The Site of Ribonucleic Acid Synthesis in the Isolates Nucleus

Abstract

There is still controversy concerning the primary site of RNA synthesis in the cell.' When tissues are radioautographed after administration of radioactive inorganic phosphorus or radioactive RNA2 precursors such as tritiated uridine or cytidine, it is commonly observed that radioactivity appears in the nucleus first and in the cytoplasm later. It has furthermore been shown that isolated nuclei of both animal' and plant4 material are able to synthesize RNA in vitro but that enucleated cytoplasm is unable to carry on such synthesis., Although the problem of whether or not RNA is synthesized in both nucleus and cytoplasm is not completely understood,6-8 there is increasing cytological evidence that a substantial portion of the cytoplasmic RNA is synthesized in the nucleus9-'7 and subsequently migrates to the cytoplasm.The site of RNA synthesis in the nucleus, however, is variously proposed to be the nucleolus,'3 16, 18 the chromatin,'9-2' or both.22 23 It has been suggested that cytoplasmic RNA is derived from either the nucleolus'3 16, 18 or from the chromatin after passage through the nucleolus'9' 20 or from both.'3 The autoradiographic data heretofore obtained are inconclusive on these points. Autoradiographs of nuclei labeled with radioactive RNA precursors are only rigorously interpretable in the case of isolated nuclei or nucleoli because of the background otherwise created by radioactive cytoplasm or by chromatin closely associated with the nucleolus. In the present work the more direct approach, of the physical fractionation of previously labeled, isolated nuclei has been used. Large quantities of nuclei have. bten isolated, incubated under suitable conditions with labeled precursors of RNA, the subnuclear components then separated from one another, and the amounts of radioactivity contained in the RNA of chromatin, nucleoli, and other nuclear components estimated directly. It has in this way been possible to arrive at reasonably firm conclusions concerning the site of RNA synthesis in the nucleus.Materials and Methods.-Plant tissues: Pea seedlings, Pisum sativum, were grown in vermiculite in the dark for 4 days at 250C. From these seedlings, apical tips, 1.0-~l.0 cm in length, were cut, freed of vermiculite, and then washed with 0.5% clorox for 3 min. These stem sections were then used for isolation of nuclei.Chemicals: Cytidine-H3, specific activity: 4,900 mc per mM, used as RNA precursor and obtained from New England Nuclear Corporation, Boston, was added to the incubation medium in a final concentration of 25 Itc per ml. Phosphocreatine kinase was prepared from rabbit muscle according to the procedure described by Kuby et al.24 Penicillin powder, specific activity 1,000 units per mg was used as manufactured by Chas. Pfizer & Co., Inc., New York. Other chemicals were of reagent grade.Isolation and incubation of nuclei: Nuclei were isolated from pea seedlings by a new method which consists in passing the plant tissue through a set of counter-rotating rollers with simultaneous addition of a sucrose-CaCl2 ...

THE BASE COMPOSITION OF NUCLEIC ACIDS IN CHROMOSOMES, PUFFS, NUCLEOLI, AND CYTOPLASM OF CHIRONOMUS SALIVARY GLAND CELLS

“…The results of several investigations on different kinds of material, including giant chromosomes, indicate that the chromatin or chromosomes is the source of nucleolar RNA (14,8,22,20,18,16). In the present study, there is no indication of a common origin for any of the investigated samples of chromosomal RNA and nucleolar RNA.…”

THE BASE COMPOSITION OF RIBONUCLEIC ACID IN LAMPBRUSH CHROMOSOMES, NUCLEOLI, NUCLEAR SAP, AND CYTOPLASM OF TRITURUS OOCYTES

“…Incorporation experiments which demonstrate an early labeling of the RNA in the chromosomes provide the main experimental arguments for a local origin of chRNA (McMaster-Kaye and Taylor, 1958;Pelling, 1959;Rho and Bonner, 1961). The findings of Gall and Callan (1962) on uridine incorporation and RNA movement in one of the giant loops of the lampbrush chromosomes would be difficult to reconcile with a non-chromosomal origin of chRNA.…”

Plant Virus-Host Cell Relations

“…In spite of these uncertainties there seems to be little doubt that the onset of nonnucleolar, nuclear RNA-synthesis preceeds that of the nucleolus. Interestingly, pulse labelling experiments with isolated pea embryo nuclei indi cate that RNA synthesis on the chromatin part of the nucleus precedes that in the nucleolus (100).…”

“…The widths of the bars in each sequence are proportional to the values determined experimentally. Each bar represents an average of the data obtained from many (occasionally more than100) cells. Data represented by hatched bars are less reliable due to technical difficulties.-Below the wavy line: numbering begins with the cell adjacent to the inoculated one and proceeds towards the base of the hair.…”