Circular YAC vectors containing short mammalian origin sequences are maintained under selection as HeLa episomes

pYACneo, a 15.8-kb plasmid, contains a bacterial origin, G418-resistance ge ne, and yeast ARS, CEN, and TEL elements. Three mammalian origins have been cloned into this circular vector: 343, a 448-bp chromosomal origin from a transcribed region of human chromosome 6q; X24, a 4.3-kb element containing the hamster DHFR origin of bidirectional replication (ori beta), and S3, a 1.1-kb human anti-cruciform purified autonomously replicating sequence. Th e resulting constructs have been transfected into HeLa cells, and G418-resi stant subcultures were isolated. The frequency of G418-resistant transforma tion was 1.7-8.7 times higher with origin-containing YACneo than with vecto r alone. After >45 generations under G418 selection, the presence of episom al versus integrated constructs was assessed by fluctuation assay and by PC R of supercoiled, circular, and linear genomic cellular DNAs separated on e thidium bromide-cesium chloride gradients. In stable G418-resistant subcult ures transfected with vector alone or with linearized constructs, as well a s in some subcultures transfected with circular origin-containing construct s, resistance was conferred by integration into the host genome. However, s everal examples were found of C418-resistant transfectants maintaining the Y.343 and the YAC.S3 circular constructs in a strictly episomal state after long-term culture in selective medium, with 80-90% stability per cell divi sion. The episomes were found to replicate semiconservatively in a bromodeo xyuridine pulse-labeling assay for less than or equal to 130 cell generatio ns after transfection. Furthermore, alter less than or equal to 172 cell ge nerations rescued episomal DNA could be isolated intact and unrearranged, a nd could be used to retransform bacteria. These versatile constructs, conta ining mammalian origins, have the capacity for further modification with hu man telomere or large putative centromere elements, in an effort to move to wards construction of a human artificial chromosome. (C) 2000 Wiley-Liss, I nc.