Biochemical genetic analysis of pyrimidine biosynthesis in mammalian cells: I. Isolation of a mutant defective in the early steps of de novo pyrimidine synthesis

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Abstract
The isolation and characterization of a new mutant of Chinese hamster ovary cells is described. This mutant, Urd−A, shows an absolute requirement for exogenously added pyrimidines for growth. Complementation analysis indicates that the lesion in this mutant is recessive. Revenants can be isolated at frequencies suggesting that it is a single gene alteration. Biochemical analysis of cellfree extracts of CHO-K1 (Urd+) and Urd−A revealed that Urd−A possesses no more than 10% of wildtype levels of carbamyl phosphate synthetase (EC 2.7.2.9) activity, no more than 1% of wildtype levels of aspartate transcarbamylase (EC 2.1.3.2) activity, and undetectable levels of dihydroorotase (EC 3.5.23) activity. Thus, this mutant appears simultaneously to possess marked or complete deficiencies in the activities of the first three enzymes of pyrimidine biosynthesis. Activities of the other enzymes of the pathway appear normal. The use of this mutant for biochemicalgenetic studies of pyrimidine biosynthesis is discussed.