Administration of estraciol-17B to male Xenopus laevis induces liver cell differentiation and the synthesis of massive amounts of the egg yolk precursor protein, vitellogenin, apo very low density lipoprotein II (apo VLDLII), and several other proteins. We propose to examine the role of 5' flanking sequences in transcription using the relatively simple apo VLDLII gene system, rather than the large and structurally complex vitellogenin gene family. Our approach will be examine the transcription, after microinjection into Xenopus oocyte nuclei, of a series of cloned deletions in the 5' flanking regions of the DNA and to develop an assay for transcription regulatory factors, by coinjection into Xenopus oocyte nuclei, of intact nuclei from unstimulated cells and extracts from estrogen stimulated cells. cDNA clones of apo VLDLII will be selected by plus-minus hybridization. Genomic clones will be selected from our Xenopus library. Using solution hybridization and transcription rate measurements, the essential features of th estrogen induction of apo VLDLII mRNA will be characterized. The 5' flanking sequences in chromatin will be examined for sites hypersensitive to DNase I digestion. DNA methylation patterns of transcriptionally active and inactive apo VLDLII genes and their flanking regions will be determined. The 5' flanking sequences required for efficient and accurate transcription will be identified by construction of a series of 5' and 3' deletion mutants using the Bal 31 digestion method, and injecting these deletions into homologous Xenopus oocyte nuclei. Blot hybridization and moderate probe excess solution hybridization will be used to quantitate the accuracy and efficiency of transcription. An assay for cellular proteins which mediate estrogen regulated gene expression will be established by microinjecting 500-1,000 intact liver cell nuclei, from unstimulated or estrogen stimulated cells, into each Xenopus ooxyte nucleus. If the original liver cell transcription pattern is perserved in oocyte nuclei (this is the case for somatic 5S genes), extracts from estrogen stimulated nuclei and nuclei from unstimulated cells will be coinjected into oocytes in an effort to activate transcription of the apo VLDLII genes.
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