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

taka -淀粉酶A （Taa）是由米曲霉诱导合成和分泌的α -淀粉酶之一。本文以中性曲霉为中间寄主，研究了米曲霉Taa基因的表达调控机制。通过分析从淀粉和葡萄糖培养的携带a.m oryzae Taa基因的A. nidulans细胞核中分离的Taa基因合成特异性转录本，表明Taa的诱导在转录水平上受到调节。Taa基因特异性转录物仅在可诱导条件下生长的细胞（即淀粉生长细胞）的细胞核中检测到。淀粉诱导性必需的潜在候选序列元件存在于转录起点的上游；一个CCAAT盒子，一个GC盒子，一个类似图案的序列。只要CCAAT序列完整，直到缺失-362，诱导性都保持完整。进一步分析表明，含有CCAAT序列的55bpDNA片段足够长，可以使Taa基因具有淀粉诱导性。从淀粉和葡萄糖培养的细胞中提取核提取物，检测与Taa基因启动子区域结合的蛋白质。在淀粉和葡萄糖培养的细胞的核提取物中检测到一种称为ancp1的蛋白质，并发现它与CCAAT序列结合。出乎意料的是，在葡萄糖生长细胞的核提取物中检测到另一种名为AnNP1的DNA结合蛋白，并被发现与AnCP1结合位点上游的25bp区域结合。这两个结合位点的占用似乎是相互排斥的，这暗示了Taa基因表达的负调控机制。

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.