Crystallins are the principal structural proteins of the eye lens, comprising 90% of the soluble protein. Conservation of sequence within the alpha-and beta-crystallin families suggests that stringent requirements exist for the structure of these proteins for maintenance of lens transparency. This project will examine the relationship of crystallin structure to the properties of protein stability and intermolecular associations and the effect changes in these properties produce on lens transparency. Objectives of this project include delineation of structure-function relationships for the lens crystallins and investigation into the role of crystallins in cataractogenesis. We have ligated the RSV promoter and the alphaA-crystallin promoter to the 5' end of modified betaA3/A1-crystallin sequences. These constructions will be used to express modified crystallins in tissue culture cells and to create transgenic mice expressing abnormal amounts or types of crystallin proteins respectively. In modifying the betaA3/A1-crystallin coding sequence we have taken advantage of the large amount of structural and sequence information available about this family of proteins. We have altered specific amino acids felt to be vital for maintaining the Greek key motif structures by site specific mutagenesis. The N-terminal arms, felt to be important in intermolecular associations, are being modified, deleted, and interchanged. In a related set of experiments, we have ligated 173 bp surrounding the translation termination site of betaA3/A1- crystallin downstream and antisense to the alphaA-crystallin promoter and created 6 lines of transgenic mice in which the transgene is co-inherited with autosomal dominant cataracts. We are monitoring the expression and effects of these constructions on crystallin structure and interactions by clinical ophthalmological examination, Southern blot analysis (for DNA), Northern blot and S1 analysis (for RNA) and a combination of one and two dimensional gel electrophoresis, Western blot, and histological analysis (for proteins). These studies will provide insight into the importance of crystallin structure for the functions of stability and protein interactions and delineate the role of lens crystallins in hereditary cataracts.
