For the assembly of a large human locus, overlapping regions on yeast artificial chromosomes (YACs) and cosmids were linked up using their regions 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 joining (J) segments, all constant (C) genes and the downstream enhancer. A contiguous region was first constructed from three overlapping cosmids. 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 were obtained which contained the reassembled 3′ J-C region in authentic configuration. The assembled YAC was further extended by mitotic recombination 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 three-way selection procedure was developed involving the integration of different selectable marker genes at specific sites in the left and right YAC arms. Rare recombination events between two overlapping YACs could be identified in yeast clones able to grow in lysine- and adenine-deficient medium in the presence of 5-fluoro-orotic acid which is toxic for yeast cells containing a YAC with a functional URA3 gene. This approach made it possible to assemble and extend large YACs from an unlimited number of smaller overlapping YACs by positive-negative selection.
Assembly and extension of yeast artificial chromosomes to build up a large locus
Popov, A. V.; Butzler, C.; Frippiat, J. P.; Lefranc, M. P.; Bruggemann, M.
1996-10-24 / vol 177 / pages 195-201
organization; homologous recombination; saccharomyces-cerevisiae; saccharomyces cerevisiae; transformation; lambda; light-chain locus; segments; constant region genes; DNA; kar1 mutation; meiotic recombination; mitotic recombination; overlapping cosmids; populations; selection vector; site-specific integration; vector