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 of two identical subunits, each possessing three separate domains, with flavin, heme, and molybdenum-containing cofactors. A number of mutants of nit-3, the structural gene that encodes Neurospora nitrate reductase, have been characterized at the molecular level. Amber nonsense mutants of nit-3 were found to possess a truncated protein detected by a specific antibody, whereas Ssu-1-suppressed nonsense mutants showed restoration of the wild-type, full-length nitrate reductase monomer. The mutants show constitutive expression of the truncated nitrate reductase 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 sequenced; two of these mutants were due to the deletion of a single base in the coding region for the flavin domain, the third mutant was a nonsense mutation within the amino-terminal molybdenum-containing domain. Homologous recombination was shown to occur when a deleted nit-3 gene was introduced by transformation into a host strain with a single point mutation 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.
Okamoto
P M
PM
Department of Biochemistry, Ohio State University, Columbus 43210.
Garrett
R H
RH
Marzluf
G A
GA
D000483:Alleles; D000595:Amino Acid Sequence; D001483:Base Sequence; D015153:Blotting, Western; D003433:Crosses, Genetic; D005800:Genes, Fungal; D016153:Genes, Suppressor; D008969:Molecular Sequence Data; D009154:Mutation; D009492:Neurospora crassa / Q000201:enzymology* / Q000235:genetics*; D050901:Nitrate Reductase; D009565:Nitrate Reductases / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism; D010957:Plasmids; D012091:Repetitive Sequences, Nucleic Acid
D009565:Nitrate Reductases; D050901:Nitrate Reductase