DNA sequences involved in the heavy chain switch

描述(申请人提供)：抗体生产中的重链开关对于有效预防病原体至关重要。重链开关是由开关区之间的DNA重组事件介导的。开关区是位于每个重链基因上游的2-8kb长的串联重复的简单序列[Delta除外]。两个开关区域之间的重组必须是有效的，这样才能产生大量的特定类型的抗体。开关重组的效率被认为是由位于重链恒定区3‘端的四个增强子元件调节的。在目标1和目标2中，我们将测试四个增强子元件的各种组合的缺失如何改变开关重组的效率。这七种分泌型抗体中的每一种都是为中和、清除或碾磨病原体的子集而设计的。因此，必须以重链特异的方式调节重链开关，以便产生重链，从而对给定的病原体产生最佳保护。在重链开关之前，特定重链基因的染色质打开，这是在开关重组事件之前通过生殖系转录物的表达来衡量的最佳方法。生殖系转录和开关区之间的DNA重组受细胞因子的调控。在目标3中，我们将确定细胞因子对生殖系转录和切换重组到特定基因的调控是由生殖系转录物的启动子区域的DNA序列还是由切换区序列来调节的。所有这三个目标的实验都将通过测试整个重链恒定区基因的各种突变形式来进行。在大肠杆菌中突变这种重组DNA分子，然后测试其介导生殖系转录的能力，并在正常B淋巴细胞中切换所有七个转基因重链基因的重组，与其他方法相比具有几个优点。调控的重链转换是几个实际免疫学问题的中心。疫苗的设计应该激发最大数量的最有利类型的抗体。几乎所有的过敏症都是由一种抗体的表达引起的。从拟议的实验中获得的理解可能会改进疫苗开发和过敏治疗的方法。

DESCRIPTION (provided by applicant): The heavy chain switch in antibody production is critical to effective protection against pathogens. The heavy chain switch is mediated by a DNA recombination event between switch regions. Switch regions are 2-8 kb long stretches of simple sequences repeated in tandem that lie upstream of each heavy chain gene [except delta). Recombination between two switch regions must be efficient so that large quantities of a given type of antibody can be produced. The efficiency of switch recombination is thought to be regulated by four enhancer elements that lie 3' of the heavy chain constant region locus. In Aims 1 and 2, we will test how deletion of various combinations of the four enhancer elements alter the efficiency of switch recombination. Each of the seven types of secreted antibodies are designed for the optimal neutralizing, clearing, or milling of a subset of pathogens. Hence, the heavy chain switch must be regulated in a heavy chain-specific manner in order to produce a heavy chain for the best protection against a given pathogen. The heavy chain switch is preceded by opening of the chromatin for a specific heavy chain gene, which is best measured by the expression of germline transcripts before the switch recombination event. Germline transcription and DNA recombination between switch regions are regulated by cytokines. In Aim 3, we will determine if cytokine regulation of germline transcription and switch recombination to a specific gene is regulated by DNA sequences in the promoter region for germline transcripts or by the switch region sequences. The experiments in all three Aims will be carried out by testing various mutant forms of a transgene of the entire heavy chain constant region locus. The ability to mutate this recombinant DNA molecule in E. coli, and then test its ability to mediate germline transcription and switch recombination of all seven transgenic heavy chain genes in normal B lymphocytes, offers several advantages compared to other approaches. Regulated heavy chain switching is at the center of several practical immunological problems. Vaccines should be designed to elicit the largest amounts of the most favorable types of antibodies. Almost alll allergies are caused by the expression of one type of antibody. The understanding to be gained from the proposed experiments may improve approaches toward vaccine development and toward treatment of allergy.