Vol. 10, No. 12
MOLECULAR AND CELLULAR BIOLOGY, Dec. 1990, p. 6389-6396 0270-7306/90/126389-08$02.00/0 Copyright © 1990, American Society for Microbiology
gcr2, a New Mutation Affecting Glycolytic Gene Expression in Saccharomyces cerevisiae HIROSHI UEMURAt AND DAN P. FRAENKEL* Department of Microbiology and Molecular Genetics, Harvard Medical School, Boston, Massachusetts 02115 Received 17 July 1990/Accepted 7 September 1990
Screening of a mutagenized strain carrying a multicopy ENOI-'lacZ fusion plasmid revealed a new mutation affecting most glycolytic enzyme activities in a pattern resembling that caused by gcrl: levels in the range of 10% of wild-type levels on glycerol plus lactate but somewhat higher on glucose. The recessive single nuclear gene mutation, named gcr2-1, was unlinked to gcrl, and GCRI in multiple copies did not restore enzyme levels. GCR2 was obtained by complementation from a YCp5O genomic library; the complemented strain had normal enzyme levels, as did a strain with GCR2 in multiple copies. GCR2 in multiple copies did not suppress geri. A chromosomal gcr2 null mutant was constructed; its pattern of enzyme activities resembled that of the gcr2-1 mutant and, like the gcr2-1 mutant, its growth defect on glucose was only partial (in contrast to the glucose negativity of the gcrl mutant). Northern (RNA) analysis showed that gcr2 and gcrl affect ENO] mRNA levels.
respectively. pGCR8 (21) is a derivative of YEp13 carrying GCRI. All these plasmids also carry LEU2. YEp352 (15), a multicopy shuttle vector carrying URA3, was used for the subcloning of the GCR2 gene. YIp351 (15), an integration type plasmid carrying LEU2, was used to examine the linkage between the cloned gene and the gcr2 mutation. pGCR2 is a GCR2 clone obtained from an S. cerevisiae genomic DNA library in YCp5O. Plasmids pML16-2, pML17-1, pML18-1, and pML19-1 are "dropout" derivatives of pGCR2. Plasmids pML41-1, pML41-3, pML44-1, and pML44-2 are multicopy GCR2 clones on YEp352. The structures of these plasmids are shown in Fig. 1. E. coli and yeast transformations were done as described by Cohen et al. (9) and Ito et al. (20), respectively. A genomic library of yeast DNA in YCp50 (30) was kindly supplied by F. Winston, who also gave us the actin probe DNA. The HIS5-'lacZ fusion plasmid was from S. Harashima. Media. E. coli cells were grown in LB (10). Yeast cells were grown in rich medium (R61) (12), synthetic complete medium (SC) (33), or SC lacking leucine or uracil to maintain selective pressure for plasmids. Two percent glucose or 2% glycerol plus 2% lactate was added as indicated. For P-galactosidase detection on plates, 40 ,ug of X-Gal (5-bromo-4-chloro-3-indolyl-f3-D-galactoside) per ml was included in SC, which was buffered to pH 6.8 with 0.1 M potassium phosphate buffer. Mutagenesis and isolation of mutants. Yeast strain 2845 harboring pHU15002 was grown in SC-Leu (plus glucose) to mid-log phase. The cells were centrifuged, washed once with 0.1 M potassium phosphate buffer (pH 7.0), and resuspended in the same buffer at 4 x 108 cells per ml. After incubation for 60 min at 23°C with 3% ethylmethanesulfonic acid to yield approximately 50% killing, the cells were centrifuged, washed with water, and resuspended in 6% sodium thiosulfate. After 10 min of incubation at room temperature, the cells were washed twice with water and plated for single colonies on X-Gal-containing SC-Leu (plus glucose) and SC-Leu (plus glycerol and lactate) plates at 30°C. Colonies
The glycolytic pathway is a major metabolic route in Saccharomyces cerevisiae. Most of the S. cerevisiae enzymes, which form a significant fraction of the total protein (11, 14), depend on structural genes expressed at high rates. Whether their expression is regulated may be strain dependent, with reports ranging from virtual constitutivity (1, 6) to apparent induction by glucose (24) of at least some of the enzymes. gcrl mutants were recognized as a class of mutants impaired in their growth on glucose and low in their levels of most of the glycolytic enzymes but relatively normal in their levels of other proteins (6, 7, 21). GCRJ has been cloned and sequenced (1, 17) and may be a positive, trans-acting transcriptional regulator. Most mutations affecting single glycolytic enzymes have been shown to identify the structural gene rather than specific regulatory elements. Thus, with an ENOJ-'lacZ fusion gene for screening, no mutations so far analyzed appeared to affect only ENO] (unpublished data). However, several mutations affecting more than one enzyme were obtained. The present report describes one of them, named gcr2, which resembles gcrl in its effect on the levels of glycolytic enzymes but identifies a different gene, which we have cloned and mutated.
MATERLALS AND METHODS Strains and plasmids. The S. cerevisiae strains used are listed in Table 1. Strain 2845 was the wild-type strain used to isolate mutants in this study. Escherichia coli DH5a (F- endAl hsdRJ7 supE44 thi-J recAl gyrA96 relAl A(1acU169 480 dlacZAM15) was used to propagate all plasmids (13, 31a). Plasmids pHU15002 (35) and pSH530 (27) are multicopy plasmids containing ENOJ-'lacZ and HIS5-'lacZ fusions, Corresponding author. t Present address: National Chemical Laboratory for Industry, Tsukuba Research Center, Tsukuba, Ibaraki 305, Japan. *
6389
6390
UEMURA AND FRAENKEL
MOL. CELL. BIOL.
TABLE 1. S. cerevisiae strains Strain
2845
Genotype
Comments or source
a leu2-3 leu2-112 ura3-52
R. Wickner his6 YHU2012 a leu2-3 leu2-112 ura3-52 Isogenic with 2845a NW9-19-1 a gcr2-1 leu2-3 leu2-112 gcr2 mutant of 2845 ura3-52 his6 MWGL29 a gcrl-6 leu2-3 leu2-112 gcrl mutant of 2845 ura3-52 his6 DFY67 a gcrl-l leu2-1 lysi trpl-J 7 DFY522 a Agcrl::LEU2 leu2-3 1 leu2-112 his3 can] DFY639 a/a Agcr2:: URA3/GCR2 See text leu2-3,1121leu2-3,112 ura3-521ura3-52 HIS61 his6 a leu2-3 leu2-112 ura3-52 DFY640 See text his6 GCR2 DFY641 a leu2-3 leu2-112 ura3-52 See text Agcr2:: URA3 DFY642 a leu2-3 leu2-112 ura3-52 See text GCR2 DFY643 a leu2-3 leu2-112 ura3-52 See text his6 Agcr2::URA3 DFY644 a Agcrl::LEU2 leu2-3 See text" leu2-112 ura3-52 his3 DFY645 a Agcrl::LEU2 leu2-3 See textb leu2-112 ura3-52 HIS3 YHU2012 was constructed from a His' revertant of 2845 by converting a
the mating type through transformation with a multicopy plasmid carrying the HO gene, curing of the plasmid from the diploid, and segregation. b DFY644 and DFY645 were segregants from a diploid between a spontaneous His' revertant of 2845 and DFY522.
which showed a bluer or a whiter phenotype than nonmutagenized cells were screened. If the color phenotype was stable after restreaking, pHU15002 was cured from putative mutants, which were then retransformed separately with (nonmutagenized) pHU15002 as well as with a HIS5-'lacZ fusion plasmid, pSH530. Transformants were tested on X-Gal-containing SC-Leu (plus glucose) or SC-Leu (plus glycerol and lactate) plates. If the color was affected in pHU15002 transformants but not in pSH530 transformants, it was judged that the mutation was chromosomal and of possible interest. Matings, diploid selection, sporulation, and dissection were carried out by the usual methods (26). Enzyme assays. Cultures were harvested in R61 (plus glycerol and lactate) at an A580 of about 15 to 20, in R61 (plus glucose) at an A580 of about 5 to 9, and in SC (plus glycerol and lactate [and for plasmid-containing strains, lacking leucine or uracil, as appropriate]) at an A580 of about 5 to 10. For glycolytic enzyme assays, extracts were prepared by passage through a French pressure cell and assayed as described previously (7), with the exception of aldolase, which was assayed by the method of Richards and Rutter (29). Enzyme activities were normalized to protein concentrations determined by the method of Bradford (2). ,B-Galactosidase assays were done as described previously
(35).
DNA manipulation. Standard techniques of restriction endonuclease digestion, ligation, filling of recessed ends of DNA, plasmid DNA isolation, and gel electrophoresis were done as described by Maniatis et al. (25). Isolation of plasmid DNA from yeast cells was done by the method of Hoffman and Winston (16).
Preparation of yeast RNA and Northern RNA blot analysis. Yeast cells were grown to mid-log phase (A580, 0.3) in R61 (plus glucose) and R61 (plus glycerol and lactate), and RNA was prepared as described elsewhere (22). RNAs were electrophoresed through a 1.2% agarose-1 M formaldehyde gel, and Northern blot analysis was performed as described by Maniatis et al. (25). The ECL gene detection system RPN2101 (Amersham) was used for labeling of DNA probes with peroxidase, hybridization, and signal detection.
RESULTS Original mutant. Yeast strain 2845 carrying the ENOJ'lacZ fusion plasmid pHU15002 was mutagenized and screened for putative mutations decreasing ENO] expression (see Materials and Methods). Among candidate strains, MWGL29 proved to be a new geri mutant, while NW9-19-1 was different. Both mutations were recessive, with normal growth and ,-galactosidase activities in diploids formed with the isogenic wild-type strain YHU2012. For NW9-19-1, eight tetrads examined segregated 2:2 (wild-type versus smaller colonies) on glucose and had low 3-galactosidase activity and low enolase activity, indicating a single nuclear gene mutation. Similar results were obtained with MWGL29. The expression of the EN02-'lacZ fusion and of a PYKJ'lacZ fusion was also affected in both strains, but the impaired growth of NWGL29 was complemented by a multicopy plasmid carrying GCRI, while that of NW9-19-1 was not. Furthermore, a diploid between NW9-19-1 and a gcrl-l mutant, DFY67, grew normally on glucose and expressed normal P-galactosidase levels from the ENOJ-'lacZ fusion, whereas no complementation was observed between MWGL29 and DFY67. Table 2 shows enzyme activities of these two strains, as compared with those of their isogenic parental wild-type strain, 2845. The patterns of enzyme deficiency, as expressed in the usual permissive medium, which contained glycerol plus lactate as carbon sources, were remarkably similar in the two mutant strains and resembled the patterns known for both gcrl-l and gcrl null mutants (1, 6); most enzymes of the glycolytic pathway were present at 10% or less of wild-type levels, with the largest deficiencies occurring for enolase and phosphoglycerate mutase, while phosphofructokinase and total hexokinase activities, as well as the activities of the nonglycolytic enzyme glucose-6-phosphate dehydrogenase and the gluconeogenic enzyme fructose-1,6-bisphosphate phosphatase, were relatively normal. On the basis of these results, the mutations were assigned as gcrl-6 in MWGL29 and as gcr2-1 in NW9-19-1. Although quite similar in their patterns of affected enzymes, the gcrl-6 and gcr2-1 mutant strains were different in their growth. The gcrl-6 mutant, like known gcrl mutants, was extremely defective in its growth on glucose, fructose, and mannose, while the gcr2-1 mutant grew fairly well at the usual 30°C incubation temperature (Table 3), although it could readily be scored as somewhat impaired. The growth of both mutants was almost normal on respiratory media. Cloning of GCR2. The relatively "leaky" growth deficiency of the gcr2-1 mutant at 30°C proved to be slightly less so at 37°C, and inclusion of the respiration inhibitor antimycin A provided a virtually nonpermissive medium (Table 3). An S. cerevisiae genomic DNA library in the centromere plasmid YCp5O (30) was transformed into mutant strain NW9-19-1, and Ura+ transformants were selected on non-
NEW S. CEREVISL4E GLYCOLYTIC GENE EXPRESSION MUTANT
VOL. 10, 1990
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O E o° X sion. Both on the centromere plasmid (pGCR2) and on the multicopy plasmid (pML41-1) the gene restored normal enzyme levels to the gcr2 mutant (Table 4). ° X C= URA3 substitution mutation. The 1.5-kbp XhoI-SacI frag4° 66 ment from pML18-1 was introduced into Sall-Sacl sites of a pUC18 derivative plasmid, pT7/T3a-18 (Bethesda Research 'Tt 3 EC 00 : c Laboratories); subsequently, a 1.2-kbp Hindlll fragment 02 O URA3 was inserted into the unique BamHI site in carrying 00 the XhoI-SacI fragment after filling in of the protruding ends. _ A SacI-SphI fragment (fragment B in Fig. 1; the SphI site is 0)0 in the vector 12 bp from the SalI-XhoI junction) was used to 0. X between strains 2845 and YHU2012 ?_1 oUco E o oooQ ,(GCR2IGCR2 O ^ °transform a diploid grew ura3lura3). The diploid transformants .6 tetrads examined showed 2:2 and 15 segregation of normally Ura+ Gcr2- and Ura- Gcr2+. The expected disruption of -E t oF o X N : digestion of genomic DNAs with XhoI and Sacl and probing o o =, u with the 1.5-kbp XhoI-SacI fragment of pML18-1 (data not > , shown). The fact that Agcr2::URA3, like the original gcr2-1 0.XcL Qmutation, conferred a recessive and nonlethal phenotype ^ N Q I) d °° that GCR2 is not essential. The disrupted strains 0. had a growth phenotype almost identical to that of the 0 originalgcr2-1 mutant (Table 3): a marginal effect at 30°C but a strong effect at 37°C in the presence of antimycin A. = ;° .> bi activities for one tetrad are shown in Table 5. In No - o .~. >the usual permissive medium (experiment 1), the enzyme pattern in the disrupted strains (DFY641 and DFY643) was Q > Q the same as that in the original gcr2-1 strain. These strains o 7a Hv o .s2 0.. showed fairly normal growth on glucose at 30°C (experiment N :N N 2), and most of the affected enzyme levels were higher by a 444t. , ,factor of 2 to 4; induction is also known for gcrl (1, 6). It should be noted, however, that certain enzyme levels relow (phosphoglycerate mutase and pyruvate kinase ° OQ X Q 3 site in the vector), the linearized DNA was used to transform ,@, Q X 0., ' > 3 Q °:S o the original mutant strain NW9-19-1 to Leu+. Transformants CZ were crossed with YHU2012 (GCR2 leu2), and segregants . , :5 3 3 m .- D ,_were analyzed. In four tetrads from each of three transfor-C Z> 33 mants, Leu+ cosegregated with Gcr2-, indicating that the Dz Q oo
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NEW S. CEREVISIAE GLYCOLYTIC GENE EXPRESSION MUTANT
VOL. 10, 1990
6393
TABLE 3. Growth on plates Growth (colony size, mm) on plates containing:
SCb
R61"
RelevantGlcsGuoe vate lycrol
Strain genotype Strain
Glucose
2845 NW9-19-1
MWGL29 DFY641
Wild typec
2.1
gcr2-1 gcrl-6 Agcr2::URA3C
1.7 0.2 1.6
Fructose
Mannose
1.8 1.5
2.0 1.7
0.2 1.5
0.2 1.7
plusPyruplus lacvate
Ethanol
tate
No addition
1.0
0.8
0.7
0.6
0.7 0.4 0.5
0.8 0.8
0.6 0.6 0.5
0.5 0.4 0.4
0.7
Glucose (300C) 2.1 1.5
0.2 1.5
timycin A
Glucose (37'C)
2.0 1.1
MOLECULAR AND CELLULAR BIOLOGY, Dec. 1990, p. 6389-6396 0270-7306/90/126389-08$02.00/0 Copyright © 1990, American Society for Microbiology
gcr2, a New Mutation Affecting Glycolytic Gene Expression in Saccharomyces cerevisiae HIROSHI UEMURAt AND DAN P. FRAENKEL* Department of Microbiology and Molecular Genetics, Harvard Medical School, Boston, Massachusetts 02115 Received 17 July 1990/Accepted 7 September 1990
Screening of a mutagenized strain carrying a multicopy ENOI-'lacZ fusion plasmid revealed a new mutation affecting most glycolytic enzyme activities in a pattern resembling that caused by gcrl: levels in the range of 10% of wild-type levels on glycerol plus lactate but somewhat higher on glucose. The recessive single nuclear gene mutation, named gcr2-1, was unlinked to gcrl, and GCRI in multiple copies did not restore enzyme levels. GCR2 was obtained by complementation from a YCp5O genomic library; the complemented strain had normal enzyme levels, as did a strain with GCR2 in multiple copies. GCR2 in multiple copies did not suppress geri. A chromosomal gcr2 null mutant was constructed; its pattern of enzyme activities resembled that of the gcr2-1 mutant and, like the gcr2-1 mutant, its growth defect on glucose was only partial (in contrast to the glucose negativity of the gcrl mutant). Northern (RNA) analysis showed that gcr2 and gcrl affect ENO] mRNA levels.
respectively. pGCR8 (21) is a derivative of YEp13 carrying GCRI. All these plasmids also carry LEU2. YEp352 (15), a multicopy shuttle vector carrying URA3, was used for the subcloning of the GCR2 gene. YIp351 (15), an integration type plasmid carrying LEU2, was used to examine the linkage between the cloned gene and the gcr2 mutation. pGCR2 is a GCR2 clone obtained from an S. cerevisiae genomic DNA library in YCp5O. Plasmids pML16-2, pML17-1, pML18-1, and pML19-1 are "dropout" derivatives of pGCR2. Plasmids pML41-1, pML41-3, pML44-1, and pML44-2 are multicopy GCR2 clones on YEp352. The structures of these plasmids are shown in Fig. 1. E. coli and yeast transformations were done as described by Cohen et al. (9) and Ito et al. (20), respectively. A genomic library of yeast DNA in YCp50 (30) was kindly supplied by F. Winston, who also gave us the actin probe DNA. The HIS5-'lacZ fusion plasmid was from S. Harashima. Media. E. coli cells were grown in LB (10). Yeast cells were grown in rich medium (R61) (12), synthetic complete medium (SC) (33), or SC lacking leucine or uracil to maintain selective pressure for plasmids. Two percent glucose or 2% glycerol plus 2% lactate was added as indicated. For P-galactosidase detection on plates, 40 ,ug of X-Gal (5-bromo-4-chloro-3-indolyl-f3-D-galactoside) per ml was included in SC, which was buffered to pH 6.8 with 0.1 M potassium phosphate buffer. Mutagenesis and isolation of mutants. Yeast strain 2845 harboring pHU15002 was grown in SC-Leu (plus glucose) to mid-log phase. The cells were centrifuged, washed once with 0.1 M potassium phosphate buffer (pH 7.0), and resuspended in the same buffer at 4 x 108 cells per ml. After incubation for 60 min at 23°C with 3% ethylmethanesulfonic acid to yield approximately 50% killing, the cells were centrifuged, washed with water, and resuspended in 6% sodium thiosulfate. After 10 min of incubation at room temperature, the cells were washed twice with water and plated for single colonies on X-Gal-containing SC-Leu (plus glucose) and SC-Leu (plus glycerol and lactate) plates at 30°C. Colonies
The glycolytic pathway is a major metabolic route in Saccharomyces cerevisiae. Most of the S. cerevisiae enzymes, which form a significant fraction of the total protein (11, 14), depend on structural genes expressed at high rates. Whether their expression is regulated may be strain dependent, with reports ranging from virtual constitutivity (1, 6) to apparent induction by glucose (24) of at least some of the enzymes. gcrl mutants were recognized as a class of mutants impaired in their growth on glucose and low in their levels of most of the glycolytic enzymes but relatively normal in their levels of other proteins (6, 7, 21). GCRJ has been cloned and sequenced (1, 17) and may be a positive, trans-acting transcriptional regulator. Most mutations affecting single glycolytic enzymes have been shown to identify the structural gene rather than specific regulatory elements. Thus, with an ENOJ-'lacZ fusion gene for screening, no mutations so far analyzed appeared to affect only ENO] (unpublished data). However, several mutations affecting more than one enzyme were obtained. The present report describes one of them, named gcr2, which resembles gcrl in its effect on the levels of glycolytic enzymes but identifies a different gene, which we have cloned and mutated.
MATERLALS AND METHODS Strains and plasmids. The S. cerevisiae strains used are listed in Table 1. Strain 2845 was the wild-type strain used to isolate mutants in this study. Escherichia coli DH5a (F- endAl hsdRJ7 supE44 thi-J recAl gyrA96 relAl A(1acU169 480 dlacZAM15) was used to propagate all plasmids (13, 31a). Plasmids pHU15002 (35) and pSH530 (27) are multicopy plasmids containing ENOJ-'lacZ and HIS5-'lacZ fusions, Corresponding author. t Present address: National Chemical Laboratory for Industry, Tsukuba Research Center, Tsukuba, Ibaraki 305, Japan. *
6389
6390
UEMURA AND FRAENKEL
MOL. CELL. BIOL.
TABLE 1. S. cerevisiae strains Strain
2845
Genotype
Comments or source
a leu2-3 leu2-112 ura3-52
R. Wickner his6 YHU2012 a leu2-3 leu2-112 ura3-52 Isogenic with 2845a NW9-19-1 a gcr2-1 leu2-3 leu2-112 gcr2 mutant of 2845 ura3-52 his6 MWGL29 a gcrl-6 leu2-3 leu2-112 gcrl mutant of 2845 ura3-52 his6 DFY67 a gcrl-l leu2-1 lysi trpl-J 7 DFY522 a Agcrl::LEU2 leu2-3 1 leu2-112 his3 can] DFY639 a/a Agcr2:: URA3/GCR2 See text leu2-3,1121leu2-3,112 ura3-521ura3-52 HIS61 his6 a leu2-3 leu2-112 ura3-52 DFY640 See text his6 GCR2 DFY641 a leu2-3 leu2-112 ura3-52 See text Agcr2:: URA3 DFY642 a leu2-3 leu2-112 ura3-52 See text GCR2 DFY643 a leu2-3 leu2-112 ura3-52 See text his6 Agcr2::URA3 DFY644 a Agcrl::LEU2 leu2-3 See text" leu2-112 ura3-52 his3 DFY645 a Agcrl::LEU2 leu2-3 See textb leu2-112 ura3-52 HIS3 YHU2012 was constructed from a His' revertant of 2845 by converting a
the mating type through transformation with a multicopy plasmid carrying the HO gene, curing of the plasmid from the diploid, and segregation. b DFY644 and DFY645 were segregants from a diploid between a spontaneous His' revertant of 2845 and DFY522.
which showed a bluer or a whiter phenotype than nonmutagenized cells were screened. If the color phenotype was stable after restreaking, pHU15002 was cured from putative mutants, which were then retransformed separately with (nonmutagenized) pHU15002 as well as with a HIS5-'lacZ fusion plasmid, pSH530. Transformants were tested on X-Gal-containing SC-Leu (plus glucose) or SC-Leu (plus glycerol and lactate) plates. If the color was affected in pHU15002 transformants but not in pSH530 transformants, it was judged that the mutation was chromosomal and of possible interest. Matings, diploid selection, sporulation, and dissection were carried out by the usual methods (26). Enzyme assays. Cultures were harvested in R61 (plus glycerol and lactate) at an A580 of about 15 to 20, in R61 (plus glucose) at an A580 of about 5 to 9, and in SC (plus glycerol and lactate [and for plasmid-containing strains, lacking leucine or uracil, as appropriate]) at an A580 of about 5 to 10. For glycolytic enzyme assays, extracts were prepared by passage through a French pressure cell and assayed as described previously (7), with the exception of aldolase, which was assayed by the method of Richards and Rutter (29). Enzyme activities were normalized to protein concentrations determined by the method of Bradford (2). ,B-Galactosidase assays were done as described previously
(35).
DNA manipulation. Standard techniques of restriction endonuclease digestion, ligation, filling of recessed ends of DNA, plasmid DNA isolation, and gel electrophoresis were done as described by Maniatis et al. (25). Isolation of plasmid DNA from yeast cells was done by the method of Hoffman and Winston (16).
Preparation of yeast RNA and Northern RNA blot analysis. Yeast cells were grown to mid-log phase (A580, 0.3) in R61 (plus glucose) and R61 (plus glycerol and lactate), and RNA was prepared as described elsewhere (22). RNAs were electrophoresed through a 1.2% agarose-1 M formaldehyde gel, and Northern blot analysis was performed as described by Maniatis et al. (25). The ECL gene detection system RPN2101 (Amersham) was used for labeling of DNA probes with peroxidase, hybridization, and signal detection.
RESULTS Original mutant. Yeast strain 2845 carrying the ENOJ'lacZ fusion plasmid pHU15002 was mutagenized and screened for putative mutations decreasing ENO] expression (see Materials and Methods). Among candidate strains, MWGL29 proved to be a new geri mutant, while NW9-19-1 was different. Both mutations were recessive, with normal growth and ,-galactosidase activities in diploids formed with the isogenic wild-type strain YHU2012. For NW9-19-1, eight tetrads examined segregated 2:2 (wild-type versus smaller colonies) on glucose and had low 3-galactosidase activity and low enolase activity, indicating a single nuclear gene mutation. Similar results were obtained with MWGL29. The expression of the EN02-'lacZ fusion and of a PYKJ'lacZ fusion was also affected in both strains, but the impaired growth of NWGL29 was complemented by a multicopy plasmid carrying GCRI, while that of NW9-19-1 was not. Furthermore, a diploid between NW9-19-1 and a gcrl-l mutant, DFY67, grew normally on glucose and expressed normal P-galactosidase levels from the ENOJ-'lacZ fusion, whereas no complementation was observed between MWGL29 and DFY67. Table 2 shows enzyme activities of these two strains, as compared with those of their isogenic parental wild-type strain, 2845. The patterns of enzyme deficiency, as expressed in the usual permissive medium, which contained glycerol plus lactate as carbon sources, were remarkably similar in the two mutant strains and resembled the patterns known for both gcrl-l and gcrl null mutants (1, 6); most enzymes of the glycolytic pathway were present at 10% or less of wild-type levels, with the largest deficiencies occurring for enolase and phosphoglycerate mutase, while phosphofructokinase and total hexokinase activities, as well as the activities of the nonglycolytic enzyme glucose-6-phosphate dehydrogenase and the gluconeogenic enzyme fructose-1,6-bisphosphate phosphatase, were relatively normal. On the basis of these results, the mutations were assigned as gcrl-6 in MWGL29 and as gcr2-1 in NW9-19-1. Although quite similar in their patterns of affected enzymes, the gcrl-6 and gcr2-1 mutant strains were different in their growth. The gcrl-6 mutant, like known gcrl mutants, was extremely defective in its growth on glucose, fructose, and mannose, while the gcr2-1 mutant grew fairly well at the usual 30°C incubation temperature (Table 3), although it could readily be scored as somewhat impaired. The growth of both mutants was almost normal on respiratory media. Cloning of GCR2. The relatively "leaky" growth deficiency of the gcr2-1 mutant at 30°C proved to be slightly less so at 37°C, and inclusion of the respiration inhibitor antimycin A provided a virtually nonpermissive medium (Table 3). An S. cerevisiae genomic DNA library in the centromere plasmid YCp5O (30) was transformed into mutant strain NW9-19-1, and Ura+ transformants were selected on non-
NEW S. CEREVISL4E GLYCOLYTIC GENE EXPRESSION MUTANT
VOL. 10, 1990
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UEMURA AND FRAENKEL
MOL. CELL. BIOL. 0 0 0
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O E o° X sion. Both on the centromere plasmid (pGCR2) and on the multicopy plasmid (pML41-1) the gene restored normal enzyme levels to the gcr2 mutant (Table 4). ° X C= URA3 substitution mutation. The 1.5-kbp XhoI-SacI frag4° 66 ment from pML18-1 was introduced into Sall-Sacl sites of a pUC18 derivative plasmid, pT7/T3a-18 (Bethesda Research 'Tt 3 EC 00 : c Laboratories); subsequently, a 1.2-kbp Hindlll fragment 02 O URA3 was inserted into the unique BamHI site in carrying 00 the XhoI-SacI fragment after filling in of the protruding ends. _ A SacI-SphI fragment (fragment B in Fig. 1; the SphI site is 0)0 in the vector 12 bp from the SalI-XhoI junction) was used to 0. X between strains 2845 and YHU2012 ?_1 oUco E o oooQ ,(GCR2IGCR2 O ^ °transform a diploid grew ura3lura3). The diploid transformants .6 tetrads examined showed 2:2 and 15 segregation of normally Ura+ Gcr2- and Ura- Gcr2+. The expected disruption of -E t oF o X N : digestion of genomic DNAs with XhoI and Sacl and probing o o =, u with the 1.5-kbp XhoI-SacI fragment of pML18-1 (data not > , shown). The fact that Agcr2::URA3, like the original gcr2-1 0.XcL Qmutation, conferred a recessive and nonlethal phenotype ^ N Q I) d °° that GCR2 is not essential. The disrupted strains 0. had a growth phenotype almost identical to that of the 0 originalgcr2-1 mutant (Table 3): a marginal effect at 30°C but a strong effect at 37°C in the presence of antimycin A. = ;° .> bi activities for one tetrad are shown in Table 5. In No - o .~. >the usual permissive medium (experiment 1), the enzyme pattern in the disrupted strains (DFY641 and DFY643) was Q > Q the same as that in the original gcr2-1 strain. These strains o 7a Hv o .s2 0.. showed fairly normal growth on glucose at 30°C (experiment N :N N 2), and most of the affected enzyme levels were higher by a 444t. , ,factor of 2 to 4; induction is also known for gcrl (1, 6). It should be noted, however, that certain enzyme levels relow (phosphoglycerate mutase and pyruvate kinase ° OQ X Q 3 site in the vector), the linearized DNA was used to transform ,@, Q X 0., ' > 3 Q °:S o the original mutant strain NW9-19-1 to Leu+. Transformants CZ were crossed with YHU2012 (GCR2 leu2), and segregants . , :5 3 3 m .- D ,_were analyzed. In four tetrads from each of three transfor-C Z> 33 mants, Leu+ cosegregated with Gcr2-, indicating that the Dz Q oo
0^o o
&h
X
°
_
~
-: ffisuggests
X0=>Enzyme
-
N
a
NEW S. CEREVISIAE GLYCOLYTIC GENE EXPRESSION MUTANT
VOL. 10, 1990
6393
TABLE 3. Growth on plates Growth (colony size, mm) on plates containing:
SCb
R61"
RelevantGlcsGuoe vate lycrol
Strain genotype Strain
Glucose
2845 NW9-19-1
MWGL29 DFY641
Wild typec
2.1
gcr2-1 gcrl-6 Agcr2::URA3C
1.7 0.2 1.6
Fructose
Mannose
1.8 1.5
2.0 1.7
0.2 1.5
0.2 1.7
plusPyruplus lacvate
Ethanol
tate
No addition
1.0
0.8
0.7
0.6
0.7 0.4 0.5
0.8 0.8
0.6 0.6 0.5
0.5 0.4 0.4
0.7
Glucose (300C) 2.1 1.5
0.2 1.5
timycin A
Glucose (37'C)
2.0 1.1
