- Email: [email protected]
Chapter 5 Synchronous Zygote Formation in Yeasts
Chapter 5 Sychronozcs Zygote Formation in Yeasts T. BILINSKI, J. LITWmSKA, J. ZUK,
AND
W. GAJEWSKI
Institute of Biochemistry and Biophysim, Polish Amdenry of Sdenm. Warsaw,Poland
I. General Characteristic of the Conjugation Process
.
.
.
.
.
.
. . .
. . .
. . .
. . .
. . . .
. . . .
11. Basic Pri9ciples for Synchronous Mass Production of Zygotes . . 111. Method for Poorly Synchronized Zygote Formation for Genetic Analysis
IV. Method of Synchronous Zygote Mass Production V. Limitations of the Method . . . . References. . . . . . .
.
.
89 91 92 92 93 96
I. General Characteristic of the Conjugation Process Although genetic analysis of the yeast Saccharomyces cerevisiae based on sexual fusion and tetrad analysis is one of the most advanced as regards eukaryotic organisms, knowledge of sexual processes in this organism is still very limited. It was only recently that the processes occurring during the sexual cycle were experimentally studied. For any kind of physiological or biochemical study of the sexual cycle in yeasts, synchronous mass production of zygotes is a first prerequisite. The sexual processes in haploid strains of opposite mating type (a and a) leading to zygote formation consist of many characteristic consecutive stages. Three main stages of the sexual cycle are courtship, cell fusion, and karyogamy. In each stage characteristic genetic and biochemical processes occur which prepare the cells for the next sexual reaction. As already established by Hartwell (1973), a and acells are competent for sexual fusion only at an appropriate stage of the cell cycle; competent cells are unbudded and in a cycle position just before the initiation of DNA replication. Duntze et al. (1970) and Yanagishima (1971) demonstrated that inter89
90
T. BILINSKI, J. LITWINSKA, J. ZUK, AND W. GAJEWSKI
action between haploid cells ofoppositemating typeismediated by hormonal substances released into the culture medium. According to Throm and Duntze (1970) and Bucking-Throm et al. (1973), a cells produce an afactor (peptidic in nature) which is released into the medium. This diffusible a factor inhibits the initiation of DNA replication in a cells. In this way synchronization of a cells is achieved; they stop budding and become competent for sexual fusion. Analogous processes of synchronization probably also occur in a cells. We showed recently (Bilidski et al., 1974) that, in a cells separated from the conjugation mixture on Dowex resin, DNA synthesis and budding are inhibited. The lag in DNA synthesis preceding sexual fusion of a and a cells is not only due to swelling but, according to our unpublished data, the dry synthesis and budding are resumed. The mixing of haploid cells of opposite mating type results not only in mutual synchronization, but also in other changes characteristic of the conjugation process. The cell volume increases, and agglutination of cells takes place (Sakai and Yanagishima, 1971). The increase in the cell volume is not only due to swelling but, according to our unpublished data, the dry mass content also increases owing to intensive RNA and protein syntheses. Agglutination is a decisive factor ensuring intimate contact between cells of opposite mating type. Agglutination, however, is nonspecific, it occurs among cells of different as well as the same mating type and results in the formation of large clumps. In S. cerevisiae agglutination is much weaker than, for instance, in Hansenula wingei (Crandall and Brock, 1968). In the next step cells of opposite mating type form pairs and undergo fusion and plasmogamy. As a result, young zygotes are formed in which nuclear fusion (karyogamy) takes place. Diploid zygotes proliferate mitotically or undergo meiosis and ascospore formation, depending on nutritional conditions. The capacity for sexual reaction between a and a cells is controlled by one allelic pair in the mating-type locus in yeasts. This locus is probably multicistronic and regulates the synthesis of many products involved in mating reactions. It is known that copulation and efficiency of mating reactions are highly variable, depending on the yeast strain used. Most probably the efficiency of the mating reaction depends on the interaction of many different genetic factors present in different strains. For a detailed study of the biochemical processes occurring in consecutive stages of sexual cell fusion in yeasts, the use of genetic mutants with specific blocks for different stages is necessary. To obtain such mutants, however, a general knowledge of culture conditions for each stage and of synchronization of mating processes must first be obtained. Only in this way can the genetic mechanisms governing the sexual cycle in yeasts be determined.
5.
SYNCHRONOUS ZYGOTE FORMATION IN YEASTS
91
11. Basic Principles for Synchronous Mass Production of Zygotes
The classic method of Lindegren gives a yield of zygotes usually of the order of a few percent of the haploid cells in the conjugation mixture. Moreover, the period of zygote formation is extended to several hours. Jakob (1962) greatly increased the yield of zygotes by enforcing cellular contacts between competent haploid cells by centrifugation. This procedure raised the yield of zygotes to about 15%. However, the synchronization in Jakob’s procedure is still rather poor, and the zygotes are formed in the course of more than 2 hours. Since the initial a- and ahcell cultures are unsynchronized and the synchronization through mutual interaction in the conjugation mixture is only partial and transitory, the first and most important step is the preparation of a homogeneous mixture of competent cells of opposite mating type. This can be accomplished either by separation of homogeneous a and (Y cells in suitable stages for sexual fusion, as was done by Sena et al. (1973), or by differentiating the mating procedure in such a way that conditions are optimal for synchronization but unfavorable for cell fusion (Bilidski et al., 1973). Homogeneous populations of competent cells ensure a maximal yield of zygotes for any given pair of haploid strains, whatever the method used for purification of competent cells. Synchronization of zygote formation can be achieved in several ways. We usually obtain the highest efficiency of conjugation in cultures from late logarithmic phase when the initial cell population is highly asynchronous. The time required for cells from different phases of the cell cycle to achieve competence varies greatly, and therefore a basic condition for synchronization of cell fusion is the prevention of premature zygote formation before the whole cell population becomes competent. This can be achieved by creating conditions favorable to quick competence development and by disrupting early contacts between competent cells by sonication. In this way it is possible to obtain a practically pure competent cell suspension (95%) without premature zygote formation. The next condition for synchronous zygote formation is the enforcement of contact between competent cells by centrifugation as applied by Jakob (1962). A contact enforced in a pellet during 3 hour results in restriction of the time of fusion between cells to about I hour, this being a much higher degree of synchronization as compared with that obtained by other methods. At the end of efficient conjugation, sonication should be applied again to disintegrate clumps and to stop formation of new zygotes.
92
T. BILINSKI, J. LITWINSKA, J. ZUK,AND W. GAJEWSKI
111. Method for Poorly Synchronized Zygote Formation for Genetic Analysis The cultures of a and a strains in late log phase in YPG medium (yeast extract, 1%; Bacto peptone, 1%; and glucose, 2%) are centrifuged and resuspensed in conjugation medium containing 1% yeast extract and 3% glucose with pH 4.5 (adjusted with acetic acid). The concentration of both strains should be nearly identical within the range of 107 to lo8cells/ml. The cells of both strains are mixed in a 1: 1 ratio, and the mixture is incubated for 2 hours at 280Cwithout shaking. After incubation the mixture is diluted 10 times with fresh medium, immediately centrifuged for 2 minutes at 3000 g, and left for 30 minutes at 28 oC. Then the pellet is gently agitated by manual shaking, and the preparation left for another 30 minutes at 28 OC without shaking. After this time the pellet is disintegrated by more vigorous manual shaking without formation of a homogeneous suspension. The shaking is applied only to provide better access of nutrients to the pellet; more thorough disintegration decreases markedly the efficiency of conjugation. The pellet is then further incubated at 280C. For most of the strains tested, this procedure gives maximum zygote formation 5 hours after the mixing of a and cx cells.
IV. Method of Synchronous Zygote Mass Production The basic medium for zygote formation is 3% YE consisting of 3% of glucose and 1% of yeast extract. For cultivation of haploid strains, YPG medium consisting of 1% yeast extract, 1% Bacto peptone, and 2% glucose is used. The haploid strains are cultivated in 250-ml Erlenmeyer flasks containing 100 ml of YPG medium for 18 hours at 3OoCon an orbital shaker (120 rpm). Cells are harvested at the end of the log phase by centrifugation for 5 minutes at 3000 g (Sorvall SS1) in 50-ml steel tubes sterilized with 70% ethanol. Suspensions of lo8 cells/ml of each strain in the conjugation medium (3% YE) are prepared. The pH of the medium is adjusted to 4.5 with 1 M acetic acid. The suspensions of cultures of both mating types are mixed together in a 1 :1 ratio. The samples containing 35 ml of mixture are incubated for 1 hour at 3OoC in 50-ml steel Sorvall centrifuge tubes, After incubation the cells are centrifuged under the same conditions for 5 minutes at 3000 g in a Sorvall SSl centrifuge, resuspended in 35 ml of 3% YE, adjusted to pH 8.5 with 1 M Na,HPO,, and exposed for a further incubation of 1 hour. The change in pH of the medium from 4.5 during the first hour to 8.5 during the next hour of the mating reaction is very important. It provides optimal
5.
SYNCHRONOUS ZYGOTE FORMATION IN YEASTS
93
conditions for synchronization of cells, but suboptimal conditions for cell fusion and zygote formation. After 1 hour of incubation at pH 8.5, the cells are centrifuged, resuspended in 35 ml of fresh YE medium (pH 4.9, and sonicated twice for 30 seconds (amplitude from peak to peak 2 pm with an MSE 100-w ultrasonic disintegrator). Sonication ensures maximum efficiency of synchronization without cell fusion. Samples of 20 ml of sonicated suspension are added to 250-ml steel centrifuge tubes containing 180 ml of fresh 3% YE medium (pH 4.5) and centrifuged immediately on a MSE 18 high-speed centrifuge with an angle rotor (6 x 250 ml) at room temperature for 2 minutes at 3000 rpm. The samples in the centrifuge tubes are then incubated at 300 for 30 minutes. Afterward the pellet is fragmented by gentle shaking and incubated without shaking for another 30 minutes. At the end of incubation, the samples are shaken again to disintegrate the pellet, and further incubated without shaking. With this procedure zygote formation starts after 4.5 hours of incubation following the mixing of a and o cells, and their number rises abruptly within 1 hour. During this time no buds are formed by the zygotes, which are all in the same relatively young stage. The yield of zygotes ranges from 30 to 40%. The changes observed in the cell population during the conjugation procedure under the conditions described above are shown in Fig. 1. Samples of initial cultures of a and o cells and the conjugation mixture are analyzed microscopically at 15-minute intervals for estimation of budded cells percentage. Also, the number of conjugated cells (zygotes) can be estimated in the conjugation mixture. The first zygotes appear 4.5 hours after cell mixing, and the curve of zygote formation rises very abruptly during the first hour. A further increase in the frequency of diploid cells is due to the separation of diploid buds from the zygotes. The incubation time for maximal efficiency of zygote production must be determined for each pair of haploid strains used. When the maximum is reached, the conjugation mixture should be sonicated to prevent the formation of new zygotes. If strict synchronization is required and the maximum yield of zygotes is less important, sonication may be applied much earlier.
V.
Limitations of the Method
The described method of synchronous mass production of zygotes is applicable to any pair of haploid strains, provided they are mutually well synchronized in the conjugation mixture so that the majority of haploid cells are competent to fuse and form zygotes. If the a and o cells do not
T. BILINSKI, J. LITWINSKA. J. ZVK, AND W. GAJEWSKl
94 60
50
-
.. E
140
U U W
;30
I20
n
Y)
L
0
$? 2 0
60
% ;
40
2
80
(0
h N
L
5
20 z
0
I
2
3
4
5
6
Incubation time ( h o u r s )
FIG. 1. Numerical relations among different kinds of cells during conjugation. Solid circles, percent of a cells with buds in initial culture; open circles, percent of a cells with buds in initial culture; squares, percent of a and a cells with buds in conjugation mixture; triangles, number of zygotes in 1 ml of conjugation mixture.
synchronize well and the percentage of competent cells in the conjugation mixture is low, the application of the procedure proposed by Sena et al. (1973) is recommended. This method consists of isolation of competent cells from the conjugation mixture by gradient centrifugation. For all technical details of this method, the reader is referred to Sena’s publication (Sena et al., 1973). However, if the ability of the cells to fuse is low, it is impossible to raise the yield of the zygotes. If the zygotes are being produced for biochemical or physiological studies, it is important to start with a and a strains checked beforehand for high capacity for zygote formation (see our simplified method, Section 111). It should be stressed that the proportion of a and acells in the conjugation mixture is a very important factor for high efficiency of zygote formation. As Sena et al. (1973) showed, the optimal proportion is 1: 1. Even small changes in this proportion can result in a drastic decrease in zygote production. We have already described (Bilidski et d., 1973) the inhibitory effect of increasing proportions of @mating-type cells in the conjugation mixture on zygote formation. Figure 2 shows the effect of changes in the
5.
SYNCHRONOUS ZYGOTE FORMATION IN YEASTS
95
50
40
;3 0 t
O m h
N
,\" 20
% a Cells
100
a0
60 40 % a Cells
20
0
FIG. 2. Mating efficiency at various a and a ratios. Broken line, theoretical corrected; solid line, observed.
proportions of a and a cells on the efficiency of conjugation. As seen, the efficiency curve is clearly asymmetric on the side of the a surplus and differs markedly from the symmetric theoretical curve. This phenomenon is observed very often, and the degree of asymmetry differs greatly according to the pair of strains used for conjugation. The absolute concentration of cells in the conjugation mixture is also of paramount importance for cell fusion processes. For mass production of zygotes, concentrations of lo* cells/ml in the first phase and lo7 cells/ml during the conjugation process are recommended (as described in our procedure). High concentrations of cells are very convenient for all manipulations and economize in the use of media. Lower concentrations (107 cells/ml) during the first 2 hours and lo6cells/ml, but not less, duringzygote formation may also be used if a smaller number of zygotes is required. For a high degree of synchronization, higher concentrations are recommended, but for cell fusion itself rather low concentrations are optimal. The composition of the medium also has a very strong influence on the course of conjugation. As a rule, it should be a rich medium, and the most important constituent is glucose. Any other source of carbon or energy that still permits growth practically stops zygote formation completely.
96
T. BILINSKI, J. LITWII%KA, J. ZUK, AND W. GAJEWSKl
REFERENCES Bilifiski, T., Litwidska, J., Zuk, J., and Gajewski, W. (1973). J. Gen. Microbiol. 79, 285. Bilidski, T., Jachymczyk, W., Litwihska, J., and Zuk, J. (1974). J. Gem Microbiol. 82,97. Backing-Throm, E., Duntze, W., Hartwell, H., and Manney, T. R. (1973). Exp. CellRes. 76, 99. Crandall, M. A., and Brock, T. D. (1968). Bacteriol. Rev. 32, 139. Duntze, W., MacKay, V., and Manney, T. R. (1970). Science 168, 1472. Hartwell, L. H. (1973). Exp. Cell Res. 76, 111. Jakob, H. (1%2). C. R. Amd. Sci. 254,3909. Sakai, K., and Yanagishima, N. (1971). Arch. Microbiol. 75,260. Sena, E. P., Radin, N., and Fogel, S. (1973). Proc. Nat. Amd. Sci. US.70, 1373. Throm, E., and Duntze, W. (1970). J. Bacteriol. 104, 1388. Yanagishima, N. (1971). Physiol. Plant. 24, 260.
AND
W. GAJEWSKI
Institute of Biochemistry and Biophysim, Polish Amdenry of Sdenm. Warsaw,Poland
I. General Characteristic of the Conjugation Process
.
.
.
.
.
.
. . .
. . .
. . .
. . .
. . . .
. . . .
11. Basic Pri9ciples for Synchronous Mass Production of Zygotes . . 111. Method for Poorly Synchronized Zygote Formation for Genetic Analysis
IV. Method of Synchronous Zygote Mass Production V. Limitations of the Method . . . . References. . . . . . .
.
.
89 91 92 92 93 96
I. General Characteristic of the Conjugation Process Although genetic analysis of the yeast Saccharomyces cerevisiae based on sexual fusion and tetrad analysis is one of the most advanced as regards eukaryotic organisms, knowledge of sexual processes in this organism is still very limited. It was only recently that the processes occurring during the sexual cycle were experimentally studied. For any kind of physiological or biochemical study of the sexual cycle in yeasts, synchronous mass production of zygotes is a first prerequisite. The sexual processes in haploid strains of opposite mating type (a and a) leading to zygote formation consist of many characteristic consecutive stages. Three main stages of the sexual cycle are courtship, cell fusion, and karyogamy. In each stage characteristic genetic and biochemical processes occur which prepare the cells for the next sexual reaction. As already established by Hartwell (1973), a and acells are competent for sexual fusion only at an appropriate stage of the cell cycle; competent cells are unbudded and in a cycle position just before the initiation of DNA replication. Duntze et al. (1970) and Yanagishima (1971) demonstrated that inter89
90
T. BILINSKI, J. LITWINSKA, J. ZUK, AND W. GAJEWSKI
action between haploid cells ofoppositemating typeismediated by hormonal substances released into the culture medium. According to Throm and Duntze (1970) and Bucking-Throm et al. (1973), a cells produce an afactor (peptidic in nature) which is released into the medium. This diffusible a factor inhibits the initiation of DNA replication in a cells. In this way synchronization of a cells is achieved; they stop budding and become competent for sexual fusion. Analogous processes of synchronization probably also occur in a cells. We showed recently (Bilidski et al., 1974) that, in a cells separated from the conjugation mixture on Dowex resin, DNA synthesis and budding are inhibited. The lag in DNA synthesis preceding sexual fusion of a and a cells is not only due to swelling but, according to our unpublished data, the dry synthesis and budding are resumed. The mixing of haploid cells of opposite mating type results not only in mutual synchronization, but also in other changes characteristic of the conjugation process. The cell volume increases, and agglutination of cells takes place (Sakai and Yanagishima, 1971). The increase in the cell volume is not only due to swelling but, according to our unpublished data, the dry mass content also increases owing to intensive RNA and protein syntheses. Agglutination is a decisive factor ensuring intimate contact between cells of opposite mating type. Agglutination, however, is nonspecific, it occurs among cells of different as well as the same mating type and results in the formation of large clumps. In S. cerevisiae agglutination is much weaker than, for instance, in Hansenula wingei (Crandall and Brock, 1968). In the next step cells of opposite mating type form pairs and undergo fusion and plasmogamy. As a result, young zygotes are formed in which nuclear fusion (karyogamy) takes place. Diploid zygotes proliferate mitotically or undergo meiosis and ascospore formation, depending on nutritional conditions. The capacity for sexual reaction between a and a cells is controlled by one allelic pair in the mating-type locus in yeasts. This locus is probably multicistronic and regulates the synthesis of many products involved in mating reactions. It is known that copulation and efficiency of mating reactions are highly variable, depending on the yeast strain used. Most probably the efficiency of the mating reaction depends on the interaction of many different genetic factors present in different strains. For a detailed study of the biochemical processes occurring in consecutive stages of sexual cell fusion in yeasts, the use of genetic mutants with specific blocks for different stages is necessary. To obtain such mutants, however, a general knowledge of culture conditions for each stage and of synchronization of mating processes must first be obtained. Only in this way can the genetic mechanisms governing the sexual cycle in yeasts be determined.
5.
SYNCHRONOUS ZYGOTE FORMATION IN YEASTS
91
11. Basic Principles for Synchronous Mass Production of Zygotes
The classic method of Lindegren gives a yield of zygotes usually of the order of a few percent of the haploid cells in the conjugation mixture. Moreover, the period of zygote formation is extended to several hours. Jakob (1962) greatly increased the yield of zygotes by enforcing cellular contacts between competent haploid cells by centrifugation. This procedure raised the yield of zygotes to about 15%. However, the synchronization in Jakob’s procedure is still rather poor, and the zygotes are formed in the course of more than 2 hours. Since the initial a- and ahcell cultures are unsynchronized and the synchronization through mutual interaction in the conjugation mixture is only partial and transitory, the first and most important step is the preparation of a homogeneous mixture of competent cells of opposite mating type. This can be accomplished either by separation of homogeneous a and (Y cells in suitable stages for sexual fusion, as was done by Sena et al. (1973), or by differentiating the mating procedure in such a way that conditions are optimal for synchronization but unfavorable for cell fusion (Bilidski et al., 1973). Homogeneous populations of competent cells ensure a maximal yield of zygotes for any given pair of haploid strains, whatever the method used for purification of competent cells. Synchronization of zygote formation can be achieved in several ways. We usually obtain the highest efficiency of conjugation in cultures from late logarithmic phase when the initial cell population is highly asynchronous. The time required for cells from different phases of the cell cycle to achieve competence varies greatly, and therefore a basic condition for synchronization of cell fusion is the prevention of premature zygote formation before the whole cell population becomes competent. This can be achieved by creating conditions favorable to quick competence development and by disrupting early contacts between competent cells by sonication. In this way it is possible to obtain a practically pure competent cell suspension (95%) without premature zygote formation. The next condition for synchronous zygote formation is the enforcement of contact between competent cells by centrifugation as applied by Jakob (1962). A contact enforced in a pellet during 3 hour results in restriction of the time of fusion between cells to about I hour, this being a much higher degree of synchronization as compared with that obtained by other methods. At the end of efficient conjugation, sonication should be applied again to disintegrate clumps and to stop formation of new zygotes.
92
T. BILINSKI, J. LITWINSKA, J. ZUK,AND W. GAJEWSKI
111. Method for Poorly Synchronized Zygote Formation for Genetic Analysis The cultures of a and a strains in late log phase in YPG medium (yeast extract, 1%; Bacto peptone, 1%; and glucose, 2%) are centrifuged and resuspensed in conjugation medium containing 1% yeast extract and 3% glucose with pH 4.5 (adjusted with acetic acid). The concentration of both strains should be nearly identical within the range of 107 to lo8cells/ml. The cells of both strains are mixed in a 1: 1 ratio, and the mixture is incubated for 2 hours at 280Cwithout shaking. After incubation the mixture is diluted 10 times with fresh medium, immediately centrifuged for 2 minutes at 3000 g, and left for 30 minutes at 28 oC. Then the pellet is gently agitated by manual shaking, and the preparation left for another 30 minutes at 28 OC without shaking. After this time the pellet is disintegrated by more vigorous manual shaking without formation of a homogeneous suspension. The shaking is applied only to provide better access of nutrients to the pellet; more thorough disintegration decreases markedly the efficiency of conjugation. The pellet is then further incubated at 280C. For most of the strains tested, this procedure gives maximum zygote formation 5 hours after the mixing of a and cx cells.
IV. Method of Synchronous Zygote Mass Production The basic medium for zygote formation is 3% YE consisting of 3% of glucose and 1% of yeast extract. For cultivation of haploid strains, YPG medium consisting of 1% yeast extract, 1% Bacto peptone, and 2% glucose is used. The haploid strains are cultivated in 250-ml Erlenmeyer flasks containing 100 ml of YPG medium for 18 hours at 3OoCon an orbital shaker (120 rpm). Cells are harvested at the end of the log phase by centrifugation for 5 minutes at 3000 g (Sorvall SS1) in 50-ml steel tubes sterilized with 70% ethanol. Suspensions of lo8 cells/ml of each strain in the conjugation medium (3% YE) are prepared. The pH of the medium is adjusted to 4.5 with 1 M acetic acid. The suspensions of cultures of both mating types are mixed together in a 1 :1 ratio. The samples containing 35 ml of mixture are incubated for 1 hour at 3OoC in 50-ml steel Sorvall centrifuge tubes, After incubation the cells are centrifuged under the same conditions for 5 minutes at 3000 g in a Sorvall SSl centrifuge, resuspended in 35 ml of 3% YE, adjusted to pH 8.5 with 1 M Na,HPO,, and exposed for a further incubation of 1 hour. The change in pH of the medium from 4.5 during the first hour to 8.5 during the next hour of the mating reaction is very important. It provides optimal
5.
SYNCHRONOUS ZYGOTE FORMATION IN YEASTS
93
conditions for synchronization of cells, but suboptimal conditions for cell fusion and zygote formation. After 1 hour of incubation at pH 8.5, the cells are centrifuged, resuspended in 35 ml of fresh YE medium (pH 4.9, and sonicated twice for 30 seconds (amplitude from peak to peak 2 pm with an MSE 100-w ultrasonic disintegrator). Sonication ensures maximum efficiency of synchronization without cell fusion. Samples of 20 ml of sonicated suspension are added to 250-ml steel centrifuge tubes containing 180 ml of fresh 3% YE medium (pH 4.5) and centrifuged immediately on a MSE 18 high-speed centrifuge with an angle rotor (6 x 250 ml) at room temperature for 2 minutes at 3000 rpm. The samples in the centrifuge tubes are then incubated at 300 for 30 minutes. Afterward the pellet is fragmented by gentle shaking and incubated without shaking for another 30 minutes. At the end of incubation, the samples are shaken again to disintegrate the pellet, and further incubated without shaking. With this procedure zygote formation starts after 4.5 hours of incubation following the mixing of a and o cells, and their number rises abruptly within 1 hour. During this time no buds are formed by the zygotes, which are all in the same relatively young stage. The yield of zygotes ranges from 30 to 40%. The changes observed in the cell population during the conjugation procedure under the conditions described above are shown in Fig. 1. Samples of initial cultures of a and o cells and the conjugation mixture are analyzed microscopically at 15-minute intervals for estimation of budded cells percentage. Also, the number of conjugated cells (zygotes) can be estimated in the conjugation mixture. The first zygotes appear 4.5 hours after cell mixing, and the curve of zygote formation rises very abruptly during the first hour. A further increase in the frequency of diploid cells is due to the separation of diploid buds from the zygotes. The incubation time for maximal efficiency of zygote production must be determined for each pair of haploid strains used. When the maximum is reached, the conjugation mixture should be sonicated to prevent the formation of new zygotes. If strict synchronization is required and the maximum yield of zygotes is less important, sonication may be applied much earlier.
V.
Limitations of the Method
The described method of synchronous mass production of zygotes is applicable to any pair of haploid strains, provided they are mutually well synchronized in the conjugation mixture so that the majority of haploid cells are competent to fuse and form zygotes. If the a and o cells do not
T. BILINSKI, J. LITWINSKA. J. ZVK, AND W. GAJEWSKl
94 60
50
-
.. E
140
U U W
;30
I20
n
Y)
L
0
$? 2 0
60
% ;
40
2
80
(0
h N
L
5
20 z
0
I
2
3
4
5
6
Incubation time ( h o u r s )
FIG. 1. Numerical relations among different kinds of cells during conjugation. Solid circles, percent of a cells with buds in initial culture; open circles, percent of a cells with buds in initial culture; squares, percent of a and a cells with buds in conjugation mixture; triangles, number of zygotes in 1 ml of conjugation mixture.
synchronize well and the percentage of competent cells in the conjugation mixture is low, the application of the procedure proposed by Sena et al. (1973) is recommended. This method consists of isolation of competent cells from the conjugation mixture by gradient centrifugation. For all technical details of this method, the reader is referred to Sena’s publication (Sena et al., 1973). However, if the ability of the cells to fuse is low, it is impossible to raise the yield of the zygotes. If the zygotes are being produced for biochemical or physiological studies, it is important to start with a and a strains checked beforehand for high capacity for zygote formation (see our simplified method, Section 111). It should be stressed that the proportion of a and acells in the conjugation mixture is a very important factor for high efficiency of zygote formation. As Sena et al. (1973) showed, the optimal proportion is 1: 1. Even small changes in this proportion can result in a drastic decrease in zygote production. We have already described (Bilidski et d., 1973) the inhibitory effect of increasing proportions of @mating-type cells in the conjugation mixture on zygote formation. Figure 2 shows the effect of changes in the
5.
SYNCHRONOUS ZYGOTE FORMATION IN YEASTS
95
50
40
;3 0 t
O m h
N
,\" 20
% a Cells
100
a0
60 40 % a Cells
20
0
FIG. 2. Mating efficiency at various a and a ratios. Broken line, theoretical corrected; solid line, observed.
proportions of a and a cells on the efficiency of conjugation. As seen, the efficiency curve is clearly asymmetric on the side of the a surplus and differs markedly from the symmetric theoretical curve. This phenomenon is observed very often, and the degree of asymmetry differs greatly according to the pair of strains used for conjugation. The absolute concentration of cells in the conjugation mixture is also of paramount importance for cell fusion processes. For mass production of zygotes, concentrations of lo* cells/ml in the first phase and lo7 cells/ml during the conjugation process are recommended (as described in our procedure). High concentrations of cells are very convenient for all manipulations and economize in the use of media. Lower concentrations (107 cells/ml) during the first 2 hours and lo6cells/ml, but not less, duringzygote formation may also be used if a smaller number of zygotes is required. For a high degree of synchronization, higher concentrations are recommended, but for cell fusion itself rather low concentrations are optimal. The composition of the medium also has a very strong influence on the course of conjugation. As a rule, it should be a rich medium, and the most important constituent is glucose. Any other source of carbon or energy that still permits growth practically stops zygote formation completely.
96
T. BILINSKI, J. LITWII%KA, J. ZUK, AND W. GAJEWSKl
REFERENCES Bilifiski, T., Litwidska, J., Zuk, J., and Gajewski, W. (1973). J. Gen. Microbiol. 79, 285. Bilidski, T., Jachymczyk, W., Litwihska, J., and Zuk, J. (1974). J. Gem Microbiol. 82,97. Backing-Throm, E., Duntze, W., Hartwell, H., and Manney, T. R. (1973). Exp. CellRes. 76, 99. Crandall, M. A., and Brock, T. D. (1968). Bacteriol. Rev. 32, 139. Duntze, W., MacKay, V., and Manney, T. R. (1970). Science 168, 1472. Hartwell, L. H. (1973). Exp. Cell Res. 76, 111. Jakob, H. (1%2). C. R. Amd. Sci. 254,3909. Sakai, K., and Yanagishima, N. (1971). Arch. Microbiol. 75,260. Sena, E. P., Radin, N., and Fogel, S. (1973). Proc. Nat. Amd. Sci. US.70, 1373. Throm, E., and Duntze, W. (1970). J. Bacteriol. 104, 1388. Yanagishima, N. (1971). Physiol. Plant. 24, 260.