Term paper on Test Design For Oculopharyngeal Muscular Dystrophy
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Protein Binding Studies for Expanded Poly-A Repeats and Mutant PABP2 resulting from Oculopharyngeal Muscular Dystrophy
INTRODUCTION:
Oculopharyngeal muscular dystrophy (OPMD) is an inherited neuromuscular genetic disorder. It has an autosomal dominant pattern of inheritance (Fried et al. 1975) in that the abnormal gene can be transmitted from only one parent. A child of an affected parent has a 50% chance of being affected. The disorder is found to be more prevalent among French-Canadians and is characterized by its late onset (approximately 50). Affected persons experience dropping eyelids (optosis), difficulty with swallowing (dysphagia), and some develop shoulder, hip or leg weaknesses (MDA publications 1998). Genetically, its mutation is quite unique. OPMD is caused by the expansion of a GCG (which codes for the amino acid alanine) 6 repeat (Brais et al. 1998), whereas most triplet repeat disorders are expansions of CAG (glutamine) repeats. Rare polymorphisms would be to have 7 consecutive GCG's, but the disease is mostly characterized by the mutation of having 8-15 consecutive GCG's. Other findings have shown that even the expansion of a 6 GCG repeat to 7 can also lead to OPMD (LaFontaine 1996). The severity of the disease depends on the number of extra alanines. Quite recently, scientists have found that the mutation occurs on chromosome 14 and is in the gene coding for a poly(A)-binding protein 2 gene (PABP2) (Brais et al. 1998). PABP2 was considered a good candidate for OPMD because it maps to the same location as the diseased gene, its mRNA is highly expressed in skeletal muscle, and the PAB2 protein is exclusively localized in the nucleus, where it acts as a factor in mRNA polyadenylation. The site of the additional GCG expansions in the PABP2 gene is at the polyalanine tract at the N terminus. From these findings, one may ask why this disease targets preferentially the skeletal muscle cells of the eyes and throat when the protein 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 specific DNA sequences and one that activates transcription. This modular structure is best illustrated by yeast experiments showing that the DNA-binding domains or activation domains can be exchanged from one transcription factor to the next and retain function. A crucial consequence of the modular nature of transcription activators is that the DNA-binding and activation domains do not need to be covalently attached to each other for activation to occur. Because of this, yeast transcription could be used to assay the interaction between two proteins if one of them is fused to a DNA- binding domain and the other is fused to an activation domain. The system contains three components: Yeast vectors for expression of a known protein fused to a DNA-binding domain, yeast vectors that direct expression of cDNA-encoded proteins fused to a transcription activation domain, and yeast repoter genes that contain binding sites for the DNA-binding domain. For our purposes, we can use this system to screen a cDNA library for clones expressing proteins that interact with the different forms of PABP2 (wt and varying mut forms).
METHOD:
Previous studies on other triplet repeat disorders, such as Huntington's disease and fragile-X syndrome, have used a procedure called Repeat Expansion Detection (RED) method to detect and isolate the repeats (Rifugo, G. 1997). Although these studies examined glutamine repeats, the RED method can also be used for alanine repeats. The resulting isolated alanine repeats can then be used for our binding experiments.
The affinity chromatography experiments using polystyrene beads separate proteins based on affinity for a specific ligand, in our case, the alanine repeat peptides . As illustrated in Figure 1, the polystyrene beads in the column are first coated with the alanine peptide of choice. Experiments will first be performed with synthetic 6-alanine peptides as controls because this is the number of alanine repeats that 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 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...
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