MOLECULAR CHARACTERIZATION OF CONVENTIONAL AND NEW REPEAT-INDUCED MUTANTS OF NIT-3, THE STRUCTURAL GENE THAT ENCODES NITRATE REDUCTASE IN NEUROSPORA-CRASSA

Nitrate reductase of Neurospora crassa is a dimeric protein composed o f two identical subunits, each possessing three separate domains, with flavin, heme, and molybdenum-containing cofactors. A number of mutant s of nit-3, the structural gene that encodes Neurospora nitrate reduct ase, have been characterized at the molecular level. Amber nonsense mu tants of nit-3 were found to possess a truncated protein detected by a specific antibody, whereas Ssu-1-suppressed nonsense mutants showed r estoration of the wild-type, full-length nitrate reductase monomer. Th e mutants show constitutive expression of the truncated nitrate reduct ase protein; however normal control, which requires nitrate induction, was restored in the suppressed mutant strains. Three conventional nit -3 mutants were isolated by the polymerase chain reaction and sequence d; two of these mutants were due to the deletion of a single base in t he coding region for the flavin domain, the third mutant was a nonsens e mutation within the amino-terminal molybdenum-containing domain. Hom ologous recombination was shown to occur when a deleted nit-3 gene was introduced by transformation into a host strain with a single point m utation in the resident nit-3 gene. New, severely damaged, null nit-3 mutants were created by repeat-induced point mutation and demonstrated to be useful as host strains for transformation experiments.