Current Genetics

Current Genetics (1982) 6:55-61

© Springer-Verlag 1982

6 Sequences Mediate DNA Rearrangements in Saccharomyces cerevisiae Michael Ciriacy I and Dagmar Breilmann Institut ftir Mikrobiologieder Technischen Hochschule Darmstadt, D-6100 Darmstadt, Federal Republic of Germany

Summary. A solo 6 sequence flanking the 5' end of the ADHII structural gene, ADR2, can promote a number of DNA rearrangements some of which were investigated in detail. In a selective system haploid mutants were screened in which a solo 6 sequence flanking A D R 2 had been joined to a Ty element. Three different types of events can create such a structure: Reintegration of a Ty sequence at the 6-ADR2 site, inversion of ADR2 and flanking material, and transposition of A D R 2 along with 3' flanking material. The involvement of reciprocal or nonreciprocal exchange mechanisms in creating such events are discussed.

Key words: 6 Sequences - Transposition - Inversion Recombination

Introduction In the yeast Saccharomyces cerevisiae, two types of moderately repetitive sequences have been described which were thought to be capable of transposition to other, non-homologous sites elsewhere in the genome (Cameron et al. 1979; Kingsman et al. 1981). Transpositions of such sequences, like Tyl and Ty2, have indeed been verified upon isolation of regulatory mutants carrying Ty insertions in the 5' flanking region of structural genes (HIS4: Farabaugh and Fink 1980, CYC7: Errede et al. 1981 ; ADR2: Williamson et al. 1981). Once inserted, Ty elements can cause a number of sequence rearrangements which were detected as secondary mutations. Excision

of most of the element is the preponderant event found for Ty insertions both at HIS4 and ADR2, and is causative for the observed instability of insertion mutants (Roeder and Fink 1980; Ciriacy and Williamson 1981). This event is thought to be a result of recombination between the Ty-flanking direct repeats of approximately 330 bp, the so-called 8 sequences. These repeats are obviously an inherent feature of Ty elements (Gafner and Philippsen 1980; Farabaugh and Fink 1980; Kingsman et al. 1981). Ty elements can also induce inversions, deletions and transpositions of flanking unique sequences (Roeder and Fink 1980; Liebman et al. 1981; Stiles et al. 1981). Although the mechanisms of Ty-mediated DNA rearrangements are not well understood, it is likely that the flanking 8 sequences are crucial in creating such events. In fact, Rothstein (1979) could show that only two 6 elements flanking a unique sequence are required for deletion of that sequence. While the analogy to Ty excision mentioned above is striking, the relative importance of solo 6 sequences for other rearrangements has not yet been assessed. In order to clarify this point, we have investigated genome rearrangements mediated by a solo 6 element 5' flanking the yeast ADHII structural gene, ADR2. In analogy to Ty excision one could expect a reversal of this event, or deletions and inversions upon recombination with remote Ty elements on the same chromosome. Some of those events do occur with a frequency of about 1 per 106 cells. In addition, we will present evidence for transposition of a unique sequence flanked by a single 6 element.

Material and Methods 1 Presentaddress: Institut ftir Mikxobiologie,Universit~itDiissel-

doff, D-4000 Diisseldorf, Federal Republic of Germany Offprint requests to: M. Ciriacy

Strains. The haploid yeast strain R184.4-2B (MATa trp2 ade2 ADR3-2 c ADR2) carries a Ty insertion in the regulatory site (ADR3) of the ADHII structural gene, ADR2. Strain M24 is a

0172-8083/82/0006/0055/$01A0

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M. Ciriacy and D. Breilmann: 6 Sequences Mediate DNA Rearrangements in S. cerevisiae

Table 1. ADHII activity in various strains with Ty or insertions in the regulatory site (ADR3) of the ADHII structural gene, ADR2. Strain M24 (~-ADR2) was derived from the original Tyinsertion strain R184.4-2B (ADR3-2c). All other strains were ADHII-constitutive derivatives of M24. Cells were grown either on YEP-8% glucose and harvested before depletion of glucose (repressed) or on YEP-3% ethanol (derepressed). ADHII activity is given as nmoles/min per mg protein Strain

Genotype

454 10 526 628 480 310 530 1,430 775

ADR3-2 c 6-ADR2 ADR3 c ADR3 c ADR3 c ADR3 c 6-ADR2 + ADR3 c ~-ADR2 + ADR3 e 6-ADR2 + ADR3 e

Derepressed 930 80 740 840 930 232 834 1,600 857

direct derivative of R184.4-2B. It has inserted a solo 6 sequence instead of Ty in front of ADR2 (mutant allele adr3-224; Ciriacy and Williamson 1981). CH1-25C MATa his 3 ADR3 ADR2 and strain ADE4. 1-6A MATa ade4 his4 trpl ADR3 ADR2 are pure wild types with respect to ADR3 or ADR2. All strains used are deficient in ADHI (adcl-ll) and mitochondrial ADH (adm). E. coli strain RRI was used for isolation of plasmid DNA. Hybridization Probes. Various fragments of the ADR2-ADE4 region and of a Ty element Were used as hybridization probes. These fragments were obtained from previously isolated ADR2 DNA (Williamson et al. 1981) or in one case from ADE4 DNA cloned in this laboratory (Breilmann and Ciriacy, in preparation). Media, Growth Conditions and Enzymatic Analysis. These methods were previously described (Ciriacy 1976). DNA Preparation, Southern Blotting and Hybridization. Yeast chromosomal DNA was prepared according to Olson et al. (1979). Small scale preparations of yeast DNA 2-5 ug) were performed as described previously (Ciriacy and Williamson 1981 ). Electrophoresis in agarose gels, Southern transfer and hybridization were carried out as described by Williamsonet al. (1981) with minor modifications. [a3SS]-dATP (NEN Chemicals) was used for labeling DNA by nick translation. Fluorography was routinely carried out at -70 °C overnight using an enhance spray (NEN Chemicals).

H

H I

EB II

X I

E I

B I

ADR2

?

H I

EH [] li I

ADE4

a

b

ADR2-ADE4

d

c

E,BX~H e

? I 2, 3kb

,E ,S

Results Isolation o f Mutants with Rearrangements at the A D R 2 Locus

Specific activity Repressed

R184.4-2B M24 R2 R27 R6 R49 R5 R14 R16

Enzymes. Restriction enzymes, DNA polymerase I, and T4-DNA ligase were purchased from Boehringer or BRL.

X ,H X ADR2

,E

ADR3-2C

It has previously been shown that insertion of the yeast mobile elements, Ty 1 and T y 2 , i n t o the 5' flanking region of the ADHII structural gene, A D R 2 , is causative for constitutive expression of that gene (Williamson et al. 1981). A D H I I - derivatives of these insertion mutants had in most cases lost the Ty sequence except for asingle 6 element (Ciriacy and Williamson 1981). This phenotype allows to test whether a solo 6 element is able to promote DNA rearrangements. Events which restore constitutive expression o f A D R 2 can be screened in the same manner as the original Ty-insertion mutants (Ciriacy 1979). Cells lacking ADHI acitivity (adcl) are unable to grow on glucose media when respiration is blocked by antimycin A. Consequently, antimycin A-resistant clones on glucose can either be the result of a reversion event at a d cl , or of mutations leading to constitutive expression of normally glucose-repressible ADHII. Mutations at the regulatory site ( A D R 3 ) of the ADHII structural gene can rather specifically be screened by a test for cis-dominance (Ciriacy 1979). By this method, 50 isolates were obtained from a total of 5 x 107 cells of a haploid strain, M24 (mutant allele adr3-224;for simphcity called 6-ADR2). As expected all isolates had substantial ADHII activity on glucose (selected isolates are shown in Table 1). With two exceptions, expression of ADHII was very similar to that of the original Ty-insertion mutant, A D R 3 - 2 c. Southern blot analysis of genomic EcoRI and BamHI fragments of the A D R 2 region (see map in Fig. 1) revealed a number of different rearrangements (Fig. 2). A detailed Sothem analysis using various restriction endonucleases and different DNA probes o f a Ty sequence and of the A D R 2 - A D E 4 region, as well as tetrad analysis were performed with ten isolates representing the entire mutant spectrum. Three different kinds of events were de-

wild

type

Fig. 1. Restriction maps of theADR2-ADE4 wildtype region and of a Tyl insertion adjacent to ADR2 (mutant allele ADR3-2e). Restriction sites of ADR2 and Ty sequences were described previously (Williamson et al. 1981)whereas restriction sites of ADE4 and surrounding sequences were taken from yet unpublished material (D. Breilmann, this laboratory). Probes used are indicated by letters a, b, c, d, and e. The boxed area represents Ty sequence with 6 sequences (rifled boxes) flanking the central part, e. Abbrevations: B: BamHI; E: EcoRI; H: HindIII; S:SalI; X: XhoI

M. Ciriacy and D. Breilmann: 6 Sequences Mediate DNA Rearrangements in S. cerevisiae

57

tected: Reinsertion of a Ty element, inversion o f A D R 2 and flanking sequence, and transpositions o f A D R 2 along with its 3' flanking sequence to remote Ty elements.

Fig. 2. Southern blot analysis of genomic EcoRI (left panel) and BamHI (right panel) fragments of ADR3-2 c (lane a), &ADR2 (b), and of various mutants derived from a 8-ADR2 strain, c: R2; d: R27; e: R49; f: R6; g: R16; f: R5. Numbers refer to kilobases as estimated by appropriate standards. Probe a covering ADR2 and surrounding sequences was used (cf. Fig. 1). The 7.4 kb EcoRI fragment (lanes a, c, d, f, g, h) contains ADR2 and part of the adjacent Ty sequence. This strongly labeled fragment is absent in M24 (lane b). Presence of a weakly labeled fragment of similar lenght is due to cross hybridization with ADC1 (ADHI structural gene; cf. Williamson et al. 1981). Note that only one BamHI fragment is labeled in ADR3-2c (a), 6-ADR2 (b) and in reinsertion strains R2 (c) and R27 (d) while two fragments exist in inversion mutants (e, f) and ADR2-transposition mutants (g, h)

Fig. 3. Ty hybridization spectra of strain M24 (~-ADR2, lane a) and of three different reinsertion mutants derived from M24. Lane b: R2; c: R27; d: R34. Genomic DNA was cleaved with HindlII and fragments were labeled with nick-translated, TyADR2 DNA cloned in pBR322 (probe e in Fig. 1). The spectra differ in one fragment. A 4 kb fragment of M24 is absent in R2 and R34 while a new fragment appears (in R2:3.7 kb; in R34: 10 kb). In R27, the new fragment is thought to be coincidentially similar in length as the 8-ADR2 fragment of M24

Class I: Reinsertion o f Ty. Approximately 50% of all mutants had a restriction pattern identical or very similar to original Ty-insertion mutants (Fig. 2, lanes a, c, d). Upon BamH1 cleavage, a 13 kb fragment containing A D R 2 and the entire Ty sequence appeared instead of a 7.8 kb fragment in the parent strain M24 (6-ADR2; Fig. 2, lane b). Instead o f a single 5.2 kb EcoRI fragment in M24, two fragments (7.4 kb/3.1 kb) were typical for the original Ty-insertion mutant and mutant R27, while in R2 the shorter EcoRI fragment was only 2.7 kb. This fragment covers part of Ty and flanking unique sequence distal from A D R 2 (of. map in Fig. 1). Cleavage with other restriction enzymes revealed a small deletion (400 bp) of the e-sequence in mutant R2. Tetrad analysis of mutant x wild-type diploids showed regular 2 : 2 segregation as well as normal spore viability (90% of asci with for viable spores). This suggested that extensive chromosomal aberrations were not involved in these events. The reintegration events observed here can either be a result of transposition (upon duplication) or translocation (without prior duplication) of Ty to the 8-ADR2 site. We have tried to distinguish between these altemafives by probing genomic EcoRI or HindIII fragments of reinsertion mutants with cloned Ty DNA (probe e in Fig. 1). Due to the numerous, dispersed Ty elements present in the strains used, it is rather difficult to assess every alteration of the Ty spectrum. Figure 3 illustrates these difficulties. Of the numerous HindIII fragments of the parent strain M24 (lane a), only a 4kb fragment was absent in reinsertion mutants, R2 and R34 (lanes b and d), while one novel fragment appeared (3.7 kb in R2, and 10 kb in R34). Obviously, differences in length of these fragments are caused by the presence (in R2) or the absence (in R34) of a HindlII site within the e sequence (see map in Fig. 1). In mutant R27 no change could be detected. This can be explained by the fact that the novel HindlII fragment in R27 is coincidentiaUy very similar in length to the 4 kb A D R 2 fragment of the parent strain, M24. The latter fragment is labeled due to a short unique sequence of the probe (cf. Fig. 1). In case of R2 and R27 one would expect a second novel HindlII fragment of approximately 6 kb in length because the Ty probe overlapped the Ty-internal HindlII site. This additional fragment could probably not be detected because of co-migration with other Ty fragments. Therefore, a definite proof for either translocation or transposition o f Ty is difficult to obtain in these cases. Since none o f ten independent isolates had apparently lost a Ty sequence it is very likely that Ty sequences were duplicated before or simultaneously with reinsertion. It should be noticed that reinsertion into a solo ~ sequence

58

M. Ciriacy and D. Breilmann: 5 SequencesMediate DNA Rearrangements in S. cerevisiae

Fig. 4. Southern blot hybridization of genomic BamHI fragments from M24 (5-ADR2, lane a), ADR2-transposition mutants R5, R14, and R16 (lanes b, c, and d), and ADR2-inversion mutants (lane e: R6, lane f: R49). AnADR3-2 c strain (lane g) is givenfor comparison. Hybridization with a probe containing ADR2 5' flanking sequence (left panel) or 3' flanking sequence(rightpanel) indicated that the additional BamHI fragment (~18 kb)in transposition strains contained only ADR2 and 3' flanking material. In strains R6 and R49 it is obvious that ADR2 flanking sequences axe located on different BamHI fragments

is different from transposition of an entire Ty to a unique site. Reinsertion described here requires only duplication of e along with one 6 element. In formal terms, reinsertion is therefore a correct reversal of Ty excision leaving behind a solo 6. Class 11: Inversion o f A D R 2 . It was expected that recom-

bination events between 6-ADR2 and remote Ty sequences could create rearrangements different from the insertion events described above. Provided that a rearrangement would place ADR2 in appropriate orientation adjacent to a Ty sequence the gene would be expressed, and consequently, the ADHII-constitutive phenotype would be restored. One such event would be deletion of DNA between 6-ADR2 and the proximal 6 flanking some Ty on the same chromosome, provided the 6 sequences would be orientated in the same direction. This kind of event would be analogous to Ty excision by 6-6 recombination (cf. Introduction). In haploid cells, its occurence would certainly depend on the length of the deleted material. In fact, evidence for such a deletion was not found. In case of reverse orientation of 6-ADR2 and a 6 of a remote Ty on the same chromosome, 6-6 recombination would generate an inversion of the intervening DNA. Resuits from two isolates (R6 and R49) can be explained in this way. Of the two EcoRI fragments of R6 (Fig. 2, lane f) one was in length identical to a 7.4 kb fragment of the original ADR3-2 c Ty insertion while the short fragment of R6 was dearly different from a 3.4 kb fragment in ADR3-2 c cells. Instead of a single BamHI fragment of ADR3-2 c DNA two different fragments hybridized to ADR2-DNA in mutant R6. Various cleavage reactions using SalI, HindlII or BgllI clearly confirmed that ADR2 is linked to a Ty sequence in R6 (results not shown).

Strain R49 did not show any resemblance in restriction patterns with R6 or with an ADR3-2 c strain (Fig. 2, lane e). However, it is very likely that the large, 10 kb EcoRI fragment carries a complete Ty sequence similar to the Ty 2 variant (Kingsman et al. 1981; Williamson et al. 1981). This Ty variant lacks an EcoRI site. Genomic fragments of both mutants were further analyzed by probing with cloned DNA flanking ADR2 in wild-type strains (probes b and c in Fig. 1). Figure 4, lanes e and f, shows hybridization patterns of BamHI fragments. In both revertants the larger fragments were only labeled by the probe which carried a small portion of ADR2-coding region along with 2 kb 3' flanking material. Correspondingly, the short fragments were labeled exclusively with a probe of 5' flanking DNA. This clearly suggested that gene ADR2 and its 5' flanking sequence are located on separate BamHI fragments. Further evidence for an extensive chromosomal rearrangement came from tetrad analysis of mutant x wildtype crosses. Of 35 four-spored asci dissected only four had four viable spores while the majority of asci generated only two viable spores (23 asci) or one viable spore (8 asci). In tetrads with two viable spores one was ADHIIconstitutive in most cases. When such a spore was crossed to the original mutant strain normal spore viability was obtained. This segregation behavior can most easily be explained by an extensive inversion. Lethal segregation is thought to be a corollary of cross-over events within the inverted segment (Chaleff and Fink 1980). Class IlL" Transposition o f ADR2. Southern blot analysis

of genomic DNA from three isolates (RS, R14, R16) suggested that they carded an additional ADR2 sequence along with the 6-ADR2 sequence of the parent strain (Fig. 2, lanes g and h). While the novel EcoRI fragments were identical in length to the corresponding Ty-ADR2 fragment of the original Ty insertion (Fig. 2, lane a), the additional BamHI fragments were clearly different. Probing with sequences flanking ADR2 (Fig. 4, lanes b, c, and d) moreover revealed that the large (about 18kb) BamHI fragments only contained ADR2 and 3' flanking sequence since these fragments were not labeled with the 5' flanking probe. This was taken as suggestive evidence that ADR2 along with 3' flanking material has been duplicated and transposed to a remote Ty element. This could be confirmed by genetic analysis of revertant x wild-type crosses in which segregation of the duplicated ADR2 region could be followed (results not shown). In order to assess the length of the transposed region, a short sequence (probe d, Fig. 1) flanking gene ADE4 was used as a hybridization probe for genomic DNA fragments. Gene ADE4 has recently been cloned in this laboratory and localized on the ADR2 3' flanking sequence (D. Breilmann, unpublished observation). Comparison of SalI fragments from M24 (6-ADR2), R5, R14, R16, and

M. Ciriacy and D. Breilmann: 6 Sequences Mediate DNA Rearrangements in S. cerevisiae

59

occured at different sites in these mutants. This is indicated by slight differences in length of the large BamHI fragments (Fig. 2 and Fig. 4) which carry the transposed segment.

Discussion

Fig. 5. Southern blot hybridization of genomic SalI fragments from strain M24 (allele designation adr3-224 refers to solo 6 adjacent to ADR2), and three transposition mutants. An ADR3-2c strain (original Ty-insertion mutant) is given for comparison. A cloned sequence which flanks gene ADE4 (cf. Fig. 1, probe d) was used as hybridization probe. The shorter fragments (~16 kb) carry the transposed ADR2 gene and part of Ty whereas the larger fragment which is also present in the parent strain M24 contains 6-ADR2. This indicated that the transposed segmentencompasses gene ADE4 and flanking material

from the original Ty4nsertion mutant, ADR3-2 c, indicated that the duplicated sequence must be longer than the additional, 16 kb SalI fragment which is identical to the single SalI fragment in the ADR3-2 c strain (Fig. 5). This suggested that the duplication encompassed gene (ADE4. Thus, ADE4 could be used as a marker in order to determine the approximate genetic distance between the insertion site of the transposed sequence and the wild-type ADR2-ADE4 region. Reliable data were only obtained for revertant R5. Assuming genetic linkage, one would expect predominance of parental or tetra-type segregation. In a cross ADR3-ADR2-ade4 x adr3-ADR2A D E 4 - - A D R 3 e - A D R 2 - A D E 4 23 parental di-type tetrads (2ADE + :2ADE-) and 10 tetratype tetrads (3ADE+:IADE - ) where found while non-parental ditype tetrads (4ADE ÷ :OADE-) could not be obtained. Thus the insertion site with mutant R5 would lie roughly 15 cM apart fromADR2 on the same chromosome (it could not yet clearly be established which chromosome bears ADR2 or ADE4). The transposed ADR2 gene in R5 was centromere-linked as could be shown by following segregation of a centromere marker (trpl). While R5 in crosses with wild-type strains did not show any remarkable effect on spore viability, a significant decrease in spore viability was observed in crosses which involved strains R14 and R16. Therefore, it can not be ruled out that additional chromosomal rearrangements were connected with ADR2-transposition in both cases. It should also be noted that transpositions of ADR2 probably had

By analyzing a number of spontaneous mutations which caused constitutive ADHII synthesis it was possible to provide further evidence for the involvement of 6 sequences in the generation of DNA rearrangements in yeast. Such gross alterations in chromosome structure in yeast cells Were described in a number of instances (Roeder and Fink 1980; Stiles et al. 1981 ; Liebman et al. 1981 ; Ciriacy and Williamson 1981). In all cases, Ty elements were found in close vicinity of the rearranged sequence. To date, there is only one report on deletions mediated by ~i elements not associated with Ty elements (Rothstein 1979). The properties of yeast strains which have a solo element in front of gene ADR2 allowed to test whether 6 sequences per se can promote DNA rearrangements other than deletions. The rationale of screening for such putative events based on the assumption that any rearrangement which joins the ~-ADR2 sequence in appropriate position and orientation to some Ty sequence would result in constitutive expression of gene ADR2. From known restriction sites of Ty elements and of ADR2 and its flanking sequences it could be deduced that in all mutants the ~i-ADR2 sequence was indeed linked with Ty elements in the same orientation as in the original Ty-insertion mutants, ADR3 c. Figure 6 shows a formal interpretation of the events we have analyzed. Class I is a precise reinsertion of Ty into the ~ sequence. Two different types of exchange could create this structure. First, one could assume that some remote Ty element is excised by ~-6 recombination and reintegrated at 5-ADR2 by homologous recombination. No evidence for such a reciprocal exchange was obtained in ten independently isolated mutants. Therefore, we favour a gene conversion mechanism for explaining this movement of Ty elements. Scherer and Davis (1980) have presented evidence that gene conversion in haploid cells can occur between nonhomologous strands provided short homologous sequences are present on both strands. In their experiments recombination of Ty sequences were analyzed by using plasmids which carried Ty along with a selectable marker. One should note that recombination between the Ty sequence and solo 6 elements could not unambigously be verified by this approach. In Class II events no material was found to be duplicated. The two cases investigated in detail (R6 and R49) can most easily be explained by an iversion event (Fig. 6).

M. Ciriacy and D. Breilmann: 6 Sequences Mediate DNA Rearrangements in S. cerevisiae

60

Class

I

i Class

II

Class

III

L =t w

,,

-r

-* B

%.

Fig. 6. Interpretation of 6-mediated DNA rearrangements. Ty sequences are indicated by open boxes with flanking 6 sequences (filled boxes). Relative orientation of 6 sequences is given by little arrows. Location of ADR2 is shown by long arrows. Class I and Class III are non-reciprocal eyelets (transpositions) whereas Class II is a reciprocal exchange (inversion)

The solo 6 element adjacent to ADR2 and another 5 element which is part of a Ty sequence would represent specific sites for intrastrand recombination provided the 6 elements are arranged in reverse orientation. Short inverted repeats flanking a unique sequence were found to be responsible also for frequent inversion events in yeast 2-/am DNA (HoUenberg et al. 1976; Beggs 1978). Class III events (transposition of ADR2 and 3' flanking material) are related to Ty reinsertion in so far as sequences were duplicated prior to, or simultaneously with insertion into another site, The analogy would be perfect if the transposed material would be also flanked by direct repeats of 5 elements. This could not yet be verified for the ADR2-distal end of the transposed segment due to its length (> 15 kb). In at least one case (mutant R5) it could be established that the duplicated material is located on the ADR2 cffromosome. Thus, one could also envisage non-equal sister strand exchange as a mechanism (Szostak and Wu 1980). This kind of exchange could either be reciprocal or non-reciprocal. Reciprocal, nonequal sister strand exchange does not seem to be a likely explanation. Because the transposed segment was located closer to the centromere, the order of segments in the parent strain M24 would be: c e n t r o m e r e - - T y - - 6 ADR2 (In Fig. 6 the centromere would lie next to the right margin). There is no reasonable way to envisage a reciprocal sister strand exchange which places ADR2 in front of Ty sequences, and which is not associated with lethal aberrations on both strands. Therefore, it is more

plausible to assume that either non-reciprocal sister chromatid recombination or non-reciprocal intrachromatid recombination (conversion) has created the transposition event in RS. Klein and Petes (1981) have shown that in. trachromosomal gene conversion does occur between two LEU2 genes on one chromosome. It is not clear however, whether this example is applicable to the events investigated here. For instance, there was no indication at all for frequent occurrence of ADR2-transposition in meiosis. The other two examples of Class III rearrangements may have basically been generated by the same mechanism as in R5. Frequent lethal segregation of spores from R14 (or R16) x wild type crosses might indicate that transposition events in these two cases were associated with another rearrangement which has not yet been discovered. Remarkably, one of these strains (R14) showed an extremely strong expression of ADR2 compared with other ADHII-constitutive mutants. This might suggest that two copies of ADR2 were transposed and expressed. Indeed, Stiles et al. (1981) reported on such an instance with the CYCI-OSM1-RAD7 gene cluster in yeast. However, the number of restriction fragments from genomic DNA labeled with variousADR2 probes is not compatible with the assumption of two transposed ADR2 copies. Also no genetic evidence could be obtained for the expression of the solo 6-ADR2 sequence. Although several details of the chromosomal rearrangements investigated here are not yet understood one can assume that they have been generated by interactions of 6 sequences. Whether such 6-6 recombination is controlled in the same manner as generalized recombination has to await further analysis. Interestingly, the function of gene RAD52 which is involved in mitotic and meiotic recombination is not required for Tyl-mediated deletions of CYC1 (Liebman and Downs 1980). These deletions can most easily be explained by intrastrand, 6-6 recombination (Liebman et al. 1981). As shown here, 6-6-interactions do not only allow reciprocal, intrastrand recombination (inversion or deletion) but also non-reciprocal events (transposition). This kind of mobilization and transfer of DNA sequences has some resemblance to Ty transposition. However, it should be emphasized that transposition events described in this paper require an interaction between homologous sequences on both donor and target DNA. Such homologies are apparently not required for Ty transposition. Furthermore,it should be noted that such 6-6 interactions can mobilize unique sequences and therefore, they may actively be involved in creating structural diversity of the eukaryotic genome.

Acknowledgements. Part of this work was done while M. C. was a Heisenberg Fellow sponsored by the Deutsche Forschungsgemeinschaft. Dr. A. Sledziewski (Seattle, USA) is thanked for providing probes of ADR2-flanking sequences.

M. Ciriacy and D. Breilmann: ~ Sequences Mediate DNA Rearrangements in S. cerevisiae

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Liebman SW, Downs KM (1980) Mol Gen Genet 179:703-705 Liebman SW, Shalit P, Picologlou S (1981) Cell 26:401-409 Olson MV, Loughney K, Hall BD (1979)J Mol Bio1132:387-410 Roeder GS, Fink GR (1980) Cell 21:239-249 Rothstein R (1979)Cell 17:185-190 Scherer S, Davis RW (1980) Science 209:1380-1384 Stiles JI, Friedman LR, Helms C, Consaul S, Sherman F (1981) J Mol Biol 148:331-346 Szostak JW, Wu R (1980) Nature 284:426-430 Williamson VM, Young ET, Ciriacy M (1981) Cell 23:605-614

Communicated by C. P. Hollenberg Received July 2, 1982