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

圣何塞州立大学莫斯登陆海洋实验室：利用AmpTrap技术分离线虫(Anthopleura Elegantissima)的同种识别基因乔纳森·B·盖勒登陆海洋实验室在许多多细胞生物中发现了区分自我和非我的能力，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的后生动物中都可以看到某种形式的自我/非我识别系统，但分子水平上的详细知识只存在于脊椎动物免疫系统和一些配子识别系统。盖勒将应用分子技术的最新发展来分离一种具有良好特征的海葵的同种识别基因，这是一种克隆的海葵。先前观察到的个体对非克隆体的攻击性反应表明，同种识别受体位于触须和特殊的攻击触须上，并与膜结合。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acrohagi特异性基因的cDNA库。由于acrohagi特异的cDNA库仍将是复杂的，Geller将使用新的克隆载体pAmpTrap来进一步丰富编码膜结合蛋白的基因的cDNA库。这些cDNA将通过生物信息学进行测序和评估，以消除明显无关的基因。然后，将通过Southern印迹分析筛选候选基因在海葵种群中的多态性(预计在同种识别基因中)，并筛选预测的结构特征。除了拟议方法的技术复杂性外，这种克隆战略还带来了巨大的风险。首先，该项目基于对花盘中同种异体识别受体的推断：它们是细胞表面蛋白；它们的表达在很大程度上限于触须和触角噬菌体；它们可以根据多态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有毒性，克隆就会失败。尽管存在固有的风险，但成功将在几个方向上催化新的研究：对花冠花的同种识别基因的了解将有助于理解海葵无性系之间优势关系的复杂性。花伞属和其他蛇形目动物攻击性的变异为研究攻击性的进化生态学提供了基因组和系统发育相结合的方法。此外，分离花瓣中的同种识别受体将进一步发现其他具有重要生态意义的爬行动物的同种识别基因，如珊瑚。最后，花胸的异种识别系统的特征类似于哺乳动物自然杀伤细胞上的受体系统。这可能是较年轻的哺乳动物系统向更古老的哺乳动物系统趋同的一个例子。在这种情况下，对独立衍生的哺乳动物和哺乳动物的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。或者，NK细胞受体可能确实是古老的，在基础后生动物中有祖先。这一发现将对免疫系统的古老产生深远的影响。

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.