Immunity in Transgenic Mice

描述（由申请人提供）：类别转换DNA重组对于B细胞中的同种型转换很重要，并且似乎也参与某些癌基因的染色体易位。然而，转换的机制和将这些机制靶向某些DNA区域的过程仍然未知。含有串联重复序列的开关（S）区位于所有H链抗体恒定区基因的上游，并且是开关重组机制的靶标。然而，S区在靶向中的作用尚不清楚。我们已经发现，S？串联重复区不是转换所必需的，但在提供高效转换中起作用。在缺乏DNA错配修复蛋白Msh 2的小鼠中，我们还发现S？串联重复序列对于转换是至关重要的，这表明不同的DNA区域需要不同的蛋白质来完成转换重组。最后，测量开关网站分布在小鼠缺乏S？串联重复序列或Msh 2蛋白显示开关靶向的变化。这些位移表明，4-5 kb的结构域下游的l？启动子是可访问的开关，即使S？串联重复序列不在该结构域内。此外，在不存在Msh 2的情况下，转换集中于结构域内的串联重复区域。目前的建议旨在进一步分析通过S区域的转换的靶向，使用影响同种型转换过程的各种突变小鼠品系。第一，S的作用。将在野生型和突变小鼠中分析通过可能形成R环结构或通过促进特定染色质结构的靶向开关重组中的区域序列。其次，将Mlh 1和Exo 1错配修复蛋白在开关中的作用与Msh 2的作用进行比较，以确定这些蛋白质在开关机制中影响相同还是不同的通路。第三，S的能力？我呢？通过重新定位这些序列并评估开关靶向是否也被重新定位来分析调节开关靶向的区域。最后，我们将评估AID蛋白的重要性，这是在启动开关重组，在我们的实验室中发现的u转基因染色体易位的关键。虽然开关机制对癌基因的异常靶向被认为与某些癌基因：lgH易位有关，但另一个实验室最近的研究表明这些易位不涉及AID。如果呢？转基因易位也不涉及AID，那么这将为IgH易位过程重要的序列和蛋白质的遗传分析提供方便的模型系统。

DESCRIPTION (provided by applicant): Class switch DNA recombinations are important for isotype switching in B cells and also appear to be involved in chromosomal translocations of some oncogenes. However, the mechanisms of switching and the processes that target these mechanisms to certain DNA regions are still not known. Switch (S) regions containing tandemly repeated sequences are located upstream of all H-chain antibody constant region genes and are the target of the switch recombinational machinery. However, the roles of S regions in targeting are unclear. We have found that the S? tandem repeat region is not required for switching but does play a role in providing highly efficient switching. In mice lacking the DNA mismatch repair protein, Msh2, we have also found that the S? tandem repeats are critical for switching, indicating that different DNA regions need different proteins to complete switch recombination. Finally, measurements of switch site distributions in mice that lack either the S? tandem repeats or the Msh2 protein show shifts in switch targeting. These shifts indicate that a 4-5 kb domain downstream of the l? promoter is accessible for switching even if the S? tandem repeats are not within this domain. In addition, in the absence of Msh2, switching is focused to the tandem repeat region within the domain. The current proposal seeks to further analyze the targeting of switching by S regions using a variety of mutant mouse strains that affect the isotype switching process. First, the roles of S? region sequences in targeting switch recombination through possible formation of R-loop structures, or by promoting specific chromatin structures will be analyzed in wild-type and mutant mice. Second, the roles of the Mlh1 and Exo1 mismatch repair proteins in switching will be compared to the role of Msh2 to determine whether these proteins affect the same or different pathways in the switching mechanism. Third, the abilities of S? and l? regions in regulating switch targeting will be analyzed by relocating these sequences and assessing whether switch targeting is also relocated. Finally, we will assess the importance of the AID protein, which is critical in initiating switch recombination, for the u transgene chromosomal translocations that were discovered in our laboratory. Although aberrant targeting of oncogenes by the switch mechanism has been suggested to be involved in some oncogene:lgH translocations, recent studies by another laboratory have indicated that these translocations do not involve AID. If ? transgene translocations also do not involve AID then this would provide a convenient model system for genetic analyses of the sequences and proteins important for the IgH translocation process.