The overall objective is to understand at the molecular level the regulation of a particular genetic system in the lower eukaryote Neurospora crassa which may serve as a model for understanding gene organization and regulation in eukaryotes in general. This system involves the qa gene cluster which encodes the inducible enzymes catalyzing the early reactions in the quinic acid catabolic pathway. The entire qa gene cluster has been cloned on a 17.6 kb fragment and sequenced. Present genetic, biochemical, and molecular evidence indicates that this cluster comprises five structural and two regulatory genes. The two regulatory genes (qa-1S and qa-1F) encode, respectively, a repressor and an activator protein. The actions of these two regulatory proteins, plus that of the inducer, quinic acid, control the synthesis of the inducible enzymes (encoded by the five structural genes) which catalyze the utilization of quinic acid as a carbon source by N. crassa. The four major specific aims of the present proposal are: (1) To identify the molecular target of the activator protein, which plays a positive regulatory role in facilitating the transcription by RNA polymerase II of all qa genes, including itself (autoregulation). Present data suggest that this target may be a 16 bp consensus sequence with dyad symmetry (i.e., an upstream activator sequence - UASQ) located 5' to each qa gene. This sequence can be deleted or modified both in vivo and in vitro and its significance studied using transformation procedures. (2) To identify the molecular target of the repressor protein. Present data suggest that this target may be the activator protein, and attempts will be made to isolate constitutive qa-1F mutants (qa-1Fc) in the coding region of the qa-1F gene, whose detection would support this hypothesis. Also additional repressor mutants will be cloned and sequenced to identify specific functional repressor domains. (3) To isolate significant amounts of both activator and repressor proteins by cloning the qa-1F and qa-1S genes into high expression vectors. These isolated proteins would be characterized by their DNA and protein binding characteristics and would be used in an available homologous N. crassa polymerase II transcription system. (4) To identify the presently unknown functions of the qa-x and qa-y genes in the qa gene cluster utilizing gene inactivation and transformation procedures.
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