1243 Functional characterization of vertebrate homol of drosophila Nina A protein

a Tdenzification of a 32 kDaiton protein in the bovine retinal pigment epithelium. Hethods A bovine retinal pigment epithelium cDNA library wa* constructed and immunologically screened with monoclonai antibody 21-C3/AV. A positive full-length clone, 21-C3RDH/CD, wan sequenced. Northern blot analysis were performed to analyze the tissue expression pattern. In order 'co analyze the function of the encoded protein, 21-CmDH/CD was expressed in-vitro using the bacul"virus based expression system. Enzymatic activity towards 11-cis retinaldehyde was investigated. &g&&E The complete "ucleotide sequence Of Clone 2iC3RDH/CD, including a" open reading frame consisting of 957 bPt was obtained. The deduced amino acid sequence revealed homology with the superfamily of abort-chain alcohol dehydrogenaaes. "PO" NO??ther" blot analyses a 1.2 kb transcript was oniy detected in the retinal pigment epithelium. Recombinant baculovirus infected insect cells synthesized the 21-C3/AV antigen. This protein appeared to be capable to reduce II-cis retinaldehyde to II-cis retinal. Conclusions Apparent malecular weight, homolqy with shortchain alcohol dehydrogenases and tissue specificity suggested that 21-C3RDH,CD might encode bovine retinal pigment epithelial II-cis retinol dehydrogenase, one of the keyenzymes of the vim.31 cycle. Direct proof came from in-vitro synthesized pr"tei" encoded by 21-C3RDH/CD, which wan found capable of reducing Il-cis retinaldehyde. ARE STUDIES TO LENS SPECIFIC GENE REGULATION IN LENS-DERIVED CELL LINES APPROPRUTE? KRAUSZ E., PIETROWSKI D. and GRAW 1. lnsfiturJ?~r Smgeliergenetik. GSF-Forschungszemwn Neuherberg (Germmy) Punmse: We determined lens specific gene expression in three lensderived cell lines since we wanted to test transcriptional activity of YEcrystallin (CrygE-) promotor fragments in ii reporter gene assay. Methods. RNA was isolated from the cell lines aTN4 from a lens tumor of a T-antigen transfomwd mouse, NKR-11 from N&no cataract mouse, N/N1003A from rabbit and NIH-3T3. Additionally, RNA from mouse and rabbit lenses and non-lens tissues was prepared. The expression of aA-, c&3and yAto yF-crystaltins was demonstrated by RT-PCR. We constructed a series of mouse CrygE promotor deletions extending from 634 to +37 in the reporter gene plawnid pBLCAT6 and atIer transfection into N/N1003A or NIH-3T3 cells we assayed the transcriptional activity for Chloramphenicol Acetyl Transferwe (CAT). &J&Z 1) aA-crystallin is expressed in aTN4 cells, but not in NKR-11 and N/N1003A. Beside the enormous expression in lens, we found only a very weak expression in kidney end spleen. The aB-crystallin is expressed in all cell lines and tissues analysed except liver and spleen. The transcripts of the 6 y-aystallin genes were found in lens tissue only. 2) CAT assay results showed differential activity of the CrygE promotor according to the length, Deletion of the DOTIS-element located &mstream of the Iranscription initiation $te reduced activity to 205/o. In NIH-3T3 cells all CrygE promotor fragments were inactive. Conclusiona Although the lens derived rabbit cell line NiN1003A lacks endogenous expression of aAand all y-crytiins, it n&t be used for timctional pre-analysis of promotor fragments. We demonstrated significant higher promotor activity of a gene preferentially tran.wibexl in lens tissue then in a reference cell line. Some transactivating factors are present in N/N1003A cells, but obviously additional regulatory fscton are required to activate the chromosomaJ gene. Therefore, experimeats in tmnsgenic animals have to be performed.