GAL3 Gene Product Is Required for Maintenance of the Induced State of the GAL Cluster Genes in Saccharomyces cerevisiae

Transcription

1 JOURNAL OF BACTERIOLOGY, Jan. 1986, p /86/ $02.00/0 Copyright 1986, American Society for Microbiology Vol. 165, No. 1 GAL3 Gene Product Is Required for Maintenance of the Induced State of the GAL Cluster Genes in Saccharomyces cerevisiae YASUHISA NOGI Laboratory of Molecular Genetics, Keio University School of Medicine, Shinjuku, Tokyo 160, Japan Received 23 May 1985/Accepted 21 October 1985 The activities of the first three enzymes for galactose catabolism normally become detectable within 15 min after the addition of galactose into a culture of the yeast Saccharomyces cerevisiae. In S. cerevisiae with a recessive mutation termed galb, a longer-than-normal lag is observed before the appearance of the enzyme activities (0. Winge and C. Roberts, C. R. Trav. Lab. Carlsberg Ser. Physiol. 24: , 1948). I isolated two S. cerevisiae mutants with temperature-sensitive defects in the GAL3 gene. Temperature shift experiments with one of those mutants led to the conclusion that the GAL3 function is required not only for the initiation of enzyme induction but also for the maintenance of the induced state in galactose-nonfermenting S. cerevisiae because of a defect in any of the genes for the galactose-catabolizing enzymes, such as gall or gal10. In contrast, the GAL3 function is phenotypically dispensable in galactose-metabolizing S. cerevisiae. Thus, the normal catabolism of galactose can substitute for the GAL3 function. Expression of the genes GAL], GAL7, GALIO, and MELJ encoding, respectively, galactokinase, galactose-1-phosphate uridyltransferase (transferase), UDP-glucose-4- epimerase (epimerase), and a-galactosidase is coordinately regulated at the transcriptional level in the eucaryotic microorganism Saccharomyces cerevisiae (22, 25-27). The three genes GAL], GAL7, and GALIO (collectively named GAL cluster genes) are tightly clustered on chromosome 11 (2, 8, 25). The activities of the enzymes encoded by the GAL cluster genes (collectively called Gal enzymes) are induced coordinately within 15 min after the addition of galactose to the culture (1, 4). This rapid and coordinate induction is under the control of at least three regulatory genes; GAL4 located on chromosome XVI, GAL80 located on chromosome XIII, and GAL3 located on chromosome IV (8, 10, 14, 23). The former two genes have been well characterized, and working hypotheses involving the interaction between the GAL4 product, which acts positively, and the GAL80 product, which acts negatively, have been proposed (11, 15, 19, 20, 21, 27, 30). The GAL4 gene works at the transcriptional level (12, 22, 25). In contrast, the role of the GAL3 gene is totally obscure. The known properties of the recessive mutation (gal3) in that gene can be summarized as follows. (i) The mutation, if it exists in respiratory-competent S. cerevisiae, causes a longer-than-normal lag period before the onset of galactose fermentation (29). Once the gal3 mutant adapts to galactose, it can ferment galactose as efficiently as GAL3 strains. In contrast, a respiratory-deficient gal3 mutant is entirely unable to ferment galactose (10). (ii) Doublemutants, such as gal80 gal3 or GAL4C gal3, can be isolated as galactose-fermenting revertants from the respiratorydeficient gal3 mutant, showing that the constitutive mutations suppress the gal3 mutation (9, 10). (iii) In respiratorycompetent gal3 strains, mutations in any one of the GAL cluster genes completely eliminate the expression of the other two genes (4). Although no unitary explanation for all these findings is available at this moment, it can be reasonably assumed that the GAL3 function is necessary for initiating the induction of the GAL cluster genes and also MEL] (14). However, it is 101 unclear whether the GAL3 function is required for the maintenance of the induced state of those genes. In this study, I isolated two temperature-sensitive gal3 mutants and performed temperature shift experiments with one of those mutants under the background of GAL', gall, or galll10. The results suggested that the GAL3 function is required for the maintenance of the induced state in gall or gallo S. cerevisiae and further that galactose catabolism can compensate for the gal3 deficiency. Possible roles of the GAL3 gene in the regulation of the GAL cluster genes will be discussed. MATERIALS AND METHODS Strains. The S. cerevisiae strains used in the present paper are listed in Table 1. Media. YPEth medium contained 2% Polypeptone (BBL Microbiology Systems), 1% yeast extract, and 0.5% ethanol. YPEthGal medium contained 0.8% galactose in YPEth medium. YPGly medium contained 3% glycerol in place of the ethanol in YPEth medium. YPGlyGal medium contained 2% galactose in YPGly medium. YPGal medium contained 2% galactose in place of the ethanol in YPEth medium. EBGal medium contained 0.002% ethidium bromide in YPGal medium. YPD medium contained 2% dextrose in place of the ethanol in YPEth medium. Solid media contained 2% agar. Genetic techniques. Mating, diploid isolation, sporulation, and tetrad analysis were performed as described previously (18). The presence of the gal3 mutation was scored on EBGal plates (14). Enzyme assays. Galactose-1-phosphate uridyltransferase (transferase) activity was assayed as described previously (17) with [14C]galactose 1-phosphate as the substrate. As the enzyme source, cells permeabilized for the substrate were prepared with dimethyl sulfoxide (1). One unit of transferase activity was defined as the amount of enzyme that produced 1 nmol of UDP-galactose in 1 h at 30 C. Selection of temperature-sensitive gals mutations. Respiratory-competent gallo mutants are sensitive to galactose (Gals); they can grow in YPEth medium but not in YPEthGal medium (8). Any mutation that reduces galactokinase activity will make gallo mutants resistant to galactose (Gal9). Spontaneous Galr mutants are easily isolated by plating

2 102 NOGI TABLE 1. S. cerevisiae strains used in this study Strain Genotype Origin or source 100-SB a gal/o ade2 trpl H. C. Douglas 107-1D a ga/3 ural trpl his] met thr H. C. Douglas N3-3D a ga/3 gallo trpl met thr 100-SB x 107-1D N3-13B a ga/3 gal/o trpl his] ade thr 100-SB x 107-1D N31-3-2C a gal3-31 gal/o trpl ade met thr This study N3-2B a gallo trpl ural hisi ade thr met 100-SB x 107-1D N36-7-4D a ga/3-31 gal/o trpl ade This study N31-3-2D a gal3-31 gallo trpl thr met This study N36-7-3D a ga/3-31 gallo trpl ade thr This study D273-11A a adel his] Cold Spring Harbor Laboratory 106-3D a ga/80 ural hisj H. C. Douglas N517-1OD a ga/3-31 trpl hisl ade met N31-3-2C x D273-llA N517-6D a ga/3-31 trpl ade met N31-3-2C x D273-11A N31-3-3D a ga/3-31 gal/o trpj ade thr This study 121-2D at gall trpl ade6 H. C. Douglas N516-8C a ga/3-31 gall trpl N31-3-3D x 121-2D N516-3B ao ga/3-31 gal/o trpl ade N31-3-3D x 121-2D galjo S. cerevisiae on YPEthGal medium. In addition to gal/o, any of the mutations gall, gal3, gal4, or GAL80S would cause the Galr phenotype. To select gal3 mutations efficiently, I constructed diploids carrying the gal3igal3 gal/olgal/o genotype. Galr colonies from the diploids would bear the gal3/gal3-x gal/olgal/o genotype. If the gal3-x mutation is of a temperature-sensitive nature, the mutants would be Galr at a high temperature but Gals at a low temperature. Diploid cells carrying the ga/31gal3 gal/olgal/o genotype were grown to a density of about 3 x 107/ml, and 0.1-ml portions were spread on YPEthGal plates. The plates were incubated for 5 days at 36 C. Galr colonies that appeared at a frequency of about 10-4 were tested for Gals at 36 and 25 C. Further characterization is given below. Isolation of respiratory-deficient mutants. Respiratorycompetent S. cerevisiae was grown in 2 ml of YPD medium containing 0.005% ethidium bromide for 2 days at 30 C and then plated on YPD agar after appropriate dilution. Respiratory-deficient colonies were identified by their inability to grow in YPEth medium. RESULTS Isolation of temperature-sensitive gal3 mutants. Based on the rationale described in Materials and Methods, diploids carrying the ga/31gal3 gal/olgal/o genotype were constructed: N31 and N36 were obtained by a cross of 100-5B with N3-3D and by a cross of 100-5B with N3-13B, respectively. Cultures of N31 or N36 were spread on YPEthGal plates, and the plates were incubated at 36 C for 5 days. The resultant 800 Galr colonies from N31 and 400 Galr colonies from N36 were restreaked on YPEthGal plates and incubated at 36 C. Each isolate was replica plated on YPEthGal plates and incubated at 25 C to screen the mutants that could grow on YPEthGal at 36 C but not at 25 C. Isolate N31-3 from N31 and isolate N36-7 from N36 were found to be temperature sensitive for the Galr phenotype: they could grow on YPEthGal at 36 C but not at 25 C. The two mutants were subjected to tetrad analysis after sporulation. All of the 24 asci from N31-3 and the 15 asci from N36-7 showed 4+:0- at 36 C and 2+:2- segregation at 25 C for growth on J. BACTERIOL. YPEthGal. This indicated that the genetic defect that caused Galr in N31-3 or N36-7 was allelic to the GAL3 locus, that is, the temperature-sensitive gal3 mutation. The mutant allele derived from N31-3 was denoted gal3-31, and that from N36 was denoted gal3-36. Both mutations were recessive to the wild-type counterpart because both the diploid (N31-3-2C [a gal3-31 gal/o], a meiotic segregant of N31-3, x N3-2B [a GAL3 gal/o]) and the diploid (N36-7-4D [a gal3-36 gal/o], a meiotic segregant of N36-7, x N3-2B [a GAL3 gal/o]) were Gals at either temperature. The gal3-31 allele did not complement with the gal3-36 allele; the diploid (N31-3-2D [a gal3-31 gal/o] x N36-7-3D [a gal3-36 gal/o]) was Galr at 36 C but Gals at 25 C. Since the two mutant alleles seemed phenotypically identical to each other, only the gal3-31 mutant was studied in detail. The gal3-31 mutant allele was separated from the gal/o mutant allele through meiotic segregation of the diploid N517 (N31-3-2C [a gal3-31 gal/o trpl] x D273-11A [a GAL3 GALJO TRPI]). The ratio of parental ditype/nonparental ditype/tetratype for Galr:Gals segregation at 36 C was 3:6:1 in 10 dissected asci (GAL3 and GALIO loci are linked to centromeres IV and II, respectively). The trpj marker was expected to cosegregate with the gal3-31 allele because of the close linkage between TRPI and GAL3 (8), and in fact all the gal3-31 gal/o meiotic segregants studied were trpl. Thus, two gal3-31 GALIO trpj and two GAL3 gal/o TRPI spore clones were obtained in the asci showing nonparental ditype Galr:Gals segregation. Unexpectedly, however, the assumed gal3-31 segregants could grow on EBGal plates as well as the GAL3 strains (D273-11A and 106-3D) at 36 C after pregrowth at 36 C in YPD medium. To confirm the presence of the gal3-31 allele in the segregants, I converted two presumptive gal3-31 segregants, N517-1OD and N517-6D, to respiratory deficiency with the ethidium bromide treatment. Remember that the effect of the gal3 mutation on the expression of the GAL cluster genes is more NHurs FIG. 1. Time course of transferase induction in the gal3-31 mutant. (A) Transferase activity; (B) cell growth. The abscissas show the time after the addition of galactose. Cells of strain NS17-1OD were grown at 25 C (0) or 36 C (0) in YPGly medium to an ODw of about 0.3 (at 25 C) or 0.1 (at 36 C). Galactose was added to 2% to each culture at zero hour, and shaking of the cultures was continued. Transferase activity was determined at appropriate times with permeabilized cells as the enzyme source (A), and cell growth was monitored by reading the OD60 of the culture (B).

3 VOL. 165, 1986 remarkable in respiratory-deficient mutants than in respiratory-competent mutants (10). Neither respiratory-deficient derivative grew on YPGal plates during incubation at 36 C for 2 days; they gave rise to visible colonies on day 4 after inoculation. Kinetics of induction of transferase synthesis in gab3-31 mutants at various temperatures. Cells of strain N517-1OD (gal3-31) were grown in YPGly medium at 25 or 37 C. Galactose was added to a final concentration of 2% into the logarithmically growing cultures, and then samples were taken from each culture for the determination of transferase activity at various times (Fig. 1). The enzyme activity became detectable at about 20 min after the addition of galactose in cells grown at 25 C. However, cells grown at 360C had detectable transferase activity in 2 to 3 h after the addition of galactose. Furthermore, the rate of transferase synthesis was markedly lower at 36 C than at 250C. In contrast, the lag time and the initial rate of transferase synthesis were not significantly affected by the growth temperature in the wild-type strain D273-11A (Fig. 2). Since it is known that S. cerevisiae doubly defective in the GAL3 gene and in any of the GAL cluster genes will not synthesize the enzymes encoded by the other two genes in galactose-containing medium (4), the induced synthesis of transferase was investigated in the gal3-31 gall mutant at 25 or 36 C (Fig. 3). Cells of strain N516-8C grown in YPGly medium at 36 C did not synthesize any trace activity of transferase for 24 h (four generations) after the addition of galactose, indicating that the block of gal3-31 was practically complete. As a control, mutant cells grown at 25 C produced transferase normally upon the addition of galactose. This suggests that the delayed but successful synthesis of transferase in the gal3-31 mutant seen in Fig. 1 was due to the presence of the intact GAL] gene. FIG. 2. Time course of transferase induction in the wild-type strain. (A) Transferase activity; (B) cell growth. The abscissas show the time after the addition of galactose. Note that the time between 0 and 60 min in panel A corresponds to the time between 0 and 1 h in panel B. Cells of strain D273-11A were grown at 25 C (0) or 36 C (-) in YPGly medium to an OD6w of about 0.3. Galactose was added to 2% to each culture at zero minute, and shaking of the cultures was continued. Transferase activity was determined at appropriate times with permeabilized cells as the enzyme source (A), and cell growth was monitored by reading the OD6w of the culture (B). The arrow in panel B indicates the time of addition of galactose. TEMPERATURE-SENSITIVE gal3 MUTATION 103 ISOA --A II 1100 U E IO _I 0.1 I 4 8 lz 1Z4 FIG. 3. Time course of transferase induction in the gal3-31 gall mutant. (A) Transferase activity; (B) cell growth. The abscissas show the time after the addition of galactose. Cells of strain N516-8C (gai3-31 gall) were grown at 25 C (0) or 36 C (O) in YPGly medium to an OD6w of about 0.1 (at 25 C) or 0.05 (at 36 C). Galactose was added to 2% to each culture at 0 h, and shaking of the cultures was continued. Transferase activity was determined at appropriate times with permeabilized cells as the enzyme source (A), and cell growth was,monitored by reading the QD6w of the culture (B) g50~~~~~~5 0~~~~~~0 ODg6op D los %Ml r%. -8 A - I --A ODOGpg* FIG. 4. Effect of a temperature shift on transferase synthesis in mutants carrying the gal3-31 or gal3-31 gall genotype. Transferase activity is shown as a function of cell growth in panels A and B. Note that the scale of the ordinates is different in panels A and B. Cells of strains N517-10D (gal3-31) (A and C) and N516-8C (gal3-31 gall) (B and D) were grown at 25 C in YPGlyGal medium. When the growth of each strain reached an OD660 of about 0.1 (arrows), each culture was divided into two parts. One part was kept at 25 C (0), and the other part was shifted to 36 C (0); shaking of the cultures was continued. Transferase activity was determined at appropriate times with permeabilized cells as the enzyme source (A and B), and cell growth was monitored by' reading the ODw of the culture (C and D). The abscissas in panels C and D show the time after the addition of galactose.

4 104 NOGI 30F --l 20-0.' r Nlours FIG. 5. Effect of a temperature shift on transferase synthesis in mutants carrying the gal3-31, gal3-31 gall, or gal3-31 gallo genotype. (A, B, and C) Transferase activity; (D, E, and F) cell growth. The abscissas show the time after the addition of galactose. Cells of strains N517-1OD (gal3-31), (A and D) N516-8C (gal3-31 gall) (B and E), and N516-3B (gal3-31 gallo) (C and F) were grown at 25 C in YPGly medium. When the growth of each strain reached an OD6w of about 0.3, galactose was added to 2% to each culture (at zero hour), and shaking of the cultures was continued. After 1 h of incubation (arrows), each culture was divided into two parts. One part was kept at 25 C (0), and the other part was shifted to 36 C (0); shaking of the cultures was continued. Transferase activity was determined at appropriate times with permeabilized cells as the enzyme source (A, B, and C), and cell growth was monitored by reading the OD6w of the culture (D, E, and F). Temperature-shift experiences with gal3-31 mutants previously adapted to galactose. To understand the effect of the gal3-31 mutation on the maintenance of the induced state, I grew cells of strains N517-1OD (gal3-31) and N516-8C (gal3-31 gall) in YPGlyGal medium at 25 C to an optical density at 660 nm (OD6w) of about 0.1. Each culture was split into two equal parts. One part was shifted to 36 C, and the other was kept at 25 C. The time course of transferase synthesis was monitored in each culture (Fig. 4). The inactivation of the GAL3 product in the gal3-31 mutant did not result in a marked reduction in transferase synthesis; the differential rate of transferase synthesis in the mutant culture decreased, but only slightly, as a result of the temperature shift, as compared with the culture kept at 25 C (Fig. 4A). This indicates that the GAL3 function is not a prerequisite for the maintenance of the induced state of the GAL cluster genes in galactose-catabolizing cells. A remarkable result was obtained when the gal3-31 gall mutant was shifted to 36 C. When cells of strain N516-8C grown in YPGlyGal medium at 25 C were shifted to 36 C, the production of transferase was halted rapidly after the shift, whereas the culture kept at 25 C continued to synthesize transferase at a normal rate (Fig. 4B). This result indicates that the GAL3 function is indispensable for the maintenance of the induced state in cells in which galactose catabolism is blocked by the gall mutation. One might argue that the elimination of GAL2 activity, which is also regulated by the GAL3 function (5), caused the observed deinduction. Such a possibility was unlikely because the gal3-31 mutant produced transferase under similar conditions (Fig. 4A) I 0.5. I F :I A similar experiment was carried out with the gal3-31 galjo mutant. Since the mutant was Gals at 25 C because of the defect in GALIO, cells were grown for a short ime (1 h) in the presence of galactose at 25 C and then shifted to 36 C. As a control, cells of gal3-31 or gal3-31 gall were subjected to the same temperature shift as gal3-31 galio (Fig. 5). Cells of strains N517-10D (gal3-31), N516-8C (gal3-31 gall), or N516-3B (gal3-31 gallo) were grown in YPGly medium at 25 C. When the cell density reached an OD6w of about 0.3, galactose was added to a final concentration of 2%. After 1 h, each culture was split into two equal portions, one of which was shifted to 36 C and the other of which kept at 25 C. Samples were removed from each culture for the transferase assay at various times. Figure 5A shows that the rate of transferase synthesis in the gal3-31 mutant was not affected by the temperature shift, supporting the above conclusion that the GAL3 function is dispensable in galactosecatabolizing cells. This result also shows that the induction period was sufficient for establishing the induced state. Figure 5B shows that transferase production stopped in the gal3-31 gall mutant rapidly after the shift to 36 C, again indicating that the GAL3 function is essential for the maintenance of the induced state in the gall mutant (Fig. 4B). Similarly, transferase activity leveled off in the gal3-31 gallo mutant when the temperature was shifted to 36 C, indicating that the GAL3 function is required for the maintenance of the induced state in the gallo mutant, too (Fig. SC). Effect of an immediate temperature shift after the addition of galactose on transferase induction in gal3-31 mutants. I examined whether the induction of Gal enzymes was normally initiated in S. cerevisiae with the temperaturesensitive gal3 mutation when the growth temperature was shifted to 36 C immediately after the addition of galactose. The gal3-31 mutant was grown at 25 C in YPGly medium. 303III D 20 c '-I 1C D - O.' 0 0.; 012 Ho. ur O FIG. 6. Effect of an immediate temperature shift after the addition of galactose on transferase synthesis in mutants carrying the gal3-31, gal3-31 gall, or gal3-31 ga/jo genotype. (A, B, and C) Transferase activity; (D, E, and F) cell growth. The abscissas show the time after the addition of galactose. Cells of strains N517-1OD (gal3-31), (A and D) N516-8C (gal3-31 gall) (B and E), and N31-3-3D (gal3-31 ga/jo) (C and F) were grown at 25 C in YPGly medium. When the cell density reached an ODw of about 0.3, galactose was added to 2% to each culture (at zero hour), and each culture was immediately split into equal parts. One part was kept at 25 C (0), and the other part was shifted to 36 C (0). Transferase activity was determined at appropriate times with permeabilized cells as the enzyme source (A, B, and C). Cell growth was monitored by reading the OD6w of the culture (D, E, and F). 5 - F J. BACTERIOL.

5 VOL. 165, 1986 TEMPERATURE-SENSITIVE gal3 MUTATION 105 glyc lysis glucose-6-phosphate galactokinase transferase phosphoglucoeutase galactose qalactose-l-phosphate glucose-i-phosphate ATP ADP UDP-glucose UDP-galactose epimerase FIG. 7. Pathway of galactose catabolism and the enzymes involved. Phosphoglucomutase is produced constitutively and encoded by the GALS gene in S. cerevisiae (7). Galactose was added to 2% to an exponentially growing culture, and the culture was immediately split into two equal parts. One was shifted to 36 C, and the other was kept at 25 C. The time course of transferase synthesis was followed in each culture (Fig. 6A). The culture shifted to 36 C showed normal kinetics of transferase induction, as did the culture kept at 25 C. Similar experiments were carried out with mutants bearing gal3-31 gall (Fig. 6B) or gal3-31 gallo (Fig. 6C), in both of which the galactose pathway was blocked by the additional mutations. Transferase activity was induced with a normal profile in the temperature-shifted cultures for the initial 1 or 2 h and then abruptly leveled off. In contrast, in control cultures kept at 25 C transferase activity continued to increase for at least 3 h. These results may be interpreted to imply that a normal GAL3 activity sufficient for initiating the normal induction of Gal enzymes was present at the time of the temperature shift in gal3-31 mutants pregrown at 25 C. Once induction was initiated to turn on the GAL cluster genes, the induced state was maintained without a functional GAL product at 36 C, provided that galactose was catabolized. In this context, it should be mentioned that the GAL3 transcript has been shown to be present in glucose-grown S. cerevisiae (24). In other words, GAL3 is expressed constitutively, consistent with the present results. DISCUSSION It is known that the GAL3 function is required for the initiation of the induction of the genes GAL], GAL7, and GAL1O. However, it was not clear whether the GAL3 function was necessary in S. cerevisiae in which those GAL cluster genes had been induced. The temperature-sensitive mutant (gal3-31) isolated in the present work enabled me to resolve this question. I demonstrated that upon temperature shift, synthesis of the GAL7-encoded enzyme (transferase) was rapidly shut off in mutants carrying the gal3-31 gall or gal3-31 gallo mutation and grown in galactose-containing medium at the permissive temperature. Such a deinduction was not seen in the mutant with a defect of gal3-31 alone under similar experimental conditions. These results led to the inferences that the GAL3 function is indispensable for the maintenance of the induced state of the GAL cluster genes in galactose-noncatabolizing S. cerevisiae and that galactose catabolism can compensate for the defect in the GAL3 function. What is the nature of the GAL3 function then? Broach (4) hypothesized that the GAL3 product was responsible for the conversion of intracellular galactose to an actual inducer and that this inducer was an intermediate of galactose catabolism. As a matter of fact, certain genes in yeasts and bacteria are known to be induced by products of the enzymes encoded by those genes (3, 6, 13). In S. cerevisiae, genes for the series of enzymes participating in allantoin degradation are induced by the last intermediate, allophanate (6). When cells of Escherichia coli are grown in the presence of lactose, allolactose produced from lactose by the alternate function of P-galactosidase behaves as the actual inducer for the lac operon (13). Besides the major products of the P- galactosidase reaction, that is, glucose and galactose, a small amount of allolactose, which induces the lac operon efficiently, is yielded. In the case of the S. cerevisiae GAL cluster genes, the problem is not that simple. If one of the known intermediates of galactose catabolism, except for UDP-glucose, was an actual inducer, such a rapid deinduction as that observed in Fig. SC and 6C would not occur in the gal3-31 gallo mutant upon temperature shift because the mutant would contain all the intermediates at the time of temperature shift (Fig. 5C) and continue to synthesize these intermediates afterward (Fig. 7). UDP-glucose cannot be the actual inducer because it is known to be synthesized by the action of UDP-glucose pyrophosphorylase from glucose 1-phosphate and UTP (16) but not from galactose. An alternative hypothesis suggested by Tsuyumu and Adams (28) assumes that GAL3 is responsible for the production of a coinducer, the low-molecular-weight substance necessary for the induction of the GAL cluster genes, in addition to the actual inducer, galactose. The presumptive coinducer must be a metabolite that exists in galactosemetabolizing S. cerevisiae. They further speculated that the most probable candidate for the coinducer was UDP-glucose and that GAL3 was the structural gene for one of several isozymes of UDP-glucose pyrophosphorylase. Up to now, these ideas have not been subjected to strict experimental verification. Experiments are in progress in my laboratory to test these ideas by making use of the present mutant. ACKNOWLEDGMENTS I thank Toshio Fukasawa for helpful discussions and for reviewing the manuscript and Wakana Hashimoto for typing the manuscript. LITERATURE CITED 1. Adams, B. G Induction of galactokinase in Saccharomyces cerevisiae: kinetics of induction and glucose effects. J.

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