期刊名称：Proceedings of the National Academy of Sciences
印刷版ISSN：0027-8424
电子版ISSN：1091-6490
出版年度：1977
卷号：74
期号：8
页码：3518-3522
DOI：10.1073/pnas.74.8.3518
语种：English
出版社：The National Academy of Sciences of the United States of America
摘要：A 4.8-kilobase DNA fragment carrying an immunoglobulin gene coding for a mouse lambda chain variable region (Vlambda gene) was enriched about 350-fold from a total endonuclease EcoRI digest of embryonic DNA by a combination of preparative agarose gel electrophoresis of double-stranded DNA and CsCl density gradient centrifugation of R-loops formed with a purified lambda chain mRNA. DNA fragments thus enriched for the immunoglobulin gene were inserted in vitro in the middle of the genome of the vector phage lambdagt Wam 403, Eam 100, Sam 100 by use of the EcoRI cohesive ends. Transfection of CaCl2-treated Escherichia coli 803 [rk-, mk- (lacking restriction and modification systems for K-12)] with such hybrid DNA and subsequent screening of about 4000 plaques by in situ hybridization with purified 125I-labeled lambda chain mRNA led to isolation of a clone that carries a Vlambda gene (lambdagtWES-Ig 13). Electron microscopy of R-loops confirmed the presence of sequences homologous to part of the lambda chain mRNA in its 5'-end.
