1995 Fiscal Year Final Research Report Summary
Analysis of molecular mechanisms for suppression of Thy-1 expression in ras-transformed cells.
| Project/Area Number |
06670163
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| Research Category |
Grant-in-Aid for General Scientific Research (C)
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| Allocation Type | Single-year Grants |
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| Research Field |
Pathological medical chemistry
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| Research Institution | Gifu University |
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Principal Investigator |
OKANO Yukio Gifu University, Medical School, Molecular Pathobiochemistry, Professor, 医学部, 教授 (10177066)
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| Co-Investigator(Kenkyū-buntansha) |
KIMURA Masashi Gifu University, Medical School, Molecular Pathobiochemistry, Research Associate, 医学部, 助手 (40260575)
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| Project Period (FY) |
1994 – 1995
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| Keywords | Ca oscillation / Thy-1 / PIG-A |
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| Research Abstract |
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.
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