Rapid qualitative changes in mRNA populations in cultured human lymphocytes: comparison of the effects of cycloheximide and concanavalin A

Abstract

To examine the hypothesis that the stimulation of cultured lymphocytes by lectins involves the inactivation of a protein repressor of putative "activation genes," the effects of a protein synthesis inhibitor (cycloheximide) and a lectin (concanavalin A) were compared. Qualitative changes in mRNA populations were assessed by translating RNA prepared from cycloheximide- or lectin-treated cultures in a rabbit reticulocyte lysate. [35S]Methionine-labelled translation products were analysed by two-dimensional polyacrylamide gel electrophoresis. Cycloheximide increased the radioactive labelling of cultured lymphocytes with the RNA precursor [3H]uridine, as previously reported. This was observed during the first 3 h of culture; thereafter, cycloheximide was inhibitory. The period of increased labelling with [3H]uridine coincided with a period of great increase in mRNA corresponding to an acidic protein of a relative mass of approximately 55 000. This mRNA was not detected in RNA prepared from control cultures, but was one of the most abundant mRNA species detected in RNA prepared from cycloheximide-treated cultures. Increases in certain less abundant mRNA species were also noted. However, the mRNAs were not observed in RNA prepared from lectin-treated cultures. If an increase in these mRNAs is important for lymphocyte activation, then the increase must be to an extent not detected by our current methods.