Figure 4

cdl-1 mRNA knockdown by RNAi prevents histone synthesis but does not affect steady-state histone mRNA levels. N2 animals were either cdl-1 (RNAi) or control gfp(RNAi) treated and grown to adults as described, when either RNA was prepared or animals were lysed for protein analysis. Similarly, N2 animals were grown to adults and subjected to RNA isolation or lysis for protein analysis. (A) top. Summary of phenotypes observed. Pvl refers to protruding vulva. bottom. cdl-1 mRNA knockdown was confirmed by comparison of RNA levels in cdl-1(RNAi) animals to RNA levels untreated (N2) and in gfp(RNAi) N2 animals, by reverse transcription followed by 21–23 cycles of PCR. Separate amplification of actin mRNA was used as control. Control reactions where reverse transcription was omitted (- rt) were performed in parallel. RNA levels were quantitated as described in Methods. (B) Actin mRNA, Histone H3 and H2B mRNA, and 18S rRNA levels in untreated N2 animals, cdl-1(RNAi) or gfp(RNAi) animals were analysed by Northern blotting. RNAs were analysed on different part of the same blot. The blot was stripped between probings for histone mRNAs. mRNA levels were standardised with respect to 18S rRNA levels and compared to levels of RNA in N2 animals, which was defined as 100 %. The graph shows mean data from 2 independent experiments, with the error bar indicating the maximum value. (C) Levels of actin, histone H3 and histone H2B proteins in cdl-1(RNAi), gfp(RNAi) and N2 animals were determined by Western blotting. For the graph, H3 and H2B protein levels were standardised with respect to actin levels, and compared to levels in N2 animals which were defined as 100 %. The average and maximum value of two independent measurements are shown.