Vol. 91, No. 3, 1979 December
BIOCHEMICAL
AND BIOPHYSICAL RESEARCH COMMUNICATIONS Pages 849-853
14, 1979
INDUCTION
OF SEXUAL
CELL
AGGLUTINABILITY
BY W-FACTOR
IN
OF 2 MATING
SACCHAROMYCES
TYPE
CELLS
CEREVISIAE
Syuichi Doi, Yasuyuki Suzuki and Masao Yoshimura Departments of Legal Medicine and Biochemistry, Kinki University School of Medicine Sayama, Osaka 589, Japan Received October 15, 1979 Summary Mating hormone, d-factor, causes characteristic changes in type cells, was also responsible agglutinability of a - mating type Mating
reaction
is achieved
through
events:
sexual
opposite
and nuclear
living
tester
fusion
of opposite
(constitutive
was observed.
When the
cells,
however,
induced
sexual Materials
sexual
(inducible
that
mating
which
cells
agglutination
sexual
agglutinability
cells the
of opposite
O(-factor
in the
sequential
cells
recognize to form
to form diploid
(1).
heat-killed , sexual
or
agglutination
When cells cells,
was observed Since
culture
tester a lag
phenomenon
grown at
type,
aggluti-
living after
this
of cells mating
grown at
no sexual
were mixed with
(2).
cerevisiae
(plasmogamy)
type
tester
agglutinability)
by living
was tested
of three
agglutinability).
nation
that
fusion
(karyogamy)
heat-killed
suggests
during
cell
36'C were mixed with
period
Saccharomyces
grown at 25'C were mixed with cells
was observed
cells,
of
progression
agglutination
type
When cells
strains
an orderly
cell
mating
zygote,
in haploid
which inhibits DNA synthesis and cell morphology in a mating for induction of sexual cell cells.
36°C is
the possibility
filtrate
may
induce
agglutinability. and Methods
Yeast strains, media and culture conditions: strains of Saccharomyces cerevisiae, K27-3B
Prototrophic haploid (a) and T22 (X2180-lB, 0006-291X/79/230849-05$01.00/0
849
Copyrighr All rights
@I 1979 by Academic Press, Inc. of reproduction in any form reserved.
Vol. 91, No. 3, 1979
BIOCHEMICAL
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
YPD (my, 5% glucose, 1% peptone, 0.5% rX) were used. Modified yeast extract, 0.5% KH2P04 and 0.2% MgS04.7H20), MV (3) and PG (1% glucose and 0.1% peptone) were used for yeast culture, preparation of culture filtrate and induction of agglutinability, respectively. Yeast culture was carried out at 25'C or 36'C on a reciprocal shaker. Induction of sexual agglutinability: Cells from late logarithmic phase culture of K27-3B grown in mY medium at 36'C were harvested, washed and suspended in water. Absorbance at 530 nm (A530) of the suspension was adjusted to 4. For induction of agglutinability, a half ml of the cell suspension was mixed with 9 ml of phosphate buffer (10 mM, pH 5.5) containing purified M-factor and 0.5 ml of 10 times concentrated PG and then the mixture was incubated at 36°C. 100 ml of 2 day-old culture of T22 were Purification of M-factor: inoculated into 20 liter of MV medium in jar fermenter (Ishiyama Tokyo) and cultured for 48 hours at 25'C. After Kagaku Kikai, removal of cells by centrifugation, cell-free culture filtrate was obtained by filtering culture fluid through glass fiber filter (GF/C, Whatman, England). Purification of M-factor from the filtrate was carried out essentially according to Duntze et -- a1.(4). W-Factor activity was determined in mY medium at 25'C 3 hours after incubation by testing two-fold dilution series. The most diluted test culture which showed characteristic change in cell morphology called "&moo" was determined as containing one unit of M-factor. Although direct comparison is impossible since different of w-factor had activity assay system was used, our preparation similar to that reported previously (4). Enzyme treatment: Purified u-factor (1 pg/ml) was treated with pepsin proteolytic enzymes, Pronase E (Kaken Kagaku, Tokyo), (Worthington,Biochem. Co., Freehold, N.J.), trypsin (P-L Biochem., Milwaukee) and 0(-chymotrypsin (ICN Pharmaceuticals, Cleveland) in for 60 min. at 36'C. The enzymes except pepsin were dissolved buffer (30 mM, pH 7.0) was 1 mM calcium acetate before use. Tris-HCl used for pronase, trypsin and ti-chymotrypsin, and sodium acetate buffer (10 mM, pH 3.5) for pepsin. Activity of %-factor to cause a-shmoos or to induce agglutinability was determined after inactivating enzyme by boiling for 5 min. at 1OO'C. Synthetic courtesy Osaka.
Synthetic H-factor (5) was provided u-factor: of Dr. Hiroshi Kita, Central Res. Inst., Suntory
Agglutination assay: described previously of heat-killed cells
by the Ltd.,
Agglutination assay was carried out as (2) except the use of living cells instead as a tester.
Results Induction
of sexual
agglutinability
As shown in Table sexual
agglutinability
buffer
enriched
with
by purified
1, purified
w-factor
of -a mating
type
both
glucose
850
cells
and peptone
o( -factor could
intensely
in the (PG).
induce
phosphate It
could
not
Vol. 91, No. 3, 1979
BIOCHEMICAL
Table 1. Induction
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
of sexual agglutinability
Medium')
by H-factor
Agglutination
index
PB
1.03
PE + glucose
1.04
PB + peptone
1.01
PB + glucose + peptone 2)
1.03
PB + glucose + peptone
1.34
PB + glucose + peptone 3)
1.38
Purified a-factor (1 pg/ml) was added to each medium. 1) Concentration of glucose and peptone was 1% and O.l%, respectively. 2) No H-factor was added. 3) Synthetic N-factor instead of purified one was added at the concentration of 2 pg/ml, although this value was not accurate because of small amount of it.
induce
agglutinability
same buffer
in the phosphate
supplemented
with
Agglutinability-inducing inactivated
with
no effect
activity pronase
a-shmoo-forming
activity.
on both
agglutinability d-factor of synthetic
glucose
Trypsin
which
are also
and O(-chymotrypsin
by a-factor.
also
induce
material
could
suggest
Furthermore,
agglutinability. not
allow
or in the
M-factor
These results
induced
alone,
or peptone.
of purified
and pepsin
activities.
is could
either
buffer
was
known to destroy had virtually that
sexual
synthetic
Unfortunately,
us to perform
shortage
further
experiments. Intensity
of agglutinability
as a function
of concentration
of
N-factor As shown in Fig. increased
with
concentration even at
1, the
increasing of
1 )Ig/ml.
the highest
intensity
of
amount of added In
this
concentration
851
sexual
agglutinability
H-factor
up to the
system,
-a-shmoo were not
tested,
i.e.
10 pg/ml.
formed
Vol. 91, No. 3, 1979
BIOCHEMICAL
AND BIOPHYSICAL
RESEARCH COMMUNICATIONS
x1.4w cl
0
21.3-
l
a
o
0
5 ;1.2u z
CONC.OF
30
O
TIME
6(
-
d 2 1.0
-,A
0
(min)
3
4
2
4
P"n
*
Fig.
1. Relationship between and concentration of incubated at 36*C in of purified O!-factor. washed and subjected
Fig.
2. Time course of the induction of sexual agglutinability. Cells were incubated at 36oC in PG medium which contained 1 pg/ml of purified M-factor. At time intervals, cells were withdrawn, washed and subjected to agglutination assay.
Fig.
3. pH dependency of induction of the agglutinability. Cells were incubated at 36“C in various buffer solution (10 mM) containing 1 )Ig/ml of purified ff -factor, 1% of glucose and 0.1% of peptone. After an hour, cells were harvested, washed and assayed for agglutination. 0 * citrate buffer, . phosphate buffer, a ; Trjs-HCl buffer and ; carbonate buffer.
Time course
of the
observed
15 min.
reached
maximal
at pH 3 to
pH dependency d-factor
after
the
increased
degree
similar
with
time,
and
of agglutinability of
7, and decreased is
began to be
45 minutes.
the induction
3 shows that
Fig.
This
of
agglutinablity
incubation,
the maximum level
pH dependency
of agglutinability
2, sexual
after
lo
intensity of sexual agglutinability purified M-factor. Cells were PG medium containing various anunount After an hour, cells were harvested, to agglutination assay.
induction
As shown in Fig.
of
02
(pg/mL)
g-FACTOR
El.1
sexual with
to that
agglutinability
ncreasing
was
in pH value.
of a-shmoo-forming
activity
(4).
Discussion It cerevisiae in cell
was showed that inhibits morphology
mating
hormone,
DNA synthesis of opposite
a-factor
from Saccharomyces
and causes characteristic
mating
type
cells
(4,6).
change
BIOCHEMICAL
Vol. 97, No. 3, 1979
The present mating
hormone,
study i.e.
The possibility,
since
the
M-factor
completely
the
another
induction
of
can not substance
preparation
RESEARCH COMMUNICATIONS
characteristic sexual
this
agglutinability.
be excluded is not
of
that
the
identical
used in present
sexual
with
study
d-factor,
may not be
pure.
Further different
suggests
however,
agglutinability-inducing
AND BlOPHYSlCAL
studies
are necessary
phenomena caused by
shmoo formation
and induction
to clarify
W-factor, of sexual
mechanism underlying
inhibition
of
DNA synthesis,
agglutinability.
Acknowledgments The authors are grateful to Dr. Hiroshi Kita and Dr. Isamu Takano, Cental Res. Inst., Suntory Ltd., Osaka for synthetic (X-factor and to Dr. Moritoshi Shibata, Nara Medical University for his continual encouragements. Thanks are also due to for helpful discussions Dr. Hyogo Sinohara of our university during this work and for reading manuscript. References 1. Yanagishima, N. (1973) Curr. Adv. Plant Sci. z, 55-66. 2. Doi, S., and Yoshimura, M. (1978) Molec. gen. Genet. 162, 251-257. 3. MacKey, V., and Manney, T.R. (1974) Genetics 76, 255-271. 4. Duntze, W., Ste)tzler, D., Biicking-Throm, E., glbitzer, S. (1973) Eur. J. Biochem. 35, 357-365. 5. Masui, Y., Chino, N., Sakakibara, S., Tanaka, T.m Murakami, T., and Kita, H. (1977) Biochem. Biophys. Res. Commun. 78, 534-538. 6. Bucking-Throm, E., Duntze, W., Hartwell, L.H., and Penney, T.R. (1973) Exptl. Cell Res. 76, 99-110.
8.53
BIOCHEMICAL
AND BIOPHYSICAL RESEARCH COMMUNICATIONS Pages 849-853
14, 1979
INDUCTION
OF SEXUAL
CELL
AGGLUTINABILITY
BY W-FACTOR
IN
OF 2 MATING
SACCHAROMYCES
TYPE
CELLS
CEREVISIAE
Syuichi Doi, Yasuyuki Suzuki and Masao Yoshimura Departments of Legal Medicine and Biochemistry, Kinki University School of Medicine Sayama, Osaka 589, Japan Received October 15, 1979 Summary Mating hormone, d-factor, causes characteristic changes in type cells, was also responsible agglutinability of a - mating type Mating
reaction
is achieved
through
events:
sexual
opposite
and nuclear
living
tester
fusion
of opposite
(constitutive
was observed.
When the
cells,
however,
induced
sexual Materials
sexual
(inducible
that
mating
which
cells
agglutination
sexual
agglutinability
cells the
of opposite
O(-factor
in the
sequential
cells
recognize to form
to form diploid
(1).
heat-killed , sexual
or
agglutination
When cells cells,
was observed Since
culture
tester a lag
phenomenon
grown at
type,
aggluti-
living after
this
of cells mating
grown at
no sexual
were mixed with
(2).
cerevisiae
(plasmogamy)
type
tester
agglutinability)
by living
was tested
of three
agglutinability).
nation
that
fusion
(karyogamy)
heat-killed
suggests
during
cell
36'C were mixed with
period
Saccharomyces
grown at 25'C were mixed with cells
was observed
cells,
of
progression
agglutination
type
When cells
strains
an orderly
cell
mating
zygote,
in haploid
which inhibits DNA synthesis and cell morphology in a mating for induction of sexual cell cells.
36°C is
the possibility
filtrate
may
induce
agglutinability. and Methods
Yeast strains, media and culture conditions: strains of Saccharomyces cerevisiae, K27-3B
Prototrophic haploid (a) and T22 (X2180-lB, 0006-291X/79/230849-05$01.00/0
849
Copyrighr All rights
@I 1979 by Academic Press, Inc. of reproduction in any form reserved.
Vol. 91, No. 3, 1979
BIOCHEMICAL
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
YPD (my, 5% glucose, 1% peptone, 0.5% rX) were used. Modified yeast extract, 0.5% KH2P04 and 0.2% MgS04.7H20), MV (3) and PG (1% glucose and 0.1% peptone) were used for yeast culture, preparation of culture filtrate and induction of agglutinability, respectively. Yeast culture was carried out at 25'C or 36'C on a reciprocal shaker. Induction of sexual agglutinability: Cells from late logarithmic phase culture of K27-3B grown in mY medium at 36'C were harvested, washed and suspended in water. Absorbance at 530 nm (A530) of the suspension was adjusted to 4. For induction of agglutinability, a half ml of the cell suspension was mixed with 9 ml of phosphate buffer (10 mM, pH 5.5) containing purified M-factor and 0.5 ml of 10 times concentrated PG and then the mixture was incubated at 36°C. 100 ml of 2 day-old culture of T22 were Purification of M-factor: inoculated into 20 liter of MV medium in jar fermenter (Ishiyama Tokyo) and cultured for 48 hours at 25'C. After Kagaku Kikai, removal of cells by centrifugation, cell-free culture filtrate was obtained by filtering culture fluid through glass fiber filter (GF/C, Whatman, England). Purification of M-factor from the filtrate was carried out essentially according to Duntze et -- a1.(4). W-Factor activity was determined in mY medium at 25'C 3 hours after incubation by testing two-fold dilution series. The most diluted test culture which showed characteristic change in cell morphology called "&moo" was determined as containing one unit of M-factor. Although direct comparison is impossible since different of w-factor had activity assay system was used, our preparation similar to that reported previously (4). Enzyme treatment: Purified u-factor (1 pg/ml) was treated with pepsin proteolytic enzymes, Pronase E (Kaken Kagaku, Tokyo), (Worthington,Biochem. Co., Freehold, N.J.), trypsin (P-L Biochem., Milwaukee) and 0(-chymotrypsin (ICN Pharmaceuticals, Cleveland) in for 60 min. at 36'C. The enzymes except pepsin were dissolved buffer (30 mM, pH 7.0) was 1 mM calcium acetate before use. Tris-HCl used for pronase, trypsin and ti-chymotrypsin, and sodium acetate buffer (10 mM, pH 3.5) for pepsin. Activity of %-factor to cause a-shmoos or to induce agglutinability was determined after inactivating enzyme by boiling for 5 min. at 1OO'C. Synthetic courtesy Osaka.
Synthetic H-factor (5) was provided u-factor: of Dr. Hiroshi Kita, Central Res. Inst., Suntory
Agglutination assay: described previously of heat-killed cells
by the Ltd.,
Agglutination assay was carried out as (2) except the use of living cells instead as a tester.
Results Induction
of sexual
agglutinability
As shown in Table sexual
agglutinability
buffer
enriched
with
by purified
1, purified
w-factor
of -a mating
type
both
glucose
850
cells
and peptone
o( -factor could
intensely
in the (PG).
induce
phosphate It
could
not
Vol. 91, No. 3, 1979
BIOCHEMICAL
Table 1. Induction
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
of sexual agglutinability
Medium')
by H-factor
Agglutination
index
PB
1.03
PE + glucose
1.04
PB + peptone
1.01
PB + glucose + peptone 2)
1.03
PB + glucose + peptone
1.34
PB + glucose + peptone 3)
1.38
Purified a-factor (1 pg/ml) was added to each medium. 1) Concentration of glucose and peptone was 1% and O.l%, respectively. 2) No H-factor was added. 3) Synthetic N-factor instead of purified one was added at the concentration of 2 pg/ml, although this value was not accurate because of small amount of it.
induce
agglutinability
same buffer
in the phosphate
supplemented
with
Agglutinability-inducing inactivated
with
no effect
activity pronase
a-shmoo-forming
activity.
on both
agglutinability d-factor of synthetic
glucose
Trypsin
which
are also
and O(-chymotrypsin
by a-factor.
also
induce
material
could
suggest
Furthermore,
agglutinability. not
allow
or in the
M-factor
These results
induced
alone,
or peptone.
of purified
and pepsin
activities.
is could
either
buffer
was
known to destroy had virtually that
sexual
synthetic
Unfortunately,
us to perform
shortage
further
experiments. Intensity
of agglutinability
as a function
of concentration
of
N-factor As shown in Fig. increased
with
concentration even at
1, the
increasing of
1 )Ig/ml.
the highest
intensity
of
amount of added In
this
concentration
851
sexual
agglutinability
H-factor
up to the
system,
-a-shmoo were not
tested,
i.e.
10 pg/ml.
formed
Vol. 91, No. 3, 1979
BIOCHEMICAL
AND BIOPHYSICAL
RESEARCH COMMUNICATIONS
x1.4w cl
0
21.3-
l
a
o
0
5 ;1.2u z
CONC.OF
30
O
TIME
6(
-
d 2 1.0
-,A
0
(min)
3
4
2
4
P"n
*
Fig.
1. Relationship between and concentration of incubated at 36*C in of purified O!-factor. washed and subjected
Fig.
2. Time course of the induction of sexual agglutinability. Cells were incubated at 36oC in PG medium which contained 1 pg/ml of purified M-factor. At time intervals, cells were withdrawn, washed and subjected to agglutination assay.
Fig.
3. pH dependency of induction of the agglutinability. Cells were incubated at 36“C in various buffer solution (10 mM) containing 1 )Ig/ml of purified ff -factor, 1% of glucose and 0.1% of peptone. After an hour, cells were harvested, washed and assayed for agglutination. 0 * citrate buffer, . phosphate buffer, a ; Trjs-HCl buffer and ; carbonate buffer.
Time course
of the
observed
15 min.
reached
maximal
at pH 3 to
pH dependency d-factor
after
the
increased
degree
similar
with
time,
and
of agglutinability of
7, and decreased is
began to be
45 minutes.
the induction
3 shows that
Fig.
This
of
agglutinablity
incubation,
the maximum level
pH dependency
of agglutinability
2, sexual
after
lo
intensity of sexual agglutinability purified M-factor. Cells were PG medium containing various anunount After an hour, cells were harvested, to agglutination assay.
induction
As shown in Fig.
of
02
(pg/mL)
g-FACTOR
El.1
sexual with
to that
agglutinability
ncreasing
was
in pH value.
of a-shmoo-forming
activity
(4).
Discussion It cerevisiae in cell
was showed that inhibits morphology
mating
hormone,
DNA synthesis of opposite
a-factor
from Saccharomyces
and causes characteristic
mating
type
cells
(4,6).
change
BIOCHEMICAL
Vol. 97, No. 3, 1979
The present mating
hormone,
study i.e.
The possibility,
since
the
M-factor
completely
the
another
induction
of
can not substance
preparation
RESEARCH COMMUNICATIONS
characteristic sexual
this
agglutinability.
be excluded is not
of
that
the
identical
used in present
sexual
with
study
d-factor,
may not be
pure.
Further different
suggests
however,
agglutinability-inducing
AND BlOPHYSlCAL
studies
are necessary
phenomena caused by
shmoo formation
and induction
to clarify
W-factor, of sexual
mechanism underlying
inhibition
of
DNA synthesis,
agglutinability.
Acknowledgments The authors are grateful to Dr. Hiroshi Kita and Dr. Isamu Takano, Cental Res. Inst., Suntory Ltd., Osaka for synthetic (X-factor and to Dr. Moritoshi Shibata, Nara Medical University for his continual encouragements. Thanks are also due to for helpful discussions Dr. Hyogo Sinohara of our university during this work and for reading manuscript. References 1. Yanagishima, N. (1973) Curr. Adv. Plant Sci. z, 55-66. 2. Doi, S., and Yoshimura, M. (1978) Molec. gen. Genet. 162, 251-257. 3. MacKey, V., and Manney, T.R. (1974) Genetics 76, 255-271. 4. Duntze, W., Ste)tzler, D., Biicking-Throm, E., glbitzer, S. (1973) Eur. J. Biochem. 35, 357-365. 5. Masui, Y., Chino, N., Sakakibara, S., Tanaka, T.m Murakami, T., and Kita, H. (1977) Biochem. Biophys. Res. Commun. 78, 534-538. 6. Bucking-Throm, E., Duntze, W., Hartwell, L.H., and Penney, T.R. (1973) Exptl. Cell Res. 76, 99-110.
8.53
