Estrogen regulates the number of RNA polymerase II molecules in rooster liver.

Administration of estrogen to roosters causes an increase in the activities of DNA-dependent RNA polymerases I and II in nuclei isolated from liver. In an effort to determine the cause of this increased transcriptional activity, we have examined the activity of RNA polymerase II that we have solubilized from nuclei of normal, estrogen-stimulated, and estrogen-withdrawn roosters. In addition, we have measured the actual numbers of RNA polymerase II molecules per nuclear equivalent of DNA in livers of roosters in each estrogenic state by the technique of [3H]amanitin-binding. The administration of estrogen is attended by a 2-fold increase in enzymatic activity of solubilized RNA polymerase II per liver nucleus within 24 h. In addition, there is a 2-fold increase in the number of RNA polymerase II molecules per nucleus in the livers of these animals after the administration of estrogen. During withdrawal from estrogen for 14 days, the activities of RNA polymerases I and II in isolated nuclei and the activity of solubilized RNA polymerase II return to the unstimulated levels. Moreover, the [3H]amanitin-binding capacity of nuclear extracts from the livers of roosters in various stages of hormonal stimulation closely mimics the RNA polymerase II activity of the same extracts. These observations indicate that estrogen exerts a rigid control over the population of RNA polymerase II molecules in avian liver.

Specific transcription in chicken liver chromatin by endogenous RNA polymerase II. Comparison of an estrogen-inducible gene with a constitutively expressed gene.

We have developed a system for the in vitro transcription of specific genes in rooster liver chromatin by endogenous RNA polymerase II that maintains the specificity of transcription in vivo. Radioactive transcripts synthesized in vitro were identified and quantitated by hybridization to a vast excess of cloned cDNA. The cDNA preparations employed corresponded to vitellogenin mRNA, the synthesis of which is responsive to estrogen stimulation in vivo, and chicken serum albumin mRNA, the synthesis of which is not significantly affected by estrogen stimulation in vivo. Comparing the pattern of transcription of the albumin and vitellogenin genes in chromatin from the liver of the normal rooster with the pattern in chromatin from the liver of the estrogen-stimulated rooster, we found that prior estrogen treatment of the rooster is attended by a slight decrease in the differential rate of transcription of the albumin gene and approximately a 10-fold increase in the differential rate of transcription of the vitellogenin gene. Because this pattern of transcription reflects the estrogen-induced changes in transcription observed in vivo, chromatin preparations from the livers of normal and estrogen-stimulated roosters can be used to investigate regulation of specific gene transcription at the molecular level in vitro.

Primary induction of vitellogenin mRNA in the rooster by 17beta-estradiol.

We have studied the kinetics of vitellogenin mRNA accumulation in rooster liver after a primary injection of 17beta-estradiol. The levels of vitellogenin mRNA have been determined both by hybridization of total cellular RNA to vitellogenin cDNA and by translation of vitellogenin mRNA in a wheat germ cell-free system. The results obtained by both methods of analysis are in good agreement and indicate that vitellogenin mRNA is present in the liver of normal roosters at a level of 0-5 molecules per liver cell and increases in amount during the 3 days following injection of estrogen, reaching a level of almost 6000 molecules per cell at the peak of the response. The level of vitellogenin mRNA declined exponentially during the next 14 days with a half-life of 29 hr, reaching a level of less than 10 molecules per cell at 17 days after injection of the hormone. The levels of vitellogenin mRNA after stimulation with estrogen have been correlated with the in vivo rate of synthesis of the vitellogenin polypeptide. The results indicate that the rate of vitellogenin synthesis is closely correlated with the level of vitellogenin mRNA. On the basis of these findings, we conclude that vitellogenin mRNA does not exist in the liver in an untranslated form after withdrawal from estrogen.

Induction of vitellogenin synthesis by estrogen in avian liver: relationship between level of vitellogenin mRNA and vitellogenin synthesis.

We have investigated the estrogen-mediated induction of vitellogenin synthesis in rooster liver. We compared the concentrations of vitellogenin messenger RNA (mRNA) in the liver with the concentrations of vitellogenin in the sera of roosters that had recieved various treatments with estrogen. We found no vitellogenin mRNA in the livers of the unstimulated roosters. An initial injection of estrogen was attended by de novo synthesis of vitellogenin mRNA in the liver and accumulation of vitellogenin in the serum. When vitellogenin was no longer present in the serum or liver (the "post-estrogen-serum-negative" state), the liver was found to contain appreciable amounts of vitellogenin mRNA. This mRNA was of the same size as that found in the liver of the rooster actively synthesizing vitellogenin in response to estrogen. Whereas vitellogenin mRNA was in large polysomes in the livers of the roosters actively synthesizing vitellogenin, the vitellogenin mRNA in the liver of the post-estrogen-serum-negative rooster was not associated with polysomes. The possible relevance of these findings to the fact that the rooster responds differently to a primary stimulation with estrogen than to subsequent stimulations is discussed.

Specific binding of the first enzyme for histidine biosynthesis to the DNA of histidine operon.

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.

Effect of estrogen on gene expression: purification of vitellogenin messenger RNA.

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 of 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.

Interaction between phosphoribosyltransferase and purified histidine tRNA from wild type Salmonella typhimurium and a derepressed hisT mutant strain.

We have examined the interaction between phosphoribosyltransferase and purified tRNA-His from the wild type strain of Salmonella typhimurium, LT-2, and the histidine regulatory mutant hisTl504. Histidyl-tRNA from the mutant strain functions normally in protein synthesis but is defective in its role in the repression mechanism of the histidine operon. Phosphoribosyltransferase has been suggested as a possible aporegulator for this operon and as such might be expected to interact abnormally with tRNA-His from hisT1504. In these studies we have been unable to detect any difference between the affinities of phosphoribosyltransferase for tRNA-His from LT-2 or hisT1504, and thus we conclude that if the complex between phosphoribosyltransferase and histidyl-tRNA does function in regulation, the defect in the hisT1504 mutant must influence the interaction of the complex with some other regulatory element.

RNA polymerases of maize. Purification and molecular structure of DNA-dependent RNA polymerase II.

Nuclear DNA-dependent RNA polymerase II has been purified from leaves of Zea mays by a new procedure that improves enzyme stability and thus permits more manipulation during purification. The purification procedure includes a heating step, gel filtration on Sepharose 6B and 4B, and chromatography on DEAE- and DNA-celluloses. This method of purification yields an enzyme that exhibits maximal activity when denatured DNA is used as a template. Electrophoresis of highly purified enzyme on polyacrylamide gels containing sodium dodecyl sulfate indicates that maize RNA polymerase IIa is composed of several polypeptide subunits. The most highly purified preparations contain polypeptides with molecular weights of 200,000, 160,000, 35,000, 25,000, 20,000, and 17,000.

RNA polymerases of maize: nuclear RNA polymerases.

Two DNA-dependent RNA polymerases of nuclear origin have been purified from leaves of Zea mays. The two enzymes can be separated on DEAE-cellulose columns. Enzymes I and II are eluted with 0.08 and 0.20 M (NH(4))(2)SO(4), respectively. Both enzymes prefer maize nuclear DNA as a template; they are also more active in the presence of Mg(++) than Mn(++) and are inhibited by (NH(4))(2)-SO(4) or KCl. Neither enzyme is inhibited by rifamycin SV. Enzyme II is strongly inhibited by alpha-amanitin, whereas enzyme I is not significantly affected. Their ability to use native and denatured DNA as templates varies according to the extent and method of purification of the polymerase. Furthermore, enzyme II can be resolved by DEAE-chromatography or glycerol-gradient centrifugation into two components, one of which prefers native DNA, while the other prefers denatured DNA.

Effects of N-hydroxyurethan on viability and metabolism of Escherichia coli.

Exposure of growing cells to low levels of N-hydroxyurethan (approximately 0.05 m) resulted in loss of colony-forming ability. This was accompanied by degradation of cellular deoxyribonucleic acid. When the concentration of N-hydroxyurethan was increased to 0.2 m, bacteriostasis was the primary manifestation. The lethal action of the drug was prevented by contreatment with chloramphenicol or azauracil or by uracil-deprivation of a bacterial strain requiring this pyrimidine for growth.

Recovery from N-hydroxyurethan-induced death.

Bacteria (Escherichia coli) can recover from the lethal action of N-hydroxyurethan when they are incubated in drug-free liquid medium. This recovery, which is dependent upon energy metabolism, does not occur on solid medium. Recovery is accompanied by repair of the cellular deoxyribonucleic acid (DNA). A bacterial mutant deficient in DNA polymerase was extremely sensitive to the lethal action of hydroxyurethan. Data are presented which support the concept that DNA replication and DNA repair are mediated by different enzymes and that only the former process is inhibited by hydroxyurea.