For a variety of Filamentous fungi the level of extracellular glucose has been found to regulate the production of those extracellular hydrolases which mobilize alternative carbon and energy sources (proteases and carbohydrases). These glucose-repressible extracellular hydrolytic enzymes play an important role in fungal growth. Using Neurospora crassa as a model system we propose to study the molecular mechanisms responsible for the regulated expression of glucose-repressible genes in the filamentous fungi. We propose to identify DNA sequences which are required for the regulated expression of grg-l, a Neurospora glucose-repressible gene. The regulatory sequences will be defined by creating deletions and mutations in vitro and following the in vivo expression from these altered genes in Neurospora transformants. We propose to isolate and study a number of mutants affected in the ability to regulate the expression of glucose-repressible genes. Neurospora transformants containing chimeric genes will be used to generate the mutants. These mutants will be characterized by classical genetics techniques. A major effort will be made to study those proteins which specifically bind to the DNA sequences needed for the regulated expression of grg-l. We will assess the presence or absence of these DNA binding proteins in mutants affected in the ability to express glucose-repressible genes. By characterizing the grg-l regulatory region, the proteins which bind to this region, and the presence or absence of these proteins in mutants unable to regulate grg-l transcription, we hope to elucidate the molecular mechanisms responsible for the regulation of glucose-repressible genes in the filamentous fungi. Given a choice of nutrients many simple organisms (bacteria and fungi) preferentially utilize glucose when it is available. When growing in the simultaneous presence of glucose and other nutrients, these organisms make none of the enzymes necessary to digest the other nutrients until they have exhausted the supply of glucose. The enzymes for digestion of the other nutrients are said to be repressed by glucose. The mechanism of glucose repression is very well understood in some but not all species of bacteria, although some details are still obscure even in well characterized species. The mechanism is apparently much more complex in yeast than in bacteria, and hence is only partly understood. The mechanism in the mold Neurospora crassa is not understood at all, although there is reason to believe that it is as complex as in yeast, though differing in detail. This is a proposal to elucidate the mechanism of glucose repression in N. crassa. Apart from its own considerable intrinsic interest, this work may lead to information useful for the engineering of industrially useful strains of Neurospora.
