We reported earlier that the addition of double-stranded RNA and ATP increases the endonuclease activity more in an extract of Ehrlich ascites tumor cells which have been treated with an interferon preparation than in a comparable extract from control cells. We repo here that the addition of double-stranded RNA to an extract from Ehrlich ascites tumor cells which have been treated with an interferon preparation [or with the interferon inducer poly(I).poly(C)J promotes the phosphorylation by Tris-CI at pH 7.6, 80 mM KCI, 4 mM MgCl2, and 6 mM 2-mercaptoethanol, the S30INT.s and S30c.s extracts were produced. S30 extracts from EAT cells treated with the interferon-inducer poly(I)-poly(C) (1) were prepared according to published procedures (11). The yield of vesicular stomatitis virus from infected cells in a single growth cycle was reduced by over 95% in cells treated with interferon, and by over 99% in those treated with poly(I)-poly(C). S30 extracts from EAT cells were

A region of human genomic DNA encompassing the 5' end of the thyroglobulin gene has been sequenced and the position of the transcriptional start site has been determined. The 5' non-translated portion of the mRNA displays a quasi-palindromic sequence which could allow this region to adopt a hairpin structure. The first exon of the gene encodes a 19 amino-acids signal peptide and the 3 first amino acids of the mature protein. Apart from the canonical TATA-Box and from a CAAT-Box homology, the promoter region contains a 209 bp-long poly(purine)-poly (pyrimidine) sequence located between positions-512 and -304 relative to the transcription start. When contained in a supercoiled plasmid, this sequence exhibits sensitivity to S1 nuclease at two distinct positions. A precise mapping of the borders of the sensitive regions was achieved by extending primers from both ends of the sequence after digestion by the enzyme. The resulting data can be explained by a model involving the formation of a triple helix structure.

No abstract

Extracts from interferon-treated Ehrlich ascites tumor cells differ in various biochemical characteristics from extracts from control cells. Thus, as reported earlier, double-stranded RNA (dsRNA) promotes the phosphorylation by ATP of at least two proteins in extracts from interferon-treated cells,but not,or to only a lesser extent, in extracts from control cells. Moreover reovirus mRNAs are degraded faster in reaction mixtures containing extracts from interferon-treated cells than in those containing extracts from control cells but only if the reaction mixtures are supplemented with dsRNA and ATP. The faster RNA degradation in extracts from interferon-treated cells is due to enhanced endonuclease action. We designated the agent(s) catalyzing this as endonuclease1NT. There is some enhancement of nuclease activity by dsRNA and ATP also in extracts from control cells. The extent of this enhancement is, however, much smaller than in extracts from interferon-treated cells. We report now that the promotion of mRNA cleavage in extracts from interferon-treated cells by dsRNA and ATP is apparently not a consequence of a possible impairment in the attachment of the mRNA to ribosomes since (a) the protein synthesis inhibitors (sparsomycin and edeine) do not affect the degradation and (b) dsRNA and ATP enhance RNA degradation also in the 200000 x g supernatant fraction from extracts of interferon-treated cells and this fraction is essentially free of ribosomes.(The ribosome concentration in our 200000 x g supernatant fraction is less than 1 % of that in the 30000 x g extract.) GTP or the ATP analogs AMP(CH2)PP or AMP-P(CH2)P do not substitute for ATP and dsDNA or DNA . RNA hybrids do not substitute for dsRNA in activating endonucleaseINT.The optimal concentration of ATP in activating endonucleaseINT is 1 mM or higher. The addition of dsRNA and ATP to extracts from interferon-treated cells affects the degradation of some RNAs much more than that of others. Thus it promotes the degradation of reovirus mRNA from the large size class strongly, the degradation of those from the medium size class to an intermediate extent and that of those from the small size class the least. Furthermore it enhances the degradation of mRNA from either control or interferon-treated Ehrlich ascites cells more than that of Ehrlich ascites cell ribosomal RNA or mouse globin mRNA. Reovirus dsRNA is not cleaved by endonucleaselNT. The partially purified mouse interferon preparation (specific activity 8 x lo8 NIH reference standard units/mg protein) used in several experiments was free of nuclease activity under our experimental conditions. Moreover the addition to extracts from control cells (or extracts from interferon-treated cells) of this interferon preparation together with dsRNA and ATP did not enhance nuclease activity.

Elongation factor G (EF G), bound to ribosomes either with GMPPCP or with fusidic acid and GDP, inhibits elongation factor Tu (EF Tu)-dependent binding of Phe-tRNA on the ribosome-poly(U) complex and binding of Ala-tRNA on the initiation complex formed with RNA from bacteriophage R17; GTP hydrolysis associated with Phe-tRNA binding is also inhibited. Moreover, nonenzymic binding of Phe-tRNA at high Mg++ concentration is completely blocked by EF G. Thus, EF G appears to bind at a site that overlaps or interacts with the ribosomal A-site.