Molec. gen. Genet. 162, 251 -257 (1978) © by Springer-Verlag 1978

Temperature-Dependent Conversion of Sexual Agglutinability in Saccharomyces cerevisiae Syuichi Doi and Masao Yoshimura Department of Legal Medicine, Kinki University School of Medicine, Sayama-Cho, Minamikawachi-Gun, Osaka, 589 Japan

Summary. Temperature dependency of sexual agglutinability in Saccharomyces cerevisiae was found. In almost all strains tested that were derived from several different sources, the agglutinability was constitutive when grown at 2 5 ° C but inducible when grown at 36 ° C, suggesting that the temperature-dependent conversion of sexual agglutinability is general nature in Saccharomyces. Cycloheximide and 8-hydroxyquinoline inhibited completely both cell division and the conversion of the agglutinability from constitutive to inducible type. N-Hydroxyurea and 5-fluorouracil which allowed cell growth to some extent inhibited the conversion slightly. Hence, ~.he conversion of the agglutinability from constitutive to inducible type may be achieved in cells newly born after temperature shift. The reverse conversion of the agglutinability was gradual in comparison with, the conversion from constitutive to inducible type. This conversion of the agglutinability was regulated by a single gene closely linked to mating type locus, which is recognizable by using a temperature-independent constitutive strain.

Introduction Sexual cell agglutination prior to cell fusion and nuclear fusion occuring during mating in yeast has been extensively investigated (Crandall and Brock, 1968; Calleja, 1970; Egel, 1971; Yanagishima, 1973; Itoh et al., 1976; Crandall, 197'7). In Hansenula and Saccharomyces, sexual agglutination which takes place when cells of opposite mating types are mixed is brought about by the complementary binding of sex-specific glycoproteins present on the cell surface (Brock, 1959; Crandall and Brock, 1968; Shimoda and Yanagishima, 1975; Hagiya et al., 1977). There are two types of sexual agglutinability, constitutive

and inducible, in a type strains of Saccharomyces cerevisiae (Sakai and Yanagishima, 1972). The former shows constitutive agglutinability without any stimuli from the opposite mating type whereas the latter becomes agglutinable only when mixed with cells of opposite mating type. The sexual agglutinability of inducible a type cells is known to be induced by a substance secreted by e type cells (Sakai and Yanagishima, 1972). We reported that constitutive strains at 25 ° C lost the agglutinability when grown at 36 ° C (Doi and Yoshimura, 1977). Recently we have found that temperature-sensitive loss of sexual agglutinability in constitutive strains was not due to complete loss of the agglutinability but to the change in sexual agglutinability caused by the change in growth temperature: cells grown at 2 5 ° C showed constitutive agglutinability, while they exhibited inducible agglutinability when grown at 36 ° C. Only one strain of our breeding stocks was found to be temperatureindependent constitutive for the agglutinability. This strain allowed us to do genetic analysis on the regulation of sexual agglutinability. In this paper, results obtained from physiological and genetic studies on this conversion will be described.

Materials and Methods Yeast Strains, Medium and Culture Conditions. Heterothallic strains of Saccharomyces cerevisiae were used (Table 1). As standard tester, prototrophic haploid strains, K21-1A and K21-1C were used. Medium (YEPD) used for yeast culture was composed of 20 g glucose, 20 g peptone and 10 g yeast extract in 1,000ml of distilled water. Minimal medium (MIN) contained 5 g (NH¢)2804, 5 g KH2PO4, 2 g MgSO4"7HzO and traces of vitamins in 1,000ml of distilled water. Sporulation medium (SPO) was 1% of potassium acetate. Single nutrient ommission medium used for scoring of auxotrophic markers was prepared by supplementing with appropriate nutrients to MIN medium. For the test of resistance to cycloheximide, YEPD medium supplemented with 5 mg/l of the drug was used. In the case of solid medium, 2% of agar was added.

0026-8925/78/162/0251~01.40

252

S. Doi and M. Yoshimura: Temperature-Dependent Conversion of Sexual Agglutinability in Yeast

Table 1. Yeast strains Strain

Genotype

T2 T4 7)

e ade 7 ura 1 c~his 4 trp 1 ura 4 ade 6 tyr 1 arg 4 leu 2 lys 2 a ade2 his8 a leu 1 met 14 c~ c~ a c~arg4 thr 1 7 a r g 4 t r p l his4 leu2 7 leu2 trp 1 arg9 his6 ilv 3 met 14 pet8 pet 19 rad 1 ~ a d e l hisl c~his 5 ade 2 a gal iS his trp a gal 4 his 1 ura 1 ade 6 trp 1 a/a trp 1/trp 1 ade 1/+

Hll-12A H96-15A $288C T22 T23 C3188-10D 11 HD144-1B 67 X3144-11A 21 D273-11A 31 A4840A 3) 429-6A 4) 278-1B 47 D M I s) K12-1A 6) K27-3B 6) K21-1A 6) K21-1C 6)

(~

a a c~

Original sources of strains are: 1 =T. Takahashi, 2 = Y e a s t Genetic Stock Center, 3 = C o l d Spring Harbor Lab., 4 = H . C . Douglas, 5 = N . Gunge, 6 = o u r stocks, 7 =D.C. Hawthorne, Others are described previously (Doi and Yoshimura, 1977)

Usually yeast cells were cultured in 100 ml of liquid YEPD in conical flask having a capacity of 500 ml with shaking on a reciprocal shaker at 25 ° C or 36 ° C. Tester cells were always cultured at 25 ° C.

Measurement of Cell Number. Cell number was measured using Thoma's counting chamber. Buds larger than half of mother cells in diameter were counted as cells.

Genetic Analysis. Hybridization was performed by mass mating method (Lindegren and Lindegren, 1943). Diploid hybrids were obtained by prototroph recovery method (Pomper and Burkholder, 1949) or serial incubation of a portion of mass mating culture to fresh YEPD. Sporulation was carried out by incubating diploid cells on SPO medium at 25°C for 2 or 3 days. Spore tetrads were dissected using micromanipulator after removal of ascus wall by treatment with Glusulase. Segregation of mating type and nutritional markers was scored by mass mating and replica plating of segregant cells on a single nutrient ommission medium, respectively. Resistance to cycloheximide was determined whether confluent growth on YEPD containning the drug was observed or not. Assay of Spore Culture for Sexual Agglutinability. Sexual agglutinability of haploid segregants was tested as follows. Segregants of a or c~ mating type were cultured on YEPD separately to avoid induction of agglutinability for about 20 h at 25 ° C or 36 ° C. Cells thus obtained were suspended in PB, heated in boiling water for 5 min, and was rapidly chilled in ice-cold water bath. The boiled cells were washed 3 times with PB and were mixed with boiled tester cells in PB. The mixture was shaken at 25°C for 2 h. In the case of living cells, cells to be tested were suspended in liquid YEPD and mixed with living tester cells of opposite mating type. The mixture was shaken for 2 h at the same temperature at which cells were grown. Assay for agglutinability of haploid segregants grown at 36 ° C, which showed constitutive agglutinability at 25 ° C was carried out in following combinations: living segregant and living tester, and boiled segregant and boiled tester. Sexual agglutinability was judged as constitutive when agglutination was observed in both combinations. When the agglutination was observed only in the combination, living segregant and living tester, the agglutinability of segregant was decided as temperature-dependent inducible (td-inducible).

Results

Change in S e x u a l Agglutinability in Relation to Growth Temperature

Preparation of Boiled Cells. Cells grown at late logarithmic phase of growth were harvested by centrifugation, washed 3 times with phosphate buffer (10 raM, pH 5.5) designated as PB and suspended in PB. The cell suspension was boiled at 100 ° C for 5 rain, rapidly chilled in the ice-bath and washed five or more times with ice-cold PB, suspended in the same buffer and kept at 4 ° C before use.

Sexual Agglutination Test. Assay for sexual agglutination and determination of the degree of agglutination was carried out essentially according to Shimoda and Yanagishima (1974). Cells were mixed with the same number of tester cells having opposite mating type in PB or liquid YEPD. Final optical density at 530 nm (ODs30) of the mixture was adjusted to one. Then the mixture was shaken on a reciprocal shaker. After 120 min, the degree of sexual agglutination was determined. One ml of the agglutinating mixture was withdrawn with Pasteur pipete after shaking with hand and diluted with 4 ml of PB. OD53o of the agglutinating mixture was measured before (ODus) and after (ODAs) brief sonication to disperse the agglutinating cells using Branson sonifier B-12 (40 W, 5 s). Intensity of sexual agglutination was expressed in terms of agglutination index (A.I.) obtained by dividing ODBs by ODAs. Intensely agglutinating mixture usually gave a value of A.I. 1.30 to 1.40. When a value of A.I. is under 1.10, sexual agglutination is hardly observed with eyes. When boiled cells were used, sexual agglutination test was done at 25 ° C. When living cells without boiling were used, however, the temperature at which cells were grown was employed.

W e h a v e r e p o r t e d t h a t c e l l s g r o w n a t 36 ° C l o s t t h e i r constitutive agglutinability (Doi and Yoshimura, 1977). T h e r e a r e t w o m a j o r p o s s i b i l i t i e s f o r t h e e x p l a nation for temperature-sensitive loss of constitutive a g g l u t i n a b i l i t y i n t h e c e l l s g r o w n a t 36 ° C ; 1) c o m p l e t e loss of the agglutinability at higher temperature and 2) c h a n g e in s e x u a l a g g l u t i n a b i l i t y f r o m c o n s t i t u t i v e t o i n d u c i b l e t y p e a t 36 ° C. I n o r d e r t o d i s t i n g u i s h these possibilities, sexual agglutination test of living c e l l s i n s t e a d o f b o i l e d c e l l s c u l t u r e d a t 25 ° C o r 36 ° C w a s d o n e u s i n g l i v i n g o r b o i l e d t e s t e r cells. I f c e l l s c u l t u r e d a t 36 ° C l o s e t h e i r a g g l u t i n a b i l i t y c o m p l e t e l y , they could agglutinate neither living nor boiled tester cells. A l t e r n a t i v e l y , i n t h e c a s e o f t h e s e c o n d p o s s i b i l ity, l i v i n g c e l l s c u l t u r e d at- h i g h e r t e m p e r a t u r e are not able to agglutinate with boiled tester cells but are able to agglutinate with living tester cells after a l a g p e r i o d e v e n a t 36 ° C. A s s h o w n i n T a b l e 2, c e l l s g r o w n a t 25 ° C c o u l d a g g l u t i n a t e r e g a r d l e s s o f whether tester cells were living or boiled. This indicates that the cells grown at 25°C are constitutive

S. Doi and M. Yoshimura : Temperature-Dependent Conversion of Sexual Agglutinability in Yeast i

Table 2. Effect of growth temperature on sexual agglutinability

i

253 i

i

A

Growth temperature

Agglutination w i t h

(oC)

LIT. a

B.T?

K12-1A

25 36

1.4l 1.33

1.31 1.05

td-inducible

K27-3B

25 36

1.37 1.3l

1.35 1.06

td-inducible

Strain

Agglutinability

~3 ~1.2 z ~-1.I

3

<9 <9 < II0

0

T23

25 36

1.43 1.37

1.31 1.28

2

4

constitutive

L.T. ; living tester cells B.T. ; boiled tester cells

~'

for sexual agglutinability. O n the other h a n d , cells g r o w n at 36 ° C showed a g g l u t i n a t i o n only in combin a t i o n with living tester cells. This ~Lype of agglutinability is inducible. These results suggest that these strains of Saccharomyces cerevisiae did n o t completely lose their sexual a g g l u t i n a b i l i t y at higher temperature, b u t converted their a g g l u t i n a b i l i t y from constitutive to inducible type in a c c o r d a n c e with the change in growth t e m p e r a t u r e . W e m a y call this type of agglutinability t e m p e r a t u r e - d e p e n d e n t inducible (td-inducible) agglutinability. I n a strain, T23, sexual agglutination was observed in a n y cases tested. This strain, hence, is t e m p e r a t u r e - i n d e p e n d e n t constitutive for sexual agglutinability. This type of agglutinability in T23 m a y be simply called c o n s t i t u t b e .

Time Course of Change in Sexual Agglutinability in td-Inducible Strains P r e l i m i n a r y results d e m o n s t r a t e d that constitutive agglutinability in t d - i n d u c i b l e strains was almost lost 3 h after t e m p e r a t u r e shift f r o m 25°C to 36°C. This suggests that the agglutinability in t d - i n d u c i b l e strains was converted from constitutive to inducible type after t e m p e r a t u r e shift. I n t d - i n d u c i b l e strains, competence for sexual a g g l u t i n a t i o n b e g a n to be lost soon after i n c u b a t i o n at 36 ° C a n d was almost completely lost after 4 h (Fig. 1 A a n d B). A g g l u t i n a b i l i t y in constitutive strain, T23 was slightly lowered d u r i n g i n c u b a t i o n at higher temperature. A l t h o u g h optical density of the culture increased c o n s i d e r a b l y d u r i n g i n c u b a t i o n , a g g l u t i n a b i l i t y was preserved at a slightly lower level as c o m p a r e d with the initial in constitutive strain, T23. Hence, the loss of constitutive agglutinability in t d - i n d u c i b l e strains at 3 6 ° C m a y be n o t due to cell growth. It seems likely that sexual agglutin a b i l i t y in t d - i n d u c i b l e strains b e g a n to change from constitutive to i n d u c i b l e type soon after t e m p e r a t u r e shift.

0 TIME

i

i

i

2

0

2

p

(HR

Fig. 1 A-C.

Loss of constitutive agglutinability after temperature shift. Cells of each strains were cultured at late logarithmic phase of growth at 25° C, harvested, washed twice with water and suspended in water (OD53o, 10). The cells were inoculated into fresh YEPD pre-warmed at 36° C at OD53o of 0.5 and incubated. At intervals, a part of culture was sampled and washed 3 times with PB. After preparation of boiled cells, sexual agglutination was assayed in PB using boiled tester cells at 25° C. A and B td-inducible strains, K12-1A and K27-3B, respectively. C constitutive strain, T23

Table 3. Effect of inhibitors on the loss of constitutive agglutinability at 36° C Inhibitora

K12-1A

K27-3B

T23

A.I.b cell growth c

A.I.

cell growth

A.I.

PB YEPD

1.33 100 1.04 348

1.42 1.10

100 511

1.35 100 1.27 392

CHI 1 pg/ml 10 pg/ml

1.30 1.27

100 100

1.3l 1.40

100 100

1.37 1.45

100 100

HQ 100 gg/ml 300 gg/ml

1.23 1.41

144 100

1.17 1.32

153 96

1.48 1.43

142 100

5-FU 50gg/ml 100gg/ml

1.14 275 1.17 232

1.15 239 1.17 214

1.33 320 1.36 242

HU 5x10 ; M 10x10 -2M

1.16 244 1.15 216

1.17 204 1.16 196

1.30 338 1.32 233

cell growth

a Inhibitor was added to YEPD; CHI-cycloheximide, HQ= 8-Hydroxyquinoline, 5-FU = 5-Eluorouracii, HU = N-Hydroxyurea b A.I. ; Agglutination index ° Cell growth was measured by cell number counting and is expressed as percent of the initial

Effect of Inhibition of MacromolecuIar Synthesis on the Conversion of Sexual Agglutinability from Constitutive to Inducible Type Effect of i n h i b i t i o n of m a c r o m o l e c u l a r synthesis on the c o n v e r s i o n of sexual agglutinability from constitutive to inducible type was studied (Table 3). Cells from late logarithmic phase culture g r o w n at 2 5 ° C were i n o c u l a t e d in Y E P D c o n t a i n i n g a p p r o p r i a t e inhibitor (optical density at 530 n m of 0.5) a n d incu-

254

S. Doi and M. Yoshimura : Temperature-Dependent Conversion of Sexual Agglutinability in Yeast

bated at 36 ° C. After 4 h, boiled cells were prepared. Sexual agglutination test was carried out using boiled tester cells in PB at 25 ° C. In the presense of cycloheximide and 8-hydroxyquinoline which are potent inhibitors for synthesis of protein and R N A , respectively, both cell division and loss of constitutive agglutinability were strongly inhibited. N-Hydroxyurea and 5-fluorouracil, inhibitors for D N A synthesis and R N A synthesis, respectively, inhibited slightly cell number increase and loss of the agglutinability. Moreover, constitutive agglutinability in td-inducible strains was not lost even at 36 ° C in PB which could not support cell division. Thus, cell number increase is parallel with loss of constitutive agglutinability, suggesting that cells budded off after temperature shift have no constitutive agglutinability.

i

x LU

~ 1.3

i

i

i

i

i

i

i

i

i

~1.2 z1,1

g 1.0

i

i

i

i

i

i

i

i

2 4 2 4 CELL NUMBER INCREASE

i

i

i

i

6 2 (FOLD)

q

i

i

4

Fig. 2A-C. Relationship between increase in cell number and loss of constitutive agglutinability. Cells of each strains grown at 25 ° C were incubated in fresh YEPD (initial OD530, 0.5) at 36 ° C. At intervals, cells were withdrawn and subjected to cell number count and preparation of boiled cells. Sexual agglutination was carried out in PB at 25 ° C using boiled tester cells. A K12-1A, B K27-3B, C T23

Relationship between Cell Number Increase and the Conversion of Sexual Agglutinability Relationship between cell growth and the conversion of sexual agglutinability from constitutive to inducible type was investigated. Cells grown at 25 ° C were obtained from late logarithmic phase culture and inoculated in Y E P D pre-warmed at 36°C at OD53o of 0.5. Sexual agglutination and cell number were measured (Fig. 2). Constitutive agglutinability in td-inducible strains started to lose soon after temperature shift and then was lost with cell number increase. In the constitutive strain T23, the level of constitutive agglutination was somewhat lowered in accordance with cell number increase but was maintained at A.I. of about 1.25. Figure 3 shows the effect of dilution of agglutinative cells with non-agglutinative ones on agglutination. The pattern of decrease in the degree of agglutination was similar to that of decrease in A.I. of td-inducible strains shown in Figure 2. F r o m these results, it is probable that cells divided after the temperature shift in td-inducible strains lost constitutive agglutinability.

i

A

i

i

i

i

1.3

1.2

1.1

1.0

2 3 DILUTION (FOLD)

Fig. 3. Effect of dilution of agglutinative cells with non-agglutinative cells on sexual agglutination. Agglutinative boiled cells were obtained from late log, culture of K21-1A (a). Non-agglutinative boiled cells were obtained from a 2 day-old culture of K21-1A. Each cell suspension having the same cell density was mixed at various rates. Sexual agglutinability of the mixed cell suspension was tested in PB at 25°C. When ~ type cells (K21-1C) instead of a type cells were used as test ceils, the result was essentially the same

I

I

I

I

E

cal.3

I

I

I

I

I

I

A

z

J

l

l

B

z1.2 o

Recovery of Constitutive Agglutinability after Temperature Shift from 36°C to 25°C

z1.1 o

~1.0

As shown in Figure 4, recovery of constitutive agglutinability of td-inducible cells after temperature shift from 36 ° C to 25 ° C was gradual in comparison wil:h the conversion from constitutive to inducible type, though optical density was considerably increased. However, td-inducible cells cultured at late logarithmic phase at 25 ° C, which were grown from cells

i

i

i

2

4

i

J

I

1

~

1

1

1

0 2 4 6 0 2 4 6 TIME

(HR)

Fig. 4A-C. Recovery of constitutive agglutinability in td-inducible strains after temperature shift. Cells of each strain grown at 36 ° C in late log. phase were harvested, washed, inoculated in fresh YEPD at OD530 of 0.5 and incubated at 25 ° C. At intervals, aliquots of culture were sampled, washed and subjected to preparations of boiled cells. Assay for agglutination was done in PB at 25 ° C. A K12-1A, B K27-3B, C T23

S. Doi and M. Yoshimura: Temperature-Dependent Conversion of Sexual Agglutinability in Yeast Table 4. Sexual agglutinability of cells grown at 36° C in various

strains of Saccharomyces Strain

Mating type

Agglutination with L.T. a

T2 T4 Hll-12A H96-15A $288C T22 C3188-10D HD144-1B X3144-11A D273-11A A4840A 429-6A 278-1B DM1

c~ ~ a

B.T."

+ + +

a

+

~ e e c~ e ~ ~ a a

+ + + + + + + + +

a/a

+

Late log. phase cells of each strain were used. In all strains cultured at 25° C, sexual agglutination was observed in any combinations between boiled or living cells to be tested and boiled or living tester cells L.T. =living tester, B.T. =boiled tester Table 5. Segregation of genetic markers in tetrads from the cross,

T23 x CM1-SA Segregation Mating in ascus type (a :~)

leu 1 lys 5 (+: )a ( + : - - )

cyh 2 (+ :--)

agglutinability at 36° C (c:i) b

4:0 3:1 2:2 1:3 0:4

0 0 58 2 0

0 0 59 1 0

0 0 59 1 0

0 0 59 1 0

0 2 56 2 0

" '°+" and " - " indicate confluent growth and non-growth on test medium, respectively b "C" and "i" indicate constitutive and inducible, respectively Table 6. Linkage between mating type allele and the locus, sag 1

Ascus type a PD

NPD

TT

59

0

0

255

Effect of Growth Temperature on Sexual Agglutinability in Various Strains of Saccharomyces cerevisiae We extended our survey to further strains of Saccharomyces derived from several different sources. Sexual a g g l u t i n a t i o n in all strains tested g r o w n at 36 ° C was observed only in the use of living tester cells b u t n o t in the use of boiled tester cells (Table 4). W h e n cells cultured at 25 ° C, a n y c o m b i n a t i o n between cells to be tested a n d tester cells gave a g g l u t i n a t i n g mixture. These strains, therefore, are t d - i n d u c i b l e for sexual agglutinability. Since these strains were derived from several different sources, it is p r o b a b l e that temp e r a t u r e - d e p e n d e n t c o n v e r s i o n of sexual agglutinability m a y be of general n a t u r e in Saccharomyces.

Genetic Analysis The results described above suggest that there is a genetic regulatory system o n sexual agglutinability in Saccharomyces. F o r t u n a t e l y , the use of constitutive strain, T23 o b t a i n e d f r o m our breeding stocks allows us to recognize gene(s) regulating t e m p e r a t u r e - d e p e n dent c o n v e r s i o n of sexual agglutinability. Since sexual a g g l u t i n a t i o n is hardly distinguished from asexual flocculation, p a r e n t strains from which non-flocculative segregants were derived were selected. Constitutive strain, T23 was crossed to t d - i n d u c i b l e strain, C M 1 - 8 A . This c o m b i n a t i o n of parents yielded no flocculative segregants. After s p o r u l a t i o n , spore tetrads were dissected a n d charactrized. Segregation data are s u m m a r i z e d in Table 5. M a t i n g type, nutritional m a r k e r s a n d cycloheximide resistance segregated 2 to 2 fashion. F o r sexual agglutinability, all segregants were constitutive at 25 ° C. Sexual agglutinability at 36 ° C, however, segregated 2 constitutive to 2 inducible in ascus. This indicates that temperat u r e - d e p e n d e n t c o n v e r s i o n of sexual a g g l u t i n a b i l i t y is regulated by a single gene. Besides, in gene pair, m a t i n g type allele a n d the gene, all ascus type were p a r e n t a l ditype (PD) (Table 6). Accordingly, the gene regulating sexual agglutinability was f o u n d to be tightly linked to m a t i n g type allele.

a PD=parantal ditype; NPD=non-parantal ditype; TT=tetra type Discussion

once cultured at 36 ° C, showed intense a g g l u t i n a t i o n (A.I. of 1.30 to 1.40). Hence, t d - i n d u c i b l e cells could o b t a i n n o r m a l constitutive a g g l u t i n a b i l i t y finally after t e m p e r a t u r e shift. The reason for this slower conversion is n o t clear yet.

We have f o u n d t e m p e r a t u r e - d e p e n d e n c e of conversion of sexual agglutinability in Saccharomyces cerevisiae. C o n s t i t u t i v e strains for sexual agglutinability at 25 ° C converted their agglutinability to inducible nature when i n c u b a t e d at 36 ° C. The c o n v e r s i o n of the

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S. Doi and M. Yoshimura : Temperature-Dependent Conversion of Sexual Agglutinability in Yeast

agglutinability was initiated soon after t e m p e r a t u r e shift. Since sexual a g g l u t i n a t i o n in Saccharomyces is caused by the c o m p l e m e n t a r y b i n d i n g of sex-specific glycoproteins located on the cell surface (Hagiya et al., 1977), it seems likely that synthesis of the glycoproteins m a y cease after t e m p e r a t u r e shift f r o m 25 ° C to 36 ° C. This was supported by the fact that the i n d u c t i o n of the agglutinability of t d - i n d u c i b l e cells at 36 ° C by opposite m a t i n g type cells was prevented by the i n h i b i t i o n of R N A a n d p r o t e i n synthesis (Doi, in preparation). O n the other h a n d , glycoproteins present o n the cell surface before t e m p e r a t u r e shift seem to be stable even at 36 ° C. It is, hence, likely that daughter cells newly b o r n after t e m p e r a t u r e shift were devoid of the glycoproteins a n d that the synthesis of the glycoproteins in these cells was i n d u c e d by opposite m a t i n g type cells. A l t h o u g h the c o n v e r s i o n from inducible to constitutive agglutinability in tdinducible strains after t e m p e r a t u r e shift from 3 6 ° C to 25 ° C was g r a d u a l as c o m p a r e d with the c o n v e r s i o n from constitutive to inducible type, constitutive agglutinability was finally restored. The r e a s o n for this difference between t e m p e r a t u r e - d e p e n d e n t c o n v e r s i o n of agglutinability f r o m constitutive to inducible type a n d vice versa is currently u n k n o w n . It is interesting that almost all strains of Saccharomyces cerevisiae o b t a i n e d from several different sources showed t e m p e r a t u r e d e p e n d e n c y of sexual agglutinability. A m o n g strains tested, only one strain was constitutive for agglutinability even at 36 ° C. The constitutivity for agglutinability in T23 is regulated by a single gene closely linked to m a t i n g type allele or m a t i n g type allele itself. This constitutive n a t u r e was recessive (Doi, in preparation). We m a y call this gene sag 1 (sexual a g g l u t i n a t i o n ) tentatively. It is plausible that the gene, sag 1 has a regulatory f u n c t i o n c o n t r o l l i n g structural genes for the glycoproteins responsible for sexual a g g l u t i n a t i o n , since it is reported that the m a t i n g type locus m a y have structural complexity with regulatory f u n c t i o n ( H a w t h o r n e , 1963; Hicks a n d Herskowitz, 1976; Hicks a n d Herskowitz, 1977). Y a n a g i s h i m a et al. (1976) have reported that sexual agglutinability was c h a n g e d by c a r b o n sources. R e l a t i o n between our findings a n d their ones is rem a i n e d open. A n u m b e r of studies on diffusible sex p h e r o m o n e s secreted by yeast cells were reported ( D u n t z e et al., 1970; Sakai a n d Y a n a g i s h i m a , 1972; B t i c k i n g - T h r o m et al., 1973; Hartwell, 1973; Scherer et al., 1974; W i l k i n s o n a n d Pringle, 1974; S h i m o d a et al., 1976; Betz et al., 1977). It r e m a i n e d to be determined, however, whether the substance which can induce sexual a g g l u t i n a b i l i t y in t d - i n d u c i b l e strains is identical to one of these yeast p h e r o m o n e s .

Acknowledgements. We would like to thank Dr. Yasutoshi Nogi, Keio University School of Medicine for supplying yeast strains and Dr. Hyogo Sinohara of our University for invaluable discussions and for reading the manuscript.

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Communicated by F. Kaudewitz Received February 23, 1978