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.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
5R01GM034432-05
Application #
3285379
Study Section
Molecular Cytology Study Section (CTY)
Project Start
1985-03-01
Project End
1993-03-31
Budget Start
1989-04-01
Budget End
1990-03-31
Support Year
5
Fiscal Year
1989
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