Analysis of molecular mechanisms for suppression of Thy-1 expression in ras-transformed cells.

ras 转化细胞中抑制 Thy-1 表达的分子机制分析。

基本信息

批准号：
06670163
负责人：
OKANO Yukio
金额：
$ 1.34万
依托单位：
Gifu University
依托单位国家：
日本
项目类别：
Grant-in-Aid for General Scientific Research (C)
财政年份：
1994
资助国家：
日本
起止时间：
1994 至 1995
项目状态：
已结题

来源：
https://kaken.nii.ac.jp/en/grant/KAKENHI-PROJECT-06670163/
关键词：
Ca oscillation Thy-1 PIG-A ジアシルグリセロール(DG)DGキナーゼ ras Cキナーゼ

项目摘要

Two possible mechanisms for the suppression of Thy-1 expression in ras-transformed DT cells are considered, 1)early breakdown of Thy-1 protein, 2)abnormalities in glycoconjugation with Thy-1 protein. Most of anomalous expression of glyco-phosphatidylinositol (GPI)-anchored proteins are known to be due to defects of enzymes catalyzing glycoconjugation or conjugation with PI.Eight complementation groups for GPI-anchored protein biosynthesis are known till now. Two (PIG-A and -F) of these are cloned from human, and only one (PIG-A) is cloned from mouse. However, we noticed that there is a difficult point in proceeding this research project. We learned from Dr.Junji Takeda (Research Institute of microbial diseases, Osaka University) that expression cloning technique is not useful if the defective trait is recessive in disomy mammalian cells. It is necessary to check if the defect is recessive or dominant by fusion experiments of NIH3T3 and DT cells first. If the defect is dominant, the defective gene could be cloned by complementation assay, whereas expression cloning can not be applied if the defect is recessive.We analyzed the expression of PIG-A mRNA,the molecular structure of which has been clarified in mouse, using a technique of reverse-transcriptase-PCR (RT-PCR). We designed two sets of PCR primers covering mouse PIG-A mRNA,producing 544bp and 1023bp bands. The 544bp-bands were detected in both DT and NIH3T3 cells. The 1023bp-bands were detected in DT cells alone. These results contradicts our original assumption that the defect might be in PIG-A,suggesting a defect(s) other than PIG-A gene.

考虑了在 ras 转化的 DT 细胞中抑制 Thy-1 表达的两种可能机制，1）Thy-1 蛋白的早期分解，2）与 Thy-1 蛋白的糖缀合异常。大多数糖磷脂酰肌醇（GPI）锚定蛋白的异常表达是由于催化糖缀合或与 PI 缀合的酶的缺陷所致。目前已知 GPI 锚定蛋白生物合成的八个互补基团。其中两种（PIG-A 和-F）是从人类克隆的，只有一种（PIG-A）是从小鼠克隆的。然而，我们注意到开展这项研究项目存在一个难点。我们从武田淳二博士（大阪大学微生物疾病研究所）了解到，如果缺陷性状在二倍体哺乳动物细胞中是隐性的，那么表达克隆技术就没有用处。需要先通过NIH3T3和DT细胞的融合实验来检查缺陷是隐性的还是显性的。如果缺陷是显性的，则可以通过互补分析来克隆缺陷基因，而如果缺陷是隐性的，则不能应用表达克隆。我们分析了PIG-A mRNA的表达，其分子结构已在小鼠中阐明，使用逆转录酶-PCR (RT-PCR) 技术。我们设计了两套覆盖小鼠PIG-A mRNA的PCR引物，产生544bp和1023bp条带。在 DT 和 NIH3T3 细胞中均检测到 544bp 条带。仅在 DT 细胞中检测到 1023bp 条带。这些结果与我们最初的假设相矛盾，即缺陷可能存在于 PIG-A 中，表明 PIG-A 基因之外还有缺陷。