ASSEMBLY AND EXTENSION OF YEAST ARTIFICIAL CHROMOSOMES TO BUILD UP A LARGE LOCUS

For the assembly of a large human locus, overlapping regions on yeast artificial chromosomes (YACs) and cosmids were linked up using their r egions of homology. By site-specific recombination a YAC of 410 kb was created accommodating the major part of the human lambda light chain locus in authentic configuration with 28 variable (V) genes, all joini ng (J) segments, all constant (C) genes and the downstream enhancer. A contiguous region was first constructed from three overlapping cosmid s. Each of these was linearized at unique sites in the vectors and YAC arms were ligated to the 5' and 3' ends. After cells of Saccharomyces cerevisiae were transformed with the three cosmids, YACs of 120 kb we re obtained which contained the reassembled 3' J-C region in authentic configuration. The assembled YAC was further extended by mitotic reco mbination with a YAC containing a 280-kb region of the C-proximal part of the V gene cluster with a 15-kb 3' overlap. For this, a simple thr ee-way selection procedure was developed involving the integration of different selectable marker genes at specific sites in the left and ri ght YAC arms. Rare recombination events between two overlapping YACs c ould be identified in yeast clones able to grow in lysine- and adenine -deficient medium in the presence of 5-fluoro-orotic acid which is tox ic for yeast cells containing a YAC with a functional URA3 gene. This approach made it possible to assemble and extend large YACs from an un limited number of smaller overlapping YACs by positive-negative select ion.