The goal of this application is to use the approach of molecular genetics to understand the interactions between protein components of myofibrils as they relate to function. Specifically, the intent is to understand the interactions between the myosin heavy chain and the myosin light chains as they relate to the structure of the myofibril and the activity of the myosin linked ATPase. Drosophila will be used as a model system to look at the interaction of myofibrillar proteins. Using classical mutagenesis screens, temperature sensitive myosin light chain mutations will be isolated. The effect of thes mutations upon the ultrastructrue and activity of the myofibril will be characterized. The mutations will then be mapped at the molecular level by DNA sequencing. By in vitro mutagenesis of the cloned myosin light chain-2 gene the site of phosphorylation will be determined. The role that phosphorylation plays in regulating contractile activity in vivo will be assessed by transforming the germline chromosomes with an in vitro mutagenized gene that encodes a myosin light chain that cannot be phosphorylated by myosin light chain kinas In addition the function of the calcium binding domain will be determined by engineering the protein so that it no longer can bind calcium, then the mutagenized gene will be transformed into the Drosophila germone chromosomes. Transformants will be characterized at the biochemical as well as ultrastructural level. The hypothesis that the myosin alkali light chain EF hand domain is a bindin site for either the myosin light chain-2 or the myosin heavy chain will be addressed by site-directed mutagenesis of the cloned gene. The structure of this domain will be altered by replacement of acidic amino acid side chains (GLU and ASP) with basic amino acid side chains (ARG and LYB). The mutagenized genes will be transformed into the germline and the effect of the mutations assessed.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
2R01GM033270-04
Application #
3282741
Study Section
Molecular Cytology Study Section (CTY)
Project Start
1984-06-01
Project End
1990-05-31
Budget Start
1987-06-01
Budget End
1988-05-31
Support Year
4
Fiscal Year
1987
Total Cost
Indirect Cost
Name
Ohio State University
Department
Type
Schools of Arts and Sciences
DUNS #
098987217
City
Columbus
State
OH
Country
United States
Zip Code
43210