Identification of the Sites of Action for Regulatory Genes Controlling the amdS Gene of Aspergillus nidulans

Abstract

The amdS gene of Aspergillus nidulans, which encodes an acetamidase enzyme, is positively regulated by the trans-acting genes amdR, facB, amdA, and areA. Sequence changes in several cis-acting mutations in the 5' region of the gene which specifically affect amdS regulation were determined. The amdI9 mutation, which results in increased facB-dependent acetate induction, is due to a single-base change at base pair -210 relative to the start point of translation. The amdI93 mutation, which abolishes amdR-dependent omega-amino acid induction, is a deletion of base pairs -181 to -151. The amdI66 mutation, which causes increased gene activation in strains carrying amdA regulatory gene mutations, is a duplication of base pairs -107 to -90. Transformation of A. nidulans can generate transformants containing multiple integrated copies of plasmid sequences. When these plasmids carry a potential binding site for a regulatory gene product, growth on substrates whose catabolism requires genes activated by that regulatory gene can be reduced, apparently because of titration of the regulatory gene product. Introduction of 5' amdS sequences via cotransformation into strains of various genotypes was used to localize sequences apparently involved in binding of the products of the amdR, amdA, and facB genes. The position of these sequences is in agreement with the positions of the specific cis-acting mutations. Consistent with these results, a transformant of A. nidulans derived from a plasmid deleted for sequences upstream from -111 was found to have lost amdR- and facB-mediated control but was still regulated by the amdA gene. In addition, amdS expression in this transformant was still dependent on the areA gene.

FacB, the Aspergillus nidulans activator of acetate utilization genes, binds dissimilar DNA sequences

“…Multiple copies of a plasmid carrying the amdS gene with the amdI9 mutation, but not a wild-type amdS plasmid, resulted in reduced growth on acetate medium (Kelly and Hynes, 1987), indicating that the amdI9 mutation results in increased affinity for, and therefore titration of, the facB gene product. Hynes et al (1988) localized regions of 5Ј amdS involved in titration to -219 to -111 by the transformation of an amdI9 strain with plasmids containing subcloned fragments. Multiple copies of the cloned facB gene reversed the titration of the facB gene product (Katz and Hynes, 1989).…”

Molecular Characterization of the lam Locus and Sequences Involved in Regulation by the AmdR Protein of Aspergillus nidulans

“…be of interest to investigate whether all of these CCAAT sequences bind the same protein or whether several distinct CCAAT-specific factors exist within the cell. The CCAAT sequence of the AmdR box of amdS (AGC CAAT) is within the amdI93 mutation, a 31-bp deletion which results in loss of amdR regulation (24,26; Fig. 5C).…”

“…Titration analysis of lam and gatA. Transformants carrying multiple copies of the AmdR protein-binding site from amdS grow weakly on 2-pyrrolidinone medium because of titration of the AmdR protein (26,35). Clones from the lam region were tested for the ability to titrate the AmdR protein.…”

“…This construct and the pLIR plasmids begin at position -76 in the 5' region of amdS. They contain essential promoter elements but none of the region required for amdR regulation of amdS (26). The insert of pIR22 was placed upstream of the amdS-lacZ fusion in pSKBX as a PstI-EcoRI fragment to create plasmid pSKBX-22.…”

Insertional inactivation and cloning of the wA gene of Aspergillus nidulans.

“…The prn sequences comprise three contiguous EcoRI fragments of 11.9 kbp in total which were obtained by partial digestion of lambda Charon 4A clone LAN122 (Figure 1) and cloned into the EcoRI site of pBR322 (DURRENS et al 1986);HULL et al 1989). pAMD2 1 contains the A. nidulans acetamidase (amdS) gene as an EcoRI-SmaI fragment in pUC9 (HYNES et al 1988). Plasmids pAMD21 and pFB39 (see above) were kindly provided by M. J. HYNES and F. P.…”