Measurement of ribonucleotide pool specific activities by an in vivo method: comparison with an in vitro method

Abstract

A biological assay is described for the specific activity of the nucleotides incorporated into nuclear RNA. Under labeling conditions in which the pool specific activity reaches steady state by a process described by a continuous function, this method allows the determination of pool specific activity at all times of labeling. In Drosophila cultured cells incubated with [3H]uridine, the incorporation of radioactivity into tRNA and the absolute rate of tRNA synthesis were used to obtain an absolute value for the average specific activities of the UTP and CTP precursor pools. The average specific activity of the UTP and CTP pool obtained by this in vivo method was 2- to 3-fold higher than the specific activity of the whole-cell UTP and CTP pools as assayed by an in vitro method. The ribonucleotide pools in Drosophila cultured cells probably are compartmentalized. Use of the in vivo assay of ribonucleotide pool specific activities described should avoid underestimates or overestimates which may arise when absolute rates of RNA synthesis are calculated from measurements of whole-cell ribonucleotide pool specific activities. The rate of labeling of tRNA was used to determine the rate at which the combined UTP and CTP pools equilibrate. This rate was then used to determine that the rate of processing of pre-tRNA to mature tRNA is 0.21 min-1; this corresponds to a half-life of 3.3 min.