Studies on mechanisms regulating hepatic protein synthesis have evolved into analysis of liver-specific gene function using molecular hybridization and cell-free protein synthesis. We have developed in situ hybridization to detect mRNAs of high (albumin) and low (proalpha2-collagen) abundance in individual hepatocytes, in vitro transcription with isolated nuclei to directly measure transcription rates for liver-specific genes under various experimental conditions and transfection methods for expressing foreign genes in primary rat hepatocytes and hepatoma cells. Our current specific aims are (1) to study the mechanism regulating transcription and processing of albumin mRNA in normal rat liver and an analbuminemic rat model in which abnormal albumin mRNA processing is thought to occur, (2) to study the role of an acute hepatotoxic agent (CCl4) on transcription of liver-specific versus general cellular genes, (3) to determine whether chronic CCl4 administration induces hepatocytes to synthesize collagen, and whether such induction participates in development of hepatic fibrosis, (4) to study regulation of specific gene transcription in developing and regenerating liver, and (5) to develop a method for introducing molecularly engineered plasmid vectors containing specific genes of interest into hepatocytes and/or hepatoma cells. Specific genes to be studied include albumin, Alpha-fetoprotein, apolipoprotein A1 and E, Alpha1-antitrypsin (secreted proteins synthesized in liver), ligandin and cytochrome P-450 (intracellular proteins enriched in liver), collagen, Beta-actin and Alpha-tubulin (genes of general cellular function), and Beta-globin and pBR322 as negative controls. In preliminary studies, we have observed immediate reduction in albumin gene transcription following administration of a single dose of CCl4, followed by a sharp increase in Alpha-fetoprotein transcription, and later, increased albumin transcription as the liver regenerates. In analbuminemic rats, albumin transcription responds normally to acute CCl4 administration; however, Alpha-fetoprotein transcription does not increase. This model will be used to study factors regulating transcription of these two genes in coordinate or non-coordinate fashion. Collagen gene expression and specific cells in the liver containing collagen mRNA during development of hepatic fibrosis and the role of corticosteroids in ameliorating this response will be studied by in situ hybridization together with collagen synthesis measurements. The role of cis or transacting factors in regulating albumin and Alpha-fetoprotein expression will be studied by introducing plasmid expression vectors containing these genes and/or upstream regulatory elements into hepatocytes and/or hepatoma cells in culture. Finally, attempts will be made to introduce biologically active albumin genes into analbuminemic rat hepatocytes to obtain stable expression of these genes and if clones become available, to perform similar studies for UDPGT-deficiency in the Gunn rat.
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