MOLECULAR-CLONING AND ANALYSIS OF NRE, THE MAJOR NITROGEN REGULATORY GENE OF PENICILLIUM-CHRYSOGENUM

We have isolated the Penicillium chrysogenum nre gene which is homologous to the major nitrogen regulatory genes areA from Aspergillus nidulans and nit-2 from Neurospora crassa. Overall, nre shows 60% identity to areA and 30% identity to nit-2 at the amino-acid level. The gene encodes a protein of 835 amino-acid residues and contains a single Cys(2)/Cys(2)-type zinc finger with an adjacent basic region and a putative acidic activation region. In the DNA-binding domain, 98% of the amino-acid residues are identical in nre, areA and nit-2. The nre gene has been shown to be functional in N. crassa by heterologous complementation of a nit-2 mutant. Growth tests indicated that transformants could utilize nitrate, amino-acids, purines and amides as sole nitrogen sources. Nitrate reductase activity assays performed with transformants demonstrated that nitrogen control was completely normal. Complementation of N. crassa nit-2 mutants with 5'-deletion clones of nre suggests the possible presence of an internal promoter within the coding region. Northern analysis and ribonuclease protection assays of total cellular RNA indicated that nre encodes a 3.2-kb transcript which is reduced in content under conditions of nitrogen repression.