MOLECULAR AND CELLULAR BIOLOGY, Jan. 1990, p. 217-222 0270-7306/90/010217-06$02.00/0 Copyright ©D 1990, American Society for Microbiology
Vol. 10, No. 1
Overexpression of the STE4 Gene Leads to Mating Response in Haploid Saccharomyces cerevisiaett MALCOLM WHITEWAY,* LINDA HOUGAN, AND DAVID Y. THOMAS Genetic Engineering Section, National Research Council, Biotechnology Research Institute, 6100 Royalmount Avenue, Montreal, Quebec, Canada H4P 2R2 Received 10 July 1989/Accepted 6 October 1989
The STE4 gene of Saccharomyces cerevisiae encodes the I8 subunit of the yeast pheromone receptor-coupled G protein. Overexpression of the STE4 protein led to cell cycle arrest of haploid cells. This arrest was like the arrest mediated by mating pheromones in that it led to similar morphological changes in the arrested cells. The arrest occurred in haploid cells of either mating type but not in MATa/MATTa diploids, and it was suppressed by defects in genes such as STE12 that are needed for pheromone response. Overexpression of the STE4 gene product also suppressed the sterility of cells defective in the mating pheromone receptors encoded by the STE2 and STE3 genes. Cell cycle arrest mediated by STE4 overexpression was prevented in cells that either were overexpressing the SCGI gene product (the a subunit of the G protein) or lacked the STE18 gene product (the -y subunit of the G protein). This finding suggests that in yeast cells, the i subunit is the limiting component of the active ,(y element and that a proper balance in the levels of the G-protein subunits is critical to a normal mating pheromone response. The Saccharomyces cerevisiae mating pheromone response pathway has a remarkable similarity to G-proteinmediated signal transduction systems. Such systems were initially described in mammalian cells (see references 4, 8, 24, and 29 for recent reviews), where they were shown to consist of tripartite (a, 1, and -y subunits) guanine nucleotide-binding proteins (G proteins) coupled to cell surface receptor proteins. In S. cerevisiae, the STE2 and STE3 genes encode the mating pheromone receptor proteins, which have seven potential membrane-spanning regions and thus are similar to mammalian G-protein-linked receptors (3, 9, 23). The GPAI (SCGI), STE4, and STE18 genes encode, respectively, the a, 1, and -y subunits of a G protein that is functionally coupled to these receptors (6, 22, 31). A current model for G-protein function in mammalian cells is that an interaction between the agonist and the receptor leads to guanine nucleotide exchange on the a subunit and the activated a subunit dissociates from the f3y subunit. The dissociated a subunit may then interact with an intracellular effector protein, such as adenylyl cyclase or cyclic GMP phosphodiesterase, and modulate its activity (8, 24, 29). Another model for some systems is that the free 13y subunit plays a role in effector modulation. This latter idea has been controversial (16, 33), but current evidence suggests that Py acts at least in the modulation of some phospholipase A2-mediated pathways (12, 13). Subsequent hydrolysis of the GTP on the a subunit allows reconstitution of the inactive atpy heterotrimer and turns the signaling pathway off (24). In yeast cells, the genetic evidence suggests that the 13y element acts positively in the mating pheromone response pathway. Disruption of either STE4 (I) or STE18 (-y) eliminates the ability of cells to respond to pheromones (33), whereas loss of the GPAJ (SCGI) protein (a) causes a constitutive mating response and a haploid-specific cell cycle
arrest (6, 11, 22). Both ste4 and stel8 mutations suppress this cell cycle arrest (33). Although there is currently no biochemical data on the mechanism of G-protein action in yeast cells, the structural similarities in the yeast and mammalian subunits and the functional distinctions between STE4 and STE18 on one hand and GPAI (SCGI) on the other make it likely the yeast mating response pathway functions similarly to the mammalian systems. Biochemical assessments of the roles of the various Gprotein subunits in mammalian systems have treated the 1 and y proteins mainly as a single unit because they remain tightly associated throughout purification (24). In yeast cells, it was impossible to clone the STE4 gene from 2,um-based libraries (17), whereas the STE18 gene was readily obtained from such libraries (L. Hougan, unpublished data). This failure to isolate high-copy-number plasmids containing the STE4 gene suggests that high levels of the STE4 gene product may be detrimental to the cell, whereas high levels of the STE18 product are tolerated. Here, we investigate the phenotypic consequences of regulated overexpression of the STE4 gene by placing the STE4 coding sequence under the control of the GAL] promoter. Use of the GAL] promoter allows for facile manipulation of STE4 expression, since the promoter is efficiently induced when cells are grown on galactose but tightly repressed when cells are grown on glucose. Thus, cells can be grown in the absence of a potentially detrimental gene product and then induced to allow analysis of the results of the expression of this gene product.
MATERIALS AND METHODS Strains. See Table 1 for a description of the yeast strains used. Plasmids. Plasmids pC3, which is a YEp13 plasmid containing the SCGI gene (6), and pJK6, which is a centromere plasmid containing the MFaJ gene under GAL] control (32; J. Kurjan, personal communication), were gifts of Janet Kurjan. Plasmid pSUL16 is YIp5 containing a LEU2 disruption of STE12 (7) and was obtained from Stan Fields. Plasmids pSL376, which is pBR322 with a LEU2 disruption
* Corresponding author. t National Research Council Canada publication no. 30671. t This article is dedicated to the memory of Allen P. James, yeast geneticist.
217
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MOL. CELL. BIOL.
plated to minimal plates to detect the formation of prototrophic diploids. Plasmid stability. Plasmid stability was measured by growing strains overnight in YEPD or YEPGAL medium and then spreading suitable dilutions on YEPD plates. After 2 days of growth, the colonies were replica plated to drop-out medium to detect colonies that retained the selectable marker characteristic of the plasmid under study. This procedure detects the loss of plasmids producing toxic proteins, since cells containing these plasmids will be at a growth disadvantage. RNA analysis. Total RNA was isolated from galactoseinduced and uninduced strains as described previously (19). Induced strains were grown in the presence of galactose as the sole carbon source for 6 h; uninduced strains were grown for an identical time in glucose-containing medium. The RNA was denatured by boiling and run on a 1% agarose gel in TBE buffer, followed by direct hybridization with an end-labeled oligonucleotide probe to the dried gel (19). Oligonucleotide-directed mutagenesis. Single-stranded DNA was isolated from strain CJ236 (15) transformed with pTZ18R (20)-derived plasmids after infection with the helper bacteriophage M13KO7 as described elsewhere (30). Introduction of the 5' EcoRI and BamHI sites was achieved by annealing the oligonucleotide 5'-GGTACACATTACGiA
TABLE 1. Yeast strains Strain
Markers
Source
Sc225 Sc252 L1-3B L1-14D DL41
MATa adel leu2-3,2-112 ura3-52 MATa adel leu2-3,2-112 ura3-52 MATa ura3 leu2-3,2-112, his3-11,3-15 MATa ura3 leu2-3,2-112, his3-11,3-15 + MMa adel ura3-52 leu2-3.2-112 MATa + ura3 leu2-3,2-112 his3-11,3-15
J. Hopper J. Hopper This work This work This work
of STE3 (9), and pAB506, which is pBR322 containing a LEU2 disruption of STE2 (14), were obtained from Karen Clark and Cathy Jackson, respectively. Plasmid YCp5O is a centromere-containing plasmid carrying the URA3 selectable marker (25). Plasmid M65pl is pTZ18R containing a LEU2 disruption of STE18 (31). Plasmid pL19 is a centromere plasmid with STE4 under the control of the GAL] promoter and was constructed as follows. Plasmid M88p5, which consists of a 4.0-kilobase EcoRI fragment containing the STE4 gene inserted into the EcoRI site of plasmid pTZ18R (20), was modified by oligonucleotide-directed mutagenesis. This placed an EcoRI and a BamHI site at the 5' end of the gene and a SalI site at the 3' end (Fig. 1). The BamHI-to-Sall fragment was excised from this modified plasmid (M88L18) and cloned into the BamHI-to-SalI region of plasmid pJK6 (Kurjan, personal communication). This replaces the MFal sequences of pJK6 with STE4 and generates pLl9, a centromere plasmid carrying the STE4 gene linked to the GALl promoter. Plasmid pL18-31 is a YEp213 plasmid containing the STE18 gene (L. Hougan, unpublished data). Recombinant DNA manipulations were performed by using standard protocols (18). Media and genetic methods. The media used have been described previously (28, 32), and standard procedures were used for strain constructions (28). Transformation. Cells were transformed by recombinant plasmids or by DNA fragments by the lithium cation protocol (10). Disruptions of STE2, STE3, STE12, and STE18 by the LEU2 gene were constructed by one-step gene replacement (26). The STE2 disruption was created using a BamHI fragment containing a LEU2 replacement of STE2 obtained from plasmid pAB506. The STE3 disruption fragment was a HindIll fragment from plasmid pSL376, the STE12 disruption fragment was a SacI-to-SphI fragment from pSUL16, and the STE18 disruption used a PstI-to-HindIII fragment containing a LEU2 replacement of STE18 derived from M65pl. Patch matings. Matings of strains containing plasmid pLl9 were performed by growing the strains as patches on uracillacking (-Ura) glucose plates and then replica plating them to mating tester lawns on either yeast extract-peptonegalactose (YEPGAL) (for GALl promoter induction) or yeast extract-peptone-dextrose (YEPD) medium. These plates were incubated overnight at 30°C and then replica
ATTCTFGGATCCATGGCAGCACAT-3' to plasmid M88p5 and extending it with T4 DNA polymerase as described previously (15). This generated plasmid M88L17, which was then mutagenized with oligonucleotide 5'-CTTCGAATTGG AGTCGACATTACTGTGAGC-3' to introduce a 3' Sall site and create plasmid M88L18. Oligonucleotides were synthesized on an Applied Biosystems 380A DNA synthesizer and purified as described previously (27). Correctly mutated plasmids were identified by screening for the presence of the created restriction sites. RESULTS
Construction of a galactose-inducible STE4 gene. We constructed a derivative of the STE4 gene that replaced the normal promoter and 5' regulatory sequences of this gene with the promoter of the GAL] gene. Northern (RNA) analysis was used to investigate the expression levels and regulation of the GAL] STE4-containing plasmid, designated pL19. Total RNA was isolated from strain Sc252 containing either pLl9 or YCp50 grown in medium containing either glucose or galactose. Growth in the presence of galactose induced a high level of the ST4-specific message only from cells containing pL19 (Fig. 2). Expression of STE4 under GAL control leads to haploidspecific cell cycle arrest. Plasmid pL19 was transformed into a variety of ura3- GAL' strains, including Sc252, L1-3B, and L1-14D, and the phenotype of the resulting transformants was checked after growth on medium containing either glucose or galactose. When maintenance of the plasmid was selected by growth on -Ura medium, glucose-
STE4
5'GGTA CAC ATT ACG ATG GCA GCA CAT
I
TAG CTT CGA ATT GGA AAT ACT GTG AGC
......
I
GTC GAC Sall FIG. 1. Position of the newly created restriction sites in the modified STE4 gene. Oligonucleotides were used to direct the insertion of EcoRl and BamHI sites at the 5' end of the STE4 gene and a Sall site downstream of the termination codon. The STE4 ATG and TAG codons
GAATTC T GGATCC EcoRI BamHl
are shown in boldface.
STE4 OVEREXPRESSION CAUSES CELL CYCLE ARREST
VOL. 10, 1990 5
6
3
4
2
1
26 S
18S FIG. 2. Galactose induction of the STE4 message. Lanes: 1 and 2, RNA from strain Sc252(YCp5O) grown in -Ura glucose medium; 3, RNA from strain Sc252(YCp5O) grown in -Ura galactose medium; 4 and 6, RNA from Sc252(pL19) grown in -Ura glucose medium; 5, RNA from strain Sc252(pL19) grown in -Ura galactose medium. The RNA was hybridized with an end-labeled oligonucleotide (5'-TTGGGTGACATTATTAGA-3') complementary to the STE4 message. Hybridization was at 45°C in 6X SSPE, and washes were done at 50°C in 2X SSPE (18). Ethidium bromide staining and nonspecific hybridization to the 18S rRNA were used to ensure that similar amounts of RNA were loaded in each lane. The arrow marks the STE4 message. grown cells formed healthy colonies, whereas galactosegrown cells formed tiny colonies containing primarily large,
abnormally shaped cells (Fig. 3A and B). When plasmid maintenance was not forced by growth on -Ura medium, plasmid pLl9 was rapidly lost from cultures growing in galactose-containing medium, which is unusual for a centromere plasmid. This plasmid was stably maintained, as expected for a centromere plasmid (5), when the cultures were grown in glucose medium (Table 2).
A
B A)
D)
Cl\6(2(
-!g \:
D
( 7I
C _
.-
-.
.-
.._ ' ._
._.._
_
Ct __ .-
l _
3
.__ ._
F
3I f0>
0A.,,
FIG. 3. Morphology of strain L1-3B(pLl9) and derivatives grown on different carbon sources. (A) L1-3B(pL19) grown on - Ura glucose medium; (B) the same strain grown on -Ura galactose
medium; (C and D) L1-3B(pL19) containing, respectively, the stel2::LEU2 and stel8::LEU2 disruptions and grown on -Ura galactose medium; E L1-3B(pLl9) containing plasmid pC3 and grown on -Ura galactose medium; F, L1-3B(pLl9) containing plasmid pL18-31 and grown on -Ura galactose medium. All panels are shown at the same magnification.
219
TABLE 2. Stability of plasmid pL19 in uracil-containing medium % URA3+ Plasmid Sugar Strain 72 Glucose YCp50 Sc252 72 Galactose YCp50 77 Glucose pL19
Vol. 10, No. 1
Overexpression of the STE4 Gene Leads to Mating Response in Haploid Saccharomyces cerevisiaett MALCOLM WHITEWAY,* LINDA HOUGAN, AND DAVID Y. THOMAS Genetic Engineering Section, National Research Council, Biotechnology Research Institute, 6100 Royalmount Avenue, Montreal, Quebec, Canada H4P 2R2 Received 10 July 1989/Accepted 6 October 1989
The STE4 gene of Saccharomyces cerevisiae encodes the I8 subunit of the yeast pheromone receptor-coupled G protein. Overexpression of the STE4 protein led to cell cycle arrest of haploid cells. This arrest was like the arrest mediated by mating pheromones in that it led to similar morphological changes in the arrested cells. The arrest occurred in haploid cells of either mating type but not in MATa/MATTa diploids, and it was suppressed by defects in genes such as STE12 that are needed for pheromone response. Overexpression of the STE4 gene product also suppressed the sterility of cells defective in the mating pheromone receptors encoded by the STE2 and STE3 genes. Cell cycle arrest mediated by STE4 overexpression was prevented in cells that either were overexpressing the SCGI gene product (the a subunit of the G protein) or lacked the STE18 gene product (the -y subunit of the G protein). This finding suggests that in yeast cells, the i subunit is the limiting component of the active ,(y element and that a proper balance in the levels of the G-protein subunits is critical to a normal mating pheromone response. The Saccharomyces cerevisiae mating pheromone response pathway has a remarkable similarity to G-proteinmediated signal transduction systems. Such systems were initially described in mammalian cells (see references 4, 8, 24, and 29 for recent reviews), where they were shown to consist of tripartite (a, 1, and -y subunits) guanine nucleotide-binding proteins (G proteins) coupled to cell surface receptor proteins. In S. cerevisiae, the STE2 and STE3 genes encode the mating pheromone receptor proteins, which have seven potential membrane-spanning regions and thus are similar to mammalian G-protein-linked receptors (3, 9, 23). The GPAI (SCGI), STE4, and STE18 genes encode, respectively, the a, 1, and -y subunits of a G protein that is functionally coupled to these receptors (6, 22, 31). A current model for G-protein function in mammalian cells is that an interaction between the agonist and the receptor leads to guanine nucleotide exchange on the a subunit and the activated a subunit dissociates from the f3y subunit. The dissociated a subunit may then interact with an intracellular effector protein, such as adenylyl cyclase or cyclic GMP phosphodiesterase, and modulate its activity (8, 24, 29). Another model for some systems is that the free 13y subunit plays a role in effector modulation. This latter idea has been controversial (16, 33), but current evidence suggests that Py acts at least in the modulation of some phospholipase A2-mediated pathways (12, 13). Subsequent hydrolysis of the GTP on the a subunit allows reconstitution of the inactive atpy heterotrimer and turns the signaling pathway off (24). In yeast cells, the genetic evidence suggests that the 13y element acts positively in the mating pheromone response pathway. Disruption of either STE4 (I) or STE18 (-y) eliminates the ability of cells to respond to pheromones (33), whereas loss of the GPAJ (SCGI) protein (a) causes a constitutive mating response and a haploid-specific cell cycle
arrest (6, 11, 22). Both ste4 and stel8 mutations suppress this cell cycle arrest (33). Although there is currently no biochemical data on the mechanism of G-protein action in yeast cells, the structural similarities in the yeast and mammalian subunits and the functional distinctions between STE4 and STE18 on one hand and GPAI (SCGI) on the other make it likely the yeast mating response pathway functions similarly to the mammalian systems. Biochemical assessments of the roles of the various Gprotein subunits in mammalian systems have treated the 1 and y proteins mainly as a single unit because they remain tightly associated throughout purification (24). In yeast cells, it was impossible to clone the STE4 gene from 2,um-based libraries (17), whereas the STE18 gene was readily obtained from such libraries (L. Hougan, unpublished data). This failure to isolate high-copy-number plasmids containing the STE4 gene suggests that high levels of the STE4 gene product may be detrimental to the cell, whereas high levels of the STE18 product are tolerated. Here, we investigate the phenotypic consequences of regulated overexpression of the STE4 gene by placing the STE4 coding sequence under the control of the GAL] promoter. Use of the GAL] promoter allows for facile manipulation of STE4 expression, since the promoter is efficiently induced when cells are grown on galactose but tightly repressed when cells are grown on glucose. Thus, cells can be grown in the absence of a potentially detrimental gene product and then induced to allow analysis of the results of the expression of this gene product.
MATERIALS AND METHODS Strains. See Table 1 for a description of the yeast strains used. Plasmids. Plasmids pC3, which is a YEp13 plasmid containing the SCGI gene (6), and pJK6, which is a centromere plasmid containing the MFaJ gene under GAL] control (32; J. Kurjan, personal communication), were gifts of Janet Kurjan. Plasmid pSUL16 is YIp5 containing a LEU2 disruption of STE12 (7) and was obtained from Stan Fields. Plasmids pSL376, which is pBR322 with a LEU2 disruption
* Corresponding author. t National Research Council Canada publication no. 30671. t This article is dedicated to the memory of Allen P. James, yeast geneticist.
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plated to minimal plates to detect the formation of prototrophic diploids. Plasmid stability. Plasmid stability was measured by growing strains overnight in YEPD or YEPGAL medium and then spreading suitable dilutions on YEPD plates. After 2 days of growth, the colonies were replica plated to drop-out medium to detect colonies that retained the selectable marker characteristic of the plasmid under study. This procedure detects the loss of plasmids producing toxic proteins, since cells containing these plasmids will be at a growth disadvantage. RNA analysis. Total RNA was isolated from galactoseinduced and uninduced strains as described previously (19). Induced strains were grown in the presence of galactose as the sole carbon source for 6 h; uninduced strains were grown for an identical time in glucose-containing medium. The RNA was denatured by boiling and run on a 1% agarose gel in TBE buffer, followed by direct hybridization with an end-labeled oligonucleotide probe to the dried gel (19). Oligonucleotide-directed mutagenesis. Single-stranded DNA was isolated from strain CJ236 (15) transformed with pTZ18R (20)-derived plasmids after infection with the helper bacteriophage M13KO7 as described elsewhere (30). Introduction of the 5' EcoRI and BamHI sites was achieved by annealing the oligonucleotide 5'-GGTACACATTACGiA
TABLE 1. Yeast strains Strain
Markers
Source
Sc225 Sc252 L1-3B L1-14D DL41
MATa adel leu2-3,2-112 ura3-52 MATa adel leu2-3,2-112 ura3-52 MATa ura3 leu2-3,2-112, his3-11,3-15 MATa ura3 leu2-3,2-112, his3-11,3-15 + MMa adel ura3-52 leu2-3.2-112 MATa + ura3 leu2-3,2-112 his3-11,3-15
J. Hopper J. Hopper This work This work This work
of STE3 (9), and pAB506, which is pBR322 containing a LEU2 disruption of STE2 (14), were obtained from Karen Clark and Cathy Jackson, respectively. Plasmid YCp5O is a centromere-containing plasmid carrying the URA3 selectable marker (25). Plasmid M65pl is pTZ18R containing a LEU2 disruption of STE18 (31). Plasmid pL19 is a centromere plasmid with STE4 under the control of the GAL] promoter and was constructed as follows. Plasmid M88p5, which consists of a 4.0-kilobase EcoRI fragment containing the STE4 gene inserted into the EcoRI site of plasmid pTZ18R (20), was modified by oligonucleotide-directed mutagenesis. This placed an EcoRI and a BamHI site at the 5' end of the gene and a SalI site at the 3' end (Fig. 1). The BamHI-to-Sall fragment was excised from this modified plasmid (M88L18) and cloned into the BamHI-to-SalI region of plasmid pJK6 (Kurjan, personal communication). This replaces the MFal sequences of pJK6 with STE4 and generates pLl9, a centromere plasmid carrying the STE4 gene linked to the GALl promoter. Plasmid pL18-31 is a YEp213 plasmid containing the STE18 gene (L. Hougan, unpublished data). Recombinant DNA manipulations were performed by using standard protocols (18). Media and genetic methods. The media used have been described previously (28, 32), and standard procedures were used for strain constructions (28). Transformation. Cells were transformed by recombinant plasmids or by DNA fragments by the lithium cation protocol (10). Disruptions of STE2, STE3, STE12, and STE18 by the LEU2 gene were constructed by one-step gene replacement (26). The STE2 disruption was created using a BamHI fragment containing a LEU2 replacement of STE2 obtained from plasmid pAB506. The STE3 disruption fragment was a HindIll fragment from plasmid pSL376, the STE12 disruption fragment was a SacI-to-SphI fragment from pSUL16, and the STE18 disruption used a PstI-to-HindIII fragment containing a LEU2 replacement of STE18 derived from M65pl. Patch matings. Matings of strains containing plasmid pLl9 were performed by growing the strains as patches on uracillacking (-Ura) glucose plates and then replica plating them to mating tester lawns on either yeast extract-peptonegalactose (YEPGAL) (for GALl promoter induction) or yeast extract-peptone-dextrose (YEPD) medium. These plates were incubated overnight at 30°C and then replica
ATTCTFGGATCCATGGCAGCACAT-3' to plasmid M88p5 and extending it with T4 DNA polymerase as described previously (15). This generated plasmid M88L17, which was then mutagenized with oligonucleotide 5'-CTTCGAATTGG AGTCGACATTACTGTGAGC-3' to introduce a 3' Sall site and create plasmid M88L18. Oligonucleotides were synthesized on an Applied Biosystems 380A DNA synthesizer and purified as described previously (27). Correctly mutated plasmids were identified by screening for the presence of the created restriction sites. RESULTS
Construction of a galactose-inducible STE4 gene. We constructed a derivative of the STE4 gene that replaced the normal promoter and 5' regulatory sequences of this gene with the promoter of the GAL] gene. Northern (RNA) analysis was used to investigate the expression levels and regulation of the GAL] STE4-containing plasmid, designated pL19. Total RNA was isolated from strain Sc252 containing either pLl9 or YCp50 grown in medium containing either glucose or galactose. Growth in the presence of galactose induced a high level of the ST4-specific message only from cells containing pL19 (Fig. 2). Expression of STE4 under GAL control leads to haploidspecific cell cycle arrest. Plasmid pL19 was transformed into a variety of ura3- GAL' strains, including Sc252, L1-3B, and L1-14D, and the phenotype of the resulting transformants was checked after growth on medium containing either glucose or galactose. When maintenance of the plasmid was selected by growth on -Ura medium, glucose-
STE4
5'GGTA CAC ATT ACG ATG GCA GCA CAT
I
TAG CTT CGA ATT GGA AAT ACT GTG AGC
......
I
GTC GAC Sall FIG. 1. Position of the newly created restriction sites in the modified STE4 gene. Oligonucleotides were used to direct the insertion of EcoRl and BamHI sites at the 5' end of the STE4 gene and a Sall site downstream of the termination codon. The STE4 ATG and TAG codons
GAATTC T GGATCC EcoRI BamHl
are shown in boldface.
STE4 OVEREXPRESSION CAUSES CELL CYCLE ARREST
VOL. 10, 1990 5
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18S FIG. 2. Galactose induction of the STE4 message. Lanes: 1 and 2, RNA from strain Sc252(YCp5O) grown in -Ura glucose medium; 3, RNA from strain Sc252(YCp5O) grown in -Ura galactose medium; 4 and 6, RNA from Sc252(pL19) grown in -Ura glucose medium; 5, RNA from strain Sc252(pL19) grown in -Ura galactose medium. The RNA was hybridized with an end-labeled oligonucleotide (5'-TTGGGTGACATTATTAGA-3') complementary to the STE4 message. Hybridization was at 45°C in 6X SSPE, and washes were done at 50°C in 2X SSPE (18). Ethidium bromide staining and nonspecific hybridization to the 18S rRNA were used to ensure that similar amounts of RNA were loaded in each lane. The arrow marks the STE4 message. grown cells formed healthy colonies, whereas galactosegrown cells formed tiny colonies containing primarily large,
abnormally shaped cells (Fig. 3A and B). When plasmid maintenance was not forced by growth on -Ura medium, plasmid pLl9 was rapidly lost from cultures growing in galactose-containing medium, which is unusual for a centromere plasmid. This plasmid was stably maintained, as expected for a centromere plasmid (5), when the cultures were grown in glucose medium (Table 2).
A
B A)
D)
Cl\6(2(
-!g \:
D
( 7I
C _
.-
-.
.-
.._ ' ._
._.._
_
Ct __ .-
l _
3
.__ ._
F
3I f0>
0A.,,
FIG. 3. Morphology of strain L1-3B(pLl9) and derivatives grown on different carbon sources. (A) L1-3B(pL19) grown on - Ura glucose medium; (B) the same strain grown on -Ura galactose
medium; (C and D) L1-3B(pL19) containing, respectively, the stel2::LEU2 and stel8::LEU2 disruptions and grown on -Ura galactose medium; E L1-3B(pLl9) containing plasmid pC3 and grown on -Ura galactose medium; F, L1-3B(pLl9) containing plasmid pL18-31 and grown on -Ura galactose medium. All panels are shown at the same magnification.
219
TABLE 2. Stability of plasmid pL19 in uracil-containing medium % URA3+ Plasmid Sugar Strain 72 Glucose YCp50 Sc252 72 Galactose YCp50 77 Glucose pL19
