AUTONOMOUS REPLICATION OF A DNA FRAGMENT CONTAINING THE CHROMOSOMAL REPLICATION ORIGIN OF THE HUMAN C-MYC GENE

被引:119
作者
MCWHINNEY, C [1 ]
LEFFAK, M [1 ]
机构
[1] WRIGHT STATE UNIV,DEPT BIOCHEM,DAYTON,OH 45435
关键词
D O I
10.1093/nar/18.5.1233
中图分类号
Q5 [生物化学]; Q7 [分子生物学];
学科分类号
071010 ; 081704 ;
摘要
The c-myc genes of HeLa cells are preferentially replicated in the transcriptional direction, from chromosomal origin sequences which display cell type-specific activity. Using a run-off replication assay involving in vitro extension of replication forks initiated in intact HeLa cells, bidirectional replication was observed to begin within a 3.5 kb domain 5′ to the c-myc gene. To characterize the replication origin further a 2.4 Hindlll-Xhol subfragment of the c-myc 5′ flanking DNA was cloned in a selectable vector and transfected into HeLa cells. The resulting pNeo.Myc-2.4 construct persisted as a circular extrachromosomal element for more than 300 cell generations under selection, with recovery of approximately 500-1000 times the mass of plasmid initially introduced into the cells. Extrachromosomal circular pNeo.Myc-2.4 monomer was reisolated in supercoiled form, along with oligomeric and miniplasmid variants which had been generated in vivo; however, chromosomally integrated copies of the plasmid were not detectable in cultures containing extrachromosomal pNeo.Myc-2.4. The recovered pNeo.Myc-2.4 plasmid was resistant to Dpnl digestion and sensitive to Mbol digestion. After transfection with pNeo.Myc-2.4 BrUdR pulse labeling of long-term or short-term cultures demonstrated that the plasmid replicated semiconservatively, under controls similar to those imposed on chromosome replication. Bisection of the pNeo.Myc-2.4 insert suggested that c-myc 5′ flanking DNA within 1.2 kb 5′ to promoter P1 was sufficient to confer autonomously replicating sequence activity on the plasmid vector In transient replication assays. © 1990 Oxford University Press.
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页码:1233 / 1242
页数:10
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