The main objectives of the proposed research are to investigate the molecular cloning and the characterization of the genes coding for amylolytic enzymes and glucose oxidase from strains of the filamentous fungi Aspergillus niger. Our approach to cloning the genes of these enzymes entails the rapid synthesis of cDNA probes directed by amino acid sequence of the NH2-terminal peptide by mixed oligonucleotide primed amplification of cDNA (MOPAC) procedure; the construction of cDNA and genomic DNA libraries and their screening. The specific cDNA probes and two anti-body probes are used to screen the A. niger full-length cDNA library and genomic DNA library constructed in lambda vectors. The desirable recombinant clones are amplified, rescued, sub-cloned and sequenced. The promoters for each of the genomic DNA clones are identified by chloramphenicol acetyl transferase assay. The know- ledge and techniques gained from this research should provide the foundation for the study of regulation of gene expression and molecular biology of A. niger and other filamentous fungi including the pathogenic fungi. This information and these techniques should also assist medical mycologists in studying pathogenic fungi both as unique organisms and as agents of infection.
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