CIRCULAR YAC VECTORS CONTAINING A SMALL MAMMALIAN ORIGIN SEQUENCE CANASSOCIATE WITH THE NUCLEAR MATRIX

Three different mammalian origins of DNA replication, 343, S3, and X24 , have been cloned into a 15.8 kb circular yeast vector pYACneo. Subse quent transfection into HeLa cells resulted in the isolation of severa l stably maintained clones. Two cell lines, C343e2 and CS3e1, were fou nd to have sequences maintained as episomes in long-term culture with a stability per generation of approximately 80%. Both episomes also co ntain matrix attachment region (MAR) sequences which mediate the bindi ng of DNA to the nuclear skeleton and are thought to play a role in DN A replication. Using high salt extraction of the nucleus and fluoresce nt in situ hybridization, we were able to demonstrate an association o f the 343 episome with the nuclear matrix, most probably through funct ional MAR sequences that allow an association with the nuclear matrix and associated regions containing essential replication proteins. The presence of functional MARs in small episomal sequences may facilitate the replication and maintenance of transfected DNA as an episome and improve their utility as small episomal constructs, potential microchr omosomes. J. Cell. Biochem. 67:439-450, 1997. (C) 1997 Wiley-Liss, Inc .