克隆溶酶体α-葡萄糖苷酶的互补DNA。

Cloning a cDNA for the lysosomal alpha-glucosidase.

作者信息

Konings A, Hupkes P, Versteeg R, Grosveld G, Reuser A, Galjaard H

出版信息

Biochem Biophys Res Commun. 1984 Feb 29;119(1):252-8. doi: 10.1016/0006-291x(84)91645-0.

Abstract

Messenger RNA was isolated from monkey testes and size-fractionated on sucrose gradients. In vitro translation of these mRNA fractions resulted in nascent, labeled alpha-glucosidase that could be precipitated with anti human alpha-glucosidase antiserum. A cDNA library was constructed from the most enriched fraction. The library was screened with cDNA made from mRNA obtained from immunoselected polysomes. Five cross-hybridizing clones were isolated and identified by their selection of alpha-glucosidase mRNA, as shown by hybrid released translation and further by their ability to hybridize with DNA from human chromosome 17, on which the gene coding for acid alpha-glucosidase is located.

摘要

从猴睾丸中分离信使核糖核酸（mRNA），并在蔗糖梯度上进行大小分级分离。对这些mRNA级分进行体外翻译，产生了可被抗人α-葡萄糖苷酶抗血清沉淀的新生标记α-葡萄糖苷酶。从最富集的级分构建了一个互补DNA（cDNA）文库。用从免疫选择的多核糖体获得的mRNA制备的cDNA对文库进行筛选。分离出五个交叉杂交克隆，并通过它们对α-葡萄糖苷酶mRNA的选择进行鉴定，如杂交释放翻译所示，进一步通过它们与人类17号染色体DNA杂交的能力进行鉴定，编码酸性α-葡萄糖苷酶的基因位于该染色体上。