Transformation-associated recombination (TAR) cloning of tumor-inducing xmrk2 gene from Xiphophorus maculatus

We modified the TAR methodology of YAC clone construction for application t o fish genomic DNA isolated from Xiphophorus maculatus. YAC libraries were developed using the XIR1 repeat sequence as the recombinational hook. Const ruction of these libraries demonstrates that Xiphophorus DNA sequences can function as hooks in the yeast recombination system and that X. maculatus g enomic DNA contains sequences that provide origin of replication function i n yeast. By screening a subset of Xiphophorus YAC clones, we isolated a clo ne harboring the Xmrk2 locus that is associated with spontaneous and induce d melanomagenesis. Modifications of the TAR technique allowed the targeted cloning of specific genes from genomic regions ranging in size from cDNAs t o several hundred kilobases. Specific genomic regions can be isolated in a directional manner from fixed map locations to saturate these areas with ph ysical markers. We discuss the applications of these and other yeast recomb inational processes to fish genetics.