Duck Antibody Genes: Unusual Organization and Expression

超出提供的空间。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的，这对人类健康具有重要意义，因为鸭子(和其他水禽)是人类传染病的携带者(或蓄水池)。例如，鸭子是A型禽流感病毒全年唯一的宿主，这种病毒是导致新的对人类致病的流感病毒株出现的基因变异的来源。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调控的进化，这是比较基因组学的一个重要方面。这只鸭有一个不同寻常的免疫球蛋白重链(IGH)基因座，它的生殖系组织发生了显著的重排。3个恒定(C)区基因排列为Mu-Alpha-upsilon，α基因为反向转录方向。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。这些都是在这个提案的特定目标中解决的，需要检验的假设可以表述为三个问题：1)α基因是否有自己的I-外显子，或者它是否依赖双向I-外显子启动子来启动类别切换到IgA？2)通过从I-外显子启动子转录控制Ig类切换的信号转导通路在脊椎动物进化中是保守的吗？3)哺乳动物的IGH基因座在C-基因簇的Jh到Mu内含子和3‘端都含有转录增强子。在鸭免疫球蛋白基因座中，增强子在哪里，它们包含哪些转录因子结合基序，它们是如何发挥作用的？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转换信号的响应。所涉及的特定基序将通过定点突变来鉴定，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加深我们对鸭子异常抗体反应的分子基础的理解，并为脊椎动物免疫系统中转录调控的进化提供洞察力。表演网站========================================Section End===========================================

EXCEED THE SPACE PROVIDED. 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. PERFORMANCE SITE ========================================Section End===========================================