B CELL REGULATION BY INTERLEUKIN 5 PLUS ANTIGEN

抗体基因在一种细胞类型中被调节的机制- 对细胞因子和抗原刺激的特异性反应很差 明白了。我们已经开发了一个模型系统来研究这些机制 其中的抗原，磷胆碱，和细胞因子，白介素5， 影响B细胞分化并发现IL-5+抗原 导致MU免疫球蛋白基因转录增加。几种DNA- 在有丝分裂原刺激或基础刺激中可能重要的结合蛋白 免疫球蛋白转录被证明结合在200个碱基以内 转录起始点的对。IL-5+抗原诱导的升高 在B细胞特异的迁移率移位的蛋白质-DNA复合体中，结合到 A+T富含序列200至500个碱基对的转录起始 地点。这些蛋白质-DNA复合体可能含有不止一个 蛋白。在拟议的研究中，这些蛋白质将被表征 并对它们的功能进行调查。 这项研究的广泛目标是确定一些 B细胞对IL-5+抗原的反应发生的调节事件。 具体的目标是：(1)确定B的分子基础 先前确定的IL-5+抗原的细胞特异性迁移率 可诱导的DNA-蛋白质复合体，(2)确定蛋白质是否 在这个复合体中，通过发挥拓扑异构酶功能来调节转录， DNA弯曲蛋白，或核基质的成分，以及(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。蛋白质将进一步表征 采用紫外光交联法和迁移率移动法。完全了解 这些蛋白质在转录中所起的作用可能需要在体外 系统。因此，单个蛋白质将通过常规的 方法用亲和层析柱进行纯化。编码蛋白质的基因 似乎对IL-5+反应重要的抗原将被克隆 使用常规和/或表达文库。 初步数据表明，从T细胞提取的蛋白质复合体 也与这些序列结合，并且这些复合体包含一些 与B细胞复合体中发现的蛋白质不同。其他 在B细胞和T细胞提取液中似乎都发现了蛋白质。检视 B细胞特有的蛋白质是否在细胞中发挥作用 免疫球蛋白、T细胞转染体的类型特异性表达 将生产和检测包含克隆的B细胞特异性基因 因为他们有能力表达和调控B细胞特异性基因。 这些研究应该有助于深入了解蛋白质 以细胞类型特异性的方式调节转录增加，并可能 导致鉴定所需的其他重要的B细胞蛋白 用于细胞内信号对IL-5和抗原的反应。

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.