Expression and V(D)J recombination activity of mutated RAG-1 proteins.
The products of the RAG-1 and RAG-2 genes are essential for the recombination of the DNA encoding the antigen receptors of the developing immune system. Little is known of the specific role these genes play. We have explored the sequences encoding mouse RAG-1 by deleting large parts of the gene and by introducing local sequence changes. We find that a RAG-1 gene with 40% of the coding region deleted still retains its recombination function. In addition, a series of small deletions within the strongly conserved remaining 60% of the coding region was tested. Nine out of ten of these prove unable to provide RAG-1 activity, but one is quite active. Certain peptide sequences were also specifically targeted for mutagenesis. The RAG-1 protein generated from this expression system is transported to the nucleus and is degraded with a 15 minute half-life. The fate of the proteins made by the deletion mutants were also assessed. Transport of RAG-1 protein to the nucleus was found even with the most extensive deletions studied. The functionality of the deleted proteins is discussed with relation to an alignment of RAG-1 sequences from five animal species.
Sadofsky
M J
MJ
Laboratory of Molecular Biology, NIDDK, NIH, Bethesda, MD 20892.
Hesse
J E
JE
McBlane
J F
JF
Gellert
M
M
D016475:3T3 Cells; D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D001692:Biological Transport; D002461:Cell Line, Transformed; D002467:Cell Nucleus / Q000378:metabolism; D004247:DNA; D004264:DNA Topoisomerases, Type I / Q000378:metabolism; D015322:Gene Rearrangement, B-Lymphocyte; D016654:Genes, RAG-1; D018398:Homeodomain Proteins; D006801:Humans; D051379:Mice; D008969:Molecular Sequence Data; D009154:Mutation; D011233:Precipitin Tests; D011506:Proteins / Q000235:genetics / Q000378:metabolism*; D017386:Sequence Homology, Amino Acid
D018398:Homeodomain Proteins; D011506:Proteins; C064658:RAG-1 protein; D004247:DNA; D004264:DNA Topoisomerases, Type I