The Study of Shark γ-Crystallin Gene

• Main Authors: Chuang,Ming-Hong, 莊明��
• Other Authors: Chiou,Shyh-Horng
• Format: Others
• Language: zh-TW
• Online Access: http://ndltd.ncl.edu.tw/handle/10564403646545304841

碩士 === 國立臺灣大學 === 生化科學研究所 === 82 === Gamma-Crystallin is a ubiquitous lens protein of most vertebrate eye lenses and the major component in lenses of fishes and many mammals, especially during embryonic and neonatal stages. To date it seems to be absent in the birds and some reptiles. To facilitate the cloning and sequencing ofγ- crystallin gene,cDNA was constructed from poly(A)+RNA of shark lenses (Chiloscyllium colax), amplified by the polymerase chain reaction (PCR), using the 5'- and 3'- coding regions of carp γ- crystallins(γs,γm1,γm2) as forward and reverse primers. The PCR products pretreated with Klenow fragment and polynucleotide kinase was subcloned into pUC18 vector and then transformed into E. coli strain JM109. Four positive γ-cry clones of shark were obtained and processed for nucleotide seq- uencing. Through sequencing by Sanger's termination method, we have found these cDNAs can be seperated into two classes; one is mammalian-like γ-cry, which comprises γs-1,γs-2,γm2 (about 65% homology in protein sequence with bovine γB) with low Met content (5-6%).The other class is teleostean γ-cry ( about 80% homology in protein sequence with carp γm1),that isγm1, unlike γs-1,γs-2,and γm2, which contains high Met con- tent(-16%). The structural analysis of the crystallins deduces from these cDNAs is carried out in order to shed some light on the molecu- lar evolution of these structurally homologous proteins.Pre- liminary study on the genomic structure encoding these crystall ins were also presented.