MOLECULAR BASIS OF IMMUNOGLOBULIN HEAVY CHAIN SWITCH

免疫球蛋白重链开关的分子基础

• 批准号: 2413524
• 负责人: Janet M. Stavnezer
• 金额: $ 31.56万
• 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 1999-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2413524
• 关键词: ADP ribosylation; B lymphocyte; DNA binding protein; endonuclease; enzyme activity; gene expression; gene rearrangement; gene targeting; genetic transcription; immunoglobulin A; immunoglobulin genes; immunoglobulin structure; laboratory mouse; messenger RNA; molecular cloning; nucleic acid sequence; polymerase chain reaction; protein structure function; southern blotting; tissue /cell culture; transfection; transforming growth factors

When mature B lymphocytes, which express IgM and IgD on their surface, are activated by antigen and receive accessory signals, they switch to express downstream heavy chain constant region (CH) genes while maintaining the same expressed variable region domain. This class switch changes the ability of the antibody to clear various pathogens from various sites in the body since the CH region determines the effector function of the antibody. Antibody class switching is effected by a DNA recombination event called switch recombination, which occurs within or near 2 to 10 kb switch (S) regions containing tandemly repeated sequences located 5' of each CH gene, except Cdelta. The means by which the DNA is cut, aligned and rejoined is unknown, as are the roles of several nuclear proteins that have been shown to bind to S regions. it has become clear that switching is directed by induction of accessibility of particular CH genes prior to class switch recombination since the unrearranged, or germline, CH genes to which a B cell will subsequently undergo class switching are transcribed prior to switching to that particular CH gene. Furthermore, this transcription is induced by cytokines that direct switching to particular CH genes and promoters for these germline CH transcripts are regulated by cytokines. This grant is a proposal to continue studies that have been funded for 9 years on the mechanism and regulation of antibody class switching, concentrating on the switch to IgA, using a mouse cell line l.29mu that undergoes this process in culture. Mouse splenic B cells will be used to verify the results with I.29mu cells, when feasible. Gene targeting will be used to determine the function of germline transcription, asking whether the RNA itself or protein binding sites 5' to tandem repeated switch sequences are required in addition to transcription. Experiments to create transfectable plasmids that can undergo class switching will be continued in order to define DNA sequences required for switching and to assay the function of genes that affect class switching. Effort will be directed toward the characterization and cloning of the gene encoding a protein(s) that binds to novel TGFbeta-response elements in the promoter for germline Calpha transcripts that were identified since the last competitive renewal. Genes encoding mRNAs that are up or downregulated in cells induced to undergo class switching will be cloned and characterized. During the previous cycle this group has shown that inhibitors of the nuclear enzyme which aids in DNA repair, poly (ADP-ribose) polymerase increase switch recombination and efforts will now be directed toward understanding the mechanism of this stimulation. The sites and kinetics of induction of the endonuclease cutting events that must be required to initiate switch recombination will be examined.

当在其表面表达IgM和IgD的成熟B淋巴细胞 被抗原激活并接收辅助信号，它们切换到表达 下游重链恒定区(CH)基因同时维持 表达相同的可变区结构域。此类开关更改 抗体清除不同部位不同病原体的能力 从CH区域开始的身体决定了 抗体。抗体类别转换受DNA重组事件的影响 称为交换机重组，发生在2到10 kb的交换机内部或附近 (S)包含位于每个CH的5‘端的重复序列的区域 吉恩，除了CDelta。DNA被切割、比对和 重新连接是未知的，也是几个核蛋白的作用 已被证明与S地区有联系。很明显，切换是 通过诱导特定CH基因的可及性在 类开关重组，因为未重排，或生殖系，CH基因 B细胞随后将经历类别转换的哪些被转录 在切换到那个特定的CH基因之前。此外，这一点 转录是由细胞因子诱导的，细胞因子直接切换到特定的 这些生殖系CH转录本的CH基因和启动子受 细胞因子。 这笔赠款是一项继续研究的建议，这些研究已经被资助了9年 多年来关于抗体类别转换的机制和调控， 专注于向IgA的转换，使用的是小鼠细胞系1.29 在文化上经历了这个过程。小鼠脾B细胞将用于 在可行的情况下，用I.29微米电池验证结果。基因打靶将是 用来确定生殖系转录的功能，询问是否 RNA本身或蛋白质结合部位5‘端串联重复开关 除了转录外，还需要序列。要创造的实验 可以进行类别转换的可转化质粒将继续 为了定义转换所需的DNA序列并分析 影响班级转换的基因的功能。努力的方向将是 蛋白质编码基因的鉴定与克隆(S) 与生殖系启动子中的新的转化生长因子β反应元件结合 自上次竞争续签以来确定的卡尔法成绩单。 编码在细胞中上调或下调的mRNA的基因诱导 进行班级转换将被克隆和表征。在.期间 在之前的循环中，这个小组已经证明了核酶的抑制剂 帮助DNA修复，多聚(ADP-核糖)聚合酶增加开关 重组和努力现在将指向理解 这种刺激的机制。诱导的部位和动力学 启动开关必须需要的核酸内切事件 我们将研究重组问题。