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Expression cloning of the murine interferon gamma receptor cDNA
1Department of Biochemistry and Molecular Biology, Harvard University, Cambridge, MA 02138.
Insights
Researchers identified the gene for the mouse interferon gamma (IFN-gamma) receptor. This discovery aids in understanding immune responses and developing targeted therapies for inflammatory diseases.
Area of Science:
- Immunology
- Molecular Biology
- Genetics
Background:
- Interferon gamma (IFN-gamma) is a crucial cytokine in immune regulation.
- Understanding the IFN-gamma receptor is vital for deciphering its signaling pathways.
- Previous research focused on the human IFN-gamma receptor.
Purpose of the Study:
- To isolate and characterize the cDNA encoding the murine interferon gamma receptor.
- To enable further studies on IFN-gamma signaling in mice.
- To compare the murine receptor with its human counterpart.
Main Methods:
- Construction of a murine thymocyte cDNA expression library.
- Transfection of monkey COS cells and screening with radiolabeled IFN-gamma.
- Autoradiography and direct plasmid rescue for clone identification.
- Isolation of full-length cDNA using conventional methods.
- Expression of cDNA in COS cells to confirm receptor function.
Main Results:
- Successfully isolated a cDNA encoding the murine IFN-gamma receptor.
- Expressed receptor in COS cells demonstrated specific binding of murine IFN-gamma.
- The binding affinity constant was comparable to that of the native receptor.
- Sequence analysis revealed homology to the human IFN-gamma receptor.
- Identified less than 60% amino acid identity between murine and human receptors, particularly in extracellular and intracellular domains.
Conclusions:
- The study successfully identified the murine IFN-gamma receptor cDNA.
- The murine and human IFN-gamma receptors share homology but have significant sequence divergence.
- This finding provides a basis for investigating species-specific differences in IFN-gamma signaling and function.
Abstract:
A cDNA encoding a receptor for murine interferon gamma (IFN-gamma) was isolated from an expression library made from murine thymocytes. The clone was identified by transfecting the library into monkey COS cells and probing the transfected monolayer with radiolabeled murine IFN-gamma. Cells expressing the receptor were identified by autoradiography and plasmids encoding the receptor were directly rescued from those cells producing a positive signal. A partial cDNA so obtained was used to isolate a full-length cDNA from mouse L929 cells by conventional means. When this cDNA was expressed in COS cells it produced a specific binding site for murine IFN-gamma with an affinity constant similar to that of the receptor found on L929 cells. The predicted amino acid sequence of the murine IFN-gamma receptor shows homology to that previously reported for the human IFN-gamma receptor. However, although the two proteins are clearly related, they show less than 60% identity in both the putative extracellular domain and the intracellular domain.