Two DNA-dependent RNA polymerases of nuclear origin have been purified from leaves of Zea mays. The discovery that the DNA-dependent RNA polymerase of Escherichia coli consists of a core enzyme plus various protein factors that confer specificity in transcription (1, 2) stimulated efforts to purify RNA polymerase from a wide variety of eucaryotes in an attempt to determine whether this enzyme has the potential for controlling transcription of specific types of genes in higher organisms. Although purification of RNA polymerase from eucaryotic organisms has provided few indications of specificity factors (3, 4), investigations with animals (5-7) and with plants (8, 9) have indicated the existence of several forms of RNA polymerase. At least two enzymes have been purified and characterized from calf thymus (7, 10). Enzyme I is believed to be of nucleolar origin and is involved in ribosomal RNA synthesis, while enzyme II has been isolated from the nucleoplasm and presumably is responsible for the synthesis of DNA-like RNA (5). In addition to their chromatographic behavior on DEAE-cellulose and their salt requirements, these two enzymes can be distinguished by their sensitivity to the toxin a-amanitin that is isolated from the basidiomycete Amanita phalloides (11), and their template specificity. Enzyme II is completely inhibited by a-amanitin, whereas enzyme I is not. Enzyme I prefers native DNA, while enzyme II prefers denatured DNA (5).We have studied the properties of RNA polymerases of nuclear origin from corn leaves (Zea mays) as a first step in determining the involvement of these enzymes in the control of transcription. In addition, the relationships among RNA polymerases of DNA-containing organelles is of interest with respect to the nature of the mechanisms controlling the cell as a whole. The study of nuclear RNA polymerases reported here is part of an effort to elucidate the relationship among RNA polymerases of nuclei and chloroplasts (12). MATERIALS AND METHODSPreparation of DNA-Dependent RNA Polymerases. Maize plants (Z. mays, WF9TMS X B 37, Illinois Foundation Seeds, Inc., Champaign, Ill.) were grown in darkness at 280C for 7 days and exposed to light 12 hr before harvest.The leaves were ground in two volumes per unit weight of a solution of 0.25 M Tris(pH 8.0), 0.50 M sucrose, 0.01 MI MgCI2, 0.01 M 2-mercaptoethanol for 15 see at low speed in a Waring Blendor. The homogenate was filtered through several layers of cheesecloth and two layers of Mliracloth (Chicopee Mills, New York), after which it was centrifuged at 100,000 X g for 90 mm in a Type 30 rotor in a Spinco preparative ultracentrifuge. The specific activity of the supernatant was 35 pmol of AMP incorporated per mg of protein.It was brought to 30% of saturation with ammonium sulfate (Schwarz BioResearch, ultrapure). The precipitate (0-30% ammonium sulfate fraction) was collected by centrifugation at 12,000 X g for 20 min, the supernatant was brought to 50% saturation with (NH4)2S0o4, and the resulting precipitate (30-50% ammonium sulf...

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We report initial studies on estrogen-mediated regulation of egg yolk protein synthesis in the rooster. Egg yolk proteins are normally synthesized as a large precursor, vitellogenin, in the liver o the laying hen; roosters synthesize vitellogenin only when treated with estrogen. Polysomal RNA from the liver of estrogen-treated roosters was translated in a reticulocyte cell-free system, and the newly synthesized proteins were identified by a highly specific and sensitive indirect immunoprecipitation reaction. The messenger RNA that specifies vitellogenin has been purified more than 800-fold from rooster liver polysomal RNA by a combination of methods, including immunoprecipitation of polysomes and chromatography of RNA on poly(U)Sepharose.In recent years an important approach has been developed for studying the molecular mechanisms by which hormones regulate gene expression in eukaryotic organisms. The goal of this approach is to study the regulatory effects of hormones in vitro, and involves the following steps: isolation of a specific messenger RNA (mRNA) from the target tissue after administration of hormone; identification of this mRNA by its ability to program the synthesis of a specific protein in vitro; purification of the specific mRNA to homogeneity; use of the pure mRNA as a template for synthesis of complementary DNA; and, finally, use of the complementary DNA as a probe for hybridization studies of in vitro transcripts from purified components with RNA polymerase and chromatin (1-14). Studies successfully utilizing this approach have been limited to specialized tissues in which thepotential for gene expression is severely limited. We have recently begun a study along the general lines of this approach but have chosen to study specific regulation of gene expression in liver, a tissue with diverse genetic potential. Our studies involve an investigation of the induction of vitellogenin synthesis by estrogen in rooster liver.Avian vitellogenin is a phosphoprotein that is normally synthesized in the liver of laying hens but is synthesized in roosters only after administration of estrogen. Vitellogenin was first identified in the plasma of estrogen-treated male Xenopus laevis (15, 16). After its synthesis in the liver, this phosphoprotein is transported through the plasma to the oviduct where it is deposited in the developing oocyte as lipovitellin and phosvitin, the two amphibian egg yolk proteins that are formed by cleavage of vitellogenin (16). Bergink et al. (17) have suggested that vitellogenin might also exist in the plasma of laying hens and estrogen-treated roosters.

Studies were done to examine direct binding of the first enzyme of the histidine biosynthetic pathway (phosphoribosyltransferase) to 32P-labeled phi80dhis DNA and competition of this binding by unlabeled homologous DNA and by various preparations of unlabeled heterologous DNA, including that from a defective phi80 bacteriophage carrying the histidine operon with a deletion of part of its operator region. Our findings show that phosphoribosyltransferase binds specifically to site in or near the regulatory region of the histidine operon. The stability of the complex formed by interaction of the enzyme with the DNA was markedly decreased by the substrates of the enzyme and was slightly increased by the allosteric inhibitor, histidine. These findings are consistent with previous data that indicate that phosphoribosyltransferase plays a role in regulating expression of the histidine operon.