Molecular mechanisms for immunoglobulin class switching

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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