DNA-dependent RNA polymerase II from calf thymus has been successfully purified using polythylenimine precipitation. Thus, 5-6 mg of nearly homogeneous homogeneous trna polymerase II (greater than 96% pure) can be prepared from 1 kg of calf thymus with three chromatography steps following extraction and precipitation of the enzyme from the polyethylenimine pellet. This procedure eliminates the high salt extraction of chromatin previously used in purification of this enzyme and makes possible the large scale preparation of mammalian RNA polymerase II. Calf thymus polymerase II prepared by this method is greater than 90% form IIb and consists of ten different subunits having the following molecular weights: 180 000; 145 000; 36 000; 25 000; 20 000; 18 500; 16 000; 15 000; 12 000; 11 500. The homologous enzyme isolated from wheat germ is greater than 90% form IIa and contains subunits of the following molecular weights: 206 000; 145 000; 44 000-47 000; 24 500; 21 000; 19 000; 17 000; 14 000; 13 500. The wheat germ and calf thymus enzymes exhibit similar subunits structures, but the molecular weights of individual subunits are clearly different between the enzymes. Wheat germ RNA polymerase II is 50% inhibited by 0.271 microng/mL of alpha-amanitin, a level 30-fold higher than that found for calf thymus RNA polymerase II. These enzymes are further distinguished by the absence of antigenic cross reactivity.

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A cDNA library, prepared from poly(A)+ RNA isolated from quiescent AKR-2B cells 4 hr after stimulation with epidermal growth factor in the presence of cycloheximide, was screened to identify RNA transcripts whose abundance is specifically increased as a primary response to growth stimulation. Approximately 40% of the inducible clones detected by this procedure corresponded to either cytoskeletal 13-or y-actin genes. One nonactin clone, designated c99, was found to be derived from an 8.5-kilobase RNA whose abundance began to increase as early as 30 min after stimulation. DNA sequencing established the identity of this RNA as fibronectin. Several additional mitogens were then tested and found to efficiently induce fibronectin mRNA. These included fetal calf serum, platelet-derived growth factor, and transforming growth factor type P. For at least one inducer, fetal calf serum, the increase in mRNA was preceded by an increase in fibronectin gene transcription. This increase was rapid, reaching maximal levels within 10 min, and was accompanied by near-coordinate increases in both c-fos and 13-actin transcription. These results indicate that fibronectin is a member of a class of "early-response" genes, typified by c-fos and including j3-actin, whose rapid expression may be important in mediating cellular responses to peptide growth factors.Peptide growth factors govern a wide range of cellular activity including embryonic development and differentiation, maintenance of cell-type homeostasis, and wound healing. Genetic and biochemical evidence indicates that cellular responses to growth factors are mediated, in part, through the regulation of specific gene expression. Thus, one approach to understanding how growth factors influence cell behavior is to clone and identify responsive genes. Estimates derived from such endeavors are inexact (1) but suggest that growth factors exert their influence through the action of perhaps 50-100 genes.In studies of cell growth control, particular attention has been focused on genes expressed soon after growth-factor stimulation of quiescent cells in culture. Such genes presumably encode proteins important to growth stimulation and have been shown to include the c-fos and c-myc protooncogenes (2, 3). Interestingly, enhanced expression of the cytoskeletal actin genes is also a prominent and rapid response to mitogenic stimuli in several cell types (4-7). This contrasts with the traditional view of the cytoskeletal actins as passive structural proteins but is broadly consistent with other data suggesting an important role for actin microfilaments in mediating cellular responses to growth factors (8) and in neoplastic transformation (9).Although the exact role of actin in cell growth control is not clear, the degree of anchorage of cells to the extracellular matrix has long been known to influence both cell division and cell differentiation (reviewed in ref. 10). Among the major mediators of cell adhesion are transmembrane glycoproteins that link actin microfilaments on the cytop...

DNA-dependent RNA polymerase III (nucleosidetriphosphate: RNA nucleotidyltransferase, EC 2.7.-7.6) has been isolated and partially purified from calf thymus tissue. Significant amounts of enzyme III are present in this tissue (up to 15% of the total activity of thymus homogenates). This enzyme has been characterized with respect to its chromatographic properties, broad ammonium sulfate optimum (0.04-0.2 M), template requirements, divalent metal optima, and its unique alpha-amanitin sensitivity (50% inhibition of activity occurring at an alpha-amanitin concentration of 10 mug/ml).

DNA-dependent RNA polymerase II has been purified to high specific activity and apparent homogeneity from both calf thymus and rat liver. Two form II enzymes are present in rat-liver preparations, one with the molecular structure 1(190,000)i(150,000),(35,000)1(25,000)dl, the other with a molecular structureof [(170,000),(150,000) -(35,000),(25,000),] (molecular weights are within ±5% but the absolute values are approximate). Inclusion of a proteolytic inhibitor during the isolation procedure decreases the proportion of the molecule containing the 170,000 subunit. Calf-thymus RNA polymerase preparations typically exhibit four components on polyacrylamide gels that contain sodium dodecyl sulfate, with an apparent molecular structure of [(190,000)1(150,000),(35,000)1-(25,000)i]. In addition, some calf-thymus polymerase II preparations contain small quantities of the [(170,OO)1-(150,000)i(35,000)1(25,000)iJ species; the quantity of this species may also be increased from less than 5% in the normal preparation to at least 40% in an "aged" preparation. Thus, the 170,000 subunit may be derived from the 190,000 subunit in both tissues. Until unequivocal evidence is obtained on this point, however, the possibility that the large subunits are unique species should not be eliminated. The general structural similarity of the eukaryotic RNA polymerase II with that of the prokaryotic polymerase suggests that the modes of action and regulation may be analogous.Multiple forms of DNA-dependent RNA polymerases have been shown to exist in eukaryotic cells (1, 2). Polymerase I is localized in the nucleolus (2); its RNA product has the base composition and hybridization behavior expected of ribosomal RNA (3). These data indicate that the major role of this enzyme is to synthesize ribosomal RNA, although it may transcribe a limited number of other RNA species. Polymerase II, on the other hand, is found in the nucleoplasm (2). Its product has a more DNA-like base composition and is competed well in hybridization-competition experiments by whole nuclear RNA (3), suggesting that this polymerase synthesizes the bulk of the nucleoplasmic RNA species. A role for polymerase III, believed to be present in the nucleoplasm, has yet to be elucidated. The mushroom toxin, aamanitin, selectively inhibits polymerase II, while forms I and III are not affected (4,5