JOURNAL OF BACTERIOLOGY, Jan. 1976, p. 379-381 Copyright 0 1976 American Society for Microbiology
Vol. 125, No. 1 Printed in U.S.A.
Effect of GAL4 Gene Dosage on the Level of Galactose Catabolic Enzymes in Saccharomyces cerevisiae AMAR J. S. KLAR' AND HARLYN 0. HALVORSON* Department of Biology and Rosenstiel Basic Medical Sciences Research Center, * Brandeis University, Waltham, Massachusetts 02154 Received for publication 11 August 1975
Lack of GAL4 gene dosage on the level of uridine diphosphogalactose epimerase (EC 5.1.3.2) activity suggests the positive regulatory role for this locus on the control of galactose catabolic enzymes in Saccharomyces cerevisiae. In Saccharomyces cerevisiae, a regulatory system for galactose catabolic enzymes has been proposed in which a "positive" regulatory gene (GAL4) affects the expression of galactose1-phosphate uridyl transferase (EC 2.7.7.10), uridine diphosphogalactose epimerase (EC 5.1.3.2), and galactokinase (EC 2.7.1.6) activities. These activities are coded for by GAL7, GAL10, and GALl gene cluster unlinked to the GAL4 locus (1). Mutants in the GAL4 locus (gal4 strains) lack the above-mentioned enzyme activities and these activities simultaneoulsy reappear in the revertants obtained from the gal4 strains. Two kinds of models have been proposed to explain the role of the GAL4 locus. First, the GAL4 could be a regulatory gene similar to araC locus in Escherichia coli (2); second, it could code for a structural component (subunit) of above enzymes (1, 4). Our previous studies on a strain containing a conditional mutation (thermosensitive) mapping in or very close to the GAL4 locus argued against the hypothesis that the GAL4 gene codes for a protein component of these enzymes since the galactose catabolic enzymes extracted from the conditional mutant did not differ quantitatively in thermolability or temperature optima from that of the wild type (7). A proportional relationship between the number of wild-type genes and the amount of enzyme activity synthesized is a common effect of structural genes (3, 6, 8). By contrast a lack of gene dosage effect has been observed for two positive regulatory loci, ma-i+ and lxd+, which affect xanthine dehydrogenase, pyridoxal oxidase, and aldehyde oxidase in Drosophila melanogaster (3). Also, Helling and Weinberg (5) showed that the rate-limiting step for Larabinose isomerase induction was the araA but not the araC locus in E. coli, suggesting a positive regulatory role of the araC locus. If we I Present address: Department of Genetics, University of California, Berkeley, Calif. 94720.
accept the above generalization to be correct, i.e., only structural genes show the gene dosage effect, then we can distinguish the structural or the regulatory role of the GAL4 locus by studying the effect of the GAL4 gene dosage on the level of galactose catabolic enzymes. We expect that if the GAL4 locus codes for a structural component of the galactose catabolic enzymes, a reduction of the GAL4 gene protein level in vivo should reduce the level of these enzymes. To investigate this possibility the effect of the GAL4 gene dosage on the level of epimerase activity was studied. Strains with different ploidy, each containing only one wild-type GAL4 allele but wild type for all other galactose catabolic loci, were constructed. These cultures were grown to 100 Klett units in galactose medium and assayed for epimerase-specific activity. The specific activity of epimerase in diploid (GAL4/gal4) and triploid (GAL4/gal4/ gal4) strains heterozygous for the GAL4 locus is approximately the same as that of a wild-type haploid strain (Fig. 1). Similar results (Table 1) were obtained from tetraploid strains containing two or more than two GAL4 wild-type alleles: they contained the same level of epimerase activity. In contrast, Nelson and Douglas (8) in a similar experiment observed a definite gene dosage effect for the GALl locus, structural gene for galactokinase. The GAL4 gene dosage effect was observed only in the tetraploid (GAL4/ gal4/gal4/ga14) strain which has decreased specific activity of epimerase (Fig. 1). Probably in this strain the level of the GAL4 protein has fallen below a critical level so that it becomes a rate-limiting factor for the synthesis of epimerase. The specific activity of alcohol dehydrogenase in all these strains is constant (Fig. 1), indicating that the increase in ploidy and the GAL4 gene dosage do not affect the level of an enzyme unrelated to the galactose pathway. The growth rate of the haploid, diploid, and triploid strains in medium containing galactose 379
J. BACTERIOL.
NOTES
380
300
5
0.3
t
.4
02
3
4
~~~~~~~~~~~~~~~~~~~0.1 .E0
0~~~~~~~~~~~~~~ Ploidy Genotype
N +
2N +/-
3N +/-/-
4N +/-/-/-
FIG. 1. Effect of GAL4 gene dosage on the level of epimerase and alcohol dehydrogenase activity. Strains with different ploidy (IN to 4N) were grown in 100 ml of yeast extract peptone galactose medium to 100 Klett units (mid-exponential phase) at 30 C and were assayed for epimerase activity as reported earlier (7) and for alcohol dehydrogenase activity according to the method used by Vallee and Hoch (10). The specific activity (units/milligram of protein) of epimerase (filled bars) and alcohol dehydrogenase (open bars) in the cell-free extract is plotted. One unit of epimerase activity was defined as the amount which caused an increase in optical density at 340 nm by 0.001 per min at 30 C. One unit of alcohol dehydrogenase was defined as the amount that reduces 1 /Lmol of nicotinamide adenine dinucleotide per min at 30 C. IN, Haploid wild-type; 2N, diploid GAL4/gal4; 3N, triploid GAL4/gal4/gal4; 4N, tetraploid GAL4/gal4/gal4/gal4. + and Wild-type GAL4 and its mutant allele gal4, respectively.
44
c
0
yg
-,
0
as the only carbon source was the same (generation time 2 h and 20 min) but the tetraploid strain grew more slowly (generation time 7 h and 30 min, Fig. 2A). This observation agrees with the decrease in the specific activity of epimerase observed only in tetraploid strain. All these strains grew with similar growth rate in medium containing glucose as the only carbon source (Fig. 2B). By analyzing the deadaptation kinetics of the galactose-induced cultures of yeast, Tsuyumu and Adams (9) suggested that the Leloir pathway enzymes are aggregated in vivo and the
5
10
15
20
25
30
Growth (hr )
FIG. 2. Growth of strains with different ploidy and GAL4 gene dosage on galactose- and glucose-containing media. Yeast strains (described in Fig. 1 legend) of different ploidy and GAL4 gene dosage were grown in (A) complete medium with galactose or (B) complete medium with glucose at 30 C as described earlier (7). At intervals Klett units for each culture were measured. Symbols: 0, 1N; 0, 2N; A, 3N; A, 4N.
product of the GAL4 locus functions to facilitate the function of this aggregate presumably by forming an aggregate with Leloir pathway enzymes. Their results showed that in a diploid
TABLE 1. Epimerase and alcohol dehydrogenase activity and the effect of carbon source on the growth rate of tetraploid strains of yeasta Strain no.
Genotype
Epimerase sp act
Alcohol dehydrogenase sp act
1 2 3 4
GAL4/GAL4/GAL4/GAL4 GAL4/GAL4/GAL4/gal4 GAL4/GAL4/gal4/gal4 GAL4/gal4/gal4/gal4
4,200 4,500 3,750 1,050
0.329 0.332 0.350 0.350
a
For details see the legend to Fig. 1 and 2, and the text.
Generation time (h) Galactose Glucose
2.10 2.10 2.10 7.30
1.55 1.55 1.55 1.55
VOL. 125, 1976
strain heterozygous for the GAL4 locus the average number of functional aggregates (induction units) become 1/64th of the units contained in homozygous GAL4 wild-type diploid strains. Our results, however, on a heterozygous diploid and a triploid strain, each containing only one wild-type GAL4 allele, showed the same specific activity of epimerase as that of the wild-type haploid strain. Lack of GAL4 gene dosage on the level of epimerase activity favors the positive regulatory role for this locus on the control of galactose catabolic enzymes in S. cerevisiae but does not rule out the unlikely possibility that this locus codes for a structural component of the galactose catabolic enzymes in excess. The results in this paper are consistent with the previous conclusions (7) that the GAL4 locus codes for a positive regulatory protein as originally proposed by Douglas and Hawthorne (1).
This investigation was supported by a Public Health Service research grant (AI10610) from the National Institute of Allergy and Infectious Diseases.
NOTES
381
LITERATURE CITED 1. Douglas, H. C., and D. C. Hawthorne. 1966. Regulation of genes controlling synthesis of the galactose pathway enzymes in yeast. Genetics 54:911-916. 2. Englesberg, E., and G. Wilcox. 1974. Regulation: positive control. Annu. Rev. Genet. 8:219-242. 3. Glassman, E. 1965. Genetic regulation of xanthine dehydrogenase in Drosophila melanogaster. Fed. Proc. 24:1243-1251. 4. Hartwell, L. H. 1970. Biochemical genetics of yeast. Annu. Rev. Biochem. 39:373-396. 5. Helling, R. B., and R. Weinberg. 1963. Complementation studies of arabinose genes in Escherichia coli. Genetics 48:1397-1410. 6. Horiuchi, R., S. Horiuchi, and A. Novick. 1963. The genetic basis of hyper synthesis of beta-galactosidase. Genetics 48:157-167. 7. Klar, A. J. S., and H. 0. Halvorson. 1974. Studies on the positive regulatory gene, GAL4, in regulation of galactose catabolic enzymes in Saccharomyces cerevisiae. Mol. Gen. Genet. 125:203-212. 8. Nelson, N. M., and H. C. Douglas. 1963. Gene dosage and galactose utilization by Saccharomyces tetraploids. Genetics 48:1585-1591. 9. Tsuyumu, S., and B. G. Adams. 1974. Dilution kinetic studies of yeast populations: in vivo aggregation of galactose utilizing enzymes and positive regulator molecules. Genetics 77:491-505. 10. Vallee, B. L., and F. L. Hoch. 1955. Zinc, a component of yeast alcohol dehydrogenase. Proc. Natl. Acad. Sci. U.S.A. 68:1555-1559.
Vol. 125, No. 1 Printed in U.S.A.
Effect of GAL4 Gene Dosage on the Level of Galactose Catabolic Enzymes in Saccharomyces cerevisiae AMAR J. S. KLAR' AND HARLYN 0. HALVORSON* Department of Biology and Rosenstiel Basic Medical Sciences Research Center, * Brandeis University, Waltham, Massachusetts 02154 Received for publication 11 August 1975
Lack of GAL4 gene dosage on the level of uridine diphosphogalactose epimerase (EC 5.1.3.2) activity suggests the positive regulatory role for this locus on the control of galactose catabolic enzymes in Saccharomyces cerevisiae. In Saccharomyces cerevisiae, a regulatory system for galactose catabolic enzymes has been proposed in which a "positive" regulatory gene (GAL4) affects the expression of galactose1-phosphate uridyl transferase (EC 2.7.7.10), uridine diphosphogalactose epimerase (EC 5.1.3.2), and galactokinase (EC 2.7.1.6) activities. These activities are coded for by GAL7, GAL10, and GALl gene cluster unlinked to the GAL4 locus (1). Mutants in the GAL4 locus (gal4 strains) lack the above-mentioned enzyme activities and these activities simultaneoulsy reappear in the revertants obtained from the gal4 strains. Two kinds of models have been proposed to explain the role of the GAL4 locus. First, the GAL4 could be a regulatory gene similar to araC locus in Escherichia coli (2); second, it could code for a structural component (subunit) of above enzymes (1, 4). Our previous studies on a strain containing a conditional mutation (thermosensitive) mapping in or very close to the GAL4 locus argued against the hypothesis that the GAL4 gene codes for a protein component of these enzymes since the galactose catabolic enzymes extracted from the conditional mutant did not differ quantitatively in thermolability or temperature optima from that of the wild type (7). A proportional relationship between the number of wild-type genes and the amount of enzyme activity synthesized is a common effect of structural genes (3, 6, 8). By contrast a lack of gene dosage effect has been observed for two positive regulatory loci, ma-i+ and lxd+, which affect xanthine dehydrogenase, pyridoxal oxidase, and aldehyde oxidase in Drosophila melanogaster (3). Also, Helling and Weinberg (5) showed that the rate-limiting step for Larabinose isomerase induction was the araA but not the araC locus in E. coli, suggesting a positive regulatory role of the araC locus. If we I Present address: Department of Genetics, University of California, Berkeley, Calif. 94720.
accept the above generalization to be correct, i.e., only structural genes show the gene dosage effect, then we can distinguish the structural or the regulatory role of the GAL4 locus by studying the effect of the GAL4 gene dosage on the level of galactose catabolic enzymes. We expect that if the GAL4 locus codes for a structural component of the galactose catabolic enzymes, a reduction of the GAL4 gene protein level in vivo should reduce the level of these enzymes. To investigate this possibility the effect of the GAL4 gene dosage on the level of epimerase activity was studied. Strains with different ploidy, each containing only one wild-type GAL4 allele but wild type for all other galactose catabolic loci, were constructed. These cultures were grown to 100 Klett units in galactose medium and assayed for epimerase-specific activity. The specific activity of epimerase in diploid (GAL4/gal4) and triploid (GAL4/gal4/ gal4) strains heterozygous for the GAL4 locus is approximately the same as that of a wild-type haploid strain (Fig. 1). Similar results (Table 1) were obtained from tetraploid strains containing two or more than two GAL4 wild-type alleles: they contained the same level of epimerase activity. In contrast, Nelson and Douglas (8) in a similar experiment observed a definite gene dosage effect for the GALl locus, structural gene for galactokinase. The GAL4 gene dosage effect was observed only in the tetraploid (GAL4/ gal4/gal4/ga14) strain which has decreased specific activity of epimerase (Fig. 1). Probably in this strain the level of the GAL4 protein has fallen below a critical level so that it becomes a rate-limiting factor for the synthesis of epimerase. The specific activity of alcohol dehydrogenase in all these strains is constant (Fig. 1), indicating that the increase in ploidy and the GAL4 gene dosage do not affect the level of an enzyme unrelated to the galactose pathway. The growth rate of the haploid, diploid, and triploid strains in medium containing galactose 379
J. BACTERIOL.
NOTES
380
300
5
0.3
t
.4
02
3
4
~~~~~~~~~~~~~~~~~~~0.1 .E0
0~~~~~~~~~~~~~~ Ploidy Genotype
N +
2N +/-
3N +/-/-
4N +/-/-/-
FIG. 1. Effect of GAL4 gene dosage on the level of epimerase and alcohol dehydrogenase activity. Strains with different ploidy (IN to 4N) were grown in 100 ml of yeast extract peptone galactose medium to 100 Klett units (mid-exponential phase) at 30 C and were assayed for epimerase activity as reported earlier (7) and for alcohol dehydrogenase activity according to the method used by Vallee and Hoch (10). The specific activity (units/milligram of protein) of epimerase (filled bars) and alcohol dehydrogenase (open bars) in the cell-free extract is plotted. One unit of epimerase activity was defined as the amount which caused an increase in optical density at 340 nm by 0.001 per min at 30 C. One unit of alcohol dehydrogenase was defined as the amount that reduces 1 /Lmol of nicotinamide adenine dinucleotide per min at 30 C. IN, Haploid wild-type; 2N, diploid GAL4/gal4; 3N, triploid GAL4/gal4/gal4; 4N, tetraploid GAL4/gal4/gal4/gal4. + and Wild-type GAL4 and its mutant allele gal4, respectively.
44
c
0
yg
-,
0
as the only carbon source was the same (generation time 2 h and 20 min) but the tetraploid strain grew more slowly (generation time 7 h and 30 min, Fig. 2A). This observation agrees with the decrease in the specific activity of epimerase observed only in tetraploid strain. All these strains grew with similar growth rate in medium containing glucose as the only carbon source (Fig. 2B). By analyzing the deadaptation kinetics of the galactose-induced cultures of yeast, Tsuyumu and Adams (9) suggested that the Leloir pathway enzymes are aggregated in vivo and the
5
10
15
20
25
30
Growth (hr )
FIG. 2. Growth of strains with different ploidy and GAL4 gene dosage on galactose- and glucose-containing media. Yeast strains (described in Fig. 1 legend) of different ploidy and GAL4 gene dosage were grown in (A) complete medium with galactose or (B) complete medium with glucose at 30 C as described earlier (7). At intervals Klett units for each culture were measured. Symbols: 0, 1N; 0, 2N; A, 3N; A, 4N.
product of the GAL4 locus functions to facilitate the function of this aggregate presumably by forming an aggregate with Leloir pathway enzymes. Their results showed that in a diploid
TABLE 1. Epimerase and alcohol dehydrogenase activity and the effect of carbon source on the growth rate of tetraploid strains of yeasta Strain no.
Genotype
Epimerase sp act
Alcohol dehydrogenase sp act
1 2 3 4
GAL4/GAL4/GAL4/GAL4 GAL4/GAL4/GAL4/gal4 GAL4/GAL4/gal4/gal4 GAL4/gal4/gal4/gal4
4,200 4,500 3,750 1,050
0.329 0.332 0.350 0.350
a
For details see the legend to Fig. 1 and 2, and the text.
Generation time (h) Galactose Glucose
2.10 2.10 2.10 7.30
1.55 1.55 1.55 1.55
VOL. 125, 1976
strain heterozygous for the GAL4 locus the average number of functional aggregates (induction units) become 1/64th of the units contained in homozygous GAL4 wild-type diploid strains. Our results, however, on a heterozygous diploid and a triploid strain, each containing only one wild-type GAL4 allele, showed the same specific activity of epimerase as that of the wild-type haploid strain. Lack of GAL4 gene dosage on the level of epimerase activity favors the positive regulatory role for this locus on the control of galactose catabolic enzymes in S. cerevisiae but does not rule out the unlikely possibility that this locus codes for a structural component of the galactose catabolic enzymes in excess. The results in this paper are consistent with the previous conclusions (7) that the GAL4 locus codes for a positive regulatory protein as originally proposed by Douglas and Hawthorne (1).
This investigation was supported by a Public Health Service research grant (AI10610) from the National Institute of Allergy and Infectious Diseases.
NOTES
381
LITERATURE CITED 1. Douglas, H. C., and D. C. Hawthorne. 1966. Regulation of genes controlling synthesis of the galactose pathway enzymes in yeast. Genetics 54:911-916. 2. Englesberg, E., and G. Wilcox. 1974. Regulation: positive control. Annu. Rev. Genet. 8:219-242. 3. Glassman, E. 1965. Genetic regulation of xanthine dehydrogenase in Drosophila melanogaster. Fed. Proc. 24:1243-1251. 4. Hartwell, L. H. 1970. Biochemical genetics of yeast. Annu. Rev. Biochem. 39:373-396. 5. Helling, R. B., and R. Weinberg. 1963. Complementation studies of arabinose genes in Escherichia coli. Genetics 48:1397-1410. 6. Horiuchi, R., S. Horiuchi, and A. Novick. 1963. The genetic basis of hyper synthesis of beta-galactosidase. Genetics 48:157-167. 7. Klar, A. J. S., and H. 0. Halvorson. 1974. Studies on the positive regulatory gene, GAL4, in regulation of galactose catabolic enzymes in Saccharomyces cerevisiae. Mol. Gen. Genet. 125:203-212. 8. Nelson, N. M., and H. C. Douglas. 1963. Gene dosage and galactose utilization by Saccharomyces tetraploids. Genetics 48:1585-1591. 9. Tsuyumu, S., and B. G. Adams. 1974. Dilution kinetic studies of yeast populations: in vivo aggregation of galactose utilizing enzymes and positive regulator molecules. Genetics 77:491-505. 10. Vallee, B. L., and F. L. Hoch. 1955. Zinc, a component of yeast alcohol dehydrogenase. Proc. Natl. Acad. Sci. U.S.A. 68:1555-1559.
