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Growth and Manipulation of S. pombe

Susan L. Forsburg1

1The Salk Institute for Biological Studies, La Jolla, California

Publication Name: 
Current Protocols in Molecular Biology
Unit Number: 
Unit 13.16
DOI: 
10.1002/0471142727.mb1316s64
Online Posting Date: 
November, 2003
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Abstract

This unit presents aspects specific to genetic and cytological manipulation of fission yeast, including mating type testing, crosses, preparingmaking diploids, and analysis of meiotic products, and basic methods of cell cycle synchronization and analysis. These methods are different from those used in budding yeast because they depend upon the distinct biology of S. pombe, particularly its unwillingness to be a diploid in normal growth conditions. Similarly, the different cell shape and growth behavior of S. pombe require different approaches to basic cell cycle analysis.

     
 
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Table of Contents

  • Unit Introduction
  • Crosses, Tetrads, and Diploids
  • Tetrad Dissection and Random Spore Analysis
  • Basic Protocol 1: Mating Type Testing
  • Basic Protocol 2: Crossing Strains for Tetrad or Random Spore Analysis
  • Basic Protocol 3: Working with Diploids
  • Basic Protocol 4: Tetrad Analysis
  • Alternate Protocol 1: Random Spore Analysis
  • Alternate Protocol 2: Nonsporulating Diploids
  • Alternate Protocol 3: Endoreduplication
  • Basic Protocol 5: Cell-Cycle Synchronization by Nitrogen Starvation
  • Support Protocol 1: Ethanol Fixation
  • Alternate Protocol 4: Cell Cycle Block and Release
  • Basic Protocol 6: Lactose Gradient Centrifugation
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
     
 
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Materials

Basic Protocol 1: Mating Type Testing

 Materials
  • Known h+ and h– tester strains
  • Rich medium plates (e.g., YES plates; UNIT 13.15)
  • Characterized (positive control) and uncharacterized strains
  • Mating plates (ME, SPAS, or EMM –N; UNIT 13.15)
  • Iodine crystals (Sigma)
  • 25°C incubator
  • Empty petri dish
  • Additional equipment and reagents for replica plating (UNITS 1.3 & 13.2)

CAUTION: Iodine is toxic and the vapors from the crystals or the exposed tester plate should not be inhaled.

Basic Protocol 2: Crossing Strains for Tetrad or Random Spore Analysis

 Materials
  • Strains to be crossed (i.e., strains 1 and 2) growing robustly on YES plates (EMM if selection is required; UNIT 13.15)
  • Mating plates (ME or SPAS plates; UNIT 13.15)
  • H2O, sterile
  • Sterile toothpicks
  • 25° to 29°C incubator

Basic Protocol 3: Working with Diploids

 Materials
  • Strains to be crossed (complementing mating types and ade6 markers) growing robustly on YES plates (EMM if required for selection; UNIT 13.15)
  • EMM plates lacking adenine (UNIT 13.15)
  • YES plates containing phloxin B (UNIT 13.15)
  • YES plates (UNIT 13.15)
  • Mating plates (ME or SPAS; UNIT 13.15)
  • Sterile toothpicks
  • 25° or 32°C incubator
  • Additional reagents and equipment for mating and testing the mating type of S. pombe (see Basic Protocol 1)

Basic Protocol 4: Tetrad Analysis

 Materials
  • Mating/sporulation plate grown for 2 to 3 (haploid) or 1 to 2 (diploid) days
  • YES plates (UNIT 13.15)
  • Sterile toothpicks
  • Tetrad dissecting microscope
  • 36°C incubator (optional)
  • 17°C incubator (optional)
  • 25° or 32°C incubator
  • Additional reagents and equipment for preparation and dissection of tetrads (UNIT 13.2)

NOTE: Azygotic asci from previously isolated diploids form more quickly than zygotic asci from haploids, which must mate first.

Alternate Protocol 1: Random Spore Analysis

 Materials
  • Mating/sporulation plate
  • H2O, sterile
  • 5% glusulase (DuPont NEN) in sterile water
  • PBS (APPENDIX 2; optional), or EMM –N –G (UNIT 13.15; optional)
  • YES plates (UNIT 13.15)
  • Sterile toothpick
  • Additional reagents and equipment for counting cells using a hemacytometer (APPENDIX 3F)

Alternate Protocol 3: Endoreduplication

 Materials
  • YES plates containing phloxin B (UNIT 13.15)
  • Liquid culture of strain of interest

Basic Protocol 5: Cell-Cycle Synchronization by Nitrogen Starvation

 Materials
  • Cells from the strain of interest
  • EMM and EMM –N (UNIT 13.15)
  • Complete medium (e.g., EMM with supplements, YES; UNIT 13.15)
  • 25°C incubator
  • Incubator set at growth temperature (i.e., 32° to 36°C)

Support Protocol 1: Ethanol Fixation

 Materials
  • Exponentially growing culture
  • 70% ethanol, 4°C
  • H2O, sterile
  • Mounting medium with stain (see recipe; optional)
  • Positively charged microscope slides

Alternate Protocol 4: Cell Cycle Block and Release

 Materials
  • Temperature-sensitive cell cycle mutants (e.g., cdc25-22; ATCC# 90337)
  • 25° and 36°C water baths
  • Thermometer cleaned with ethanol

Basic Protocol 6: Lactose Gradient Centrifugation

 Materials
  • Cells
  • 70% ethanol
  • YES (UNIT 13.15) with 0%, 10%, and 40% lactose, 32°C
  • Gradient maker
  • 20- and 50-ml conical tubes
Looking for materials?  Suppliers Guide
     
 
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Figures

  • Figure 13.16.1
    Fission yeast mating. (A) Zygotes and asci as seen under the microscope. (B) Mating type testing on plates.

    View Image
  • Figure 13.16.2
    Determining septation index for synchronous cultures.

    View Image
  • Figure 13.16.3
    Assembling a lactose gradient.

    View Image

Literature Cited

Literature Cited
    Alfa, C., Fantes, P., Hyams, J., McLeod, M., and Warbrick, E. 1993. Experiments With Fission Yeast. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.
    Bodi, Z., Gysler-Junker, A., and Kohli, J. 1991. A quantitative assay to measure chromosome stability in Schizosaccharomyces pombe. Mol. Gen. Genet. 229:77-80.
    Costello, G., Rodgers, L., and Beach, D. 1986. Fission yeast enters the stationary phase G0 state from either mitotic G1 or G2. Curr. Genet. 11:119-125.
    Edwards, R.J. and Carr, A.M. 1997. Analysis of radiation-sensitive mutants of fission yeast. Methods Enzymol. 283:471-493.
    Egel, R. 2000. Fission yeast on the brink of meiosis. BioEssays 22.9:854-860.
    Egel, R. and Egel-Mitani, M. 1974. Premeiotic DNA synthesis in fission yeast. Exp. Cell. Res. 88:127-134.
    Fernandez Sarabia, M.-J., McInerny, C., Harris, P., Gordon, C., and Fantes, P. 1993. The cell cycle genes cdc22+ and suc22+ of the fission yeast Schizosaccharomyces pombe encode the large and small subunits of ribonucleotide reductase. Mol. Gen. Genet. 238:241-251.
    Gould, K.L. (ed.) 2003. Methods for Schizosaccharomyces pombe. Methods Vol In Press.
    Hirano, T., Hiraoka, Y., and Yanagida, M. 1988. A temperature-sensitive mutation of the Schizosaccharomyces pombe gene nuc2+ that encodes a nuclear scaffold-like protein blocks spindle elongation in mitotic anaphase. J. Cell Biol. 106:1171-1183.
    Klar, A.J.S. 1992. Molecular genetics of fission yeast cell type: Mating type and mating type interconversion. In The Molecular and Cellular Biology of the Yeast Saccharomyces: Gene Expression (E. Jones, J. Pringle, and J. Broach, eds.) pp. 745-777. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.
    MacNeill, S.A. and Nurse, P. 1997. Cell cycle control in fission yeast. In The Molecular and Cellular Biology of the Yeast Saccharomyces: Cell Cycle and Cell Biology (J. Pringle, J. Broach, and E. W. Jones, eds.) pp. 697-763. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.
    Moreno, S., Hayles, J., and Nurse, P. 1989. Regulation of p34cdc2 protein kinase during mitosis. Cell 58:361-372.
    Moreno, S., Klar, A., and Nurse, P. 1991. Molecular genetic analysis of the fission yeast Schizosaccharomyces pombe. Meth. Enzymol. 194:795-823.
    Moser, B.A. and Russell, P. 2000. Cell cycle regulation in Schizosaccharomyces pombe. Curr. Opin. in Microbiol. 3:631-636.
    Nasmyth, K. 1977. Temperature-sensitive lethal mutants in the structural gene for DNA ligase in the yeast Schizosaccharomyces pombe. Cell 12:1109-1120.
    Russell, P. and Nurse, P. 1986. cdc25+ functions as an inducer in the mitotic control of fission yeast. Cell 45:145-153.
    Umesono, K., Toda, T., Hayashi, S., and Yanagida, M. 1983. Two cell division cycle genes NDA2 and NDA3 of the fission yeast Schizosaccharomyces pombe control microtubular organisation and sensitivity to anti-mitotic benzimidazole compounds. J. Molec. Biol. 168:271-284.
    Verkade, H.M. and O'Connell, M.J. 1998. Cut5 is a component of the UV-responsive DNA damage checkpoint in fission yeast. Mol. Gen. Genet. 260:426-433.
    Willer, M., Hoffmann, L., Styrkarsdottir, U., Egel, R., Davey, J., and Nielsen, O. 1995. Two-step activation of meiosis by the mat1 locus in Schizosaccharomyces pombe. Mol Cell Biol 15:4964-4670.
    Yamamoto, M. 1996. The molecular control mechanisms of meiosis in fission yeast. Trends Biochem. Sci. 21:18-22.
     
 
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