Abstract
Estrogen regulates the synthesis of the egg yolk precursor protein, vitellogenin, by causing both a 20-60-fold increase in the absolute rate of total nuclear RNA synthesis and a selective increase of at least several thousand fold in the absolute rate of vitellogenin gene transcription. Vitellogenin gene transcription is undetectable in unstimulated and withdrawn Xenopus laevis liver cells and in cultured Xenopus kidney cells allowing us to set a very low upper limit (less than 1 transcript/vitellogenin gene/day) on potential basal rates of vitellogenin gene transcription. The elevated rates of vitellogenin mRNA accumulation previously observed during restimulation of withdrawn liver cells (secondary estrogen stimulation) appear to be due to an increased rate of vitellogenin gene transcription. Both the maximum transcription rate and the rapidity of the early response increase on secondary estrogen stimulation. Relative transcription rates were determined by hybridization of pulse-labeled nuclear RNA to vitellogenin cDNA clones immobilized on nitrocellulose filters. The conversion of relative transcription rates to absolute transcription rates, was facilitated by development of a sensitive high performance liquid chromatography method for quantitation of the specific radioactivity of the cellular UTP pool.
Highlights
Estrogen regulates the synthesis of the egg yolk pre- stimulation [8, 9, 15].In order to determine whether these cursor protein, vitellogenin, by causing both a 20-60- effects on cytoplasmic vitellogeninmRNA accumulation rates fold increase in the absolute rate of total nuclReaNrA reflect changes in vitellogeningene transcription rates or postsynthesis and a selective increase of at least several transcriptional effects at the levels of RNA processing or thousand fold in the absolute rate ofvitellogenin gene mRNA turnover, the relative and absoluteof vraiteelslogenin transcription
Vitellogenin gene transcriptionis unde- gene transcriptionwere measured during theperiods of viteltectable in unstimulatedand withdrawnXenopus laevis logenin mRNA appearance and accumulatioinn primary and liver cells and in cultured Xenopus kidney cells allow- secondary stimulation
Ing us to set averylowupperlimit (
Summary
Genes-Relativetranscription rates weredeterminedby pulse-labeling the cells, andhybridizing the pulse-labeled nuclear RNA to vitellogenin cDNA clones immobilized on nitrocellulose filters [30].In order to measure the relative rate of gene transcription accurately, it is necessary to label the cells foar shorter periodthan the time requiredthe synthesize, process, and transport the RNAout of the nucleus. Preliminary experiments demonstratetdhat no labeled RNA appears in the cytoplasm for at least 45 min after adding the label, and this period was chosen as the pulse-labeling time. This represents a labeling periodof approximately twice the time requiredtotranscribeeach 18-20-kb vitellogenin gene, as the maximum rate of gene transcription inX . In order to demonstrate that the efficiency of hybridization was constant ovearbroad rangeof input RNA concentrations, the experimentshownin Fig. 1 wasperformed.[3H]RNA, isolated from nuclei labeled in organ culture, containing increasing amountsof [3H]vitellogenin m R N A of known specific radioactivity(seelegend)w, ashybridizedtovitellogenin cDNAcloneismmobilized on nitrocellulose filters. These data demonstrate that the immobilizedcloned vitellogenin DNA sequences were present in sufficient excess
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