TRANSCRIPTIONAL REGULATION OF AMYLASES AND GLUCOSE OXIDASE FROM ASPERGILLUS NIGER

丝状真菌尼日尔产生不同的集合， 淀粉分解酶的量取决于碳水化合物的类型 存在于生长培养基中。 这些酶的控制基因没有 被识别。 拟议研究的主要目标是 研究编码α- 葡萄糖苷酶、葡萄糖淀粉酶和葡萄糖氧化酶在转录水平 水平 我们的方法需要从cDNA中筛选克隆 图书馆A。用标记的寡核苷酸探针检测黑尼日尔。 的积极 然后通过PCR分别扩增每种酶的cDNA克隆， 标记的，并用于探测含有该基因的基因组DNA片段。 从基因组DNA文库中筛选所需基因。 各方面的作用 启动子和其他调控区的序列元件， 基因组DNA片段将通过凝胶移位DNA结合来确定 DNA酶I足迹分析和返回改变的基因 与报告基因融合到细胞中。 的核苷酸序列 来自cDNA和基因组DNA的各种DNA片段将被 主要通过PCR方法确定。 DNA结合蛋白 从A.尼日尔和特点。 DNA结合蛋白与细胞内特定位点的相互作用 启动子和其他调控区域的？-葡糖苷酶、葡糖淀粉酶和 检测葡萄糖氧化酶基因。 知识和技术 从这项研究中获得的信息应该有助于理解 基因表达调控和这一重要分子生物学 丝状真菌 这项研究还将提供出色的 为代表性不足的学生提供以下领域的培训机会 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.