Sequential gel mobility shift scanning of 5′ upstream sequences of the Neurospora crassa am (GDH) gene

Abstract

We have used gel mobility shift assays to scan 1.7 kb of 5′ non-coding sequence of the am (glutamate dehydrogenase) gene of Neurospora crassa for binding by partially fractionated Neurospora proteins. Using genetic analysis this region had been shown to play an important role in the control of glutamate dehydrogenase (GDH) expression. Gel mobility shift analysis identified three regions to which Neurospora proteins bind specifically. Two of these corresponded to the two elements previously defined by genetic analysis (URSamα and URSamβ). The third protein binding site appears to be unrelated to am gene expression. Competition experiments showed that the proteins that bind to the URSamα and URSamβ elements are different. The URSamα element was shown to contain two independent binding sites for the URSamα binding protein(s). Both fragments contain a CCAAT motif, suggesting that URSamα binding protein(s) may be members of one of the CCAAT-binding protein families. The effect of deletion of either the URSamα or URSamβ elements on catabolite induction of am expression was also determined. Both elements appear to act as constitutive enhancers of gene expression.