Creation of a yeast artificial chromosome fragmentation vector based on lysine-2

Yeast artificial chromosome (YAC) fragmentation vectors have proven to be useful reagents for mapping and modifying YAC clones through homologous recombination. Such vectors can be employed to remove a noncontiguous sequence present in chimeric YAC clones. However, previous fragmentation vectors have been based on auxotrophies not present in the background of most recombinant YAC libraries, which therefore makes them difficult to use. We have constructed an acentric YAC deletion vector pBCL that is fully compatible with AB 1380, the yeast host used in the majority of human total genomic YAC libraries. Transformation of three unrelated YACs with linearized pBCL yielded derivative clones with the expected phenotype at rates of 27%-49%. Analysis of a random cohort of these clones by pulsed-field gel electrophoresis and Southern blotting revealed single deleted structures in 31 of 34 clones. Using this vector, a nested set of deletion derivatives can efficiently and reliably be generated from human recombinant YAC clones.