Cloning of the arg-12 gene of Neurospora crassa and regulation of its transcript via cross-pathway amino acid control

Abstract

The arg-12 locus of Neurospora crassa encodes ornithine carbamoyl transferase, which is one of many amino acid synthetic enzymes whose activity is regulated through cross-pathway (or general) amino acid control. We report here the use of probes derived from the functionally equivalent arg-B gene of Aspergillus nidulans to identify and clone a 10 kb Neurospora DNA fragment carrying the arg-12 gene. Short Neurospora DNA probes derived from this fragment were used to identify a 1.5 kb polyA+ transcript of the arg-12 region. Arg-12 transcript levels increased approximately 20 fold under conditions of arginine or histidine limitation in strains having normal cross-pathway regulation (cpc-1+) but showed no such response in a cpc-1 mutant strain impaired in this regulation. Time course studies in cpc-1+ strains revealed a rapid response (within 10 m) of arg-12 transcript levels following inhibition of histidine synthesis by 3 amino 1,2,4 triazole, but a delayed response following arginine deprivation of an arginine requiring strain. In contrast to the behaviour of arg-12 mRNA, the level of the Neurospora am gene transcript (specifying NADP dependent glutamate dehydrogenase) was unaffected either by amino acid limitation or by the cpc-1 mutation. A possible role for the cpc-1+ product as a positive regulator of transcription of genes subject to cross-pathway control is discussed.