A NEW VECTOR FOR RECOMBINATION-BASED CLONING OF LARGE DNA FRAGMENTS FROM YEAST ARTIFICIAL CHROMOSOMES

The functional analysis of genes frequently requires manipulation of l arge genomic regions embedded in yeast artificial chromosomes (YACs). We have designed a yeast-bacteria shuttle vector, pClasper, that can b e used to clone specific regions of interest from YACs by homologous r ecombination. The important feature of pClasper is the presence of the mini-F factor replicon. This leads to a significant increase in the s ize of the plasmid inserts that can be maintained in bacteria after cl oning by homologous recombination in yeast. The utility of this vector lies in its ability to maintain large fragments in bacteria and yeast , allowing for mutagenesis in yeast and simplified preparation of plas mid DNA in bacteria. Using PCR-generated recombinogenic fragments in p Clasper we cloned a 27 kb region from a YAC containing the Hoxc cluste r and a 130 kb region containing the entire Hoxb cluster. No rearrange ments were seen when the recombinants in the shuttle vector were trans ferred to bacteria. We outline the potential uses of pClasper for func tional studies of large genomic regions by transgenic and other analys es.