yeast mating

[biosci-request at net.bio.net]

	> I have data on yeast mating that I am making available to anyone
that would
	> be able to use the information.  I have found two proteins that
inhibit
	> mating by S. cerevisiae but am unable to follow up as I am
retiring and
	> moving away from my lab.  The data are attached in the form of a
manuscript.
	> I am not looking for a collaboration or co-authorship if someone
follows up
	> on this work.  My interest is getting the information out to those
who are
	> working on yeast mating as I am unaware of any other data like
these.  As I
	> will not be at my lab it will be difficult for me to get cultures
to anyone
	> needing them but my collaborator on a cell cycle project is the
source of
	> the strains and would be willing to provide them to investigators
requesting
	> them.  Contact Michael Stark, Department of Biochemistry, Dundee
University,
	> Dundee, Scotland (M.J.R.STARK at dundee.ac.uk
<mailto:M.J.R.STARK at dundee.ac.uk> )
	> <mailto:M.J.R.STARK at dundee.ac.uk)
<mailto:M.J.R.STARK at dundee.ac.uk)> > .  If you have any questions contact me
	> and I will be glad to answer them.  

TITLE:  Inhibition of the yeast mating pathway by Kluyveromyces lactis  and
Pichia acaciae toxins

ABSTRACT

The mating pathway of Saccharomyces cerevisiae is inhibited by the toxins
produced by Kluyveromyces lactis and Pichia acaciae.  This effect is
independent of the cell cycle arrest characteristic of these toxins.
Addition of K. lactis toxin during mating of cells that can divide in its
presence causes an immediate arrest in all stages of the mating pathway.
Similar treatment with P. acaciae toxin results in mating cells arresting as
prezygotes.
INTRODUCTION

Proteinaceous toxins produced by the yeasts Kluyveromyces lactis and Pichia
acaciae share many similarities with both being characterized by causing
sensitive yeast strains to arrest in G1 of the cell cycle (Stark et al.,
1990; McCracken et al., 1994).  Saccharomyces cerevisiae cells treated with
K. lactis toxin were also shown to be incapable of mating (Butler et al.,
1991).  This defect was attributed to toxin-induced cell cycle arrest at a
point in G1 before cells were competent to respond to pheromone.  As part of
my investigation of the cellular responses to the P. acaciae toxin, I was
interested to determine if P. acaciae toxin also had an effect on S.
cerevisiae mating.  

In their investigation using K. lactis toxin, Butler et al. (1991) mated a
sensitive strain with a dominant toxin-insensitive strain in the presence of
toxin.  Using the same approach, I confirmed that K. lactis toxin completely
inhibited mating, as did P. acaciae toxin.  This experimental design,
however, does not distinguish between failure to mate because of cell cycle
arrest and failure to mate because of a mating pathway block.  In subsequent
experiments I treated only the dominant toxin-insensitive strains with
toxin, then washed the cells to remove unbound toxin.  These cells were
subsequently mated with toxin-sensitive cells.  Under these conditions K.
lactis toxin still prevented mating completely while P. acaciae toxin
reduced mating to 50% of control.  Since I could detect no carry-over of
toxin to the mating mix, these results indicated that the toxins were having
a direct effect on the mating pathway.

This report documents the specific effect of these toxins on the mating
pathway separate from any effect in the cell cycle.  I will also present
evidence that the toxins differ in where in the mating pathway the arrest
occurs.

MATERIALS AND METHODS

General methods
Strains used in this work are described in Table I.  All general yeast
methods and growth media were as described by Sherman et al. (1986). Yeast
strains were maintained on YPD agar plates, and liquid cultures were grown
in YPD medium unless otherwise stated.  Diploid cultures were grown on SD
(0.67 g yeast nitrogen base, 2 g dextrose per 100 ml) plus specific required
nutrients.  Toxin was produced by growing either K. lactis or P. acaciae in
100 ml YPD for two days at 30*C.  The cell-free culture medium was
subsequently concentrated 30-fold by Amicon stirred cell ultrafiltration
(YM30 membrane).  

Mating
Cells to be mated were grown overnight in YPD.  About 1x106 cells of each
mating type were transferred to 1 ml YPD in an Eppendorf tube.  After
mixing, 500ml of the mating mix were transferred to a fresh tube and 50ml of
toxin added.  Control tubes received 50ml H2O.  Mating was allowed to
proceed for 6 h before 100ml aliquots were plated on SDhis medium.  Diploid
colonies were counted after 48 h at 30*C.  

To study the effect of toxin on different stages of the mating pathway,
toxin was added to separate mating mixes at 0, 1, 2, 3, 4, and 5 h after
mating commenced.  Aliquots were plated 6 h after mating was initiated as
described above.  

Mating can be blocked just prior to nuclear fusion.  When this occurs the
fused cell produces a bud containing one of the nuclei and a portion of the
common cytoplasm (a cytoductant).  This process provides a means of
determining if toxin-sensitive mating is blocked at the nuclear fusion
stage.  To investigate this possibility a rho° nystatin-resistant kti10a
strain was prepared. After the resulting strain was crossed with kti10a,
cells were plated on SDhis (diploid selection) and YPglycerol containing 2
mg nystatin/ml (cytoductant selection medium). 

Microscopy
Mated cells were centrifuged, fixed in Carnoy fixative for at least 45 min
before being washed 4 times with 0.85% saline, and then stained with 4´,
6-diamidino-2-phenylindole (DAPI) (Kurihara et al., 1994).  Cells were
observed with a Leitz microscope using both fluorescence and differential
interference contrast (DIC) optics.

Toxin-insensitive mating mutants
Diploid colonies produced as the result of mating in the presence of toxin
were collected, sporulated and vortexed with diethyl ether at 4*C for 1 min
to kill vegetative cells (Dawes and Hardie, 1974). The surviving spores were
plated to produce haploid colonies derived from individual spores.  These
colonies were sorted as to mating type and nutrient requirement before being
mated to determine the genetic basis for their toxin insensitivity in
mating.
RESULTS

Toxin inhibition of mating
In their characterization of K. lactis toxin, Butler and co-workers observed
that toxin-arrested cells could not mate with dominant toxin-insensitive
cells (Butler et al., 1991).  They interpreted this to indicate that the
toxin-induced arrest in the cell cycle occurred before cells became
competent to mate.  I made a similar observation using P. acaciae toxin and
the dominant strain of GS1731 whose cell cycle is insensitive to this toxin.


In order to investigate the possibility that the toxins were actually
blocking the mating pathway independently of cell cycle effects, I altered
the experimental protocol.  Instead of exposing sensitive cells to toxin
before mating, I exposed only the dominant toxin-insensitive strains to
their respective toxin.  After three hours exposure to toxin the cells were
washed to remove unbound toxin and then mated with the appropriate sensitive
strain.  I confirmed that there was no toxin carry-over or release from the
treated cells by growing the toxin-treated, washed toxin-insensitive cells
in the same medium with toxin sensitive cells of the same mating type.  The
growth curves for cultures containing toxin-treated cells were identical to
the control curves.

Using this protocol K. lactis toxin still reduced mating success to less
than 1% of that in the control cross. P. acaciae toxin, however, only
reduced mating success to about 50% of control.  

Both of these experiments indicate that there is a toxin effect in the
mating pathway that is separate from any effect in the cell cycle because
the sensitive cell is only exposed to toxin once it fuses with a
toxin-treated cell.  Furthermore, the toxin effect is restricted to the
mating pathway because the diploid that would result from nuclear fusion
would be toxin insensitive as it has the dominant toxin-insensitivity
allele.  

I.	K. lactis Toxin

	Variability in sensitivity to K. lactis toxin
	There are thirteen different recessive kti alleles that prevent cell
cycle arrest (Butler et al., 1994).  Sensitivity to toxin in the mating
pathway was determined for all of these except for kti2 (chitin deficient
mutant).  Mating is reduced to *5% of control for two of the mutants, but,
for the remainder, mating in the presence of toxin is *25% of control with
most mating at greater than 50% of control levels (table II).  Unless
indicated otherwise the experiments described below were done using either
kti6 or kti10.   

	Time course of sensitivity to K. lactis toxin during mating.
Standard protocols for studying S. cerevisiae mating call for the mated
cells to be plated after 6 h.  Therefore I mated kti10a and a cells for six
hours, but added toxin at hourly intervals during the mating.  From table
III it is obvious that all phases of the mating pathway are sensitive to the
effects of toxin.  The addition of toxin at time 0 not only prevents the
formation of diploid cells, but it also prevents the appearance of any of
the pre-zygote fused cells.  At 5 h fused cells are plentiful, but the
addition of toxin prevents most of them from becoming zygotes and giving
rise to diploid colonies.  

	Since these experiments were performed using strains that were very
sensitive to K. lactis toxin, I repeated the assay of K. lactis toxin
sensitivity after 5 h of mating using the various kti strains that showed
less sensitivity to this toxin.  As indicated in table II all strains except
for kti6 and kti10 are insensitive to toxin addition after 5 h of mating.  

	Sensitive phase of the yeast mating pathway
	There are specific landmarks in the yeast mating pathway that can be
used to localize the effects of K. lactis toxin on mating.  Once cells are
exposed to the appropriate mating pheromone they will respond by producing
projections that have specific agglutinins in the cell wall.  Cells of
opposite mating type that encounter each other in this condition will stick
together and then fuse the cell walls of the projections.  After the cell
walls have fused, the septum between the two parts of the conjugation tube
is dissolved leaving the two haploid nuclei in a common cytoplasm.  Nuclear
fusion follows with subsequent production of diploid buds by the zygote.  

	Addition of toxin at time 0 of mating essentially prevents the
formation of fused cells, indicating that K. lactis toxin blocks the
earliest phases of the mating pathway ("shmooing" and cell adhesion).  K.
lactis toxin blocks the final phases of the mating pathway as well, since
addition of toxin after 5 h of mating prevents fused kti6 or kti10 cells
from becoming diploid zygotes.  If cells arrest in the dikaryotic condition,
then fused cells would be expected to give rise to haploid buds.  This is
not observed as control and toxin matings produce the same very small number
of cytoductant colonies.  

	Progress through the events occurring in fused cells can be followed
microscopically using DIC optics to visualize the partition cell wall and
DAPI staining to visualize the nuclei. When kti10 cells are mated for 5 h
before the addition of toxin all of the phases, from fused cells with a
partition wall to pre-bud zygotes containing a single nucleus in the
conjugation tube, are seen.  Furthermore, the distribution of cells between
the various phases is the same in control and treated samples with the
exception that budding zygotes are quite rare in treated samples.  

	K. lactis toxin-insensitive mating mutants
	Even though mating of both kti6 and kti10 is largely arrested by K.
lactis toxin treatment, some diploid colonies form.  The only way that these
colonies can form is for one or both of the parent cells to carry a mutation
that allows mating to proceed in the presence of toxin.  Haploids derived
from kti6 diploids were mated, in the presence of toxin, with all of the kti
strains to eliminate the possibility that the mating insensitivity was due
to a known kti gene.  Seven of the ascospore-derived haploids mated with all
of the kti strains, suggesting that these cells contained a dominant
toxin-insensitivity gene.  This was confirmed by mating these haploids with
LL20, the toxin sensitive parental strain.  For this I used the mating
protocol in which the cells that are insensitive to the effects of toxin on
the cell cycle are treated with toxin before mating is initiated.  LL20 is
not exposed to toxin and there is no free toxin in the tube where mating
occurs.  The mating efficiency for cells that had been pre-treated with
toxin ranged from 5-40%.  Using this same protocol with a dominant cell
cycle toxin-insensitive strain both Butler et al. (1991) and McCracken et
al. (1994) had observed essentially zero mating efficiency.  This confirmed
that these cells possess a dominant mutation that allows mating to occur in
the presence of toxin.  The other ascospore-derived haploid strains must
carry a recessive allele and so there must be at least two different
complementation groups involved in the sensitivity of mating to toxin. 

	The strains that carried the dominant allele allowing mating in the
presence of toxin were tested to see if this allele also conferred
resistance to toxin in the cell cycle.  This could not be done using the
haploid cells themselves, as they were already insensitive to toxin in the
cell cycle because they were derived from kti6 parents.  Therefore I tested
diploid colonies derived from crosses between these haploid dominant strains
and LL20 which is a sensitive strain.  None of these diploids grew in the
presence of K. lactis toxin

II.	P. acaciae toxin

Sensitivity of kti strains to P. acaciae toxin

Of the kti strains only kti1 and kti10 are insensitive to the effects of P.
acaciae toxin in the cell cycle (McCracken et al., 1994) and so are the only
strains that can be studied.  Of these, kti1 is insensitive to K. lactis
toxin during mating whereas P. acaciae toxin almost completely blocks mating
by kti1 (1.6% of control).  kti10 mating is very sensitive to K. lactis
toxin but only partially inhibited by P. acaciae toxin (35% of control). 

Sensitive phase of the mating pathway

P. acaciae toxin added after five hours of mating has no effect on the
mating efficiency of both  kti1 and 10, in contrast to the results with
toxin present at the beginning of mating (table IV).  Mating by kti10 is
sensitive to both K. lactis and P. acaciae toxins but mating efficiency in
P. acaciae toxin is considerably higher than in K. lactis toxin.  This could
result from either a lower effective concentration of the P. acaciae toxin
or a different mode of action of the P. acaciae toxin.  In order to
eliminate one of these possibilities kti10 cells were mated in K. lactis
toxin diluted to the same effective concentration as P. acaciae toxin based
on the ID50 for cell cycle arrest.  At this concentration both toxins reduce
mating efficiency to between 20 and 30% of control levels when added at time
0 of mating.  The addition of the toxins at 5 h of mating produces very
different results.  Mating is unaffected by P. acaciae toxin at this time
but is still inhibited by K. lactis toxin (table V).  

Although the production of diploid colonies by kti1 exposed to toxin
throughout mating is very low, the number of fused cells is high (42% of the
untreated control).  This is not observed for strains whose mating is
strongly inhibited by K. lactis toxin.  Examination of these fused cells
with DIC optics and DAPI staining reveals that the majority of these fused
cells arrest with an intact septum dividing the gametes.  Even in those
cases where there is an elongated bridge between the cells the septum
persists.  There are also a considerable number of fused cells that appear
to be the result of tripartite conjugation.

DISCUSSION

Although they were first described as cell cycle inhibitors, it is clear
from the above results that the toxins produced by both K. lactis and P.
acaciae also independently block the mating pathway in S. cerevisiae.  All
of the strains tested were insensitive to the effects of one or both of
these toxins on the cell cycle but most showed at least some degree of
sensitivity to these toxins in mating (table).  With the exception of kti6
and 10, all of the kti strains whose mating is inhibited by K. lactis toxin
are only affected if the toxin is present at the beginning of mating (table
II).  This suggests that all of the kti mutations, except for kti6 and
kti10, protect cells that are mating even though additional cells may be
prevented from entering the mating pathway.  

Toxin treatment of kti6 and kti10 cells at the start of mating blocks the
appearance of both fused cells and of diploid colonies (table III).  In fact
there is no time during the six hour mating period that the toxin does not
inhibit mating.  Even after five hours of mating the addition of toxin
prevents the formation of new fused cells and significantly reduces the
number of diploid colonies.  After five hours a culture contains about
two-thirds as many fused cells as it will at six hours, but microscopic
examination of these fused cells indicates that they arrest in the mating
pathway in whatever stage they are at when exposed to the toxin.  

It is clear that K. lactis toxin inhibits all of the stages of the mating
pathway with no accumulation of cells at a particular stage of the pathway.
This is in marked contrast to the effect of this toxin on the cell cycle
where all of the cells are arrested at the end of G1(Butler et al., 1991).
The absence of one specific stage of the mating pathway that is inhibited by
the toxin suggests that the molecular target of the K. lactis toxin is
directly involved in progression through all stages of the mating pathway.
Furthermore, the cells arrested in the mating pathway are apparently truly
arrested by the toxin, just as cell cycle arrested cells are, as
cytoductants do not form in response to toxin treatment.  

The two kti strains that are resistant to the effects of P. acaciae toxin on
the cell cycle were tested for mating sensitivity to P. acaciae toxin.
Mating by both strains is reduced by the presence of the toxin, but kti1
cells were much more sensitive to the effects of the toxin. 

Despite being very sensitive to the effects of P. acaciae toxin on formation
of diploid colonies, kti1 cells were able to initiate the mating process and
produce almost 50% as many fused cells as controls.  It is apparent from DIC
microscopy and DAPI staining that cell fusion arrests with the nuclei far
apart and before the septum between gametes disappears.  It would appear,
therefore, that P. acaciae toxin, unlike K. lactis toxin, regulates a
specific step in the mating pathway.  The apparent ineffectiveness of toxin
added after 5 h of mating by both kti1 and kti10 cells is due to the
formation of diploid colonies from cells that have passed the sensitive
stage.  There is no reason to expect new matings after the addition of toxin
to occur at a higher frequency than that observed when toxin is added at
time 0 of mating.  

These data and those from previous reports suggest that the control of
mating and the cell cycle by both of these toxins is by means of either a
branched signal transduction pathway or two separate pathways that share
components.  In either model, kti1, 3, 4, 7, 8, 9, 11, 12, 13, 14 block
signal transduction for both cell cycle and mating arrest caused by K.
lactis toxin.  kti6 and kti10 only block cell cycle arrest.  

Both kti1 and kti10 block signal transduction for P. acaciae toxin-induced
cell cycle arrest.  kti10, but not kti1, is involved in control of the
mating pathway by P. acaciae toxin.  Since only kti1 and kti10 protect cells
against the cell cycle arrest induced by both toxins, it is not possible to
determine if other kti gene products are involved in the signal transduction
pathway utilized in P. acaciae toxin-induced mating arrest. 

While it is not possible to identify the signal transduction pathways
involved in the response of cells to these toxins, it is possible to
consider some likely candidates.  Both of these toxins cause an arrest in
START at the end of G1 of the cell cycle (Butler et al., 1994; McCracken et
al., 1994).  In S. cerevisiae, START is the major checkpoint in the cell
cycle with the activity of the master cell cycle regulatory protein kinase
(p34cdc28 ) being a target of regulation by signal transduction pathways
such as mating factor and nutrient sensor (Reed, 1992).  Furthermore, there
appears to be an undetermined role for the p34cdc28 kinase in the mating
pathway (Rose, 1996).  Thus this kinase seemed to be a likely target of
toxin action in both the cell cycle and mating.  I have shown that the K.
lactis and P. acaciae toxins induced in vivo, but not in vitro, inhibition
of this protein kinase in S. cerevisiae sensitive cells (McCracken,
unpublished results) supporting the hypothesis that the toxins act through a
signal transduction pathway to inhibit this kinase.  

This toxin-initiated signal transduction pathway cannot involve nutrient
deficiency or pheromones, however.  The presence of a potential mate or low
levels of an essential nutrient will cause a G1 arrest mediated through the
kinase complex.  If toxin were to activate the pheromone signal pathway,
toxin-insensitive strains would then be expected to have defective
components of this signal transduction pathway and therefore to have low
mating efficiencies.  Most of the kti strains mated very efficiently with
only kti7, 9, 11, and 14 having low mating efficiency compared to the
parental, toxin-sensitive strains.  Furthermore, Far1p is the component of
the mating factor signal pathway that inhibits the p34cdc28 protein
kinase-cyclin complex (Peter and Herskowitz, 1994) yet a far1D strain was as
sensitive to both of the toxins as the FAR1 strain (McCracken, unpublished
results) indicating that Far1p was not required for inhibition by toxin.
Similarly, if the toxins activate the nutrient sensor signal transduction
pathway, toxin insensitivity would be expected to result in nutrient
deficiency insensitivity.  Microscopic examination of cultures of the kti
strains showed that, with the exception of kti14, all of them arrested in G1
in response to nutrient deficiency (McCracken, unpublished results).  kti14
cells (McCracken, unpublished results) exhibited the classic signs of a
non-functional nutrient sensor pathway, i.e. multiply budded cells at
stationary phase, no glycogen accumulation, and reduced sporulation
frequency (Cannon and Tatchell, 1987; Toda et al., 1987).  Therefore the
kti14 gene product may be a component shared between the K. lactis toxin and
nutrient sensor signal transduction pathways.  

Consequently my results indicate that the toxins are not co-opting either of
these signal transduction pathways for cell cycle control and that an
unknown pathway must be involved which may share components with either or
both of these pathways as has been observed for other signal transduction
pathways in yeast.  Although the target of the toxin-induced signal
transduction pathway in dividing cells appears to be the p34cdc28 kinase,
this does not appear to be the target in mating cells.  The p34cdc28 kinase
is required for nuclear fusion in mating (Rose, 1996) but neither toxin
causes a mating arrest only prior to nuclear fusion.  

The target of the K. lactis toxin-induced signal transduction pathway must
be a cellular component that is required throughout the mating process as
this toxin causes an arrest in every stage of mating.  One possible target
is the transcription activator STE12 inhibition of which could cause cells
to arrest in all stages of mating.  STE12 is the target of the mating factor
signal transduction pathway and is responsible for induction of genes
necessary for mating: components of the mating factor signal transduction
pathway, such as pheromone receptors and subunits of the G protein; the
agglutinins necessary for cell-cell interactions; FUS1 and FUS3, which are
necessary for cell fusion; and nuclear fusion (Marsh et al., 1991;
Herskowitz, 1995).  Recently more genes have been shown to be pheromone
regulated and have an effect on different stages of the mating pathway
(Erdman et al., 1998).  Transcription of these genes could also be induced
by STE12.  The activity of STE12 is positively regulated by association with
other proteins (Marsh et al., 1991), which suggests that K. lactis
toxin-induced regulation of STE12 could be the result of either interference
with activation of STE12 or binding of an inhibitory protein.  

Mating arrest by P. acaciae toxin is stage specific and has the
characteristics of a FUS mutation, i.e. arrest as a pre-zygote with an
intact septum dividing the two fused cells (McCaffrey et al., 1987;
Trueheart et al., 1987; Elion et al., 1990).  Furthermore, tripartite
conjugation is observed with both FUS mutation (Trueheart et al., 1987) and
P. acaciae toxin arrest of mating.  This suggests that the target of P.
acaciae toxin is one of the FUS genes or its protein product.  

One of the fascinating questions raised by studies of these two toxins is
how they can share so many biochemical features, cause the same effects, and
yet utilize different cellular pathways to accomplish these effects.  The
next goal will be to identify the genes represented by the different kti
strains in order to elucidate the toxin-initiated signal transduction
pathways.
REFERENCES

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of Saccharomyces cerevisiae cells to Kluyveromyces lactis toxin. J. Gen.
Microbiol. 137:1749-1757.
Butler, A.R., J.H. White, Y. Folawiyo, A. Edlin, D. Gardiner, and M.J.R.
Stark. 1994. Two Saccharomyces cerevisiae genes which control sensitivity of
G-1 arrest induced by Kluyveromyces lactis toxin. Mol. Cell. Biol.
14:6306-6316.
Cannon, J. and K. Tatchell. 1987. Characterization of Saccharomyces
cerevisiae genes encoding subunits of cyclic AMP-dependent protein kinase.
Mol. Cell Biol. 7:2653-2663.
Dawes, I.W. and I.D. Hardie. 1974. Selective killing of vegetative cells in
sporulated yeast cultures by exposure to diethyl ether. Mol. Gen. Genet.
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Elion, E.A., P.L. Grisafi, and G.R. Fink. 1990. FUS3 encodes a
cdc2+/CDC28-related kinase required for the transition from mitosis into
conjugation.  Cell 60:649-664.
Erdman, S., L. Lin, M. Malczynski, and M. Snyder. 1998. Pheromone-regulated
genes required for yeast mating differentiation.  J. Cell Biol. 140:461-483.
Herskowitz, I. 1995. MAP kinase pathways in yeast: for mating and more.
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Kurihara, L.J., C.T. Beh, M. Latterich, R. Schekman, and M.D. Rose. 1994.
Nuclear congression and membrane fusion: two distinct events in the yeast
karyogamy pathway. J. Cell Biol. 126:911-923.
McCaffrey, G., F.J. Clay, K. Kelsay, and G.F. Sprague. 1987. Identification
and regulation of a gene required for cell fusion during mating of the yeast
Saccharomyces cerevisiae.  Mol. Cell. Biol. 7:2680-2690.
McCracken, D.A., V.J. Martin, M.J.R. Stark, and P.L. Bolen. 1994. The
linear-plasmid-encoded toxin produced by the yeast Pichia acaciae:
characterization and comparison with the toxin of Kluyveromyces lactis.
Microbiol. 140:425-431.
Peter, M. and I. Herskowitz. 1994. Direct inhibition of the yeast
cyclin-dependent kinase Cdc28-Cln by Far1.  Science 265:1228-1231.
Reed, S.I. 1992. The role of p34 kinases in the G1 to S-phase transition.
Ann. Rev. Cell Biol. 8:529-561.
Rose, M.D. 1996. Nuclear fusion in the yeast Saccharomyces cerevisiae.  Ann.
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Stark, M.J.R., A. Boyd, A.J. Mileham, and M.A. Romanos. 1990. The
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Table I.  Yeast strains used in this study

Species/strain	Genotype	Source	
Kluyveromyces lactis IFO1267		NCYC 1368*	
Pichia acaciae		NRRL Y-18665*	
Saccharomyces cerevisiae LL20	MATa leu2-3 leu2-112 his3-11 his3-15	NCYC
1445*	
S. cerevisiae KY117	MATa ura3-52 trp1-D1 lys2-801am ade2-101 his3-D200
M.A. Romanos	
S. cerevisiae K. lactis toxin insensitive strains	kti1-14 derived from
both LL20 and KY117	M.J.R. Stark	
S. cerevisiae GS1731 dominant P. acaciae toxin insensitive	MATa ade1
his	This study	

*NCYC: National Collection of Yeast Cultures, AFRC Institute of Food
Research, Colney Lane, Norwich, UK
NRRL: National Center for Agricultural Utilization Research, Peoria IL
61604, USA

Table II.  Effects of K. lactis toxin on mating efficiency

kti	Mating efficiency when toxin added at time 0 (% of control)
Mating efficiency when toxin added at 5 h (% of control)	
1	100	N.D.	
3	  78	100	
4	  34	  92	
6	    0.5	  17	
7	  38	100	
8	  65	100	
9	  65	100	
10	    5	  11	
11	  35	  96	
12	  70	100	
13	  35	100	
14	  22	  96	


Table III.  Effects of addition of K. lactis toxin to kti10 mating mixtures
at various times

Time of toxin addition (h)	Fused cells as % of control	Mating
efficiency as % of control	
0	0	1.2	
1	0	1.4	
2	1.4	2.5	
3	10	4.5	
4	27	5.9	
5	66	9.3	


Table IV.  Effects of P. acaciae toxin on mating efficiency

Kti	Mating efficiency when toxin added at time 0 (% of control)
Mating efficiency when toxin added at 5 h (% of control)	
1	2	95	
10	25	100	

Table V.  Comparison of the effects of K. lactis and P. acaciae toxin on
kti10 mating

Time of toxin addition (h)	K. lactis	P. acaciae	
0	27	25	
5	57	100	





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