One of the mediators of interferon action is a latent endoribonuclease (ribonuclease L) that is activated by (2'-5')oligoadenylates. Among the homopolymers of the four common ribonucleotides, activated ribonuclease L degrades at an appreciable rate only polyuridylic acid. In two natural RNA's tested the most frequent ribonuclease L cleavages occur after UA, UG, and UU (A, adenine; U, uracil; and G, guanine) and much less frequent cleavages after CA and AC (C, cytosine).
Double-stranded RNA inhibits protein synthesis in at least two ways. It activates a protein kinase that blocks peptide chain initiation by phosphorylating the peptide chain initiation factor eIF-2 and also activates an endonuclease that inactivates different mRNAs at different rates. The protein kinase and the endonuclease have been partially purified from interferon-treated Ehrlich ascites tumor cells. The 2',5'-oligoadenylates [pppA(2'p5'A)n, found earlier to be mediators in the activation o[the endonuc ase by double-stranded RNA, are not mediators in the activation of the protein kinase by double-stranded RNA.
cDNAs encoding poly(ADP-ribose) polymerase from a human hepatoma lambda gt11 cDNA library were isolated by immunological screening. One insert of 1.3 kilobases (kb) consistently hybridized on RNA gel blots to an mRNA species of 3.6-3.7 kb, which is consistent with the size of RNA necessary to code for the polymerase protein (116 kDa). This insert was subsequently used in both in vitro hybrid selection and hybrid-arrested translation studies. An mRNA species from HeLa cells of 3.6-3.7 kb was selected that was translated into a 116-kDa protein, which was selectively immunoprecipitated with anti-poly (ADP-ribose) polymerase. To confirm that the 1.3-kb insert from lambda gt11 encodes for poly(ADP-ribose) polymerase, the insert was used to screen a 3- to 4-kb subset of a transformed human fibroblast cDNA library in the Okayama-Berg vector. One of these vectors [pcD-p(ADPR)P; 3.6 kb] was tested in transient transfection experiments in COS cells. This cDNA insert contained the complete coding sequence for polymerase as indicated by the following criteria: A 3-fold increase in in vitro activity was noted in extracts from transfected cells compared to mock or pSV2-CAT transfected cells. A 6-fold increase in polymerase activity in pcD-p(ADPR)P transfected cell extracts compared to controls was observed by "activity gel" analysis on gels of electrophoretically separated proteins at 116 kDa. A 10- to 15-fold increase in newly synthesized polymerase was detected by immunoprecipitation of labeled transfected cell extracts. Using pcD-p(ADPR)P as probe, it was observed that the level of poly(ADP-ribose) polymerase mRNA was elevated at 5 and 7 hr of S phase of the HeLa cell cycle, but was unaltered when artificial DNA strand breaks are introduced in HeLa cells by alkylating agents.
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