2-5A is a series of oligonucleotide effectors that regulates the breakdown of RNA in cells through the activation of 2-5A-dependent RNase (subsequently the nuclease). Clones of cDNAs for the nuclease will be isolated from a cDNA library by an immunological screening method and then identified by hybridization selection of RNA and translation in vitro. The cDNAs will be sequenced to determine if they contain the entire coding sequence of the nuclease; if they do not, such clones may be obtained either by rescreening the original library or by preparing new cDNA using mRNA selected by hybridization to the partial cDNA clones. The full-length cDNA will be inserted in both orientations into eukaryotic expression vectors which contain the dominant selectable marker, neo. Transforming cells with the cDNA in its correct orientation will result in elevated levels of the nuclease; whereas reverse orientation cDNA will produce antisense mRNA that should result in decreased levels of the nuclease. Different vector systems will be used that will place the nuclease cDNA under the control of either a constitutive (retroviral LTR) or an inducible (metallothionein) promoter. The effects of the transfected nuclease cDNA will be studied in murine L cells with regard to: cell viability and growth rate, levels of the nuclease and stability of rRNA, and sensitivity to the antiviral and cell growth inhibitory activities of interferon. The murine neuroblastoma cell line, N1E 115, and the embryonal carcinoma cell line, PC 13 clone 5, both lack detectable levels of the nuclease and are resistant to interferon action. It will be determined whether introduction of the nuclease cDNA (in correct orientation) will result in detectable levels of the nuclease in these cells; and, if so, whether the cells will: (1) become responsive to interferon and (2) undergo differentiation. The cDNA clones will be used to select the nuclease gene(s) from mouse and human genomic libraries. The nuclease gene(s) will be sequenced, and the number of genes per genome and the chromosomal locations of the gene(s) will be established. The levels and rates of synthesis of the nuclease mRNA will be determined prior to and during interferon treatment, cell growth arrest, and cell differentiation. Finally, we will attempt to regulate cell growth rates, oncogene expression, and the neoplastic phenotype by manipulating levels of the nuclease in intact cells.
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