Gene Expression In Chick Oviduct Research Articles

Regulation of Gene Expression in Chick Oviduct

Regulation of Gene Expression in Chick Oviduct

Effects of estrogen on gene expression in chick oviduct. The role of chromatin proteins in regulating transcription of the ovalbumin gene.

Histones, extractable non-histone proteins, and a tightly bound non-histone protein DNA complex were fractionated from chromatins which were isolated from estrogen-stimulated and hormone-withdrawn chick oviducts. Reconstitution of homologous constituents was performed and RNA was transcribed from these reconstituted chromatins using Escherichia coli RNA polymerase. DNA complementary to ovalbumin mRNA was then used as a hybridization probe to estimate the concentration of ovalbumin messenger RNA sequences in these in vitro transcripts. Our results demonstrated that 0.011% of the in vitro RNA transcripts produced from reconstituted estrogen-stimulated chromatin was ovalbumin mRNA sequences as compared to 0.0015% obtained for reconstituted hormone-withdrawn chromatin. Thus, the ratio of ovalbumin mRNA sequences in the in vitro transcripts of reconstituted stimulated and withdrawn chromatins was 8 to 1, identical with the value obtained from native estrogen-stimulated and hormone-withdrawn chromatins. Furthermore, the number of initiation sites for RNA synthesis on reconstituted and native chromatins are indistinguishable. Thus, the specificity of transcription of isolated chromatins appears to be conserved upon dissociation and fractionation of the chromatin proteins followed by reconstitution of these constituents to DNA. The effect of chromatin proteins on gene expression was further examined by reconstituting components from different development stages. Reconstitution of extractable non-histone proteins from estrogen-stimulated chromatins to tightly bound non-histone protein. DNA complexes from hormone-withdrawn chromatins resulted in the synthesis of a substantial amount of ovalbumin mRNA sequences. Conversely, when extractable non-histone proteins from withdrawn chromatins were reconstituted to tightly bound non-histone protein DNA complexes from estrogen-stimulated chromatins, very low levels of mRNAov sequences were detected. In contrast, interchange of the histones and tightly bound non-histone protein DNA complexes from hormone-withdrawn and estrogen-stimulated chromatins during reconstitution did not affect the level of mRNAOV sequences produced. Therefore, the extractable non-histone proteins of chromatin appear to be extremely important in regulating specific gene expression.

Effects of estrogen on gene expression in chick oviduct: nuclear receptor levels and initiation of transcription.

Estrogen (diethylstilbesterol) was administered in vivo to chicks for various time periods. Chromatin was then prepared from oviduct nuclei and assayed for its capacity to support initiation of RNA chain synthesis in vitro in the presence of saturating levels of Escherichia coli RNA polymerase (RNA nucleotidyltransferase; nucleosidetriphosphate:RNA nucleotidyltransferase; EC 2.7.7.6). These same nuclei were also assayed by a [3H]estradiol exchange assay for their endogenous receptor content. The number of available initiation sites for RNA synthesis on chromatin was shown to correlate with the endogenous levels of nuclear estrogen receptor. A decrease in the nuclear concentration of estrogen receptor molecules and the concentration of initiation sites for RNA synthesis occurred during withdrawal of estrogen from previously stimulated chicks. Both parameters declined with a similar half-life. When estrogen was readministered to withdrawn chicks, the number of initiation sites increased 2-fold as early as 30 min and approached a maximal level (3-fold) by 1 hr. During the same period of restimulation with estrogen, the number of estrogen receptor molecules bound to nuclei increased to a maximum at 20 min and then declined at 1 hr to a steady-state level 2-fold higher than the withdrawn chicks. Simultaneous measurements of RNA chain length and RNA chain propagation rate demonstrated that parameters remained relatively constant throughout estrogen withdrawal as well as secondary stimulation. The temporal correlation between changes in the levels of nuclear-bound estrogen receptor and the number of RNA chain initiation sites on chromatin prepared from these same nuclei strongly suggested that the hormone receptor complexes act on chromatin to mediate these changes in genetic transcriptional activity.