Autonomous replication in human cells of multimers of specific human and bacterial DNA sequences.
Using modules of a specific 2,712-bp human DNA sequence and a specific 2,557-bp Escherichia coli DNA sequence, we created plasmids containing between 1 and 12 modules of single or chimeric sequence composition and tested them in human cells for their autonomous replication ability. We found that replication efficiency per generation increased with successive addition of human modules, to essentially 100% by six copies. Although a single copy of the bacterial module had negligible replication ability, the replication efficiency per generation of 12 bacterial modules was 66%. Chimeras composed of human and bacterial modules displayed intermediate replication levels. We also used two-dimensional gel electrophoresis to physically map where replication initiated on a half human-half E. coli plasmid. Our results suggest that autonomous replication in human cells is stimulated by simple sequence features which occur frequently in human DNA but are more rare in bacterial DNA.
Department of Genetics, Stanford University School of Medicine, California 94305.
P J
PJ
Krysan
J G
JG
Smith
M P
MP
Calos
D000668:Ampicillin Resistance / Q000235:genetics*; D000956:Antigens, Viral; D002460:Cell Line; D004247:DNA / Q000235:genetics*; D004261:DNA Replication; D004269:DNA, Bacterial / Q000235:genetics*; D004268:DNA-Binding Proteins; D019309:Epstein-Barr Virus Nuclear Antigens; D004926:Escherichia coli / Q000201:enzymology / Q000235:genetics*; D006801:Humans; D007668:Kidney; D010770:Phosphotransferases / Q000235:genetics*; D017853:Phosphotransferases (Alcohol Group Acceptor); D010957:Plasmids; D015183:Restriction Mapping; D014162:Transfection; D001616:beta-Galactosidase / Q000235:genetics*
D000956:Antigens, Viral; D004269:DNA, Bacterial; D004268:DNA-Binding Proteins; D019309:Epstein-Barr Virus Nuclear Antigens; D004247:DNA; D010770:Phosphotransferases; D017853:Phosphotransferases (Alcohol Group Acceptor); C063242:hygromycin-B kinase; D001616:beta-Galactosidase