THE POSITIVELY ACTING AMDA GENE OF ASPERGILLUS-NIDULANS ENCODES A PROTEIN WITH 2 C2H2 ZINC-FINGER MOTIFS

Semi-dominant mutations in the amdA gene lead to elevated expression of the gene encoding acetamidase, amdS, These mutations also cause constitutive expression of the acetate-inducible gene, aciA. In the amdS 5' regulatory region, two cis-acting mutations, amdI66 and amdI666, have been isolated which specifically affect amdA activation of amdS. These mutations are a duplication and a triplication of an 18 bp GA-rich sequence, thought to define the amdA site of action within the amdS promoter region, Similar GA-rich sequences have also been found in the 5' region of aclA, This paper describes the cloning and initial functional characterization of the amdA gene and two of its mutant alleles, The wild-type amdA gene has been cloned by a chromosome walk from genes gatA and alcC on linkage group VII and localized by complementation of an amdA loss-of-function mutation, Transcriptional analysis reveals that the gene is expressed constitutively at low levels under growth conditions which affect expression of amdS and aciA, The gene is predicted to encode an 880-amino-acid protein which contains two C2H2 zinc fingers, a nuclear localization sequence and two transcriptional activation domains, The amdA7 semi-dominant gain-of-function mutation results In a glycine to aspartate substitution which would increase the acidity of one of these regions. Analysis of in vitro generated mutations in the 5' region of amdS using an amdS::lacZ reporter has been used to localize the site of action of AmdA. The C2H2 zinc-finger motifs identified in the protein are similar to those found in the carbon catabolite repressor protein, CreA, which also regulates amdS and recognizes sequences which overlap with the proposed site of action for AmdA.