There are two long range goals of research proposed in this grant application: A. To determine the lens-specific genes and their mRNA sequences in order to establish the structural functional relationship for the information transfer processes from lens genes to protein. B. To elucidate the molecular mechanisms leading to the control of specific lens gene expression in order to develop better understanding of the genetic regulation which governs the normal differentiation and pathological development of the lens. The proposed project represents an extension of continuing studies on the lens molecular biology as related to lens gene's structure and controlling mechanisms. In an effort to establish the basic molecular parameters and to elucidate the regulatory mechanism of lens gene expression, we are planning, over the next five years, to concentrate on: I. Identification and characterization of a regulatory gene product; II. Determination of chemical and regulatory properties; III. Immunological and molecular cloning studies of regulatory gene's expression. I. In order to identify a regulatory gene product, specific affinity chromatography such as DNA-cellulose, heparin-agarose and chromatofocusing, as well as various physicochemical techniques for nucleic acid binding assay, will be employed for the initial isolation and characterization. Polyclonal and monoclonal antibodies will be prepared and used for: 1. mRNA isolation; 2. evaluation of cell-free product by Western blotting, 3. Immunological screening of cDNA expression libraries. II. Conformational changes in DNA superhelical structure induced by regulatory element will be measured in terms of linking number change by agarose electrophoresis and density gradient. Effect on lens chromatin and nucleosome in terms of structuralfunction relationship will be examined by transcriptional and hybridization studies. Molecular mechanisms of translational effect on ternary and initiation complexes will be studied by using purified elF-2 and GEF systems. III. Immunohistological localization of regulatory protein in the lens topological region will be examined by immunofluorescent and PAP methods. Molecular cloning of cDNA and genomic DNA sequences will be performed by the well established cloning techniques. DNA sequences will be analysed by the chemical and Dideoxy chain termination in M-13 sequencing system.
| Jornvall, H; Persson, B; Du Bois, G C et al. (1993) Zeta-crystallin versus other members of the alcohol dehydrogenase super-family. Variability as a functional characteristic. FEBS Lett 322:240-4 |
| Gagna, C E; Mitchell, O G; Chen, J H (1991) Fixation and immunolocalization of left-handed Z-DNA sequences in the calf lens. Lens Eye Toxic Res 8:489-509 |
| Chen, J H; Tong, T C; Zhang, L (1991) Rapid enhancement of ""MIP"" 26kDa protein phosphorylation by RF-36 nucleic acid binding protein in lens cells. Lens Eye Toxic Res 8:469-87 |
| Gagna, C E; Chen, J H; Lavers, G C et al. (1991) The presence of Z-helical conformation in DNA of the calf lens. Lens Eye Toxic Res 8:27-42 |
