Function of DADl Protein in Programd Cell Death

我们从仓鼠BHK 21细胞系中分离到一系列温度敏感生长突变体。在这些ts突变体中，我们发现了在限制性温度下进入细胞凋亡的突变体。在其中一个tsBN 7中，染色体DNA以典型的方式片段化，如依赖于蛋白质合成的光性细胞死亡。我们已经克隆了一个人基因互补tsBN 7突变，并指定为DAD 1编码的蛋白质的12.5 kDa，这是很好的保守通过进化。作为合作，我们克隆了秀丽隐杆线虫的dadl同源物（Ce-dadl）。Ce-dadl在氨基酸水平上与脊椎动物DAD 1的同源性超过60%，并且与tsBN 7突变互补良好。通过过量表达，人DAD 1抑制了优雅假丝酵母细胞的程序性死亡，最近发现DAD 1是酵母OST 2的同源物，参与N-连接糖基化。基于这一发现，我们发现在tsBN 7细胞中p450的糖基化是温度敏感的。据报道，N-连接糖基化发生在进入高尔基体之前，DAD 1似乎位于内质网中。我们不知道N-连接糖基化的缺陷是如何引起细胞凋亡的。为了阐明这一问题，我们采用基因打靶的方法从小鼠染色体上删除DAD 1基因。

We have isolated a series of temperature sensitive growth mutant from the hamster BHK21 cell line. Among those ts mutants, we found the mutants which enter apoptotic cell death at the restrictive temperature. In one of them tsBN7, chromosomal DNA was fragmented with a typical manner like apoptptic cell death depending on protein synthesis. We have cloned a human gene complementing tsBN7 mutation and designated as DAD1 encoding a protein of 12.5 kDa which is well conserved through evolution. As a co-work, we have cloned a dadl homologue of Caenorhabditis elegans (Ce-dadl). Ce-dadl is more than 60% identical to vertebrate DAD1 in amino acid level and well complemented tsBN7 mutation. By overexpression, human DAD1 suppressed programd cell death in C.elegance.Recently, DAD1 was found to be a homologue of yeast OST2 involved in N linked glycosylation. Based on this finding, we found that a glycosylation of p450 is temperature sensitive in tsBN7 cells. As reported that N linked glycosylation occurs prior to entering Golgi apparatus, DAD1 seems to be located in endoplasmic reticulum. We don't know how a defect of N linked glycosylation causes apoptotic cell death. In order to clarify this issue, we are deleting DAD1 gene from mouse chromosome by gene targeting.