Based on preliminary and published data from my laboratory, it seems likely that both metabolism of the myosin heavy chain (MHC) polypeptide and expression of the MHC gene are deranged in muscle cell cultures prepared from the breast muscle of line 307 dystrophic chick embryos. Thur, the long-term goal of this research is to define the molecular events that result in aberrant expression in dystrophic muscle of the gene for the predominant contractile protein.
The specific aims of the present project are as follows: 1) To quantitatively compare normal and dystrophic muscle cell cultures for their abilities to synthesized, accumulate and degrade myosin heavy chain. These experiments require measurement of the specific radioactivity of [3H]Leu associated with trna and assessment of reutilization of amino acids from protein breakdown. 2) To measure the transit times of ribosomes along MHC mRNA in normal and dystrophic muscle cells. 3) Using pBR322 clones containing partial sequences of the MHC gene, to quantitate the amount of MHC mRNA associated with active polysomes (and therefore undergoing active translation) and the amount of MHC mRNA that is nonpolysomal (and therefore not actively involved in translation) in normal and dystrophic muscle cells. 4) If data from the above experiments indicate that dystrophic muscle cells contain higher levels of MHC mRNA, an attempt will be made to determine if higher MHC mRNA concentration in dystrophic cells results from a faster initiation rate of transcription by RNA polymerase II. This research should ultimately result in identification of the exact cellular level of aberrant myosin gene expression in line 307 dystrophic chicks. The myosin molecule has been selected for study because its unique size and solubility characteristics permit large numbers of experiments, and because it alone makes up almost a thire of the total protein in skeletal muscle tissue. Additionally, it is felt that a more meaningful understanding of the aberration can be gained by concentrating on one specific myofibrillar component, rather than measuring average metabolism of a heterogenous protein mixture. In summary, successful completion of the present research will provide significant new information about the nolecular biology of genetic muscular dystrophy.
