Characterization of coding ends in thymocytes of scid mice: implications for the mechanism of V(D)J recombination.
We previously identified possible intermediates in V(D)J recombination at the TCR delta locus and characterized molecules with signal ends and with covalently sealed (hairpin) coding ends in thymocytes of scid mice by Southern blotting. Here, we use a sensitive ligation-mediated PCR assay to demonstrate that all coding ends detected in scid thymocytes are covalently sealed. Neither coding nor signal ends exhibit loss or addition of nucleotides. These data imply that hairpin formation is coupled to the initial cleavage at the signal/coding border, and that the cleavage step in V(D)J recombination is conservative. In scid/+ or wild-type thymocytes, hairpin coding ends are at least 1000-fold less abundant than signal ends. These results provide insight into the mechanism of V(D)J recombination.
Zhu
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Department of Microbiology and Immunology, Baylor College of Medicine, Houston, Texas 77030, USA.
Roth
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D000818:Animals; D001483:Base Sequence; D004247:DNA / Q000235:genetics / Q000378:metabolism; D004254:DNA Nucleotidyltransferases / Q000378:metabolism*; D016694:Gene Rearrangement, delta-Chain T-Cell Antigen Receptor; D006579:Heterozygote; D006720:Homozygote; D051379:Mice; D016513:Mice, SCID / Q000235:genetics* / Q000276:immunology; D008957:Models, Genetic; D018448:Models, Immunological; D008969:Molecular Sequence Data; D009690:Nucleic Acid Conformation; D016133:Polymerase Chain Reaction; D016692:Receptors, Antigen, T-Cell, gamma-delta / Q000235:genetics*; D011995:Recombination, Genetic; D016176:T-Lymphocyte Subsets / Q000276:immunology* / Q000473:pathology; D013950:Thymus Gland / Q000276:immunology / Q000473:pathology; D045567:VDJ Recombinases
D016692:Receptors, Antigen, T-Cell, gamma-delta; D004247:DNA; D004254:DNA Nucleotidyltransferases; D045567:VDJ Recombinases