The physical state of newly synthesized RNA.

We have earlier reported the enzymatic synthesis of RNA from the four riboside triphosphates by a DNA-containing nucleoprotein of pea embryos.I Since the nucleoprotein contains the bulk of the tissue DNA and since it resembles in composition and properties the chromatin obtained directly from the nuclei of pea embryos,2 we refer to the synthetically active material as chromatin. The RNA newly formed by our system is not liberated as free polynucleotide chains but remains associated with the DNA-containing nucleoprotein from which it may be liberated by heat. The present paper is concerned with the nature of this complex. Materials and Methods.-Preparation of chromatin: Enzymatically active chromatin (DNAcontaining nucleoprotein) was obtained from the embryonic axes of germinating pea seeds. Pea seeds, variety Alaska, were germinated for ca. 46 hr, at ca. 20'C. The cotyledons were then removed from the growing embryonic axes, which were immediately chilled, sterilized with 100 X diluted Clorox, and ground for 1 min in a Waring Blendor with 1.5 volumes of homogenizing medium (0.25 M sucrose, 0.001 M MgCl2, and 0.05 M tris buffer, pH 8.0). The homogenate was filtered twice through miracloth and the resultant filtrate centrifuged at 4,000 X g for 30 min. The chromatin of the pellet was scraped from the underlying hard-packed starch, resuspended in grinding medium, and washed twice by centrifugation at 10,000 X g for 10 min. This was followed by two further washings in 0.25 M sucrose and one in 0.05 M tris buffer, pH 8. The final pellet was suspended in 0.05 M tris buffer, pH 8.0, and, frequently, dialyzed against the same buffer for 16 hr. Each kilogram fresh weight of tissue treated in this way yields 400 to 500 milligrams of nucleoprotein, composed of protein, DNA, and RNA, in the ratios of approximately 10:1:0.25. Determination of RNA, DNA, and protein: Quantitative determination of RNA was generally according to Ts'o and Sato.3 From the alkaline hydrolysate (0.3 N KOH, 18 hr, 370C), the DNA was precipitated with perchloric acid (0.5 N). RNA was determined upon the perchloric-soluble supernatant by optical density and checked by the orcinol method of Dische and Schwartz.4 DNA was determined by the diphenylamine method of Burton5 or by optical density of the DNA hydrolysate (0.5 N perchloric acid, 90'C, 10 min). Preparation of nucleoprotein complex containing freshly synthesized C14-labeled RNA: For the preparation of material containing freshly synthesized RNA, 1 ml of enzyme prepared as above and containing approximately 1 mg DNA was incubated for 10 min in an incubation mixture containing tris buffer, pH 8, 200 micromoles; 'MgCl2, 20 micromoles; cysteine, 20 micromoles; GTP, CTP, and UTP, each 2 micromoles; ATP (8-C"4), 3 micromoles (4 microcuries), in a total volume of 2 ml. In each preparation, 20 to 40 incubation mixtures such as that described above were separately incubated and combined at the end of the incubation period. It was found unnecessary to include sodium fluoride in the incubation mixture, since the present preparation has negligible ATPase activity. At the end of the incubation period, the reaction mixture was immediately cooled to 00 and centrifuged for 30 min at 12,000 X g, and the pellet was resuspended in standard saline citrate (NaCl 0.15 M, Na citrate 0.015 M) and dialyzed for 48 hr against continued changes of external solution to remove the bulk of the unincorporated labeled ATP. Standard assay for amount of label contained in RNA: The amount of C14 label contained in RNA was determined by a standard procedure as follows. To the reaction mixture, or aliquot, was added 5 ml cold 5% TCA per ml reaction mixture. The acid-insoluble precipitate was washed once with cold 5% TCA and once with ethanol ether (3: 1) and was subsequently extracted twice with 5 ml portions of 10% NaCl, at 100°C, for 30 min. At each extraction, 4 mg of carrier RNA was added. The combined NaCl extracts were then equilibrated with 2 mg of unlabeled ATP for 30 min at room temperature. The RNA was next precipitated by addition of absolute alcohol, final concentration 70%, and the precipitate washed twice with cold 5% TCA. The