t correspond to the poly-A tract at the N terminus of normal PABP2 proteins. Then, various homogenized muscle tissues are packed into the column. Since OPMD is known to affect mostly skeletal muscles of the eye, throat, hip, leg and shoulder, binding tests involving the alanine repeat peptides will be done on such muscles. As well, tests using smooth muscles are used as contols because it is known that the disease does not affect this type of muscle. After the tissues pass through the column, only the proteins with high affinity for the alanine peptides will bind. All the nonbinding proteins will flow through the column. The bound protein will be dislodged from the alanine peptide coated beads and eluted with a concentration solution containing the alanine peptide. The experiments will be repeated using varying number of repeated-alanine peptides, from 7 to 14. These tests will represent the mutated forms for the disease. The expected results are shown on Tables 1 & 2.To investigate if there are proteins that bind to the OPMD induced mutated forms of PABP2 proteins, the yeast-two-hybrid system is used. As shown in Figure 2, the bait' plasmid will contain a DNA sequence encoding the Gal4 DNA-binding domain fused to the coding sequence for the type of PABP2 protein being examined. Again, different experiments will be performed using genes encoding for PABP2 proteins with 6 (control) to 14 alanine repeats. The Gal4 binding domain is used because it is efficiently localized to the yeast nucleus where it binds with high affinity to the well-defined upstream activating sequence (UAS) which is placed upstream of the reporter gene (lacZ). The prey' plasmids include individual cDNAs from a library fused to the coding sequence for Gal 4 activation domain. The fused genes in both the prey' and bait' plasmids can be achieved by recombinant DNA technology. Each type of plasmid also contains a wild-type selection gene (TRP1 or LEU2) so ...
