Cloning and functional expression of a novel human connexin-25 gene
Cloning and functional expression of a novel human connexin-25 gene
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DOI：
10.3109/15419060109080718
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发表时间：
2001-01-01
影响因子：
--
通讯作者：
Bertram, JS
中科院分区：
文献类型：
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作者：
Bondarev, I;Vine, A;Bertram, JS
间隙连接是细胞间充满水的通道，由称为连接蛋白的跨膜蛋白组成，其中六个呈放射状排列，并与相邻细胞中的六个同源蛋白对接，形成大约 16 A 的孔。通过这种孔，水溶性小分子沿着浓度梯度可在细胞间转移，最高可达约 1000 道尔顿。连接蛋白包含一个多基因家族，它们在跨膜结构域以及第一和第二细胞外环中共享共有序列。将已知人类连接蛋白的蛋白质序列与人类基因组的核苷酸序列草图进行比较，发现来自 6 号染色体的两个克隆与高度保守的连接蛋白序列表现出高度相似性。详细分析显示这些克隆中存在 672 nt 开放阅读框。编码223个氨基酸的多肽，预计分子量约为25kD。这比其他已知的人类连接蛋白要小。使用人类基因组DNA作为模板，通过半巢式PCR扩增潜在的connexin25的ORF。为了证实这个新基因编码连接蛋白，将 Cx25 转染到人类 HeLa 细胞系的间隙连接缺陷亚克隆中，选择转化体后，将荧光染料 Lucifer Yellow 显微注射到细胞中。转染子（而非对照）成功转移了染料，证明该新基因编码功能性连接蛋白。
Gap junctions are intercellular, water-filled channels composed of transmembrane proteins called connexins, six of which are arranged radially and dock with six homologous proteins in an adjacent cell to form an approximate 16 A pore. Through this pore cell-to-cell transfer of small water-soluble molecules up to about 1000 daltons occurs along concentration gradients. Connexins comprise a multigene family that share consensus sequences in the trans-membrane domains and the first and second extracellular loops. Comparison of the protein sequences of known human connexins with the draft nucleotide sequence of the human genome revealed two clones from chromosome 6 which showed strong similarity to highly conserved connexin sequences. Detailed analysis revealed the presence of a 672 nt open reading frame in these clones. encoding a 223 amino acid polypeptide with a predicted molecular weight of about 25 kD. This is smaller than other known human connexins. The ORF of the potential connexin25 was amplified by semi-nested PCR using human genomic DNA as a template. To confirm that this new gene encodes a connexin, Cx25 was transfected into a gap junction deficient subclone of the human HeLa cell line, After selection of transformants, cells were microinjected with the fluorescent dye Lucifer yellow. Transfectants but not controls successfully transferred dye, demonstrating that this new gene encodes a functional connexin.