Ovalbumin messenger RNA was purified 67-fold from hen oviduct polysomal RNA by sucrose gradient centrifugation and oligo(dT)-cellulose hybridization. Ovalbumin messenger RNA activity was coincident with the main peak of absorbance after acrylamide gel electrophoresis of the purified RNA. Each purified messenger was translated over 20 times in a rabbit reticulocyte lysate, protein-synthesizing system. An apparent molecular weight of approximately 875 000 was obtained by electrophoresis under both native and denaturing (formamide) conditions. I n contrast, on sucrose gradients this messenger sedimented a t approximately 15 S, corresponding to a molecular weight of about 550000. Under certain conditions, ovalbumin messenger RNA activity was observed on both gels and gradients a t a position approximately double these molecular weights. The larger messenger RNA species could be dissociated to the smaller one by either heating a t 65 "C or formamide treatment, thus demonstrating messenger RNA aggregation.We conclude that ovalbumin messenger RNA contains between 1670 and 2640 nucleotides; a value significantly greater than the number (1161) that are translated into ovalbumin.

1. DNA complementary to ovalbumin mRNA was synthesized using reverse transcriptase from avian myelobastosis virus and ovalbumin mRNA, purified 60 -70-fold with respect to polysomal RNA, as template.2. The quantitative distribution of ovalbumin mRNA sequences in hen and chick oviduct was determined by hybridization with ovalbumin complementary DNA (cDNA). In hen, we calculate that there are approximately 36000 ovalbumin mRNA molecules per oviduct tubular gland cell.The polysome fraction contains 93 % of ovalbumin mRNA ; nuclear and non-polysomal cytoplasmic RNA fractions contain 5 and 2 % respectively. In the nucleus, the majority of sequences exist as the 16-S "translatable" form when analyzed on formamide gradients. High-molecular-weight RNA species (> 30 S) were detected, and contained less than 2 % of total nuclear mRNA sequences. .The appearance of ovalbumin mRNA sequences in total, nuclear and polysomal RNA fractions from chick oviduct was measured in response to estradiol. Prior to hormone treatment, low levels of sequences were present in all fractions, approximately one mRNA molecule per gland cell being detected in nuclei. Up to 2 h after treatment a small increase in ovalbumin mRNA concentration was observed in nuclear and, to a lesser extent, in polysomal RNA. Between 3 and 6 h ovalbumin mRNA accumulates rapidly in the polysome fraction and after 9 h the rate of appearance of this mRNA is approximately 7 molecules per min per gland cell.4. Our results indicate that the induction by estradiol of ovalbumin synthesis in oviduct is mediated either by the synthesis of new ovalbumin gene transcripts or the stabilization of ovalbumin gene products.The response of chick oviduct to steroid hormones provides a model system for studying the regulation of gene expression in eukaryotes [1,2]. Current evidence indicates that estradiol initiates the de novo synthesis of egg white proteins (namely ovalbumin, conalbumin, lysozyme and ovomucoid) by increasing the availability in the cytoplasm of their specific mRNAs [ 3 ] . The steroid also binds to a specific receptor located in the nucleus [4] and modifies the template properties of chromatin with respect to RNA synthesis [5]. These findings suggest that estradiol regulates mRNA transcription in the oviduct.Recently, techniques have been described for synthesizing DNA complementary to globin mRNA [6] and using this as a genetic probe for quantifying globin mRNA sequences in cellular RNA samples [7]. In this report we describe the synthesis of labeled DNA complementary to ovalbumin mRNA which has been extensively purified from hen oviduct polysomes [El, and the kinetics of hybridization of this DNA product to oviduct RNA fractions. The distribution of ovalbumin mRNA sequences in hen oviduct and evidence for the existence of high-molecularweight ovalbumin mRNA sequences in nuclei is described. Furthermore, we have analyzed the reEur. J. Biochem. 49 (1974)

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1.Within 2 h of administration of estradiol in vivo to immature chickens, RNA synthesis by both form A and B RNA polymerases is stimulated in isolated oviduct nuclei.2. 24 h after hormone treatment this effect is enhanced, and is accompanied by four-and two-fold increases in the level of extractable form A and B enzymes in the nucleus.3. The endogenous RNA polymerase activity associated with nuclear chromatin is also stimulated by estradiol. 9001, of this activity, as shown by the use of the drugs a-amanitin and rifampicin AF/O-13, represents from B enzymes elongating RNA chains.4. Analysis of RNA chains synthesised by the endogenous RNA polymerases of chromatin from estradiol-treated animals (E-chromatin) or control animals (C-chromatin) indicates that there is 2 to 3-times the number of elongating RNA chains in E-chromatin than in C-chromatin.6. E-chromatin is twice as efficient as template for purified hen magnum form B RNA polymerase compared with C-chromatin. Two independent methods demonstrate that this effect is not due to differential ribonuclease content in the chromatins.6. The results suggest that at early times during the differentiation of oviduct cells by estradiol the chromatin template is modified, thus providing additional binding sites for RNA polymerase molecules.One of the earliest effects of estrogenic steroids on their target tissues occurs at the level of transcription [l]. In rat uterus, the synthesis of nuclear RNA is apparently stimulated within 2 min of hormone administration [2], and in both rat uterus and chick oviduct, increases in RNA synthesis can be detected in isolated nuclei following hormone treatment [3,4]. Furthermore, chromatin isolated from target tissues of hormone-stimulated animals serves as a more active template for bacterial RNA polymerase [5] and there is evidence indicating that estrogens modify chromatin such that new species of RNA are transcribed [6,7].These data suggest that estrogens modify mechanisms of nuclear RNA synthesis, although important reservations must accompany such a conclusion. The interpretation of experiments using bacterial RNA polymerases to measure template activity of chromatins is in doubt, due to recent data suggesting that bacterial and mammalian RNA polymerases transcribe different regions of the chromatin DNA [S]. Furthermore, many studies on RNA Enzymes. DNA-dependent RNA polymerase or nucleoside triphosphate : RNA nucleotidyltransferase (EC 2.7.7.6).34 Enr. J. Biochem., Vo1.32 synthesis, both in vivo and in vitro do not account adequately for differential uptake of RNA precursors, which can mask the real effects of the hormone [9, lo], nor for contamination of assays by ribonucleases in vitro. I n one case when ribonuclease activity was measured [ll], apparent effects of estradiol on transcription were negated. Claims that estrogenreceptor complexes stimulate RNA synthesis in vitro [12,13] must also be treated with caution in view of the impurity of the 'receptor' fractions.The situation has been further complicated by the recent dem...

1. The activities of NMN adenylyltransferase and an enzyme that synthesizes poly (ADP-ribose) from NAD were investigated in the various classes of rat liver nuclei fractionated by zonal centrifugation. 2. The highest specific activities of these two nuclear enzymes occur in different classes of nuclei. In very young and in mature rats it was shown that a correlation exists between DNA synthesis and NMN adenylyltransferase activity, but in rats of intermediate age this correlation is less evident. The highest activities of the enzyme that catalyses formation of poly (ADP-ribose) are in the nuclei involved in the synthesis of RNA. 3. The significance of these results in relation to NAD metabolism is discussed.