Transient expression of human adenosine deaminase cDNAs: identification of a nonfunctional clone resulting from a single amino acid substitution.
Transient expression of human adenosine deaminase cDNAs: identification of a nonfunctional clone resulting from a single amino acid substitution.
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人腺苷脱氨酶 cDNA 的瞬时表达：鉴定由单个氨基酸取代产生的非功能性克隆。
DOI：
10.1128/mcb.5.4.762-767.1985
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发表时间：
1985
影响因子：
5.3
通讯作者：
Daddona,PE
中科院分区：
文献类型：
--
作者：
Orkin,SH;Goff,SC;Kelley,WN;Daddona,PE
人腺苷脱氨酶（ADA）是一种重要的嘌呤分解代谢酶，可不可逆地脱腺苷和脱氧腺苷。ADA的严重遗传缺陷导致免疫缺陷状态，其中T淋巴细胞被毒性水平的脱氧腺苷和脱氧ATP的积累选择性地破坏。在准备将ADA序列转移到各种细胞类型中时，我们探索了在基于猿猴病毒40的表达载体内转染到培养细胞中的ADA cDNA的表达。转染到猴肾（COS）细胞后，ADA cDNA涵盖整个编码区的蛋白质产生人ADA活性。然而，一个意想不到的发现是鉴定出一个cDNA克隆，该克隆不能产生人酶活性或免疫反应性ADA蛋白。由于这种模式是典型的许多自然发生的突变ADA等位基因，我们的特点是在这个克隆的分子缺陷。DNA序列分析显示在氨基酸位置50（甘氨酸-缬氨酸）处的单核苷酸取代。用独特的17-mer寡核苷酸进行的北方印迹证明在mRNA中不存在突变序列，从该mRNA构建产生突变cDNA的cDNA文库。因此，在克隆过程中产生了变体cDNA中的取代。这些数据定义了人ADA蛋白分子的一个关键区域，并提出了一种方便的策略，用于表征与天然突变等位基因相关的表型。
Human adenosine deaminase (ADA) is an important purine catabolic enzyme which irreversibly deaminates adenosine and deoxyadenosine. Severe genetic deficiency of ADA leads to an immunological deficiency state in which T-lymphoid cells are selectively destroyed by the accumulation of toxic levels of deoxyadenosine and deoxy-ATP. In preparation for transfer of ADA sequences into a variety of cell types, we explored expression of ADA cDNAs transfected into cultured cells within a simian virus 40-based expression vector. After transfection into monkey kidney (COS) cells, ADA cDNA encompassing the entire coding region of the protein generated human ADA activity. An unexpected finding, however, was the identification of a cDNA clone that failed to produce either human enzyme activity or immunoreactive ADA protein. As this pattern is typical of many naturally occurring mutant ADA alleles, we characterized the molecular defect in this clone. DNA sequence analysis revealed a single nucleotide substitution in amino acid position 50 (glycine-valine). Northern blotting with a unique 17-mer oligonucleotide demonstrated the absence of the mutant sequence in the mRNA from which the cDNA library giving rise to the mutant cDNA was constructed. Therefore, the substitution in the variant cDNA was created during cloning. These data define one critical region of the human ADA protein molecule and suggest a convenient strategy for characterization of the phenotypes associated with naturally occurring mutant alleles.