In vitro RNA synthesis in oocyte nuclei of the newt Notophthalmus

Abstract

An incubation medium is described which supports RNA synthesis in isolated oocyte nuclei of the newt Notophthalmus, and which permits subsequent autoradiographic examination of the lampbrush chromosomes and nucleoli. By using different concentrations of alpha-amanitin we distinguish RNA synthesis due to RNA polymerases I, II and III. All RNA synthesis on loops is inhibited by 0.5 microgram/ml of alpha-amanitin and is therefore due to polymerase II. Polymerase III is responsible for RNA synthesis at a small number of discrete sites in condensed chromatin. These include the centromere bars of three of four chromosomes, which probably represent 5S RNA synthesis, as well as 15-20 lesser sites scattered elsewhere. Polymerase I activity is confined to the nucleoli.