Selection and characterization of a glucokinase-deficient mutant of Tetrahymena thermophila.

Abstract

We have isolated a mutant of Tetrahymena thermophila that is resistant to inhibition of growth by the glucose analog 2-deoxyglucose. The mutant exhibits a deficiency in a cytoplasmic glucokinase. This enzymatic defect and the attendant inability to convert 2-deoxyglucose to toxic phosphorylated derivatives is apparently the sole basis for the mutant phenotype since transport of glucose and 2-deoxyglucose is unimpaired; there is no elevation of glucose-6-phosphatase activity, which could decrease the level of toxic 2-deoxyglucose metabolites. Genetic analyses have shown that the mutant allele is recessive and inherited as a single Mendelian mutation. The glucokinase-deficient strain described here is useful for the selection of other mutants in this organism and for the investigation of various cellular processes initiated or modulated by glucose and its analogs. We have exploited the molecular defect in this strain to investigate the initial steps in the cyclic AMP-mediated repression of galactokinase gene expression which is caused by glucose.During the course of our initial studies on the regulation of galactose metabolism in Tetrahymena (20), we found that high concentrations of the sugar analogs 2-deoxygalactose and 2-deoxyglucose (dglc) inhibited growth of wild-type cells in galactose-supplemented minimal medium. Since the elucidation of the molecular mechanisms responsible for the expression of the genes controlling galactose metabolism would be aided by the study of appropriate mutants, we isolated a series of strains resistant to the inhibitory effects of these compounds.Several 2-deoxygalactose-resistant mutants were found to be deficient in galactokinase (ATP:D-galactose-1-phosphotransferase, EC 2.7.1.6), presumably due to lesions in the galactokinase structural gene (21). This represented one of the mutant classes to be expected on the basis of the metabolism necessary for the toxicity of this analog (26) and the results of similar selections in bacterial (2) and mammalian cells (27).We then examined dglc-resistant strains in the hope of identifying mutants with specific defects in the metabolism of glucose. In analogy to the selection of the galactokinase-deficient mutants, a major mutant class was expected to be deficient in glucose-phosphorylating ability; such strains would aid in investigating the repression of the galactokinase gene by glucose.In this paper we describe the isolation and the biochemical and genetic characterization of a mutant resistant to dglc by virtue of a deficiency in the soluble glucokinase (ATP:D-glucose-6-phosphotransferase, EC 2.7.1.2) required for conversion of dglc to toxic metabolites. In a forthcoming communication