R. Pelletier et al., Functional genomic mapping of an early-activated centromeric mammalian origin of DNA replication, J CELL BIOC, 74(4), 1999, pp. 562-575
Ors12, a mammalian autonomously replicating sequence (812 bp), was previous
ly isolated by extrusion of African green monkey (CV-1 cells) nascent DNA f
rom active replication bubbles. It contains a region of a-satellite extendi
ng 168-bp from the 5'-end, and a nonrepetitive portion extending from nucle
otide position 169 to nucleotide 812 that is present in less than nine copi
es per haploid genome. Ors12 is capable of transient autonomous DNA replica
tion in vivo and in vitro, associates with the nuclear matrix in a cell cyc
le-dependent manner, and hybridizes at the centromeric region of six CV-1 c
ell chromosomes as well as a marker chromosome. To demonstrate that DNA rep
lication initiates at ors12 at a native chromosomal locus, a 14.2 kb Africa
n green monkey genomic clone was isolated and sequence information was obta
ined that allowed us to generate eight sets of PCR primers spanning a regio
n of 8 kb containing ors12. One set of primers occurred inside ors12. These
primers were used to amplify nascent DNA strands from asynchronously growi
ng CV-1 and African green monkey kidney (AGMK) cells, using noncompetitive
and competitive PCR-based mapping methodologies. Both assays showed that DN
A replication in vivo initiates preferentially in a 2.3 kb region containin
g ors12, as well as at a second site located 1.7 kb upstream of ors12. This
study provides the first demonstration of genomic function for a centromer
ic mammalian origin of DNA replication, originally isolated by nascent stra
nd extrusion. (C) 1999 Wiley-Liss, Inc.