Molecular and General Genetics MGG

, Volume 203, Issue 1, pp 29–35

Molecular cloning and characterization of the STA2 glucoamylase gene of Saccharomyces diastaticus

  • Isak S. Pretorius
  • Thomas Chow
  • Daniela Modena
  • Julius Marmur
Article

Summary

The Saccharomyces diastaticus structural gene STA2, encoding an extracellular glucoamylase (1,4-α-d-glucan glycohydrolase, EC 3.2.1.3.), has been cloned by complementation of a stao strain. A genomic library was initially constructed from a STA2 yeast strain in the yeast Escherichia coli shuttle cosmid vector pYCl. The Sta+ complementing function was further delimited to an 8.3 kb BglII fragment whose restriction map was found to be similar to related genomic regions of STA1 and STA3. Fusions of several DNA fragments derived from the 8.3 kb BglII fragment with a truncated E. coli β-galactosidase gene resulted in two overlapping fragments that could direct the production of large fusion proteins in E. coli. These fusion proteins were immunoprecipitable by anti-glucoamylase II antibodies, confirming that the Sta+ complementing fusion was due to the expression of a gene that coded for a yeast glucoamylase. Measurements of the STA1, STA2 and STA3 RNA transcripts by RNA-DNA hybridization using an internal fragment of the cloned STA2 gene as the probe indicated that a common transcript of 2.5 kb is produced by each of the STA genes. Integrative disruption of the STA2 gene through homologous recombination was achieved by transforming a STA2 yeast strain to Sta using an in vitro constructed donor DNA fragment that has the URA3 gene inserted within the coding region of the cloned glucoamylase gene. This was confirmed by tetrad analysis of crosses between strains carrying a disrupted STA2 and a functional STA2. Southern blot analysis using BamHI digested genomic DNA from 15 tetrads demonstrated consistent co-segregation and Mendelian inheritance of the Sta phenotype with STA2::URA3. These data further confirm that the cloned DNA that showed Sta+ complementing activity carries a functional STA2 gene that encodes the yeast extracellular glucoamylase II.

Key words

Glucoamylase S. diastaticus STA Cloning Gene fusion 

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References

  1. Clancy MJ, Smith LM, Magee PT (1982) Developmental regulation of a sporulation-specific enzyme activity in Saccharomyces cerevisiae. Mol Cell Biol 2:171–178Google Scholar
  2. Cryer DR, Eccleshall R, Marmur J (1975) Isolation of yeast DNA. Methods Cell Biol 12:39–44Google Scholar
  3. Fogarty WM, Kelly CT (1979) Starch degrading enzymes of microbial origin. In: Bull MJ (ed) Progress in industrial microbiology, vol 15. Elsevier Scientific Publishing Company, New York, pp 87–150Google Scholar
  4. Guerry P, LeBlanc DJ, Falkow S (1973) General method for the isolation of plasmid deoxyribonucleic acid. J Bacteriol 116:1064–1066Google Scholar
  5. Guo LH, Stepien PP, Tso JY, Brousseau R, Narang S, Thomas DY, Wu R (1984) Synthesis of human insulin gene, VIII. Construction of expression vectors for fused proinsulin production in Escherichia coli. Gene 29:251–254Google Scholar
  6. Hinnen A, Hicks JB, Fink GR (1978) Transformation in yeast. Proc Natl Acad Sci USA 75:1929–1933Google Scholar
  7. Hohn B, Hinnen A (1980) Cloning with cosmids in E. coli and yeast. In: Setlow IK, Hollaender A (ed) Genetic engineering — Principles and methods, vol 2. Plenum Press, New York, pp 169–183Google Scholar
  8. Ingle MB, Erickson RJ (1978) Bacterial α-amylases. Adv Appl Microbiol 24:257–278Google Scholar
  9. Innis MA, Holland MJ, McCabe PC, Cole GE, Wittman VP, Tal R, Watt KWK, Gelfand DH, Holland JP, Meade JH (1985) Expression, glycosylation, and secretion of Aspergillus glucoamylase by Saccharomyces cerevisiae. Science 228:21–26Google Scholar
  10. Ish-Horowicz D, Burke JF (1981) Rapid and efficient cosmid vector cloning. Nucl Acids Res 9:2989–2998Google Scholar
  11. Ito H, Fukuda Y, Murata K, Kimura A (1983) Transformation of intact yeast cells treated with alkali cations. J Bacteriol 153:163–168Google Scholar
  12. Laemmli UK (1970) Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature (London) 27:680–685Google Scholar
  13. Lemmel SA, Heimsch RC, Korus RA (1980) Kinetics of growth and amylase production of Saccharomycopsis fibuligera on potato processing wastewater. Appl Environ Microbiol 39:387–393Google Scholar
  14. Maniatis T, Fritsch EF, Sambrook J (1982) Molecular cloning: a laboratory manual. Cold Spring Harbor Laboratory, Cold Spring Harbor, New YorkGoogle Scholar
  15. Meaden P, Ogden K, Bussey H, Tubb RS (1985) A DEX gene conferring production of extracellular amyloglucosidase on yeast. Gene 34:325–334Google Scholar
  16. Meister M, Strahler J, Wiebauer K, Thomsen KK (1983) Multiple genes encode mouse pancreatic amylases. In: Scandlios J, Siciliano M (eds) Isozymes: Current topics in biological and medical research. Arthur R. Liss, New YorkGoogle Scholar
  17. Murray NE, Bruce SA, Murray K (1979) Molecular cloning of the DNA ligase gene from the bacteriophage T4. II. Amplification and preparation of the gene product. J Mol Biol 132:493–505Google Scholar
  18. Nunberg JH, Meade JH, Cole G, Lawyer FC, McCabe P, Schweickart V, Tal R, Wittman VP, Flatgaard JE, Innis MA (1984) Molecular cloning and characterization of the glucoamylase gene of Aspergillus awamori. Mol Cell Biol 4:2306–2315Google Scholar
  19. Orr-Weaver TL, Szostak JW, Rothstein RJ (1981) Yeast transformation: a model system for the study of recombination. Proc Natl Acad Sci USA 78:6354–6358Google Scholar
  20. Polaina J, Wiggs MY (1983) STA10: a gene involved in the control of starch utilization by Saccharomyces. Curr Genet 7:109–112Google Scholar
  21. Pretorius IS, Chow T, Marmur J (1986) Identification and physical characterization of yeast glucoamylase genes. Mol Gen Genet 203:36–41Google Scholar
  22. Rothstein SJ, Lazarus CM, Smith WE, Baulcombe DC, Gatenby AA (1984) Secretion of a wheat α-amylase expressed in yeast. Science 308:662–665Google Scholar
  23. Scherer G, Telford J, Baldari C, Pirrotta V (1981) Isolation of cloned genes differentially expressed at early and late stages of Drosophila embryonic development. Dev Biol 86:438–447Google Scholar
  24. Sherman F, Fink GR, Lawrence CW (1972) Laboratory manual for methods in yeast genetics. Cold Spring Harbor Laboratory, Cold Spring Harbor, New YorkGoogle Scholar
  25. Tamaki H (1978) Genetic studies of ability to ferment starch in Saccharomyces: gene polymorphism. Mol Gen Genet 164:205–209Google Scholar
  26. Ueda S (1981) Fungal glucoamylase and raw starch digestion. Trends Biochem Sci 89-90Google Scholar
  27. Veda (1981)Google Scholar
  28. Webb E, Spencer-Martins I (1983) Extracellular endodextranase from the yeast Lipomyces starkeyi. Can J Microbiol 29:1092–1095Google Scholar
  29. Wilson JJ, Khachatourians GG, Ingledew WM (1982) Schwanniomyces: SCP and ethanol from starch. Biotechnol Lett 4:333–338Google Scholar
  30. Yamashita I, Fukui S (1983) Molecular cloning of a glucoamylase-producing gene in the yeast Saccharomyces. Agric Biol Chem 47:2689–2692Google Scholar
  31. Yamashita I, Maemura T, Hatano T, Fukui S (1985a) Polymorphic extracellular glucoamylase genes and their evolutionary origin in yeast Saccharomyces diastaticus. J Bacteriol 161:574–582Google Scholar
  32. Yamashita I, Suzuki K, Fukui S (1985b) Nucleotide sequence of the extracellular glucoamylase gene STA1 in the yeast Saccharomyces diastaticus J Bacteriol 161:567–573Google Scholar

Copyright information

© Springer-Verlag 1986

Authors and Affiliations

  • Isak S. Pretorius
    • 1
  • Thomas Chow
    • 1
  • Daniela Modena
    • 1
  • Julius Marmur
    • 1
  1. 1.Department of BiochemistryAlbert Einstein College of MedicineBronxUSA
  2. 2.Department of MicrobiologyUniversity of the Orange Free StateBloemfonteinSouth Africa

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