Factors and DNA Motifs Involved in Ig Class Switch

参与 Ig 类别转换的因素和 DNA 基序

• 批准号: 6727685
• 负责人: Amy L Kenter
• 金额: $ 38.41万
• 依托单位: UNIVERSITY OF ILLINOIS AT CHICAGO
• 依托单位国家: 美国
• 财政年份: 2003
• 资助国家: 美国
• 起止时间: 2003-04-01 至 2007-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6727685
• 关键词: B lymphocyte; DNA binding protein; antibody formation; gel mobility shift assay; gene mutation; gene rearrangement; genetic mapping; immunoglobulin genes; laboratory mouse; plasmids

DESCRIPTION (provided by applicant): Immunoglobulin (Ig) isotype switching occurs by a looping-out and deletion process which is focused on switch regions located upstream of the constant regions, with the exception of Cdelta. Little is known regarding the mechanism of switch recombination (SR). We recently devised a plasmid-based transient transfection assay for SR. Using this assay we showed that there are distinct switching activities which mediate mu->gamma3, mu->alpha mu->epsilon and mu->gamma1 switching. In preliminary results reported here we show that the switch plasmid assay is AID dependent. This observation strengthens the conclusion that the switch plasmid assay faithfully recapitulates physiological SR. These studies suggest that there is isotype specific recognition of S regions by switching activities. However, essentially nothing is known regarding how switching activities identify S regions. Aim I is designed to test whether the results obtained with the switch substrates can be confirmed in vivo. We will exchange the endogenous Sgamma3 region with Sgamma1 sequence using targeted homologous recombination. This experiment will allow us to determine whether the absence of the Sgamma1 specific switching factor will lead to an inability of the Sgamma3 endogenous locus to undergo SR when S?1 sequence is present in the S region. Such an outcome would confirm the existence of isotype specific switching factors in a physiological setting. Aim II in this application is focused on defining the nature of molecular recognition of S regions. Using the plasmid assay we plan to manipulate the size and sequence of the S regions to delineate a minimal target for SR. We will also create synthetic switch regions which are mutated at specific residues and we will locate the position of double strand breaks in the S DNA. Taken together, these studies will allow us to map functional recombination motifs (FRMs) which are located in the tandem repeats. In the third Aim we will use the FRMs defined in Aim II and ask whether DNA binding proteins interact with these motifs and mediate SR using gel shift and competition binding analyses. If the FRMs overlap with previously described SNIP and SNAP recognition motifs then we will ask whether SNIP and/or SNAP are functionally involved in SR. We will also explore the involvement of the FRM binding proteins by modulating their expression levels and determining whether recombination is affected as a consequence.

说明书(由申请人提供)：免疫球蛋白(Ig)同型转换通过环出和缺失过程发生，该过程集中在位于恒定区上游的开关区，但CDelta除外。关于开关重组(SR)的机制，人们知之甚少。我们最近设计了一种基于质粒的SR瞬时转染实验。利用这一方法，我们发现存在着明显的转换活性，它们分别介导Mu-Gt；Gamma3、Mu-&gt；α-Mu-&gt；epsilon和Mu-&gt；Gamma1的转换。在这里报告的初步结果中，我们表明开关质粒试验是依赖于AID的。这一观察结果强化了开关质粒法准确概括生理性SR的结论。这些研究表明，S区域存在通过转换活动进行的同型特异性识别。然而，关于转换活动如何识别S区域，基本上一无所知。目的测试开关底物的实验结果是否能在体内得到证实。我们将利用靶向同源重组将内源性SGamma3区与SGamma1序列互换。这项实验将使我们能够确定当S区域存在S？1序列时，SGamma1特异性开关因子的缺失是否会导致SGamma3内源基因座不能进行SR。这样的结果将证实在生理环境中存在同种类型特定的开关因子。目的II在本申请中，重点是定义S区域的分子识别的性质。利用质粒法，我们计划操纵S区域的大小和序列，以描绘SR的最小靶点。我们还将创建在特定残基处突变的人工开关区域，并定位S DNA中双链断裂的位置。综上所述，这些研究将使我们能够定位位于串联重复序列中的功能重组基序(FRM)。在第三个目的中，我们将使用AIM II中定义的FRMS，并通过凝胶位移和竞争结合分析来询问DNA结合蛋白是否与这些基序相互作用并介导SR。如果FRM与先前描述的SNIP和SNAP识别基序重叠，那么我们将询问SNIP和/或SNAP是否在功能上参与SR。我们还将通过调节FRM结合蛋白的表达水平，并确定重组是否因此受到影响，来探索FRM结合蛋白的参与。