c 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 tha...
