otein of the wild- type form of the mutant gene (PABP2) is expressed in all cells. In order to answer this probing question, binding studies involving the abnormal poly A stretches and the mutant PABP2 protein need to be performed. This will determine what other proteins (if any) are involved in this particular type of muscular dystrophy. In this project, I hypothesize that there are proteins from affected tissues which bind to the expanded poly A stretches as well as the mutant PABP2 protein. These proteins may also bind to them in varying amounts depending on the length of the expanded GCG repeats.. To find out if any proteins bind with extended repeats of the corresponding mutant PABP2 protein, affinity chromatography experiments can be done. This type of experiment will involve polystyrene beads, coated with synthetically made poly-A peptide representing the mutant protein domain, and are packed with various homogenated human muscle tissues. Human tissues will be used because the disease only seem to affect humans. The synthetic peptides will be of varying repeats of alanine, thus testing for the different severities of the disease. Other molecular studies investigating OPMD examined up to 13 repeated GCGs. Therefore, for the polystyrene beads experiment, repeats of 6 to 14 will be used. A test with 14 repeats will determine whether there is still a continual increase in severity after 13 repeats or whether the effects will be abolished past the 13 repeat mark. An experiment to elucidate potential proteins that bind to the mutant PABP2 protein involved in OPMD is the yeast-two-hybrid system. The method uses the transcription of yeast reporter genes as a synthetic phenotype to detect protein-protein interactions. The approach takes advantage of the modular domain structure of eukaryotic transcription factors. Many transcription activators have at least two distinct functional domains, one that directs binding to specifi...
