An enzyme present in extracts from calf thymus degrades specifically the RNA moiety of DNA-RNA hybrids. Other nucleic acids, such as single- or double-stranded DNA and single- or double-stranded RNA, are not affected to a comparable degree. If prepared free of the hybrid-degrading enzyme, RNA polymerase from calf thymus shows a fivefold increase in activity on denatured DNA as compared to native DNA.

Extracts from calf thymus contain an enzyme degrading the RNA moiety of DNA-RNA hybrids. An assay for this enzyme and several characteristics of the enzyme reaction are described. The specificity of the enzyme for DNA-RNA hybrids is indicated by several lines of evidence : The RNA of the hybrid is rendered resistant to the action of this enzyme upon heating. Nucleic acids other than DNA-RNA hybrids are not affected to a comparable degree. The enzyme rapidly degrades polyribouridylic acid if it is hybridized to polydeoxyriboadenylic acid, but not if it is hybridized to polyriboadenylic acid. Experiments with double labelledDNA-RNA hybrids show that specifically the RNA moiety of the hybrid and not the DNA is attacked by the enzyme.I n experiments on the purification of RNA polymerase from calf thymus we observed that RNA synthesis on denatured DNA is possible only when highly purified enzyme preparations are used, whereas crude extracts are inactive in this respect. Since both preparations are effectively synthesizing RNA on native DNA, this result indicated that a factor is present in the crude extract which specifically interferes with RNA synthesis on denatured DNA. Further experiments showed that the crude extracts contained an enzyme which rapidly degrades the RNA synthesized on denatured DNA but leaves the product of the polymerase reaction on native DNA virually unaffected. Since it is known that the RNA synthesized by RNA polymerase on denatured DNA forms a hybrid with the template [I-31, the above observations indicated the presence of an enzyme which specifically degrades RNA if the latter is hybridized to DNA. This specificity was confirmed in different experiments described in this communication. An enzyme with these properties has, to the best of our knowledge, not been described before. For this reason we propose the name ribonuclease H (H standing for hybrid) for this enzyme. A preliminary report of these observations has already been published [4]. these preparations was purified from calf thymus by a method described in the accompa.nying paper [5]. The purification procedure included extraction of the enzyme at low salt concentration ; precipitation of deoxyribonucleoprotein by 0.1 M (NH,),SO,; precipitation of the enzyme by (NH,),SO, and chromatography on a DEAE-cellulose column. All characteristics of the purified enzyme were similar to those described for other preparations from bacterial or mammalian sources. For some experiments DNA-RNA hybrids were prepared using RNA polymerase from Escherichia coli. Their behaviour was in all respect identical to that of hybrids obtained with calf thymus RNA polymerase. MATERIALS AND METHODS SubstratesFor the synthesis of hybrid I00 pg of calf thymus DNA were denatured by heating to 100" for I0 min followed by rapid cooling. The DNA was incubated together with 2 pmoles of ATP, GTP, and CTP, I pC [14C]UTP (56 pC/pmole) and 250 pg of RNA polymerase in 10 ml of 2 mM MnCl,, 0.1 M (NH4),S04, 0.03 M Tris pH 7.8, 5 mM mercaptoethanol. After 30 min at 37" sodium d...

Conversion of uridine to UTP is enhanced in lymphocytes under the influence of phytohemagglutinin concomitant with the induction of uridine kinase. The uridine kinase activity in the induced cells decreases if the cells are treated with antibodies against phytohemagglutinin. The phosphorylation of uridine may be the limiting step in the incorporation of uridine into RNA. Uridine incorporation can thus not be taken as a direct measure of RNA synthesis.

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