Summary
The cellular β-galactosidase activities produced by thelac'Z gene ofEscherichia coli, cloned on YEp, YRp, or YCp-type plasmids in host cells ofSaccharomyces cerevisiae with different ploidies, and which was expressed by a modified jeastHIS5 promoter, showed characteristic differences depending on the plasmid. But for any given plasmid, the isogenic diploid and tetraploid transformants showed slightly lower enzyme activities than their respective haploid transformants. This was due to the similar copy numbers of the plasmids in host cells. Since the cell number per unit volume of the culture decreased with increasing cell ploidy, the enzyme activity per unit volume of the culture decreased significantly. The holding stability of plasmids increased with increasing ploidy of the host cell, especially that of the YRp plasmid. On the YRp plasmid, thelac'Z gene showed higher productivity withTRP1 thanLEU2 as the selection marker for the plasmid.
Similar content being viewed by others
References
Andreadis A, Hsu Y-P, Kohlhaw GB, Schimmel P (1982) Nucleotide sequence of yeastLEU2 shows 5′-noncoding region has sequences cognate to leucine. Cell 31:319–325
Araki H, Jearnpipatkul A, Tatsumi H, Sakurai T, Ushio K, Muta T, Oshima Y (1985) Molecular and functional organization of yeast plasmid pSR1. J Mol Biol 182:191–203
Bachmann BJ (1983) Linkage map ofEscherichia coli K-12, edition 7. Microbiol Rev 47:180–230
Broach JR (1981) Genes ofSaccharomyces cerevisiae. In: Strathern JN, Jones EW, Broach JR (eds) The molecular biology of the yeast saccharomyces: life cycle and inheritance, Cold Spring Harbor Laboratory, Cold Spring Harbor, NY, pp 653–727
Broach JR, Strathern JN, Hicks JB (1979) Transformation in yeast: development of a hybrid cloning vector and isolation of theCAN1 gene. Gene 8:121–133
Casadaban MJ, Martinez-Arias A, Shapira SK, Chou J (1983) β-Galactosidase gene fusions for analyzing gene expression inEscherichia coli and yeasts. Methods Enzymol 100:293–308
Clarke L, Carbon J (1978) Functional expression of cloned yeast DNA inEscherichia coli: specific complementation of argininosuccinate lyase (argH) mutations. J Mol Biol 120:517–532
Dobson MJ, Tuite MF, Mellor J, Roberts NA, King RM, Burke DC, Kingsman AJ, Kingsman SM (1983) Expression inSaccharomyces cerevisiae of human interferon-alpha directed by theTRP1 5′ region. Nucleic Acids Res 11:2287–2302
Donahue TF, Daves RS, Lucchini G, Fink GR (1983) A short nucleotide sequence required for regulation ofHIS4 by the general control system of yeast. Cell 32:89–98
Erhart E, Hollenberg CP (1983) The presence of a defectiveLEU2 gene on 2 μ DNA recombinant plasmids ofSaccharomyces cerevisiae is responsible for curing and high copy number. J Bacteriol 156:625–635
Harashima S, Takagi A, Oshima Y (1984) Transformation of protoplasted yeast cells is directly associated with cell fusion. Mol Cell Biol 4:771–778
Hereford L, Fahrner K, Woolford J, Rosbash M, Kaback DB (1979) Isolation of yeast histone genes H2A and H2B. Cell 18:1261–1271
Hinnebusch AG, Fink GR (1983a) Repeated DNA sequences upstream fromHIS1 also occur at several other co-regulated genes inSaccharomyces cerevisiae. J Biol Chem 258:5238–5247
Hinnebusch AG, Fink GR (1983b) Positive regulation in the general amino acid control ofSaccharomyces cerevisiae. Proc Natl Acad Sci USA 80:5374–5378
Hsu Y-P, Kohlhaw G, Niederberger P (1982) Evidence that α-isopropylmalate synthase ofSaccharomyces cerevisiae is under the “general” control of amino acid biosynthesis. J Bacteriol 150:967–972
Jeffreys AJ, Flavell RA (1977) A physical map of the DNA regions flanking the rabbit β-globin gene. Cell 12:429–439
Miller JH (1972) Experiments in molecular genetics. Cold Spring Harbor Laboratory, Cold Spring Harbor, New York, pp 352–355; p 431
Miozzari G, Niederberger P, Hütter R (1978) Tryptophan biosynthesis inSaccharomyces cerevisiae: control of the flux through the pathway. J Bacteriol 134:48–59
Miyajima A, Miyajima I, Arai K, Arai N (1984) Expression of plasmid R388-encoded type II dihydrofolate reductase as a dominant selective marker inSaccharomyces cerevisiae. Mol Cell Biol 4:407–414
Morrison DA (1977) Transformation inEscherichia coli: cryogenic preservation of competent cells. J Bacteriol 132:349–351
Rigby PWJ, Dieckmann M, Rhodes C, Berg P (1977) Labeling deoxyribonucleic acid to high specific activityin vitro by nick translation with DNA polymerase I. J Mol Biol 113:237–251
Rose M, Casadaban MJ, Botstein D (1981) Yeast genes fused to β-galactosidase inEscherichia coli can be expressed normally in yeast. Proc Natl Acad Sci USA 78:2460–2464
Schürch A, Miozzari J, Hütter R (1974) Regulation of tryptophan biosynthesis inSaccharomyces cerevisiae: mode of action of 5-methyl-tryptophan and 5-methyl-tryptophan-sensitive mutants. J Bacteriol 117:1131–1140
Southern EM (1975) Detection of specific sequences among DNA fragments separated by gel electrophoresis. J Mol Biol 98:503–517
Stinchcomb DT, Mann C, Davis RW (1982) Centromeric DNA fromSaccharomyces cerevisiae. J Mol Biol 158:157–179
Struhl K, Stinchcomb DT, Scherer S, Davis RW (1979) High-frequency transformation of yeast: autonomous replication of hybrid DNA molecules. Proc Natl Acad Sci USA 76:1035–1039
Takagi A, Harashima S, Oshima Y (1983) Construction and characterization of isogenic series ofSaccharomyces cerevisiae polyploid strains. Appl Environ Microbiol 45:1034–1040
Takagi A, Harashima S, Oshima Y (1985) Hybridization and polyploidization ofSaccharomyces cerevisiae strains by transformation-associated cell fusion. Appl Environ Microbiol 49:244–246
Toh-e A, Tada S, Oshima Y (1982) 2-μm DNA-like plasmids in the osmophilic haploid yeastSaccharomyces rouxii. J Bacteriol 151:1380–1390
Tschumper G, Carbon J (1980) Sequence of a yeast DNA fragment containing a chromosomal replicator and theTRP1 gene. Gene 10:157–166
Wahl GM, Stern M, Stark GR (1979) Efficient transfer of large DNA fragments from agarose gels to diazobenzyloxymethyl-paper and rapid hybridization by using dextran sulfate. Proc Natl Acad Sci USA 76:3683–3687
Author information
Authors and Affiliations
Rights and permissions
About this article
Cite this article
Takagi, A., Chua, E.N., Boonchird, C. et al. Constant copy numbers of plasmids inSaccharomyces cerevisiae hosts with different ploidies. Appl Microbiol Biotechnol 23, 123–129 (1985). https://doi.org/10.1007/BF01982728
Received:
Revised:
Issue Date:
DOI: https://doi.org/10.1007/BF01982728