An electron microscopic method for localization of ribosomal proteins during transcription of ribosomal DNA: a method for studying protein assembly.

We describe a method for the localization of ribosomal proteins on electron microscopic spreads of active rRNA genes. The method consists of raising antibodies against Drosophila melanogaster proteins and allowing these antibodies to react with lysates of D. melanogaster egg chambers. The locations of the bound IgGs in the active transcripts are detected with goat anti-rabbit IgGs that have been labeled with electron-dense polymethacrylate spheres. By statistical analysis we can generate a confidence interval for the initial point of protein assembly for a particular protein. The first point of protein assembly for S14 is located near the 5' end of the pre-18S rRNA. In contrast, the first point of protein assembly for L4 is at 0.38 unit from the initiation point (a unit being the length of a ribosomal transcription unit). The binding patterns of S14 and L4 are consistent with the 5' proximal and the 5' distal orientations of the pre-18S and the pre-28S rRNAs. The method described here provides an approach to the elucidation of the assembly of eukaryotic ribosomal proteins in vivo.