| Retinitis pigmentosa (RP) is a genetically heterogeneous disorder characterized by progressive loss of photoreceptors by apoptosis. In 1998, a Spanish group performed linkage analysis in a large nuclear Spanish family with autosomal recessive retinitis pigmentosa, and fine mapped the RP26 locus in this family to a 2.5-Mb region on chromosome 2q31.2-q32.3. They identified a novel gene, CERKL, in the RP26 critical linkage region and found a homozygous mutation in exon 5 of the gene in two Spanish families. However, the physiological functions of CERKL in retina and molecular mechanism by which CERKL mutation causing RP remains unclear. There have been only 5 publications on CERKL reported until now. In order to study biology function of CERKL and reveal its pathway in the retina, we cloned the full-length CERKL gene and screened the proteins interacted with CERKL by using Yeast Two-Hybrid System.We firstly cloned the full-length CERKL gene using nested PCR from HeLa cell cDNA library. Secondly, the Yeast Two-Hybrid System (Clontech) was used to screen a human pancreas GAL4-AD-cDNA library with human CERKL cDNA fused to GAL4-BD in plasmid pGBKT7 as bait. Then we transformed the pGBKT7-CERKL into yeast AH109 to examine the toxicity and self-activation of pGBKT7-CERKL plasmid. After that, pGBKT7-CERKL and the library plasmids were transformed sequentially into yeast strain AH109. The transformants were plated on SD agar to screen for interacting partners according to the manufacturer's instructions.560 preliminary positive clones were further checked by phenotypes retesting, clones sorting andα-galactosidase activity detecting to exclude the false positive clones. Plasmids recovered from positive clones were amplified by PCR for the inserts and sequenced. The sequencing results of the candidate clones were analyzed using the BLAST database to find the potential interaction of proteins. Two proteins, CTRC(Homo sapiens chymotrypsin C preproprotein) and EIF3S9(Homo sapiens eukaryotic translation initiation factor 3 subunit 9), were identified as the candidate interacting proteins. In conclusion, we screened the proteins interacted with CERKL using Yeast Two-Hybrid System and found two potential candidates. It is the first time that proteins which may interact with CERKL were reported in the world. However, further studies on their biological function of these interactions are needed, which will shed more light on the understanding of the structure and function of CERKL in retina, and also of the pathogenesis by which mutations of CERKL caused retinitis pigmentosa. |
