Molecular cloning of rat brain Na,K-ATPase alpha-subunit cDNA.
Molecular cloning of rat brain Na,K-ATPase alpha-subunit cDNA.
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大鼠脑 Na,K-ATP 酶 α 亚基 cDNA 的分子克隆。
DOI：
10.1073/pnas.82.18.6357
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发表时间：
1985
影响因子：
11.1
通讯作者：
Levenson,R
中科院分区：
文献类型：
--
作者：
Schneider,JW;Mercer,RW;Caplan,M;Emanuel,JR;Sweadner,KJ;BenzJr,EJ;Levenson,R
我们分离出大鼠脑 Na,K-ATP 酶 α 亚基的 cDNA 克隆。使用与大鼠脑 Na,K-ATP 酶反应的抗体筛选由 1 周龄和 2 周龄大鼠脑的 mRNA 构建的 lambda gt11 cDNA 表达文库。阳性噬菌体克隆 lambda rb5 含有 1200 个碱基对 cDNA 插入片段，表达 β-半乳糖苷酶-cDNA 融合蛋白，该蛋白通过与 Na,K-ATPase 抗体进行免疫印迹反应而发生反应。该融合蛋白在 ELISA 中也能与针对 Na,K-ATP 酶 α 亚基的单克隆抗体发生反应。在大鼠脑、肾、肝以及狗肾中鉴定出与 lambda rb5 cDNA 插入片段表现出序列杂交的 27S mRNA 种类。该27S mRNA表现出与体内Na,K-ATP酶多肽的相对丰度一致的组织特异性丰度模式：肾大于脑大于肝。在耐哇巴因的 HeLa 细胞系 C+ 中，该细胞系含有微小的染色体和比亲代 HeLa 细胞多至少 10 倍数量的钠泵，与 lambda rb5 cDNA 互补的 DNA 序列被扩增约 40 倍。对 lambda rb5 cDNA 序列的分析表明，该 cDNA 的一部分与 Na,K-ATP 酶 α 亚基异硫氰酸荧光素结合位点的氨基酸序列之间存在完美的核苷酸序列匹配。总而言之，此处提供的数据表明 lambda rb5 cDNA 克隆是编码大鼠脑 Na,K-ATP 酶 α 亚基的基因的一部分。 ATP酶基因似乎在大鼠和人类基因组中以一个或很少的拷贝存在，并且在不同的大鼠组织中受到转录调节。另一方面，在乌巴因抗性人类细胞系中，乌巴因抗性似乎涉及编码Na,K-ATP酶的基因拷贝数的增加。
We have isolated a cDNA clone for the rat brain Na,K-ATPase alpha subunit. A lambda gt11 cDNA expression library constructed from mRNA of 1- and 2-week-old rat brains was screened with an antibody reactive with rat brain Na,K-ATPase. A positive phage clone, lambda rb5, containing a 1200-base-pair cDNA insert expressed a beta-galactosidase-cDNA fusion protein that was reactive by immunoblotting with the Na,K-ATPase antibody. This fusion protein was also reactive in ELISA with a monoclonal antibody directed against the alpha subunit of the Na,K-ATPase. A 27S mRNA species exhibiting sequence hybridization to the cDNA insert of lambda rb5 was identified in rat brain, kidney, and liver, as well as in dog kidney. This 27S mRNA exhibited a tissue-specific pattern of abundance consistent with the relative abundance of Na,K-ATPase polypeptides in vivo: kidney greater than brain greater than liver. In a ouabain-resistant HeLa cell line, C+, which contains minute chromosomes and at least a 10-fold greater number of sodium pumps than parental HeLa cells, DNA sequences complementary to lambda rb5 cDNA were amplified approximately 40-fold. Analysis of the lambda rb5 cDNA sequence demonstrated a perfect nucleotide sequence match between a portion of the cDNA and the amino acid sequence of the Na,K-ATPase alpha-subunit fluorescein isothiocyanate binding site. Taken together, the data presented here demonstrate that the lambda rb5 cDNA clone is a portion of the gene coding for the rat brain Na,K-ATPase alpha subunit. The ATPase gene appears to be present in one or very few copies in the rat and human genomes and to be transcriptionally regulated in different rat tissues. In a ouabain-resistant human cell line, on the other hand, ouabain resistance appears to involve an increase in the number of gene copies coding for the Na,K-ATPase.