Construction of a yeast artificial chromosome (YAC). A YAC vector includes an origin of replication (ori), a centromere (CEN), two telomeres (TEL), and selectable markers (here designated X and Y). Two separate DNA arms are generated by digestion with BamHI and EcoRI, each arm having a telomeric end and one selectable marker. A large DNA fragment, produced by EcoRI digestion, is ligated to the two arms, creating a YAC. The YAC is transferred into yeast cells (which have been prepared by removing the cell wall to form spheroplasts). The transformed cells are selected for X and Y, and the surviving cells propagate the DNA insert.