amanitins and Lymphoma--Non-Hodgkin

Bovine lymphosarcoma tissue has been extracted with low- and high-salt buffers [0.05 M Tris-C1 plus or minus 0.3 M (NH4) 2S04]. Diethylaminoethyl-Sephadex chromatography of both the high-salt and low-salt extracts yields RNA polymerases I and II, although low-salt extraction releases only one-third as much activity. Extraction by high salt of the residue from the low-salt extract, followed by diethylaminoethyl-Sephadex chromatography, yields additional enzyme activity with properties of Form II. Purification of the low-salt extract by protamine precipitation, elution with sodium succinate, and phosphocellulose chromatography yields a preparation of RNA polymerase (RNAP) with hybrid properties, combining the salt optimum of Form I, diethylaminoethyl-Sephadex elution pattern of form II, and alpha-amanitin sensitivity of Form III. RNAP. transcribes native D,A and chromatin efficiently. More RNAPL is recovered from lymphosarcoma tissue than from calf thymus.

Topics: Amanitins; Animals; Cattle; Cattle Diseases; Chemical Precipitation; Chromatin; Chromatography, DEAE-Cellulose; DNA-Directed RNA Polymerases; In Vitro Techniques; Lymphoma, Non-Hodgkin; Protamines; Succinates; Templates, Genetic; Thymus Gland; Transcription, Genetic; Tritium

DNA-dependent RNA polymerase was solubilized from nuclei of Ehrlich ascites carcinoma (EAC) cells by sonic disruption in the presence of 0.3 M (NH4)2 SO4, and the multiple RNA polymerases were separated by chromatography on DEAE-Sephadex A-25. Elution with a nine-step gradient of (NH4)2 SO4 yielded five peaks of activity designated RNA polymerases Ia, Ib, IIa, IIb, and III, of which IIb was the most prominent. Linear-gradient elusion also yielded five peaks of the same designation, but Ia and Ib, as well as IIa and IIb, were not well separated. IIa and IIb were inhibited completely by 0.1 mug alpha-amanitin/ml, whereas the other forms were not. EAC RNA polymerases Ia, Ib, IIa, and IIb possessed Mg2+ ion, Mn2+ ion, and (NH4)2 SO4 optima, molecular weights, and thermal sensitivities similar to those reported for other mammalian DNA-dependent RNA polymerases. As measured by relative ribonucleoside monophosphate incorporation, with native calf thymus DNA template, EAC RNA polymerases Ia and Ib synthesized ribosomal RNA-like products, whereas forms IIa, IIb, and the parent enzyme mixture synthesized compounds that were more similar to DNA. No species specificity was found for DNA templates, and denatured DNA was consistently preferred to the native template by RNA polymerases IIa and IIb; the two kinds of template were about equally efficient for RNA polymerases Ia and Ib. Although EAC RNA polymerases Ia, IIa, and IIb were inhibited by daunomycin, form IIa was preferentially affected. 3',5'-Cyclic AMP, 3',5'-Cyclic GMP, and gibberellic acid, implicated as RNA polymerase regulators in other systems, were generally ineffective. The levels of nuclear RNA polymerase activities, per mg DNA, of 3 mouse ascites tumors (EAC, 6C3HED lymphosarcoma, and TA3 adenocarcinoma) were compared with those from 3 normal mouse tissues (kidney, liver, and spleen). On the average, the tumor cell nuclei contained (per mg of DNA) 8.9, 1.5, 2.7, 20.0, and 3.8 times the activities of RNA polymerases Ia, Ib, IIa, IIb, and III, respectively, as the normal cells, but the difference was significantly only for IIb.

Topics: Adenocarcinoma; Amanitins; Animals; Carcinoma, Ehrlich Tumor; Cell Nucleus; Chromatography, Ion Exchange; Daunorubicin; DNA-Directed RNA Polymerases; DNA, Neoplasm; Kidney; Liver; Lymphoma, Non-Hodgkin; Magnesium; Male; Manganese; Mice; Molecular Weight; Neoplasms, Experimental; RNA, Ribosomal; Species Specificity; Spleen; Temperature; Templates, Genetic