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

San Jose State University Foundation, San Jose CA

Investigators

Abstract

SGER: Isolation of Allorecognition Genes from a Cnidarian (Anthopleura elegantissima) using AmpTrap Technology Jonathan B. Geller Moss Landing Marine Laboratories San Jose State University The 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.