In vitro transcription by isolated nuclei of Rhynchosciara americana salivary glands. Characteristics of incorporation and inhibition by alpha-amanitin.
A method for the isolation of polytene nuclei from salivary glands cells of the Diptera Rhynchosciara americana is described. The stage-specific morphological pattern of the chromosome is maintained during the isolation. The isolated nuclei show two distinct RNA polymerase activities, namely I and II, characterized on the basis of ionic requirements and alpha-amanitin sensitivity. Studies of the product under the incubation conditions show that the system allows the synthesis of high-molecular weight RNA, beside a low molecular weight peak which may comprise pre-4S and 5S RNAs. Autoradiographic studies carried out in the presence or absence of the toxin alpha-amanitin showed that micronucleoli contain products of RNA polymerase type I activity (ribosomal RNA) and that the DNA puffs are engaged in alpha-amanitin sensitive RNA synthesis and thus are sites of polymerase type II activity.
D000546:Amanitins / Q000494:pharmacology*; D000818:Animals; D001345:Autoradiography; D002458:Cell Fractionation / Q000379:methods; D002466:Cell Nucleolus / Q000201:enzymology; D002467:Cell Nucleus / Q000378:metabolism*; D012321:DNA-Directed RNA Polymerases / Q000032:analysis / Q000037:antagonists & inhibitors; D004175:Diptera / Q000166:cytology*; D009994:Osmolar Concentration; D012313:RNA / Q000096:biosynthesis; D012335:RNA, Ribosomal / Q000096:biosynthesis; D012469:Salivary Glands / Q000648:ultrastructure; D014158:Transcription, Genetic / Q000187:drug effects
D000546:Amanitins; D012335:RNA, Ribosomal; D012313:RNA; D012321:DNA-Directed RNA Polymerases