Quantitative analysis of mRNA synthesis during early cortisol action on rat thymocytes: Restricted size of a possible hormone response

The cytoplasmic accumulation of non-polyadenylated RNA in rat thymocytes is differentially changed at an early time of protein synthesis inhibition

“…The significance of this result was tested by conversion of the amount of radioactivity represented by 2 SD of the isotope ratios to number of RNA molecules per cell. In a previous study it was found, that about 41,000 molecules of tRNA and 6,200 molecules of other RNA species accumulate in the cytoplasm during 45 min (9). From the distribution of radioactivity shown in Figure 1, poly(A) § was then found to represent 2,400 molecules per cell, and deviations of the isotope ratios corresponding to + 2 SD could thus be calculated to represent between _ 2 and + 20 molecules per cell.…”

“…In view of the 41 hour half-life estimated for tRNA in rat thymocytes (9), as well as the very short delay between synthesis and migration to the cytoplasm, this effect is most probably explained as an increased production rate of tRNA. This observation confirms an earlier work, in which the effects of polysome stabilizing and destabilizing inhibitors were compared (22~ The stimulatory effect of emetin but not of p'actamycin adds to their list of inhibitors showing this correlation, and supports their hypothesis that tRNA synthesis may be regulated by a factor, whose effective concentration is determined by an equilibrium between a free and a polysome bound form.…”

“…Rat thymocytes were prepared and cultivated as previously described (9). Briefly, the cells were incubated at 1.5x 107/ml in medium 199 supplemented with 5 mM-TES and 5 mM-Hepes, 0.1 mg/ml streptomycin and 5% bovine serum albumin.…”

“…The RNA was precipitated and recollected as described above, and analyzed by electrophoresis on cylindrical 8 cm 2.5% polyacrylamide gels, as described by LOENING (15), using diethylpyrocarbonate (1 ml/l) instead of SDS in the electrophoresis buffer. After scanning at 260 rim, the gels were sliced and counted as previously described (9).…”

“…Deviations of the isotope ratio in tingle gel slices from the integrated mean ratio were measured in terms of standard deviations, denoted by SD, using a statistical test of significance, as previously described (9). For a series of isotope ratios, one-third would be expected to deviate 1 SD or more from the mean.…”