Changes in RNA synthesis of endogenous “free” and template-engaged RNA polymerases in isolated nuclei of highly synchronized coleopteran embryos

Abstract

Nuclei were isolated from Bruchidius obtectus embryos at well-defined stages during blastoderm formation and gastrulation and their capacity for in vitro transcription was investigated. It is shown that RNA synthesis in vitro is DNA dependent and due to chain elongation, not to chain initiation. Furthermore, the ionic requirements of this system during the time of development are investigated. The absolute rate of transcription of nuclei is raised twofold during the late blastoderm stage. Concomitantly, the ratio of template-engaged RNA polymerases of classes I and II is altered; this is indicated by changes of α-amanitin sensitivity of RNA synthesis. After high sensitivity (70% inhibition) during early developmental stages up to the first late blastoderm stage, RNA synthesis becomes rather insensitive to α-amanitin (about 10% inhibition) during the latest blastoderm stage and at gastrulation. Besides the activities of template-engaged RNA polymerases, a pool of “free” RNA polymerases is found, presumably not engaged in RNA synthesis in vivo. This pool decreases from high values in the early blastoderm stage to low levels during middle and late blastoderm stages and rises again to some extent in gastrulation. These free RNA polymerases are totally α-amanitin resistant during early blastoderm, whereas at gastrulation about 90% of measured poly(AU) synthesis is inhibited by α-amanitin.