The Cellular Sites of Synthesis of Ribosomal and 4s Rna.

In previous publications,'-4 we presented evidence which indicates that the major portion of cytoplasmic RNA (C-RNA) is derived from RNA which is synthesized in the nucleolar region of the nucleus (n-RNA). Furthermore, it was proposed that n-RNA and the RNA of the extranucleolar parts of the nucleus (N-RNA) are synthesized independently of each other. These studies, carried out on the level of the individual cell, did not distinguish between the different molecular species of RNA such as ribosomal, amino acid-accepting (transfer), and messenger RNA. A general relationship, such as C-RNA is mostly ribosomal RNA, can readily be inferred but there is as yet no description of RNA synthesis which adequately comprehends both the cytological and molecular terms. Some progress along these lines has been made by Georgiev and collaborators.5 6 These investigators used different phenol extraction procedures to separate partially the various classes of RNA and deduced from an analysis of incorporation kinetics that an RNA, from what they term "the chromosomal-nucleolar apparatus," not extractable by phenol at pH 6.0-0.14 M NaCl, is a precursor of "high polymeric" cytoplasmic RNA. Other nuclear fractions were described, one of which is "low polymeric" and capable of an in vivo incorporation of P32 which is 20 times faster than a similar "low polymeric" cytoplasmic fraction. Unfortunately, no clear distinction is made between nucleolar and chromosomal RNA. An approach to this problem which allows direct comparison with autoradiographic studies was suggested when it was found that actinomycin D, when used at sufficiently low concentrations, greatly suppresses the incorporation of nucleosides into nand C-RNA of growing tissue culture cells with little or no depression of the rate of incorporation into N-RNA.7 Using this agent, together with a radioactively labeled RNA precursor, one may obtain relatively large quantities of cells in which the intracellular pattern of RNA synthesis is altered in a well-defined way. From such cells, one can extract and analyze the newly formed RNA and then correlate the alterations in the different molecular species of RNA with the changes observed in the various cytological classes. One of the advantages of this approach is that it allows a comparison between autoradiographic and biochemical data without risking the pitfalls of cellular fractionation (cf. ref. 8). In addition, it allows conclusions concerning precursor product relationships among the RNA fractions which are stronger than deductions made solely on the basis of kinetic data. Materials and Methods-General: Exponentially growing I cell fibroblasts, of a strain originally obtained from A. F. Graham, were cultured in Eagle's basal medium with 10 per cent calf serum on Leighton Tube coverslips for autoradiography and in 400 and 800 ml spinner cultures for RNA extraction. Actinomycin D (Merck-56R4094) was used at a concentration of 0.04 /Ag/ml (3.3 X 10-8 M). H3-cytidine (1.84 C./mmole) was used at 0.2 ,c/ml for the coverslip cultures and 0.15 4lc/ml for the spinner cultures. Chase incubations were done in media containing unlabeled cytidine at a concentration of 0.1 mg/ml. Autoradiography: Details of the autoradiographic technique have been described in a previous publication.2 However, the following important points should be mentioned. (1) After fixation in Carnoy's and before application of the radioautographic emulsion, the cells were treated