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Synchronization of Yeast

  • Jessica Smith
  • Arkadi Manukyan
  • Hui Hua
  • Huzefa Dungrawala
  • Brandt L. Schneider
Protocol
Part of the Methods in Molecular Biology book series (MIMB, volume 1524)

Abstract

The budding yeast Saccharomyces cerevisiae and fission yeast Schizosaccharomyces pombe are amongst the simplest and most powerful model systems for studying the genetics of cell cycle control. Because yeast grows very rapidly in a simple and economical media, large numbers of cells can easily be obtained for genetic, molecular, and biochemical studies of the cell cycle. The use of synchronized cultures greatly aids in the ease and interpretation of cell cycle studies. In principle, there are two general methods for obtaining synchronized yeast populations. Block-and-release methods can be used to induce cell cycle synchrony. Alternatively, centrifugal elutriation can be used to select synchronous populations. Because each method has innate advantages and disadvantages, the use of multiple approaches helps in generalizing results. An overview of the most commonly used methods to generate synchronized yeast cultures is presented along with working Notes: a section that includes practical comments, experimental considerations and observations, and hints regarding the pros and cons innate to each approach.

Key words

Yeast Cell cycle Synchronization Block-and-release Centrifugal elutriation 

References

  1. 1.
    Forsburg SL (2003) Overview of Schizosaccharomyces pombe. Curr Protoc Mol Biol Chapter 13:Unit 13.14PubMedGoogle Scholar
  2. 2.
    Forsburg SL (2005) The yeasts Saccharomyces cerevisiae and Schizosaccharomyces pombe: models for cell biology research. Gravit Space Biol Bull 18(2):3–9PubMedGoogle Scholar
  3. 3.
    Sherman F (2002) Getting started with yeast. Methods Enzymol 350:3–41CrossRefPubMedGoogle Scholar
  4. 4.
    Forsburg SL, Rhind N (2006) Basic methods for fission yeast. Yeast 23(3):173–183CrossRefPubMedPubMedCentralGoogle Scholar
  5. 5.
    Broach JR, Pringle JR, Jones EW (1991) The Molecular and cellular biology of the yeast Saccharomyces. Cold Spring Harbor monograph series, vol 21. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NYGoogle Scholar
  6. 6.
    Guthrie C, Fink GR (1991) Guide to yeast genetics and molecular biology, vol 194. Methods Enzymol Academic Press, Inc, Pasadena, CACrossRefGoogle Scholar
  7. 7.
    Murray AW, Hunt T (1993) The cell cycle: an introduction. W.H. Freeman, New YorkGoogle Scholar
  8. 8.
    Jorgensen P, Tyers M (2004) How cells coordinate growth and division. Curr Biol 14:R1014–R1027CrossRefPubMedGoogle Scholar
  9. 9.
    Schneider BL, Zhang J, Markwardt J, Tokiwa G, Volpe T, Honey S, Futcher B (2004) Growth rate and cell size modulate the synthesis of, and requirement for, G1-phase cyclins at start. Mol Cell Biol 24:10802–10813CrossRefPubMedPubMedCentralGoogle Scholar
  10. 10.
    Fantes P, Brooks R (1993) The cell cycle: a practical approach. The practical approach series. IRL Press at Oxford University Press, Oxford, New YorkGoogle Scholar
  11. 11.
    Johnston JR (1994) Molecular genetics of yeast: a practical approach. The practical approach series, vol 141. IRL Press at Oxford University Press, Oxford, New YorkGoogle Scholar
  12. 12.
    Kume K (2016) Elutriation for cell cycle synchronization in fission yeast. Methods Mol Biol 1342:149–155CrossRefPubMedGoogle Scholar
  13. 13.
    Tormos-Perez M, Perez-Hidalgo L, Moreno S (2016) Fission yeast cell cycle synchronization methods. Methods Mol Biol 1369:293–308CrossRefPubMedGoogle Scholar
  14. 14.
    Gomez EB, Forsburg SL (2004) Analysis of the fission yeast Schizosaccharomyces pombe cell cycle. Methods Mol Biol 241:93–111PubMedGoogle Scholar
  15. 15.
    Green MD, Sabatinos SA, Forsburg SL (2009) Microscopy techniques to examine DNA replication in fission yeast. Methods Mol Biol 521:463–482CrossRefPubMedGoogle Scholar
  16. 16.
    Humphrey T, Brooks G (2005) Cell cycle control: mechanisms and protocols. Methods in molecular biology, vol 296. Humana Press, Totowa, NJ, p 402Google Scholar
  17. 17.
    Day A, Schneider C, Schneider BL (2004) Yeast Cell Synchronization. Cell Cycle Checkpoint Control Protocols 241:55–76Google Scholar
  18. 18.
    Luche DD, Forsburg SL (2009) Cell-cycle synchrony for analysis of S. pombe DNA replication. Methods Mol Biol 521:437–448CrossRefPubMedGoogle Scholar
  19. 19.
    Spellman PT, Sherlock G, Zhang MQ, Iyer VR, Anders K, Eisen MB, Brown PO, Botstein D, Futcher B (1998) Comprehensive identification of cell cycle-regulated genes of the yeast Saccharomyces cerevisiae by microarray hybridization. Mol Biol Cell 9:3273–3297CrossRefPubMedPubMedCentralGoogle Scholar
  20. 20.
    Futcher B (1999) Cell cycle synchronization. Methods Cell Sci 21:79–86CrossRefPubMedGoogle Scholar
  21. 21.
    Walker GM (1999) Synchronization of yeast cell populations. Methods Cell Sci 21:87–93CrossRefPubMedGoogle Scholar
  22. 22.
    Johnston LH, Johnson AL (1997) Elutriation of budding yeast. Methods Enzymol 283:342–350CrossRefPubMedGoogle Scholar
  23. 23.
    Day A, Schneider C, Schneider BL (2004) Yeast cell synchronization. Methods Mol Biol 241:55–76PubMedGoogle Scholar
  24. 24.
    Sonoda E (2006) Synchronization of cells. Subcel Biochem 40:415–418Google Scholar
  25. 25.
    Amon A (2002) Synchronization procedures. Methods Enzymol 351:457–467CrossRefPubMedGoogle Scholar
  26. 26.
    Marbouty M, Ermont C, Dujon B, Richard GF, Koszul R (2014) Purification of G1 daughter cells from different Saccharomycetes species through an optimized centrifugal elutriation procedure. Yeast 31:159–166CrossRefPubMedGoogle Scholar
  27. 27.
    Wang S, Luo C (2016) Cell cycle synchronization using a microfluidic synchronizer for fission yeast cells. Methods Mol Biol 1342:259–268CrossRefPubMedGoogle Scholar
  28. 28.
    Tian Y, Luo C, Lu Y, Tang C, Ouyang Q (2012) Cell cycle synchronization by nutrient modulation. Integr Biol (Camb) 4:328–334CrossRefGoogle Scholar
  29. 29.
    Hur JY, Park MC, Suh KY, Park SH (2011) Synchronization of cell cycle of Saccharomyces cerevisiae by using a cell chip platform. Mol Cells 32:483–488CrossRefPubMedPubMedCentralGoogle Scholar
  30. 30.
    Yang J, Dungrawala H, Hua H, Manukyan A, Abraham L, Lane W, Mead H, Wright J, Schneider BL (2011) Cell size and growth rate are major determinants of replicative lifespan. Cell Cycle 10:144–155CrossRefPubMedPubMedCentralGoogle Scholar
  31. 31.
    Bean JM, Siggia ED, Cross FR (2006) Coherence and timing of cell cycle start examined at single-cell resolution. Mol Cell 21:3–14CrossRefPubMedGoogle Scholar
  32. 32.
    Di Talia S, Skotheim JM, Bean JM, Siggia ED, Cross FR (2007) The effects of molecular noise and size control on variability in the budding yeast cell cycle. Nature 448:947–951CrossRefPubMedGoogle Scholar
  33. 33.
    Schmoller KM, Turner JJ, Koivomagi M, Skotheim JM (2015) Dilution of the cell cycle inhibitor Whi5 controls budding-yeast cell size. Nature 526:268–272CrossRefPubMedPubMedCentralGoogle Scholar
  34. 34.
    Amberg DC, Burke D, Strathern JN (2005) Methods in yeast genetics : a Cold Spring Harbor Laboratory course manual, 2005th edn. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NYGoogle Scholar
  35. 35.
    Hasek J (2006) Yeast fluorescence microscopy. Methods Mol Biol 313:85–96PubMedGoogle Scholar
  36. 36.
    Banfalvi G (2008) Cell cycle synchronization of animal cells and nuclei by centrifugal elutriation. Nat Protoc 3:663–673CrossRefPubMedGoogle Scholar
  37. 37.
    Banfalvi G (2011) Synchronization of mammalian cells and nuclei by centrifugal elutriation. Methods Mol Biol 761:25–45CrossRefPubMedGoogle Scholar
  38. 38.
    Manukyan A, Abraham L, Dungrawala H, Schneider BL (2011) Methods Mol Biol 761:173–199CrossRefPubMedGoogle Scholar
  39. 39.
    Ausubel FM (1987) Current protocols in molecular biology. Greene Publishing Associates, Brooklyn, N. Y & Media, PaGoogle Scholar
  40. 40.
    Futcher B (2002) Transcriptional regulatory networks and the yeast cell cycle. Curr Opin Cell Biol 14:676–683CrossRefPubMedGoogle Scholar
  41. 41.
    Chu S, DeRisi J, Eisen M, Mulholland J, Botstein D, Brown PO, Herskowitz I (1998) The transcriptional program of sporulation in budding yeast. Science 282:699–705CrossRefPubMedGoogle Scholar
  42. 42.
    Primig M, Williams RM, Winzeler EA, Tevzadze GG, Conway AR, Hwang SY, Davis RW, Esposito RE (2000) The core meiotic transcriptome in budding yeasts. Nat Genet 26:415–423CrossRefPubMedGoogle Scholar
  43. 43.
    Haase SB, Reed SI (2002) Improved flow cytometric analysis of the budding yeast cell cycle. Cell Cycle 1:132–136CrossRefPubMedGoogle Scholar
  44. 44.
    Sabatinos SA, Forsburg SL (2009) Measuring DNA content by flow cytometry in fission yeast. Methods Mol Biol 521:449–461CrossRefPubMedGoogle Scholar
  45. 45.
    Zhang H, Siede W (2004) Analysis of the budding yeast Saccharomyces cerevisiae cell cycle by morphological criteria and flow cytometry. Methods Mol Biol 241:77–91PubMedGoogle Scholar
  46. 46.
    Breeden LL (1997) Alpha-factor synchronization of budding yeast. Methods Enzymol 283:332–341CrossRefPubMedGoogle Scholar
  47. 47.
    Richardson HE, Wittenberg C, Cross F, Reed SI (1989) An essential G1 function for cyclin-like proteins in yeast. Cell 59:1127–1133CrossRefPubMedGoogle Scholar
  48. 48.
    Schneider BL, Patton EE, Lanker S, Mendenhall MD, Wittenberg C, Futcher B, Tyers M (1998) Yeast G1 cyclins are unstable in G1 phase. Nature 395:86–89CrossRefPubMedGoogle Scholar
  49. 49.
    Woldringh CL, Fluiter K, Huls PG (1995) Production of senescent cells of Saccharomyces cerevisiae by centrifugal elutriation. Yeast 11:361–369CrossRefPubMedGoogle Scholar
  50. 50.
    Egilmez NK, Chen JB, Jazwinski SM (1990) Preparation and partial characterization of old yeast cells. J Gerontol 45:B9–B17CrossRefPubMedGoogle Scholar
  51. 51.
    Day A, Markwardt J, Delaguila R, Zhang J, Purnapatre K, Honigberg SM, Schneider BL (2004) Cell size and Cln-Cdc28 complexes mediate entry into meiosis by modulating cell growth. Cell Cycle 3:1433–1439CrossRefPubMedGoogle Scholar
  52. 52.
    Nachman I, Regev A, Ramanathan S (2007) Dissecting timing variability in yeast meiosis. Cell 131:544–556CrossRefPubMedGoogle Scholar

Copyright information

© Springer Science+Business Media New York 2017

Authors and Affiliations

  • Jessica Smith
    • 1
  • Arkadi Manukyan
    • 2
  • Hui Hua
    • 3
  • Huzefa Dungrawala
    • 3
  • Brandt L. Schneider
    • 4
  1. 1.Department of Cell Biology and BiochemistryTexas Tech University Health Sciences CenterLubbockUSA
  2. 2.Department of Biochemistry and Molecular GeneticsUniversity of Virginia, School of MedicineCharlottesvilleUSA
  3. 3.Department of BiochemistryVanderbilt University School of MedicineNashvilleUSA
  4. 4.Department of Medical EducationTexas Tech University Health Sciences CenterLubbockUSA

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