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Functional genomic mapping of an early‐activated centromeric mammalian origin of DNA replication
Author(s) -
Pelletier Richard,
Price Gerald B.,
ZannisHadjopoulos Maria
Publication year - 1999
Publication title -
journal of cellular biochemistry
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.028
H-Index - 165
eISSN - 1097-4644
pISSN - 0730-2312
DOI - 10.1002/(sici)1097-4644(19990915)74:4<562::aid-jcb6>3.0.co;2-2
Subject(s) - biology , dna replication , control of chromosome duplication , dna , genomic dna , eukaryotic dna replication , microbiology and biotechnology , genetics , origin of replication , genome , origin recognition complex , gene
Ors12, a mammalian autonomously replicating sequence (812 bp), was previously isolated by extrusion of African green monkey (CV‐1 cells) nascent DNA from active replication bubbles. It contains a region of α‐satellite extending 168‐bp from the 5′‐end, and a nonrepetitive portion extending from nucleotide position 169 to nucleotide 812 that is present in less than nine copies per haploid genome. Ors12 is capable of transient autonomous DNA replication in vivo and in vitro, associates with the nuclear matrix in a cell cycle‐dependent manner, and hybridizes at the centromeric region of six CV‐1 cell chromosomes as well as a marker chromosome. To demonstrate that DNA replication initiates at ors12 at a native chromosomal locus, a 14.2 kb African green monkey genomic clone was isolated and sequence information was obtained that allowed us to generate eight sets of PCR primers spanning a region of 8 kb containing ors12 . One set of primers occurred inside ors12 . These primers were used to amplify nascent DNA strands from asynchronously growing CV‐1 and African green monkey kidney (AGMK) cells, using noncompetitive and competitive PCR‐based mapping methodologies. Both assays showed that DNA replication in vivo initiates preferentially in a 2.3 kb region containing 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 centromeric mammalian origin of DNA replication, originally isolated by nascent strand extrusion. J. Cell. Biochem. 74:562–575, 1999. © 1999 Wiley‐Liss, Inc.