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细胞阶段特异性及其功能特性 负责抗体(Ab)类别切换的系统。我们的新战略 很大程度上是基于使用免疫球蛋白(Ig)重链常数 (CH)区基因开关(S)区作为重组底物 被改造成逆转录病毒。两性逆转录病毒携带多种 将S地区的基板引入各种开关-- 能力B细胞：300-18株Pre-B和1.29株成熟B细胞，EBV- 永生化生发中心B细胞，长期培养 分化的抗体分泌B细胞，刺激脾和Peyer‘s斑 B细胞。促有丝分裂素(LPS)和重组蛋白的重组效应 分化刺激(内毒素和II-4)对B细胞的特异性 S底物逆转录病毒的重组将被确定。遗传 携带嵌合体的反选择逆转录病毒技术 潮霉素-胸苷激酶(Hytk)基因将被用于尝试 分离具有上调和下调其组成成分的B细胞 开关重组酶活力。这种开关可变线路将是 不仅对于破译重组酶的调控，而且对于作为 克隆重组酶活性的潜在关键试剂。在……里面 第二个目的，BSAP/NFSmuB1(B细胞特异性)的作用 转录/开关区结合因子)在开关重组中 被调查。高亲和力BSAP的重组活性研究 S底物中插入的结合位点及其建立的影响 以及抑制重组酶功能的BSAP活性将被确定 在各种B细胞背景下。在第三个目标中，BSAP附件或合作伙伴 可以调节其DNA结合位点特异性的蛋白质，影响 S区域无障碍和/或代表开关的组件- 通过筛选lambda-gt11基因的表达克隆重组酶 含谷胱甘肽S的重组酶阳性B细胞文库 转移酶-BSAP融合蛋白。最后，一种基因拯救--选择 将开发策略来克隆编码B细胞特异性的基因 开关-重组酶的组成部分。一种哺乳动物表达载体基因的构建 将介绍由重组酶阳性B细胞mRNA制备的文库 脂质体介导转染重组酶阴性抗体 分泌杂交瘤细胞系A39RGamma1.1tk-，携带重组 插入开关-反转矢量。溴脱氧尿苷的选择将允许 稀有缺失S逆转录载体胸腺嘧啶核苷转染体的分离 融合蛋白介导的开关缺失对激酶基因功能的影响 位于SMU和SGamma2b序列两侧。这些抵抗BUDR的细胞将是 开关重组酶激活基因的候选接受者，这将 极大地方便了它们的克隆和功能鉴定。这些 目标将使我们能够破译分子基础和 抗体成熟过程中一个关键过程的发育调控 产生多种基本效应细胞的介导性免疫反应 具体细节。

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.