This proposal represents a continuation of our efforts to investigate mechanisms regulating synthesis of specific proteins in the liver. Our long range goal is to understand at the molecular level how various forms of hepatocellular injury, pathophysiologic states, and genetic diseases cause alterations in hepatic protein synthesis. Using recombinant DNA, molecular hybridization and methods of gene transfer, we will use the rat genetic model of analbuminemia to study 1) events controlling the albumin mRNA steady-state level, 2) factors responsible for increased expression of the albumin gene in this mutant during liver regeneration and 3) to identify the specific abnormality in albumin mRNA biogenesis. We have observed that a single dose of CCl4 causes a marked increase in the albumin mRNA steady-state level in the liver of analbuminemic rats. We hypothesize that the cell type synthesizing albumin mRNA during liver regeneration in the analbuminemic rat is the specific cell type that has undergone division and is in the process of differentiating into a mature hepatocyte. This hypothesis will be tested by identifying the cell type induced to synthesize albumin mRNA in the analbuminemic rat during liver regeneration. Studies will involve 1) isolation of specific cell types from the liver, 2) molecular hybridization to identify specific cell types containing albumin mRNA, 3) in situ hybridization to identify the same phenomenon in vivo and 4) cytochemical, immunohistochemical and morphologic analysis to determine the lineage of this cell type. Plasmid vectors have been prepared with rat albumin regulatory sequences and the complete human albumin cDNA sequence. These vectors will be introduced into isolated hepatocytes, hepatoma cell lines and liver epithelial cell lines in attempts to obtain both transient and/or permanent expression of albumin RNA and protein. Finally, studies will be performed to transplant cells (hepatocytes and/or epithelial cell lines) containing introduced human albumin cDNA into analbuminemic rats to obtain expression of albumin mRNA and study its regulation in vivo compared to that of the mutated endogenous gene.
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