During terminal skeletal muscle differentiation, proliferating myoblasts withdraw irreversibly from the cell cycle and fuse to form biosynthetically active myotubes. A similar loss in proliferative activity occurs during differentiation of other cell types and during cell senescence. The transition to a postreplicative state is accompanied by a rapid decline in the activity levels of many enzymes involved in DNA precursor biosynthesis. A thorough understanding of the molecular mechanism governing replication-dependent expression of this class of gene products is an important step in defining the overall process by which the proliferative state of cells is regulated. Using mouse muscle cells transformed with the cellular chicken TK gene, the following progress has been made. First, the decline in TK enzyme activity is accompanied by a transcriptionally- mediated decline in TK mRNA levels. Second, the decline in mRNA does not account fully for the decline in enzyme activity; a translational or posttranlational process must also contribute to the overall regulatory mechanism. Third, the cis acting information causing TK mRNA levels to be regulated have been mapped to two locations within the TK gene; one within the protein-coding region introns, the other within the 3' non- translated region. Only by deleting both the introns and the 3' nontranslated region is the chicken TK gene converted from a regulated to a constitutive phenotype. During the next grant period, the following specific objectives will be met: 1) the translational or posttranslational mechanism affecting TK enzyme levels will be characterized by immunological determination of TK protein steady state level, synthetic rate, and degradation rate in proliferating and committed muscle cells; 2) the cis acting regulatory elements governing TK mRNA levels will be precisely identified by in vitro mutagenesis; once identified, the elements will be manipulated to determine if they have enhancer properties; 3) trans acting factors that recognize the cis acting elements will be investigated by DNA footprinting and gel retardation assays. The factors coordinating expression of replicative enzymes are the final mediators of events triggered by oncogene activation or mitogenic signaling. In establishing the regulatory hierarchy, important components in the process of cell growth control will be resolved.
