TRANSCRIPTIONAL REGULATION OF AMYLASES AND GLUCOSE OXIDASE FROM ASPERGILLUS NIGER

丝状真菌黑曲霉产生不同的组和 淀粉分解酶的数量取决于碳水化合物的类型 存在于生长介质中。这些酶的控制基因还没有 已被确认身份。拟议研究的主要目标是 研究编码α-干扰素基因的表达调控 葡萄糖苷酶、糖淀粉酶和葡萄糖氧化酶的转录 水平。我们的方法需要从cdna中筛选克隆。 带有标记寡核苷酸探针的黑曲霉文库。积极的一面 然后通过聚合酶链式反应分别扩增每种酶的cDNA克隆， 标记的，并用于探测包含 从基因组DNA文库中提取所需的基因。不同组织的角色 启动子和其他调节区中的序列元件 基因组DNA片段将通过凝胶移位DNA结合来确定 分析，DNase I足迹分析和返回改变的基因 与报告基因融合到细胞中。该病毒的核苷酸序列 来自cDNA和基因组DNA的各种DNA片段将被 主要由聚合酶链式反应程序决定。DNA结合蛋白是 从黑曲霉的核提取物中分离出来，并进行了表征。 DNA结合蛋白与细胞内特定位点的相互作用 -葡萄糖苷酶、糖淀粉酶和β-葡萄糖苷酶的启动子和其他调节区 将对葡萄糖氧化酶基因进行检测。知识和技术 从这项研究中获得的成果应该有助于理解 这一重要的基因表达调控和分子生物学 丝状真菌。拟议的研究也将提供极好的 在以下领域为任职人数不足的学生提供培训机会 分子遗传学，特别是重组DNA技术。

The filamentous fungus Aspergillus niger produces different sets and amounts of amylolytic enzymes depending on the types of carbohydrate present in growth medium. The control genes of these enzymes have not been identified. The main objectives of the proposed research are to investigate the regulation of the expression of the genes encoding alpha- glucosidase, glucoamylase and glucose oxidase at the transcriptional level. Our approach entails the screening for the clones from the cDNA library of A. niger with labeled oligonucleotide probes. The positive cDNA clones for each of the enzymes are then separately amplified by PCR, labeled, and used to probe the genomic DNA fragments containing the desired genes from the genomic DNA library. The roles of the various sequence elements in the promoter and other regulatory regions of the genomic DNA fragments are to be determined by gel-shift DNA-binding assay, DNase I footprinting analysis and returning the altered genes fused with a reporter gene to cell. The nucleotide sequences of the various DNA fragments derived from both cDNA and genomic DNA are to be determined mostly by PCR procedures. DNA binding proteins are to isolated from the nuclear extracts of A. niger and characterized. Interactions of the DNA binding proteins with the specific sites in the promoter and other regulatory regions of ?-glucosidase, glucoamylase and glucose oxidase genes are to be examined. The knowledge and techniques gained from this research should contribute to the understanding of regulation of gene expression and molecular biology of this important filamentous fungus. The proposed research will also provide excellent training opportunities for the underrepresented students in the area of molecular genetics and in particular the recombinant DNA technology.