as to provide a selection for cells that have taken up both plasmids.Firstly, both bait' and prey' plasmids are transfected into yeast cells with mutations in genes required for tryptophan and leucine biosynthesis and then grown in the absence of tryptophan and leucine. The cells also contain the reporter gene construct. Only cells that contain the bait plasmid and at least one prey' plasmid survive under these selection conditions. If there is protein binding, it will bring the Gal4 activation domain within reach of the start of the lacZ reporter gene and will activate transcription. If there is no protein binding, no activation will occur. A screen for the activation of the lacZ reporters is performed by plating yeast on indicator plates that contain X-Gal. On this medium yeast in which the reporters are transcribed produce beta-galactosidase and turn blue. DISCUSSION:The results for the two types of protein binding experiments can address the hypothesis of the existence of proteins binding to the extended poly-A repeats and to the mutant PABP2 proteins. In terms of the affinity chromatography experiments, there may be two possible outcomes. One set of experiments could demonstrate that there is in fact proteins that bind to the expanded poly-A peptides to cause clinical manifestations of OPMD. These results will therefore confirm the hypothesis. The results of this potential outcome are illustrated in Table 1. Under the assumption of protein binding leading to OPMD, we can expect no protein binding to all forms of the alanine peptide when testing with smooth muscles. This is because the disease is known to only affect skeletal muscles. For eye and throat skeletal muscles, we can expect no protein binding for normal poly-A repeats but find protein binding for repeats of 7-14. The amount of bound proteins also increase from 7 to 14 alanine repeats, thus accounting for the disease's increase in severity with additional rep...
