The long term goal of our research is to identify cataract mechanisms which might be manipulated to prevent or delay development of cataracts. Our studies suggested that initiation of galactose-cataracts may have been aided by downward fluctuations in mRNA concentrations, as manifested by their translational products, possibly leading to enhanced formation of the high molecular weight protein aggregates found in cataractous lens. Gene activity in galactose-cataracts was found not to be totally suppressed; e.g., MP26 mRNA was present at all stages of galactose- cataract development. Further, all of the expected gamma- crystallin mRNA products found in normal fibers were also present, although reduced, in decapsulated lens of mature galactose- cataracts. Therefore, the present studies are aimed at examining the levels of specific gene activity in both galactose-cataracts and sepile cataracts in order to better characterize the observed cooperative response of the lens genes to this disease process. The hypothesis to be tested states that the relative level of abundance of the products of several different mRNAs in lens is a determining factor in maintaining lens clarity; therefore, changes in concentrations of mRNAs will contribute significantly to initiation of cataracts. Overall, the specific aims are to quantiate mRNA levels in galactose-cataracts, reversed galactose- cataracts, sorbinil inhibited galactose-cataracts, in senile cataracts (Emory mouse), and in aging lens of rat, mouse and human. To apply these studies to human senile cataracts, we are proposing to prepare human lens cDNA expression library in lambda gt11 phage to use for the isolation of various crystallin recombinant phage to probe for these mRNA levels in both aging human lens, and possibly in human senile cataracts. The methodology we are proposing to use include contemporary molecular biology methods (hybridization) plus other immunocytochemistry procedures for visualizing the distribution of various gene products in lens undergoing the process of cataractogenesis and aging. The analyze for the various mRNAs found in lens, the mRNAs are translated in vitro, following their isolation from the various lenses, then their 35S-methionine labeled products are separated by two- dimensional gel electrophoresis, localized by fluorography, and the products quantitated by computer-assisted soft laser densitometry. Thus, our procedures will quantitate both the mRNAs and their products in normal lens and lens undergoing cataractogenesis.
