Carroll A M, Slack J K, Mu X
Department of Microbiology, Immunology and Molecular Genetics, Albany Medical College, NY 12208.
J Immunol. 1993 Mar 15;150(6):2222-30.
The standard products of V(D)J recombination are coding junctions, which encode Ag receptor polypeptides, and their commonly excised reciprocal products, signal junctions. Additional nonstandard products also have been detected, mostly in artificial recombination substrate studies. The occurrence of nonstandard products, including pseudonormal, hybrid, and open/shut junctions, indicates significant indeterminacy of the V(D)J recombinase. However, the incidence of nonstandard products for endogenous Ag receptor genes in vivo has not been specifically addressed. The data presented here show that for the TCR-delta locus, D element-associated recombination in mouse thymocytes results in a high incidence of nonstandard recombination products. D delta 1-D delta 2 rearrangements, both chromosome retained and excised episomal products, were studied by polymerase chain reaction amplification, cloning, and sequence analysis. The proximity of D delta 1 and D delta 2 elements, and the fact that both are flanked by 5' and 3' recombination signal sequences with 12-bp and 23-bp spacers, respectively, results in frequent pseudonormal joining. The resulting products are signal junctions retained on the chromosome. Excised episomal products include coding junctions, hybrid junctions formed in apparent violation of the 12/23 spacer rule, and standard signal junctions; some signal junctions show evidence of imprecise cleavage. Evidence for open/shut and/or oligonucleotide capture events was also seen. Similar rearrangements were detectable in thymocytes of mutant scid mice. These findings indicate a high degree of indeterminacy of V(D)J recombinase-mediated D delta 1-D delta 2 rearrangement in both wild-type and scid thymocytes. This indeterminacy affects the productive potential of TCR-delta loci.
V(D)J重组的标准产物是编码连接，其编码抗原受体多肽，以及它们通常被切除的相互产物，即信号连接。还检测到了其他非标准产物，主要是在人工重组底物研究中。非标准产物的出现，包括假正常、杂交和开放/关闭连接，表明V(D)J重组酶存在显著的不确定性。然而，体内内源性抗原受体基因非标准产物的发生率尚未得到具体研究。本文提供的数据表明，对于TCR-δ基因座，小鼠胸腺细胞中与D元件相关的重组会导致非标准重组产物的高发生率。通过聚合酶链反应扩增、克隆和序列分析研究了Dδ1-Dδ2重排，包括染色体保留产物和切除的游离环产物。Dδ1和Dδ2元件的接近性，以及它们分别两侧是具有12bp和23bp间隔序列的5'和3'重组信号序列这一事实，导致频繁的假正常连接。产生的产物是保留在染色体上的信号连接。切除的游离环产物包括编码连接、明显违反12/23间隔规则形成的杂交连接和标准信号连接；一些信号连接显示出不精确切割的证据。还发现了开放/关闭和/或寡核苷酸捕获事件的证据。在突变型scid小鼠的胸腺细胞中也可检测到类似的重排。这些发现表明，在野生型和scid胸腺细胞中，V(D)J重组酶介导的Dδ1-Dδ2重排具有高度的不确定性。这种不确定性影响了TCR-δ基因座的有效潜力。
