Efficiency of de Novo Centromere Formation in Human Artificial Chromosomes

In a comparative study, we show that human artificial chromosome (HAC) vectors based on α-satellite (alphoid) DNA from chromosome 17 but not the Y chromosome regularly form HACs in HT1080 human cells. We constructed four structurally similar HAC vectors, two with chromosome 17 or Y alphoid DNA (17α, Yα) and two with 17α or Yα and the hypoxanthine guanine phosphoribosyltransferase locus (HPRT1). The 17α HAC vectors generated artificial minichromosomes in 32–79% of the HT1080 clones screened, compared with only ∼4% for the Yα HAC vectors, indicating that Yα is inefficient at forming a de novo centromere. The 17α HAC vectors produced megabase-sized, circular HACs containing multiple copies of alphoid fragments (60–250 kb) interspersed with either vector or HPRT1 DNA.The 17α-HPRT1 HACs were less stable than those with 17α only, and these results may influence the design of new HAC gene transfer vectors.