Mol Gen Genet (1987) 210:485-489

© Springer-Verlag 1987

Strains of Schizosaccharomyces pombe with a disrupted swil gene still show some mating-type switching

Henning Schmidt Institut ffir Genetik, Technische Universitfit Braunschweig, Spielmannstrasse 8, D-3300 Braunschweig, Federal Republic of Germany

Summary. The swil + gene is necessary for effective mating- type (MT) switching in Schizosaccharomyces pombe. It was cloned on a 4.2 kb genomic DNA fragment. By site-directed integration into the genome and gene disruption experi- ments it was proved that the swil + gene itself and not a suppressor had been isolated. Disruption of the swil + gene causes a phenotype identical to that of the original swil mutant, i.e. the strain still shows some MT switching. The swil gene is unique in the genome and gives rise to a 3 kb mRNA.

Key words: Mating-type switching- Switching genes Dou- ble-strand break Sehizosaccharomyces pombe

involved in general recombination) cause some residual MT switching (Gutz and Schmidt 1985). The latter view was supported by the finding that three swi mutations are sup- pressible by nonsense suppressors (Schmidt et al. 1987). In order to discriminate between the above two possibilities, I isolated the swil + gene from a genomic gene bank. Trans- formation of an swil mutant with the cloned swil + gene yielded strains with normal frequencies of MT switching and normal amounts of DSBs at matl. Strains with a dis- rupted swil gene still exhibit MT switching although with a reduced frequency. The swil gene is not essential for vege- tative growth or sporulation. The mRNA originating from swil + comprises about 3 kb.

Introduction

In Schizosaccharomyces pombe and Saccharomyces cerevi- siae mating-type (MT) switching is initiated by a DNA double-strand break (DSB) at the MT locus (Beach 1983; Strathern et al. 1982). In Saccharomyces this site-specific DNA cut is made by an endonuclease coded by the HO gene (Kostriken et al. 1983). HO is under the regulation of the cell type, the cell cycle, and the age of the cell (Jensen et al. 1983; Nasmyth 1983). So far, no mutation analogous to ho has been found in fission yeast.

In homothallic (h 9°) strains of S. pombe the cells fre- quently switch their MT from Plus to Minus and vice versa: throughout the Cell cycle about 20% of the cells have DSBs at the smt (switching of mating type) signal (Beach 1983; Beach and Klar 1984). As yet, neither the DNA sequence surrounding this cleavage site nor the enzymes involved are known.

From h 9° strains mutants with a reduced frequency of MT switching were isolated; they can easily be detected because of their mottled iodine reaction (Gutz and Schmidt 1985). The underlying mutations map either in the smt sig- nal at matl or in switching (swi) genes that are not, or at least not closely, linked to the MT region. Ten swi genes are known which can be divided into three classes (Ia, Ib, and II) (Egel et al. 1984; Gutz and Schmidt 1985). The mutants of class Ia genes (swil, swi3, and swi7) show a reduced frequency of DSBs at matl while mutants from the other two classes do not.

Since all swi mutants still show some MT switching, the question arises as to whether the underlying mutations are leaky or whether the products of other genes (e.g. genes

Materials and methods

Strains and plasmids. The Escherichia coli and S. pombe strains used are listed in Table 1. An S. pombe library of partial Sau3A-digested DNA cloned into the BclI site of the yeast bacterial shuttle vector pWH5 (Wright et al. 1986) was kindly provided by Peter Schuchert (Bern). The plasmid pEP2 (matl-P) was used as a probe for the MT genes (Beach 1983). The plasmid pUC8-ura4, obtained from C. Grimm (Bern), contains the ura4 + gene originally cloned by F. Lacroute.

Media and genetic analysis. The standard genetic techniques and the media for S. pombe are described in Gutz et al. (1974). It should be noted that ascospores of S. pombe con- tain starch, therefore, sporulating colonies turn black if treated with iodine vapour (Leupold 1955). Standard meth- ods for manipulating DNA in vitro were done according to Maniatis et al. (1982).

DNA preparation. Large scale preparations of plasmid DNA were performed by the alkaline lysis procedure (Man- iatis et al. 1982). Cleared lysates were prepared as described by Birnboim and Doly (1979). The isolation of DNA from yeast is described by Beach and Klar (1984).

Transformation. E. coli cells were transformed according to the procedure of Dagert and Ehrlich (1979). Yeast cells were transformed by the lithium acetate method (Ito et al. 1983).

RNA isolation. For the isolation of RNA the regimen of Nicolet et al. (1985) was followed. Poly(A) + RNA was se-

486

Table l. Strains of Escherichia coli and Schizosaccharomyces pombe

Strain Genotype Reference

E. coli

JA221 BJ5183

S. pombe

HE34 HE123 HE298 HE307 HE318 HE364 HE370 HE384 L975(h 9°)

F- leuB6 trpE5 lacY hsdR hsdM + recA! F- recBC sbcB endo-1 gal met str thi bio hsd

Clarke and Carbon (1978) Losson and Lacroute (1983)

h 9° ade7 This study h 9° ade7 swil-1 This study h 9° leul-32 swi5-39 This study h 9° leul-32 swil-1 This study h 9° leu1-32 swil-1 swi5-39 This study h 9° leul swil-intl This study" h 9° ura4-D18 This study h 9° ura4-D18 swil:: ura4 + This study h 9° Gutz and Doe (1975)

" The whole plasmid pilE6 was integrated via homologous recombination at the chromosomal site of swil

lected with oligo(dT)-cellulose (BRL) according to the in- structions o f the suppliers.

Isolation o f D N A fragments. D N A fragments were sepa- rated by agarose gel electrophoresis and eluted from low- melting agarose (Wieslander 1979).

Recovery o f plasmids f rom yeast transformants. Plasmids were recovered from yeast cells as described by Beach et al. (1982).

Southern and Northern transfer. The transfer of D N A frag- ments from agarose gels to nitrocellulose was performed according to Southern (1975); the Northern transfer was as described by Maniatis et al. (1982).

Results

Cloning of swil ÷

For the isolation of the swil + gene an S. pombe library of partial Sau3A-digested genomic D N A cloned into the BclI site of the yeast bacterial shuttle vector pWH5 (Wright et al. 1986) was used. This vector contains the L E U 2 gene from Saccharomyces which complements the leul mutat ion of S. pombe and the 2 ~tm autonomously replicating se- quence (ARS) which allows high-frequency transformation of S. pombe (Beach and Nurse 1981). Since it is not possible to select directly for swil ÷ transformants, I first selected Leu ÷ transformants on minimal medium (MMA). In the second step the Leu + transformants were replicated on a sporulation medium (MEA) in order to screen for transfor- mants showing an increased frequency of MT switching.

To facilitate the detection of swil ÷ transformants, the hg°leul swil swi5 strain (HE318) was used as recipient. Col- onies o f this swi double mutant show virtually no iodine- positive material whereas colonies o f swil and swi5 single mutants are mottled (Schmidt et al. 1987). The use of the swil swi5 strain also offered the chance to clone swil + as well as swi5 ÷. However, I only succeeded in cloning swil +.

Strain HE318 was transformed with D N A from the gene bank. Leucine proto t roph colonies were selected on M M A medium. After 6 days incubation at 30 ° C, the Leu ÷ trans-

formants were replicated on MEA plates. These were incu- bated for 2 days at 30 ° C. Upon treatment with iodine va- pour, the plates were screened for transformants showing a mottled phenotype.

Four iodine-positive clones were found out of approxi- mately 15 000 Leu + transformants. From these clones, plas- mids were recovered and retransformed into HE307 (swil) and HE298 (swi5). The plasmids (called p i lE6 and pi lE7) isolated from two of the transformants were able to comple- ment the swil but not swi5 mutation. The plasmids which were recovered from the other two transformants could neither complement HE307, HE298, nor HE318.

p i lE6 contains a 4.2 kb insert. Its restriction map is shown in Fig. 1 a. Plasmid pi lE7 turned out to be identical to p i lE6 and was not studied further. Two HindIII frag- ments from pi lE6 were subcloned into the HindIII site of pWH5 (Fig. I b, c). Neither of the resulting plasmids pHE6- P13 and pHE6-P19 was able to complement the swil muta- tion.

Mapping the cloned fragment

To establish that p i lE6 contained the swil + gene itself and not a phenotypic suppressor the plasmid was integrated into the S. pombe chromosomes and mapped. For that pur- pose p i lE6 was cut at the unique XhoI restriction site lo- cated in the cloned D N A fragment (Fig. 1 a). Strain HE307 was transformed with the linearized plasmid. Through re- striction enzyme digestion in the region homologous to chromosomal D N A the integration o f the plasmid D N A can be targeted to a specific chromosomal site (Orr-Weaver et al. 1981). The efficiency of integration is greatly enhanced by such a digestion. Leucine prototrophs were selected on M M A medium. All ten Leu + transformants tested re- mained stable for leucine protot rophy when incubated in non-selective medium for about 20 generations. Moreover they all showed an Swil + phenotype. One of the stable Leu + Swil + transformants (HE364) was crossed to HE123 (ade7 swil) to test whether the integration had actually oc- curred at the chromosomal site of swil +. The swil gene maps on the left arm of chromosome II and is linked with ade7 (Gutz and Schmidt 1985). From this cross 15 tetrads were analysed. The swil + marker showed linkage to ade7

487

H

I

8

I kb 4

<

HH~ 0 0 - ~ ,.4 0 ,- t r~ ~

I I I I I I I 1 [ pHE6

b t pHE6-P19 H

C

t pHE6-P13

ura4 +

i I pilE6- in t21

d H E H E SH

ura4 +

! , I I . " ~ , i " I I I pHE6-int22 e HX E H E SH

Fig, 1 a--e. Restriction map of plasmid pilE6 and its derivatives, a pilE6 contains the 4.2 kb fragment isolated from the genomic gene bank. b, e The 1.3 kb and 1.9 kb HindlII fragments of pilE6 were subcloned into the HindIII site of pWH5. d, e The 1.8 kb ura4 + marker was integrated into each of the two HindIII sites of the cloned fragment. Sequences of the vector pWH5 are indicated by a thin line

as expected. This does not rule out the possibility that the cloned fragment codes for a tightly linked suppressor. To substantiate further that p i lE6 contains s w i l +, gene disrup- tion experiments were performed (see below).

Chromosomal D N A of strains HE307 and HE364 was digested with H i n d I I I and hybridized to p i lE6 (Fig. 2a). The Southern blots o f HE307 and HE364 gave three and five bands, respectively, as predicted from the restriction map. No further bands appeared indicating that s w i l is unique in the genome.

To determine the length of the s w i l + transcript a North- ern blot analysis was performed using poly (A) + R N A from the wild-type strain L975 (h9°). A weak band corresponding to an m R N A of about 3 kb could be detected (Fig. 3) indi- cating the size of the s w i l + mRNA.

Disrupt ion of swil +

With the cloned s w i l + gene it was possible to test whether or not the swi mutations are leaky. This was accomplished with the help of a gene disruption experiment. Two plas- mids were constructed in which the s w i l gene is interrupted by a 1.8 kb H i n d I I I fragment containing the ura4 + gene. The construction was performed by partial cleavage of p i lE6 D N A with H i n d I I I to an average of one cut per molecule. Full-length linear molecules were extracted from a low-melting agarose gel. The ura4 + 1.8 kb H i n d I I I frag- ment was obtained by cleaving pUC8-ura4 D N A to com-

pletion with HindI I I . The ura4 + fragment was then ligated into the partially digested p i lE6 DNA. The resulting plas- mids pHE6-int21 and pHE6-int22 (Fig. 1 d, e) were tested for ability to complement the s w i l mutation. Neither con- struct could complement sw i l . Plasmid pHE6-int22 was chosen for the gene disruption experiment because it has sequences homologous to s w i l on both sides of the ura4 + marker. Furthermore, it has restriction sites which permit an almost exact cutting out of the disrupted s w i l gene (i.e. virtually without adjoining vector sequences). Thus, pHE6- int22 fulfils the requirements for a one-step gene disruption experiment as outlined by Rothstein (1983).

pHE6-int22 D N A was digested with X h o I and S m a I yielding a 4.8 kb D N A fragment (Fig. 1 e). This fragment was transformed into HE370 (h9°ura4-D18). In this strain the ura4 1.8 kb H i n d I I I fragment is deleted on the chromo- some (Grimm 1986). Stable Ura + transformants were se- lected. The phenotype of their colonies on MEA plates after treatment with iodine vapour was examined: all ten inde- pendently isolated transformants yielded colonies with a mottled phenotype, i,e. they had an Swil phenotype. The iodine reactions of these s w i l : : ura4 + mutants were indis- tinguishable from previously isolated s w i l mutants.

In order to map the integration site by tetrad analysis, the strain HE384 (ura4-D18 s w i l : : ura4 +) was crossed with HE34 (ade7). In the progeny all mottled colonies were Ura +. F rom 60 tetrads (31 PD, 2 NPD, and 27 T) the map distance for s w i l - a d e 7 was calculated as 32.7 cM. This is

488

1 2 . 5 - -

1 2 1 2 1 2 3 4

- - 1 2 . 5 m a t 1 - - 1 0 . 4

__3.7 - - 3 . 5

2 . 4 - -

1 . 9 m - - 1 . 7

1 . 3 ~

1 . 0 - -

a b Fig. 2a, b. Analyses of chromosome insertions, a Southern blot analysis of HindIII-digested chromosomal yeast DNA. Lane 1, HE307 (swil); lane 2, HE364 (swil-intl). The 12.5 kb and 1.3 kb bands (lane 2) are due to integration of the complete plasmid pilE6 in the strain HE364. b Southern blots of chromosomal DNA cut with EeoRI. Lane 1, HE370 (swil +); lane 2, HE384 (swil:: ura4+). Both blots are probed with pilE6 DNA. Numbers indicate the sizes of DNA fragments in kb

s t a r t

- 3 k b

Fig. 3. Northern blot analysis of swil + mRNA. Schizosaccharo- myees pombe poly(A) + RNA was transferred to a nitrocellulose membrane and hybridized with nick-translated pilE6 DNA

in agreement with previous mapping data for swil (Gutz and Schmidt 1985). To verify that swi l is indeed disrupted, a Southern blot analysis was performed with D N A from HE370 and HE384. Figure 2b, lane 2 shows that in strain HE384 the 1.7 kb Eeo RI fragment is replaced by a 3.5 kb fragment.

D S B s at the M T locus

As has already been stated, MT switching in S. pombe is initiated by a D N A DSB at the smt signal, swi mutants of class Ia show a reduced frequency of DSBs. Does the cloned swi l + gene restore the normal amounts of DSBs at m a t l ? Chromosomal D N A of strains L975(h9°), HE307, HE364, and HE384 was digested with HindIII , separated on an agarose gel, and blotted to a nitrocellulose filter.

m a t 2 - - 6 . 3 s m t ( p ! - - - - 5 . 4 s m t ( d ) - - - - 5 . 0

m a t 3 - - - - 4 . 2

Fig. 4. Analysis of double-strand breaks (DSBs) in the mating-type (MT) region in strains with and without an intact swil + gene. Southern blot analysis of HindIII-digested DNA. Lane 1, L975(h 9°) (swil +); lane 2 HE307 (swil); lane 3, HE364 (swil-intl); lane 4, HE384 (swil:: ura4+), smt(p) and smt(d) are the proximal and distal fragments of the 10.4 kb matl fragment generated in vivo by the DSB at smt. mat2 and mat3 are the silent storage genes for P and M information, respectively. The blot is hybridized with pEP2 containing the 10.4 kb matl-P HindIII fragment. The 12.5 kb band (lane 3) corresponds to vector sequences since pilE6 is integrated completely in strain HE364

The filter was hybridized with a 32P-labelled MT region specific probe (Fig. 4). In the Southern blots the swi l + inte- gration strain HE364 yielded smt(p) and smt(d) bands like the homothallic wild-type strain L975(hg°). Thus, the inte- grated swi l + gene fully restores MT switching. In both the original swi l mutant (HE307) and the disruption strain (HE384) no DSBs are visible at the smt (Fig. 4, lanes 2, 4).

D i s c u s s i o n

MT switching in yeast is accomplished by transposition of genetic material from silent cassette genes to the MT locus, the actual site where the information is expressed. Homo- thallic strains o f S. pombe frequently switch their MT from Plus to Minus and vice versa. Ten different swi genes are necessary for effective MT switching. The swi genes can be grouped into three classes (Ia, Ib, and II), which concur with three successive steps in MT switching (Egel et al. 1984): (1) formation of the DSB at smt (swi genes o f class Ia); (2) utilization o f the smt cut (class Ib); and (3) resolu- tion of recombinational intermediates (class II). This classi- fication has been confirmed by the analysis of swi double mutants (Schmidt et al. 1987). Double mutants having mu- tations in swi genes of different classes show an increased reduction o f MT switching as compared with the single mutants, whereas swi genes o f the same class do not exhibit such a cumulative effect. Thus, if one of the three postulated steps is impaired by a mutation in one swi gene, it does not matter if a second swi gene of the same class is defective, too.

The clone with the swiI ÷ gene was identified by comple- mentation of an swi l mutant (class Ia). A gene disruption experiment showed that the swi l ÷ gene itself and not a phenotypic suppressor had been cloned. Furthermore, this experiment proved that the disruption of the swiI + gene in h 9° cells results in a phenotype identical to that of the original swil mutants. Therefore, a mutant having an abso- lutely defective swil gene still exhibits a mottled iodine reac- tion. F rom this observation the important conclusion fol- lows that mottled mutants are not necessarily leaky. Appar-

489

ently, the mutants possess a residual MT switching capacity which may be caused by the general recombinat ion system of the cells.

Disruption of swil + was carried out in a haploid strain implying that swil + is not essential for viability. The South- ern blot analysis indicated that swil is unique in the ge- nome.

In S. pombe no mutat ion analogous to ho of Saccharo- myces is known. The present results indicate that the swil + gene does not code for the endonuclease generating the DSB at smt. The swi genes of classes Ib and II act at later stages in the process of MT switching and are not involved in the initiation.

The genes of class Ia may act rather by stabilizing the DSB than by having a function in the formation of the DSB. The finding that swil , swi3, and swi7 mutants have a reduced frequency of DSBs at smt (Egel et al. 1984) could also mean that these mutants fail to stabilize the DSBs which normally persist throughout the length of the cell cycle (Beach 1983).

In S. cerevisiae switching genes are known, too (Haber and Garvik 1977; Stern et al. 1984; Breeden and Nasmyth 1987). The genes SWI1 to SWIIO are required for HO tran- scription. Thus the switching genes of Saccharomyces and those of Schizosaecharomyces have quite different func- tions. In Saecharomyces they are involved in two different aspects of HO regulation: the cell cycle control and the mother cell specificity (Nasmyth 1983). In Schizosaecharo- myces the swi genes of classes Ib and II at least have a function in later MT switching steps. Their gene products seem to be directly engaged in the switching process.

The cloning of swi l + is a first step towards identifying the swil gene product and should lead to a better under- standing of the mechanism of MT switching in S. pombe.

Acknowledgements. I am grateful to Peter Schuchert (Bern) for providing the S. pombe gene bank and Christian Grimm for the ura4-D18 deletion strain. I thank Herbert Gutz, Gerhard Gross, Lutz Heim, Holger Michael and Philippe Szankasi for helpful dis- cussions and/or critical reading of the manuscript. The work was supported by Deutsche Forschungsgemeinschaft, Sachbeihilfe Gu 48/9-3.

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Communicated by W. Gajewski

Received June 23, 1987