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Preparation and regeneration of protoplasts and spheroplasts for fusion and transformation ofSchizosaccharomyces pombe

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Abstract

Preparation and regeneration of protoplasts is essential for somatic hybridization and transformation of yeasts. We present conditions that were found to be optimal for preparing and regeneratingSchizosaccharomyces pombe protoplasts for cell fusion. In contrast to these conditions, genetic transformation ofS. pombe requires spheroplasts that are osmotically sensitive, but still have some wall material attached to the cell. The main finding were as follows: (a) For protoplast formation with Novozym SP234, 0.9M sorbitol was found to be the optimal osmotic milieu and β-mercaptoethanol is not necessary. (b) Embedding in soft agar yields considerably better regeneration frequencies than direct plating. (c) Cell fusion is optimal when both fusion partners are fully protoplasted, although considerable fusion occurs between spheroplasted cells as well. (d)Schizosaccharomyces pombe transformation frequencies are much higher with spheroplasts than with protoplasts. Inclusion of β-mercaptoethanol did not enhance transformation frequency.

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Literature Cited

  1. Beach D, Nurse P (1981) High-frequency transformation of the fission yeastSchizosaccharomyces pombe. Nature 290:140–142

    Google Scholar 

  2. Beach D, Piper M, Nurse P (1982) Construction of aSchizosaccharomyces pombe gene bank in a yeast bacterial shuttle vector and its use to isolate genes by complementation. Mol Gen Genet 187:326–329

    PubMed  Google Scholar 

  3. Broach JR, Strathern JN, Hicks JB (1979) Transformation in yeast: development of a hybrid cloning vector and isolation of the CAN1 gene. Gene 8:121–133

    PubMed  Google Scholar 

  4. Clarke L, Carbon J (1978) Functional expression of cloned yeast DNA inEscherichia coli: specific complementation of argino succinate lyase (argH) mutations. J Mol Biol 120:517–532

    PubMed  Google Scholar 

  5. Dickinson DP, Isenberg I (1982) Preparation of spheroplasts ofSchizosaccharomyces pombe. J Gen Microbiol 128:651–654

    Google Scholar 

  6. Duffus JH, Mitchell CJ (1969) The growth ofSchizosaccharomyces pombe in media of high osmotic pressure. Antonie van Leeuwenhoek [Suppl] 35:H25

    PubMed  Google Scholar 

  7. Foury F, Goffeau A (1973) Combination of 2-deoxyglucose and snail-gut enzyme treatments for preparing spheroplasts ofSchizosaccharomyces pombe. J Gen Microbiol 75:227–229

    Google Scholar 

  8. Harashima S, Takagi A, Oshima Y (1984) Transformation of protoplasted yeast cells is directly associated with cell fusion. Mol Cell Biol 4:771–778

    PubMed  Google Scholar 

  9. Havelková M, Křipalová J (1980) Karyokinesis in growing and reverting protoplasts ofSchizosaccharomyces pombe. Folia Microbiol (Praha) 25:219–227

    Google Scholar 

  10. Humphreys GO, Williams GA, Anderson ES (1975) A simple method for the preparation of large quantities of pure plasmid DNA. Biochim Biophys Acta 383: 457–463

    PubMed  Google Scholar 

  11. Kohli J, Hottinger H, Munz P, Strauss A, Thuriaux P (1977) Genetic mapping inSchizosaccharomyces pombe by mitotic and meiotic analysis and induced haploidization. Genetics 87:471–489

    Google Scholar 

  12. Kopecká M (1975) The isolation of protoplasts of the fission yeastSchizosaccharomyces byTrichoderma viride and snail enzymes. Folia Microbiol (Praha) 20:273–276

    Google Scholar 

  13. Losson R, Lacroute F (1983) Plasmids carrying the yeast OMP decarboxylase structural and regulatory genes: transcription regulation in a foreign environment. Cell 32:371–377

    PubMed  Google Scholar 

  14. Manners DJ, Meyer MT (1977) The molecular structures of some glucans from the cell walls ofSchizosaccharomyces pombe. Carbohydr Res 57:189–203

    Google Scholar 

  15. Nečas O (1971) Cell wall synthesis in yeast protoplasts. Bacteriol Rev 35:149–170

    PubMed  Google Scholar 

  16. Nečas O, Svoboda A (1981) Morphogenesis in protoplasts. In: Arnold WN (ed) Yeast cell envelops: biochemistry, biophysics, and ultrastructure, vol 2. Boca Raton FL: CRC Press, pp 105–127

    Google Scholar 

  17. Nečas O, Svoboda A, Havelková M (1968) Mechanism of regeneration of yeast protoplasts. V. Formation of the cell wall inSchizosaccharomyces pombe. Folia Biol (Praha) 14:80–85

    Google Scholar 

  18. Schwencke J, Nagy M (1978) Preparation of protoplasts ofSchizosaccharomyces pombe. In: Prescott DM (ed) Methods in cell biology, vol 20. New York: Academic Press, pp 101–104

    Google Scholar 

  19. Sipiczki M (1983) Diploid protoplasts inSchizosaccharomyces pombe: formation, growth and regeneration. Can J Microbiol 29:593–595

    Google Scholar 

  20. Sipiczki M, Ferenczy L (1977) Protoplast fusion ofSchizosaccharomyces pombe auxotrophic mutants of identical mating type. Mol Gen Genet 151:77–88

    PubMed  Google Scholar 

  21. Sipiczki M, Ferenczy L (1978) Enzymatic methods for enrichment of fungal mutants. I. Enrichment ofSchizosaccharomyces pombe mutants. Mutat Res 50:163–173

    Google Scholar 

  22. Sipiczki M, Creanor J, Fantes P (1983) Fusion of cdc mutants: possible effect of cell cycle on the fusion of yeast protoplasts. In: Potrykus I, Harms CT, Hinnen AH, Hütter R, Kink PJ, Shillito RD (eds) Protoplasts. Basel: Birkhäuser, pp 338–339

    Google Scholar 

  23. Sipiczki M, Kucsera J, Dobo E (1984) Homo- and heterothallic sexual types inSchizosaccharomyces pombe var.malidevorans. Curr Genet 9:263–272

    Google Scholar 

  24. Stephen ER, Nasim A (1981) Production of protoplasts in different yeasts by mutanase. Can J Microbiol 27:550–553

    PubMed  Google Scholar 

  25. Svoboda A (1978) Fusion of yeast protoplasts induced by polyethylene glycol. J Gen Microbiol 109:169–175

    Google Scholar 

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Authors and Affiliations

  1. Department of Genetics, University of Debrecen, H-4010, Debrecen, Hungary

    Mátyás Sipiczki

  2. Institute of General Microbiology, University of Bern, Bern, Switzerland

    Wolf-Dietrich Heyer & Jürg Kohli

Authors
  1. Mátyás Sipiczki
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  2. Wolf-Dietrich Heyer
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  3. Jürg Kohli
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Sipiczki, M., Heyer, WD. & Kohli, J. Preparation and regeneration of protoplasts and spheroplasts for fusion and transformation ofSchizosaccharomyces pombe . Current Microbiology 12, 169–173 (1985). https://doi.org/10.1007/BF01567671

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