MOLECULAR BASIS OF IMMUNOGLOBULIN HEAVY CHAIN SWITCH

免疫球蛋白重链开关的分子基础

• 批准号: 3135188
• 负责人: Janet M. Stavnezer
• 金额: $ 25.39万
• 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/3135188
• 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; laboratory mouse; lymphoma; molecular cloning; molecular oncology; nucleic acid sequence; protein biosynthesis; 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.

小鼠B细胞淋巴瘤I.29由表达IgM的细胞组成 或具有相同独特型和相同重(H)链的IgA 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细胞中含有 一个表达VDJ-CMU基因片段和一个未表达的DJ-CMU基因片段，以及 生殖系构型中的所有其他H链C基因。免疫球蛋白A细胞 已经从两条染色体上删除了Mu基因，并经历了典型的 与表达的染色体上的Alpha基因切换重组，以及 未表达染色体上的Gamma3基因。 当从肿瘤中纯化的IgM细胞在体外培养时，它们可以 用脂多糖或单抗诱导转成IgA或IgE 抗I.29独特型(ID)。我们建议克隆T细胞，这将有助于 切换到IgA或IgE。我们计划克隆带有该基因的DNA片段 Alpha和Mu基因重排约在诱导后3天 检查最早的DNA重组事件，以确定 是否会发生姐妹染色单体交换，以及是否涉及特定的位点。 IgM细胞似乎致力于转换为IgA或IgE，因为 Alphagene在IgM细胞中是低甲基化的(相对于肝脏DNA)， 而Gamma2b基因则不是。此外，Alpha和Epsilon基因 在IgM细胞中低水平转录，而Gamma1 而Gamma2b基因则不是。我们将进一步研究其机制。 通过对染色质结构的研究预先确定同型特异性 并试图在B细胞的杂交体中诱导同型转换 淋巴瘤、WEHI 279和I.29Mu细胞。所需的DNA序列 同种类型的转换和转换的专一性将通过 可作为底物的免疫球蛋白H链基因载体的转染 用于开关重组到I.29Mu细胞，然后将被诱导 换一下。最终，我们将尝试准备一种无细胞系统，它可以 重组开关区域序列。我们的计划还包括克隆 诱导转换时出现的Poly(A)+RNA的cDNA。我们会 试图确定这些基因编码什么蛋白质。