Transformants of Neurospora crassa with the nit-4 nitrogen regulatory gene: copy number, growth rate and enzyme activity.
nit-4 is a pathway-specific regulatory gene which controls nitrate assimilation in Neurospora crassa, and appears to mediate nitrate induction of nitrate and nitrite reductase. The NIT4 protein consists of 1090 amino-acid residues and possesses a single GAL4-like putative DNA-binding domain plus acidic, glutamine-rich, and polyglutamine regions. Several mutants with amino-acid substitutions in the putative DNA-binding domain and a nit-4 deletion mutant, which encodes a truncated NIT4 protein lacking the polyglutamine region, are functional, i.e., they are capable of transforming a nit-4 mutant strain. However, transformants obtained with most of these nit-4 mutant genes possess a markedly reduced level of nitrate reductase and grow only slowly on nitrate, emphasizing the need to examine quantitatively the affects of in vitro-manipulated genes. The possibility that some mutant genes could yield transformants only if multiple copies were integrated was examined. The presence of multiple copies of wild-type or mutant nit-4 genes did not generally lead to increased enzyme activity or growth rate, but instead frequently appeared to be detrimental to nit-4 function. A hybrid nit-4-nirA gene transforms nit-4 mutants but only allows slow growth on nitrate and has a very low level of nitrate reductase.
Yuan
G F
GF
Department of Biochemistry, Ohio State University, Columbus 43210.
Marzluf
G A
GA
D003001:Cloning, Molecular; D004268:DNA-Binding Proteins / Q000235:genetics*; D005656:Fungal Proteins; D005800:Genes, Fungal; D005809:Genes, Regulator; D009492:Neurospora crassa / Q000201:enzymology / Q000235:genetics* / Q000254:growth & development; D050901:Nitrate Reductase; D009565:Nitrate Reductases / Q000378:metabolism*; D015534:Trans-Activators; D014170:Transformation, Genetic
D004268:DNA-Binding Proteins; D005656:Fungal Proteins; D015534:Trans-Activators; C071240:NIT-4 protein, Neurospora crassa; D009565:Nitrate Reductases; D050901:Nitrate Reductase