JOURNAL OF BACTERIOLOGY, Feb. 1979, p. 1051-1052 0021-9193/79/02-1051/02$02.00/0
Vol. 137, No. 2
Microtiter Plate Assay for Sexual Agglutination in the Yeast Hansenula wingei MARJORIE CRANDALL Thomas Hunt Morgan School of Biological Sciences, University of Kentucky, Lexington, Kentucky 40506 Received for publication 12 December 1978
A fast method is described for determining the mating type of yeast spore clones independent of genetic complementation between auxotrophic markers.
The standard method in yeast genetics for determining the mating type of a spore clone is by complementation between auxotrophic markers (4). This test is performed by mixing cells from an auxotrophic spore clone with tester cells of each mating type carrying a complementary auxotrophic marker. Growth of a prototrophic diploid with one tester but not the other tester is the evidence used to determine mating type. For this method to be successful, the diploid being dissected must be homozygous for one auxotrophic allele so that prototrophic spores cannot be generated by recombination. Preparing the proper hybrids and testers requires considerable genetic engineering, and the complementation test takes several days. A simpler and quicker method for determining the mating type of spore clones is to assay for sexual agglutination in a microtiter plate. The yeast Hansenula wingei is constitutive for sexual agglutination-the two mating types clump immediately upon mixing (6). Use of disposable microtiter plates for assaying sexual agglutination has the advantages of being quick, requiring only a small amount of cells for assay, and eliminating cleanup of culture flasks, pipettes and assay tubes. In one microtiter plate with 96 wells, 48 spore clones (12 tetrads) can be assayed for mating type with both testers. A microtiter plate assay has also been used to titrate the solubilized sexual agglutination factor from strain 5 of H. wingei (7). Tester cells are grown at 30°C with aeration for 36 to 48 h in YKD broth (0.7% yeast extract, 0.5% KH2PO4, 2% dextrose) inoculated 1:100 from a 24-h preculture. The two mating types, strains 5 and 21 (1), can be wild type or genetically marked. Fernbach flasks are used to grow large culture volumes (800 ml) which are centrifuged in l-quart (ca. 0.95-liter) bottles in a Sorvall RC-3 swinging bucket head. The cells are washed once in sterile distilled water and then the cell pellet from 800 ml of culture is resuspended in 400 ml of sterile distilled water (equals
2x cell concentration). This suspension is divided into 200-ml portions in centrifuge bottles and steamed for 30 min (2). Immediately after this heat activation step, the suspensions must be quick cooled in an ice water bath. The cells are harvested in a precooled (5°C) centrifuge and then washed twice more with sterile distilled water at room temperature. The pellet from 800 ml is resuspended in 20 ml of sterile distilled water (equals 40x cell concentration). This suspension is stored in the refrigerator in screw-cap test tubes with a few drops of chloroform added to prevent contamination. These testers are stable for years. Before use, the chloroform is aspirated from the bottom, and the cells are resuspended and diluted 1:20 (2x) in 0.9% saline. Spore clones are replica plated from the dissection master to a plate of YKD plus 2% agar. After 1 to 3 days of growth, cells are removed from the growing edge of the clone with a sterile toothpick. The amount of cells added per well is not critical; usually a quantity of cells about 1 to 2 mm in diameter is removed from the spore clone. Half is suspended in 2 drops (0.04 ml) of saline in one well of a microtiter plate; the other half is suspended in a duplicate well. To one well is added two drops of 5H (strain 5 heated tester at 2x cell concentration in saline); to the duplicate well is added 2 drops of 21H. The disposable microtiter plate (3.5 by 5.25 inches [ca. 8.9 by 13.3 cm]; clear plastic with 96 round cups, each having a 0.25-ml capacity; Preiser Scientific Co.) is covered with transparent plate sealers (precut acetate sealing tape; 7.9 by 13.1 cm; Cooke Laboratory Products, Alexandria, Va.) and sealed tightly around each well. The plate is tapped to dislodge bubbles and shaken in the vertical position on a reciprocating shaker at 200 rpm for 5 min. Strength of agglutination can be scored on a scale from - to ++++ (2). However, quantitative measurements are more accurately performed with liquid cultures in test tubes (2). This microtiter plate assay for mating type saves time, sample, and money and is semiquan-
1051
1052
J. BACTERIOL.
NOTES
The technical assistance of Bob Wood and Don Cagle is titative with respect to strength of agglutination. It is especially valuable because it can be done acknowledged. on prototrophic as well as auxotrophic spore LITERATURE CITED clones. The agglutination types 5 and 21 segre- 1. Crandall, M. A., and T. D. Brock. 1968. Molecular basis gate 2:2 in tetrads, indicating that they are alof mating in the yeast Hansenula wingei. Bacteriol. Rev. 32:139-163. lelic. It might be possible to adapt this method to 2. Crandall, M. A., and J. H. Caulton. 1975. Induction of haploid glycoprotein mating factors in diploid yeasts. Saccharomyces cerevisiae because constituMethods Cell Biol. 12:185-207. tively agglutinative strains of S. cerevisiae have 3. Crandall, M. A., R. Egel, and V. L. MacKay. 1977. Physiology of mating in three yeasts. Adv. Microb. been reported (5). Alternatively, sexual agglutiPhysiol. 15:307-398. nation could be induced by mixing live tester 4. Fogel, S., and R. K. Mortimer. 1970. Fidelity of meiotic cells in growth medium with cells from spore gene conversion in yeast. Mol. Gen. Genet. 109:177clones in the microtiter plate and shaking for 185. several hours. This assay could also be used to 5. Sakai, K., and N. Yanagishima. 1972. Mating reaction in Saccharomyces cerevisiae. II. Hormonal regulation titrate a-factor and a-factor preparations for of agglutinability of a type cells. Arch. Mikrobiol. 84: their ability to induce agglutinability in a and a 191-198. cells, respectively (3). Cells could be incubated 6. Wickerham, L. J. 1956. Influence of agglutination on zygote formation in Hansenula wingei, a new species with mating factor from the opposite sex in a of yeast. C. R. Trav. Lab. Carlsberg Ser. Physiol. 26: microtiter plate, and then shaken with induced 423-443. or constitutive cells of the opposite mating type. 7. Yen, P. H., and C. E. Ballou. 1974. Partial characteriThis
work
was
supported by Public
Health
Service grant
GM21889 from the National Institutes of Health.
zation of the sexual agglutination factor from Hansenula wingei Y-2340 type 5 cells. Biochemistry 13:24282437.
Vol. 137, No. 2
Microtiter Plate Assay for Sexual Agglutination in the Yeast Hansenula wingei MARJORIE CRANDALL Thomas Hunt Morgan School of Biological Sciences, University of Kentucky, Lexington, Kentucky 40506 Received for publication 12 December 1978
A fast method is described for determining the mating type of yeast spore clones independent of genetic complementation between auxotrophic markers.
The standard method in yeast genetics for determining the mating type of a spore clone is by complementation between auxotrophic markers (4). This test is performed by mixing cells from an auxotrophic spore clone with tester cells of each mating type carrying a complementary auxotrophic marker. Growth of a prototrophic diploid with one tester but not the other tester is the evidence used to determine mating type. For this method to be successful, the diploid being dissected must be homozygous for one auxotrophic allele so that prototrophic spores cannot be generated by recombination. Preparing the proper hybrids and testers requires considerable genetic engineering, and the complementation test takes several days. A simpler and quicker method for determining the mating type of spore clones is to assay for sexual agglutination in a microtiter plate. The yeast Hansenula wingei is constitutive for sexual agglutination-the two mating types clump immediately upon mixing (6). Use of disposable microtiter plates for assaying sexual agglutination has the advantages of being quick, requiring only a small amount of cells for assay, and eliminating cleanup of culture flasks, pipettes and assay tubes. In one microtiter plate with 96 wells, 48 spore clones (12 tetrads) can be assayed for mating type with both testers. A microtiter plate assay has also been used to titrate the solubilized sexual agglutination factor from strain 5 of H. wingei (7). Tester cells are grown at 30°C with aeration for 36 to 48 h in YKD broth (0.7% yeast extract, 0.5% KH2PO4, 2% dextrose) inoculated 1:100 from a 24-h preculture. The two mating types, strains 5 and 21 (1), can be wild type or genetically marked. Fernbach flasks are used to grow large culture volumes (800 ml) which are centrifuged in l-quart (ca. 0.95-liter) bottles in a Sorvall RC-3 swinging bucket head. The cells are washed once in sterile distilled water and then the cell pellet from 800 ml of culture is resuspended in 400 ml of sterile distilled water (equals
2x cell concentration). This suspension is divided into 200-ml portions in centrifuge bottles and steamed for 30 min (2). Immediately after this heat activation step, the suspensions must be quick cooled in an ice water bath. The cells are harvested in a precooled (5°C) centrifuge and then washed twice more with sterile distilled water at room temperature. The pellet from 800 ml is resuspended in 20 ml of sterile distilled water (equals 40x cell concentration). This suspension is stored in the refrigerator in screw-cap test tubes with a few drops of chloroform added to prevent contamination. These testers are stable for years. Before use, the chloroform is aspirated from the bottom, and the cells are resuspended and diluted 1:20 (2x) in 0.9% saline. Spore clones are replica plated from the dissection master to a plate of YKD plus 2% agar. After 1 to 3 days of growth, cells are removed from the growing edge of the clone with a sterile toothpick. The amount of cells added per well is not critical; usually a quantity of cells about 1 to 2 mm in diameter is removed from the spore clone. Half is suspended in 2 drops (0.04 ml) of saline in one well of a microtiter plate; the other half is suspended in a duplicate well. To one well is added two drops of 5H (strain 5 heated tester at 2x cell concentration in saline); to the duplicate well is added 2 drops of 21H. The disposable microtiter plate (3.5 by 5.25 inches [ca. 8.9 by 13.3 cm]; clear plastic with 96 round cups, each having a 0.25-ml capacity; Preiser Scientific Co.) is covered with transparent plate sealers (precut acetate sealing tape; 7.9 by 13.1 cm; Cooke Laboratory Products, Alexandria, Va.) and sealed tightly around each well. The plate is tapped to dislodge bubbles and shaken in the vertical position on a reciprocating shaker at 200 rpm for 5 min. Strength of agglutination can be scored on a scale from - to ++++ (2). However, quantitative measurements are more accurately performed with liquid cultures in test tubes (2). This microtiter plate assay for mating type saves time, sample, and money and is semiquan-
1051
1052
J. BACTERIOL.
NOTES
The technical assistance of Bob Wood and Don Cagle is titative with respect to strength of agglutination. It is especially valuable because it can be done acknowledged. on prototrophic as well as auxotrophic spore LITERATURE CITED clones. The agglutination types 5 and 21 segre- 1. Crandall, M. A., and T. D. Brock. 1968. Molecular basis gate 2:2 in tetrads, indicating that they are alof mating in the yeast Hansenula wingei. Bacteriol. Rev. 32:139-163. lelic. It might be possible to adapt this method to 2. Crandall, M. A., and J. H. Caulton. 1975. Induction of haploid glycoprotein mating factors in diploid yeasts. Saccharomyces cerevisiae because constituMethods Cell Biol. 12:185-207. tively agglutinative strains of S. cerevisiae have 3. Crandall, M. A., R. Egel, and V. L. MacKay. 1977. Physiology of mating in three yeasts. Adv. Microb. been reported (5). Alternatively, sexual agglutiPhysiol. 15:307-398. nation could be induced by mixing live tester 4. Fogel, S., and R. K. Mortimer. 1970. Fidelity of meiotic cells in growth medium with cells from spore gene conversion in yeast. Mol. Gen. Genet. 109:177clones in the microtiter plate and shaking for 185. several hours. This assay could also be used to 5. Sakai, K., and N. Yanagishima. 1972. Mating reaction in Saccharomyces cerevisiae. II. Hormonal regulation titrate a-factor and a-factor preparations for of agglutinability of a type cells. Arch. Mikrobiol. 84: their ability to induce agglutinability in a and a 191-198. cells, respectively (3). Cells could be incubated 6. Wickerham, L. J. 1956. Influence of agglutination on zygote formation in Hansenula wingei, a new species with mating factor from the opposite sex in a of yeast. C. R. Trav. Lab. Carlsberg Ser. Physiol. 26: microtiter plate, and then shaken with induced 423-443. or constitutive cells of the opposite mating type. 7. Yen, P. H., and C. E. Ballou. 1974. Partial characteriThis
work
was
supported by Public
Health
Service grant
GM21889 from the National Institutes of Health.
zation of the sexual agglutination factor from Hansenula wingei Y-2340 type 5 cells. Biochemistry 13:24282437.
