Test Design For Oculopharyngeal Muscular Dystrophy Essay — страница 3

  • Просмотров 315
  • Скачиваний 5
  • Размер файла 18
    Кб

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 repeats. Because patients with OPMD show more effects on their eyes and throat as compared to their legs, hip and shoulders, protein binding for the latter three muscle tissues will be less than that of the former two. We would still expect an increase in protein binding for leg, hip, and shoulder muscles with increasing alanine

repeats. However, this increase is slower than that of eye/throat muscles. Table 1 also shows that for tests with normal repeats (6), there would not be binding if indeed there is protein binding for expanded repeats, no matter which muscle type. If these results occur, there will be a need for further characterizations of the newly found mutant PABP2-binding proteins. Table 1 shows a situation where a specific protein is binding to extended Ala repeats and more of it binds with increasing repeat size. There may however, be different proteins that bind to the different repeats. This possibility will nonetheless still affirm the hypothesis. The protein(s) that bind to the extended poly A stretches will be involved in the progressive destruction of muscle tissue over time (a

characteristic of muscular dystrophies). They will also lead to weakness and loss of muscular function. Because of this senescence phenotype, it can be predicted that the extended poly-A-binding protein(s) may affect(s) proper functioning of telomerases. The protein may also alter the proper functioning of PABP2 and initiate a cascade which results in degradation of muscle tissues. The other possible outcome would be that the decrease in protein binding leads to OPMD (Table 2). The two controls (tests with smooth muscles and tests with 6 Ala repeats) will show significant protein binding. With the onset of the disease, under this scenario, there will be less binding of the particular protein. For experiments with eye or throat muscles, binding is decreased as the number of

extended repeats increase. That same pertains to leg, hip and shoulder muscle tissues, but the decrease will be to a lesser extent. If these results are to occur, identification of the binding protein is needed via sequencing methods (ie. Edman degradation). A prediction is that the protein binds to the poly-A stretches for normal functioning of PABP2, perhaps assisting it to act as a factor in mRNA polyadenylation. In affected tissues, some of these binding proteins may not be able to recognize the extended repeats thus decreasing PABP2’s function. Under these results, the hypothesis would be rejected. For the yeast-two-hybrid experiments, blue colonies will signify the binding of proteins to the different forms of PABP2. If blue colonies appear for tests with the gene

encoding for mutant PABP2s, it will indicate an acceptance of the hypothesis. These positive clones will need to be analyzed further by restriction analysis, PCR, or by sequencing. These analytical methods will show if the same protein binds to all forms of the mutant or if different proteins bind to the varying mutant forms. The proteins that bind to PABP2 may bind specifically to the poly-A stretches or to other sites of PABP2. This system, however, cannot distinguish these two possibilities. On the other hand, if no colonies appear blue for tests with mutant PABP2s, there are no proteins that bind to them. This result will help reject the hypothesis. It may also show that the lack of binding to a particular protein specific for wild-type PABP2 results in the disease. In this