Tycko B, Coyle H, Sklar J
Department of Pathology, Brigham and Women's Hospital, Boston, MA 02115.
J Immunol. 1991 Jul 15;147(2):705-13.
Rearrangement of Ag receptor genes requires recognition by the lymphocyte recombinase of heptamer-nonamer signal sequences followed by two endonucleolytic cleavages and two DNA ligations to form the coding and signal joints. The phenomenon of trans-rearrangement, in which Ag receptor gene segments located on different chromosomes recombine to yield chimeric products, provides an in vivo system in which to investigate the ability of the recombinase to carry out each of these functions in trans. Trans-rearrangements between TCRG and TCRD loci, similar in structure and frequency to those observed previously in human lymphoid tissues, were demonstrated in normal mouse thymus by PCR with crossed V gamma/J delta and V delta/J gamma primer pairs. A simple mechanistic model for trans-rearrangement was then tested. This model posits an ability of the recombinase to catalyze the formation of both coding and signal joints in trans and therefore predicts that trans-rearrangements will generate chimeric signal joints. In adult thymus, chimeric D delta 2-J gamma 1 and D delta 2-J gamma 2 signal joints, containing fused heptamer-nonamer sequences, could be detected by PCR and were each present at frequencies sufficient to account for a large proportion of the corresponding TCRG/TCRD trans-rearrangements. In agreement with the predictions of the model, chimeric signal joints were found as both linear chromosomal and circular episomal DNA. The results provide a framework for understanding the formation of chromosomal translocations in normal and neoplastic lymphoid cells and support the possibility of a looping mechanism for standard gene rearrangement. To test the form of regulation of TCRG rearrangement, the frequencies of specific signal joints from standard and trans-rearrangements were compared. Although J gamma 1 and J gamma 2 segments participated with equal frequency in trans-rearrangement with D delta 2, only the J gamma 1 segment participated in standard rearrangement with V gamma 5. The results suggest that V-J recombination in the TCRG locus is regulated directly at the DNA level by cis-acting constraints which do not affect the accessibility of individual TCRG gene segments to recombination in trans.
抗原受体基因的重排需要淋巴细胞重组酶识别七聚体-九聚体信号序列，随后进行两次核酸内切酶切割和两次DNA连接，以形成编码接头和信号接头。转重排现象是指位于不同染色体上的抗原受体基因片段发生重组，产生嵌合产物，它提供了一个体内系统，用于研究重组酶在转位情况下执行这些功能的能力。通过使用交叉的Vγ/Jδ和Vδ/Jγ引物对进行PCR，在正常小鼠胸腺中证实了TCRG和TCRD基因座之间的转重排，其结构和频率与先前在人类淋巴组织中观察到的相似。然后测试了一个简单的转重排机制模型。该模型假定重组酶具有催化转位情况下编码接头和信号接头形成的能力，因此预测转重排将产生嵌合信号接头。在成年胸腺中，通过PCR可以检测到含有融合七聚体-九聚体序列的嵌合Dδ2-Jγ1和Dδ2-Jγ2信号接头，它们各自的出现频率足以解释相当一部分相应的TCRG/TCRD转重排。与模型预测一致，嵌合信号接头以线性染色体DNA和环状附加体DNA的形式存在。这些结果为理解正常和肿瘤性淋巴细胞中染色体易位的形成提供了一个框架，并支持了标准基因重排的环化机制的可能性。为了测试TCRG重排的调控形式，比较了标准重排和转重排中特定信号接头的频率。尽管Jγ1和Jγ2片段与Dδ2转重排的频率相同，但只有Jγ1片段参与了与Vγ5的标准重排。结果表明，TCRG基因座中的V-J重组在DNA水平上直接受到顺式作用限制的调控，这些限制不影响单个TCRG基因片段在转位情况下进行重组的可及性。
