MOLECULAR REQUIREMENTS FOR ANTIBODY CLASS SWITCHING

我们的长期目标是阐明机械要求， DNA重组酶的B细胞阶段特异性和功能特性 负责抗体（Ab）类别转换的系统。我们的新战略 免疫球蛋白（IG）重链恒定区的使用是主要的基础 (CH)区域基因开关（S）区域作为重组底物 在逆转录病毒中改造。携带多种双嗜性逆转录病毒 S区衬底将被引入到各种开关- 感受态B细胞：300-18个前B细胞系和1.29个成熟B细胞系，EBV- 永生化的生发中心B细胞，长期培养 分化的分泌抗体的B细胞，刺激的脾和派伊尔集合淋巴结 B细胞。促有丝分裂（LPS）和 分化（LPS和II-4）刺激对B细胞特异性 将确定S底物逆转录病毒的重组。遗传 带有嵌合体的反选择逆转录病毒技术 潮霉素-胸苷激酶（Hytk）基因将用于尝试 分离B细胞已经上调和下调其组成型 转换重组酶活性。这种开关可变线路将是 这不仅对于破译重组酶的调控机制， 用于克隆重组酶活性的潜在关键试剂。在 第二个目的，BSAP/NFSmu-B1（一种B细胞特异性 转录/开关区结合因子）在开关重组中将 追究高亲和力BSAP的重组活性 插入S底物的结合位点和建立 并测定抑制BSAP活性对重组酶功能的影响 在不同的B细胞背景中。在第三个目标中，BSAP的配件或合作伙伴 能够调节其DNA结合位点特异性的蛋白质， S区域可访问性和/或代表交换机的组件- 重组酶将通过筛选Gt 11 cDNA表达来克隆 重组酶阳性B细胞的文库与谷胱甘肽S- BSAP融合蛋白。最后，基因拯救选择 将开发策略来克隆编码B细胞特异性 开关重组酶的组分。哺乳动物表达载体cDNA 将引入从重组酶阳性B细胞mRNA制备的文库 通过脂质体介导的转染进入重组酶阴性抗体， 分泌型杂交瘤细胞系A39Rgamma1.1tk-， 插入反向转换器。溴脱氧尿苷选择将允许 缺失S-逆转录载体胸苷罕见转染子的分离 激酶基因功能通过开关缺失介导的融合 Smu和Sgamma 2b序列的侧翼。这些BudR抗性细胞 转换重组酶激活基因的候选受体， 极大地促进了它们的克隆和功能表征。这些 目标将使我们能够破译分子基础， 抗体成熟关键过程的发育调控 介导的免疫应答产生多种必需的效应细胞 特殊性

Our long-term objectives are to elucidate the mechanistic requirements, B cell stage specificity and functional properties of the DNA recombinase system responsible for antibody (Ab) class switching. Our novel strategy is largely based on the use of immunoglobulin (Ig) heavy chain constant (CH) region gene switch (S) regions as recombination substrates engineered in retroviruses. Amphotropic retroviruses harboring a variety of S region substrates will be introduced into a variety of switch- competent B cells: 300-18 pre-B and 1.29 mature B cell lines, EBV- immortalized germinal center B cells, long-term cultures of differentiated Ab secreting B cells, stimulated splenic and Peyer's patch B cells. The recombinagenic effects of mitogenic (LPS) and differentiation (LPS and II-4) stimuli on the B cell specific recombination of S substrate retroviruses will be determined. Genetic techniques with counter-selectable retroviruses harboring a chimeric hygromycin-thymidine kinase (Hytk) gene will be employed in attempts to isolate B cells which have up and down-modulated their constitutive switch-recombinase activity. Such switch-variant lines would be invaluable not only for deciphering recombinase regulation but also as potentially critical reagents for cloning the recombinase activity. In a second aim, the role of BSAP/NFSmu-B1 (a B cell specific transcription/switch region binding factor) in switch-recombination will be investigated. The recombinagenic activity of high affinity BSAP binding sites inserted in S substrates and the affects of establishing and inhibiting BSAP activity on recombinase function will be determined in various B cell backgrounds. In a third aim, BSAP accessory or partner proteins which could modulate its DNA binding site specificity, affect S region accessibility and/or represent components of the switch- recombinase will be cloned by screening lambda-gt11 cDNA expression libraries of recombinase positive B cells with a glutathione S- transferase-BSAP fusion protein. Finally, a gene rescue-selection strategy will be developed to clone genes encoding B cell specific components of the switch-recombinase. A mammalian expression vector cDNA library prepared from recombinase positive B cell mRNA will be introduced by liposome-mediated transfection into a recombinase negative, antibody secreting hybridoma line, A39Rgamma1.1tk-, harboring a recombinationally insert switch-retrovector. Bromodeoxyuridine selection will permit the isolation of rare transfectants which have lost S-retrovector thymidine kinase gene function via switch-deletion mediated by the fusion of flanking Smu and Sgamma2b sequences. These BudR resistant cells would be candidate recipients of switch-recombinase activating genes, which would greatly facilitate their cloning and functional characterization. These objectives will enable us to decipher the molecular basis and developmental regulation of a key process in the maturation of antibody mediated immune responses yielding a variety of essential effector cell specificities.