Factors and DNA Motifs Involved in Ig Class Switch

描述（由申请人提供）：免疫球蛋白（Ig）同型转换通过环出和删除过程发生，该过程集中在位于恒定区上游的开关区域，Cdelta除外。关于开关复合（SR）的机理，目前所知甚少。我们最近为sr设计了一种基于质粒的瞬时转染实验。使用这种实验，我们发现有不同的开关活动介导mu->gamma3, mu->alpha, mu->epsilon和mu->gamma1开关。在这里报告的初步结果中，我们表明开关质粒测定依赖于AID。这一观察结果加强了开关质粒测定忠实地概括生理sr的结论。这些研究表明，通过开关活动存在对S区域的同型特异性识别。然而，关于开关活动如何识别S区域，基本上一无所知。Aim I旨在测试使用开关底物获得的结果是否可以在体内得到证实。我们将使用靶向同源重组将内源性Sgamma3区域与Sgamma1序列交换。该实验将使我们能够确定Sgamma1特异性开关因子的缺失是否会导致Sgamma3内源性位点在S？1序列存在于S区。这样的结果将证实生理环境中同种型特异性转换因子的存在。本应用程序的目的II集中在定义S区分子识别的性质。利用质粒测定，我们计划操纵S区域的大小和序列，以描绘sr的最小目标。我们还将创建在特定残基上突变的合成开关区域，我们将定位S DNA中双链断裂的位置。综上所述，这些研究将使我们能够绘制位于串联重复序列中的功能重组基序（FRMs）。在第三个目标中，我们将使用目标II中定义的FRMs，并询问DNA结合蛋白是否与这些基序相互作用，并使用凝胶转移和竞争结合分析介导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来探索FRM结合蛋白的参与，并确定重组是否因此受到影响。

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.