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Biochemical genetic analysis of pyrimidine biosynthesis in mammalian cells. II. Isolation and characterization of a mutant of chinese hamster ovary cells with defective dihydroorotate dehydrogenase (E.C. 1.3.3.1) activity
Author(s) -
Stamato Thomas D.,
Patterson David
Publication year - 1979
Publication title -
journal of cellular physiology
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.529
H-Index - 174
eISSN - 1097-4652
pISSN - 0021-9541
DOI - 10.1002/jcp.1040980304
Subject(s) - dihydroorotate dehydrogenase , chinese hamster ovary cell , pyrimidine metabolism , mutant , biochemistry , biosynthesis , dehydrogenase , biology , enzyme , wild type , microbiology and biotechnology , chemistry , gene , purine , receptor
A mutant (A204) of Chinese hamster ovary cells (CHO‐K1), which is deficient in dihydroorotate (DHO) dehydrogenase (E.C. 1.3.3.1) activity, has been isolated by a replica plating procedure. The mutant does not show a requirement for exogenously added pyrimidines. Examination of intact cells shows that the mutant accumulates a large amount of carbamyl aspartate and is markedly but not totally deficient in biosynthesis of orotate from earlier precursors of pyrimidine biosynthesis, including aspartate and dihydroorotic acid, when compared to wild‐type cells. Analysis of cell‐free extracts of mutant and wild‐type cells shows that the mutant is deficient in DHO dehydrogenase activity, possessing ca. 5% of the wild‐type activity. This evidence leads to the conclusion that this mutant, A204, is in fact partially deficient in DHO dehydrogenase, and that in these cells it is this enzyme which carries out the fourth step of de novo pyrimidine biosynthesis.