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.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
5R01GM034432-03
Application #
3285378
Study Section
Molecular Cytology Study Section (CTY)
Project Start
1985-03-01
Project End
1988-03-31
Budget Start
1987-03-01
Budget End
1988-03-31
Support Year
3
Fiscal Year
1987
Total Cost
Indirect Cost
Name
Oregon State University
Department
Type
Schools of Arts and Sciences
DUNS #
053599908
City
Corvallis
State
OR
Country
United States
Zip Code
97339
Schmidt, E E; Merrill, G F (1991) Changes in dihydrofolate reductase (DHFR) mRNA levels can account fully for changes in DHFR synthesis rates during terminal differentiation in a highly amplified myogenic cell line. Mol Cell Biol 11:3726-34
Schmidt, E E; Owen, R A; Merrill, G F (1990) An intragenic region downstream from the dihydrofolate reductase promoter is required for replication-dependent expression. J Biol Chem 265:17397-400
Schmidt, E E; Merrill, G F (1989) Transcriptional repression of the mouse dihydrofolate reductase gene during muscle cell commitment. J Biol Chem 264:21247-56
Gross, M K; Merrill, G F (1989) Thymidine kinase synthesis is repressed in nonreplicating muscle cells by a translational mechanism that does not affect the polysomal distribution of thymidine kinase mRNA. Proc Natl Acad Sci U S A 86:4987-91
Schmidt, E E; Merrill, G F (1989) Maintenance of dihydrofolate reductase enzyme after disappearance of DHFR mRNA during muscle cell differentiation. In Vitro Cell Dev Biol 25:697-704
Merrill, G F (1989) Clonal derivation of a rat muscle cell strain that forms contraction-competent myotubes. In Vitro Cell Dev Biol 25:471-6
Gross, M K; Merrill, G F (1988) Regulation of thymidine kinase protein levels during myogenic withdrawal from the cell cycle is independent of mRNA regulation. Nucleic Acids Res 16:11625-43
Gross, M K; Kainz, M S; Merrill, G F (1987) The chicken thymidine kinase gene is transcriptionally repressed during terminal differentiation: the associated decline in TK mRNA cannot account fully for the disappearance of TK enzyme activity. Dev Biol 122:439-51
Gross, M K; Kainz, M S; Merrill, G F (1987) Introns are inconsequential to efficient formation of cellular thymidine kinase mRNA in mouse L cells. Mol Cell Biol 7:4576-81
Merrill, G F; Tufaro, F D (1986) Structural and functional analysis of an alternatively spliced chicken TK messenger RNA. Nucleic Acids Res 14:6281-97