DNA sequences involved in the heavy chain switch

描述（由申请方提供）：抗体生产中的重链转换对于有效抵御病原体至关重要。重链开关由开关区域之间的DNA重组事件介导。转换区是位于每个重链基因上游的串联重复的2-8 kb长的简单序列段（除delta外）。两个开关区域之间的双链化必须是有效的，以便可以产生大量的给定类型的抗体。开关重组的效率被认为是由位于重链恒定区基因座3'的四个增强子元件调节的。在目的1和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。七种分泌型抗体中的每一种都被设计用于病原体子集的最佳中和、清除或研磨。因此，必须以重链特异性方式调节重链开关，以产生针对给定病原体的最佳保护的重链。重链转换之前是特定重链基因的染色质打开，这最好通过转换重组事件之前生殖系转录物的表达来测量。细胞因子调节生殖细胞转录和开关区之间的DNA重组。在目标3中，我们将确定细胞因子对生殖系转录的调控和对特定基因的开关重组是否受生殖系转录物启动子区中的DNA序列或开关区序列的调控。通过检测整个重链恒定区基因座转基因的各种突变形式，进行所有三个目的中的实验。在E.大肠杆菌，然后测试其介导生殖系转录和正常B淋巴细胞中所有七个转基因重链基因的开关重组的能力，与其他方法相比提供了几个优点。调节重链转换是几个实际免疫学问题的中心。疫苗的设计应能激发最大数量的最有利的抗体类型。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善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.