Figure 6.

Induction of inosines in thymic mRNA during acute systemic inflammation. (a) The first TLC assay. Inosine-containing mRNA was analysed in thymuses from endotoxin-stimulated mice (n = 4) at 0–24 hr. Total poly(A)⁺ RNA (tRNA and rRNA were not visible when monitored on denature agarose gels) from each time-point was isolated and a synthetic RNA was made by in vitro transcription using a plasmid with T7 promoter. Poly(A)⁺ RNA was digested with RNase T2, labelled with polynucleotide kinase and digested again with RNase P1. The inosine-labelled mRNA was significantly increased. pA, pG, pI, pC and pU are 5′-monophosphate nucleotides; I, the standard 5′-[³²P]monophosphate inosine. (b) The second TLC assay. After the first TLC, inosine and adenosine were recovered and analysed on a new TLC. In agreement with the first TLC, inosine-labelled mRNA was significantly increased. S, synthetic RNA. (c) Quantification of the increased level of inosine-containing mRNA. Per cent of inosine was calculated using radioactive counting or an NIH Image software. Results were plotted against the duration of stimulation. Note the increase from a baseline of approximately 0·5–5% at 24 hr. (d) Labelling efficiency of 3′-monophosphate nucleotides. PNK equally labelled all 3′-monophosphate nucleotides (pA, pG, pC, pU and pI).