TRANSCRIPTIONAL REGULATION OF AMYLASES AND GLUCOSE OXIDASE FROM ASPERGILLUS NIGER

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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.