4/5/2023 0 Comments Taipan mutant short![]() The first step of V(D)J recombination, specific cleavage at the recombination signal sequence (RSS), can be carried out by the recombination activating proteins RAG1 and RAG2. In vitro V(D)J recombination: Signal joint formationĬortes, Patricia Weis-Garcia, Frances Misulovin, Ziva Nussenzweig, Andre Lai, Jiann-Shiun Li, Gloria Nussenzweig, Michel C. 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. Although these treatments decrease the level of expression an additional 10-100-fold, there is still no observable effect on V(D)J recombination. 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). We find that the substrates recombine equally well over a 100-fold range in transcriptional variation. In this system, the substrates acquire a minichromosome conformation within the first several hours after transfection. 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. We have been interested in determining whether this temporal correlation indicates a causal connection between these two processes. 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. V(D)J recombination on minichromosomes is not affected by transcription. The in vitro reaction resembled authentic V(D)J recombination in being Ku-antigen-dependent. However, RAG1 and RAG2 complemented with nuclear extracts were able to recombine an extrachromosomal substrate and form precise signal joints. RAG1 and RAG2 alone or in combination were unable to generate signal joints. We have investigated signal joint formation using deletion and inversion substrates in a cell free system. In vivo, the cleaved coding and signal ends must be rejoined to generate functional antigen receptors and maintain chromosomal integrity. In vitro V(D)J recombination: signal joint formation.Ĭortes, P Weis-Garcia, F Misulovin, Z Nussenzweig, A Lai, J S Li, G Nussenzweig, M C Baltimore, D We report that the entire Emu, the Emu core as well as its flanking 5' and 3' matrix associated regions (5' and 3' MARs) upregulate V(D)J recombination while the downstream section of the 3' MAR of Emu does not. Respectively, can markedly affect the frequency of V(D)J recombination. V(D)J recombination frequency is affected by the sequence interposed between a pair of recombination signals: sequence comparison reveals a putative recombinational enhancer element
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