Duck Antibody Genes: Unusual Organization and Expression

描述（由申请人提供）：拟议研究的长期目标是了解控制鸭免疫球蛋白基因表达的遗传机制。鸭子的免疫反应可以被描述为无效，鉴于鸭子（和其他水禽）作为人类传染性疾病的携带者（或宿主）的作用，这对人类健康具有重要意义。例如，鸭子是甲型禽流感病毒唯一的全年储存库，甲型禽流感病毒是基因变异的一个来源，与出现对人类致病的新型流感毒株有关。了解鸭抗体表达的分子基础将有助于理解它们的无效抗体反应，也有助于我们理解转录控制的进化，这是比较基因组学的一个重要方面。鸭有一个不寻常的Ig重链（IgH）位点，在其种系组织中发生了显著的重排。恒定(C)区的3个基因排列为mu- α -upsilon， α基因呈逆转录取向。这导致了转录控制的独特问题，特别是与类开关重组的控制有关。这些都在本提案的具体目标中得到了解决，需要测试的假设可以用3个问题来表述：1)α基因是否有自己的i-外显子，或者它是否依赖于双向I-upsilon启动子来启动类转换到IgA？2)通过i -外显子启动子转录控制Ig类转换的信号转导通路在脊椎动物进化中是否保守？3)哺乳动物的IgH位点在JH - to - mu内含子和c基因簇的3′上都含有转录增强子。在鸭的IgH基因座中，增强子在哪里？它们包含哪些转录因子结合基序？它们是如何起作用的？该方法包括通过功能分析识别转录控制元件（即它们驱动报告基因转录的能力），以及i -外显子启动子对细胞因子诱导的类别转换信号的响应性。所涉及的特定基序将通过位点定向诱变来识别，结合这些位点的转录因子将通过EMSA/抗体超移方法来识别。这些研究结果将增强我们对鸭异常抗体反应的分子基础的理解，并为脊椎动物免疫系统转录控制的进化提供见解。

DESCRIPTION (provided by applicant): The long-term goal of the proposed studies is to understand the genetic mechanisms controlling the expression of duck immunoglobulin genes. The duck has an immune response that can be characterized as ineffectual, which has significance for human health given the role of ducks (and other waterfowl) as carriers (or reservoirs) of diseases infectious for man. E.g., ducks constitute the only year-round reservoir of avian influenza A viruses, which are a source of genetic variation implicated in the emergence of novel influenza strains pathogenic to man. Understanding the molecular basis of the expression of duck antibodies will permit an understanding of their ineffectual antibody response and also contribute significantly to our understanding of the evolution of transcriptional control, an important aspect of comparative genomics. The duck has an unusual Ig heavy chain (IgH) locus, which has undergone significant rearrangement in its germline organization. The 3 constant (C) region genes are arranged mu-alpha-upsilon, with the alpha gene in reverse transcriptional orientation. This leads to unique problems of transcriptional control associated particularly with the control of class switch recombination. These are addressed in the Specific Aims of this proposal, and the hypotheses to be tested can be phrased as 3 questions: 1) Does the alpha gene have its own I-exon or does it rely on the bi-directional I-upsilon promoter to initiate class switching to IgA? 2) Have the signal transduction pathways that control Ig class-switching, by transcription from I-exon promoters, been conserved in vertebrate evolution? 3) The mammalian IgH locus contains transcriptional enhancers in both the JH to mu intron and 3' of the C-gene cluster. Where, in the duck IgH locus are the enhancers, what transcription factor-binding motifs do they contain, and how do they function? The approach involves identifying transcriptional control elements through functional analysis (i.e. their ability to drive transcription of a reporter gene), and the responsiveness of I-exon promoters to cytokine-induced class-switch signals. The specific motifs involved will be identified by site-directed mutagenesis, and the transcription factors binding these sites will be identified through EMSA/antibody supershift approaches. The results of these proposed studies will enhance our understanding of the molecular basis of the unusual antibody response of the duck, and provide insight into the evolution of transcriptional control in the vertebrate immune system.