STUDIES OF THE CONTROL OF IMMUNOGLOBULIN GENE EXPRESSION

免疫球蛋白基因表达控制的研究

• 批准号: 3445629
• 负责人: Huntington Potter
• 金额: $ 6.43万
• 依托单位: HARVARD MEDICAL SCHOOL
• 依托单位国家: 美国
• 财政年份: 1985
• 资助国家: 美国
• 起止时间: 1985-03-01 至 1988-02-29
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/3445629
• 关键词: B lymphocyte; antibody formation; bacteria; gene expression; genetic transcription; human tissue; immune response genes; immunogenetics; immunoglobulin genes; messenger RNA; molecular oncology; multiple myeloma; nucleic acid sequence; plasmids; tissue /cell culture

Specialized nucleic acid elements are involved in the expression of antibody genes. These elements are 'enhancers' which act positively to promote transcription. The DNA rearrangements that occur during the construction of an antibody gene bring the enhancer near an otherwise weak promoter, thus activating the promoter. Specially significant is the fact that unlike other enhancers, which are carried on viruses, the immunoglobulin enhancers are tissue specific - they only function in B lymphocytes. This suggests that B cells contain as a result of their differentiation, proteins which are designed to function as specific transcriptional factors at this site. This application proposes two general approaches for identifying and isolating the transcriptional factors involved in the expression of immunoglobulin genes. These factors and the control of their synthesis during differentiation may serve as prototypes for intracellular regulatory factors involved in other aspects of development. The first proposed approach is biochemical and involves trying to isolate the enhancer recognition protein directly, by assuming that it will specifically bind to enhancer DNA. In addition, an in vitro transcription system will be developed in which messenger RNA synthesis from DNA fragments - containing immunoglobulin genes with or without the enhancer - is asked to be stimulated by the addition of a protein fraction derived from B lymphocytes. The second general strategy is to seek mutants in the gene which codes for the enhancer recognition protein, and then use the mutants to isolate the gene itself. The fact that the immunoglobulin enhancer works only in B cells has allowed the design of three specific experimental strategies. Each is based on being able to construct and introduce into cells a drug resistance gene whose expression is dependent on an active immunoglobulin promoter and enhancer. With both the protein and nucleic acid components involved in immunoglobulin gene expression in hand, it should be possible to study this example of a regulated eukaryotic gene with the same depth that has been possible with, for example, the lactose and tryptophan operons in bacteria. A deeper goal is to understand how the irreversible regulatory switch is thrown at the start of the differentiation of B lymphocytes, and how this switch is related to the initiation of transformation in B cell myelomas. Ultimately, purification of the enhancer recognition protein and analysis of mutations in the enhancer site that affect the proteins' ability to bind to the DNA will begin to provide such a basis molecular description of immunoglobulin regulation and provide key insights into the processes of development and differentiation in general.

专门的核酸元件参与表达 抗体基因 这些元素是“增强剂”， 促进转录。 DNA重排发生在 抗体基因的构建使增强子接近原本较弱的 启动子，从而激活启动子。 特别重要的是， 与其他病毒携带的增强子不同， 免疫球蛋白增强子是组织特异性的-它们仅在B中起作用 淋巴细胞 这表明，B细胞含有作为其结果， 分化，蛋白质被设计为作为特异性功能 转录因子在这个位置。 本申请提出了两种用于识别和 分离参与表达的转录因子， 免疫球蛋白基因。 这些因素及其合成的控制 在分化过程中可以作为细胞内调节的原型， 其他发展方面的因素。 第一种方法是生物化学的， 增强子识别蛋白直接，通过假设它将 特异性结合增强子DNA。 此外，体外转录 将开发一种系统，其中信使RNA从DNA合成 片段-含有免疫球蛋白基因，有或没有增强子- 被要求通过添加蛋白质级分来刺激， 来自B淋巴细胞。 第二种一般策略是在编码以下基因的基因中寻找突变体 增强子识别蛋白，然后使用突变体来分离增强子识别蛋白。 基因本身 免疫球蛋白增强子只在B中起作用的事实 cells允许设计三种特定的实验策略。 每一个都是基于能够构建并将药物引入细胞 依赖于活性免疫球蛋白表达的抗性基因 启动子和增强子。 蛋白质和核酸成分都参与了 免疫球蛋白基因的表达，应该有可能研究这一点， 一个受调控的真核基因的例子，其深度与已经被 例如，可能与乳糖和色氨酸操纵子一起， 细菌 更深层次的目标是了解不可逆转的监管如何 开关在B淋巴细胞分化开始时被触发， 这种开关如何与B细胞转化的启动相关 骨髓瘤 最终，纯化增强子识别蛋白， 分析增强子位点的突变，这些突变影响蛋白质的 与DNA结合的能力将开始提供这样的基础分子 免疫球蛋白调节的描述，并提供关键的见解， 一般的发展和分化过程。