No abstract

No abstract

No abstract

Poly(A)-containing messenger RNA was isolated from polysomes of Ehrlich ascites tumor cells, and analyzed for sequence complexity by hybridization to its complementary DNA. The results indicate the presence of about 27000 diverse mRNA species in mouse Ehrlich ascites tumor cells. Total nuclear RNA was also hybridized to cDNA transcribed from ,polysomal poly(A)-containing mRNA up to an rot of 3000 M . s. It was found that all classes of the polysomal poly(A)-containing mRNA sequences were also present in the nucleus, although the distribution varied. About 2 % of the total nuclear RNA sequences were expressed as total polysomal poly(A)-containing mRNA. We also report that the total percentage of the haploid mouse genome transcribed in Ehrlich cells is significantly higher than that found in other mouse cells previously examined for poly(A)-containing mRNA sequence complexity. [6] and 10000 for rat liver [7].In this communication we report the kinetic complexity of Ehrlich ascites tumor cell polysomal poly(A)-containing mRNA, and its relative abundance in total nuclear RNA by hybridization studies. The experiments reported show (a) the approximate number of genes transcribed in Ehrlich cells and (b) the proportion of the mouse haploid genome that is transcribed into poly(A)-containing mRNA, which is assumed to be responsible for synthesis of functioning RNA, and appears to be of a higher level than that found in normal cells. EXPERIMENTAL PROCEDURE Isolation of Polysomal Poly(A)-Containing m R N ATo maximize the yield of RNA, glassware and plasticware were rinsed with 0.1 % diethylpyrocarbonate and autoclaved or incubated at 60°C for 30 min to destroy the reagent; all solutions were treated with 0.1 % diethylpyrocarbonate and then autoclaved. Operations were carried out at 4 "C unless otherwise indicated. Biochemicals were purchased from Calbiochem-Behring unless otherwise indicated.Ehrlich ascites tumor cells propagated in Swiss Webster mice were harvested, washed in deionized water, and lysed in 0.2 % Triton X-100. The nuclei and mitochondria were pelleted by centrifugation at 20000 x g for 15 min. The post-mitochondria1 supernatant was made 0.1 M in MgClz [8], and the mixture was left in the cold for 1 h. The precipitated polysomes were collected by centrifugation at 20000 x g for 20 min and dissolved in 10 mM Tris-HC1, pH 7.5, containing 0.5 M NaCl and 0.5% sodium dodecylsulfate, the running buffer for oligo(dT)-cellulose chromatography. The polysome solution, which had an absorbance of 2.5 at 260 nm, was loaded on a sterile oligo(dT)-cellulose (PL-Biochemicals) and cellulose type 38 (1: 1) column with 0.5 ml Chelex 100 at the bottom. The column was washed with running buffer until the eluant had zero absorbance at 260 nm. The column was then washed with two additional column volumes of running buffer. Poly(A)-containing RNA was eluted with 10 mM Tris-HC1, pH 7.5, containing 0.05 % sodium dodecylsulfate. The eluant was made 1 % in sodium dodecylsulfate, 5 M in CsC1, and then centrifuged at 5000 rev./min at 2...

Novikoff hepatoma cells, grown in monolayer cultures, when permeabilized by treatment with lysolecithin, incorporated 3H-UTP at a high rate for 2 hours at 25 degrees. The incorporation was inhibited by initiation inhibitors such as rifampicin AF/013, heparin and aurintricarboxylic acid. About 75% of RNA polymerase II, and 45% of RNA polymerase I activities were inhibited by rifampicin AF/013. In contrast, transcription in isolated nuclei was not inhibited either by rifampicin AF/013 or heparin. The permeabilized cells apparently retain the mechanisms for reinitiation in vitro and may be a useful model for studies on the effects of proteins on gene transcription.