The isolation of thiol containing RNA in the study of RNA metabolism
The separation of newly-synthesised RNA on mercurated cellulose, after pulsing tissue culture cells with 4-thiouridine was investigated. It was hoped to apply this technique to the measurement of rates of synthesis and turnover of specific mRNA species. The Friend cell, where globin production is induced by treatment with dimethylsulphoxide (DMSO) was utilised. This inducible cell, clone 707, was characterised and the relative rates of synthesis of globin mRNA, after induction with DMSO and pulsing with [3H]adenosine, measured for ultimate comparison with measurements made using the physical isolation approach. Friend cells were incubated with various concentrations of 4-thiouridine and the effects upon binding of isolated RNA to mercurated cellulose and Friend cell viability monitored. Incubation with 200µM 4-thiouridine for 2 hrs allowed the isolation of 70% of the newly synthesised poly(A)+ RNA on mercurated cellulose although only 40% of the poly(A)- RNA would bind. Concentrations up to 200µM had little effect upon cell growth and RNA synthesis although concentrations over 400µM appeared to affect the appearance of poly(A)- RNA in the cytoplasm. Inconsistent binding of RNA to the organomercurial led to the investigation of 4-thiouridine uptake into Friend cells. Several conditions for mercurated cellulose chromatography were studied. The incorporation of 4-thiouridine into bacterial RNA was used to estimate the degree of 4-thiouridine substitution required for binding to mercurated cellulose. The 4-thiouridine content was measured by spectrophotometry.