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Cell. 1978 Sep;15(1):1-14.

A complete immunoglobulin gene is created by somatic recombination.

Abstract

Using a pCRI plasmid containing an enzymatically synthesized, full-length DNA transcript of immunoglobulin lambda chain mRNA as the hybridization probe in the Southern gel blotting experiments (Southern, 1975), we identified three DNA fragments of 8.6, 4.8 and 3.5 kb in Eco RI-digested total DNA from BALB/c mouse embryos. A fourth fragment of 7.4 kb was found in addition to these three fragments in similarly digested total DNA from a lambda chain-secreting myeloma (HOPC 2020). We have cloned the four DNA fragments in an EK-2 phage vector, lambdaWES, and characterized them with respect to size, type of lambda gene sequences contained and position of these sequences in the fragments, using agarose gel electrophoresis, the gel blotting technique and electron microscopic R loop mapping. The embryonic DNA clones Ig 99 lambda, Ig 25lambda and Ig 13lambda contain one copy each of V lambdaI, C lambdaI and V lambdaII sequences, respectively, while the myeloma DNA clone Ig 303lambda contains one copy each of V lambdaI and C lambdaI sequences that are separated by a 1.2 kb nontranslated DNA segment. Ig 25lambda was also shown to contain a DNA segment of approximately 40 base pairs (bp) (J sequence) that lies 1.2 kb away from the C lambdaI sequence and is homologous to the V-C junction region of a lambdaI mRNA. Heteroduplex analysis of the three lambdaI DNA clones revealed that Ig 303lambda DNA is composed of two parts, one of which is entirely homologous to one end of Ig 99lambda, and the other to one end of Ig 25lambda DNA. The sequence arrangement observed in the cloned DNA is the same as that in the corresponding cellular DNA. This was shown by identifying certain restriction enzyme cleavage sites on the cloned DNAs and demonstrating the presence of these sites in the total cellular DNA by the gel blotting technique. The site of the homology switch is at the boundary of the V sequence and the 1.2 kb nontranslated DNA segment, and corresponds to the position of the J sequence on the Ig 25lambda DNA. We consider the above experimental results the most direct evidence for somatic rearrangement in immunoglobulin genes. We discuss the significance of these findings for the origin of genes in the evolution of higher organisms and in cell differentiation.

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PMID:
100225
[Indexed for MEDLINE]

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