When tritiated ATP is incubated with a membrane-enriched fraction prepared from the eukaryotic microorganism Dictyostelium discoideum significant levels of radioactivity can be precipitated with cold, 10% trichloroacetic acid. Reaction product was formed from ATP and dATP but not from GTP, CTP and UTP. Other studies showed that the maximum amount of the acid-insoluble product was formed about 1 min after the addition of the membranes and that, with further incubation, this reaction product was degraded. The rate of degradation of the reaction product was greatly reduced when the temperature was reduced to 4 degrees C, and when either NaF, Na2SO4 or dithiothreitol was added to the reaction mixture. These additions or conditions had no effect on the product-formation reaction. The rate of degradation was also reduced following the addition of adenosine to the reaction and this result did not occur following the addition of ADP, AMP or cyclic AMP. The acid-insoluble reaction product could be solubilized with SDS and analysis by gel-filtration chromatography on Sephadex G-75 revealed that the radioactivity was associated with a macromolecule that was not sensitive to RNAase or DNAase but was degraded by pronase. The nucleotide-protein complex was stable at room temperature but radioactivity was released in hot acid, which, after analysis by thin-layer chromatography, was found to co-migrate with authentic AMP, suggesting the formation of an adenylyl-protein complex as the reaction intermediate. The complex bond was stable at neutral and alkaline pH, suggesting a phosphoamide linkage between the protein and the adenylyl moiety.

Mesenchymal cells isolated from the papilla of embryonic tooth germs of the mouse were cultured in a complex medium for five to six days. Liquid nitrogen lysates, prepared from these cells, incorporated nucleoside monophosphates into a cold acid-insoluble product. The product was sensitive to RNase and no product was formed if the lysate was pretreated with DNase. The reaction was sensitive to EDTA and, in its presence, optimum activity was obtained with 2 mM MgCl2. On sucrose gradients, the reaction product was distributed between two broad peaks; one centered about 18S and the other above 28S. The RNA polymerase inhibitor alpha-amanitin inhibited approximately 50% of the activity at a concentration of 10 microgram/ml.

In this study we report procedures for the isolation of a transcriptionally active fraction from Dictyostelium discoideum. Chemical analysis of this fraction shows that it contains DNA, RNA, protein, and lipid. The fraction exhibits less than 5% of the alkaline phosphatase-specific activity associated with plasma membranes and less than 10% of the succinate dehydrogenase-specific activity associated with purified mitochondria. Two RNA polymerase activities associated with the fraction have been resolved using DEAE-Sephadex chromatography. One activity is sensitive to low concentrations (25 μ/ml) of the toxin α-amanitin. The total protein and basic protein components of the transcriptionally active fraction have been fractionated on polyacrylamide gels. Eleven major polypeptides were obtained from electrophoresis of the transcriptionally active fraction proteins in sodium dodecyl sulfate-urea polyacrylamide gels, several of which had molecular weights above 50,000. The basic proteins associated with the transcriptionally active fraction after fractionation on polyacrylamide gels appeared similar in mobility to calf thymus histones and also to histones isolated from D. discoideum nuclei. The transcriptional activity of the reaction has been shown to require ATP, GTP, CTP, and UTP. The reaction is inhibited by EDTA, which can be reversed in the presence of either Mg2+ or Mn2+ ions. Activity is abolished by treatment of the fraction with DNase or actinomycin D, while the product is susceptible to both RNase and KOH. The reaction is insensitive to rifampicin and is not affected following the addition of DNA or RNA. An analysis of the α-amanitin sensitivity of the transcriptional reaction suggests that the two polymerase activities isolated from the complex are both functioning during the reaction. The transcriptional reaction product, which has been analyzed by sucrose gradient centrifugation, ranges between 4 and 20 S. The profile is lost after treatment of the fractions with RNase. Transcriptional assays run in the presence of α-amanitin yielded a sucrose gradient profile slightly larger than the untreated transcriptionally active fraction product profile, while isolated poly(A) containing in vitro transcripts synthesized by the transcriptionally active fraction appeared similar to the original transcriptionally active fraction product profile. The rate of transcription observed in the present study is comparable within a factor of 2 to that of other endogenous transcriptional systems. As shown by hybridization analysis, the in vitro transcript anneals specifically with D. discoideum DNA. Both the α-amanitin-insensitive and the poly(A)-containing in vitro transcripts showed greater than 80% hybridization with D. discoideum DNA. In the presence of D. discoideum polysomal RNA, the amount of in vitro transcript hybridized is reduced by approximately 90%.