Biochemical studies on the Nit mutants of Neurospora crassa

Abstract

One allele at each of the five nit loci in Neurospora crassa together with the wild type strain have been compared on various nitrogen sources with regard to (i) their growth characteristics (ii) the level of nitrate reductase and its associated activities (reduced benzyl viologen nitrate reductase and cytochrome c reductase) (iii) the level of nitrate reductase and (iv) their ability to take up nitrite from the surrounding medium. Results are consistent with the hypothesis that nit-3 is the structural gene for nitrate reductase, nit-1 specifies in part of molybdenum containing moiety which is responsible for the nit-3 gene product dimerising to form nitrate reductase, nit-4 and nit-5 are regulator genes whose products are involved in the induction of both nitrate reductase and nitrite reductase and nit-2 codes for a generalised ammonium activated repressor protein. Studies on the induction of nitrate reductase (and its associated activities) and nitrite reductase in wild type, nit-1 and nit-3 in the presence of either nitrate or nitrite suggest that each enzyme may be regulated independently of the other and that nitrite could be true co-inducer of the assimilatory pathway. Nitrite uptake experiments with nit-2, nit-4 and nit-5 strains show that whereas nit-4 and nit-5 are freely permeable to this molecule, it is unable to enter the nit-2 mycelium.

Nitrate Reductase Deficient Mutants of Chlamydomonas reinhardii. Biochemical Characteristics

“…extent. This mutant grows well on hypoxanthine and this finding supports the view that the BVH(FMNH,)-NR activity is associated with the molybdenum-containing sub-unit of nitrate reductase (Coddington, 1976). The nitrate reductase from the nitA mutant elutes later than nitrate reductase from wild-type from a Sepharose column.…”

“…It is therefore presumably of lower molecular weight than the wild-type enzyme and it is reasonable to suggest that it has lost the sub-unit of the enzyme which combines with NADH (Barea et ul., 1976). Phenotypically our nitA mutant corresponds closely to the nit-3 mutant of Neurospora crassa (Coddington, 1976) which also produces a BVH-nitrate reductase of lower molecular weight than enzyme from wild-type and which has also lost NAD(P)H-NR and NAD(P)H-CR activities. In both mutants, BVH-nitrate reductase activity, under inducing conditions, was higher than that of wild-type under similar conditions and in both, the nitrate reductase, like that of the wild-type strains, was repressed in the presence of ammonium.…”

A Nitrate Reductase-less Variant Isolated from Suspension Cultures of Datura innoxia (Mill.)

“…was obtained from the cell culture stocks of the National Research Council of Canada, Prairie Regional Laboratory in Saskatoon. This culture, designated DI-6, was routinely maintained by transferring 10 to 15 ml cell suspension every 7 days into 250-ml Erlenmeyer flasks containing 60 ml either fresh B5 medium (9) (3,4,16 Five-ml aliquots of DI-6 cell suspension, containing approximately 1.5 x 106 cells ml-', which had been growing in B5 medium for 5 days, were transferred into each of 50 125-ml Erlenmeyer flasks containing 50 ml of medium A and 49 mm KC103. After 6 weeks continuous shaking, growth was observed in one flask.…”

Molecular cloning and analysis of the regulation of nit-3, the structural gene for nitrate reductase in Neurospora crassa.

“…The nitrogen control circuit of Neurospora has been studied extensively and is comprised of a set of genes that encode enzymes that enable this fungus to use various secondary nitrogen sources, e.g., nitrate, nitrite, purines, or amino acids, when preferred nitrogen sources such as ammonia or glutamine are unavailable (1). These nitrogen-related enzymes include nitrate and nitrite reductase, several purine catabolic enzymes, as well as various enzymes, including L-amino acid oxidase, a general amino acid permease, and an extracellular protease involved in the use of proteins and amino acids as nitrogen sources (1,(4)(5)(6)(7)(8). The various nitrogen-related structural genes of the nitrogen circuit are unlinked, yet their expression is controlled in a parallel fashion by major and minor control genes as well as metabolic inducers and repressors.…”