title: Synthesis and processing of RNA by isolated human placental nuclei.
abstract: Conditions for isolating intact and active nuclei from human term pacenta and for studying their transcription products are described. The isolated nuclei can synthesize cell-free RNA for a prolonged period at 29 degree C in a medium containing 100 mM KCl and 5 mM MgCl2. Actinomycin D inhibited 92 per cent of RNA synthesis, whereas approximately 60 per cent of RNA synthesis was sensitive to alpha-amanitin. When nuclei were incubated at 29 degrees C for 1 h, about 27 per cent of the newly synthesized RNA was released into the medium outside the nucleus. Analysis of this released material by affinity chromatography on an oligo(dT)-cellulose column revealed that 2.4 per cent of the total released RNA was adsorbed at high salt concentration. Most of this fraction was eluted with a low-salt buffer at 45 degrees C and the remainder by 50 per cent formamide, conditions that are necessary for elution of poly(A)-containing mRNP particles from oligo(dT)-cellulose. These results show that placental nuclei incubated in vitro synthesize poly(A)-containing RNA, which is released as a protein-bound complex. This procedure allows exploration of changes in mRNA release during placental development.
mesh_terms: D002458:Cell Fractionation; D002467:Cell Nucleus / Q000502:physiology*; D004247:DNA / Q000032:analysis; D012321:DNA-Directed RNA Polymerases / Q000037:antagonists & inhibitors; D005260:Female; D006801:Humans; D010920:Placenta / Q000166:cytology / Q000502:physiology*; D011247:Pregnancy; D012313:RNA / Q000032:analysis / Q000096:biosynthesis*; D012333:RNA, Messenger / Q000096:biosynthesis; D013696:Temperature; D014544:Uridine Triphosphate / Q000378:metabolism
chemical_list: D012333:RNA, Messenger; D012313:RNA; D004247:DNA; D012321:DNA-Directed RNA Polymerases; D014544:Uridine Triphosphate