The interferons are essential for host defense against viral infection, modulate the immune response and may be important in preventing tumorigenesis. These pleiotropic effects of interferon are mediated by specific enzymes whose de novo synthesis is induced by interferon treatment. One of these mediators of interferon action is a (2'-5')oligoadenylate-dependent endonuclease (RNase L) which is responsible for the observed degradation of viral mRNA seen in virus-infected, interferon- treated cells. In the proposed research RNase L will be studied in detail. The effect of interferon treatment on the levels of this enzyme in cultured murine cells and in mice will be determined in order to identify cell and tissue types which show the greatest fold induction and the greatest overall specific activity of RNase L. The effect of interferon inducers on the level of RNase L will also be determined. Tissue sources from treated animals will provide material for the purification of RNase L. Purified RNase L will be characterized with regard to its activation by (2'- 5')oligoadenylates, its substrate specificity, and its sensitivity to sulfhydryl reagents. Antibody to RNase L will be produced and used to facilitate purification and functional characterization. Purification of RNase L to homogeneity will be followed by peptide analysis, amino acid analysis and the determination of a partial amino acid sequence. The partial amino acid sequence will be used as a basis for the synthesis of oligodeoxyribonucleotides encoding RNase L. These synthetic oligonucleotides will be used to screen murine recombinant cDNA and/or genomic DNA libraries for the gene encoding RNase L. Alternative methods for isolating the gene encoding RNase L will also be attempted. When the encoding RNase L has been obtained, its chromosomal location, intron-exon structure and sequence will be determined. Increased knowledge of RNase L and the gene encoding it will increase scientific understanding of enzymatic regulation and the regulation of gene expression by interferons. This knowledge may be of value to medical researchers developing new treatments for cancer, viral diseases or immune deficiency conditions.
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