Studies on the molecular cloning, characterization and expression of a DNA binding protein (RF-36

A molecular clone containing the partial gene of a GC-rich binding protein (RF-36), was isolated from a bovine lens cDNA library. The cDNA-encoded protein was expressed as a beta-galactosidase fusion product. Selection was by in situ immunoassay followed by Northern blotting analysis and hybridization-selection. Northern hybridization indicated that the DNA of the antibody-screened lambda clone bound with a lens 18S RNA. This DNA was subsequently expressed by in vitro translation, producing products recognized by RF-36-specific monoclonal antibodies. In vivo expression in E. coli was regulated by the beta-galactosidase gene; gene expression was observed only upon induction by IPTG and recombinant-encoded peptide bands (36-KDa) were identified using RF-36 monoclonal antibody. Therefore, the antibody-screened clone contained cDNA that expressed RF-36 protein as a product of about 36-KDa, cleaved from a beta-galactosidase fusion product. The RF-36 cDNA sequence was analyzed by the dideoxy method.;The deduced amino acid sequence of RF-36 was shown to be about 79% similar to zeta-crystallin, which is related to alcohol dehydrogenase. Immunoblot analysis showed immuno-crossreaction between RF-36 and zeta-crystallin, corroborating a homogeny relationship between RF-36 and zeta-crystallin. In contrast to RF-36, zeta-crystallin does not cooperatively enhance DNA conformational changes from supercoiled to relaxed forms. Thus, these two homologous proteins have a distinct functional dissimilarity. If RF-36 had the potential to be a structural protein in the bovine lens, as suggested by its similarities to the structural zeta-crystallin of the guinea pig lens, some crucial differences in structure have destined both proteins to serve quite different functions in the lens of each species. By that distinction RF-36 is not a zeta-crystallin, but it may be a divergent form derived from the alcohol dehydrogenase family, which has come to serve specific functions for bovine lens cell differentiation and development. This new hypothesis remains to be tested.