Human artificial chromosomes generated by modification of a yeast artificial chromosome containing both human alpha satellite and single-copy DNA sequences

A human artificial chromosome (HAC) vector was constructed from a 1-Mb yeas t artificial chromosome (YAC) that was selected based on its size from amon g several YACs identified by screening a randomly chosen subset of the Cent re d'Etude du Polymorphisme Humain (CEPH) (Paris) YAC library with a degene rate alpha satellite probe. This YAC, which also included non-alpha satelli te DNA, was modified to contain human telomeric DNA and a putative origin o f replication from the human beta-globin locus, The resultant HAC vector wa s introduced into human cells by lipid-mediated DNA transfection, and HACs were identified that bound the active kinetochore protein CENP-E and were m itotically stable in the absence of selection for at least 100 generations. Microdissected HACs used as fluorescence in situ hybridization probes loca lized to the HAC itself and not to the arms of any endogenous human chromos omes, suggesting that the NAC was not formed by telomere fragmentation. Our ability to manipulate the HAC vector by recombinant genetic methods should allow us to further define the elements necessary for mammalian chromosome function.