The overall goal of the proposed research is to determine the molecular basis for tissue specific gene regulation. We have shown that activation of a number of liver specific human genes occurs when somatic cell hybrids are made between a differentiated mouse hepatoma line and non-hepatic human diploid cells. These studies and others suggest that trans-acting factors are responsible, at least in part, for the tissue specificity of gene expression. It has also been established that cis-acting sequences or elements play a role in differentiated gene expression. We propose to examine the genetic and biochemical basis for the regulation of genes that function only in certain cell lineages. We will utilize gene transfer of flanking and internal regions of genomic DNA for human albumin and complement C3 coupled to chloramphenical acetyltransferase. This bacterial gene will serve as a marker of transcription promoting activity of the human DNA segments following DEAE-dextran mediated gene transfer into human hepatoma and non-hepatic cell recipients. Various treatments of the hepatoma cells containing promotor regions of albumin and C3 will enable us to define sequences that direct liver specific expression, density dependent expression of albumin, inhibition of albumin and stimulation of C3 by interleukin-1 (Il-l).
The second aim of the proposal is to generate mutants that are deficient in transacting factors required for albumin expression. We will employ techniques of gene transfer, mutagenesis and cell hybridization to identify variants that meet the criteria for a trans-acting factor mutant. These mutants will be characterized genetically by complementation and gene expression analyses. They will also be examined biochemically for alterations in the pattern of DNA binding proteins. A third major effort will be devoted to identifying DNA binding proteins that are specific to the cis-acting regions identified by gene transfer. Thus we will search for proteins that are unique to liver and that may modulate in response to cell density and Il-l. The final goal of this proposal is to clone genes responsible for the production of transacting factors. The scheme for this aim utilizes variant mouse hepatoma cell lines that behave like trans-acting factor mutants and transfer of human genomic DNA. Transfected cells will be selected for the presence of a human gene that compliments the mutant phenotype.
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