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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The experimental conditions to study lymphocyte transformation in cultures derived from bovine lymph nodes are described. Both deoxyribonucleoprotein-bound and soluble RNA polymerase can conveniently be tested in cell lysates. During the course of transformation the activities of the two enzymes begin to rise a t the same time when the cells begin to accumulate stable RNA. At 30 h after addition of phytohemagglutinin both enzyme activities are enhanced by a factor of two over the controls.The transformation of small lymphocytes to large pyroninophilic cells under tho stimulus of phytohemagglutinin provides an excellent model system to study the conversion of resting cells to actively growing cells. The assumption that the synthesis of RNA may play a key role in this process has focused attention on the RNA metabolism in transforming lymphocytes [ 1 -81. A drastic increase in the incorporation of radioactive uridine into RNA soon after the addition of the stimulating agent phytoheniagglutinin indicated a rapid induction of RNA synthesis. The interpretation of these labelling experiments is difficult since the uptake of the labeled precursor into the intracellular nucleotide pool is increased considerably in lymphocytes treated with phytohemagglutinin [9,10]. Therefore it was decided to investigate the RNA synthesizing capacity of cell free systems derived from transforming lymphocytes. This approach avoids the discussion of "pool quesstions" and might give information as to what extent the RNA synthesizing apparatus of the cell is changed after stimulation.Different methods for studying RNA synthesis in cell free systems derived from animal cells have been described. Using bacterial RNA polymerase isolated chromatin has a template activity which changes with the physiological state of the cell [ll]. At low salt concentration isolated nuclei show RNA synthesizing activity which possibly reflects the activity of ribosomal operons within the nucleolus [12-151. The activity of cellular RNA polymerase bound to the chromatin can be tested at high salt concentration [16,17]. Finally, a soluble enzyme which uses exogeneous DNA as template for RNA synthesis can be extracted from the cells [is--211. The activities observed in these different assay systems probably reflect different aspects of tbe i n vivo conditions. Studies on the activity of chromatin-bound and of the soluble RNA polymerase will be described in this communication.I n order to perform such experiments large amounts of lymphocytes are needed. The limited supply of human blood used earlier [lo] prompted the investigation of other sources for lymphocytes. Bovine lymph node cells were found to respond to phytohemagglutinin in a similar way as human blood lymphocytes. These cells have been used throughout the studies. Culture conditions and the changes in leucine and uridine labelling as well as the net synthesis of DNA, RNA and protein after phytohemagglutinin stimulation will be described in the first section of this communication.Lymphocytes are particular...

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A large scale purification procedure of RNA polymerase from calf thymus is described. A factor stimulating synthesis of RNA by this enzyme has been extracted from the same tissue. Addition of the factor to the enzyme assay results in a 10-fold increased enzyme activity. This effect is only seen when native and not when denatured DNA is used as template. Several lines of evidence exclude trivial explanations of the stimulation effect and indicate a direct action of the factor on the enzyme. The active agent is probably a protein with a sedimentation constant of about 3 S.Studies on the synthesis of RNA by DNA-dependent RNA polymerase in vitro have contributed considerably to the understanding of the control of gene activity in bacterial and bacteriophage systems. Thus it has been found that the enzyme is able to discriminate different regions of phage DNA and that this specificity can be influenced by protein factors contained in the phage infected and in the uninfected cell [l-41. These data stress the importance of the properties of the RNA polymerase in the control of gene activity. An absolute prerequisite for experiments bearing on these questions has been the use of enzyme preparations of high purity. With systems prepared from cells of higher organisms all attempts to perform similar investigations have as yet proven unsatisfactory mainly due to the fact that the corresponding RNA polymerase can only be obtained in limited purity and quantities. If factors influencing the activity of this RNA polymerase are present in the cells, they can only be detected after the enzyme has been purified to such a degree that the putative factors are separated from the enzyme preparation. The response of the enzyme to cell extracts would then allow the detection of such substances.In this communication a method to purify RNA polymerase from calf thymus on a large scale is described, which yields enzyme preparations of sufficient purity to demonstrate that a factor is present in calf thymus extracts, which stimulates the activity of this enzyme approximately 10-fold. This stimulating factor has been considerably purified. Some data on its physical-chemical properties as well as changes in characteristics of the RNA polymerase reaction Unwml Abbreviation. Tris-glycerol buffer: 0.05 M Tria pH 7.8,2 mM MnC12, 15 mM mercaptoethanol, 30°/, glycerol. under the influence of the factor will be described. This work is based on observations described earlier [5]. MATERIALS AND METHODSXtandard Assay of RNA Polymerase RNA polymerase activity was determined by the incorporation of [14C]UTP into RNA. The standard assay contained in a total volume of 0.1 ml: 0.2 mM each of GTP, CTP, and ATP, 10 pg of calf thymus DNA, 0.03 M Tris pH 7.8, 1 mM MnC1, , 0.02 M KC1, 0.1 pC [14C]UTP (50 pC/pmole) and enzyme. After incubation at 37" the samples were chilled in an ice bath and 5 drops of saturated Na4Pz07, pH 7.8, and 100 pg of yeast RNA as carrier were added. Acid insoluble material was precipitated by addition of 5 ml of ice-cold 4 trichloroac...