MOLECULAR-CLONING AND FUNCTIONAL-CHARACTERIZATION OF CHICK LENS FIBER CONNEXIN-45.6
MOLECULAR-CLONING AND FUNCTIONAL-CHARACTERIZATION OF CHICK LENS FIBER CONNEXIN-45.6
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DOI：
10.1091/mbc.5.3.363
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发表时间：
1994-03-01
影响因子：
3.3
通讯作者：
PAUL, DL
中科院分区：
文献类型：
--
作者：
JIANG, JX;WHITE, TW;PAUL, DL
鸟类透镜是研究细胞间隙连接通讯的理想系统。实验表明，与其他生物相比，发育中的鸡胚胎中的透镜更容易接近，并且鸡透镜细胞在原代培养物中分化良好。然而，在鸟类透镜中只发现了两个连接蛋白基因家族成员，而在哺乳动物系统中已知有三个。我们在这里报告的第三个透镜连接蛋白，鸡连接蛋白45.6（ChCx 45.6），预测分子量为45.6 kDa的蛋白质的分子克隆和表征。ChCx 45.6由单拷贝基因编码，在透镜中特异性表达。mRNA有6.4kb和9.4kb两种。ChCx 45.6是一种功能性连接蛋白，因为在非洲爪蟾卵母细胞对中的表达导致了具有特征性电压敏感性的高水平电导的发展。针对ChCx45.6和鸡连接蛋白56（ChCx56），另一种禽晶状体特异性连接蛋白，提出了抗血清，允许检查这两种蛋白质的分布。免疫荧光定位显示，透镜纤维中ChCx 45.6和ChCx 56均较丰富。用碱性磷酸酶处理透镜膜导致电泳迁移率变化，表明ChCx 45.6和ChCx 56在体内都是磷蛋白。
The avian lens is an ideal system to study gap junctional intercellular communication in development and homeostasis. The lens is experimentally more accessible in the developing chick embryo than in other organisms, and chick lens cells differentiate well in primary cultures. However, only two members of the connexin gene family have been identified in the avian lens, whereas three are known in the mammalian system. We report here the molecular cloning and characterization of the third lens connexin, chick connexin45.6 (ChCx45.6), a protein with a predicted molecular mass of 45.6 kDa. ChCx45.6 was encoded by a single copy gene and was expressed specifically in the lens. There were two mRNA species of 6.4 kilobase (kb) and 9.4 kb in length. ChCx45.6 was a functional connexin protein, because expression in Xenopus oocyte pairs resulted in the development of high levels of conductance with a characteristic voltage sensitivity. Antisera were raised against ChCx45.6 and chick connexin56 (ChCx56), another avian lens-specific connexin, permitting the examination of the distribution of both proteins. Immunofluorescence localization showed that both ChCx45.6 and ChCx56 were abundant in lens fibers. Treatment of lens membranes with alkaline phosphatase resulted in electrophoretic mobility shifts, demonstrating that both ChCx45.6 and ChCx56 were phosphoproteins in vivo.