Molecular Genetics of a Lens Intrinsic Membrane Protein

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DESCRIPTION (provided by applicant): The ocular lens is an avascular, semisolid organ whose function is to refract incoming light upon the retina. Concomitant with this function is a necessity for the lens to remain transparent. A loss of transparency of the lens is usually permanent and is termed a cataract. Cell and organ functions, such as obtaining nutrients necessary for maintaining the healthy, transparent lens state, for handling of cell waste removal, and for maintenance of overall membrane stability are carried out through select and specific proteins or groupings of proteins within the lens cell membrane. Proper controls on expression of these proteins and their subsequent function is essential for lens survival, and maintenance of transparency. The mechanisms regulating these membrane proteins in the lens are presently unknown. The long term goals of this project are to understand the function of the lens fiber cell intrinsic membrane protein MP19 and the regulation of the gene coding for this protein (LIM2). To achieve this goal, we will investigate the following topics: a). Using knockout mouse technology, to ablate the Lim2 gene in order to study the function of MP19. b). Using mammalian expression vectors, the investigate the synthesis of MP19 and various mutations of this coding sequence. Truncations of the MP19 expressed protein will be made to investigate the membrane topology of MP19. Finally, we will use these expression vectors to investigate the putative signal residing in the MP19 molecule which effects transport of MP19 to the cell membrane. c). Investigate functional consequences of point mutations in the Lim2 gene using site-specific RNA/DNA chimeric oligonucleotides (CO). Specific CO will be injected into mouse pronuclei and subsequently implanted in foster mothers. Pups containing successful mutations will be investigated to determine the effect of the mutation on lens development and integrity. These studies will give a better understanding of the role of MP19 in the maintenance of lens transparency and its possible role in cataract formation.