Molecular mechanisms for immunoglobulin class switching

我们提出了免疫球蛋白类转换的分子机制假说，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基因C_H基因的转录，并通过转录使含有靶基因的染色体区域开放并为重组酶系统所可及。我们还提出未重排的C_H基因本身作为反式剪接反应的底物之一，同时表达多个同型。我们做了实验来验证我们的假设，得到的结果支持我们的假设如下：1。在人C_<gamma3>基因的种系转录调控区发现了一个响应白细胞介素-4 （IL-4）激活转录的增强子。在LPS和IL-4刺激下，从脾细胞中分离出S-S重组的反产物环状DNA。这为S-S重组过程中的分子内环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本身对C_<gammal>基因的重组2。虽然在我们使用的转基因小鼠中，人重排的mu转基因基因的表达排除了大部分内源性mu链的表达，但在LPS和IL-4的刺激下，有相当一部分脾B淋巴细胞可以同时表达转基因人IgM和内源性小鼠IgG。facs纯化的人IgM^+/小鼠IgG^+细胞群体表达由转基因V_HDJ_H和内源性小鼠C_< γ >基因（trans-mRNA）正确拼接的mRNA，以及转基因人mu mRNA和小鼠C_< γ >基因的种系转录本组成的mRNA，无明显的转基因重排。我们还发现一个淋巴瘤肿瘤，来源于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的转基因小鼠，在转基因和内源性小鼠开关区均表达了相同水平的反式mRNA和转基因mu mRNA，没有DNA重排。这些发现有力地支持了我们关于多重同型表达的反式剪接假说。目前，我们正试图从LPS_+IL-4刺激的小鼠脾细胞中制备和筛选表达型cDNA文库，分离出与种系转录调控有关的基因。为了鉴定参与s - s重组的基因（重组酶基因），我们试图从诱导类转换的小鼠脾细胞中，利用lamdba gtll表达载体构建cDNA文库，并利用西南法筛选该文库，分离出编码每个C_H基因开关s区特异性结合的蛋白的基因。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