION

VOL. 12, 1992

DISCUSSION In the present study we have shown that Ty cDNA can be involved in homologous recombination with chromosomal copies of Ty elements. Our results may explain the paucity of chromosomal translocations associated with recombination between Tys. They do not, however, rule out the existence of recombination events involving chromosomal copies of Tys. Chromosomal translocations following conversion events between Tys have been observed, although their frequency is extremely low (reviewed in reference 23;

19). We have shown that growth of cells that carry a GalTy element under conditions that induce transcription causes an increase in homologous recombination (gene conversion). This increase is not due to the presence of transposed copies that can serve as donors in the conversion event. In the conditions we used, transposition occurred at relatively low levels, and most of the cells that had undergone a gene conversion event did not carry additional copies of TylNeos in their genomes that could act as donors of information. Both Tyls and Ty2s in the genome can interact with the marked TylNeo; the increase observed in MK104/pM97 (86-fold) seems to be higher than the one seen in Ty2Uracontaining strains (up to 10-fold), suggesting a homology requirement for this type of recombination. We have shown that an intron can be precisely spliced out of TylNeo before the conversion event, thus providing positive evidence for the existence of a RNA intermediate in the recombination process. Mutations in the Ty that reduce reverse transcription abolish the induction of gene conversion as well. Thus, Ty cDNA appears to be the main molecule involved in homologous recombination with the chromosomal Ty elements. We propose that after being reverse transcribed in the VLP, the Ty cDNA returns to the nucleus. There it can participate in a nonhomologous integration in the chromosome (driven by the Ty integrase [2]) or can be available to the general cellular recombinational machinery for an omega type of recombination (33). We have used a system in which high levels of Ty mRNA and protein products are created upon induction. In strains carrying only the normal load of Ty elements, these interactions between Ty cDNA and chromosomal copies of Tys take place at much lower frequencies. In fact, the frequency of mitotic and meiotic recombination between Tys appears to be low compared with the frequency of recombination between similarly sized genes. The introduction of additional Ty elements on high-copy-number plasmids does not elevate the rate of recombination (Tables 2 and 3, compare results on glucose with and without plasmid). These results imply that the copy number of potential donor sequences in the cell does not affect the level of recombination between Tys. Several explanations can be given for this observation. It is possible that chromosomal or plasmid copies of the Ty do not usually participate as donors in homologous recombination, because of a specific trans-acting repressing system or some special feature(s) of Ty sequence or chromatin. Alternatively, the level of recombination between two sequences could be determined by the rate at which recombination is initiated, by a double-strand break, in the recipient copy (16, 36). This rate would be usually very low for Tys, but in our experiments the presence of large amounts of cDNA carrying homologous ends could bypass this first limiting step and induce a high level of recombination, similar to a transformation event with linearized plasmids (27, 33). Most eukaryotes contain degenerate copies of genes called

1619

pseudogenes (38). These are usually copies of expressed genes containing mutations in the coding region. They lack promoter sequences and introns and contain poly(A) stretches at their 3' end. They are probably created by reverse transcription of cellular RNAs, with subsequent integration into the genome. The yeast S. cerevisiae has few, if any, processed pseudogenes. Yeast genes also usually lack introns, and whenever those are found, they are located at the very 5' end of the gene. Fink (12) has proposed an explanation for these intriguing features of the yeast genome: because of the very efficient homologous recombination of yeast, reverse transcripts of expressed genes will tend to recombine back by homologous recombination, instead of integrating elsewhere in the genome. In this way, all of the yeast genes will eventually be replaced by cDNA, eliminating the introns (except when they are located at the terminus of a gene, where not enough homology is left to carry out the gene conversion). We have shown that this is indeed the case for Ty elements. An intron inserted in the marker within the Ty sequence is removed before the Ty sequence is converted into the genome. Does this process take place with non-Ty transcripts? Reverse transcription is carried out within the VLP, and thus the reaction is isolated from other cellular components. Although no mRNA from overexpressed lacZ (41) or URA3 (15) genes could be detected inside VLPs, TRP1 and HIS3 mRNAs were seen (40). Recently, Derr and coworkers (9) have shown that an intron-containing copy of the HIS3 gene, when highly expressed, can be reverse transcribed and become part of the genome. In their experiments, in approximately half of the cases the new gene had transposed to a new location and in the other half it had converted the homologous copy of the gene (carried by a plasmid). This process was dependent on the level of Ty products in the cell and was associated with Ty reverse transcription. Families of repeated sequences can usually retain homogeneity by means of gene conversion between different members of the family (10). However, the association of crossover with gene conversion introduces the possibility of deleterious chromosomal rearrangements. Conversion events involving cDNA dissociate in fact both processes and provide an evolutionarily useful mechanism of sequence conservation. We assume that an omega type of conversion event usually occurs; a crossover associated with the conversion of a short cDNA molecule would be lethal. Further investigation is needed in order to elucidate the actual mechanism of conversion between Ty cDNA and chromosomal copies of Ty elements. It is interesting to note that if gene conversion usually takes place through a cDNA intermediate, highly expressed copies will tend to be the main donors of information among the family members, thus creating an ever-increasing level of expression. In fact, the level of Ty mRNA has been estimated to reach as much as 50% of the poly(A) mRNA level of the yeast cell (8). ACKNOWLEDGMENTS We thank T. Petes, in whose lab this study was started, S. Rozenblat's lab members for help with sequencing, and J. Boeke for generously providing us with plasmids and information. We thank Rivka Steinlauf for excellent technical assistance and T. Petes, D. Nag, Y. Koltin, and the members of the Kupiec lab for critically reading the manuscript. This work was supported by grants to M.K. from the Council for Tobacco Research and the Israeli Cancer Research Foundation.

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MELAMED ET AL.

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