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

Taka-amylase A（Taa）是一种α-淀粉酶，由曲霉Aspergillus sp.以构巢曲霉为中间宿主，研究了黑鳃金龟Taa基因的表达调控机制。通过分析从淀粉和葡萄糖培养的细胞中分离的Taa基因合成双核特异性转录本，表明Taa的诱导在转录水平上受到调控。携带A. tagaa. Taa基因的构巢菌。Taa基因特异性转录物仅在从在诱导条件下生长的细胞（即淀粉生长的细胞）分离的核中检测到。潜在的候选者的序列元件必不可少的淀粉诱导存在上游的转录起始点; CCAAT盒，GC盒，octermer基序样序列。只要CCAAT序列是完整的，完整的诱导序列被保留到缺失-362。进一步分析表明，含有CCAAT序列的55 bpDNA片段足够长，可以使Taa基因具有淀粉诱导作用。从淀粉和葡萄糖生长的细胞的核提取物进行了分析的蛋白质结合到Taa基因的启动子区域。在淀粉和葡萄糖生长的细胞的核提取物中检测到一种名为AnCP 1的蛋白质，并发现其与CCAAT序列结合。出乎意料的是，在葡萄糖生长的细胞的核提取物中检测到另一种称为AnNP 1的DNA结合蛋白，并发现其结合于AnCP 1结合位点上游的25 bp区域。这两个结合位点的占据似乎是相互排斥的，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.