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Regulation of the Rat Oxytocin Gene by Estradiol

Oxytocin (OT) plays a role in reproduction at the level of the pituitary and mammary glands and uterus. This OT is synthesized in the hypothalamo‐neurohypophyseal system (HNS). A number of observations have suggested that estrogens regulate the production of OT in the HNS. In this study the effect of 17β‐estradiol on the activity of the OT gene promoter was examined as well as the effect of 17β‐estradiol in vivo on OT messenger ribonucleic acid (mRNA) and peptide revels in the rat HNS. Vasopressin (VP) and its mRNA were also determined in the in vivo studies. The direct transcriptional stimulation of OT gene expression by 17β‐estradiol was studied in two different heterologous expression systems. When a plasmid having nucleotides −363 to +16 of the rat OT gene fused to the firefly luciferase reporter gene was co‐transfected with an estrogen receptor expression vector in P19 embryonal carcinoma cells, luciferase activity was stimulated 80‐fold by 17β‐estradiol. In estrogen receptor containing MCF‐7 cells transfected with a plasmid having nucleotides −188 to +16 of the rat OT gene fused to the chloramphenicol acetyl transferase gene, 17β‐estradiol induced the expression of the chloramphenicol acetyl transferase gene through the cloned promoter element. After in vivo treatment of ovariectomized rats with 17β‐estradiol, levels of OT mRNA and VP mRNA were measured in microdissected supraoptic and paraventricular nuclei as well as VP and OT levels in these nuclei and the pituitary gland. As compared to non‐treated ovariectomized rats there was no difference in contents of OT mRNA and VP mRNA in these hypothalamic nuclei and in levels of the peptides in paraventricular nuclei and the pituitary gland. A 30% reduction of the OT content of the supraoptic nuclei only was found, while the VP content did not change. To explain the results immunocytochemical analyses of the hypothalamus were performed, showing that the estrogen receptor was absent in the magnocellular neurons of the supraoptic and paraventricular nuclei. The results demonstrate that the 5’flanking region of the OT gene confers estrogen‐sensitivity to transcription of the OT gene. This potential to respond to estrogens is not used in the OT‐producing neurons of supraoptic and paraventricular nuclei probably due to the absence of the estrogen receptor.

Chromosomal targeting of replicating plasmids in the yeast Hansenula polymorpha.

Using an optimized transformation protocol we have studied the possible interactions between transforming plasmid DNA and the Hansenula polymorpha genome. Plasmids consisting only of a pBR322 replicon, an antibiotic resistance marker for Escherichia coli and the Saccharomyces cerevisiae LEU2 gene were shown to replicate autonomously in the yeast at an approximate copy number of 6 (copies per genome equivalent). This autonomous behaviour is probably due to an H. polymorpha replicon-like sequence present on the S. cerevisiae LEU2 gene fragment. Plasmids replicated as multimers consisting of monomers connected in a head-to-tail configuration. Two out of nine transformants analysed appeared to contain plasmid multimers in which one of the monomers contained a deletion. Plasmids containing internal or flanking regions of the genomic alcohol oxidase gene were shown to integrate by homologous single or double cross-over recombination. Both single- and multi-copy (two or three) tandem integrations were observed. Targeted integration occurred in 1-22% of the cases and was only observed with plasmids linearized within the genomic sequences, indicating that homologous linear ends are recombinogenic in H. polymorpha. In the cases in which no targeted integration occurred, double-strand breaks were efficiently repaired in a homology-independent way. Repair of double-strand breaks was precise in 50-68% of the cases. Linearization within homologous as well as nonhomologous plasmid regions stimulated transformation frequencies up to 15-fold.

Estrogen-induced phosphoprotein synthesis in roosters: Kinetics of induction

Abstract The synthesis of the lipovitellin-phosvitin phosphoprotein complex induced in the rooster liver by estradiol and its removal from the blood were investigated. The complex is detectable in the blood 10 h after the first estradiol dose and its concentration increases linearly from about 25 h on for several days, followed by a decline to the original undetectably low level. The half-life of complex in the blood of estrogenized roosters is 8.4 h and in that of untreated control roosters 5.2 h as could be shown by the degradation of intravenously injected radioactive complex. In the period of detectable concentrations the rate of synthesis is calculated from the accumulation and degradation data. From at least 25 h after estradiol injection, the rate of synthesis increases linearly for several days. This is interpreted as the synthesis of long-lived mRNA(s) at a constant rate. These data agree with the finding that the amount of lipovitellin-synthesizing polysomes per liver increases linearly in the same period. From both observations a constant translation rate of 6–10 amino acids · s−1 per ribosome can be calculated. A “memory effect” is observed after a second hormone dose given some time after the disappearance of the phosphoprotein complex from the blood. Detectable accumulation starts sooner and its increase is more rapid in the initial period. This phenomenon is not due to a shorter lag period, for the start of induced synthesis, measured by incorporation of 32P1 into phosphoprotein complex, is 3–4 h after the first as well as after the second injection. From that moment the rate of synthesis after the first injection increases only gradually and becomes a linear function of time after about 15 h, whereas after a second injection the rate of synthesis increases linearly immediately after the lag period.

Specific inhibition of ribosomal RNA synthesis in vitro by guanosine 3' diphosphate, 5' diphosphate.

CONSIBERABLE evidence has accumulated that guanosine 3′-diphosphate, 5′-diphosphate (ppGpp)1 is involved in the regulation of ribosomal RNA (rRNA) synthesis in Escherichia coli in vivo (see ref. 2). The involvement of ppGpp is most convincingly indicated by results obtained under conditions of amino acid starvation. A strong increase in ppGpp accumulation is always accompanied by a concomitant decrease in stable RNA accumulation3. In vitro, a specific effect of ppGpp has not been found, neither in a purified4, nor in a crude system5. Travers et al.6,7 have proposed that ppGpp specifically affects rRNA synthesis by counteracting the effects of the protein elongation factor EF-Tu. Here, we show that ppGpp can specifically inhibit rRNA synthesis in vitro using purified RNA polymerase with two templates—DNA prepared by phenol extraction, and nucleoids prepared using methods developed by Pettijohn et al.8.

Cloning and characterisation of a nuclear, site specific ssDNA binding protein.

Estradiol inducible, liver-specific expression of the apoVLDL II gene is mediated through the estrogen receptor and a variety of other DNA-binding proteins. In the present study we report the cloning and characterisation of a single-strand DNA binding protein that interacts with the lower strand of a complex regulatory site, which includes the major estrogen responsive element and a site that resembles the rat albumin site D (apoVLDL II site D). Based on its binding specificity determined with electro-mobility shift assays, the protein is named single-strand D-box binding factor (ssDBF). Analysis of the deduced 302 amino acid sequence revealed that the protein belongs to the heteronuclear ribonucleoprotein A/B family (hnRNP A/B) and resembles other known eukaryotic single-strand DNA binding proteins. Transient transfection experiments in a chicken liver cell-line showed that the protein represses estrogen-induced transcription. A protein with similar binding characteristics is present in liver nuclear extract. The relevance of the occurrence of this protein to the expression of the apoVLDL II gene is discussed.

Estradiol-induced synthesis of vitellogenin. IV. The isolation of non-degraded polysomes from avian liver using an endogenous ribonuclease inhibitor.

A procedure allowing the isolation of intact polysomes from rooster liver is described. Good recovery of polysomes is achieved by the presence of Triton X-100 in the homogenization and centrifugation steps since the detergent prevents the sedimentation of microsomes with the nuclear fraction. This sedimentation of microsomes leads to considerable losses of polysomes, especially the larger ones. In the detergent-treated homogenate the integrity of the polysomes is threatened by various ribonucleases, some of which can be effectively inhibited by the addition of both heparin and yeast RNA. The remaining nuclease activity is counteracted by the endogenous ribonuclease inhibitor of the liver. In estradiol-treated roosters, sufficient endogenous inhibitor is present to inhibit its specific ribonuclease, but in control roosters there is not. This difference is due to a hormone-mediated increase in inhibitor level and decrease in nuclease level. Consequently, for an estrogenized rooster, the addition of both heparin and yeast RNA to the homogenate suffices to stabilize the polysomes, whereas control rooster liver homogenate needs supplementation with endogenous ribonuclease inhibitor. The cytosol of estrogenized rooster liver can be used as a crude inhibitor preparation. Rat liver cytosol is only partially effective; this may indicate a certain degree of species specificity of the inhibitor. The isolation procedure described also yields large polysomes from the livers of duck and Xenopus.

Estradiol-induced synthesis of vitellogenin II. Immunochemical characterization of vitellogenin polysomes

Abstract Polysomes from estrogenized rooster liver were subfractionated on sucrose gradients. The size distribution of the vitellogenin polysomes was determined with in vitro labelling of the nascent chains followed by immunochemical determination of the elongated vitellogenin chains. Vitellogenin polysomes contain 30–40 ribosomes.

DIFFERENTIAL ESTROGEN RESPONSIVENESS OF THE VITELLOGENIN AND APO VERY LOW-DENSITY LIPOPROTEIN-II GENES IN THE ROOSTER LIVER

The primary transcript of the chicken apo Very Low Density Lipoprotein II (apoVLDL-II) gene is formed almost immediately after a first estrogen administration, contrary to the appearance of the vitellogenin primary transcript which has a lag of at least 4 h. However, after a second estrogen administration the vitellogenin gene transcription shows no detectable lag (memory effect). After estrogen withdrawal, the primary transcripts of both genes rapidly decline to undetectably low levels. In the presence of estrogen, the half-lives of the mRNAs of apoVLDL-II and vitellogenin are 15 and at least 70 h, respectively, whereas in the absence of hormone they are only 3.5 and 5.5 h, respectively. Thus estrogen not only controls the transcription of both genes, but also the turnover of their mRNAs. In short, there appears to be a quantitative difference in the response of both genes.

Estradiol-induced synthesis of vitellogenin I. The isolation of large polysomes from estrogenized rooster liver

Conventional procedures for the isolation of polysomes, applied to estrogenized rooster liver, fail to yield polysomes containing 30 or more ribosomes, the size expected for polysomes synthesizing the estradiol-induced protein vitellogenin. A new procedure characterized by early and selective removal of cell components ribonucleases allowed the isolation of polysomes with up to 55 ribosomes. Electron microscopy was used for the determination of polysome size and showed that the large polysomes were not aggregates.

Post-translational phosphorylation of phosvitin

is synthesized in tke liver of laying heps and, after estradiot injection, al50 in rooster liver. It is secreted into the biood as a complex with lipovitelIin f l.--33 . Mypo-ee than half of the amino acids czf phosviFin am zxeriries, v&ich are practicslly all present as phosphate esters. Phosvitiut-phospha;te comprises at least 7’5% of the protein phosphate in the cumples with &poviteHin [I ] _ While the @~l~s~~~~~at~~~ of other proteins is genes&y assumed to occur after translation [49 some authors have sk~estrcl a specific phosphoseTine-tRNA S. a possible intermediate in the synthesis of phosvitin [S, 6) _ We have studied in what phase of the synthesis of phostitin the se,rines are phosphorplated: befqre tr;insiatian, &t the nascent chain, or after compktion of the gqtide chain. Ex this purpose we have measured the incorporation oftabeitfed teucine and ph&phate rir ~ilpo (slices) and if2 viva. and studied the effect of cycloheximide. Our resutts show that the ~~~s~ho~~at~o~ occurs mainly after the release of the peptide chain from the po!~~omes, probabfy during transport to the .:-_;ood.