DNA Sequences Involved in the Heavy Chain Switch

描述（由申请人提供）：重链开关是由与供体（通常是mu）和受体(gamma (g), epsilon （e)或alpha (a)）重链基因相关的开关(S)区域之间的重组事件介导的。在伯基特淋巴瘤、艾滋病相关淋巴瘤和其他淋巴样肿瘤中，异常的开关重组事件可导致染色体易位。因此，了解重链开关对于提高对病原体的免疫反应和我们对淋巴样肿瘤发生的理解都是重要的。开关重组之前通过S区和常数区进行种系转录。虽然开关重组被认为是一种开始和结束于S区的DNA缺失，但对其机制几乎一无所知。开关重组也受到调节，因为它是由细胞外信号引导到一个或有时两个重链基因。重链位点上的顺式作用元件必须在基因特异性开关重组中发挥作用。为了了解开关重组的机制，以及了解对调控和重组事件本身都很重要的顺式元件，我们应用新技术对小鼠重链位点的克隆版本进行了基因修饰。在本实验中，我们将在具有组装可变区和整个重链恒定区位点的细菌人工染色体上修饰一个特定的重链基因，并测试各种重链基因在转基因小鼠中的表达。我们将测试一个特定的蛋白质结合位点或启动子区域的作用，一般来说，小鼠γ重链基因在种系转录和开关重组中的作用（目的1和2）。我们将测试开关区域的删除或修改如何改变开关重组（目标3）。在Aim 4中，我们将研究开关序列取向的作用，以及种系转录本和开关区DNA之间不寻常复合物的作用。

DESCRIPTION (provided by applicant): The heavy chain switch is mediated by recombination event between switch (S) regions associated with the donor (usually mu) and recipient (gamma (g), epsilon (e), or alpha (a)) heavy chain genes. Aberrant switch recombination events lead to the chromosomal translocations found in Burkitt's lymphoma, AIDS-associated lymphoma, and perhaps other lymphoid tumors. Hence, understanding the heavy chain switch is important for both improving immune responses to pathogens and to our understanding of the genesis of lymphoid tumors. Switch recombination is preceded by germline transcription through the S region and the constant region. Although switch recombination is understood to be a DNA deletion beginning and ending in S regions, almost nothing else is known about its mechanism. Switch recombination is also regulated in that it is directed by extracellular signals to one, or sometimes, two heavy chain genes. Cis-acting elements in the heavy chain locus must play a role in gene-specific switch recombination. To understand the mechanism of switch recombination, and to understand the cis elements that are important to both regulation and the recombination event itself, we have applied new technology to modify genes in a cloned version of the murine heavy chain locus. In the proposed experiments we will modify a specific heavy chain gene on a bacterial artificial chromosome with an assembled variable region and the entire heavy chain constant region locus, and test expression of various heavy chain genes in transgenic mice. We will test the role of a specific protein binding site or promoter regions, in general, for murine gamma heavy chain genes in both germline transcription and switch recombination (Aims 1 and 2). We will test how deletion or modification of the switch region changes switch recombination (Aim 3). We will examine the role of the orientation of switch sequences, and hence the role of the unusual complexes between germline transcripts and switch region DNA, in Aim 4.