B CELL REGULATION BY INTERLEUKIN 5 PLUS ANTIGEN

抗体基因在细胞类型中的调节机制- 对细胞因子和抗原刺激的特异性应答方式较差 明白我们已经开发了一个模型系统来研究机制， 其中抗原，磷酸胆碱，和细胞因子，白细胞介素-5， 影响B细胞分化，并已发现IL-5 +抗原 导致mu免疫球蛋白基因转录增加。几个DNA- 结合蛋白，可能是重要的有丝分裂原刺激或基础 免疫球蛋白转录已显示在200个碱基内结合 转录起始位点对。IL-5+抗原诱导的增加 在B细胞特异性迁移率改变的蛋白质-DNA复合物中， 富含A+T的序列，转录起始点5'的200至500个碱基对 绝佳的价钱这些蛋白质-DNA复合物可能含有不止一个 蛋白在拟议的研究中，这些蛋白质将被表征 并对其功能进行研究。 这项研究的主要目标是确定一些 在B细胞中响应IL-5+抗原而发生的调节事件。 具体目标是：（1）确定B的分子基础 先前鉴定的IL-5+抗原的细胞特异性迁移率 可诱导的DNA-蛋白质复合物，（2）以确定蛋白质是否 在这种复合物中， DNA弯曲蛋白，或核基质的组分，和（3） 测试这些蛋白质是否参与维持B细胞特异性 免疫球蛋白转录。蛋白质将被进一步表征 使用UV交联和迁移率变化测定。全面了解 这些蛋白质在转录中所起作用可能需要在体外 系统.因此，单个蛋白质将通过常规方法富集。 方法并用亲和柱纯化。编码蛋白质的基因， 将克隆在对IL-5+抗原的应答中似乎很重要的 使用常规和/或表达文库。 初步数据表明，T细胞提取物中的蛋白质复合物 也与这些序列结合，并且这些复合物含有一些 与在B细胞复合体中发现的蛋白质不同。其他 在B和T细胞提取物中似乎都发现了蛋白质。审查 B细胞特有的蛋白质是否在细胞中发挥作用 免疫球蛋白的类型特异性表达，T细胞转染子 将产生并检查含有克隆的B细胞特异性基因的 表达和调节B细胞特异性基因的能力。 这些研究应该提供深入了解蛋白质 以细胞类型特异性的方式调节增加的转录，并且可以 导致鉴定所需的其他重要B细胞蛋白 用于响应IL-5和抗原的细胞内信号传导。

The mechanisms by which antibody genes are regulated in a cell type- specific manner in response to cytokines and antigenic stimuli is poorly understood. We have developed a model system to study the mechanisms by which the antigen, phosphocholine, and the cytokine, interleukin-5, influence B cell differentiation and have found that IL-5 + antigen caused increased transcription of mu immunoglobulin genes. Several DNA- binding proteins that may be important in mitogen-stimulated or basal immunoglobulin transcription have been shown to bind within 200 base pairs of the transcription start site. IL-5+ antigen induced increases in B cell-specific mobility-shifted protein-DNA complexes that bound to A+T rich sequences 200 to 500 base pairs 5' of the transcription start site. These protein-DNA complexes probably contain more than one protein. In the proposed studies, these proteins will be characterized and their functions will be investigated. The broad objective of this research is to identify some of the regulatory events that occur in B cells in response to IL-5+ antigen. The specific goals are: (1) to determine the molecular basis for the B cell-specific mobility of the previously identified IL-5+ antigen inducible DNA-protein complexes, (2) to determine whether the proteins in this complex regulate transcription by functioning as topoisomerases, DNA-bending proteins, or components of the nuclear matrix, and (3) to test whether these proteins are involved in maintaining B cell-specific immunoglobulin transcription. Proteins will be further characterized using UV crosslinking and mobility shift assays. Total understanding of the role these proteins play in transcription may require in vitro systems. Therefore, individual proteins will be enriched by conventional methods and purified with affinity columns. Genes encoding proteins that appear to be important in the response to IL-5+ antigen will be cloned using conventional and/or expression libraries. Preliminary data indicates that protein complexes from T cell extracts also bind to these sequences, and that these complexes contain some proteins different than those found in the B cell complexes. Other proteins appear to be found in both B and T cell extracts. To examine whether the proteins that are unique to B cells play a role in the cell type-specific expression of immunoglobulin, T cell transfectants containing cloned B cell-specific genes will be produced and examined for their ability to express and regulate B cell-specific genes. These studies should provide insight into mechanisms by which proteins regulate increased transcription in a cell type-specific manner, and may lead to the identification of other important B cell proteins required for intracellular signaling in response to IL-5 and antigen.