SGER: Isolation of Allorecognition Genes from a Cnidarian (Anthopleura Elegantissima) Using AmpTrap Technology

SGER：利用AmpTrap技术从刺胞动物（Anthopleura elegantissima）中分离异源识别基因Jonathan B. GellerMoss Landing海洋生物圣何塞州立大学区分自我与非自我的能力在许多多细胞生物中可见，并且对于个体之间种内和种间接触的反应至关重要。这些反应的例子包括对病原体的免疫反应、攻击行为或形态的诱导、交配行为和配子融合。在从多孔动物到脊索动物的后生动物中可以看到某种形式的自我/非自我识别系统，但分子水平上的详细知识只存在于脊椎动物的免疫系统和一些配子识别系统中。 盖勒将应用分子技术的最新发展，以分离异源识别基因在一个良好的特点刺胞动物，克隆海葵花侧elegantissima。以前的观察对非克隆个体的侵略性反应表明，allorecognition受体位于触角和acrorhagi（专门的触角侵略）和膜结合。这表明了一种分子克隆策略：消减方法可以产生富含同种识别基因的acrorhagi特异性基因的cDNA库。因为一个acrorhagi特异性的cDNA库仍然很复杂，Geller将使用新的克隆载体pAmpTrap来进一步丰富cDNA文库中编码膜结合蛋白的基因。将对这些cDNA进行测序并通过生物信息学进行评估，以消除明显不相关的基因。候选基因，然后将被筛选的多态性在海葵人口（预期在异源识别基因）通过Southern印迹分析，并筛选预测的结构特征。除了所提出的方法的技术复杂性之外，这种克隆策略还带来了巨大的风险。首先，该项目是基于对花侧耳属中的同种识别受体的推断：它们是细胞表面蛋白;它们在很大程度上局限于触角和acrorhagi的表达;它们可以根据多态性和预测的蛋白质结构进行识别。其次，pAmpTrap之前的测试有限。例如，如果表达的同种识别基因对细菌宿主具有毒性，则克隆将失败。尽管存在内在风险，但成功将在几个方向上催化新的研究：对优雅花侧耳中的同种识别基因的了解将有助于理解海葵克隆之间优势关系的复杂性。花侧刺胞动物和其他刺胞动物在攻击性上的差异为研究攻击性的进化生态学提供了基因组学和系统发育学相结合的方法。同样，在花侧刺胞动物中分离同种识别受体将进一步发现其他生态重要的刺胞动物（如珊瑚）中的同种识别基因。最后，花侧耳属同种异体识别系统的特征类似于在哺乳动物自然杀伤细胞上发现的受体系统。这可能是年轻哺乳动物系统在更古老的刺胞动物系统上趋同的一个例子。在这种情况下，独立衍生的刺胞动物和哺乳动物NK样受体系统的比较提供了一个平台，研究免疫相关分子的结构和功能的限制。或者，NK细胞受体可能确实是古老的，并且在基底后生动物中具有祖先。这一发现将对免疫系统的古老性产生深远的影响。

SGER: Isolation of Allorecognition Genes from a Cnidarian (Anthopleura elegantissima) using AmpTrap TechnologyJonathan B. GellerMoss Landing Marine LaboratoriesSan Jose State UniversityThe ability to distinguish self from non-self is seen in many multicellular organisms, and is critical for dictating responses to intraspecific and interspecific contact between individuals. Examples of these responses include immune reactions to pathogens, induction of aggressive behaviors or morphologies, mating behaviors and gamete fusion. Some form of a self/nonself recognition system is seen in metazoans from Porifera to Chordata, yet detailed knowledge at the molecular level exists only for vertebrate immune systems and some gamete recognition systems. Geller will apply recent developments in molecular technology to isolate allorecognition genes in one well characterized cnidarian, the clonal sea anemone Anthopleura elegantissima. Previous observations of aggressive responses toward non-clonemates by individual Anthopleura indicate that allorecognition receptors are located on tentacles and acrorhagi (specialized tentacles for aggression) and are membrane-bound. This suggests a molecular cloning strategy: subtractive methods can produce cDNA pools of acrorhagi-specific genes that are enriched for allorecognition genes. Because an acrorhagi-specific cDNA pool will still be complex, Geller will use the new cloning vector pAmpTrap to further enrich the cDNA library for genes encoding membrane-bound proteins. These cDNAs will be sequenced and evaluated by bioinformatics to eliminate clearly irrelevant genes. Candidate genes will then be screened for polymorphism in an anemone population (expected in allorecognition genes) by Southern blot analysis, and screened for predicted structural features. This cloning strategy entails significant risk, aside from the technical complexities of the proposed methods. First, the project is based on inferences about allorecognition receptors in Anthopleura: they are cell surface proteins; they are largely confined in their expression to tentacles and acrorhagi; and they can be recognized on the basis of polymorphism and predicted protein structures. Second, pAmpTrap has had limited prior testing. For example, cloning will fail if the expressed allorecognition genes have toxicity to the bacterial host. Despite intrinsic risks, success would catalyze new research in several directions: Knowledge of allorecognition genes in Anthopleura elegantissima will help make comprehensible the complexity of dominance relationships among anemone clones. Variation in aggressiveness in the genus Anthopleura and other cnidarians provide for combined genomic and phylogenetic approaches to study the evolutionary ecology of aggression. Too, isolation of allorecognition receptors in Anthopleura will further discovery of allorecognition genes in other ecologically important cnidarians, such as corals. Finally, features of the Anthopleura allorecognition system resemble the receptor system found on mammalian natural killer cells. This could be an example of convergence of the younger mammalian system upon the more ancient cnidarian system. In that case, comparisons of independently derived cnidarian and mammalian NK-like receptor systems provide a platform to study structural and functional constraints on immunity-related molecules. Alternatively, the NK cell receptors may indeed be ancient and have ancestry in basal metazoans. This discovery would have profound implications for the antiquity of immune systems.