Identification and localization of DNA alteration in Chinese hamster ovary cell mutants (Urd-) defective in the first three enzymes of de novo pyrimidine synthesis.

ABSTRACT

In animals, the first three enzymatic steps of de novo pyrimidine synthesis, carbamyl phosphate synthetase, aspartate transcarbamylase, and dihydroorotase, comprise the multifunctional protein known as the CAD protein. Mutants of Chinese hamster ovary cells (CHO-K1, pro-) deficient in CAD protein activities require uridine for growth and are designated Urd-A mutants. To examine further the nature of the genetic alterations in Urd-A mutants and revertants, we have performed a detailed Southern blot hybridization analysis of DNA from wild-type, Urd-A, and revertant cells using as hybridization probes cDNAs complementary to CAD mRNA isolated from Syrian hamster. This has allowed us to identify an apparent alteration in the CAD gene in DNA from Urd-A cells. This alteration is in a region of the gene which appears to correspond to the region of the protein which is hypersensitive to proteases and which seems to be altered in the mutants. Only one of the two CAD alleles present appears to be altered in this way. Study of certain revertants of Urd-A strongly suggests that in some cases reversion has occurred by amplification of the mutant CAD allele.