JOURNAL OF BACTERIOLOGY, Mar. 1979, p. 1200-1207
Vol. 137, No. 3
0021-9193/79/03-1200/08$02.00/0
Gene Dosage Effects on the Synthesis of Maltase in Yeast DEBORAH BERNHARDT MOWSHOWITZ Department of Biological Sciences, Coluinbia University, New York, New York 10027 Received for publication 4 December 1978
Inbred strains of Saccharomyces cerevisiae carrying MALl, MAL2, or MAL6 in a common background were used to construct (i) homo- or heterozygous diploids carrying one or two active alleles of a single MAL locus (MALl, MAL2, or MAL6) and (ii) triploids carrying one, two, or three active alleles of MAL2. The diploid and triploid strains were used to investigate gene dosage effects on the differential rate of maltase synthesis (A enzyme activity/A growth) and the kinetics of induction (for MAL2). All three MAL loci exhibited a gene dosage effect on the differential rate of maltase synthesis; MAL2 also exhibited a gene dosage effect on the kinetics of induction. The dosage effects of the MALl and MAL6 loci were additive, but the effects of the MAL2 locus were not; the magnitude of the MAL2 gene dosage effect decreased with increasing dosage. These results are compatible with the current genetic evidence that the MAL genes are regulatory loci if the product(s) of the MALl and MAL6 locus is produced in limiting amounts but the product(s) of the MAL2 locus is produced in excess, except at very low gene dosages.
There are (at least) five unlinked genes that control the ability of yeast to produce maltase (and maltase permnease) in response to maltoseMALl, MAL2, MAL3, MAL4, and MAL6 (reviewed in 1). Strains carrying an active allele of any one of these loci are inducible; strains carrying inactive alleles at all loci are uninducible but produce basal levels of maltase (4, 12). The role of the MAL genes is not understood. It has been suggested that the MAL loci are (i) structural genes for maltase (9), (ii) regulatory genes controlling maltase and the other enzymes of maltose metabolism (4, 13, 14) or (iii) complex loci containing both regulatory and structural elements (7, 8). The suggestion that the MAL genes are structural genes for maltase is based primarily on the findings that the MAL genes exhibit additive gene dosage effects on the level of maltase produced in fully induced cultures (9) and on the number of periods of maltose synthesis per cell cycle (11). The suggestion(s) that the MAL loci are regulatory elements is based on the more recent observations that regulatory mutations causing both constitutive synthesis (13, 14) and temperature-sensitive induction (12) have been mapped in or close to several of the MAL loci and none of these mutations causes production of an altered maltase. It has been proposed that the MAL loci are complex to explain the existence of complementing noninducible mutations that map in the same or different MAL loci (7, 8).
Given the current information about the role of the MAL loci, we decided to reexamine the gene dosage effects because (i) it is generally assumed that regulatory genes do not exhibit gene dosage effects (5) (because their products are present in excess), (ii) structural genes for inducible and repressible enzymes do not necessarily show strictly additive gene dosage effects (3), and (iii) it has not been demonstrated that the differences in maltase levels are actually due to differences in the MAL gene dosage. Therefore, we have constructed a series of inbred haploid strains carrying a single active MAL locus (MALI, MAL2, or MAL6) in a common background, and the regulation of maltase synthesis has been examined in appropriate diploids and triploids derived from these standard strains. Our results confirm previous findings that the MAL loci exhibit gene dosage effects (9), but the magnitude of the effect for at least one of the loci is not additive. MATERIALS AND METHODS Strain construction and genetic analysis. Haploid strains carrying MAL1, MAL2, or MAL6 were obtained from the sources given in Table 1. To standardize the backgrounds of the strains, especially with regard to the MGL genes, which control fermentation of a-methylglucoside and may affect regulation of aglucosidases in general (1), the strains were crossed three times to one of a small number of closely related standard strains of genotype maWO (no active MAL loci), mgll, mgl2, mgla, MGLB, MGLC. Since the roles of the individual MGL genes are not well under-
1200
VOL. 137, 1979
GENE DOSAGE EFFECTS ON MALTASE SYNTHESIS
1201
TABLE 1. Saccharomyces strains used' Strain DC-5A
J. Marmur
a trpl-I his4 leu2 MAL2
Source of MAL2 allele
1315-2C
R.
Needleman
a MALl hisl trp met ade2
Source of MALl allele
77
R.
Needleman
a MAL6 adel
Source of MAL6 allele
1323-1B
N. Eaton
1412-EK-6B
R.
a gal SUC' mal ura lysl MGLI mgl2 mgla MGLB | MGLC a MAL3 SUC3 leul adel mtgll MGL2 MGLA MGLB MGLC
Source of malO (and MGLI, MGL2 testers)
Source
Needleman
Genotype
Importance
M127-l1A
Cross in this a MAL2 his4 leu2 thr4 trp5b Haploids used for trilaboratory ploid construction M119-7A Cross in this a mal his4 leu2 adel lysl laboratory M59-5B Cross in this a MAL2 his4 leu2 lysl laboratory HL-5 Cross in this a MAL2 + leu2 thr4 trp5 Diploids used for trilaboratory ploid construction a MAL2his4 + thr4 trp5 l HL-6 Cross in this a mal his4 + Iysl adel laboratory a mal + leu2 lysl adel J T10 HL6 x M127MAL2/mal/mal Triploids used for gene 11A dosage Tll HL5 x M119MAL2/MAL2/mal 7A T12 HL5 x M59MAL2/MAL2/MAL2 J 5B All Saccharomyces strains are S. cerevisiae except 77, which is derived from S. carlsbergensis. bAll strains used for triploid construction are mgll, mg12, mgla, MGLB, MGLC. a
stood, the choice of a standard background with regard to the MGL genes was, in part, arbitrary. This particular genotype was chosen because (i) it most closely approximates the mgl genotypes used in previous gene dosage experiments (9) and (ii) it has been reported that active alleles of MGLI and MGL2 may interfere with the gene dosage effects controlled by the MAL loci (9). MGLA is equivalent in function to MGL2 (A. M. A. ten Berge, Ph.D. thesis, University of Utrecht, Utrecht, The Netherlands, 1973). The standard genetic methods and media described by Sherman et al. (10) were used for mating, sporulation, and tetrad analysis, except that maltose and amethylglucoside fermentation were scored on indicator plates (1% yeast extract, 2% peptone, 2% sugar, 0.033% bromocresol purple indicator, 1.5% agar) supplemented with 1 ug of ethidium bromide/ml to prevent residual growth of nonfermenting strains. MGL genotypes were established by complementation tests with strains obtained from Norm Eaton (MGLI, MGL2 testers) or Richard Needleman (MGLA, MGLB, MGLC testers). The complementation tests were performed on plates and scored by replica plating to a-methylglucoside indicator plates containing ethidium bromide. The resulting haploid strains of genotype MALI (or 2 or 6), mgll, mgl2, mgla, MGLB, MGLC were used
to construct diploid strains homo- or heterozygous for each individual MAL locus. To increase the probability that any differences found between heterozygotes and homozygotes could be attributed to the differences in gene dosage and not to other factors, some of the strains were constructed from two tetrads as follows. One tetrad was picked from a cross between each MAL strain and a standard malO strain. The four segregants, two MAL+ and two mal, were crossed to two MAL' segregants from a second tetrad of the same cross to produce two heterozygotes and two homozygotes. All four diploids were analyzed to be sure that any apparent gene dosage effects corresponded to the number of active MAL alleles and not to other factors segregating in the cross. Triploids were constructed by selecting prototrophs resulting from the mating of a doubly auxotrophic diploid with a doubly auxotrophic haploid with complementary requirements. The genotypes of all the strains used are listed in Table 1. Diploids can mate by loss of one copy of chromosome III (which carries both the mating type locus and MAL2) or by achieving homozygosity at the mating type locus. To obtain the desired gene dosages, it was necessary to be sure that the rare diploids which mated retained both copies of chromosome III. Therefore, both copies were marked (see Table 1) so that prototrophic growth required
1202
MOWSHOWITZ
either retention of both chromosomes or a rare recombinational event (mitotic crossing over between the two linked markers or a gene conversion in one of the two loci), and triploids were selected on the basis of their prototrophic growth. Retention of all copies of chromosome III in these triploids was considered likely since prototrophic triploid formation by chromosome loss required two relatively rare events (a recombinational event plus loss). Prototrophic triploid formation by achieving homozygosity at the mating type locus required only one relatively rare eventmitotic gene conversion or mitotic crossing over between the locus and its centromere. To confirm that the triploids so constructed carried one, two, or three active MAL2 loci, the triploids were subjected to random spore analysis. A culture of each triploid was sporulated, digested with glusulase to release the spores, diluted (but not sonically treated), and spread on YPD plates by standard methods as described by Sherman et al. (10). Aneuploid segregants were distinguished from unsporulated triploids on the basis of their slow growth (small colony size) and pink color (if adel was segregating in the cross). The presumed aneuploid segregants were then scored by replica plating for all auxotrophic requirements and for the ability to ferment maltose; only strains exhibiting at least one auxotrophic requirement were counted as segregants. The number of active MAL alleles carried by each triploid was deduced from the proportions of segregants recovered that were able to ferment maltose; the number of prototrophs recovered for each auxotrophic marker served as a control (see Table 4). Media and growth conditions. Minimal medium contained 0.67% nitrogen base without amino acids (Difco) plus a carbon source as indicated. When necessary, it was supplemented with 60 mg of lysine hydrochloride per liter of medium. Maltose broth consisted of the semidefined medium of Van Wijk (2) (2 g of KH2PO4, 6 g of (NH4)2SO4, 2.5 g of yeast extract, and 0.25 g of MgSO4. 7H20 per liter of medium) containing 2% maltose and supplemented with 20 mg each of adenine sulfate, uracil, tryptophan, and histidine hydrochloride, and 30 mg each of tyrosine, leucine, and lysine hydrochloride per liter. Cell cultures were grown on a roller at 30°C, and cell growth was monitored with a Klett-Summerson colorimeter equipped with a red filter. In the range used, one Klett unit corresponds to approximately 2 Ag of protein/nil for both diploids and triploids. For differential growth curves, cells were grown overnight to log phase and either followed without further manipulations (method A) or diluted and grown for two generations before use (method B). Results with both methods were indistinguishable. For induction, cells were pregrown in minimal medium containing 3% glycerol and 2% ethanol to late log or early stationary phase; then they were diluted in the same medium and grown overnight to log phase. Maltose was added to 1% to begin induction. Maltase assays. Cell suspensions were spotted onto filter-paper disks, dried, and assayed for a-glucosidase activity by hydrolysis of p-nitrophenyl-a-Dglucoside as previously described (6). The activity is expressed in arbitrary units: 1 U - absorbance at 410 nm of 4 optical density units per min. The a-glucosi-
J. BACTERIOL.
dase activity measured in this way has been equated with maltase on the basis of previous findings that most of the a-glucosidase in maltose-grown strains with our standard MGL genotype is maltase (J. Gorman, Ph.D. thesis, University of Wisconsin, Madison, 1963; A. M. A. ten Berge, Ph.D. thesis, University of Utrecht, Utrecht, The Netherlands, 1973) and on direct measurements on extracts of our standard strains (results not shown).
RESULTS Dosage effect of the MAL2 locus on steady-state synthesis. Homozygous diploids carrying two active MAL2 alleles and heterozygotes carrying one active MALl allele were constructed by standard genetic methods. The strains were grown in maltose broth to log phase, and growt' and maltase levels were monitored as described in Materials and Methods. The results for a typical strain of each genotype are shown in Fig. 1 as differential growth curvesenzyme activity per milliliter of culture plotted against growth. The slopes of the differential growth curves represent the rate of accumulation of maltase per unit of growth; if degradation is insignificant, the slopes represent the rate of enzyme synthesis. It is assumed that degradation is not significant because the enzyme activity is stable in
60
U
0-4
E 20
KLETT
UNITS
FIG. 1. Maltase synthesis in MAL2 strains. Homozygous (+/+) and heterozygous (+/-) MAL2 strains were grown to logphase in maltose broth containing 4% maltose by method A. Maltase activity and growth were monitored as described in Materials and Methods. Symbols: 0, MAL2/MAL2; U, MAL2/mal.
VOL. 137, 1979
GENE DOSAGE EFFECTS ON MALTASE SYNTHESIS
the absence of protein synthesis and after removal of inducer (R. Chandra, Ph.D. thesis, Columbia University, New York, N.Y., 1977; D. Mowshowitz, unpublished data). All MAL2 homozygotes and heterozygotes tested showed the same pattern as that in Fig. 1; the homozygote carrying two active MAL2 alleles produced more maltase than the heterozygote carrying only one active MAL2 allele, but the difference was much less than the factor of two reported in the literature (9). Segregation of enzyme levels with gene dosage. Since the difference seen in Fig. 1 was so small, four additional diploid strains, two homozygotes and two heterozygotes, were constructed by crossing all spores of a single tetrad to the two MAL2 spores of a second tetrad as described in Materials and Methods. The rates of maltase synthesis were measured in all four diploids to determine whether the differences found corresponded with the MAL2 gene dosage. The slopes of the differential growth curves are presented in Table 2. Both homozygotes produced more maltase than both heterozygotes, as expected if the difference seen in Fig. 1 was due to a gene dosage effect. If the difference in Fig. 1 was due to factors unlinked to the MAL genes, then the levels of enzyme synthesis would not be expected to correspond to the gene dosage because the factors should have segregated independently of the MAL loci in one or both of the tetrads used for strain construction. Therefore, the differences in the levels of enzyme synthesis are most reasonably attributed to a gene dosage effect and not to random differences between the strains. This conclusion is supported by the results obtained with triploids and diploids carrying other MAL genes as described below. Effects of different growth conditions. Since the size of the MAL2 gene dosage effect was so different from that previously reported, different conditions of growth were tried to see if they would affect the magnitude of the dosage effect. A pair of MAL2 strains, one homozygous
1203
and one heterozygous, were grown in minimal medium for different lengths of time to be sure that the steady-state conditions were achieved. The two strains were also grown with different amounts of maltose in the medium because it
seemed possible that this might alter the intracellular levels of maltose which might otherwise be limited by the supply of permease. Since maltase synthesis is subject to both induction by maltose and catabolite repression by glucose (1), it seemed possible that altering the levels of maltose might affect either the extent of feedback repression (especially in the homozygote with its high enzyme levels) and/or the extent of induction, especially in the heterozygote. Table 3 summarizes the rates of maltase production observed in the two strains after growth for different periods of time in minimal medium containing 0.2 to 4% maltose. As shown in Table 3, the rates of enzyme synthesis in minimal medium were lower than that found in broth but were unaffected by the length of growth in the medium, suggesting that steady-state conditions had been achieved under all growth conditions employed. (This is probably not the case for some other strains grown in broth, as explained below.) The absolute rates of maltase production did not seem to be significantly affected by alterations in the amount of maltose in the growth medium, although the magnitude of the gene dosage effect did appear to increase slightly at lower maltose concentrations. The results suggest that the increase in the gene dosage effect could be due to a slightly higher level of enzyme synthesis in the homozygote (caused by the release of repression from the glucose produced from maltose) combined with a lower level of synthesis in the heterozygote. However, none of the conditions tested greatly increased or abolished the gene dosage effect. MAL2 dosage effects on induction. Since induction of maltase is controlled by the MAL loci, and rate of induction might be even more dependent on the activity of the MAL gene product(s) than the rate of steady-state synthesis, MAL2 dosage effects on induction were investigated. A pair of strains, one homozygous TABLE 2. Maltase production in MAL2 strains and one heterozygous for MAL2, were pregrown grown in maltose broth to log phase in minimal medium without malA Maltase/A growthb MAL2 gene dosage' tose, maltose was added, and growth and maltase levels were monitored as described. The ++ 6.75 results of two experiments are plotted in Fig. 2 ++ 6.75 5.2 and 3 as enzyme activity per milliliter (corrected 5.6 for growth) versus time; Fig. 2 shows the results Strains constructed from two tetrads as described at relatively early times after induction, and Fig. 3 shows the results at later times. The results in in Materials and Methods. b A Units of maltase x 10/A Klett units; obtained Fig. 2 demonstrate a gene dosage effect on the from slopes of differential curves similar to those in lag time before maltase synthesis begins; the results in Fig. 3 demonstrate an additional effect Fig. 1. a
J . BACTrERIOL.
1204 MOWSHOWITZ TABLE 3. Malose production in MAL2 strains grown in minimal medium A Maltase/A growth"
Expt no.
Maltose in medium (%)
MAL2/MAL2
MAL2/mal
MAL2/MAL2
6.0
5.0
1.2
5.0 4.7 4.4 5.4 4.4 4.5 4.73 ± 0.40
4.2 3.85 3.6 4.2 3.8 4.0 3.94 ± 0.24
1.22 1.22 1.28 1.16 1.12 1.20 ± 0.05
6.0 5.2 5.0 5.40 ± 0.53
5.0 3.8 3.3 4.03 ± 0.87
1.20 1.37 1.52 1.34 ± 0.16
5.2
3.8 2.8 3.8 4.0 3.6
MAL2/mal lb
4
2b 3b 4c 5d 6d
2
7d
Avg
lb
0.4
8e
gd Avg
8e 6d 7d
0.2
4.1
6.2 5.4 5.22
0.82
1.19
1.37 1.46
0.54
1.63 1.35 1.45
0.13
Units as in Table 2. b Grown by method A as described in Materials and Methods. Grown to late log phase twice, then grown by method A. d Grown by method B as described in Materials and Methods. Grown overnight in 0.4% maltose, diluted into 0.4 or 0.2%, and grown for two generations before monitoring.
a
c
on the maximum rate per unit of time finally achieved. MAL2 dosage effects in triploids. Triploids carrying one, two, or three active MAL alleles were constructed as described in Materials and Methods to see whether varying the gene dosage over a wider range would produce a wider variation of the rate of maltase synthesis. To be sure that the strain construction procedure had actually produced triploid strains carrying the proper number of active MAL alleles, the three presumptive triploids were subjected to random spore analysis as described in Materials and Methods. The results are summarized in Table 4, which presents the presumed genotype, the number of segregants scored, the percentage of the segregants prototrophic for each nutrilite requirement, and the percentage of the segregants able to ferment maltose for each (presumed) triploid strain. The triploid strains were of two presumed genotypes for all the nutritional markers-either +-- or ++-. As shown in Table 4, 46 to 62% (average, 58%) of the segregants derived from each presumed triploid +-- were prototrophic (for that marker); 87 to 96% of the segregants derived from each presumed ++- were prototrophic. These are the expected proportions if most of the segregants were disomic for the
chromosomes scored. The recovery of prototrophs in two different proportions demonstrates that the strains are triploids since it shows that two types of heterozygosity are possible (+-and ++-). The proportions recovered indicate that the gene dosages (+-- and ++-) match the input for all auxotrophic markers, including those on chromosome III (leu, his, thr). Therefore, the triploids have retained all three copies of chromosome III. The pattern of segregation of the ability to ferment maltose was the same as that of the segregation of auxotrophic markers; as shown in Table 4, 61% of the segregants derived from the presumed +-- and 83% of the segregants of the presumed ++- were fermenters. These results indicate that the triploids are of the expected (input) genotype for MAL2, in other words, that the strains T10, Tll, and T12 carry one, two, and three copies of the active MAL2 allele, respectively. Maltase synthesis was examined in the triploid strains during induction and after long-term growth on maltose in the same fashion as for the diploid strains. The results of the induction experiment are shown in Fig. 4; the results of the differential growth curves are presented in Table 5 as a function of gene dosage along with the analogous results with diploids. The induction
GENE DOSAGE EFFECTS ON MALTASE SYNTHESIS
VOL. 137, 1979
1205
strains carrying these two active MAL loci produce less maltase than haploids with the same background carrying MAL2. Diploid strains homozygous or heterozygous for MALl or MAL6 were constructed from the four spores of a single tetrad as described in Materials and Methods; the background was the same as that of., the MAL2 strains discussed | z above. All the strains were first grown in maltose broth to see if a gene dosage effect could be demonstrated and if it segregated with the MAL / gene dosage. Then at least one strain of each * genotype was grown in minimal medium to measure the magnitude of the gene dosage effect 4 / / E under defined conditions. The results are presented in Table 6. Both the MALl and the MAL6 locus exhibit a gene dosage effect which f is approximately additive in minimal medium. / 4 2 /The larger dosage effects in broth are probably / z due to an underestimation of the rates of syn, / 4 y by heterozygotes, because the heterozythesis 6 gotes may not be fully induced under the growth ________'___*_______,__ b,
1
2
3
TIME (HOURS)
FIG. 2. Induction of MAL2 diploids. Kinetics at relatively early times. Homozygous (+/+) and heterozygous (+/-) MAL2 strains were grown on nonfermentable carbon sources. Maltose was added at time zero to start induction, and maltase activity and growth were monitored as described in Materials and Methods. Symbols: 0, MAL2/MAL2; U, MAL2/ mal.
40/
30
experiments show that there is a gene dosage effect on the length of the lag period before / maltase synthesis reaches its steady state; the length of the lag decreases with increasing dosage. Both the induction and steady-state experiments demonstrate that there is little if any , 20 difference in the rate of fully induced maltase , synthesis between the two strains carrying two or three active MAL2 alleles; this is clear E whether the rates are compared per unit of time ' / (slopes of the induction curves) or per unit of growth (slopes of the differential growth curves). lo / However, there is a large difference between / these two strains and the one carrying only a '-i single active MAL2 allele. When the rates of z fully induced enzyme synthesis are compared '/ over the entire range of gene dosage tested in ° both diploids and triploids (Table 5), it is clear 4 6 2 that the MAL2 locus exhibits a gene dosage TIME (HOURS) effect but that the effect plateaus at higher FIG. 3. Induction of MAL2 diploids. Kinetics at dosages. Dosage effects of other MAL loci. Since relatively late times. Homozygous (+/+) and hetero(+/-) MAL2 strains were grown on nonferstrains carrying different active MAL loci appear zygous carbon sources. Maltose was added at time mentable maltase synthesis and maltase activity and zero to start induction, totodiffer in their regulation ofmanucrp (9, 11; D. B.B. Mowshowitz, manuscript in prepa- growth were monitored as described in Materials ration), gene dosage effects were investigated in and Methods. Symbols: 0, MAL2/MAL2; I, MAL2/ strains carrying MALl or MAL6. Haploid mal. -
regulatzo
inthepas
J. BACTERIOL.
1206 MOWSHOWITZ TABLE 4. Random spore analysis of triploid strains Marker scored Strain ade
lys
leu
trp
thr
MAL
95.6
61.4 +--
his
T10 58.7 60.3 91.3 56 62 Percent recoverya +-+-+++-Genotype ..+-T1l 54.5 54.5 46.3 89.6 85.1 Percent recoverya +-+-+-++++Genotype T12 56.5 62.3 59.4 86.8 NTb Percent recovery. +-+-+-+++++ Genotype ... a Number of prototrophic or fermenting segregants/total x 100; total number scored T10, Tll, and T12, respectively. b Not tested.
++-
58.2 +--
82.8 ++-
100 +++ = 184, 134, and 69 for 61.6
+--
low dosages. Two other MAL loci, MALl and MAL6, exert an approximately additive gene dosage effect on the rate of fully induced maltase
z
synthesis. Both types of gene dosage effects reported here, one strictly additive and the other cumulative but not strictly additive at high dosages, have been found for both repressible structural genes (3) and for positive regulatory genes (2, 5). In the case of regulatory genes, additive effects have been attributed to positive regulatory products that are present in limiting amounts (2) whereas nonadditive effects have been attributed to regulatory products present in excess (5). In the case of inducible or repressible structural genes, the nature of the gene dosage effects has been attributed to the ability of the enzymes to regulate the supply of the small molecules which are the inducers and/or corepressors of the enzymes involved (3). For example, if the gene dosage is reduced and the resulting activity of 6 3 the corresponding enzyme is insufficient to TIME (HOURS) the supply of end product which acts maintain Induction of MAL2 triploids. Triploid
FIG. 4. strains carrying one, two, or three active MAL2 alleles were grown on nonfermentable carbon sources. Maltose was added at time zero to start induction, and maltase activity and growth were monitored as described in Materials and Methods. Symbols: 0, T12, MAL2/MAL2/MAL2; G, Tll, MAL2/MAL2/ mal; , T10, MAL2/mal/mal.
TABLE 5. Effects of gene dosage on maltase synthesis in MAL2 diploids and triploids Strain
DISCUSSION The results presented in this paper show that the MAL2 locus exerts a gene dosage effect on the kinetics of maltase induction and on the rate of fully induced enzyme synthesis. The effects are not additive; adding an additional active MAL2 allele has little or no effect at relatively high gene dosages but a large effect at relatively
No. of MAL2
alleles
Gene
dosagea
A Maltase/A growthb
Expt 1 Expt 2
5.2 1 4.5 3 3N 1 2 4.7c 2N 1 2 4.2 2N 2 5.6 4.8 3N 2/3 T1l 1 3.9c 1/2 2N x1lc 1 3.6 2N 1/2 HL7d 1 3.6 2N 1/2 HL8d 1 2.7 2.8 3N 1/3 T10 a Number of MAL2 alleles/N genome. b Units as in Table 2. c Strains and average values from Table 3. d HL7 and HL8 were constructed from parents of HL5 and HL6.
T12
conditions used. (This is indicated by nonlinear differential growth curves.)
Ploidy
XL3C HL5
VOL. 137, 1979
GENE DOSAGE EFFECTS ON MALTASE SYNTHESIS
TABLE 6. Maltase synthesis in MALl and MAL6 strains cus lo-Genotype
MAL lo-
MALIb
MAL6b
MAL6
A Maltase/A growtha
Broth
Minimal medium
++
4.6
++
4.4 1.2 1.2
3.7 3.7
cus
1.4 1.6
++ ++
2.5 2.5 0.4 0.4
Not tested Not tested 1.7 1.8
++
3.0
+_
1.25
2.85 ± 0.27c 1.44 ± 0.23d
Units as in Table 2. constructed from two tetrads as described in Materials and Methods. c Average of five experiments. d Average of four experiments. a
b Strains
as a corepressor, then the synthesis of the enzyme will be derepressed and a nonadditive gene dosage effect will be observed. On the other hand, if the activity of the enzyme at low gene dosages is still sufficient to maintain the supply of end product, derepression will not occur and an additive gene dosage effect will be observed. It is possible to apply either line of reasoning given above to reconcile the observed gene dosage effects with a regulatory and/or a structural role for the MAL genes. If the MAL genes are regulatory loci, then the results of the gene dosage experiments suggest that the MAL genes code for a positive regulatory product whose activity and/or amount limits the rate of maltase synthesis; they also imply that the MAL2 locus codes for a product whose activity and/or amount exceeds that of MALl and MAL6. This would explain why the MAL2 strains both produce more maltase and exhibit a less pronounced gene dosage effect at high dosages. If the MAL genes code for maltase, then the results presented here suggest that these structural genes are fully induced at low gene dosages but that the MAL2 loci are partially repressed at higher gene dosages. It is known that maltase is subject to glucose repression as well as to induction by maltose (1), and high levels of maltase could lead to overproduction of glucose. This would explain why only the strains with relatively high maltase levels show nonadditive gene dosage effects. The first explanation, which assigns a regulatory role to the MAL gene product(s), seems the most likely explanation at the current time,
1207
given the genetic evidence that the MAL genes do not code for maltase but do encode a product with a regulatory function. The best genetic evidence for this position comes from the analysis of MAL6 by Berge and his associates (12, 13); they have found that both noninducible and constitutive mutations map in the MAL6 locus and that two strains that are temperature sensitive for induction contain maltase indistinguishable from wild type. ACKNOWLEDGMENTS This work was supported by grant PCM 73-02023-AOl from the National Science Foundation. LITERATURE CITED 1. Barnett, J. A. 1976. The utilization of sugars by yeasts. Adv. Carbohydr. Chem. Biochem. 32:126-234. 2. Cove, D. J. 1969. Evidence for a near limiting intracellular concentration of a regulator substance. Nature (London) 224:272-273. 3. Hilger, F., M. Culot, M. Minet, A. Pierard, M. Grenson, and J. M. Wiame. 1973. Studies on the kinetics of the enzyme sequence mediating arginine synthesis in Saccharomyces cerevisiae. J. Gen. Microbiol. 75:33-41. 4. Khan, N. A., F. K. Zimmerman, and N. R. Eaton. 1973. Genetic control of maltase formation in yeast. II. Evidence for a gene regulating the level of maltase production. Mol. Gen. Genet. 124:365-367. 5. Klar, A. J. S., and H. 0. Halvorson. 1976. Effect of GALA gene dosage on the level of galactose catabolic enzymes in Saccharomyces cerevisiae. J. Bacteriol. 125:379-381. 6. Mowshowitz, D. B. 1976. Permeabilization of yeast for enzyme assays: an extremely simple method for small samples. Anal. Biochem. 70:94-99. 7. Naumov, G. I. 1971. Comparative genetics of yeasts. V. Complementation in maltose MA, locus in hybrids of wild maltose-negative species of Saccharomyces. Genetika 7:141-148. 8. Oshima, Y. 1967. The inter-cistronic complementation of the polymeric genes for maltose fermentation in Saccharomyces. J. Fermen. Technol. (Japan) 45:550-565. 9. Rudert, F., and H. 0. Halvorson. 1962. The effect of gene dosage on the level of a-glucosidase in yeast. Bull. Res. Counc. Isr. llA4:337-344. 10. Sherman, F., G. R. Fink, and C. W. Lawrence. 1972. Laboratory manual for methods in yeast genetics. Cold Spring Harbor Laboratory for Quantitative Biology, Cold Spring Harbor, N.Y. 11. Tauro, P., and H. 0. Halvorson. 1966. Effect of gene position on the timing of enzyme synthesis in synchronous cultures of yeast. J. Bacteriol. 92:652-661. 12. ten Berge, A. M. A., G. Zoutewelle, and K. W. van de Poll. 1973. Regulation of maltose fermentation in Saccharomyces carisbergensis. I. The function of the gene MAL6 as recognized by mal6 mutants. Mol. Gen. Genet. 123:233-246. 13. ten Berge, A. M. A., G. Zoutewelle, K. W. van de Poll, and H. P. J. Bloemers. 1973. Regulation of maltose fennentation in Saccharomyces carlsbergensis. II. Properties of a constitutive mal6-mutant. Mol. Gen. Genet. 125:139-146. 14. Zimmerman, F. K., and N. R. Eaton. 1974. Genetics of induction and catabolite repression of maltase synthesis in Saccharomyces cerevisiae. Mol. Gen. Genet. 134: 261-272.
Vol. 137, No. 3
0021-9193/79/03-1200/08$02.00/0
Gene Dosage Effects on the Synthesis of Maltase in Yeast DEBORAH BERNHARDT MOWSHOWITZ Department of Biological Sciences, Coluinbia University, New York, New York 10027 Received for publication 4 December 1978
Inbred strains of Saccharomyces cerevisiae carrying MALl, MAL2, or MAL6 in a common background were used to construct (i) homo- or heterozygous diploids carrying one or two active alleles of a single MAL locus (MALl, MAL2, or MAL6) and (ii) triploids carrying one, two, or three active alleles of MAL2. The diploid and triploid strains were used to investigate gene dosage effects on the differential rate of maltase synthesis (A enzyme activity/A growth) and the kinetics of induction (for MAL2). All three MAL loci exhibited a gene dosage effect on the differential rate of maltase synthesis; MAL2 also exhibited a gene dosage effect on the kinetics of induction. The dosage effects of the MALl and MAL6 loci were additive, but the effects of the MAL2 locus were not; the magnitude of the MAL2 gene dosage effect decreased with increasing dosage. These results are compatible with the current genetic evidence that the MAL genes are regulatory loci if the product(s) of the MALl and MAL6 locus is produced in limiting amounts but the product(s) of the MAL2 locus is produced in excess, except at very low gene dosages.
There are (at least) five unlinked genes that control the ability of yeast to produce maltase (and maltase permnease) in response to maltoseMALl, MAL2, MAL3, MAL4, and MAL6 (reviewed in 1). Strains carrying an active allele of any one of these loci are inducible; strains carrying inactive alleles at all loci are uninducible but produce basal levels of maltase (4, 12). The role of the MAL genes is not understood. It has been suggested that the MAL loci are (i) structural genes for maltase (9), (ii) regulatory genes controlling maltase and the other enzymes of maltose metabolism (4, 13, 14) or (iii) complex loci containing both regulatory and structural elements (7, 8). The suggestion that the MAL genes are structural genes for maltase is based primarily on the findings that the MAL genes exhibit additive gene dosage effects on the level of maltase produced in fully induced cultures (9) and on the number of periods of maltose synthesis per cell cycle (11). The suggestion(s) that the MAL loci are regulatory elements is based on the more recent observations that regulatory mutations causing both constitutive synthesis (13, 14) and temperature-sensitive induction (12) have been mapped in or close to several of the MAL loci and none of these mutations causes production of an altered maltase. It has been proposed that the MAL loci are complex to explain the existence of complementing noninducible mutations that map in the same or different MAL loci (7, 8).
Given the current information about the role of the MAL loci, we decided to reexamine the gene dosage effects because (i) it is generally assumed that regulatory genes do not exhibit gene dosage effects (5) (because their products are present in excess), (ii) structural genes for inducible and repressible enzymes do not necessarily show strictly additive gene dosage effects (3), and (iii) it has not been demonstrated that the differences in maltase levels are actually due to differences in the MAL gene dosage. Therefore, we have constructed a series of inbred haploid strains carrying a single active MAL locus (MALI, MAL2, or MAL6) in a common background, and the regulation of maltase synthesis has been examined in appropriate diploids and triploids derived from these standard strains. Our results confirm previous findings that the MAL loci exhibit gene dosage effects (9), but the magnitude of the effect for at least one of the loci is not additive. MATERIALS AND METHODS Strain construction and genetic analysis. Haploid strains carrying MAL1, MAL2, or MAL6 were obtained from the sources given in Table 1. To standardize the backgrounds of the strains, especially with regard to the MGL genes, which control fermentation of a-methylglucoside and may affect regulation of aglucosidases in general (1), the strains were crossed three times to one of a small number of closely related standard strains of genotype maWO (no active MAL loci), mgll, mgl2, mgla, MGLB, MGLC. Since the roles of the individual MGL genes are not well under-
1200
VOL. 137, 1979
GENE DOSAGE EFFECTS ON MALTASE SYNTHESIS
1201
TABLE 1. Saccharomyces strains used' Strain DC-5A
J. Marmur
a trpl-I his4 leu2 MAL2
Source of MAL2 allele
1315-2C
R.
Needleman
a MALl hisl trp met ade2
Source of MALl allele
77
R.
Needleman
a MAL6 adel
Source of MAL6 allele
1323-1B
N. Eaton
1412-EK-6B
R.
a gal SUC' mal ura lysl MGLI mgl2 mgla MGLB | MGLC a MAL3 SUC3 leul adel mtgll MGL2 MGLA MGLB MGLC
Source of malO (and MGLI, MGL2 testers)
Source
Needleman
Genotype
Importance
M127-l1A
Cross in this a MAL2 his4 leu2 thr4 trp5b Haploids used for trilaboratory ploid construction M119-7A Cross in this a mal his4 leu2 adel lysl laboratory M59-5B Cross in this a MAL2 his4 leu2 lysl laboratory HL-5 Cross in this a MAL2 + leu2 thr4 trp5 Diploids used for trilaboratory ploid construction a MAL2his4 + thr4 trp5 l HL-6 Cross in this a mal his4 + Iysl adel laboratory a mal + leu2 lysl adel J T10 HL6 x M127MAL2/mal/mal Triploids used for gene 11A dosage Tll HL5 x M119MAL2/MAL2/mal 7A T12 HL5 x M59MAL2/MAL2/MAL2 J 5B All Saccharomyces strains are S. cerevisiae except 77, which is derived from S. carlsbergensis. bAll strains used for triploid construction are mgll, mg12, mgla, MGLB, MGLC. a
stood, the choice of a standard background with regard to the MGL genes was, in part, arbitrary. This particular genotype was chosen because (i) it most closely approximates the mgl genotypes used in previous gene dosage experiments (9) and (ii) it has been reported that active alleles of MGLI and MGL2 may interfere with the gene dosage effects controlled by the MAL loci (9). MGLA is equivalent in function to MGL2 (A. M. A. ten Berge, Ph.D. thesis, University of Utrecht, Utrecht, The Netherlands, 1973). The standard genetic methods and media described by Sherman et al. (10) were used for mating, sporulation, and tetrad analysis, except that maltose and amethylglucoside fermentation were scored on indicator plates (1% yeast extract, 2% peptone, 2% sugar, 0.033% bromocresol purple indicator, 1.5% agar) supplemented with 1 ug of ethidium bromide/ml to prevent residual growth of nonfermenting strains. MGL genotypes were established by complementation tests with strains obtained from Norm Eaton (MGLI, MGL2 testers) or Richard Needleman (MGLA, MGLB, MGLC testers). The complementation tests were performed on plates and scored by replica plating to a-methylglucoside indicator plates containing ethidium bromide. The resulting haploid strains of genotype MALI (or 2 or 6), mgll, mgl2, mgla, MGLB, MGLC were used
to construct diploid strains homo- or heterozygous for each individual MAL locus. To increase the probability that any differences found between heterozygotes and homozygotes could be attributed to the differences in gene dosage and not to other factors, some of the strains were constructed from two tetrads as follows. One tetrad was picked from a cross between each MAL strain and a standard malO strain. The four segregants, two MAL+ and two mal, were crossed to two MAL' segregants from a second tetrad of the same cross to produce two heterozygotes and two homozygotes. All four diploids were analyzed to be sure that any apparent gene dosage effects corresponded to the number of active MAL alleles and not to other factors segregating in the cross. Triploids were constructed by selecting prototrophs resulting from the mating of a doubly auxotrophic diploid with a doubly auxotrophic haploid with complementary requirements. The genotypes of all the strains used are listed in Table 1. Diploids can mate by loss of one copy of chromosome III (which carries both the mating type locus and MAL2) or by achieving homozygosity at the mating type locus. To obtain the desired gene dosages, it was necessary to be sure that the rare diploids which mated retained both copies of chromosome III. Therefore, both copies were marked (see Table 1) so that prototrophic growth required
1202
MOWSHOWITZ
either retention of both chromosomes or a rare recombinational event (mitotic crossing over between the two linked markers or a gene conversion in one of the two loci), and triploids were selected on the basis of their prototrophic growth. Retention of all copies of chromosome III in these triploids was considered likely since prototrophic triploid formation by chromosome loss required two relatively rare events (a recombinational event plus loss). Prototrophic triploid formation by achieving homozygosity at the mating type locus required only one relatively rare eventmitotic gene conversion or mitotic crossing over between the locus and its centromere. To confirm that the triploids so constructed carried one, two, or three active MAL2 loci, the triploids were subjected to random spore analysis. A culture of each triploid was sporulated, digested with glusulase to release the spores, diluted (but not sonically treated), and spread on YPD plates by standard methods as described by Sherman et al. (10). Aneuploid segregants were distinguished from unsporulated triploids on the basis of their slow growth (small colony size) and pink color (if adel was segregating in the cross). The presumed aneuploid segregants were then scored by replica plating for all auxotrophic requirements and for the ability to ferment maltose; only strains exhibiting at least one auxotrophic requirement were counted as segregants. The number of active MAL alleles carried by each triploid was deduced from the proportions of segregants recovered that were able to ferment maltose; the number of prototrophs recovered for each auxotrophic marker served as a control (see Table 4). Media and growth conditions. Minimal medium contained 0.67% nitrogen base without amino acids (Difco) plus a carbon source as indicated. When necessary, it was supplemented with 60 mg of lysine hydrochloride per liter of medium. Maltose broth consisted of the semidefined medium of Van Wijk (2) (2 g of KH2PO4, 6 g of (NH4)2SO4, 2.5 g of yeast extract, and 0.25 g of MgSO4. 7H20 per liter of medium) containing 2% maltose and supplemented with 20 mg each of adenine sulfate, uracil, tryptophan, and histidine hydrochloride, and 30 mg each of tyrosine, leucine, and lysine hydrochloride per liter. Cell cultures were grown on a roller at 30°C, and cell growth was monitored with a Klett-Summerson colorimeter equipped with a red filter. In the range used, one Klett unit corresponds to approximately 2 Ag of protein/nil for both diploids and triploids. For differential growth curves, cells were grown overnight to log phase and either followed without further manipulations (method A) or diluted and grown for two generations before use (method B). Results with both methods were indistinguishable. For induction, cells were pregrown in minimal medium containing 3% glycerol and 2% ethanol to late log or early stationary phase; then they were diluted in the same medium and grown overnight to log phase. Maltose was added to 1% to begin induction. Maltase assays. Cell suspensions were spotted onto filter-paper disks, dried, and assayed for a-glucosidase activity by hydrolysis of p-nitrophenyl-a-Dglucoside as previously described (6). The activity is expressed in arbitrary units: 1 U - absorbance at 410 nm of 4 optical density units per min. The a-glucosi-
J. BACTERIOL.
dase activity measured in this way has been equated with maltase on the basis of previous findings that most of the a-glucosidase in maltose-grown strains with our standard MGL genotype is maltase (J. Gorman, Ph.D. thesis, University of Wisconsin, Madison, 1963; A. M. A. ten Berge, Ph.D. thesis, University of Utrecht, Utrecht, The Netherlands, 1973) and on direct measurements on extracts of our standard strains (results not shown).
RESULTS Dosage effect of the MAL2 locus on steady-state synthesis. Homozygous diploids carrying two active MAL2 alleles and heterozygotes carrying one active MALl allele were constructed by standard genetic methods. The strains were grown in maltose broth to log phase, and growt' and maltase levels were monitored as described in Materials and Methods. The results for a typical strain of each genotype are shown in Fig. 1 as differential growth curvesenzyme activity per milliliter of culture plotted against growth. The slopes of the differential growth curves represent the rate of accumulation of maltase per unit of growth; if degradation is insignificant, the slopes represent the rate of enzyme synthesis. It is assumed that degradation is not significant because the enzyme activity is stable in
60
U
0-4
E 20
KLETT
UNITS
FIG. 1. Maltase synthesis in MAL2 strains. Homozygous (+/+) and heterozygous (+/-) MAL2 strains were grown to logphase in maltose broth containing 4% maltose by method A. Maltase activity and growth were monitored as described in Materials and Methods. Symbols: 0, MAL2/MAL2; U, MAL2/mal.
VOL. 137, 1979
GENE DOSAGE EFFECTS ON MALTASE SYNTHESIS
the absence of protein synthesis and after removal of inducer (R. Chandra, Ph.D. thesis, Columbia University, New York, N.Y., 1977; D. Mowshowitz, unpublished data). All MAL2 homozygotes and heterozygotes tested showed the same pattern as that in Fig. 1; the homozygote carrying two active MAL2 alleles produced more maltase than the heterozygote carrying only one active MAL2 allele, but the difference was much less than the factor of two reported in the literature (9). Segregation of enzyme levels with gene dosage. Since the difference seen in Fig. 1 was so small, four additional diploid strains, two homozygotes and two heterozygotes, were constructed by crossing all spores of a single tetrad to the two MAL2 spores of a second tetrad as described in Materials and Methods. The rates of maltase synthesis were measured in all four diploids to determine whether the differences found corresponded with the MAL2 gene dosage. The slopes of the differential growth curves are presented in Table 2. Both homozygotes produced more maltase than both heterozygotes, as expected if the difference seen in Fig. 1 was due to a gene dosage effect. If the difference in Fig. 1 was due to factors unlinked to the MAL genes, then the levels of enzyme synthesis would not be expected to correspond to the gene dosage because the factors should have segregated independently of the MAL loci in one or both of the tetrads used for strain construction. Therefore, the differences in the levels of enzyme synthesis are most reasonably attributed to a gene dosage effect and not to random differences between the strains. This conclusion is supported by the results obtained with triploids and diploids carrying other MAL genes as described below. Effects of different growth conditions. Since the size of the MAL2 gene dosage effect was so different from that previously reported, different conditions of growth were tried to see if they would affect the magnitude of the dosage effect. A pair of MAL2 strains, one homozygous
1203
and one heterozygous, were grown in minimal medium for different lengths of time to be sure that the steady-state conditions were achieved. The two strains were also grown with different amounts of maltose in the medium because it
seemed possible that this might alter the intracellular levels of maltose which might otherwise be limited by the supply of permease. Since maltase synthesis is subject to both induction by maltose and catabolite repression by glucose (1), it seemed possible that altering the levels of maltose might affect either the extent of feedback repression (especially in the homozygote with its high enzyme levels) and/or the extent of induction, especially in the heterozygote. Table 3 summarizes the rates of maltase production observed in the two strains after growth for different periods of time in minimal medium containing 0.2 to 4% maltose. As shown in Table 3, the rates of enzyme synthesis in minimal medium were lower than that found in broth but were unaffected by the length of growth in the medium, suggesting that steady-state conditions had been achieved under all growth conditions employed. (This is probably not the case for some other strains grown in broth, as explained below.) The absolute rates of maltase production did not seem to be significantly affected by alterations in the amount of maltose in the growth medium, although the magnitude of the gene dosage effect did appear to increase slightly at lower maltose concentrations. The results suggest that the increase in the gene dosage effect could be due to a slightly higher level of enzyme synthesis in the homozygote (caused by the release of repression from the glucose produced from maltose) combined with a lower level of synthesis in the heterozygote. However, none of the conditions tested greatly increased or abolished the gene dosage effect. MAL2 dosage effects on induction. Since induction of maltase is controlled by the MAL loci, and rate of induction might be even more dependent on the activity of the MAL gene product(s) than the rate of steady-state synthesis, MAL2 dosage effects on induction were investigated. A pair of strains, one homozygous TABLE 2. Maltase production in MAL2 strains and one heterozygous for MAL2, were pregrown grown in maltose broth to log phase in minimal medium without malA Maltase/A growthb MAL2 gene dosage' tose, maltose was added, and growth and maltase levels were monitored as described. The ++ 6.75 results of two experiments are plotted in Fig. 2 ++ 6.75 5.2 and 3 as enzyme activity per milliliter (corrected 5.6 for growth) versus time; Fig. 2 shows the results Strains constructed from two tetrads as described at relatively early times after induction, and Fig. 3 shows the results at later times. The results in in Materials and Methods. b A Units of maltase x 10/A Klett units; obtained Fig. 2 demonstrate a gene dosage effect on the from slopes of differential curves similar to those in lag time before maltase synthesis begins; the results in Fig. 3 demonstrate an additional effect Fig. 1. a
J . BACTrERIOL.
1204 MOWSHOWITZ TABLE 3. Malose production in MAL2 strains grown in minimal medium A Maltase/A growth"
Expt no.
Maltose in medium (%)
MAL2/MAL2
MAL2/mal
MAL2/MAL2
6.0
5.0
1.2
5.0 4.7 4.4 5.4 4.4 4.5 4.73 ± 0.40
4.2 3.85 3.6 4.2 3.8 4.0 3.94 ± 0.24
1.22 1.22 1.28 1.16 1.12 1.20 ± 0.05
6.0 5.2 5.0 5.40 ± 0.53
5.0 3.8 3.3 4.03 ± 0.87
1.20 1.37 1.52 1.34 ± 0.16
5.2
3.8 2.8 3.8 4.0 3.6
MAL2/mal lb
4
2b 3b 4c 5d 6d
2
7d
Avg
lb
0.4
8e
gd Avg
8e 6d 7d
0.2
4.1
6.2 5.4 5.22
0.82
1.19
1.37 1.46
0.54
1.63 1.35 1.45
0.13
Units as in Table 2. b Grown by method A as described in Materials and Methods. Grown to late log phase twice, then grown by method A. d Grown by method B as described in Materials and Methods. Grown overnight in 0.4% maltose, diluted into 0.4 or 0.2%, and grown for two generations before monitoring.
a
c
on the maximum rate per unit of time finally achieved. MAL2 dosage effects in triploids. Triploids carrying one, two, or three active MAL alleles were constructed as described in Materials and Methods to see whether varying the gene dosage over a wider range would produce a wider variation of the rate of maltase synthesis. To be sure that the strain construction procedure had actually produced triploid strains carrying the proper number of active MAL alleles, the three presumptive triploids were subjected to random spore analysis as described in Materials and Methods. The results are summarized in Table 4, which presents the presumed genotype, the number of segregants scored, the percentage of the segregants prototrophic for each nutrilite requirement, and the percentage of the segregants able to ferment maltose for each (presumed) triploid strain. The triploid strains were of two presumed genotypes for all the nutritional markers-either +-- or ++-. As shown in Table 4, 46 to 62% (average, 58%) of the segregants derived from each presumed triploid +-- were prototrophic (for that marker); 87 to 96% of the segregants derived from each presumed ++- were prototrophic. These are the expected proportions if most of the segregants were disomic for the
chromosomes scored. The recovery of prototrophs in two different proportions demonstrates that the strains are triploids since it shows that two types of heterozygosity are possible (+-and ++-). The proportions recovered indicate that the gene dosages (+-- and ++-) match the input for all auxotrophic markers, including those on chromosome III (leu, his, thr). Therefore, the triploids have retained all three copies of chromosome III. The pattern of segregation of the ability to ferment maltose was the same as that of the segregation of auxotrophic markers; as shown in Table 4, 61% of the segregants derived from the presumed +-- and 83% of the segregants of the presumed ++- were fermenters. These results indicate that the triploids are of the expected (input) genotype for MAL2, in other words, that the strains T10, Tll, and T12 carry one, two, and three copies of the active MAL2 allele, respectively. Maltase synthesis was examined in the triploid strains during induction and after long-term growth on maltose in the same fashion as for the diploid strains. The results of the induction experiment are shown in Fig. 4; the results of the differential growth curves are presented in Table 5 as a function of gene dosage along with the analogous results with diploids. The induction
GENE DOSAGE EFFECTS ON MALTASE SYNTHESIS
VOL. 137, 1979
1205
strains carrying these two active MAL loci produce less maltase than haploids with the same background carrying MAL2. Diploid strains homozygous or heterozygous for MALl or MAL6 were constructed from the four spores of a single tetrad as described in Materials and Methods; the background was the same as that of., the MAL2 strains discussed | z above. All the strains were first grown in maltose broth to see if a gene dosage effect could be demonstrated and if it segregated with the MAL / gene dosage. Then at least one strain of each * genotype was grown in minimal medium to measure the magnitude of the gene dosage effect 4 / / E under defined conditions. The results are presented in Table 6. Both the MALl and the MAL6 locus exhibit a gene dosage effect which f is approximately additive in minimal medium. / 4 2 /The larger dosage effects in broth are probably / z due to an underestimation of the rates of syn, / 4 y by heterozygotes, because the heterozythesis 6 gotes may not be fully induced under the growth ________'___*_______,__ b,
1
2
3
TIME (HOURS)
FIG. 2. Induction of MAL2 diploids. Kinetics at relatively early times. Homozygous (+/+) and heterozygous (+/-) MAL2 strains were grown on nonfermentable carbon sources. Maltose was added at time zero to start induction, and maltase activity and growth were monitored as described in Materials and Methods. Symbols: 0, MAL2/MAL2; U, MAL2/ mal.
40/
30
experiments show that there is a gene dosage effect on the length of the lag period before / maltase synthesis reaches its steady state; the length of the lag decreases with increasing dosage. Both the induction and steady-state experiments demonstrate that there is little if any , 20 difference in the rate of fully induced maltase , synthesis between the two strains carrying two or three active MAL2 alleles; this is clear E whether the rates are compared per unit of time ' / (slopes of the induction curves) or per unit of growth (slopes of the differential growth curves). lo / However, there is a large difference between / these two strains and the one carrying only a '-i single active MAL2 allele. When the rates of z fully induced enzyme synthesis are compared '/ over the entire range of gene dosage tested in ° both diploids and triploids (Table 5), it is clear 4 6 2 that the MAL2 locus exhibits a gene dosage TIME (HOURS) effect but that the effect plateaus at higher FIG. 3. Induction of MAL2 diploids. Kinetics at dosages. Dosage effects of other MAL loci. Since relatively late times. Homozygous (+/+) and hetero(+/-) MAL2 strains were grown on nonferstrains carrying different active MAL loci appear zygous carbon sources. Maltose was added at time mentable maltase synthesis and maltase activity and zero to start induction, totodiffer in their regulation ofmanucrp (9, 11; D. B.B. Mowshowitz, manuscript in prepa- growth were monitored as described in Materials ration), gene dosage effects were investigated in and Methods. Symbols: 0, MAL2/MAL2; I, MAL2/ strains carrying MALl or MAL6. Haploid mal. -
regulatzo
inthepas
J. BACTERIOL.
1206 MOWSHOWITZ TABLE 4. Random spore analysis of triploid strains Marker scored Strain ade
lys
leu
trp
thr
MAL
95.6
61.4 +--
his
T10 58.7 60.3 91.3 56 62 Percent recoverya +-+-+++-Genotype ..+-T1l 54.5 54.5 46.3 89.6 85.1 Percent recoverya +-+-+-++++Genotype T12 56.5 62.3 59.4 86.8 NTb Percent recovery. +-+-+-+++++ Genotype ... a Number of prototrophic or fermenting segregants/total x 100; total number scored T10, Tll, and T12, respectively. b Not tested.
++-
58.2 +--
82.8 ++-
100 +++ = 184, 134, and 69 for 61.6
+--
low dosages. Two other MAL loci, MALl and MAL6, exert an approximately additive gene dosage effect on the rate of fully induced maltase
z
synthesis. Both types of gene dosage effects reported here, one strictly additive and the other cumulative but not strictly additive at high dosages, have been found for both repressible structural genes (3) and for positive regulatory genes (2, 5). In the case of regulatory genes, additive effects have been attributed to positive regulatory products that are present in limiting amounts (2) whereas nonadditive effects have been attributed to regulatory products present in excess (5). In the case of inducible or repressible structural genes, the nature of the gene dosage effects has been attributed to the ability of the enzymes to regulate the supply of the small molecules which are the inducers and/or corepressors of the enzymes involved (3). For example, if the gene dosage is reduced and the resulting activity of 6 3 the corresponding enzyme is insufficient to TIME (HOURS) the supply of end product which acts maintain Induction of MAL2 triploids. Triploid
FIG. 4. strains carrying one, two, or three active MAL2 alleles were grown on nonfermentable carbon sources. Maltose was added at time zero to start induction, and maltase activity and growth were monitored as described in Materials and Methods. Symbols: 0, T12, MAL2/MAL2/MAL2; G, Tll, MAL2/MAL2/ mal; , T10, MAL2/mal/mal.
TABLE 5. Effects of gene dosage on maltase synthesis in MAL2 diploids and triploids Strain
DISCUSSION The results presented in this paper show that the MAL2 locus exerts a gene dosage effect on the kinetics of maltase induction and on the rate of fully induced enzyme synthesis. The effects are not additive; adding an additional active MAL2 allele has little or no effect at relatively high gene dosages but a large effect at relatively
No. of MAL2
alleles
Gene
dosagea
A Maltase/A growthb
Expt 1 Expt 2
5.2 1 4.5 3 3N 1 2 4.7c 2N 1 2 4.2 2N 2 5.6 4.8 3N 2/3 T1l 1 3.9c 1/2 2N x1lc 1 3.6 2N 1/2 HL7d 1 3.6 2N 1/2 HL8d 1 2.7 2.8 3N 1/3 T10 a Number of MAL2 alleles/N genome. b Units as in Table 2. c Strains and average values from Table 3. d HL7 and HL8 were constructed from parents of HL5 and HL6.
T12
conditions used. (This is indicated by nonlinear differential growth curves.)
Ploidy
XL3C HL5
VOL. 137, 1979
GENE DOSAGE EFFECTS ON MALTASE SYNTHESIS
TABLE 6. Maltase synthesis in MALl and MAL6 strains cus lo-Genotype
MAL lo-
MALIb
MAL6b
MAL6
A Maltase/A growtha
Broth
Minimal medium
++
4.6
++
4.4 1.2 1.2
3.7 3.7
cus
1.4 1.6
++ ++
2.5 2.5 0.4 0.4
Not tested Not tested 1.7 1.8
++
3.0
+_
1.25
2.85 ± 0.27c 1.44 ± 0.23d
Units as in Table 2. constructed from two tetrads as described in Materials and Methods. c Average of five experiments. d Average of four experiments. a
b Strains
as a corepressor, then the synthesis of the enzyme will be derepressed and a nonadditive gene dosage effect will be observed. On the other hand, if the activity of the enzyme at low gene dosages is still sufficient to maintain the supply of end product, derepression will not occur and an additive gene dosage effect will be observed. It is possible to apply either line of reasoning given above to reconcile the observed gene dosage effects with a regulatory and/or a structural role for the MAL genes. If the MAL genes are regulatory loci, then the results of the gene dosage experiments suggest that the MAL genes code for a positive regulatory product whose activity and/or amount limits the rate of maltase synthesis; they also imply that the MAL2 locus codes for a product whose activity and/or amount exceeds that of MALl and MAL6. This would explain why the MAL2 strains both produce more maltase and exhibit a less pronounced gene dosage effect at high dosages. If the MAL genes code for maltase, then the results presented here suggest that these structural genes are fully induced at low gene dosages but that the MAL2 loci are partially repressed at higher gene dosages. It is known that maltase is subject to glucose repression as well as to induction by maltose (1), and high levels of maltase could lead to overproduction of glucose. This would explain why only the strains with relatively high maltase levels show nonadditive gene dosage effects. The first explanation, which assigns a regulatory role to the MAL gene product(s), seems the most likely explanation at the current time,
1207
given the genetic evidence that the MAL genes do not code for maltase but do encode a product with a regulatory function. The best genetic evidence for this position comes from the analysis of MAL6 by Berge and his associates (12, 13); they have found that both noninducible and constitutive mutations map in the MAL6 locus and that two strains that are temperature sensitive for induction contain maltase indistinguishable from wild type. ACKNOWLEDGMENTS This work was supported by grant PCM 73-02023-AOl from the National Science Foundation. LITERATURE CITED 1. Barnett, J. A. 1976. The utilization of sugars by yeasts. Adv. Carbohydr. Chem. Biochem. 32:126-234. 2. Cove, D. J. 1969. Evidence for a near limiting intracellular concentration of a regulator substance. Nature (London) 224:272-273. 3. Hilger, F., M. Culot, M. Minet, A. Pierard, M. Grenson, and J. M. Wiame. 1973. Studies on the kinetics of the enzyme sequence mediating arginine synthesis in Saccharomyces cerevisiae. J. Gen. Microbiol. 75:33-41. 4. Khan, N. A., F. K. Zimmerman, and N. R. Eaton. 1973. Genetic control of maltase formation in yeast. II. Evidence for a gene regulating the level of maltase production. Mol. Gen. Genet. 124:365-367. 5. Klar, A. J. S., and H. 0. Halvorson. 1976. Effect of GALA gene dosage on the level of galactose catabolic enzymes in Saccharomyces cerevisiae. J. Bacteriol. 125:379-381. 6. Mowshowitz, D. B. 1976. Permeabilization of yeast for enzyme assays: an extremely simple method for small samples. Anal. Biochem. 70:94-99. 7. Naumov, G. I. 1971. Comparative genetics of yeasts. V. Complementation in maltose MA, locus in hybrids of wild maltose-negative species of Saccharomyces. Genetika 7:141-148. 8. Oshima, Y. 1967. The inter-cistronic complementation of the polymeric genes for maltose fermentation in Saccharomyces. J. Fermen. Technol. (Japan) 45:550-565. 9. Rudert, F., and H. 0. Halvorson. 1962. The effect of gene dosage on the level of a-glucosidase in yeast. Bull. Res. Counc. Isr. llA4:337-344. 10. Sherman, F., G. R. Fink, and C. W. Lawrence. 1972. Laboratory manual for methods in yeast genetics. Cold Spring Harbor Laboratory for Quantitative Biology, Cold Spring Harbor, N.Y. 11. Tauro, P., and H. 0. Halvorson. 1966. Effect of gene position on the timing of enzyme synthesis in synchronous cultures of yeast. J. Bacteriol. 92:652-661. 12. ten Berge, A. M. A., G. Zoutewelle, and K. W. van de Poll. 1973. Regulation of maltose fermentation in Saccharomyces carisbergensis. I. The function of the gene MAL6 as recognized by mal6 mutants. Mol. Gen. Genet. 123:233-246. 13. ten Berge, A. M. A., G. Zoutewelle, K. W. van de Poll, and H. P. J. Bloemers. 1973. Regulation of maltose fennentation in Saccharomyces carlsbergensis. II. Properties of a constitutive mal6-mutant. Mol. Gen. Genet. 125:139-146. 14. Zimmerman, F. K., and N. R. Eaton. 1974. Genetics of induction and catabolite repression of maltase synthesis in Saccharomyces cerevisiae. Mol. Gen. Genet. 134: 261-272.
