Cloning of an immunoglobulin variable region gene from mouse embryo.

Proceedings of the National Academy of Sciences of the United States of America ·Vol. 74 ·No. 8 ·1977-08-00 ·Pages 3518-22

Tonegawa S, Brack C, Hozumi N, Schuller R

Abstract

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.

MeSH Terms

Animals Binding Sites, Antibody Chromosome Mapping Clone Cells Coliphages/metabolism DNA Ligases/metabolism DNA, Recombinant/metabolism Embryo, Mammalian Escherichia coli/metabolism Genes Immunoglobulin Light Chains/biosynthesis Immunoglobulin Variable Region Immunoglobulin lambda-Chains/biosynthesis Mice Microscopy, Electron Nucleic Acid Hybridization Transfection

Chemicals

DNA, Recombinant Immunoglobulin Light Chains Immunoglobulin Variable Region Immunoglobulin lambda-Chains DNA Ligases

Authors & Affiliations

4 authors, click to expand affiliations / ORCID

Tonegawa S

Brack C

Hozumi N

Schuller R

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26 references, click to expand

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Article Info

Journal

Proceedings of the National Academy of Sciences of the United States of America

Abbr.

Proc Natl Acad Sci U S A

ISSN

0027-8424

DOI

10.1073/pnas.74.8.3518

Published

1977-08-00

Pages

3518-22

Language

English

Region

United States

NLM ID

7505876

PMCID

PMC431622

Subset

External Links

PubMed source DOI: 10.1073/pnas.74.8.3518 PMC Full Text View journal details

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Cloning of an immunoglobulin variable region gene from mouse embryo.

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