DNA Sequences Involved in the Heavy Chain Switch

描述（由申请人提供）：重链转换由与供体（通常为mu）和受体（γ（g）、γ（e）或α（a））重链基因相关的转换（S）区域之间的重组事件介导。异常的开关重组事件导致伯基特淋巴瘤、艾滋病相关淋巴瘤和可能其他淋巴肿瘤中发现的染色体易位。因此，了解重链开关对于提高对病原体的免疫应答和我们理解淋巴肿瘤的发生都很重要。开关重组之前是通过S区和恒定区的种系转录。虽然开关重组被理解为在S区开始和结束的DNA缺失，但对其机制几乎一无所知。开关重组也受到调节，因为它由细胞外信号引导至一个或有时两个重链基因。重链基因座中的顺式作用元件必须在基因特异性开关重组中发挥作用。为了理解开关重组的机制，并理解对调控和重组事件本身都很重要的顺式元件，我们应用了新技术来修饰小鼠重链基因座的克隆版本中的基因。在拟定的实验中，我们将用组装的可变区和整个重链恒定区基因座修饰细菌人工染色体上的特定重链基因，并测试转基因小鼠中各种重链基因的表达。我们将测试特定蛋白质结合位点或启动子区的作用，一般而言，对于鼠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缺失或修饰如何改变开关重组（目的3）。我们将在目标4中研究开关序列方向的作用，从而研究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.