Summary
Carbon catabolite repression in yeast depends on catalytic active hexokinase isoenzyme PII (Entian 1980a). A yeast strain lacking hexokinase isoenzymes PI and PII was transformed, using a recombinant pool with inserts of yeast nuclear DNA up to 10 kbp in length. One hundred transformants for hexokinase were obtained. All selected plasmids coded for hexokinase isoenzyme PII, none for hexokinase isoenzyme PI, and carbon catabolite repression was restored in the transformants. Thirty-five independently isolated stable plasmids were investigated further. Analysis with the restriction enzyme EcoRI showed that these plasmids fell into two classes with different restriction behaviour. One representative of each class was amplified in Escherichia coli and transferred back into the yeast hexokinase-deficient strain with concomitant complementation of the nuclear mutation. The two types of insert were analysed in detail with 16 restriction enzymes, having 0–3 cleavage sites on transformant vector YRp7. The plasmids differed from each other by the orientation of the yeast insert in the vector. After yeast transformation with fragments of one plasmid the hexokinase PII gene was localised within a region of 1.65 kbp.
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References
Anderson CM, Stenkamp RE, Steitz TA (1978) Sequencing a protein by X-ray crystallography. II. Refinement of yeast hexokinase B co-ordinates and sequence at 2.1 Å resolution. J Mol Biol 123:15–33
Barnett JA, Kornberg HL, (1960) The utilization by yeast of acids of the glycarboxylic acid cycle. J Gen Microbiol 23:65–82
Beggs JD (1978) Transformation of yeast by a replicating hybrid plasmid. Nature 275:104–109
Bergmeyer HU (1970) Methoden der enzymatischen Analyse, 2 ed Verlag Chemie, Weinheim/Bergstraße
Ciriacy M (1975) Genetics of alcohol dehydrogenase in Saccharomyces cerevisiae. I. Isolation and genetic analysis of mutants. Mutat Res 29:315–326
Clewell DB, Helinski DR (1970) Properties of a supercoiled deoxyribonucleic acid protein relaxation complex and strand specifity of the relaxation event. Biochemistry 9:4428–4440
Entian K-D (1980a) Genetic and biochemical evidence for hexokinase PII as a key enzyme involved in carbon catabolite repression in yeast. Mol Gen Genet 178:633–637
Entian K-D (1980b) A defect in carbon catabolite repression associated with uncontrollable and excessive maltose uptake. Mol Gen Genet 179:169–175
Entian K-D (1981) A carbon catabolite repression mutant of Saccharomyces cerevisiae with elevated hexokinase activity: Evidence for a regulatory control of hexokinase PII synthesis. Mol Gen Genet 186:278–282
Entian K-D, Mecke D (1982) Genetic evidence for a role of hexokinase isozyme PII in carbon catabolite repression in Saccharomyces cerevisiae. J Biol Chem 257:870–874
Entian K-D, Zimmermann FK (1980) Glycolytic enzymes and intermediates in carbon catabolite repression mutants of Saccharomyces cerevisiae. Mol Gen Genet 177:345–350
Entian K-D, Zimmermann FK (1982) New genes involved in carbon catabolite repression and derepression in the yeast Saccharomyces cerevisiae. J Bacteriol 151:1123–1128
Gancedo C, Salas ML, Giner A, Sols A (1965) Reciprocal effects of carbon sources on the level of an AMP-sensitive fructose-1,6-bisphosphatase and phosphofructokinase in yeast. Biochem Biophys Res Commun 20:15–20
Gancedo C, Schwerzmann N (1976) Inactivation by glucose of phosphoenolpyruvate carboxykinase from Saccharomyces cerevisiae. Arch Microbiol 109:221–225
Gascon C, Neumann NP, Lampen JO (1968) Comparative study of the properties of the purified internal and external invertases from yeast. J Biol Chem 243:1573–1577
Holmes DS, Quigley M (1981) A rapid boiling method for the preparation of bacterial plasmids. Anal Biochem 114:193–197
Kopetzki E, Entian K-D (1982) Purification of yeast hexokinase isoenzymes using affinity chromatography and chromatofocusing. Anal Biochem 121:181–185
Lobo Z, Maitra PK (1977) Resistance to 2-deoxyglucose in yeast: A direct selection of mutants lacking glucose-phosphorylating enzymes. Mol Gen Genet 157:297–300
Magasanik B (1961) Cold Spring Harbor Symp Quant Biol 26:249–256
Maitra PK (1970) A glucokinase from Saccharomyces cerevisiae. J Biol Chem 245:2423–2431
Mandel M, Higa A (1970) Calcium-dependent bacteriophage DNA infection. J Mol Biol 53:159–162
Nasmyth KA, Reed SI (1980) Isolation of genes by complementation in yeast: molecular cloning of a cell-cycle gene. Proc Natl Acad Sci USA 77:2119–2123
Polakis ES, Bartley W (1965) Changes in enzymes activities in Saccharomyces cerevisiae during aerobic growth on different carbon sources. Biochem J 97:284–297
Polakis ES, Bartley W, Meek GA (1965) Changes in the activities of respiratory enzymes during the aerobic growth on different carbon sources. Biochem J 97:298–302
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
Wijk R van, Quwehand J, Bos T, van den Koningsberger VV (1969) Induction and catabolite repression of alpha-glucosidase synthesis in protoplasts of Saccharomyces carlsbergensis. Biochim Biophys Acta 186:178–191
Witt J, Kronau R, Holzer H (1966) Isoenzyme der Malatehydrogenase und ihre Regulation in Saccharomyces cerevisiae. Biochim Biophys Acta 128:63–73
Zamenhoff S (1957) Methods enzymol 3:696–704
Zimmermann FK, Kaufmann I, Rasenberger H, Haußmann P (1977) Genetics of carbon catabolite repression in Saccharomyces cerevisiae: Genes involved in the derepression process. Mol Gen Genet 151:95–103
Zimmermann FK, Scheel I (1977) Mutants of Saccharomyces cerevisiae resistant to carbon catabolite repression. Mol Gen Genet 154:75–82
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Communicated by H. Böhme
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Fröhlich, KU., Entian, KD. & Mecke, D. Cloning and restriction analysis of the hexokinase PII gene of the yeast Saccharomyces cerevisiae . Molec. Gen. Genet. 194, 144–148 (1984). https://doi.org/10.1007/BF00383509
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DOI: https://doi.org/10.1007/BF00383509