The molecular basis of mammary specific gene expression is being studied through analysis of cis-acting regulatory elements in milk protein genes and their cognate trans-acting factors. Applying mobility shift assays, Exonuclease III and DNase I protection, it was shown that nuclear proteins from mammary epithelial cells form a multiple nucleoprotein complex with the whey acidic protein (WAP) gene promoter/upstream region. Whereas some of the DNA sequences were recognized by proteins present in a variety of different cell types, other sequences were recognized by proteins preferentially or exclusively present in mammary gland nuclear extract. Furthermore, a promoter fragment of the WAP gene, encompassing the sites of protein-DNA interaction was found to confer the expression of 'non-mammary' genes in lactating mammary glands of transgenomic animals. This suggested a physiological role of the protein binding sites. In the second project an in vitro system is being established which mimics the in vivo activation of the human cytomegalovirus (HCMV). This might allow the study of molecular mechanisms of viral gene activation . Upon virus infection in HCMV, immediate early gene 1 (IEI) is the first viral gene to be expressed. It appears that the IEI gene enhancer mediated transcriptional stimulation in vitro involves its recognition by specific nuclear trans-acting factors. DNase I protection analyses revealed at least 13 sites in the enhancer/promoter region that interact specifically with nuclear proteins. A correlation was made between protein binding to specific DNA sequences in the enhancer/promoter region and transcriptional stimulation in vitro.
