| Budget Amount *help |
¥2,000,000 (Direct Cost: ¥2,000,000)
Fiscal Year 1988: ¥900,000 (Direct Cost: ¥900,000)
Fiscal Year 1987: ¥1,100,000 (Direct Cost: ¥1,100,000)
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| Research Abstract |
The ret transforming gene was activated by the joining of a tyrosine kinase sequence to part of a "finger"-containing gene (designated rfp). Analysis of the amino acid sequence indicated that the first 315 amino acids of rfp were fused to the amino terminus of the 5' truncated ret tyrosine kinase. To further analyze the structure and function of the ret and frp proteins, we have developed a monoclonal antibody against a synthetic peptide corresponding to amino acids 148 to 163 of the proteins. The isolated MoAb, designated RFP-1 (IgM), was reactive with the immunizing peptide but not with a 95 kD in vitro translated ret protein. In contrast, RFP-1 recognized a 60 kD in vitro translated rfp product and a 85 kD native protein in extracts made from HL-60 promyelocytic leukemia cell line in which rfp is highly expressed. This might be due to the difference in conformations between the ret and rfp proteins. By the avidin -biotin complex immunoperoxidase method, RFP-1 strongly stained over 90% of the neclei of human spermatogenic cells, except mature spermatozoon, and of human testicular tumor cells. In addition, when its reactivity with other normal adult tissues were examined, 20-40% of cells were stained in epithelial cells of the gastroineteinal tract mucosae, the kidney tubules, and the thyroid gland follicles; lymphocytes of the thymus, the spleen, and the lymph nodes; and hepatocytes. All of RFP-1 positive cells showed nuclear staining. Since the results of immunological study is consistent with the previous study on expression of rfp mRNA, it is likely that RFP-1 MoAb recognizes the native rfp protein. To further elucidate the nature and function, sufficient amounts of the native protein will be required. We believe that the availabilty of the anti-rfp monoclonal antibody could facilitate purification of the rfp protein from cells and tissues.
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