Cytoplasmic zone analysis. RNA flow studied by micromanipulation

A technique is described, cytoplasmic zone analysis, by which it is possible to study the flow of different RNA-containing components in the cytoplasm after their release from the nucleus. After a pulse of RNA precursors, the salivary glands of the insect Chironomus tentans are fixed and microdissected for the isolation of three zones of cytoplasm situated at increasing distances from the nucleus. The RNA from each zone is isolated and analyzed by gel electrophoresis. The three ribosomal RNA components, 18 S, 28 S and 5 S RNA, appear in steep, specific radioactivity gradients (exit gradients) during the time interval 2-3 h after a precursor injection, the 18 S RNA gradient lying 30-50 mum peripheral to that of the 28 S or 5 S RNA gradient. Administration of puromycin led to the complete disappearance of the 28 S RNA and most of the 28 S RNA gradient within 45 min, suggesting that the gradients are caused by an engagement of the ribosomal subunits into polysomes close to the nucleus immediately or soon after their exit from the nucleus. The gradients may offer a unique model for the study of polysome formation and maintenance in vivo.