A method for analyzing transcription using permeabilized cells.

Abstract

Brief exposure of Friend cells to a buffered hypotonic solution containing 1% Tween 80 caused permeabilization and allowed incorporation of [3H]UTP into RNA. The incorporation was inhibited 85-97% by 20 micrograms/ml actinomycin D and the reaction product was completely hydrolyzed by 0.1 M KOH. UMP incorporation was nearly linear for 60 min at 23 degrees C; however, at 37 degrees C it ceased after 15-20 min of rapid incorporation. The inhibition of UMP incorporation by 2 micrograms/ml alpha-amanitin was much greater at 23 degrees C than at 37 degrees C. The molecular weight of the RNA synthesized in permeabilized cells is broadly distributed with about 83% larger than 18 S. In vitro transcription of the mouse beta-major globin gene was studied by hybridizing 32P-labeled nascent RNA to filter-bound DNA sequences representing this gene and its flanking regions. After induction by hexamethylene-bisacetamide, Friend cells exhibited more than fivefold increases in the rate of transcription for the beta-major globin gene as compared to the uninduced control cells. Induction also caused an increase in the transcription rate of the 3'-flanking region located downstream from the poly(A) addition site. Thus, the primary transcription unit of beta-major globin gene is essentially the same in permeabilized cells as that previously reported for nuclei isolated from the same cell line. In addition, permeabilized cells actively initiate RNA synthesis as determined by the incorporation of a thiol group at the 5' initiating nucleotide, when synthesis was in the presence of [gamma-S]-labeled nucleoside triphosphates. Permeabilized cells are about 7-11 times more active than isolated nuclei in the synthesis of both in vitro-initiated and total RNA.