Hydractinia symbiolongicarpus displays an unambiguous and well-characterized allorecognition response. It has recently been established that the allorecognition response segregates as a single co-dominant Mendelian trait. Two inbred lines and a line near-isogeneic for fusibility for each inbred line, heretofor unavailable, have been developed in this laboratory and using them, a genetic and molecular characterization of the chromosomal region that includes the locus will be done. That information will yield sufficient positional information to clone the gene encoding allorecognition determinants from this lower metazoan. The near-isogenic animals will be used to generate pools of offspring homozygous for different alleles at the fusibility locus. These offspring pools will allow identification of amplified fragment length polymorphisms (AFLP) linked within a 1cM interval containing the locus of interest (arl). A high resolution genetic map of these markers will be generated and the markers most tightly flanking arl will be determined in a large recombinant test cross. A bacterial artificial chromosome (BAC) library will be generated, screened for positives using flanking marker sequences and a high resolution map of the set of contiguous BAC's generated. BAC clones will be shed of the highly repetitive fraction by reverse Southerns and resulting fragments will be used to probe a cDNA library to isolate candidate gene(s). The ability to confer fusibility phenotype by transformation in a simple adhesion assay will be preformed to confirm the isolation of the correct locus.
Colonial animals typically display the capacity to discriminate between their own tissues and those of unrelated members of their own species, with the recognition event culminating in either fusion or rejection. Such allorecognition systems are often claimed to reflect the evolutionary origins of the vertebrate immune response and, for unrelated reasons have played a prominent role in problems in evolution theory. Despite their widespread occurrence and importance, the molecular basis of invertebrate allorecognition remains obscure. The products of this effort, novel genetic lines, a DNA collection derived from a large recombinant test cross and a BAC library, will be available to the community and the demand is expected to be high. The genetic map and large insert library emanating from this research will be the first such resources for any eumetazoan animal.
