[56]
M O L E C U L A R B I O L O G Y O F T H E FISSION
YEAST S.
pombe
795
[56] Molecular Genetic Analysis of Fission Yeast
Schizo~accharomycespombe By SERGIOMOR~NO,AMARKLAR,and PAULNURSE The fission yeast Schizosaccharomyces pombe is proving increasingly attractive as an experimental system for investigating problems of eukaryotic cell and molecular biology. Many of the powerful molecular genetic procedures developed for Saccharomyces cerevisiae can now be applied to S. pombe. In this chapter we describe a range of techniques concerned with classical and molecular genetics, cell biology, and biochemistry which can be used with S. pombe and are in routine operation in o u r laboratories. Schizosaccharomyces pombe is a simple unicellular eukaryote with a genome size of 14 megabases (Mb), about 4 times that of the Escherichia coll. Despite being an Ascomycete fungus ~ke S. cerevisiae, fission yeast is not closely related to budding yeast. Protein comparisons between homologous genes in the two yeasts have revealed identities of between 60 and 90o/0 in amino acid type and position, values close to those found in similar comparisons between yeast and mammalian genes. Such divergence means that if a function is conserved between the two yeasts it is likely that an equivalent function will also be found in other eukaryotes. Also, sequence comparisons of homologous genes in the two yeasts are useful for identifying those regions of proteins which are conserved and probably essential for function. Evolution has provided us with an extensive mutagenesis experiment to examine the effects of specific amino acid changes on protein functions. Comparison of gene sequences has suggested that S. pombe may be slightly more similar to mammalian cells than is S. cerevisiae. Certain features such as cell cycle, chromosome structure, and RNA splicing are likely to be more similar between mammalian cells and S. pombe than mammalian cells and S. cerevisiae, but this need not be the case for all problems of cell and molecular biology. The fission yeast has been used successfully to study mating type, recombination, translation, RNA splicing, chromosome structure, meiosis, mitosis, and cell cycles. Most of these topics are covered in The Molecular Biology of the Fission Yeast (A. Nasim, P. Young, and B. F. Johnson, eds.), publish~ by Academic Press in 1989. In general there are good genetic, cytological, biochemical, and molecular genetic techniques available for S. pombe, although molecular genetics can be more di~cult than with S. cerevisiae. We hope that this review of techniques will attract more groups to work on this very attractive and amenable organism. METHODS IN ENZYMOLOGY, VOL. 194
Co~t O 1991 by Academic Pre~ Inc. All rights ofrepmduetion in any form re~erved.
796
FISSION YEAST
[56]
Introduction to Biology of Fission Yeast
Schizosaccharomyces pombe grows as a cylinder around 3 - 4 / t m in diameter and 7 - 1 5 / t i n in length. It has a typical enkaryotic cell cycle with discrete Gl, S, (32, and M Iihases) In normal minimal or complex media the generation time is between 2 and 4 hr, (32 is about 0.7 of a cell cycle, and the remaining phases are each of about 0.1 of a cell cycle length. Schizosaccharomyces pombe is not very versatile in using different carbon sources, and it is difficult to vary the generation time substantially in batch culture. However, in chemostat cultures generation times can be increased to over 10 hr, and in these circumstances most ofthe expansion in the cell cycle occurs in GI phase. Normally S. pombe cells are haploid and may be of two mating types known as h+ and h- (Fig. 1). Starvation induces the haploid cells of opposite mating types to mate in pairs, forming diploid zygotes which are heterozygous at the mating type locus (h+/h-). The zygotes then undergo meiosis to form four haploid spores, which germinate when nutrient conditions are improved to produce haploid clones.2 Thus, normally the haploid phase predominates. However, h+/h- diploid strains can also be maintained, using procedures we describe later, thus, mutations can easily be isolated in a haploid strain and then tested for complementation and dominance in a diploid strain. Isolates of S. pombe from the wild are homothallic (h9°) strains; that is, they can switch their mating type between h+ and h- every other generation. This means that a single cell gives rise to a colony containing h+ and h- cells which then can mate with one another when nutritional conditions become limiting. Mutations and rearrangements at the mating-type locus give rise to h + and h- strains which either cannot switch or switch rarely. These are called heterothallic strains. Hcterothallic h +~ and h-t~ strains do revert at low frequency (10-4) to hg°; h-s strains, however, are the result of a deletion and are therefore stable, and so it is convenient to use h-s strains for experimental analysis.3 More than 270 S. pombe genes have been mapped genetically, and they define three linkage groups with a meiotic map length of 1200 centimorgans (cM). 4,5 Three chromosomes can be visualized microscopically,e Ret M. Mitchison, in "Methods in Cell Physiology" (D. M. Prescott,cd.), Vol. 4, p. 131. Academic Press,N e w York, 1970. 2 U. Leupold, in "Methods in Cell Physiology" (D. M. Prescott, od.), Vol. 4, p. 169. Academic Press,N e w York, 1970. 3 H. Gutz, H. Hcslot, U. Lcupold, and N. Lopricno, in "Handbook of Genetics" (R. C. King, cd.),Vol. I, p. 395, Plenum, N e w York, 1974. 4 A. Gygax and P. Thuriuax, Curt. Genet, 8, 85 (1984).
[56]
MOLECULAR BIOLOGY OF THE FISSION YEAST
S. pombe
797
mating
I
h2ap:oi:2~h r ~
diyp~;~
~
~11
porulation
aSCUS
FIG. 1. Schizosaccharomyces pombe mitoticand meioticcellcycles.
cently S. pombe chromosomes have been separated using pulsed-field gel electrophoresis (PFGE);7 they have a size of 5.7, 4.6, and 3.5 Mb. There is also available a NotI macrorestriction map of the S. pombe genome with 14 detectable NotI sites, s Therefore, it is now possible to map a cloned gene within a particular region of any of the three chromosomes by using PFGE and Southern blotting. s j. Kohli, Curt. Genet. II, 575 (1987). e C. F. Robinow, Genetics 87, 491 (1977). 7 C. L. Smith, T. Matsumoto, O. Niwa, S. Klco, L-B. Fan, M. Yanagida, and C. R. Cantor,
Nucleic Acids Res. 15,4481(1988). 8J.-B.Fan,Y. Chikashi~,C. L. Smith,O. Niwa,M.Yanagida,and C. R. Cantor,Nucleic Acids Res. 17,2801(1989).
798
FISSIONrE^ST
[56]
Scl~zosaccharomycespombe S t r a i n s A standard collection of S. pombe mutant strains has been established by Alan Coddington in Norwich, England. A catalog is available and strains can be obtained, for a small fee, from Peter J. H. Jackman, National Collection of Yeast Cultures, AFRC Institute of Food Research, Colney Lane, Norwich NR4 7UA, UIC The nomenclature for S. pombe strains and genes differs from that of S. cerevisiae and follows lowercase italic symbols.5 All S. pombe strains have been derived from the haploid wild-type strains 972h-, 975h+ and 968h 9° and are generally isogenic. They grow on media as described in Table I. ~,3,9Long-term storage of S. pombe strains is on glycerol stocks at - 7 0 * . These are prepared as follows: 1. Grow up cells in 1 ml YES medium at 25" -32" for 2 days. 2. Mix with 1 ml of YES containing 30% glycerol in a cryotube. Snapfreeze on liquid nitrogen or dry ice/ethanol. The cells can then be stored a t - 70*. Strains kept in this way remain viable for several years at least. It is wise to make a duplicate each time and store it in a different freezer. For short-term storage cells can be kept as patches on YES slants or agar plates at 4" for up to 2 months. Strains do not store well on minimal medium or phloxin B-supplemented medium.
Reisolation of Fission Yeast Strains For strains stored on glycerol at - 7 0 " , the following procedure is recommended: 1. With a sterile spatula serape off a small amount of frozen glycerol stock and then transfer to a YES plate. 2. Incubate at 25* -32* for 1-4 days, depending on the strain. 3. When colonies are visible streak out to single colonies on a YEP (YES + phloxin B) plate and incubate at 25" -32" for 2 - 3 days. Strains stored on slants or patches are streaked out onto YEP plates directly, and incubated at 25"-32* as appropriate. Before any genetic or molecular procedure is carded out the phenotype of the strain should be checked.
Testing the Phenotype of a Strain Haploid~Diploid. It is important to check the ploidy because certain strains of S. pombe diploidize at a high frequency. Haploid cells divide at 9 p. Nurse, Nature (London) 256, 547 (1975).
[56]
MOLECULAR BIOLOGYOF THE FISSIONYEAST S.
pombe
799
approximately 12-15/zm in length and are 3-4/~m in width. Diploid cells are both longer (20- 25/~m at division)and wider (4- 5/~m); they are also lessviablethan haploid cells,and a diploidcolony contains more dead cells (I - 5%). Phloxin B is a stainthat accumulates in dead cells,which become dark red. By growing a strain in Y E P plates it is possible to screen for haploid colonies,which willbc stained lightpink whereas diploid colonies will be darker pink? ° This can be confirmed by microscopic examination of the cells. Mating Type. To test for the presence of homothallic h ~ the strain is streaked out to single colonies on YE and then replica plated to malt extract, incubated below 30 ° for 3 days to allow conjugation and sporulation to occur, and then held over a petri dish containing iodine crystals for about 1- 5 min. h 9° colonies will be stained black owing to the presence of starch in the spores. Often sectored colonies arc seen. To check mating type, the strain should be crossed to h+ and h- tester grains (see later) and tested as above. Temperature Sensitivity. Many temperature-sensitive mutants (ts) have been isolated in S. pombe. They can Ix checked by replica plating onto YEP (YES + phloxin B) and incubating at the restrictive temperature. Phloxin B will stain the dead cells and these can be examined under the light microscope for checking the ts phenotypc. Auxotrophy. The auxotrophic markers most commonly used in S. pombe are adenine, glutamic acid, histidine, leucine, lysine, and uracil, although others arc available. To test for auxotrophy the strain is grown up to single colonies on YES and then replica plated to minimal medium with and without the appropriate supplement. The plates are incubated for 1 - 2 days and then examined for growth under these conditions. After testing a strain in these various ways, it can be stored as a patch at 4 ° and generally used for 2 - 4 weeks without further testing. Growing Schizosaccharomyces p o m b e Cells Haploid strains of S. pombe grow with the generation times shown in Table II. As S. pombe cells enter stationary phase the cells generally accumulate in G~ or (}2, depending on whether they are deprived of nitrogen or glucose, respectively,l~ and the cells become rounder and more refractile under phase microscopy. In supplemented yeast extract medium and minimal medium, glucose is usually limiting and cells accumulate inG2 .11 to j. Kohli, H. Hottinger, P. Munz, A. Strauss, and P. Thuriaux, Genetics 87, 471 (1977). '~ P. Nurse, P. Thuriaux, and IC Nasmyth, Moi. Gen. Genet. 146, 167 (1976).
800
[56]
FISSION YE^Sr
8
"~.m
~
~o:~.~'~o ! ~.,,.'s~ ~ z x~u o...
z
8 0
.~
0
o; °°
~.~
[56]
MOLECU~R BXOLO~Y OF ThE ~SSION YEAST S. porabe
801
z Z
o
~q Z O
~o 6,
v
802
[56]
FISSION YEAST
TABLE II GENERATIONTIMESFORFISSIONYEAST
Schizosaccharomycespombe Medium YE
EMM
Temperature(*C)
Generationtime
25 29 32 35.5 25 29 32 35.5
3 hr 2 hr ,30rain 2 hr I0 m/n 2 hr 4 hr 3 hr 2 hr 30 rain 2 hr 20 rain
For physiological experiments it is important that cultures be mainrained in mid-exponential growth between 2 X 106 and 1 X 10T cells/ml. The optical density (OD) of a culture can be used to measure the concentration of cells. It is necessary to establish a ratio between OD59s and cell concentration. In our hands for wild-type strains, an OD59s of 0.1 is equivalent to 2 × 106 cells/ml, but this will vary between strains, from one spectrophotometer to another, and among different growth conditions. To generate cultures and mid-exponential growth, use a fresh patch of a strain of checked phenotype to inoculate 10 ml YES (or minimal medium) and incubate for 1 - 2 days at the appropriate temperature until cells are in early stationary phase. At this point no shaking is required as S. pombe can grow in partially anaerobic conditions. This preculture can then be used to inoculate a larger culture, taking into consideration the generation times shown in Table II. A preculture normally contains 2 - 5 X 107 cells/ml, and one generation is necessary for cells to recover from stationary phase and reenter exponential growth. Typically a culture in 100 ml minimal medium grown at 25 ° overnight for 16 hr (i.e., 4 generations) to an OD59s of 0.25 (5 × 10~ cells/ml) will require an inoculum of 1 ml from the preculture.
When growing liquid cultures for physiological experiments gentle shaking is advisable to maintain uniform growth conditions. Generally minimal medium is used, as the growth conditions can be more accurately defined. Phthalate is used as a buffer as it reduces clumping, which can be a problem with some strains. The media for growing S. pombe strains are given in Table I. YES liquid or agar-containing solid medium is used for vegetative growth. Yeast extract from most sources when used in YES inhibits conjugation and
[56]
MOLECULAR BIOLOGY OF THE FISSION YEAST S.
pombe
803
sporulation. However S. pombe cells can grow, mate, and sporulate in the YEPD rich medium commonly used for growth of S. cerevisiae. For physiological experiments cells are generally cultured in EMM with the required supplements added. Crosses are carried out on ME, with each supplement added, or in YEPD.
Sehizo~accharomyces p o m b e Classical Genetic Techniques Genetic Crosses Conjugation and sporulation cannot take place in S. pombe except under conditions of nutrient starvation. ME medium is generally used for genetic crosses. This medium also supports some vegetative growth; mixed strains will undergo several rounds of cell division before running out of nutrients. The cells will then be able to conjugate and sporulate. Alternatively, cells can be grown and sporulated in YEPD [1% yeast extract, 2% peptone, 2% glucose (all w/v)]. Several crosses can be carried out on a 9-cm ME plate. To cross two strains, a loopful of h- and a loopful of h + are mixed together on a ME plate. A loopful of sterile distilled water is then used to thoroughly mix the cells on the afar plate to an area of about 1 em 2. The cross is left to dry and is then incubated below 30", as conjugation is severely reduced above this temperature. Fully formed four-spore asci can be seen after 2 - 3 days of incubation (see morphology of the vegetative cells, ascus, and spores in Fig. 1). When carrying out genetic crosses for recombination mapping, it is useful to have unlinked markers to ensure that recombination has occurred° A cross between a homothailic and a heterothallic strain mostly generates asci of the liomothallie parent. This is because yeast cells prefer to mate with sister cells which are of different mating type owing to homothallic switching. It is preferable to first select a diploid hybrid between these strains (see below) and then subject this to tetrad or random spore analysis.
Tetrad Analysis A 2
M O L E C U L A R B I O L O G Y O F T H E FISSION
YEAST S.
pombe
795
[56] Molecular Genetic Analysis of Fission Yeast
Schizo~accharomycespombe By SERGIOMOR~NO,AMARKLAR,and PAULNURSE The fission yeast Schizosaccharomyces pombe is proving increasingly attractive as an experimental system for investigating problems of eukaryotic cell and molecular biology. Many of the powerful molecular genetic procedures developed for Saccharomyces cerevisiae can now be applied to S. pombe. In this chapter we describe a range of techniques concerned with classical and molecular genetics, cell biology, and biochemistry which can be used with S. pombe and are in routine operation in o u r laboratories. Schizosaccharomyces pombe is a simple unicellular eukaryote with a genome size of 14 megabases (Mb), about 4 times that of the Escherichia coll. Despite being an Ascomycete fungus ~ke S. cerevisiae, fission yeast is not closely related to budding yeast. Protein comparisons between homologous genes in the two yeasts have revealed identities of between 60 and 90o/0 in amino acid type and position, values close to those found in similar comparisons between yeast and mammalian genes. Such divergence means that if a function is conserved between the two yeasts it is likely that an equivalent function will also be found in other eukaryotes. Also, sequence comparisons of homologous genes in the two yeasts are useful for identifying those regions of proteins which are conserved and probably essential for function. Evolution has provided us with an extensive mutagenesis experiment to examine the effects of specific amino acid changes on protein functions. Comparison of gene sequences has suggested that S. pombe may be slightly more similar to mammalian cells than is S. cerevisiae. Certain features such as cell cycle, chromosome structure, and RNA splicing are likely to be more similar between mammalian cells and S. pombe than mammalian cells and S. cerevisiae, but this need not be the case for all problems of cell and molecular biology. The fission yeast has been used successfully to study mating type, recombination, translation, RNA splicing, chromosome structure, meiosis, mitosis, and cell cycles. Most of these topics are covered in The Molecular Biology of the Fission Yeast (A. Nasim, P. Young, and B. F. Johnson, eds.), publish~ by Academic Press in 1989. In general there are good genetic, cytological, biochemical, and molecular genetic techniques available for S. pombe, although molecular genetics can be more di~cult than with S. cerevisiae. We hope that this review of techniques will attract more groups to work on this very attractive and amenable organism. METHODS IN ENZYMOLOGY, VOL. 194
Co~t O 1991 by Academic Pre~ Inc. All rights ofrepmduetion in any form re~erved.
796
FISSION YEAST
[56]
Introduction to Biology of Fission Yeast
Schizosaccharomyces pombe grows as a cylinder around 3 - 4 / t m in diameter and 7 - 1 5 / t i n in length. It has a typical enkaryotic cell cycle with discrete Gl, S, (32, and M Iihases) In normal minimal or complex media the generation time is between 2 and 4 hr, (32 is about 0.7 of a cell cycle, and the remaining phases are each of about 0.1 of a cell cycle length. Schizosaccharomyces pombe is not very versatile in using different carbon sources, and it is difficult to vary the generation time substantially in batch culture. However, in chemostat cultures generation times can be increased to over 10 hr, and in these circumstances most ofthe expansion in the cell cycle occurs in GI phase. Normally S. pombe cells are haploid and may be of two mating types known as h+ and h- (Fig. 1). Starvation induces the haploid cells of opposite mating types to mate in pairs, forming diploid zygotes which are heterozygous at the mating type locus (h+/h-). The zygotes then undergo meiosis to form four haploid spores, which germinate when nutrient conditions are improved to produce haploid clones.2 Thus, normally the haploid phase predominates. However, h+/h- diploid strains can also be maintained, using procedures we describe later, thus, mutations can easily be isolated in a haploid strain and then tested for complementation and dominance in a diploid strain. Isolates of S. pombe from the wild are homothallic (h9°) strains; that is, they can switch their mating type between h+ and h- every other generation. This means that a single cell gives rise to a colony containing h+ and h- cells which then can mate with one another when nutritional conditions become limiting. Mutations and rearrangements at the mating-type locus give rise to h + and h- strains which either cannot switch or switch rarely. These are called heterothallic strains. Hcterothallic h +~ and h-t~ strains do revert at low frequency (10-4) to hg°; h-s strains, however, are the result of a deletion and are therefore stable, and so it is convenient to use h-s strains for experimental analysis.3 More than 270 S. pombe genes have been mapped genetically, and they define three linkage groups with a meiotic map length of 1200 centimorgans (cM). 4,5 Three chromosomes can be visualized microscopically,e Ret M. Mitchison, in "Methods in Cell Physiology" (D. M. Prescott,cd.), Vol. 4, p. 131. Academic Press,N e w York, 1970. 2 U. Leupold, in "Methods in Cell Physiology" (D. M. Prescott, od.), Vol. 4, p. 169. Academic Press,N e w York, 1970. 3 H. Gutz, H. Hcslot, U. Lcupold, and N. Lopricno, in "Handbook of Genetics" (R. C. King, cd.),Vol. I, p. 395, Plenum, N e w York, 1974. 4 A. Gygax and P. Thuriuax, Curt. Genet, 8, 85 (1984).
[56]
MOLECULAR BIOLOGY OF THE FISSION YEAST
S. pombe
797
mating
I
h2ap:oi:2~h r ~
diyp~;~
~
~11
porulation
aSCUS
FIG. 1. Schizosaccharomyces pombe mitoticand meioticcellcycles.
cently S. pombe chromosomes have been separated using pulsed-field gel electrophoresis (PFGE);7 they have a size of 5.7, 4.6, and 3.5 Mb. There is also available a NotI macrorestriction map of the S. pombe genome with 14 detectable NotI sites, s Therefore, it is now possible to map a cloned gene within a particular region of any of the three chromosomes by using PFGE and Southern blotting. s j. Kohli, Curt. Genet. II, 575 (1987). e C. F. Robinow, Genetics 87, 491 (1977). 7 C. L. Smith, T. Matsumoto, O. Niwa, S. Klco, L-B. Fan, M. Yanagida, and C. R. Cantor,
Nucleic Acids Res. 15,4481(1988). 8J.-B.Fan,Y. Chikashi~,C. L. Smith,O. Niwa,M.Yanagida,and C. R. Cantor,Nucleic Acids Res. 17,2801(1989).
798
FISSIONrE^ST
[56]
Scl~zosaccharomycespombe S t r a i n s A standard collection of S. pombe mutant strains has been established by Alan Coddington in Norwich, England. A catalog is available and strains can be obtained, for a small fee, from Peter J. H. Jackman, National Collection of Yeast Cultures, AFRC Institute of Food Research, Colney Lane, Norwich NR4 7UA, UIC The nomenclature for S. pombe strains and genes differs from that of S. cerevisiae and follows lowercase italic symbols.5 All S. pombe strains have been derived from the haploid wild-type strains 972h-, 975h+ and 968h 9° and are generally isogenic. They grow on media as described in Table I. ~,3,9Long-term storage of S. pombe strains is on glycerol stocks at - 7 0 * . These are prepared as follows: 1. Grow up cells in 1 ml YES medium at 25" -32" for 2 days. 2. Mix with 1 ml of YES containing 30% glycerol in a cryotube. Snapfreeze on liquid nitrogen or dry ice/ethanol. The cells can then be stored a t - 70*. Strains kept in this way remain viable for several years at least. It is wise to make a duplicate each time and store it in a different freezer. For short-term storage cells can be kept as patches on YES slants or agar plates at 4" for up to 2 months. Strains do not store well on minimal medium or phloxin B-supplemented medium.
Reisolation of Fission Yeast Strains For strains stored on glycerol at - 7 0 " , the following procedure is recommended: 1. With a sterile spatula serape off a small amount of frozen glycerol stock and then transfer to a YES plate. 2. Incubate at 25* -32* for 1-4 days, depending on the strain. 3. When colonies are visible streak out to single colonies on a YEP (YES + phloxin B) plate and incubate at 25" -32" for 2 - 3 days. Strains stored on slants or patches are streaked out onto YEP plates directly, and incubated at 25"-32* as appropriate. Before any genetic or molecular procedure is carded out the phenotype of the strain should be checked.
Testing the Phenotype of a Strain Haploid~Diploid. It is important to check the ploidy because certain strains of S. pombe diploidize at a high frequency. Haploid cells divide at 9 p. Nurse, Nature (London) 256, 547 (1975).
[56]
MOLECULAR BIOLOGYOF THE FISSIONYEAST S.
pombe
799
approximately 12-15/zm in length and are 3-4/~m in width. Diploid cells are both longer (20- 25/~m at division)and wider (4- 5/~m); they are also lessviablethan haploid cells,and a diploidcolony contains more dead cells (I - 5%). Phloxin B is a stainthat accumulates in dead cells,which become dark red. By growing a strain in Y E P plates it is possible to screen for haploid colonies,which willbc stained lightpink whereas diploid colonies will be darker pink? ° This can be confirmed by microscopic examination of the cells. Mating Type. To test for the presence of homothallic h ~ the strain is streaked out to single colonies on YE and then replica plated to malt extract, incubated below 30 ° for 3 days to allow conjugation and sporulation to occur, and then held over a petri dish containing iodine crystals for about 1- 5 min. h 9° colonies will be stained black owing to the presence of starch in the spores. Often sectored colonies arc seen. To check mating type, the strain should be crossed to h+ and h- tester grains (see later) and tested as above. Temperature Sensitivity. Many temperature-sensitive mutants (ts) have been isolated in S. pombe. They can Ix checked by replica plating onto YEP (YES + phloxin B) and incubating at the restrictive temperature. Phloxin B will stain the dead cells and these can be examined under the light microscope for checking the ts phenotypc. Auxotrophy. The auxotrophic markers most commonly used in S. pombe are adenine, glutamic acid, histidine, leucine, lysine, and uracil, although others arc available. To test for auxotrophy the strain is grown up to single colonies on YES and then replica plated to minimal medium with and without the appropriate supplement. The plates are incubated for 1 - 2 days and then examined for growth under these conditions. After testing a strain in these various ways, it can be stored as a patch at 4 ° and generally used for 2 - 4 weeks without further testing. Growing Schizosaccharomyces p o m b e Cells Haploid strains of S. pombe grow with the generation times shown in Table II. As S. pombe cells enter stationary phase the cells generally accumulate in G~ or (}2, depending on whether they are deprived of nitrogen or glucose, respectively,l~ and the cells become rounder and more refractile under phase microscopy. In supplemented yeast extract medium and minimal medium, glucose is usually limiting and cells accumulate inG2 .11 to j. Kohli, H. Hottinger, P. Munz, A. Strauss, and P. Thuriaux, Genetics 87, 471 (1977). '~ P. Nurse, P. Thuriaux, and IC Nasmyth, Moi. Gen. Genet. 146, 167 (1976).
800
[56]
FISSION YE^Sr
8
"~.m
~
~o:~.~'~o ! ~.,,.'s~ ~ z x~u o...
z
8 0
.~
0
o; °°
~.~
[56]
MOLECU~R BXOLO~Y OF ThE ~SSION YEAST S. porabe
801
z Z
o
~q Z O
~o 6,
v
802
[56]
FISSION YEAST
TABLE II GENERATIONTIMESFORFISSIONYEAST
Schizosaccharomycespombe Medium YE
EMM
Temperature(*C)
Generationtime
25 29 32 35.5 25 29 32 35.5
3 hr 2 hr ,30rain 2 hr I0 m/n 2 hr 4 hr 3 hr 2 hr 30 rain 2 hr 20 rain
For physiological experiments it is important that cultures be mainrained in mid-exponential growth between 2 X 106 and 1 X 10T cells/ml. The optical density (OD) of a culture can be used to measure the concentration of cells. It is necessary to establish a ratio between OD59s and cell concentration. In our hands for wild-type strains, an OD59s of 0.1 is equivalent to 2 × 106 cells/ml, but this will vary between strains, from one spectrophotometer to another, and among different growth conditions. To generate cultures and mid-exponential growth, use a fresh patch of a strain of checked phenotype to inoculate 10 ml YES (or minimal medium) and incubate for 1 - 2 days at the appropriate temperature until cells are in early stationary phase. At this point no shaking is required as S. pombe can grow in partially anaerobic conditions. This preculture can then be used to inoculate a larger culture, taking into consideration the generation times shown in Table II. A preculture normally contains 2 - 5 X 107 cells/ml, and one generation is necessary for cells to recover from stationary phase and reenter exponential growth. Typically a culture in 100 ml minimal medium grown at 25 ° overnight for 16 hr (i.e., 4 generations) to an OD59s of 0.25 (5 × 10~ cells/ml) will require an inoculum of 1 ml from the preculture.
When growing liquid cultures for physiological experiments gentle shaking is advisable to maintain uniform growth conditions. Generally minimal medium is used, as the growth conditions can be more accurately defined. Phthalate is used as a buffer as it reduces clumping, which can be a problem with some strains. The media for growing S. pombe strains are given in Table I. YES liquid or agar-containing solid medium is used for vegetative growth. Yeast extract from most sources when used in YES inhibits conjugation and
[56]
MOLECULAR BIOLOGY OF THE FISSION YEAST S.
pombe
803
sporulation. However S. pombe cells can grow, mate, and sporulate in the YEPD rich medium commonly used for growth of S. cerevisiae. For physiological experiments cells are generally cultured in EMM with the required supplements added. Crosses are carried out on ME, with each supplement added, or in YEPD.
Sehizo~accharomyces p o m b e Classical Genetic Techniques Genetic Crosses Conjugation and sporulation cannot take place in S. pombe except under conditions of nutrient starvation. ME medium is generally used for genetic crosses. This medium also supports some vegetative growth; mixed strains will undergo several rounds of cell division before running out of nutrients. The cells will then be able to conjugate and sporulate. Alternatively, cells can be grown and sporulated in YEPD [1% yeast extract, 2% peptone, 2% glucose (all w/v)]. Several crosses can be carried out on a 9-cm ME plate. To cross two strains, a loopful of h- and a loopful of h + are mixed together on a ME plate. A loopful of sterile distilled water is then used to thoroughly mix the cells on the afar plate to an area of about 1 em 2. The cross is left to dry and is then incubated below 30", as conjugation is severely reduced above this temperature. Fully formed four-spore asci can be seen after 2 - 3 days of incubation (see morphology of the vegetative cells, ascus, and spores in Fig. 1). When carrying out genetic crosses for recombination mapping, it is useful to have unlinked markers to ensure that recombination has occurred° A cross between a homothailic and a heterothallic strain mostly generates asci of the liomothallie parent. This is because yeast cells prefer to mate with sister cells which are of different mating type owing to homothallic switching. It is preferable to first select a diploid hybrid between these strains (see below) and then subject this to tetrad or random spore analysis.
Tetrad Analysis A 2
