EXPRESSION OF IMMUNOGLOBULIN HEAVY CHAIN GENES

这个应用的长期目标是定义分子 免疫球蛋白重链常数的要求和调节 B淋巴样细胞中的区域基因开关(S)重组 发展。我们的战略在很大程度上基于引入 携带免疫球蛋白基因重组的逆转录病毒载体 序列分为淋巴和非淋巴细胞系。重组 将使用DNA技术来确定分子 S区域重组的约束条件和最低要求。 还将准备适当的载体来筛选假定的 CH同位素特异性开关重组酶。CH的存在 将在细胞系中检测开关重组酶的活性 B细胞发育不同阶段的代表和 脂多糖和白介素类在致病中的作用 B细胞分化，如IL-4也将被确定。在……里面 第二个特定目标，我们将研究B细胞特异性核 与S区域序列相结合的因子，以确定其 在开关重组中的潜在作用(S)。第三个目标是， 核连续转录技术将被用于 直接决定CH基因的转录激活 预先决定了B细胞分化过程中的CH开关重组。 我们还将调查正义和反义的调节 未重排CH基因座的转录本及其潜力 开关复合机制的意义。最后，我们 哺乳动物免疫球蛋白开关重组酶基因(S)的克隆计划 一种方便、选择性抢救表达载体cDNA库的方法 策略。在这里，胸苷激酶阴性的成纤维细胞系 拥有稳定整合的，尽管重组不活跃， 逆转录病毒IgH基因重组底物将被转染 构建了一个哺乳动物表达载体cDNA文库， 一株免疫球蛋白重组能力强的前B细胞系的mR-NA。损失 一种胸苷激酶标记基因插入我们的 反转录载体的免疫球蛋白重组序列将被选择用于 溴脱氧尿嘧啶核苷(BUR)培养液。通过这种选择策略，cDNA. 能够编码负责TK的开关重组酶的克隆 基因缺失将被识别和表征。这些 目标将使我们能够剖析分子基础和 免疫应答中一个关键过程的发育调控 成熟。

The long term goals of this application are to define the molecular requirements and regulation of immunoglobulin heavy chain constant region gene switch (S) recombination in B lymphoid cell development. Our strategy is largely based on the introduction of retroviral vectors harboring immunoglobulin gene recombination sequences into lymphoid and non-lymphoid cell lines. Recombinant DNA techniques will be employed to determine the molecular constraints and minimal requirements for S region recombination. Appropriate vectors will also be prepared to screen for putative CH isotope specific switch-recombinases. The presence of CH switch-recombinase activity will be assayed in cell lines representative of various stages of B cell development and the effects of lipopolysaccharide (LPS) and interleukins which cause B cell differentiation, such as IL-4 will also be determined. In a second specific aim, we will study B cell specific nuclear factors, which bind to S region sequences, to determine their potential role(s) in switch recombination. In a third objective, the nuclear run-on transcription technique will be employed to directly determine if transcriptional activation of CH genes predetermines CH switch-recombination in differentiating B cells. We will also investigate the regulation of sense and anti-sense transcripts of unrearranged CH loci and their potential significance for the switch recombination mechanism. Finally, we plan to clone Igh switch-recombinase gene(s) from a mammalian expression vector cDNA library by a convenient, selectable rescue strategy. Here, thymidine kinase negative fibroblast cell lines harboring stably integrated, though recombinationally inert, retroviral Igh gene recombination substrates will be transfected with a mammalian expression vector cDNA library prepared from the mRNA of an Igh recombination competent pre B cell line. The loss of a thymidine kinase marker gene inserted in between our retrovector's Igh recombination sequences will be selected for in bromodeoxyuridine (BudR) media. By this selection strategy, cDNA clones which could encode a switch recombinase responsible for TK gene deletion will be identified and characterized. These objectives will enable us to dissect the molecular basis and developmental control of a key process in immune response maturation.