Current Genetics

Curt Genet (1992)21:285-289

9 Springer-Verlag 1992

Cloning and mapping of the CYS4 gene of Saccharomyces cerevisiae Bun-ichiro Ono 1, Chinatsu Heike 1, Yukie Yano 1, Toyomi Inoue 1, Kazuhide Naito 1, Satoru Nakagami 2, and Akio Yamane 2 1 Laboratory of Environmental Hygiene Chemistry, Faculty of Pharmaceutical Sciences, Okayama University, Okayama 700, Japan 2 Institute for Biotechnology Research, Wakunaga Pharmaceutical Co. Ltd., Koda-cho, Takata-gun, Hiroshima 729-64, Japan Received November 30, 1991

Summary. A D N A fragment containing the C YS4 gene of Saccharomyces cerevisiae was isolated from a genomic library. The cloned fragment hybridized to the transverse-alternating-field-electrophoresis band corresponding to chromosomes VII and XV. According to the 2 ~tm D N A chromosome-loss procedure, the cys2 and cys4 mutations, which are linked together and co-operatively confer cysteine dependence, were assigned to chromosome VIL By further mapping involving tetrad analysis, the cys2-cys4 pair was localized between SUP77 (SUP166) and ado3 on the right arm of chromosome VII.

Key words: Saccharomyces cerevisiae - Cysteine biosynthetic

CYS4

blocks in both pathways are cysteine-dependent (Ono et al. 1984, 1988 a). Moreover, the genes involved in these pathways are arranged in a unique manner (Ono et al. 1984, 1988a). That is, cysl and cys2, each of which confers SATase deficiency (Halos cited in Jones and Fink 1982; Ono et al. 1988 a), are unlinked to one another, but each of them is linked to cys3 (7-CTLase deficiency) or cys4 ([3-CTSase deficiency). Although the cysl-cys3 pair has been mapped on the left arm of chromosome I (Ono et al. 1984), the map position of the cys2-cys4 pair remains unknown. We, therefore, attempted to map the cys2-cys4 pair. Here, we present evidence that the pair is on the right arm of chromosome VII.

Mapping

Materials and methods Introduction The budding yeast Saccharomyces cerevisiae has two cysteine biosynthetic pathways. Serine receives sulphur from /IS - in one pathway and from homocysteine in the other. The former pathway consists of serine O-acetyltransferase (SATase) [EC 2.3.1.30] and O-acetylserine sulphhydrylase (OAS SHLase) [EC 4.2.99.8]. Similar pathways are present in bacteria (see Soda 1987) and in plants (see Giovanelli 1987). This is regarded as the autotrophic pathway for sulphur utilization. On the other hand, the latter pathway, which consists of cystathionine [~-synthase (13-CTSase) [EC 4.2.1.22] and cystathionine ~,-lyase (7-CTLase) [EC 4.4.1.1], is considered as the heterotrophic pathway for sulphur utilization because it is a part of the pathway in which mammals convert methionine-sulphur to cysteine-sulphur (see Griffith 1987). Although mammals synthesize homocysteine only by demethylation of methionine, S. cerevisiae forms homocysteine by sulphhydrylation of homoserine (via O-acetylhomoserine) as well (Yamagata 1971). The two S. cerevisiae cysteine biosynthetic pathways function simultaneously because only strains that have Offprint requests to." B.-i. Ono

Strains. S. cerevisiae strain OK312-7C (MATa, cys2, cys4, his3, leu2, trpl) was used as a cloning host. Strain IS66-4C was used as a control for enzymatic activities (Ono et al. 1988a). Strain OK3613D (MATs, hisT, lys2, tyro was used as a chromosome standard for transverse alternating field electrophoresis (TAFE). This strain gave rise to 14 distinct bands, including one at the origin, by TAFE, and the band-chromosome correspondence was established using appropriate cloned markers (Ono, unpublished data). Strain NA123C (MATer, cys2, cys4) was used for the 2 ~tm DNA chromosomeloss procedure and for the tetrad analysis. Tester strains for the 2 I-tmDNA chromosome-loss procedure and the experimental protocol were as described in the Yeast Genetic Stock Center Catalogue (Mortimer 1987). For meiotic mapping, the standard strains described by Ono et al. (1985), or their derivatives, were used. Genomic library. The S. cerevisiae genomic library used in this study was constructed by T. Ohya (Tokyo University, Tokyo, Japan). For library construction, S. cerevisiae Strain A5-8-IA genomic DNA was partially digested with Sau3AI, and DNA fragments of 5-15 kbp were isolated and inserted into the BamHI site of YEpI3 (Broach et al. 1979). The library consisted of 4 000 independent clones. Yeast growth media and growth conditions. Standard yeast growth media were used (Sherman etal. 1986). Genetic markers were scored as described by Ono et al. (1985). Manipulation of DNA. Plasmids were propagated in Escherichia coli K12 strain DH1 (Hanahan 1983). E. coli transformation, extraction

286 of plasmid from E. coli, purification of plasmid by CsC1 density gradient centrifugation, digestion of DNA with restriction endonucleases, and separation of DNA fragments by agarose gel electrophoresis were as described by Sambrook et al. (1986). Transformation of S. cerevisiaewas carried out by the method of Ito et al. (1983). Extraction of plasmid from S. cervisiaefollowed the technique of Sherman et al. (1986).

Enzyme assay. 13-CTSase and SATase were assayed as described previously (Ono et al. 1988a). The enzyme activity unit (U) was expressed as ktmoles of product formed (cystathionine for 13-CTSase), or substrate consumed (acetyl-CoA for SATase), per min. Protein was measured by the method of Lowry et al. (1951) using bovine serum albumin as a control.

Chromosome separation by TAFE. Chromosome DNAs were separated by TAFE using a Geneline apparatus (Beckman, Instruments, Inc., Palo Alto, CA, USA). Cells of strain OK361-3D were grown to the early stationary phase, harvested and embedded in agarose. Sample gels were prepared as described by Ono and Ishino-Arao (1988). Electrophoresis was carried out by a two-stage mode (4 sec intervals for 30 min at 170 mA and 60 sec intervals for 18 h at 150 mA). DNA-DNA hybridization was monitored by the universal probe method (Yamane et al. 1988). After separation, chromosome DNAs were blotted onto a nitrocellulose membrane (Southern 1975). The membrane was challenged firstly with a primary probe (plasmid of interest) and secondly with the universal probe (i.e., biotinated single-stranded pUCRfl). The membrane was treated with the streptavidin-alkaline phosphatase conjugate (BRL, Cosmo Bio, Co., Ltd., Tokyo, Japan). Color was then developed by treating the membrane with a mixture of 5-bromo-4-chloro-3-indolylphosphate p-toluidine salt and nitroblue tetrazolium chloride (BRL, Cosmo Bio, Co., Ltd., Tokyo, Japan).

2#m DNA chromosome-loss mapping. Strain NAI2-3C (eys2-cys4) was crossed with the tester strains. Zygotes were selected on an SD plate, and they were grown on YPD medium for 3 days by transferring to fresh medium every day. The cells were then spread on a YPD plate, and an appropriate number of clones were picked from each zygote. The colonies were tested for cysteine requirement.

Tetrad analys&. Strains to be crossed were mixed on YPD medium and incubated overnight. The resultant zygotes were selected by complementation of included markers and were sporulated, and the asci dissected. Linkage was established by a statistically significant deviation from P:N = 1 : 1 (Perkins 1949). The genetic distance was expressed in centi-Morgan units (cM) (Mortimer and Hawthorn 1975).

Results and discussion

Cloning of the CYS4 gene Strain O K 3 1 2 - 7 C was transformed with an S. cerevisiae genomic library (see Materials and methods), and three independent, Leu +, Cys § colonies, T - l , T-2 and T-3, were obtained. After g r o w t h on Y P D m e d i u m for 2 days, all o f them gave rise to L e u - clones. The L e u - clones derived f r o m strain T-2 were C y s - , while those f r o m strains T-1 and T-3 were all Cys § . F r o m these results, we conclude that only strain T-2 carries a plasmid o f interest. In order to test which mutation, cys2 or cys4, o f strain T-2 was suppressed by t r a n s f o r m a t i o n , we assayed SATase and 13-CTSase activities. As s h o w n in Table 1, strain T-2 h a d 13-CTSase activity but n o t SATase activity; the parental strain and the L e u - , C y s - colonies derived f r o m strain T-2 did n o t have 13-CTSase activity or SATase activity. F r o m this result, we conclude that we have

Table 1. Enzymatic activities of S. eerevisiaestains Strain

Phenotype

Specific activity a (mU per mg protein)

Given or deduced genotype

/~-CTSase SATase IS66-4C Leu § Cys + OK312-7C Leu- CysT-2 b Leu + Cys + T-2-1 c T-2-2 ~

Leu- CysLeu- Cys-

56.8___ 9.8 503_+134 Wild-type ND ND eys2 cys4 113_+13.6 ND cys2 cys4 [YEp13-C YS4] ND ND eys2 eys4 ND ND eys2 eys4

" ND represents not detectable b Strain T-2 was a Leu + Cys + clone obtained after transformation of strain OK321-7C with a genomic library (see text) c Strains T-2-1 and T-2-2 were Leu- Cys- clones derived from strain T-2 (see text)

cloned CYS4 rather than CYS2 in strain T-2. The fact that strain T-2 had a b o u t twice higher [3-CTSase activity than strain IS66-4C (wild-type) strongly supports our contention that CYS4 is on the plasmid. We then extracted plasmid D N A f r o m strain T-2. By transforming E. coli K I 2 strain D H 1 with the plasmid D N A , we obtained A m p r colonies. A plasmid (p19) was recovered f r o m one o f the A m p r colonies. Plasmid p19 gave rise to Leu +, Cys + colonies when t r a n s f o r m e d to strain OK312-7C, and the t r a n s f o r m a n t s h a d 13-CTSase activity but n o t SATase activity (data n o t shown). A restriction m a p o f plasmid p19 is shown in Fig. 1. A c c o r d ing to subcloning experiments, the CYS4 gene was confined to the 5.6 kbp BamHI-XbaI fragment.

Chromosome assignment of CYS4 by TAFE W h e n plasmids p19 and Y E p l 3 were used as probes, several T A F E bands in addition to those c o r r e s p o n d i n g to c h r o m o s o m e H (LEU2) and 2 ~m D N A showed positive signals (data n o t shown). To eliminate unnecessary ambiguity, we constructed a plasmid, p76(pBR322), by inserting the 7.6 k b p BamHI-BamHI f r a g m e n t o f p19 into the BamHI site o f p B R 3 2 2 (Sutcliffe 1978). Plasmid p76(pBR322) hybridized only to the b a n d c o r r e s p o n d i n g to c h r o m o s o m e s VII and X V (Fig. 2).

Chromosome assignment of the cys2-cys4 pair by the 2 #m DNA chromosome-loss procedure Using strain N A 1 2 - 3 C (cys2-eys4), we carried out the 2 ~tm D N A c h r o m o s o m e - l o s s procedure. As shown in Table 2, only the c h r o m o s o m e VII tester (B-7592) gave rise to C y s - colonies, indicating that the cys2-eys4 pair is on c h r o m o s o m e VII. This result is in accord with the result obtained by the T A F E procedure.

Meiotic mapping of the cys2-cys4 pair on the right arm of chromosome VII Based on the above results, we examined linkage o f the cys2-cys4 pair to markers on c h r o m o s o m e VII (Table 3).

287

(CYS4)

p19 B

H

H

r

H

B

xE II I

0.0

Sa

I

(kbp)

10.2

Function

]

Fig. 1. A restriction map of plasmid p19. The restriction enzymes used were BamHI (B), BglII (Bg), EcoRI (E), HindlII (H), SalI (S), Sau3A1 (Sa) and XbaI (X); PstI, PvulI and XhoI did not cut the cloned fragment. The subfragments shown below the map were inserted into vector YEpl3 and tested for their function; + and - indicate presence and absence, respectively, of Leu § Cys § colonies after transformation to strain OK312-7C

The cys2-cys4 pair showed linkage to SUP77(SUP166) and ade3. Since SUP77(SUP166) and ade3 were not pair should localize between the two linked, the cys2-r markers. By comparing the present result with the previously reported linkage relationships among genetic markers in this region, we place the eys2-cys4 pair between fro2 and SUP76 (Fig. 3). It should be mentioned here that C YS2 and C YS4 are distinct genes but they never recombine (Ono et al. 1988; this study). Consequently the distance between the two genes and their orientation relative to the flanking markers have not been determined.

Conclusions Genetic mapping in S. cervisiae by meiotic recombination is a laborious and time-consuming task. If a gene (mutation) is assigned to a chromosome, genetic mapping of it is greatly facilitated. There are now two powerful methods for chromosome assignment, the 2 ~tm D N A chromosome-loss procedure and the TAFE procedure. The former is applicable to all genes whose mutations are phenotypically detectable, but it requires the handling of several strains. The latter is much faster although it is applicable only to cloned genes. In this work, we adopted a non-radioactive probe method to detect DNA-DNA hybridization for the TAFE procedure because it is advantageous over the conventional radioactive probe method in that one does not need to worry about irradiation from, and decay of the radioactive probe.

Fig. 2A, B. Chromosome assignment by the TAFE procedure. Chromosomes of strain OK361-3D were separated by TAFE (A); Roman numerals indicate chromosomes corresponding to each band. The separated chromosomes were blotted to nitrocellulose membrane and were hybridized with plasmid p76 (pBR322); (B) hybridization was monitored by the universal probe method (see Materials and methods)

SUP77 (SUP166) cys2 cys4 SUP155 fro2 SUP76 - .

-0.

o

: :

: ;

I

( I I I

I

ade3

frol ,I

I

22.0 27.2

15.3 23.0

---

(Mortimer and Schild 1980) (Mortimer and Schild 1985)

m

4.2

(Ono et a l . 1988b) 26.0

47.8

(This study)

Fig. 3. Map position of the cys2cys4 pair. The present results is compared with the previously reported linkage relationships among genes in the vicinity. The figures represent genetic distances (cM)

288 Table 2. Assignment of cys2-cys4 to chromosome VII by the 2 Ixm DNA chromosome-loss procedure Test strain

B-7588 B-7170 B-7171 B-7589 B-7590 B-7591 B-7592 B-7174 a

Chromosome tested

No. of colonies

I II III IV V VI VII VIII

Tested a

Cys-

16 16 71 16 16 16 109 16

0 0 0 0 0 0 11 0

Test strain

Chromosome tested

B-7175 B-7593 B-7594 B-7595 B-7255 B-7596 B-7597 B-7598

IX X XI XII XIII XIV XV XVI

No. of colonies Tested a

Cys-

16 16 16 16 16 102 112 16

0 0 0 0 0 0 0 0

For all strains, 16 colonies each were examined first, then additional ones were examined for strains B-7171, B-7592, B-7596 and B-7598

Table 3. Mapping of cys2-cys4 to the right arm of chromosome VII Cross

Segregation" of cys2-cys4 against

ade6

SUP77 (SUP166)

P

N

T

IS415-16D x NA12-3C IS416-3C x NA12-3C OK372-10B x NA12-3C KN15-8A x KN14-7D

0

2

10

Total

0

2

10

P

N

T

cM

30

1

21

30

1

21

" Since SUP166 used in this study is very likely to be identical with SUP77 (Ono et al. 1988b), SUP77(SUP166) was used to designate the corresponding gene. P, N and T represent parental ditype, nonparental ditype and tetratype tetrads, respectively. The genetic distances (cM) were calculated only when the deviation from

O f five genes i n v o l v e d in cysteine biosynthesis in S.

cervisiae, four have n o w been m a p p e d . C Y S 1 a n d C Y S 3 are linked together a n d are o n the left a r m of c h r o m o some I ( O n o et al. 1984); C Y S 2 a n d C Y S 4 are linked together a n d are o n the right a r m of c h r o m o s o m e VII (this study). T h e r e m a i n i n g gene, M E T 1 7 , has n o t yet been m a p p e d . O u r a t t e m p t to m a p it b y linkage analysis using a set o f strains c o n t a i n i n g v a r i o u s genetic m a r k e r s was so far unsuccessful (Ono, u n p u b l i s h e d data). A prel i m i n a r y a t t e m p t to assign it to a c h r o m o s o m e by the 2 g m D N A c h r o m o s o m e - l o s s p r o c e d u r e yielded a n a m b i g u o u s result; i.e., n o tester strain gave rise to M e t colonies w h e n crossed to a m e t l 7 strain. T h e r e a s o n for this result is n o t k n o w n . Since M E T 1 7 has already been cloned ( S a n g s o d a et al. 1985; D ' A n d r e a e t a l . 1987), c h r o m o s o m e a s s i g n m e n t o f it m a y be m o r e easily achieved by the T A F E procedure. We are n o w u s i n g this a p p r o a c h to m a p MET17.

Acknowledgements. We thank Dr. Y. Ohya (Tokyo University, Tokyo, Japan) for his kind gift of the genomie library used in this study. We are grateful to the Yeast Genetic Stock Center (University of California, Berkeley, California, USA) for supplying a set of strains for the 2 Ixm DNA chromosome-loss procedure. We thank the Gene Research Laboratory of Okayama University for allowing us to use the Geneline apparatus.

ade3

26.0

rad2

P

N

T

16 19

4 2

38 33

35

6

71

cM

47.8

P

N

T

12 8

4 8

18 44

20

12

62

P: N = 1 : 1 was statistically significant at a 1% level; cys2-cys4 and SUPI66 were scored by cysteine dependence and suppression of leu2-2, respectively. The eys2-cys4 pair segregated independently from ade5, lys5, cyh2, and trp5, on the left arm of chromosome VII (data not shown)

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