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Erythroid cells constitute a model particularly suitable for study of the relationship between cell maturation and nucleic acid and protein synthesis. Circulating reticulocytes, nonnucleated cells capable of prolonged protein synthesis, are derived from cells in the bone marrow that proceed from an apparently multipotential nucleated stem cell through various stages of increasingly mature or differentiated cell types (2-5). Protein synthesis in reticulocytes proceeds on ribosomes (6, 7), in particular those ribosomes characterized by sedimentation coefficients in excess of 110 S, referred to as polyribosomes (8-10). In addition to ribosomal RNA, transfer RNA and, presumably, "messenger" RNA are required for protein synthesis in reticulocytes. It vitro studies have shown that reticulocytes cannot synthesize RNA (8). The present investigations were designed to determine when, in the course of in vivo maturation of erythroid cells, the RNA of reticulocytes is synthesized.In these experiments, the synthesis of RNA in rabbit erythroid cells in vivo was evaluated by following the time course of incorporation of administered PS2 into cellular RNA. The findings indicate that RNA of reticulocytes is synthesized in a relatively early precursor cell stage of erythroid cell maturation. RNA, once synthesized, can remain functional through the reticulocyte stage. Our study also provides evidence that nucleated leukocytes, in contrast to reticulocytes, can synthesize RNA.*Submitted for publication January 3, 1964; accepted February 17, 1964. This work was supported in part by grants CY 2332 and GM 07368 from the U. S. Public Health Service and by grant NSF GB 310 from the National Science Foundation.A portion of these data was presented at the Annual Meeting of the American Federation for Clinical Investigation, Atlantic City, N. J., May 1963, and appeared in abstract form (1). MethodsInduction of reticulocytosis in rabbits. Two-to 3-kg rabbits were made anemic by subcutaneous injection of 25 mg phenylhydrazine per day for 4 days (11). This regularly resulted in a reticulocytosis of 60 to 90%.In vivo labeling of RNA with P". Twenty-four hours after the last injection of phenylhydrazine, 3 mc of carrier-free P's (approximately 3 mc per ml) was injected intravenously. Samples of blood were then obtained at intervals of from 30 minutes to 17 days. A total of 14 rabbits was studied. In 10 animals, a single sample of blood was obtained at the times indicated below. In 3 experiments, blood was obtained from the same animal at two different time intervals, and in 1 experiment, at three different time intervals. Reticulocyte ribosomal RNA was purified and analyzed for each of these blood samples. The RNA of cells remaining unlysed after "shock" lysis was analyzed for 7 of the blood samples. These samples were drawn at 30 minutes and 1 (twice), 3,20,22, and 42 hours.Preparation of blood samples. Blood was obtained by cardiac puncture and collected into heparinized syringes.A sample was removed for enumeration of the erythrocytes, le...

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Miany lines of evidence indicate that all specialized cells contain the total genetic information characteristic of the organism, but the mechanisms involved in the selective expression of this total information are unknown. Stedman and Stedman' suggested that the histones, the basic proteins of the nucleus, could serve as the controlling mechanism. Most cells of an organism, however, have the same histones,2' 3 and since the histones of individual organs such as liver and brain do not change appreciably during developments or during carcinogenesis,5' 6 it seems unlikely that the histones alone possess sufficient specificity for the control of gene function.Sypherd and Strauss7 suggested that an associated RNA species complementary to particular sequences of DNA could provide a mechanism for the histones to act as repressors by conferring additional specificity to these proteins. Huang and Bonner8 have recently described an RNA-histone complex in the pea seedling. We have concurrently carried out experiments on the nucleoprotein fraction isolated from normal rat liver nuclei and have found an RNA species with a high adenine and uridine content which is intimately associated with the nucleoprotein fraction and is resistant to ribonuclease. After extraction from the basic protein with phenol and detergent, this RNA species is RNase-sensitive and is heterogeneous by sucrose density gradient analysis.Materials and Methods.-Male Wistar rats weighing 100-120 gm from the Columbia University Colony were used in these experiments. They were fed rat pellets (A. E. Staley Manufacturing Co., Rockland Ill.) ad libitum.Labeled nucleoprotein: In the experiments to be reported, either the P3I-labeled RNA-nucleoprotein complex or the nucleoprotein labeled with C'4-lysine was used. For each preparation 2-5 animals were injected intraperitoneally with neutral isotonic solutions containing either 2 me of carrier-free p32 (Radiochemical Centre, Amersham, England) or 50 Muc lysine-UL-C4 (Nuclear-Chicago Corp., Des Plaines, Ill.) per 100-gm rat. The animals were allowed free access to food and water at all times. Three hours following p32 administration-snd 4 hr after the injection of, lysine, liver nuclei were prepared and purified by a modification of the method of Maggio et al.9With a Potter-Elvehjem homogenizer 9-12 gm of liver were homogenized in 3 vol of ice-cold buffer, pH 7.5, which consisted of 5 X 10-3 M Tris, 0.25 M sucrose, 5 mM MgCl2, 1 mM CaC12, and 30 mM KCl. The homogenate was filtered through cheesecloth and centrifuged at 800 X g at 00 for 10 min. The crude nuclear pellet was washed two times with buffer and was repelleted after each wash at 800 X g for 10 min. After washing, the pellet was resuspended in a small volume of similar buffer containing 2 M sucrose and then layered over additional hypertonic buffer in a centrifuge tube. The contents of the upper third of the tube were stirred to distribute the nuclear 'suspension, which was then centrifuged for 1 hr in an SW 25:1 head at 25,000 rpm in a model L2 ultracentr...