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Isolated nuclei, prepared from myeloblasts of chicks infected with avian myeloblastosis virus, synthesize RNA sequences present in avian myeloblastosis viral RNA. These sequences are also formed during transcription of chromatin, isolated from myeloblasts, by DNA-dependent RNA polymerases purified from Escherichia coli or calf thymus. In the latter case, transcription is a-amanitin sensitive. Formation of hybrids between RNA and avian myeloblastosis virus DNA probes has been monitored by the combined use of ribonucleases A, T1, and H, and ribonucleases specific for single strands.

We have isolated from vaccinia virus cores an enzyme, 5'-phosphate-polyribonucleotide kinase, that in the presence of ATP and Mg2+ catalyzes the conversion of 5'-phosphate and 5'-diphosphate termini of RNA to the 5'-triphosphate species. With the exception of dATP, other nucleoside triphosphates were inactive as hosphate donors; activity with dATP was 10%o of that observed with ATP The purified enzyme did not phosphorylate 5'-hydroxyl-or 5'-monophosphate-terminated polydeoxyribonucleotides, although a variety of 5'-monophosphateterminated RNA chains were active as phosphate acceptors. By using a coupled system of 5'-phosphatepolyribonucleotide kinase and guanylyltransferase in the presence of ATP, GTP, Mg2+, and S-adenosylmethionine, capping of 5'-P-,,5-PP-, and 5'-PPP-RNA was demonstrated; in the absence of 5'-phosphate-polyribonucleotide kinase only 5'-PPP-RNA was capped by guanylyltransferase. Vaccinia virus, a large DNA-containing virus that replicates in the cytoplasm of its host cells (1), contains a large number of enzymes, many of which are involved in the synthesis and processing of RNA (2). Virions made permeable in the presence of the four rNTPs synthesize 26S RNA that contains 5'-terminal cap structures and 3'-terminal poly(A). Current evidence suggests that this RNA is cleaved to yield 6-12S RNA products that are also capped and polyadenylylated (2).Previous studies showed that synthetic poly-and oligonucleotides were capped by purified guanylyltransferase, isolated from vaccinia virus cores, provided the 5'-ends of the RNA chains contained 5'-PPP termini (3, 4). RNA chains containing terminal 5'-OH, 5'-P (5), or 5'-PP (4) were not guanylylated.Thus, the viral guanylyltransferase provides a specific reagent for detecting small amounts of 5'-PPP-terminated RNA chains. Using this method, we observed that 5'-P-RNA was guanylylated after incubation with ATP, GTP, and crude fractions prepared from vaccinia virions. We have purified the enzyme activity responsible for the activation of 5' termini of RNA. This activity, 5'-phosphate-polyribonucleotide kinase (5'-P-PR kinase), catalyzes the ATP-dependent phosphorylation of 5'-P-RNA chains, yielding 5'-PP-and 5'-PPP-RNAs.MATERIALS AND METHODS Virus. Vaccinia virus (strain WR) was purified from infected HeLa cells by sedimentation through a sucrose cushion and two sucrose gradient sedimentations as described by Joklik (6).Enzyme Assay. Two methods were used interchangeably to measure 5'-P-PR kinase; the methods gave similar results. various amounts of 5'-P-PR kinase were incubated at 370 for 30 min. Reactions were halted by addition of 2 Mig of proteinase K in the presence of 0.1% sodium dodecyl sulfate; mixtures were incubated at 370 for 30 min, after which 0.2 ml of a solution containing bovine serum albumin (1 mg/ml) and denatured salmon sperm DNA (1 mg/ml) was added. The mixture was treated with 3 ml of 5% trichloroacetic acid; acid-insoluble material was collected by centrifugation, redissolved in 0.2 ml of a solution containing 0.1 M sodium pyr...

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RNA transcribed in vitro from myeloblast chromatin by exogenously added RNA polymerase B predominantly consists of short chains that remain in hybrid structure with the template; the remainder of the product is free RNA of heterogeneous size. Addition of polyanions during synthesis caused an increase in the size and amount of free RNA with a concomitant decrease in the proportion of small RNA. The large molecular weight RNA is derived from the short RNA chains, which are synthesized de novo during the reaction in vitro. The effect of polyanions on the size and nature of the product may be related to structural changes induced in the template rather than to an inhibition of nuclease activity.Cells infected with RNA tumor viruses contain virus-specific information in the form of DNA in their genome (1); transcription of this (proviral) DNA is thought to be an obligatory step in viral replication. Since progeny RNA is probably synthesized by host nucleoplasmic RNA polymerase (2, 3) and viral RNA in transcript products can be detected with complementary probe DNA, these cells offer a system for studying the expression of specific genes in eukaryotes. Recently, we reported the presence of virus-specific RNA in products synthesized in vitro from myeloblast chromatin by exogenously added eukaryotic RNA polymerase B (2). This chromatin was isolated from myeloblasts of chickens infected with avian myeloblastosis virus (AMV). The proportion of virus-specific sequences in transcript products (1-2%) was approximately the same as that in isolated infected cells (4) and that synthesized in isolated myeloblast nuclei (2). Since the amount of viral RNA in the product was 102-to 103-fold higher than expected from random transcription, it appeared that the RNA synthesizing system in vitro maintained some of the specificity exhibited during transcription in vivo. For

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