CHIMERIC IG MRNAS AND HUMAN ISOTYPE SWITCHING

描述：(改编自调查人员的摘要)总体 这项提案的目的是阐明 免疫球蛋白(Ig)异型转换： 确定并最终实现了Ig同型开关的特异性。我们 将检验嵌合免疫球蛋白生殖系mRNA转录本作为 决定Ig类开关特异性的具体模板 重组，重点是转向IgE。我们已经证明了这样的 嵌合的mRNA来自于C的种系Ig前-mRNAs的反式剪接 Mu和下游同种异型的胚系前mRNAs(S)。这些嵌合Ig 生殖系mRNAs，通过Hoogsteen碱基配对到其相应的双 链状DNA区域是理想的模板，可以起到桥接作用 这种桥接将把MU Switch和相关的下游 非常接近的同型开关区域，从而直接进行免疫球蛋白开关 重组。为了验证这一假设，我们将在人类初选中确定 B细胞是否转染产生特异性嵌合免疫球蛋白的构建体 反义定位中的生殖系mRNA转录本阻断特异性Ig 转换型重组和同型生产以及是否转染人 正义定位的嵌合Ig生殖系mRNA特异性增强开关 重组和免疫球蛋白生产。我们将定量地确定 嵌合免疫球蛋白种系mRNA转录本的出现动力学及其意义 与Ig同型转换的关系。为了更好地定义嵌合体的作用 在Ig胚系转录本中，我们将确定Ig切换的能力 调节反式剪接和由此产生的产物的已知开关信号 嵌合免疫球蛋白生殖系的mRNAs。作为开关的选择和模式 重组位点(即初级重组与二级重组)具有重要的作用 对免疫球蛋白同型生产和稳定的影响，我们将延长我们的 通过检验嵌合免疫球蛋白种系的特定结构是否为假说 转录本(3‘-5’与5‘-3’嵌合体)具有不同的中介能力 初级或次级重组预测3‘-5’嵌合体(例如I Epsilon-C Mu转录本)将介导初级重组，而5‘-3’ 嵌合体(例如I Mu-C epsilon转录本)将介导第二次 重组。最后，我们将检验重复G基的假设 在Ig开关区发现的RNA(“G4-RNA”)在 促进免疫球蛋白种系前转录产物的反式剪接。用人 一个无细胞的体外剪接系统，我们将分析G4-RNA的作用 Ig反式剪接的形成。此外，我们将确定是否有能力 嵌合免疫球蛋白生殖系mRNAs体外配对到相应区域 通过Hoogsteen碱基配对获得双链DNA。

DESCRIPTION: (Adapted from the investigator's abstract) The overall objective of this proposal is to elucidate a critical process in immunoglobulin (Ig) isotype switching: the mechanism by which the specificity of Ig isotype switch is determined and ultimately achieved. We will test the hypothesis that chimeric Ig germline mRNA transcripts serve as the specific templates that determine the specificity of Ig class switch recombination with emphasis on switching to IgE. We have shown that such chimeric mRNA are derived from trans-splicing of germline Ig pre-mRNAs of C mu and the germline pre-mRNAs of downstream isotype(s). These chimeric Ig germline mRNAs, by Hoogsteen base-pairing to their corresponding double stranded DNA region, are ideal templates to function as "bridging templates". This bridging will bring mu switch and the involved downstream isotype switch regions in close proximity and thereby direct Ig switch recombination. To test this hypothesis, we will determine in human primary B cells whether transfection of constructs producing specific chimeric Ig germline MRNA transcripts in anti-sense orientation blocks specific Ig switching recombination and isotype production and whether transfection of chimeric Ig germline MRNA in sense orientation specifically enhances switch recombination and Ig production. We will quantitatively determine the kinetics of appearance of chimeric Ig germline MRNA transcripts and their relationship to Ig isotype switching. To better define the role of chimeric Ig germline transcripts in Ig switching, we will determine the ability of known switch signals to regulate trans-splicing and the resulting production of chimeric Ig germline MRNAS. As the selection and pattern of switch recombination sites (i.e. primary vs. secondary recombination) has important implications for Ig isotype production and stabilization, we will extend our hypothesis by testing if the specific structure of chimeric Ig germline transcripts (3'-5' vs 5'-3' chimeras) have distinct abilities to mediate primary or secondary recombination predicting that 3'-5' chimeras (e.g. I epsilon-C mu transcripts) will mediate primary recombination whereas 5'-3' chimeras (e.g. I mu-C epsilon transcripts) will mediate secondary recombination. Finally, we will test the hypothesis that repetitive G bases in RNA ("G4-RNA") as found in Ig switch regions plays a key role in promoting the trans-splicing of Ig germline pre-mRNA transcripts. Employing a cell free in vitro splicing system, we will analyze the effects of G4-RNA formation on Ig trans-splicing. Furthermore we will determine the ability of chimeric Ig germline mRNAs to pair in vitro to the corresponding region of double stranded DNA through Hoogsteen base pairing.