Yu C W, Chiou S H
Laboratory of Crystallin Research, National Taiwan University and Institute of Biological Chemistry, Taipei.
Biochem Biophys Res Commun. 1993 Apr 30;192(2):948-53. doi: 10.1006/bbrc.1993.1507.
To facilitate the cloning of delta-crystallin gene, the product of which is a major lens protein present in the avian and reptilian lenses, polymerase chain reaction (PCR) was employed to amplify cDNAs constructed from the poly(A)+RNA of goose lenses. The PCR product was then subcloned into pUC19 vector and transformed in E. coli strain JM109. Plasmids purified from the positive clones were prepared for nucleotide sequencing by dideoxynucleotide chain-termination method. Sequencing several clones containing 1.4 kb DNA inserts encoding delta-crystallin constructed a complete and unambiguous full-length reading frame of 1401 base pairs covering a deduced protein sequence of 465 amino acids excluding the universal translation-initiating methionine. The goose delta-crystallin shows 88, 94, 88 and 69% sequence identity to pigeon delta, duck delta 2, chicken delta 1 crystallins and human argininosuccinate lyase respectively. It is also shown that, similar to duck delta 2 and in contrast to pigeon delta crystallin, goose delta-crystallin appears to possess high argininosuccinate lyase activity despite the fact that a highly homologous structure is shared among these homologous proteins. Structural analysis and comparison of these closely related delta-crystallin homologues with or without enzymatic activity should be of value in unraveling the intriguing evolutionary process leading to the development and evolution of active enzymatic crystallins in the lenses of certain avian species.
为便于克隆δ-晶体蛋白基因（其产物是鸟类和爬行动物晶状体中的一种主要晶状体蛋白），采用聚合酶链反应（PCR）扩增从鹅晶状体的聚腺苷酸加尾RNA（poly(A)+RNA）构建的cDNA。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。对几个含有编码δ-晶体蛋白的1.4 kb DNA插入片段的克隆进行测序，构建了一个完整且明确的1401个碱基对的全长阅读框，涵盖了一个推导的465个氨基酸的蛋白质序列，不包括通用的翻译起始甲硫氨酸。鹅δ-晶体蛋白与鸽δ-晶体蛋白、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%、94%、88%和69%。还表明，与鸭δ2-晶体蛋白相似，与鸽δ-晶体蛋白不同，鹅δ-晶体蛋白似乎具有较高的精氨琥珀酸裂解酶活性，尽管这些同源蛋白之间共享高度同源的结构。对这些具有或不具有酶活性的密切相关的δ-晶体蛋白同源物进行结构分析和比较，对于揭示导致某些鸟类晶状体中活性酶晶体蛋白发展和进化的有趣进化过程应该具有价值。
