Positional cloning of a cnidarian allorecognition locus

Many sessile, colonial marine invertebrates are capable of allorecognition, the ability to distinguish between their own tissues and those of conspecifics. Yet despite a history of interest from ecologists, evolutionary theorists, and comparative immunologists, the molecular basis of invertebrate allorecognition remains largely unknown. This thesis describes the positional cloning of the first allorecognition locus in a lower metazoan, the cnidarian Hydractinia symbiolongicarpus. Chapter 1 reviews the general significance of allorecognition phenomena before briefly summarizing its occurrence in poriferans, cnidarians, bryozoans, and colonial ascidians, paying particular attention to what is known about the genetics of allorecognition in these groups. This chapter includes a detailed account of the only two model systems for the study of invertebrate allorecognition, the ascidian Botryllus schlosseri and the hydroid Hydractinia symbiolongicarpus. Chapter 2 describes the construction of Bacterial artificial chromosome (BAC) and fosmid Hydractinia genomic libraries and their use in a chromosome walk to isolate and sequence a 350 kb region containing alr2, one of two allorecognition loci in the Hydractinia allorecognition complex. Chapter 3 describes the analysis of this region to identify candidate genes for alr2. This work revealed that the alr2 genomic region encodes a predicted type I transmembrane receptor with 4 extracellular domains that are similar to Ig-like domains, one of which was highly polymorphic between inbred strains. This result made this gene the primary candidate for alr2, prompting further investigation to determine (a) whether the gene displayed a pattern of expression consistent with its putative role in allorecognition phenomena, (b) whether the gene displayed the expected level of polymorphism in field-collected colonies, and (c) whether alleles at this locus could predict allorecognition responses in field-collected colonies. These experiments are described in Chapter 4, and the results provide strong circumstantial evidence that this gene is alr2.