Molecular biological char acterization of regulatory mechanism of gene expression in filamentous fungi

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Taka-amylase A (Taa), one of alpha -amylases,is inducibly synthesized and secreated by Aspergillus oryzae. We investigated the expression regulatory mechanism of the Taa gene from Aspergill us oryzae by using Aspergillus nidulans as an intermediate host. The induction of Taa was shown to be regulated at the transcription level by analyzing the transcripts specific for Taa gene synthesize dinnuclei isolated from starch-and glucose-grown cells, A. nidulans carrying the A.oryzae Taa gene. Taa gene specific transcript was detected only in the nucl ei isolated from cells grown under the inducible conditions, namely starch-grown cells. Potential candidates for sequence elements essential for starch inducibility are present upstream of the transcription start point;a CCAAT box, a GC box, an octermer motif-like sequence. Full inducibility was preserved up to deletion-362,as long as the CCAAT sequence was intact. Further analysis showed that a 55bpDNA fragment containing the CCAAT sequence was long enough to confer starch inducibility on the Taa gene. Nuclear extracts from starch-and glucose-grown cells were assayed for proteins which bind to the promoter region of the Taa gene. A protein designated AnCP 1 was detected in nuclear extracts of both starch-and glucose-grown cells and was found to bind to the CCAAT sequence. Unexpectedly,the other DNA binding protein designated AnNP1 was detected in the nuclear extract of glucose grown cells and was found to bind to the 25bp region just upstream of the AnCP1 binding site. Occupancy of the two binding sites appeared to be mutually exclusive, which is suggestive of a negative regulatory mechanism for Taa gene expression.