The Regulation of a Neurosopora Glucose-repressible Gene

对于各种丝状真菌来说，胞外葡萄糖的水平被发现调节那些胞外水解酶的产生，这些水解酶可以调动替代的碳源和能源(蛋白酶和碳水解酶)。这些耐葡萄糖的胞外水解酶在真菌生长中起着重要作用。以粗糙脉孢菌为模型系统，研究丝状真菌中葡萄糖抑制基因表达调控的分子机制。我们建议确定脉孢子菌葡萄糖抑制基因grg-L调控表达所需的DNA序列。调控序列将通过在体外产生缺失和突变，并在体内跟踪这些改变的基因在脉孢子菌转化体中的表达来定义。我们建议分离和研究一些影响葡萄糖抑制基因表达能力的突变体。含有嵌合基因的脉孢菌转化子将被用来产生突变体。这些突变体将以经典遗传学技术为特征。将致力于研究那些与GRG-L调控表达所需的DNA序列特异结合的蛋白质。我们将评估这些DNA结合蛋白在影响葡萄糖抑制基因表达能力的突变体中的存在与否。通过对GRG-L调控区、与该区域结合的蛋白以及这些蛋白在不能调控GRG-L转录的突变体中的存在与否的研究，我们希望能够阐明丝状真菌中葡萄糖抑制基因调控的分子机制。如果有营养可供选择，许多简单的生物体(细菌和真菌)会优先利用可利用的葡萄糖。当在葡萄糖和其他营养物质同时存在的情况下生长时，这些生物体在耗尽葡萄糖供应之前不会产生任何消化其他营养物质所需的酶。其他营养物质的消化酶据说被葡萄糖抑制。葡萄糖抑制的机制在一些但不是所有种类的细菌中都很清楚，尽管一些细节仍然不清楚，即使在特征很好的物种中也是如此。显然，酵母中的这种机制比细菌中的复杂得多，因此人们对此只有部分了解。虽然有理由相信它和酵母一样复杂，但在细节上有所不同，但对粗糙脉孢菌的作用机制却完全不了解。这是一项旨在阐明粗枝藻葡萄糖抑制机制的建议。除了它本身相当大的内在兴趣之外，这项工作可能会带来对工业有用的脉孢菌菌株工程有用的信息。

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.