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）中分离异体识别基因jonathan B. GellerMoss Landing Marine laboratory, san Jose State university)在许多多细胞生物中都可以看到区分自我和非自我的能力，这对于决定个体对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。从Porifera到Chordata，在后生动物中可以看到某种形式的自我/非自我识别系统，但在分子水平上的详细知识仅存在于脊椎动物免疫系统和一些配子识别系统中。Geller将应用分子技术的最新进展，从一种已被充分表征的刺胞动物——克隆海葵Anthopleura elegantissima中分离出同种异体识别基因。先前对单个胸膜虫对非克隆生物的攻击反应的观察表明，同种异体识别受体位于触须和触角（用于攻击的特殊触须）上，并且是膜结合的。这提示了一种分子克隆策略：减法方法可以产生富含同种异体识别基因的自噬特异性基因的cDNA库。由于核糖核酸自噬特异性cDNA库仍然很复杂，Geller将使用新的克隆载体pAmpTrap进一步丰富编码膜结合蛋白基因的cDNA文库。这些cdna将被生物信息学测序和评估，以消除明显不相关的基因。然后通过Southern blot分析筛选候选基因在海葵群体中的多态性（预计在同种异体识别基因中），并筛选预测的结构特征。除了所提出的方法的技术复杂性外，这种克隆策略还需要承担重大风险。首先，该项目是基于对胸膜动物同种异体识别受体的推断：它们是细胞表面蛋白；它们在表达上主要局限于触手和食轴；它们可以根据多态性进行识别并预测蛋白质结构。其次，pAmpTrap之前的测试有限。例如，如果表达的同种异体识别基因对细菌宿主有毒性，克隆就会失败。尽管存在内在的风险，但成功将在几个方向上催化新的研究：了解优雅胸膜的异体识别基因将有助于理解海葵克隆间优势关系的复杂性。刺胸属和其他刺胞动物的攻击性变异为研究攻击的进化生态学提供了基因组学和系统发育相结合的方法。此外，在胸膜动物中分离异体识别受体将进一步发现其他生态重要的刺胞动物（如珊瑚）的异体识别基因。最后，胸膜异体识别系统的特征类似于哺乳动物自然杀伤细胞的受体系统。这可能是较年轻的哺乳动物系统向较古老的刺胞动物系统趋同的一个例子。在这种情况下，独立衍生的刺胞动物和哺乳动物nk样受体系统的比较为研究免疫相关分子的结构和功能限制提供了一个平台。另外，NK细胞受体可能确实是古老的，在基础后生动物中有祖先。这一发现将对古老的免疫系统产生深远的影响。

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.