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標題: | 鯊魚γ-水晶體蛋白基因定序分析與演化特性之研究 The Study of Sharkγ-Crystallin Genes |
作者: | MING-HONG CHUANG 莊明恆 |
出版年 : | 1994 |
學位: | 碩士 |
摘要: | 除了鳥類和一些爬蟲類外,在大多數脊椎動物眼球內的水晶體中,γ水晶體蛋白是一種十分常見的蛋白質。特別是魚類與哺乳類的胚胎幼兒時期,γ會被大量表現出來。為了要定出鯊魚的γ水晶體蛋白基因序列並瞭解此蛋白質的特性,首先從鯊水晶體中抽取poly(A)+RNA,並將此反轉錄成cDNA混合液。然後使用經由已知的鯉魚γ水晶體蛋白基因序列合成所需的正、反引子,利用聚合?連鎖反應(PCR)技術增殖鯊魚的γ水晶體蛋白基因。 增殖後的產物先與Klenow fragment及聚核?酸激?(PNK)進行磷酸化反應,再將其接上pUC18載體,然後植入大腸桿菌JM109繁殖。經過菌落篩選及利用Sange’s dideoxynucleotide lermination方法定序後得到四條不同的γ水晶體白基因序列(γs-1,γs-2,γm1,γm2)。此四條基因所轉錄的蛋白質發現可分成兩類:一是經胺基酸比對後與哺乳動物較為類似的γ水晶體蛋白,如γs-1,γs-2,γm2(與牛的γII相似性大約為65%)且其甲硫胺酸(methionine)的含量亦較少(5?6%);另一種是與魚類較為類似的γ水晶體蛋白,如γm1(與鯉魚γm1相似性高達80%),其甲硫胺酸含量也相當高(約占16%)。而γs-1,γs-2, γm2這三條彼此的相似性有85%,但與γm1卻都只有56?58%的相似性。 另外本篇論文也對此四種蛋白質結構與演化特性及部分genomic DNA序列和結構加以研究與探討。 γ-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 positiveγ-crystallin clones of shark were obtained and processed for nucleotide sequencing. Through sequencing by Sanger’s dideoxynucleotide chain-termination method, we have found these cDNAs can be separated into two classes; one is mammalian-likeγ-Crystallin, which comprises γs-1, γs-2, and γm2 (about 65% homology in protein sequence with bovine γB) with low methionine content (5?6%). The other class is teleostean γ-crystallin (about 80% homology in protein sequence with carp γm1), that is γm1, unlike γs-1, γs-2 and γm2, which contains high methionine content (?16%). The amino acid sequence homology between γs-1, γs-2 and γm2 is about 85%, but these, three show only about 56-58% homology with γm1. The structural analysis of the crystallins deduced from these cDNAs is carried out in order to shed some light on the molecular evolution of these structurally homologous proteins. Preliminary study on the genomic structure encoding these crystallins were also presented. |
URI: | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/76018 |
全文授權: | 未授權 |
顯示於系所單位: | 生化科學研究所 |
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