GENETICAL AND BIOCHEMICAL CHARACTERIZATION OF QA-3 MUTANTS AND REVERTANTS IN THE QA GENE CLUSTER OF NEUROSPORA CRASSA

Mary E. Case ¹, Carmen Pueyo ¹, J. Lopez Barea ¹, and Norman H. Giles ¹

¹ Program in Genetics and Department of Zoology, University of Georgia, Athens, Georgia 30602

The qa-3 gene, one of the four genes in the qa gene cluster, encodes quinate (shikimate) dehydrogenase (quinate: NAD oxidoreductase, ER 1.1.1.24), the first enzyme in the inducible quinic acid catabolic pathway in Neurospora crassa. Genetic analyses have localized 26 qa-3 mutants at 11 sites on the qa-3 genetic map on the basis of prototroph frequencies. Certain mutants, e.g., 336–3–10 and 336–3–3, are located at opposite ends of the qa-3 gene. Data from four-point crosses (qa-1^S mutant 124 x five different qa-3 mutants in triple mutants qa-3, qa-4, qa-2) indicate the following orientation of the qa-3 gene within the qa cluster: qa-1, qa-3 mutant 336–3–10 ("left" end) qa-3 mutant 336–3–3 ("right" end), qa-4, qa-2. Ultraviolet-induced revertants have been obtained from 14 of the qa-3 mutants. The revertable mutants fall into two major classes: those that revert by changes either at the same site or at a second site within the qa-3 gene, and those that revert by unlinked suppressor mutations. The intragenic revertants can be further distinguished by quantative and/or qualitative differences in their quinate dehydrogenase activities. Some revertants with activities either equivalent to or less than wild type produce a thermostable enzyme, and others an enzyme which is thermolabile in vitro at 35°. A concentration of quinic acid or shikimic acid as low as 50 µm protects the enzyme markedly from heat inactivation. The genetic organization and the orientation of the qa-3 gene are discussed with respect to its direction of transcription and to the possible localization of a promoter (initiator) region(s) within the qa gene cluster.