NEGATIVE SELECTION AT THE PRO-B TO PRE-B CELL TRANSITION

描述(由申请人提供)：初步研究已确定 一组不同寻常的人类MU重链可以在细胞上表达 表面作为Pre-BCR的一部分，在没有替代光或常规光的情况下 锁链。这些异常的MU重链，最初是在一种 BCR前信号有缺陷的患者似乎能够激活细胞 并诱导轻链基因重排，但不能促进扩张 或B细胞前体细胞的长期存活。轻链独立链 具有预期的相对分子质量，并具有完整的VH和CH1结构域。 在VH3和VH4中都可以找到这些多重链的转录本 家人。它们约占重新排列的VH3的10% 在正常人前B细胞中发现多重链基因；然而，它们在 正常的前B细胞或成熟的B细胞。拟议中的实验将 对这些异常的重链进行生化表征，并检查 由他们引起的反应。具体目标是：1)确定具体目标 V(D)J重排中允许细胞表面表达或 在没有轻链的情况下分泌。异常的MuHeavy的结合 还将检查内质网伴侣BIP的链；2)使用两种培养方法 RAG2缺陷的前B细胞和四环素的逆转录病毒感染系统 Jurkat T细胞BCR的诱导性重组及其效果评价 细胞表面异常粘重链的表达 激活标记物与信号转导的磷酸化变化 分子和转录本数量的变化，这是表征 前B细胞或前B细胞分化阶段；以及3)产生转基因 表达正常或异常MU重链的小鼠及其B细胞特性 这些小鼠的发育。转基因小鼠将与下列小鼠杂交 所涉及的信号转导蛋白或分子为空或转基因 在细胞凋亡中。更好地了解这些不寻常的生物化学本质 MU重链及其去除机制应得到澄清 要求正常的前B细胞向前B细胞转变。建议数 研究还将提供有关发展的宝贵信息 正常和异常抗体谱系。

DESCRIPTION (provided by applicant): Preliminary studies have identified an unusual group of human mu heavy chains that can be expressed on the cell surface as part of a pre-BCR in the absence of surrogate or conventional light chains. These aberrant mu heavy chains, which were first identified in a patient with a defect in pre-BCR signaling, appear to be able to activate cells and induce rearrangement of light chain genes but they do not promote expansion or long-term survival of B-cell precursors. The light chain independent chains are of the expected molecular weight and have intact VH and CH1 domains. Transcripts for these muheavy chains can be found in both the VH3 and VH4 families. They constitute approximately 10 percent of the rearranged VH3 muheavy chain genes in normal human pro-B-cells; however, they are not found in normal pre-B-cells or mature B-cells. The proposed experiments will biochemically characterize these aberrant heavy chains and examine the responses elicited by them. The specific aims are 1) determine the particular sequences within the V(D)J rearrangement that allow cell surface expression or secretion in the absence of light chains. The binding of the aberrant muheavy chains to the ER chaperone BiP will also be examined; 2) use two culture systems, retroviral infection of RAG2 deficient pro-B-cells and a tetracycline inducible reconstitution of the BCR in Jurkat T-cells, to evaluate the effects of expression of the aberrant muheavy chain on cell surface expression of activation markers, alterations in phosphorylation of signal transduction molecules and shifts in the amount of transcripts that characterize the pro-B-cell or pre-B-cell stage of differentiation; and 3) produce transgenic mice expressing the normal or aberrant mu heavy chain and characterize B-cell development in these mice. The transgenic mice will be crossed with mice that are null or transgenic for signal transduction proteins or molecules involved in apoptosis. A better understanding of the biochemical nature of these unusual mu heavy chains and the mechanisms by which they are removed should elucidate requirements for the normal pro-B-cell to pre-B-cell transition. The proposed studies will also provide valuable information about the development of the normal and abnormal antibody repertoire.