The measurement of the production of during erythroid differentiation of the Friend erythroleukemia cell

Abstract

The production of [Formula: see text] during Friend cell erythroid differentiation has been studied. In vitro measurements of total nuclear RNA synthesis in nuclei isolated from Friend cells at different stages of differentiation show the total RNA synthesis increases 1.5-fold at day 1 of induction and then decreases through days 2 and 3 to approximately 75% of its rate of synthesis in the nuclei of uninduced cells. The synthesis of RNA polymerase III transcripts undergoes a similar fluctuation through day 2 of induction, but increases again at day 3. The specific synthesis of [Formula: see text] was measured by hybridization of labelled nuclear RNA to a [Formula: see text] gene probe. During erythroid differentiation the percentage of nuclear RNA represented by [Formula: see text] remains constant (0.065%), so that the absolute synthesis of [Formula: see text] fluctuates during differentiation, in parallel with the fluctuations in the synthesis of total nuclear RNA. The relative synthesis of [Formula: see text]in vivo was studied by labelling cells with 35Pi, isolating the resulting radioactive tRNA – 5S RNA population, and hybridizing this population to a [Formula: see text] gene probe. The ratio of [Formula: see text] in newly synthesized cytoplasmic RNA remains similar throughout differentiation (averaging 0.0171), implying that the fluctuations observed in the nuclear synthesis of [Formula: see text] during differentiation probably also occur for the nuclear synthesis of most tRNA and 5S RNA species. Attempts were made to measure the relative steady-state concentration of [Formula: see text] using both aminoacylation and in vitro end labelling of tRNA followed by hybridization to a [Formula: see text] gene probe. These two methods gave different results and we discuss the possible pitfalls of using enzymatic methods for quantitating tRNA concentrations in the cell.