Summary
The HEX2 gene which is necessary for glucose repression and is involved in the regulation of hexokinase PII synthesis and maltose uptake, has been cloned by complementation of a hex2 mutant, and selection for restored growth on maltose. Glucose repression in the transformants was like that in the wild type. The HEX2 gene was localized within a 2.15 kb fragment. The restriction map was confirmed by Southern hybridization of genomic DNA. Based on 30 tetrads, the linkage between HEX2 and TRP1 was determined as 10 cM. Plasmid integration directed to the genomic site of the cloned gene also gave a similar linkage distance between the amino acid auxotroph plasmid marker and genomic TRP1. Gene disruption of HEX2 yielded nonrepressible transformants with elevated hexokinase PII activity showing inhibition by maltose; this provides clear evidence that the HEX2 gene has been isolated.
This is a preview of subscription content, log in via an institution to check access.
Similar content being viewed by others
References
Beggs JD (1978) Transformation of yeast by a replicating hybrid plasmid. Nature 275:104–109
Bergmeyer HU (1970) Methoden der enzymatischen Analyse, 2nd edition. Verlag Chemie, Weinheim, FRG, p 432
Carlson M, Osmond BC, Botstein D (1981) Mutants of yeast defective in sucrose utilization. Genetics 98:25–40
Ciriacy M (1975) Genetics of alcohol dehydrogenase in Saccharomyces cerevisiae. Isolation and genetic analysis of mutants. Mutat. Res. 29:315–325
Clewell DB, Helinski DR (1970) Supercoiled cicular DNA-protein complex in Escherichia coli: Purification and induced conversion to an open cicular DNA form. Proc Natl Acad Sci USA 62:1159–1166
Cohen SN, Chang ACY, Hsu L (1972) Nonchromosomal antibiotic resistance in bacteria. Genetic transformation of Escherichia coli by R-factor DNA. Proc Natl Acad Sci USA 69:2110–2114
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 controle of hexokinase PII synthesis. Mol Gen Genet 186:278–282
Entian K-D (1986) Glucose repression: A complex regulation system in yeast. Microbiol Sci 3:366–371
Entian K-D, Mecke D (1982) Genetic evidence for a role of hexokinase isoenzyme 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
Fröhlich K-U, Entian K-D, Mecke D (1984) Cloning and restriction analysis of the hexokinase PII gene of the yeast Saccharomyces cerevisiae. Mol Gen Genet 194:144–148
Gancedo JM, Gancedo C (1986) Catabolite repression mutants of yeast. FEMS Microbiol Lett 32:179–187
Holmes DS, Quigley M (1981) A rapid boiling method for the preparation of bacterial plasmids. Anal Biochem 114:193–197
Ito H, Fukuda Y, Murata K, Kimura A (1983) Transformation of intact yeast cells treated with alkali cations. J Bacteriol 153:163–168
Kelly RB, Cozzarelli NR, Deutscher MP, Lehmann JR, Kornberg A (1970). Enzymatic synthesis of deoxyribonucleic acid. XXXII. Replication of duplex ribonucleic acid by polymerase at a single strand break. J Biol Chem 245:39–45
Maniatis T, Fritsch EF, Sambrook (1982) Molecular cloning. A laboratory manual. Cold Spring Harbor Laboratory, Cold Spring Harbor, New York
Matsumoto K, Tadanori Y, Yasuji O (1983) Recessive mutations conferring resistance to carbon catabolite repression of galactokinase synthesis in Saccharomyces cerevisiae. J Bacteriol 153:1405–1414
Nasmyth KA, Reed SJ (1980) Isolation of genes by complementation in yeast. Molecular cloning of a cell-cycle gene. Proc Natl Acad Sci USA 77:2119–2123
Nasmyth KA, Tatchell K (1980) The structure of transposable yeast mating type loci. Cell 19:753–764
Neigeborn L, Carlson M (1984) Genes affecting the regulation of SUC2 gene expression by glucose repression in Saccharomyces cerevisiae. Genetics 108:845–858
Orr-Weaver TL, Szostak JW, Rothstein RJ (1981) Yeast transformation: A model system for the study of recombination. Proc Natl Sci USA 78:6354–6358
Southern EM (1975) Detection of specific sequences among DNA fragments seperated by gel electrophoresis. J Mol Biol 98:503–517
Tautz D, Renz M (1983) An optimized freeze-squeeze method for recovery of DNA fragments from agarose gels. Anal Biochem 132:114–119
Zamenhoff F (1957) Preparation and assay of deoxyribonucleic acid from animal tissue. 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
Author information
Authors and Affiliations
Additional information
Communicated by C.P. Hollenberg
Rights and permissions
About this article
Cite this article
Niederacher, D., Entian, KD. Isolation and characterization of the regulatory HEX2 gene necessary for glucose repression in yeast. Mol Gen Genet 206, 505–509 (1987). https://doi.org/10.1007/BF00428892
Received:
Issue Date:
DOI: https://doi.org/10.1007/BF00428892