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Genetical and biochemical studies on physiological function of intracellular nuclease of Aspergillus oryzae

米曲霉胞内核酸酶生理功能的遗传学和生化研究

基本信息

批准号：
10660075
负责人：
UOZUMI Takeshi
金额：
$ 1.98万
依托单位：
Meiji University (1999)The University of Tokyo (1998)
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (C)
财政年份：
1998
资助国家：
日本
起止时间：
1998 至 1999
项目状态：
已结题

项目摘要

Aspergillus oryzae has nuclease O and its specific inhibitor protein in the cell. The nuclease and the inhibitor combine to make an inactive complex with a Ki of 3.2 x 10^<-12> (T.Uozumi et al., J.Biol. Chem., 251, 2808 (1976)). In the course of senescence of the fungi, especially under the deficiency of oxygen, the inhibitor protein is decomposed by protease, resulting in the activation of the nuclease, which in turn digests cellular nucleic acids. This study was intended to clarify the physiological function of intracellular nucleases including nuclease O.We reported the cloning of nuclease O gene (nucO)(M.Sano et al., Curr. Genet., 30, 312 (1996)). In the present study, a mutant strain of A.oryzae with disrupted nucO gene was constructed. The mutant showed an apparently normal growth comparable to the wild strain, which suggested that nuclease O is not essential for growth of the fungi.Recently, a caspase-activated DNase (CAD) and the specific inhibitor (ICAD) have been reported in animal cells. In the process of apoptosis, caspase degrades ICAD leading to the activation of CAD, which in turn degrades the cellular DNA.Physical properties of nuclease O (isoelectric point, pI=10.0) and the inhibitor (heat-stable, pI=4.1) are very similar to those of CAD and ICAD.So, there is a high possibility that the phenomenon of autolysis of A.oryzae corresponds to apoptosis of animal cells. More researches should be done to clarify the possibility.

黑曲霉细胞内含有核酸酶O及其特异性抑制蛋白。核酸酶和抑制剂联合收割机组合以产生Ki为3.2 × 10 - 6的无活性复合物<-12>（T.Uozumi等人，J. Biol. Chem.，251，2808（1976））。在真菌衰老过程中，特别是在缺氧条件下，抑制蛋白被蛋白酶分解，导致核酸酶激活，进而使细胞核酸降解。本研究旨在阐明包括核酸酶O在内的细胞内核酸酶的生理功能。我们报道了核酸酶O基因（nucO）的克隆（M.佐野等人，Curr. Genet.，30，312（1996））。本研究构建了一株nucO基因被破坏的A. pylori突变株。该突变株的生长与野生株相当，表明核酸酶O不是真菌生长所必需的。最近，在动物细胞中发现了一种半胱天冬酶激活的DNA酶（CAD）及其特异性抑制剂（ICAD）。在细胞凋亡过程中，caspase降解ICAD，导致CAD活化，进而降解细胞DNA，核酸酶O（等电点pI=10.0）和抑制剂（热稳定性pI=4.1）的物理性质与CAD和ICAD非常相似，因此，A. acetylacetate的自溶现象很可能与动物细胞的凋亡相对应。需要进一步的研究来阐明这种可能性。