Lin C W, Chiou S H
Laboratory of Crystallin Research, National Taiwan University, Taipei.
FEBS Lett. 1992 Oct 26;311(3):276-80. doi: 10.1016/0014-5793(92)81119-7.
delta-Crystallin is a major lens protein present in the avian and reptilian lenses. To facilitate the cloning of the delta-crystallin gene, cDNA was constructed from the poly(A)+ RNA of pigeon lenses, amplified by the polymerase chain reaction (PCR). The PCR product was then subcloned into pUC19 vector and transformed into E. coli strain JM109. Plasmids purified from the positive clones were prepared for nucleotide sequencing by the dideoxynucleotide chain-termination method. Sequencing two clones, containing 1.4 kb DNA inserts coding for delta-crystallin allowed the construction of a complete, full-length reading frame of 1,417 bp covering a deduced protein sequence of 466 amino acids, including the universal translation-initiating methionine. The pigeon delta-crystallin shows 88, 83 and 69% sequence identity to duck delta 2, chicken delta 1 crystallins and human argininosuccinate lyase respectively. It is also shown that, in contrast to duck delta 2 crystallin which has a high argininosuccinate lyase activity, pigeon delta-crystallin appears to contain very low activity of this enzyme, despite the fact that they share a highly homologous structure. A structural comparison of delta-crystallins with or without enzymatic activity suggested several amino acid replacements which may account for the loss of argininosuccinate lyase activity in the lenses of certain avian species.
δ-晶体蛋白是鸟类和爬行类晶状体中的一种主要晶状体蛋白。为便于克隆δ-晶体蛋白基因，从鸽晶状体的聚腺苷酸加尾RNA构建了互补DNA（cDNA），通过聚合酶链反应（PCR）进行扩增。然后将PCR产物亚克隆到pUC19载体中，并转化到大肠杆菌JM109菌株中。从阳性克隆中纯化的质粒通过双脱氧核苷酸链终止法进行核苷酸测序准备。对两个含有编码δ-晶体蛋白的1.4kb DNA插入片段的克隆进行测序，从而构建了一个1417bp的完整全长阅读框，涵盖了一个推导的466个氨基酸的蛋白质序列，包括通用的翻译起始甲硫氨酸。鸽δ-晶体蛋白与鸭δ2晶体蛋白、鸡δ1晶体蛋白和人精氨琥珀酸裂解酶的序列同一性分别为88%、83%和69%。还表明，与具有高精氨琥珀酸裂解酶活性的鸭δ2晶体蛋白相反，鸽δ-晶体蛋白似乎含有非常低的该酶活性，尽管它们具有高度同源的结构。对具有或不具有酶活性的δ-晶体蛋白进行结构比较，发现了几个氨基酸替换，这可能解释了某些鸟类晶状体中精氨琥珀酸裂解酶活性的丧失。
