Active role of a human genomic insert in replication of a yeast artificialchromosome

Yeast artificial chromosomes (YACs) are a common tool for cloning eukaryoti c DNA. The manner by which large pieces of foreign DNA are assimilated by y east cells into a functional chromosome is poorly understood, as is the rea son why some of them are stably maintained and some are not, We examined th e replication of a stable YAC containing a 240-kb insert of DNA from the hu man T-cell receptor beta locus. The human insert contains multiple sites th at serve as origins of replication. The activity of these origins appears t o require the yeast ARS consensus sequence and, as with yeast origins, addi tional flanking sequences. In addition, the origins in the human insert exh ibit a spacing, a range of activation efficiencies, and a variation in time s of activation during S phase similar to those found for normal yeast chro mosomes. We propose that an appropriate combination of replication origin d ensity, activation times, and initiation efficiencies is necessary for the successful maintenance of YAC inserts.