V(D)J recombination and allelic exclusion of a TCR beta-chain minilocus occurs in the absence of a functional promoter.
Transcriptional activation of rearranging Ag receptor gene segments has been hypothesized to regulate their accessibility to V(D)J recombination. We analyzed the role of a functional promoter in the rearrangement of the murine TCR beta-chain locus using two transgenic minilocus constructs. These miniloci each contain an unrearranged V beta 8.3 gene. One has a wild-type V beta 8.3 gene, but the other has a V beta 8.3 gene with a promoter mutation that was previously shown to abrogate transcription in tissue culture. FACS analysis of thymus and lymph node cells from transgenic mouse lines showed that only the lines with the wild-type V beta 8.3 gene promoter express an 8.3 TCR beta-chain. Consistent with the protein expression data, V beta 8.3 gene transcripts were found only in the transgenic lines with the wild-type promoter. Using a quantitative PCR-based assay, it was shown that both types of transgenic lines recombine the V beta 8.3 gene at similar levels. Rearrangement of the transgenes was normal with respect to thymic development and junctional reading frame. Interestingly, both types of miniloci also underwent allelic exclusion in that recombination was blocked by the expression of a rearranged TCR beta-chain transgene. We conclude that a functional V beta gene promoter is not necessary for proper V(D)J recombination to occur.
Alvarez
J D
JD
Howard Hughes Medical Institute, Department of Medicine, Genetics, and Molecular Microbiology, Washington University School of Medicine, St. Louis, MO 63110, USA.
Anderson
S J
SJ
Loh
D Y
DY
D000483:Alleles; D000818:Animals; D001483:Base Sequence; D004254:DNA Nucleotidyltransferases / Q000378:metabolism*; D005260:Female; D015333:Gene Rearrangement, beta-Chain T-Cell Antigen Receptor; D005813:Genes, Synthetic; D051379:Mice; D008807:Mice, Inbred BALB C; D008809:Mice, Inbred C3H; D008810:Mice, Inbred C57BL; D008822:Mice, Transgenic; D008969:Molecular Sequence Data; D016133:Polymerase Chain Reaction; D011401:Promoter Regions, Genetic; D016693:Receptors, Antigen, T-Cell, alpha-beta / Q000235:genetics*; D016415:Sequence Alignment; D014158:Transcription, Genetic; D045567:VDJ Recombinases
D016693:Receptors, Antigen, T-Cell, alpha-beta; D004254:DNA Nucleotidyltransferases; D045567:VDJ Recombinases