DNA SEQUENCES INVOLVED IN THE HEAVY CHAIN SWITCH

在免疫反应的早期，主要的免疫球蛋白重链 产生的是Mu，后来在免疫反应中占主导地位的免疫球蛋白 重链是伽马、阿尔法或埃西隆。这种重链使用的变化 是通过DNA重排移动重链变量来实现的 从Mu恒定区到α恒定区的区域基因， Epsilon恒定区，或四个伽马1，伽马2b， Gamma2a，或小鼠中的Gamma3)恒定区。这种DNA重排 涉及每个常量上游的DNA片段(称为开关片段) 区域基因。免疫球蛋白产生细胞有六种不同的选择 换位重排。这个选择是一个重要的选择；七个 不同的重链恒定区对不同的效应器起中介作用 功能，并因此处理传染性有机体或其有毒产品 完全不同的方式。此前的结果表明，这一选择 在免疫球蛋白产生细胞中不是随机的，而是 受监管的。此外，监管似乎很可能是由 特定开关区域染色质局部结构的变化。 拟议中的实验的总体目标是了解分子 基因特异性调控开关重组的机制 道路。我们计划鉴定可能控制重链的核蛋白 基因特异性重排，并鉴定它们所针对的DNA序列 捆绑。候选的调控DNA序列将被测试其 通过测量核染色质/DNA结构的变化来改变其结构的能力 由DNA序列介导的转录。DNA序列将会是 通过瞬时转染产生和不产生的细胞进行测试 免疫球蛋白，并通过瞬时转染将被治疗的细胞 以各种方式诱导基因特异性重组。我们将测试 相同序列调控DNA分子转录的能力 植入转基因小鼠体内。最后，我们将尝试测试 序列调控DNA开关重组的能力 被引入转基因小鼠的分子。我们将研究两只重的小鼠 链基因，Gamma1和Gamma2a，以及开关重组的两个诱导子， 干扰素-伽玛1和干扰素-伽马2a。

Early in an immune response, the predominant immunoglobulin heavy chain produced is mu, later in the immune response the predominant immunoglobulin heavy chain is gamma, alpha, or epsilon. This change in heavy chain usage is accomplished by a DNA rearrangement that moves the heavy chain variable region gene from the mu constant region to the alpha constant region, epsilon constant region, or to one of the four gamma gamma1, gamma2b, gamma2a, or gamma3 in mouse) constant regions. This DNA rearrangement involves segments of DNA (called switch segments) upstream of each constant region gene. Immunoglobulin producing cells have a choice of six different switch rearrangements. This choice is an important one; the seven different heavy chain constant regions mediate very different effector functions and so handle an infectious organism or its toxic products in very different ways. Previous results have demonstrated that this choice is not random in the immunoglobulin producing cell, but rather is regulated. Furthermore, it seems likely that the regulation is mediated by changes in the local structure of chromatin of specific switch regions. The overall goal of the proposed experiments is to understand the molecular mechanisms by which switch recombination is regulated in a gene specific way. We plan to identify nuclear proteins that might control heavy chain gene-specific rearrangement and to identify the DNA sequences to which they bind. The candidate regulatory DNA sequences will be tested for their ability to alter chromatin/DNA structure by measuring changes in transcription mediated by the DNA sequences. The DNA sequences will be tested by transient transfection of cells that produce and do not produce immunoglobulin, and by transient transfection of cells that will be treated in various ways to induce gene-specific recombination. We will test the ability of the same sequences to regulate transcription from DNA molecules inserted into transgenic mice. Finally, we will attempt to test the ability of the sequences to regulate switch recombination from DNA molecules introduced into transgenic mice. We will study two murine heavy chain genes, gamma1 and gamma2a, and two inducers of switch recombination, IL4 for gamma1 and interferon-gamma for gamma2a.