MOLECULAR BASIS OF IMMUNOGLOBULIN HEAVY CHAIN SWITCH

免疫球蛋白重链开关的分子基础

• 批准号: 3135187
• 负责人: Janet M. Stavnezer
• 金额: $ 24.31万
• 依托单位: UNIV OF MASSACHUSETTS MED SCH WORCESTER
• 依托单位国家: 美国
• 财政年份: 1985
• 资助国家: 美国
• 起止时间: 1985-08-01 至 1989-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/3135187
• 关键词: B lymphocyte; endonuclease; endoribonucleases; gene complementation; gene expression; genetic manipulation; genetic mapping; hybridomas; immunofluorescence technique; immunogenetics; immunoglobulin G; immunoglobulin M; immunoglobulin genes; immunoglobulins; immunoregulation; lymphoma; molecular cloning; molecular oncology; nucleic acid sequence; tissue /cell culture

The murine B cell lymphoma, I.29, consists of cells expressing either IgM or IgA with the identical idiotype and with the identical heavy (H) chain variable (V) region sequence. By DNA blotting experiments and by examination of cloned H chain genes, we found that the IgM cells contain one expressed VDJ-CMu gene and one non-expressed DJ-CMu gene segment, and all the other H chain C genes in the germline configuration. The IgA cells have deleted Mu genes from both chromosomes, and have undergone a typical switch recombination with the Alpha gene on the expressed chromosomes, and the Gamma3 gene on the non-expressed chromosome. When purified IgM cells from the tumor are cultured in vitro they can be induced to switch to IgA or to IgE with LPS or with monoclonal anti-IgM or anti-I.29 idiotype (Id). We propose to clone T cells which will help the switch to either IgA or to IgE. We plan to clone DNA fragments bearing the rearranged Alpha and Mu genes about 3 days after induction of switching to examine the earliest DNA recombination events, to determine whether specific sites are involved and whether sister-chromatid exchange may occur. The IgM cells appear to be committed to switch to either IgA or IgE, as the Alphagene is hypomethylated (relative to liver DNA) in the IgM cells, whereas the Gamma2b gene is not. Furthermore, the Alpha and Epsilon genes are being transcribed at a low level in the IgM cells, whereas the Gamma1 and Gamma2b genes are not. We will further examine the mechanism of predetermination of isotype specificity by studies of chromatin structure and by attempting to induce isotype switching in hybrids of the B cell lymphoma, WEHI 279, and I.29Mu cells. The DNA sequences required for isotype switching and the specificity of switching will be studied by transfection of Ig H chain gene constructs which can serve as substrates for switch recombination into I.29Mu cells, which will then be induced to switch. Eventually we will attempt to prepare a cell-free system which can recombine switch region sequences. Our plans also include the cloning of cDNAs for poly(A)+ RNAs which appear when switching is induced. We will attempt to determine what proteins these genes encode.

鼠B细胞淋巴瘤I. 29由表达IgM或IgM的细胞组成。 或具有相同独特型和相同重（H）链的伊加 可变（V）区序列。 通过DNA印迹实验和 对克隆的H链基因进行检测，我们发现IgM细胞含有 一个表达的VDJ-CMu基因和一个非表达的DJ-CMu基因片段，和 所有其他的H链C基因在生殖系的配置。 伊加细胞 从两条染色体上删除了Mu基因，并经历了一个典型的 与表达染色体上的Alpha基因进行切换重组，以及 非表达染色体上的Gamma 3基因。 当在体外培养来自肿瘤的纯化的IgM细胞时，它们可以是 用LPS或单克隆抗IgM诱导转换为伊加或IgE，或 抗I.29独特型（Id）。 我们建议克隆T细胞，这将有助于 换用伊加或IgE 我们计划克隆带有 诱导转换后约3天， 检查最早的DNA重组事件，以确定是否 是否涉及特定位点以及是否可能发生姐妹染色单体交换。 IgM细胞似乎致力于转换为伊加或IgE，因为IgM细胞可能是IgA细胞。 Alphagene在IgM细胞中是低甲基化的（相对于肝脏DNA）， 而Gamma 2b基因不是。 此外，阿尔法和Epoxy基因 在IgM细胞中以低水平转录，而γ 1 而Gamma 2b基因则不是。 我们会进一步研究 通过染色质结构研究预先确定同种型特异性 并通过尝试诱导B细胞杂交体中的同种型转换 淋巴瘤、WEHI 279和I.29Mu细胞。 所需的DNA序列 同种型转换和转换的特异性将通过 可作为底物的IG H链基因构建体的转染 用于开关重组到I.29Mu细胞中，然后将诱导I.29Mu细胞 开关. 最终我们将尝试制备一种无细胞系统， 重组开关区序列。 我们的计划还包括克隆 当诱导转换时出现的poly（A）+RNA的cDNA。 我们将 试图确定这些基因编码的蛋白质。