During terminal skeletal muscle differentiation, proliferating myoblasts withdraw irreversibly from the cell cycle and fuse to form multinucleated myotubes. A similar loss in proliferative activity occurs during differentiation of other cell types and during cell senescence. Despite its widespread occurrence in developmental systems and its involvement in disease states such as cancer, the mechanism by which a cell selectively represses its replicative machinery is rarely studied and poorly understood. Thymidine kinase (TK) is one of several """"""""replication-associated"""""""" gene products that rapidly declines during muscle cell differentiation. This regulation is mediated by a rapid and specific decline in the abundance of TK mRNA. TK mouse myoblasts transformed with the cloned chicken TK gene also appropriately regulate TK enzyme activity during differentiation. Conversely, myoblasts transformed with the cloned herpes simplex virus (HSV) TK gene continue to express high levels of TK activity during differentiation. Using an approach based on in vitro modification of the gene prior to its transfection, our experiments have thus far eliminated upstream promoter elements or downstream polyadenylation signals as playing a necessary role in regulation of cellular TK activity. The regulatory information resides entirely within an intragenic region bounded by the translation start and stop codons. The two-fold goal of the experiments described in this research proposal is to precisely locate the regulatory information within this intragenic region and to determine at what level that information is expressed. This will lead to a greater understanding of how the replicative state of a cell as a whole is regulated.
