We selected a 5-fluorouracil-resistant, thermosensitive mutant of the uridine monophosphokinase step in Saccharomyces cerevisiae. The mutant displays very weak thermolabile uridine monophosphokinase activity and wild-type uridine diphosphokinase activity. Growth of the mutant at the non-permissive temperature causes immediate reduction of pyrimidine triphosphate pools to 10% of the wild-type level as well as significantly lowering total RNA and protein synthesis. These conditions also provoke derepression of the first gene of the pathway, URA2, at both the levels of enzymatic activity and transcription. The mutation segregates independently of all known genes of the pyrimidine biosynthetic pathway. The corresponding gene has been isolated on a 4.8 kb fragment by complementation of the mutant phenotype. The new gene, named URA6, codes for a 2.2 kb polyadenylated messenger RNA, exists in a single copy per haploid genome, and was mapped to the centromere of chromosome XI.
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