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。鸭的免疫应答可以被表征为无效的，鉴于鸭（和其他水禽）作为人类传染性疾病的携带者（或贮存者）的作用，这对人类健康具有重要意义。鸭是唯一一个全年携带A型禽流感病毒的宿主，而A型禽流感病毒是导致人类致病的新型流感病毒株出现的遗传变异的来源。了解鸭抗体表达的分子基础将有助于了解其无效的抗体应答，也有助于我们了解转录控制的进化，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）基因座，它经历了显着的重排，在其种系组织。3个恒定（C）区基因排列为μ-α-上调，α基因处于逆转录方向。这导致了转录控制的独特问题，特别是与类别转换重组的控制相关的问题。这些在本提案的具体目的中得到了解决，并且待检验的假设可以表述为3个问题：1）α基因是否具有其自身的I-外显子或其是否依赖于双向I-上调启动子来启动向伊加的类别转换？2)控制IG类转换的信号转导途径，通过I-外显子启动子的转录，在脊椎动物进化中是保守的吗？3)哺乳动物IgH基因座在JH至μ内含子和C-基因簇的3'中都含有转录增强子。在鸭IgH基因座中，增强子在哪里，它们包含什么样的转录因子结合基序，它们是如何起作用的？该方法包括通过功能分析（即它们驱动报告基因转录的能力）和I-外显子启动子对甘氨酸诱导的类别转换信号的响应性来鉴定转录控制元件。所涉及的特定基序将通过定点诱变来鉴定，并且结合这些位点的转录因子将通过EMSA/抗体超移位方法来鉴定。这些拟议的研究结果将提高我们对鸭的不寻常的抗体反应的分子基础的理解，并提供深入了解脊椎动物免疫系统中的转录控制的演变。

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.