Genetic Analysis of Haploids from Industrial Strains of Baker's Yeast

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1 APPLIED AND ENVIRONMENTAL MICROBIOLOGY, JUlY 1989, p /89/ $02.00/0 Copyright ) 1989, American Society for Microbiology Vol. 55, No. 7 Genetic Analysis of Haploids from Industrial Strains of Baker's Yeast YUJI ODA* AND KOZO OUCHI Tokyo Research Laboratories, Kyowa Hakko Kogyo Co., Ltd., Machida-shi, Tokyo 194, Japan Received 3 October 1988/Accepted 19 April 1989 Strains of baker's yeast conventionally used by the baking industry in Japan were tested for the ability to sporulate and produce viable haploid spores. Three isolates which possessed the properties of baker's yeasts were obtained from single spores. Each strain was a haploid, and one of these strains, YOY34, was characterized. YOY34 fermented maltose and sucrose, but did not utilize galactose, unlike its parental strain. Genetic analysis showed that YOY34 carried two MAL genes, one functional and one cryptic; two SUC genes; and one defective gal gene. The genotype of YOY34 was identified as MATot MALl MAL3g SUC2 SUC4 gall. The MALl gene from this haploid was constitutively expressed, was dominant over other wild-type MAL tester genes, and gave a weak sucrose fermentation. YOY34 was suitable for both bakery products, like conventional baker's yeasts, and for genetic analysis, like laboratory strains. Saccharomyces cerevisiae strains used in fundamental biological studies are laboratory yeasts and are dissimilar to industrial strains. These laboratory strains are useful for classical and molecular genetic analyses (2), but cannot usually be applied to industries such as baking, brewing, distilling, and wine making. On the other hand, basic techniques in yeast genetics are often unavailable for industrial strains since most of these yeasts are homothallic and aneuploid or polyploid and have poor sporulation and low spore viability (7, 16). We have shown previously that baker's yeasts were clearly differentiated from laboratory and other industrial strains by principal component analysis (11). The strains used in the baking industry were indeed more suitable for dough leavening. To investigate the genes governing the characteristics of baker's yeasts, it was necessary to obtain haploid strains which were applicable to both baking and genetic studies. In the present paper we report the isolation and some properties of three strains derived from single spores of baker's yeasts and the genotype of one of these strains. MATERIALS AND METHODS Organisms. Yeast strains used in the present experiment are listed in Table 1. The genetic notation for the MAL strains and the meaning of MALp, MALg, and map have been reported (9). Five unlinked related genes, MAL], MAL2, MAL3, MAL4, and MAL6, have been identified in different isolates of S. cerevisiae (9, 10). Each functional MAL gene is composed of two complementary genes: MALp, encoding the positive regulatory protein, and MALg, encoding both maltose permease and ot-glucosidase. Strains containing either MALp or MALg alone are defective in maltose fermentation. MALI(mal) indicates that there are no MALp and MALg genes other than the MALI gene. MAL(maP) strains without auxotrophs were constructed by crossing wild-type maltose-fermenting MAL tester strains and mal strains kindly provided by R. B. Needleman (Wayne State University, Detroit, Mich.). Media. Growth media used were YP (1% yeast extract, 2% peptone) supplemented with either 2% glucose (YPD), 3% * Corresponding author. glycerol plus 2% ethanol (YPGE), 2% maltose (YPM), or 2% sucrose plus 0.05% adenine sulfate (YPSA). Sporulation medium employed was SPO (0.1% yeast extract, 1% potassium acetate, 0.05% glucose). Media were solidified by the addition of 2% agar. For the determination of leavening ability, yeast cells were cultured in a molasses medium containing 3% sugar as cane molasses, 0.193% urea, and 0.046% KH2PO4 (11). Fermograph studies of sponge dough. The ingredients of sponge dough, containing 100 g of flour, 0.5 g of salt, 0.66 g of yeast cells as a dry matter, and 65 ml of distilled water, were mixed for 2 min with a National Mixer (National Mfg. Co., Lincoln, Nebr.). A piece of dough based on 20 g of flour was cut and placed in a bottle of a Fermograph (Atto Co. Ltd., Tokyo, Japan), and the rate of CO2 gas production was recorded automatically at 30 C (4). Baking test. Both sweet dough and white dough were made to test the baking quality of yeast by sponge and dough methods according to the standard method (1). Table 2 shows the composition of two doughs. Compressed yeast was prepared from cells cultured in a 5-liter jar fermentor (5), and its moisture was adjusted to 67% (wt/wt). Sponges were mixed for the desired period and fermented at 28 C and 90 to 95% relative humidity. After 2.5 h for sweet dough and 4 h for white dough, each sponge was mixed with the rest of the ingredients to optimum development. Mixing was carried out with a Kanto Mixer (Kanto Mixer Co. Ltd., Tokyo, Japan). The mixed doughs were given a 20-min floor time at 28 C and 90 to 95% relative humidity. At the end of the fermentation, sweet dough was divided into 50-g and 100-g pieces and proofed at 38 C and 85% relative humidity. The larger pieces were put into a cylinder and used for the measurement of proof time (the period required for proofing to the defined dough volume). When the volume of 100-g doughs had increased threefold, the 50-g pieces of dough were baked at 220 C for 10 min. White dough was cut into pieces of 450 g, proofed to 7.5 cm high at 38 C and 85% relative humidity after molding and panning, and then baked at 220 C for 25 min. Weight and volume of baked goods were measured immediately after baking. Volume was determined by the displacement of rape seed. Genetic analysis. Standard techniques of mating, sporula- 1742

2 VOL TABLE 1. List of S. cerevisiae strains used Strain Genotype Source' YOY34 MATax MALI' MAL3g SUC2 This study SUC4 gall 349-6A MATa malo leu2 ura3-51 trpl R. Needleman 3/14-2 MATot malo ura3-52 ade2 R. Needleman D MATa MALI SUCI MGL adel T. Mizunaga ade2 leul A MATa MAL2 suc MEL] his4 leu2 YGSC D MATa MAL3 SUC3 MELI MGL2 YGSC MGL3 ade A MATa MAL4' suc MGL3 gal3 YGSC gal4 ura3 C9 MATa MAL6 SUC MEL adel YGSC trp5 ade6 (or ade7) X2180-1A MATa SUC2 mal gal2 CUPI YGSC SS-4A MATa SUC4 mal adel YGSC C MATa SUCS mal ade6 YGSC NIH 1267 MATa gall petl7 arg4 aro7 his2 K. Ouchi his6 trpl adel asp5 cdc D MATa gal3 ural hisi trpl thr met Y. Nogi G4-a MATa gal4 met] ural ade2 ade6 YGSC 156-SC MATa gals his] Y. Nogi N77-1C MATa gal7 ade met trpl Y. Nogi N72-16D MATa gallo ade tyri ural Y. Nogi 108-3A MATa gal80 ade6 thr4 trpl his3 YGSC [rho-] a YGSC, Yeast Genetic Stock Center, University of California, Berkeley. tion, spore isolation, dissection, and tetrad analysis were used (17). Diploid cells were transferred to an SPO plate and incubated at 25 C for 3 to 7 days. After sporulation, the ascus walls were treated with lytic enzyme and the spore suspension was lightly sonicated and spread or dissected by micromanipulation on YPD plates. Colonies derived from single spores after growth for 2 to 3 days were picked and used for subsequent experiments. Mating type was determined by separately mixing each isolate in YPD medium with the two tester strains X2180-1A and X2180-1B. Zygote formation in the conjugation mixture was observed under a microscope after incubation at 28 C for 5 to 6 h. Fermentation of sugar was observed by the production of gas and acid from a medium containing 0.5% yeast extract, 1.0% peptone, 2.0% sugar, and 30 mg of bromthymol blue per liter, 1 to 3 days after inoculation (18). Enzyme assays. The activities of oa-glucosidase and invertase were assayed as described previously (11). RESULTS Isolation of haploids from baker's yeasts. Yeasts used in baking possess higher dough-leavening ability than other TABLE 2. Dough formula for bakery products Amt of ingredient (g) Ingredient Sweet dough White dough Sponge Dough Sponge Dough Flour Water Yeast (compressed) Yeast food Salt Shortening Sugar GENETIC ANALYSIS OF BAKER'S YEAST 1743 E Tnme(min) FIG. 1. Changes of CO, production rate from sponge dough by the three strains. Symbols: C, YOY34; E, FSC6007; A, X2180-1A. industrial yeasts (11). In the present experiment, we obtained isolates derived from single spores of baker's yeasts conventionally used in Japan. Five of 10 strains tested did not sporulate in any sporulation medium used in the taxonomic studies (18). After 3 to 5 days on SPO plates, FSC6012 sporulated well, with a spore viability of 43% but no complete tetrads. FSC6007, FSC6008, FSC6009, and IF02044 produced low numbers of two- and three-spored asci, and the spore viabilities were less than 1%. Microdissection of spores from FSC6012 yielded 15 MATa and 12 MATot isolates, and random plating of spores from FSC6007 yielded 7 MATa and 11 MATo- isolates, indicating that these two strains were heterothallic. Leavening abilities of doughs containing 0, 5, and 30% sucrose were determined for all the isolates. Most of the isolates could not leaven the dough higher than their parents. Three isolates, YOY50 (MATa) and YOY51 (MATot) from FSC6012 and YOY34 (MAToa) from FSC6007, were selected as those with the highest leavening abilities. When these three isolates were cultured in the molasses medium (11), cell yields were about 80% of their parents. Characterization of the selected haploid strains as baker's yeasts. According to the previous paper (11), 10 properties desirable in baker's yeasts were assayed in the three isolates, and the data obtained were applied to principal component analysis with those of 56 industrial and 2 laboratory strains (11). The first and second principal components were extracted as described in previous experiments (11). The three isolates were mapped in the cluster of baker's yeasts in a scattergram of the first and second principal components. Since these isolates were all quite similar in their properties as baker's yeasts, only one strain, YOY34, was selected for further study. Bread TABLE 3. Effect of three yeast strains on the quality of bakery products Yeast Specific Proof time" strain vol (min) Sweet goods YOY FSC X2180-1A White bread YOY FSC X2180-1A "The period required for proofing to the defined dough volume.

3 1744 ODA AND OUCHI APPL. ENVIRON. MICROBIOL. YOY34 maltose+ )Y A (MATa malo) maltose- malose+:maltose- 4+:- 3+: MALa:malP 2+:0-1+: I I 1IA 1OB YOY107 1~C YOY108 10D 349-6A (MATa mal ) A MA) (MATa malo) (MATa) (MATa malo) (luio) (JhjaO) maltose- maltose- maltose- maltose+ maltose- maltose+ maltose- YOY287 YOY288 YOY428 Figure 1 compares the Fermogram curves of YOY34, FSC6007 (the parental strain of YOY34), and X2180-1A (a laboratory yeast). This curve depicts the rate of CO2 production (milliliters per 5 min) from sponge dough and is equivalent to the Zymotachygram (14). The curves for YOY34 and FSC6007 had two distinct peaks. The first peak represents the fermentation of sugars which preexisted in the flour, and the second, occurring after 60 to 90 min, corresponds to the fermentation of maltose liberated from the starch of the flour by amylases (14). The rate of CO2 production by X2180-1A remained low, and there was no second peak because X2180-1A cannot ferment maltose. YOY34, FSC6007, and X2180-1A were compared with respect to the quality of their backing products. Table 3 shows specific volumes and proof times of sweet goods and white breads. Although no difference was found in sweet goods produced by the three strains, an unpleasant odor was detected in the products of X2180-1A. In white bread the proof time of YOY34 was 6 min longer than that of FSC6007, but much shorter than that of X2180-1A. Specific volumes of YOY34 and FSC6007 were higher than that of X2180-1A. :malo maltose+:maltose- maltos4:maftose- 4+:O- 3+:1-2+:2-4+:0-3+:1-2+:2-4+:0-3+:1-2+:2- o MALa:maP MALg:malO 22+:0-1+:1-0+:2-2+:0-1+:1-0+: MALg MALa MALaMALBO FIG. 2. Genetic analysis of maltose fermentation genes in YOY34. These findings showed that YOY34 could be used to study the properties required for baker's yeasts. Genetic analysis. Since industrial yeasts are often polyploid and some of these have extra copies of particular chromosomes (8, 12), it was necessary to determine the ploidy of YOY34 before analyzing for specific nuclear genes. YOY34 was crossed to strains with the following chromosome-specific markers (number of chromosome): adel (I), his7 (II), MAT (III), trpl (IV), hisi (V), his2 (VI), leul (VII), arg4 (VIII), lysi (IX), ura2 (X), ural (XI), ura4 (XII), rnal-i (XIII), pet8 (XIV), ade2 (XV), aro7 (XVI). Complete tetrads from each hybrid gave essentially 2+:2- segregation, suggesting that YOY34 was a haploid. The other two isolates, YOY50 and YOY51, were also found to be haploids (data not shown). By mutagenesis of YOY34 with UV irradiation, auxotrophic mutants were obtained for adenine, uracil, histidine, phenylalanine, tyrosine, tryptophan, methionine, arginine, lysine, and threonine. When these mutant strains were crossed to X2180-1A, about 70% of the dissected tetrads had Segregation" TABLE 4. Allelism test of MAL genes from YOY34 Segregation' Combination PD T NPD Combination PD T NPD (4+:O-) (3+:1-) (2+:2-) (2+:O-) (1+:1-) (0+:2-) MALa-MALI MALfg-MALI MALot-MAL MALpg-MAL MALu-MAL MALpg-MAL MALot-MAL4C MALpg-MAL4" MALot-MAL MALpg-MAL apd, Parental ditype; T, tetratype; NPD, nonparental ditype.

4 VOL. 55, 1989 GENETIC ANALYSIS OF BAKER'S YEAST 1745 YOY34 L8256 YOY10 sucrose+ I sucrose- sucrose+:sucrose- 4+:O- 3+: A- YOY85 13B L8256 icl 13D YOY85 (MATa) (MATa suco) (MATa) (MATa suco) (MATa) (MATa suco) (MAT) (MATa suco) sucrose+ sucrose- sucrose+ sucrose- sucrose+ sucrose- sucrose+i sucrose- SUCB I 1-10A 156-T1l1 1OB (MATa) (MATa suco) (MATa) sucrose+ sucrose- sucrose+ YOY314 2sucrose+ 2sucrose- SUCI YONY315 2sucrose. +2sucrose- SU )Ca 156-T15 110C (MATa suco) sucrose- (MATa) sucrose+ SUCa YOY155 YOY153 YOY154 YOY156 sucrose+:sucrose : YOY316 SJCa FIG. 3. Genetic analysis of sucrose fermentation genes in YOY34. IOD (MATa) sucrose+ YOY T3 (MATa suco) 156-Ti (MATa suco) sucrose- sucrose- Sijoy Downloaded from four viable spores. These results show that classical techniques in yeast genetics are applicable to YOY34. YOY34 fermented maltose and sucrose but not galactose, while the parental FSC6007 fermented all three sugars. Genetic analyses of sugar fermentations were carried out as described below. Maltose fermentation genes were analyzed in detail according to the procedure of Needleman and Michels (10) (Fig. 2). YOY34 was crossed to strain 349-6A, a non- TABLE 5. oa-glucosidase activities of MAL strains a-glucosidase activity (nmol/min per mg of cells) Strain Genotype in medium: Molasses YPGE YPM YOY107 MA Ta malo ND" YOY108 MATot nalo ND YOY416 MATa MAL] YOY328 MATot MALI YOY334 MATa MALot YOY329 MATot MALot YOY413 MA Ta MAL4' YOY374 MAToa MAL4' yoy349b MATa/a MALoaIMALI YOU383' MATa/oa MALoaIMALI "ND, Not determined. b Hybrid of YOY329 and YOY416. c Hybrid of YOY334 and YOY328. maltose-fermenting mal" strain, to obtain the diploid YOY67. In 12 tetrads resulting from this diploid, maltose fermentation segregated 2+:2-. The non-maltose-fermenting segregants in each tetrad were mated to MALJp tester strains. When maltose fermentation was tested in the resulting diploids, it was found that in 2 tetrads both nonfermenting spores produced maltose-fermenting diploids and in 10 tetrads one of the two non-maltose-fermenting spores produced maltose-fermenting diploids. Further analysis of MAL genes in tetrad 10 showed that spore 10A was malo, 10B was cryptic MALg, 10C was functional MAL, and 10D was both functional MAL and cryptic MALg. Thus, YOY34 apparently contained functional MAL and cryptic MALg genes, which we have tentatively designated as MALot and MAL3g, respectively. Strains carrying either MALot or MALfg were crossed to MAL tester strains (Table 4). In tetrads from the cross between the MALot strain and the MAL] strain, maltose fermentation segregated 4+ :0-, indicating that MALot was allelic to MALI. All of the non-maltose-fermenting segregants derived from the cross between MAL3g and MAL3 strains were complemented by MALJp to give maltose fermentation, showing that MALf3g was allelic to MAL3. Therefore, the MAL genotype of YOY34 is MALI MAL3g. Table 5 compares the cx-glucosidase activities of the strains carrying MAL genes when cultured in the three media. In the molasses medium the activities of MALI tester strains were similar to the basal levels observed in the non-maltose-fermenting malo strains, and the MALot strains on August 31, 2018 by guest

5 1746 ODA AND OUCHI APPL. ENVIRON. MICROBIOL. TABLE 6. Allelism test of SUC genes from YOY34 Segregation Segregation Segregation Combination PD T NPD Combination PD T NPD Combination PD T NPD 4+:O- 3+:1-2+:2-4+:0-3+:1-2+:2-4+:O- 3+:1-2+:2- SUCo-SUCJ SUCI-SUCl SUCy-SUCI SUCot-SUC SUCP-SUC SUC-y-SUC SUCot-SUC SUCI-SUC SUCy-SUC SUCct-SUC SUCP-SUC SUCy-SUC SUCa-SUCS SUCP-SUC SUCy-SUCS a PD, Parental ditype; T, tetratype; NPD, nonparental ditype. had activities as high as those in constitutive MAL4' strains. Activities of MALot strains when grown in YPGE were similar to those of MALI tester strains and different from MAL4C strains. The MALot strains were crossed with the MALI tester strain to obtain diploid heterozygotes at MAL]. The ot-glucosidase activities of these hybrids were similar to those of the MALot strains (Table 5). Activities of hybrids between the strains carrying the MALot and other wild-type MAL genes were comparable to those of MALot strains (data not shown). A diagram of the genetic analysis of SUC genes is given in Fig. 3. YOY34 was crossed to strain L8256, a non-sucrosefermenting suc strain. In 16 tetrads resulting from this cross (cross YOY10), sucrose fermentation segregated 4+:0- in 12 tetrads and 3+:1- in 4 tetrads, suggesting the involvement of more than two SUC genes. Further pedigree analysis in sucrose-fermenting segregants showed that three unlinked SUC genes were present in YOY34 (Fig. 3). The SUC genes in YOY10-13C, YOY156-1OB, and YOY156-1OC were allelic, and this gene was tentatively designated SUCco. Similarly, the SUC gene in YOY10-13A and those in YOY156-1OA and YOY156-1OD were designated SUCP and SUC-y, respectively. The strains carrying one of three genes derived from YOY34 were crossed to wild-type SUC tester strains, and the resulting tetrads were dissected. The hybrid in the combination of SUCot-SUC2, SUCQ-SUC4, and SUCy-SUCI produced only the parental ditype tetrad, but neither the nonparental ditype tetrad nor the tetratype tetrad, indicating that SUCot, SUCP, and SUCy were allelic to SUC2, SUC4, and SUCJ, respectively (Table 6). All of SUC strains actively fermented sucrose in 2 days except for the SUC-y segregants derived from YOY34, which only produced detectable amounts of gas after 7 days. The strains carrying SUCot and SUC3 derived from YOY34 and those carrying one of the five wild-type SUC genes had extracellular invertase, whereas a segregant containing a SUC-y gene TABLE 7. Invertase activities of SUC strains Strains SUC genotype Invertase activity (nmol/min per mg of cells) YOY156-1OC sucoa 326 YOY1O-13A sucra 1,530 YOY156-1OA SUCYa NDb L8256 suc ND D SUCI 513 X2180-1A SUC D SUC3 458 SS-4A SUC4 1, C SUC5 616 a Gene derived from YOY34. b ND, Not detected. derived from YOY34 showed no detectable activity (Table 7). The genotype of galactose fermentation was analyzed as follows. YOY34 was crossed to a galactose-fermenting strain, giving diploid YOY67. Dissected asci from YOY67 gave 22 complete tetrads which all segregated 2+:2- for galactose fermentation, indicating the involvement of a single gal gene. When YOY34 was mated to eight gal tester strains, gal from YOY34 was not complemented by gall. Therefore, the gal genotype of YOY34 is gall. DISCUSSION There have been some variations of standard constitutive MAL genes designated as MALC. ox-glucosidase activities of MALJC and MAL2C strains when grown on maltose were higher than those on sucrose (15, 19). A MAL4C strain showed higher activity and remained constant in the presence or absence of maltose (3). Since the MALot strains derived from YOY34 and the diploids from crosses between the MALot strains and other wild-type MAL strains showed a constitutive phenotype in the molasses medium, we defined this MALot gene as a constitutive and dominant allele. Perkins and Needleman (13) recently selected constitutive MAL mutants from a suco MAL6 strain as sucrose fermenters, in which sucrose was transported into the cells by the maltose carrier with subsequent hydrolysis by maltase. Since a SUC-y strain, YOY156-1OA, lacking extracellular invertase can grow on sucrose as a sole carbon source, sucrose might be hydrolyzed inside these cells as in the constitutive MAL" strains. Considering that the chromosomal position of SUCJ is tightly linked to MAL], the apparent sucrose-fermenting phenotype of YOY156-1OA could be based on MALl'. Consequently, the genotype of the isolated haploid, YOY34, which mimicked a conventional baker's yeast, was identified as MATot MALJC MAL3g SUC2 SUC4 gall. Cryptic MALlg and MAL3g are also found in the wild-type MAL tester strains (9). For galactose fermentation, YOY34, in contrast to FSC6007, lacked the structural gene for galactokinase. This means that FSC6007, the parental strain of YOY34, is heterozygous for a galactose fermentation gene, which was probably the result of a spontaneous mutation as found in some industrial yeasts (6). Lower leavening ability and worse baking quality make X2180-1A a poor choice of a model strain as a baker's yeast. YOY34 gave a higher percentage of four-spored asci when crossed with laboratory strains and contained the desired properties required for baker's yeast. In conclusion, YOY34 is a strain which can be applicable to genetic and biochemical analyses. Elucidation of genes responsible for dough leavening is in

6 VOL. 55, 1989 progress. The role of the MAL' gene in flour dough fermentation will be reported elsewhere. ACKNOWLEDGMENTS We thank R. B. Needleman, T. Mizunaga, and Y. Nogi for providing standard strains, K. Ohsawa for her technical assistance, and T. Kanetoki for his helpful advice. LITERATURE CITED 1. Approved Methods Committee, American Association of Cereal Chemists, Inc Baking quality of wheat bread flour, p American Association of Cereal Chemists, St. Paul. Minn. 2. Botstein, D., and G. R. Fink Yeast: an experimental organism for modern biology. Science 240: Charron, M. J., and C. A. Michels The constitutive, glucose-repression-insensitive mutation of the yeast MAL4 locus is an alteration of the MAL43 gene. Genetics 116: Hino, A., H. Takano, N. Kitabayashi, F. Nitta, T. Ohishi, and Y. Tanaka Automatic measuring system for dough testing: design, construction and reproducibility. Nippon Syokuhin Kogyo Gakkaishi 35: (In Japanese.) 5. Hino, A., H. Takano, and Y. Tanaka New freeze-tolerant yeast for frozen dough preparations. Cereal Chem. 64: Jimenez, J., and T. Benitez Genetic analysis of highly ethanol tolerant wine yeasts. Curr. Genet. 12: Johnston, J. R., and H. Oberman Yeast genetics in industry. Prog. Ind. Microbiol. 15: Keiding, A. K Genetic and molecular characterization of a distiller's yeast. Carlsberg Res. Commun. 50: Michels, C. A., and R. B. Needleman A genetic and GENETIC ANALYSIS OF BAKER'S YEAST 1747 physical analysis of the MALI and MAL3 standard strains of Saccharomvces cerev'isiae. Mol. Gen. Genet. 191: Needleman, R. B., and C. Michels Repeated family of genes controlling maltose fermentation in Saccharomynces cairlsbergensis. Mol. Cell. Biol. 3: Oda, Y., and K. Ouchi Principal component analysis of the characteristics desirable in baker's yeasts. Appl. Environ. Microbiol. 55: Pedersen, M. B DNA sequence polymorphisms in the genus Saccharomnyces. IV. Homeologous chromosome III of Saccharotnvces bavanus, S. ccarlsbergensis. and S. uvarumn. Carlsberg Res. Commun. 51: Perkins, E. L., and R. B. Needleman MAL64' is a global regulator of ox-glucoside fermentation: identification of a new gene involved in melezitose fermentation. Curr. Genet. 13: Ponte, J. G., Jr., and G. Reed Bakery foods, p In G. Reed (ed.), Prescott & Dunn's industrial microbiology, 4th ed. AVI Publishing Co., Inc., Westport, Conn. 15. Rodicio, R Insertion of non-homologous DNA sequences into a regulatory gene cause a constitutive maltase synthesis in yeast. Curr. Genet. 11: Spencer, J. F. T., and D. M. Spencer Genetic improvement of industrial yeasts. Annu. Rev. Microbiol. 37: Spencer, J. F. T., and D. M. Spencer Yeast genetics, p In I. Campbell and J. H. Duffus (ed.). Yeast: a practical approach. IRL Press, Oxford. 18. van der Walt, J. P., and D. Yarrow Methods for the isolation, maintenance, classification and identification of yeasts, p In N. J. W. Kreger-van Rij (ed.), The yeasts: a taxonomic study, 3rd ed. Elsevier Science Publisher B. V., Amsterdam. 19. Zimmermann, F. K., and N. R. Eaton Genetics of induction and catabolite repression of maltase synthesis in Saccharomyces cerevisiae. Mol. Gen. Genet. 134: Downloaded from on August 31, 2018 by guest