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的细胞组成 或具有相同惯用型和相同重（H）链的IgA 变量（V）区域序列。 通过DNA印迹实验和 克隆H链基因的检查，我们发现IgM细胞包含 一个表达的VDJ-CMU基因和一个非表达的DJ-CMU基因段，并且 种系构型中的所有其他H链C基因。 IgA细胞 已经从两个染色体中删除了MU基因，并且经历了典型的 与α基因的开关重组在表达的染色体上，并且 非表达染色体上的γ3基因。 当从肿瘤中纯化的IgM细胞在体外培养时可以是 诱导切换到IgA或使用LPS或单克隆抗IGM或 抗I.29白痴（ID）。 我们建议克隆T细胞，这将有助于 切换到IgA或IgE。 我们计划克隆DNA片段带有 诱导切换到3天后，重新排列的α和MU基因 检查最早的DNA重组事件，以确定是否 涉及特定的站点以及是否可能发生姊妹染色剂交换。 IgM单元似乎致力于切换到IgA或IgE，因为 字母是甲基化的（相对于肝DNA），在IgM细胞中， 而γ2b基因不是。 此外，Alpha和Epsilon基因 在IgM细胞中以低水平转录，而γ1 和γ2b基因不是。 我们将进一步研究 通过染色质结构的研究对同种型特异性进行预先确定 并试图诱导同种异体类型切换B的杂种 淋巴瘤，WEHI 279和I.29MU细胞。 DNA序列需要 同型切换和切换的特异性将由 可以用作底物的Ig H链基因构建体的转染 对于开关重组到I.29MU单元，然后将其诱导为 转变。 最终，我们将尝试准备一个可以的无单元系统 重组开关区域序列。 我们的计划还包括克隆 诱导开关时出现的poly（a）+ RNA的cDNA。 我们将 尝试确定这些基因编码的蛋白质。