Incorporation of 13C,15N-labeled nucleosides and measurement of RNA synthesis and turnover in sea urchin embryos

The uptake of nucleosides into sea urchin embryos and their subsequent incorporation into RNA increases with increasing external nucleoside concentration. When embryos are incubated with high concentrations of ¹³C,¹⁵N-labeled nucleosides, newly synthesized RNA becomes sufficiently labeled with heavy isotope to be separated from unlabeled RNA on cesium formate equilibrium gradients. High concentrations of nucleosides do not affect development of embryos or rates of RNA synthesis. The extent of density-labeling of precursor pools increases with incubation time, and only levels off after many hours. During incubations with high concentrations of nucleosides, ATP pools expand up to twofold. Using density-labeling to circumvent precursor pool measurements, a method is presented to study the synthesis and decay of pulse-labeled RNA. The instantaneous rate of synthesis of total RNA at the blastula stage is 9.3 × 10^?15 mol of total nucleotide/embryo per minute and the average half-life of total RNA is 23 minutes.