鯉魚水晶體γ－水晶蛋白基因的結合蛋白質研究

Abstract

利用鯉魚gamma－水晶蛋白基因 gamma－ml及gamma－ml '製成的各探針，與鯉魚成體水晶體細胞核萃取物進行結合反應藉以初步瞭解鯉魚成體水晶體中，gamma－水晶蛋白基因結合蛋白的活性與種類，以及和探針形成複合體後對探針的影響。由膠體延滯分析（Mobility shirt DNA binding assay）得知，鯉魚成體水晶體細胞核萃取物會造成探針於電泳膠中加速移動的現象，並證明應該不是由於探針被核酸分解酵素（DNase）分解，可能是蛋白質造成去氧核糖核酸（DNA）立體結構改變，而影響在電泳膠中的泳動力。另外在西南方點墨分析（Southwestern blotting assay) 中測得一個20KD蛋白質可和所有gamma－水晶蛋白基因gamma－ml, gamma－ml,探針結合，推測20KD蛋白質為 gamma-水晶蛋白基因共同結合蛋白，其蛋白質特性與結合去氧核糖核酸(DNA)序列仍待進一步鑑定。抽取同科中體型較小錦鯉的水晶體細胞核萃取物，發現其gamma－水晶蛋白基因結合蛋白活性及種類和鯉魚系統有所不同，由此提供一個訊息：不同年齡鯉魚其水晶蛋白基因調控有所不同。

In this study, we used the nuclear extract of adult carp lens to bind with the probes that were derived from carp γ -crystallin genes, γ-ml and γ-ml’. From the binding assays, we investigated the basic properties of the γ-crystallin gene binding proteins from carp lens, and studies observed the effect of protein binding on these probes. In mobility shift assay, we observed that nuclear extract from carp lens caused the probes to move faster than free probes in the agarose gel electrophoresis. It might be due to DNA conformational change caused by the proteins binding. The phenomenon was not caused by DNase digestion. In southwestern blotting assay, we found a 20 KD protein could bind to all probes of γ-crystallin genes, γ-ml and γ-ml’. The 20 KD protein is suggested to be a common binding factor of carp lens γ-crystallin genes. However, we have not identified the binding sequence of target genes. When comparing the lens nuclear extract of older carps with that of younger carps, it was found that the classes and activity of γ-crystallin gene binding proteins seem to be different. It implies that the regulation of γ-crystallin gene is age-dependent.