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人类腺苷脱氨酶基因序列的分子克隆

Molecular cloning of human adenosine deaminase gene sequences.

作者信息

Orkin S H, Daddona P E, Shewach D S, Markham A F, Bruns G A, Goff S C, Kelley W N

出版信息

J Biol Chem. 1983 Nov 10;258(21):12753-6.

PMID:6688808
Abstract

Using a mixture of synthetic 17-mer oligonucleotides encoding the 64 possible sequences for a peptide of adenosine deaminase as probe, we have isolated a clone for adenosine deaminase mRNA sequences from a collection of T-cell cDNA recombinants. This cDNA clone, phADA-1, contains an insert of 0.8 kilobase. In addition to the peptide chosen for synthesis of the oligonucleotide probe, the complete DNA sequence predicts 16 other experimentally determined peptides. Mapping of total cellular human DNAs with several restriction enzymes revealed relatively simple patterns of hybridization with phADA-1 as probe, including a polymorphism for PvuII cleavage. In agreement with previous studies, the adenosine deaminase gene was localized by blot hybridization to chromosome 20 in a hybrid cell mapping panel. Using the cDNA as probe, an 18-kilobase EcoRI fragment of human cellular DNA was also cloned in bacteriophage Charon 4A. These adenosine deaminase clones will prove valuable in the full characterization of the cellular gene, molecular analysis of inherited enzyme deficiency associated with immunodeficiency, and regional mapping of human chromosome 20.

摘要

我们以一组编码腺苷脱氨酶肽段的64种可能序列的合成17聚体寡核苷酸混合物作为探针,从T细胞cDNA重组体文库中分离出一个腺苷脱氨酶mRNA序列的克隆。这个cDNA克隆,即phADA - 1,含有一个0.8千碱基的插入片段。除了用于合成寡核苷酸探针的肽段外,完整的DNA序列还预测了另外16个经实验确定的肽段。用几种限制酶对人细胞总DNA进行图谱分析,以phADA - 1作为探针显示出相对简单的杂交模式,包括PvuII切割的多态性。与先前的研究一致,通过杂交细胞图谱分析,腺苷脱氨酶基因通过印迹杂交定位到20号染色体上。以该cDNA为探针,人细胞DNA的一个18千碱基的EcoRI片段也被克隆到噬菌体Charon 4A中。这些腺苷脱氨酶克隆在细胞基因的全面表征、与免疫缺陷相关的遗传性酶缺乏的分子分析以及人类20号染色体的区域图谱绘制中将被证明是有价值的。

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