Abstract
From a mutagenized population of mouse T-lymphoma cells (S49) in continuous culture a cell line has been isolated (Ullman, B., Gudas, L. J., Clift, S. M., Martin, D. W., Jr. (1979) Proc. Natl. Acad. Sci. U. S. A. 76, 1074-1978) with ribonucleotide reductase activity that is inhibited only 50% by concentrations of dGTP which abolish wild type enzyme activity. Ribonucleotide reductase activity from this dGuo-L cell line retains its normal sensitivity to dATP. The partial sensitivity/partial resistance of the ribonucleotide reductase suggests that the dGuo-L cell line is heterozygous for ribonucleotide reductase, possessing one normal allele and one allele which codes for a dGTP-resistant enzyme. Both homologous and heterologous mixing experiments between the separated nonidentical subunits of ribonucleotide reductase, protein M1 and protein M2, from wild type and dGuo-L cells showed that the dGTP- feedback sensitivity was governed by the source of the protein M1. A partial resolution of two dGuo-L protein M1 components was achieved by chromatography on dextran blue-Sepharose. In order to resolve the two dGuo-L protein M1 components more completely, we introduced into dGuo-L cells a second mutation which conferred resistance of the ribonucleotide reductase to dATP, while the original dGTP resistance was maintained. The chromatography of protein M1 from this latter clone, dGuo-L-Aphid-G5, on dATP-Sepharose resolved two kinetically distinct protein M1 components. The first component was sensitive to dGTP inhibition but stimulated by dATP; the second was absolutely refractory to dGTP but sensitive to dATP inhibition. This confirms the hypothesis that the dGuo-L parent is heterozygous for protein M1, containing one wild type and one mutant allele.
Highlights
From a mutagenized population of mouseT-lym- heterozygous €or protein M1, containing one wild type phoma cells (-9) in continuous culture a cell line has and one mutant allele
Studies on the metabolic pathogenesis of the human immunodeficiency disease associated with the inherited deficiency of purine nucleoside phosphorylase have led to the selection, isolation, and characterization of a mutant mouse T-lymphoma (549) cell in which the ribonucleotide reductase gests that the dGuo-L cell line is heterozygous for ri- exhibits decreased sensitivity to feedback inhibition by dGTP
In order to resolve the two dGuo-L protein M 1 components more completely, we introduced into dGuo-L cells a second mutation which conferred resistance of the ribonucleotide reductase to dATP, while the original dGTP resistance was maintained
Summary
Materials-The dGTP-Sepharose was synthesized by the method of Pfeuffer (4). The sources and specifics of all other materials are described in the preceding report (2). From a mutagenizedpopulation of dCuo-Lcells, we isolated a series of secondary mutants resistant to deoxyadenosine These clones were selected for resistance to 0.4 pg/ml of aphidicolin, a tetracyclic diterpenoidcompoundwhichistoxic to mammaliancellsandis known to inhibit eukaryoticDNA polymerasea in vitro(8,9).Mutant mammalian cell lines resistant to aphidicolin have been describbeyd Ayusawa et el. In some experiments,the protein M1 preparations from the dGuo-L and HAT-1.5A cell lines were eluted from dextran blue-Sepharose witha linear 0 to 0.4 M KC1 gradient. With[50] mM Tris, pH 7.5, and 2 mM dithiothreitol (buffer B) This dilutedprotein M1 preparation wasapplied to a dATP-Sepharose for t h e dGuo-200-1 cells (2, 15), the alteration in the ribonucolumn that had been previously equilibrated with bufferB contain- cleotide reductase of each cell line resides in protein MI. Eluted from dextran blue-Sepharose wliothw (0.25 M) concentrations of KC1 tended to exhibit greater sensitivitoy dGTP
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