Autonomous replication of a DNA fragment containing the chromosomal replication origin of the human c-myc gene.
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 HindIII-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.
McWhinney
C
C
Department of Biochemistry, Wright State University, Dayton, OH 45435.
Leffak
M
M
D002877:Chromosomes, Human / Q000378:metabolism*; D004247:DNA / Q000378:metabolism; D004261:DNA Replication; D004262:DNA Restriction Enzymes / Q000378:metabolism; D005822:Genetic Vectors; D006367:HeLa Cells; D006801:Humans; D008745:Methylation; D010957:Plasmids; D011519:Proto-Oncogenes; D015183:Restriction Mapping; D014162:Transfection; D014170:Transformation, Genetic
D004247:DNA; D004262:DNA Restriction Enzymes