CreA is the major regulatory protein involved in carbon catabolite repression in Aspergillus nidulans. Previously we have reported the molecular characterization of a number of in vivo selected mutant alleles and showed that they were unlikely to represent total loss of function alleles (Shroff et al., 1996) and that a deletion of the creA gene and surrounding DNA has an extremely severe effect on morphology under both carbon catabolite repressing and carbon catabolite nonrepressing conditions (Dowzer and Kelly, 1991). Here we present an analysis of in vivo selected creA mutations with an extreme morphological phenotype and show that some of these alleles would be predicted to result in no functional CreA. The most extreme of these alleles resulted in a truncation of the protein within the first zinc finger. Precise gene disruptions, leaving the flanking sequences intact, show essentially the same phenotype as this truncated allele. Thus, a strain containing a null allele is viable, and the leaky-lethal phenotype of previous deletion alleles (Dowzer and Kelly, 1991) must be due to the deletion of additional 3' genomic sequence. A strain containing an allele that results in a deletion of the final 80 amino acids shows reduced sensitivity to carbon catabolite repression for a number of systems, thus localizing a region of the protein involved in repression. Surprisingly, the phenotypically most extreme allele studied is not a null allele, but results in an amino acid substitution that would disrupt the zinc finger region and abolish binding to DNA. This is the only allele that produces a full-length protein, predicted to be nuclear localized, but which completely abolishes DNA binding. The phenotype may be more extreme than the null alleles due to the nuclear located CreA protein titrating interacting proteins.
Copyright 1997 Academic Press.