V(D)J recombination on minichromosomes is not affected by transcription.
It has been shown previously by others that transcription is temporally correlated with the onset of V(D)J recombination at the endogenous antigen receptor loci. We have been interested in determining whether this temporal correlation indicates a causal connection between these two processes. We have compared V(D)J recombination minichromosome substrates that have transcripts running through the recombination zone with substrates that do not in a transient transfection assay. In this system, the substrates acquire a minichromosome conformation within the first several hours after transfection. We find that the substrates recombine equally well over a 100-fold range in transcriptional variation. In additional studies, we have taken substrates that have low levels of transcription and inhibited transcription further by methylating the substrate DNA or by treating the cells with a general transcription inhibitor (alpha-amanitin). Although these treatments decrease the level of expression an additional 10-100-fold, there is still no observable effect on V(D)J recombination. Based on these results, we conclude that transcription is not necessary for the V(D)J reaction mechanism and does not alter substrate structure at the DNA level or at the simplest levels of chromatin structure in a way that affects the reaction.
Hsieh
C L
CL
Department of Pathology, Stanford University School of Medicine, California 94305-5324.
McCloskey
R P
RP
Lieber
M R
MR
D000011:Abelson murine leukemia virus / Q000235:genetics; D000818:Animals; D001402:B-Lymphocytes / Q000276:immunology; D002461:Cell Line, Transformed; D002875:Chromosomes / Q000502:physiology*; D004261:DNA Replication; D015321:Gene Rearrangement; D005803:Genes, Immunoglobulin; D007700:Kinetics; D008156:Luciferases / Q000235:genetics / Q000378:metabolism; D051379:Mice; D008957:Models, Genetic; D010957:Plasmids; D011948:Receptors, Antigen, T-Cell / Q000235:genetics*; D011994:Recombinant Proteins / Q000378:metabolism; D011995:Recombination, Genetic; D015183:Restriction Mapping; D013601:T-Lymphocytes / Q000276:immunology; D014158:Transcription, Genetic; D014162:Transfection
D011948:Receptors, Antigen, T-Cell; D011994:Recombinant Proteins; D008156:Luciferases