KINETICS OF NUCLEOSIDE INCORPORATION INTO NUCLEAR AND CYTOPLASMIC RNA

Abstract

HeLa and conjunctiva tissue culture cells were incubated for various intervals with tritiated nucleosides and the incorporation into RNA was localized in different parts of the cell by means of autoradiography. In order to obtain quantitative measurements of incorporation from grain count data the influence of cell geometry on the absorption of the tritium [beta] ray was considered. Relative correction factor, E = g/g*, relating an idealized grain count in the absence of absorption g, to the actual grain count, g*, were derived for the different cell compartments. For the average HeLa cell the factors for the nucleolus, n, non-nucleolar parts of the nucleus, N, and the cytoplasm, C, are in the ratio En/EN/EC= 2.3/1.6/1.0. The kinetics of incorporation for cytidine and adenosine are similar. The n and N curves are characterized by a rapid rise and early saturation, whereas the C curves show an appreciable lag and no evidence of saturation for intervals as long as one generation time. Estimates of the relative amounts of RNA in each compartment were obtained from ultraviolet micro absorption measurements and used together with the kinetic data to calculate specific activities. For incubation periods of short duration the ratio of specific activities n/N for cytidine is approximately twice that for adenosine.. Three hypotheses for the mechanism of ribonucleoside incorporation and RNA synthesis are discussed, and arguments favoring a transport of RNA or an RNA by-product from the nucleus and nucleolus to the cytoplasm are presented.