Premium
Deletion analysis of ors12, a centromeric, early activated, mammalian origin of DNA replication
Author(s) -
Pelletier Richard,
Mah David,
Landry Suzanne,
Matheos Diamanto,
Price Gerald B.,
ZannisHadjopoulos Maria
Publication year - 1997
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(19970701)66:1<87::aid-jcb10>3.0.co;2-a
Subject(s) - biology , microbiology and biotechnology , ter protein , dna replication , dna , replication factor c , origin of replication , mutant , genetics , control of chromosome duplication , gene
We have generated a panel of deletion mutants of ors12 (812‐bp), a mammalian origin of DNA replication previously isolated by nascent strand extrusion from early replicating African Green monkey (CV‐1) DNA. The deletion mutants were tested for their replication activity in vivo by the bromodeoxyuridine substitution assay, after transfection into HeLa cells, and in vitro by the DpnI resistance assay, using extracts from HeLa cells. We identified a 215‐bp internal fragment as essential for the autonomous replication activity of ors12. When subcloned into the vector pML2 and similarly tested, this subfragment was capable of autonomous replication in vivo and in vitro. Several repeated sequence motifs are present in this 215‐bp fragment, such as TGGG(A) and G(A)AG (repeated four times each); TTTC, AGG, and CTTA (repeated 3 times each); the motifs CACACA and CTCTCT, and two imperfect inverted repeats, 22 and 16 bp long, respectively. The overall sequence of the 215‐bp fragment is G/C‐rich (50.2%), by comparison to the 186‐bp (33.5% G/C‐rich) minimal sequence required for the autonomous replication activity of ors8, another functional ors that was similarly isolated and characterized. J. Cell. Biochem. 66:87–97, 1997. © 1997 Wiley‐Liss, Inc.