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）1）THY-1蛋白的早期分解，2）与THY-1蛋白质糖结偶联性异常。已知糖 - 磷脂酰肌醇（GPI）锚定蛋白的大多数异常表达是由于酶的糖化糖偶联性酶的缺陷或与pi.eight互补基团催化GPI蛋白质生物合成的缺陷。它们的两个（pig-a和-f）是从人克隆的，只有一个（pig-a）是从小鼠克隆的。但是，我们注意到该研究项目很难。我们从大阪大学微生物疾病研究所Junji Takeda博士那里了解到，如果缺陷性状在疾病哺乳动物细胞中具有隐性性，则表达克隆技术是无用的。首先，必须首先通过NIH3T3和DT细胞的融合实验来检查缺陷是隐性还是显性的。如果缺陷占主导地位，则可以通过互补测定法克隆缺陷的基因，而如果缺陷是隐性的，则表达克隆无法应用。我们分析了PIG-A mRNA的表达，使用了一种反向转录酶-PCR（RT-RT-PCR），在小鼠中阐明了分子结构的分子结构。我们设计了两组PCR引物，涵盖了小鼠Pig-A mRNA，产生了544bp和1023bp频段。在DT和NIH3T3细胞中都检测到544BP波段。仅在DT细胞中检测到1023BP波段。这些结果与我们的原始假设相矛盾，即缺陷可能是猪-A，表明除了PIG-A基因以外的缺陷。