Molecular mechanisms for immunoglobulin class switching

我们提出了一个关于免疫球蛋白类转换的分子机制的假说，即淋巴因子或细胞因子刺激B淋巴细胞在DNA重组(S-S重组)之前诱导类转换的靶C_H基因的转录，并通过转录使含有目的基因的染色体区域开放并可供重组酶系统使用。我们还提出，未重排的C_H基因本身的转录本可以作为反式剪接反应的底物之一，同时表达多种异型。我们已经做了实验来验证我们的假设，并获得了支持我们的假设的结果，如下所示。在人类C&lt；Gamma3&gt；基因的生殖系转录调控区域，发现了一种激活IL-4基因转录的增强子。从Splee…中分离到S-S重组的副产物环状DNA在内毒素和IL-4刺激下，n细胞数量增加。这为S-S重组过程中分子内环出缺失提供了第一个直接证据。这也表明，IL-4直接激活了S-S本身到C_lt；Gammal&gt；基因2的重组。虽然我们使用的转基因小鼠中大部分内源性MU链的表达被人重排的MU转基因排除，但在脂多糖和IL-4的刺激下，很大一部分脾B淋巴细胞可以同时表达转基因的人IgM和内源性的小鼠免疫球蛋白。FACS纯化的人IgM~+/小鼠免疫球蛋白G~+细胞群表达了由转基因V_hdj_H和内源性小鼠C_(lt；)γ&gt；基因的正确剪接序列组成的mRNA，以及转基因的人Mu基因和小鼠C_(lt；)我们还发现了一种淋巴瘤肿瘤，起源于TG之间的交叉。SA小鼠和另一只携带Ig H链增强子驱动的c-myconcogene基因的转基因小鼠在转基因或内源性切换区都表达了与转基因小鼠相同水平的反式和转基因mumRNA，而没有DNA重排。这些发现有力地支持了我们对多种异型表达的反式剪接假说。目前，我们正试图通过构建和筛选内毒素+IL-4刺激的小鼠脾细胞中表达型基因文库，来分离与生殖系转录调控有关的基因(S)。为了确定S-S重组所涉及的基因(S)(重组酶基因(S))，我们试图用Lamdba gtll表达载体从诱导类转换的小鼠脾细胞中分离出与每个C_H基因的开关(S)区特异结合的编码蛋白(S)的基因(S)，并用西南法筛选该文库。较少

We proposed a hypothesis concerning the molecular mechanisms for the immunoglobulin class switching that lymphokines or cytokines stimulate B lymphocytes to induce the transcription from the target C_H gene of the class switch prior to the DNA recombination (S-S recombination), and to make the chromosomal region containing the target gene open and accessible for the recombination-enzyme system through the transcription. We also proposed that the transcripts from un-rearranged C_H genes per se act as one of the substrates of trans-splicing reaction to express multiple isotypes simultaneously. We have done the experiments to verify our hypotheses, and obtained the results which supported our hypothesis as listed below.1. An enhancer which activate the transcription in response to interleukin-4 (IL-4) was identified with in the region regulating the germ line transcription of human C_<gamma3> gene. Circular DNA which was the counter-product of the S-S recombination was isolated from splee … More n cells stimulated by LPS and IL-4. This provided the first direct evidence for the intra-molecular looping-out deletion during the S-S recombination. This also showed that IL-4 directly activated the S-S recombination per se to C_<gammal> gene.2. Though most of the endogenous mu chain expression was excluded by the expression of the human rearranged mu transgene in the transgenic mouse we used, a significant portion of splenic B lymphocytes could express the transgenic human IgM and endogenous mouse IgG simultaneously after stimulation with LPS and IL-4. The FACS-purified population of the human IgM^+/mouse IgG^+ cells expressed mRNA which consisted of properly spliced sequences of the transgenic V_HDJ_H and the endogenous mouse C_<gamma> genes (trans-mRNA), together with the transgenic human mu mRNA and germ line transcripts of the mouse C_<gamma> gene, without apparent rearrangement of the transgene. We also found that a lymphoma tumor, derived from the cross between the TG. SA mouse and another transgenic mouse carrying Ig H chain enhancer-driven c-myconcogene, expressed about equal levels of the trans-mRNA and the transgenic mu mRNA without DNA rearrangement in either the transgene or the endogenous mouse switch region. These findings strongly support our trans-splicing hypothesis for the multiple isotype expression. Now we are trying to isolate the gene (s) which are responsible to the regulation of germ line transcription, by making and screening an expression-type cDNA library from LPS_+IL-4 stimulated mouse spleen cells.3. In order to identify the gene (s) involved in the S-S recombination (gene (s) for recombinase), we are trying to isolate gene (s) encoding protein (s) which specifically binds to switch (S) region of each C_H gene by making cDNA library using lamdba gtll expression vector from mouse spleen cells which are induced class switching, and by screening this library using South-western method. Less