Isolation of the ace1 gene encoding a Cys2-His2 transcription factor involved in regulation of activity of the cellulase promoter cbh1 of Trichoderma reesei
Isolation of the ace1 gene encoding a Cys2-His2 transcription factor involved in regulation of activity of the cellulase promoter cbh1 of Trichoderma reesei
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DOI：
10.1074/jbc.275.8.5817
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发表时间：
2000-02-25
影响因子：
4.8
通讯作者：
Penttilä, M
中科院分区：
文献类型：
--
作者：
Saloheimo, A;Aro, N;Penttilä, M
建立了一种克隆酿酒酵母正作用转录调控基因的遗传选择方法。应用该方法分离了里氏木霉纤维素酶激活因子基因。激活基因是从T. reesei表达cDNA文库中分离出来的，基于它们的翻译产物能够激活T. reesei cbh1全长启动子与S. cerevisiae HIS3基因偶联的转录，并且在缺乏组氨酸的情况下支持酵母菌落的生长。在获得的克隆中，ace1基因编码一种新的多肽ACEI，该基因含有三个Cys(2)-His(2)型锌指基序。在曲霉和神经孢子菌表达的序列标签中发现了可能的ACEI同源物。在酵母单杂交系统中进一步证实了ACEI与cbh1启动子结合的能力。体外结合和凝胶迁移实验揭示了cbh1启动子中ACEI蛋白的几个结合位点。在含有纤维素的培养基上，纤维素酶通常是高度表达的，而在这种培养基上，破坏了T. reesei的ace1基因会导致真菌生长迟缓。
A genetic selection method was developed for the cloning of positive-acting transcriptional regulatory genes in Saccharomyces cerevisiae. The method was applied for the isolation of activators of Trichoderma re reesei (Hypocrea jecorina) cellulase genes. Activator genes were isolated from a T. reesei expression cDNA library on the basis of the ability of their translation products to activate transcription from the full-length T. reesei cbh1 promoter coupled to the S. cerevisiae HIS3 gene and to support the growth of the yeast colonies in the absence of histidine. Among the clones obtained was the ace1 gene encoding a novel polypeptide, ACEI, that contains three zinc finger motifs of Cys(2)-His(2) type. Possible ACEI homologues were found among expressed sequence tags of Aspergillus and Neurospora. The ability of ACEI to bind to the cbh1 promoter was further confirmed in the yeast one-hybrid system. In vitro binding and gel mobility shift assays revealed several binding sites for the ACEI protein in the cbh1 promoter. Disruption of the ace1 gene in T. reesei resulted in retarded growth of the fungus on a cellulose-containing medium, on which cellulases are normally highly expressed.