EXPRESSION OF IMMUNOGLOBULIN HEAVY CHAIN GENES

我们的目标是关注分子机制和要求 免疫球蛋白（IG）重链恒定区（CH）基因开关 以及B细胞特异性反式作用对IG基因表达的调控 因素 更具体地说，我们计划继续我们的分子研究， 在MPC-11类别转换变体中连续的C-Gama基因转换。 我们也 描述了一种用于转换重组酶活性的新的体内选择测定 在淋巴细胞系中。 质粒和逆转录病毒载体携带不同的 将S区底物引入具有孔的前B细胞系中 定义的CH切换能力。 开关重组事件将是 通过遗传标记（HSV-胸苷激酶基因）的缺失进行选择 位于不同的S区域之间。 在最后一个具体目标中，我们将 研究IG H链基因转录的调控机制 增强子（Igh-E）通过反式作用的组织特异性因子。 我们再次 将采用体内选择测定。 在这里，我们将联合收割机 标记（新霉素抗性基因）置于Igh-E控制下， 体细胞杂交技术拯救携带这种染色体的B细胞染色体 反式作用因子 我们的战略将依赖于观察， 在Igh-E控制下用neo基因转化的成纤维细胞不获得 对新霉素的抗性。 因此，我们将尝试拯救新的表达 通过与骨髓瘤细胞系融合。 细胞杂交将被选择与 独立的遗传标记。 如果这种方法成功，我们将 使用该生物测定分离编码这些因子的基因 来自在哺乳动物表达载体中制备的B细胞cDNA文库。 这 技术具有分离和表征基因的潜力， 调节H和L链基因表达。

Our objectives are concerned with the molecular mechanism and requirements for the Immunoglobulin (Ig) Heavy chain Constant Region (CH) gene switch and the regulation of IG gene expression by B cell specific trans-acting factors. More specifically, we plan to continue our molecular studies of successive, C-Gama gene switches in MPC-11 class switch variants. We also describe a novel in vivo selection assay for switch recombinase activities in lymphoid cell lines. Plasmid and retroviral vectors harboring different S region substrates will be introduced into pre B cell lines with well defined CH switching capability. Switch-recombination events will be selected by the loss of a genetic marker (the HSV-Thymidine Kinase gene) placed in between different S regions. In our last specific aim, we will examine the mechanism of regulation of the Ig H chain gene transcriptional enhancer (Igh-E) by trans-acting, tissue specific factors. Once again, we will employ an in vivo selection assay. Here, we will combine a selectable marker (the neomycin resistance gene) placed under Igh-E control and somatic cell hybrid techniques to rescue B cell chromosomes harboring such trans-acting factors. Our strategy will rely on the observation that fibroblasts transformed with a neo gene under Igh-E control do not acquire resistance to neomycin. Therefore we will attempt to rescue neo expression by fusion with a myeloma cell line. Cell hybrids will be selected for with independent genetic markers. If this approach is successful, we will employ this biological assay to isolate the gene(s) encoding such factors from a B cell cDNA library prepared in a mammalian expression vector. This technology has the potential to isolate and characterize genes which regulate H and L chain gene expression.