Cloning of cDNA sequences of human adenosine deaminase.
Cloning of cDNA sequences of human adenosine deaminase.
复制标题
人腺苷脱氨酶 cDNA 序列的克隆。
DOI：
10.1073/pnas.80.24.7481
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发表时间：
1983
影响因子：
11.1
通讯作者：
Hutton,JJ
中科院分区：
文献类型：
--
作者：
Wiginton,DA;Adrian,GS;Friedman,RL;Suttle,DP;Hutton,JJ
人腺苷脱氨酶（ADA;腺苷氨基水解酶，EC 3.5.4.4）的克隆cDNA序列已从噬菌体λ gt 10中构建的cDNA文库中分离。该库的cDNA是从人T淋巴母细胞系CCRF-CEM中分离的poly（A）+ RNA制备的。该库最初筛选的差异噬斑杂交标记的cDNA制备的人T-和B-淋巴母细胞细胞系的翻译ADA mRNA的水平有21倍的差异。通过杂交选择性翻译鉴定了两个含有ADA克隆cDNA序列的重组体。两个重组体都含有大约1,600个碱基对的插入人类DNA。两个插入片段的限制性酶切图谱不完全相同。其中一个含有大约40个碱基对的额外DNA，朝向cDNA的中心。克隆的cDNA特异性杂交的5个片段产生的HindIII消化的人基因组DNA。它还与长度为1.6和5.8的人淋巴母细胞RNA种类杂交。cDNA用作探针来估计人淋巴母细胞系中ADA mRNA水平。在含有结构正常ADA的细胞系中，ADA mRNA水平与ADA催化活性和ADA蛋白水平密切相关。白血病T淋巴母细胞系产生的ADA蛋白和ADA mRNA是转化B淋巴母细胞系的6至9倍。两个突变的B淋巴母细胞系的遗传性ADA缺乏症患者含有不稳定的ADA蛋白，但有3至4倍的正常水平的ADA mRNA。
Cloned cDNA sequences of human adenosine deaminase (ADA; adenosine aminohydrolase, EC 3.5.4.4) have been isolated from a cDNA library constructed in bacteriophage lambda gt10. The cDNA for the library was prepared from poly(A)+ RNA isolated from a human T-lymphoblast cell line, CCRF-CEM. The library was initially screened by differential plaque hybridization to labeled cDNA prepared from human T- and B-lymphoblast cell lines with a 21-fold difference in levels of translatable ADA mRNA. Two recombinants containing cloned cDNA sequences for ADA were identified by hybridization-selected translation. Both recombinants contained approximately 1,600 base pairs of inserted human DNA. Restriction maps of the two inserts were not identical. One contained approximately 40 base pairs of additional DNA toward the center of the cDNA. The cloned cDNA specifically hybridized to five fragments generated by HindIII digestion of human genomic DNA. It also hybridized to human lymphoblast RNA species 1.6 and 5.8 kilobases in length. The cDNA was used as a probe to estimate ADA mRNA levels in human lymphoblast cell lines. ADA mRNA levels correlate closely with levels of ADA catalytic activity and ADA protein in cell lines containing structurally normal ADA. A leukemic T-lymphoblast line produced 6 to 9 times as much ADA protein and ADA mRNA as transformed B-lymphoblast lines. Two mutant B-lymphoblast lines from patients with hereditary ADA deficiency contained unstable ADA protein but had 3 to 4 times the normal level of ADA mRNA.