Homologous recombination catalyzed by human cell extracts.
Two plasmids containing noncomplementing and nonreverting deletions in a bacterial phosphotransferase gene conferring resistance to neomycin (Neor) were incubated with human cell extracts, and the mixtures were used to transform recombination-deficient (recA-) Escherichia coli cells. We were able to obtain Neor colonies at a frequency of 2 X 10(-3). This frequency was 100 to 1,000 times higher than that obtained with no extracts. The removal of riboadenosine 5'-triphosphate, Mg2+, or deoxynucleoside triphosphates from the reaction mixture severely reduced the yield of Neor colonies. Examination of plasmid DNA from the Neor colonies revealed that they resulted from gene conversion and reciprocal recombination. On the basis of these results, we conclude that mammalian somatic cells in culture have the enzymatic machinery to catalyze homologous recombination in vitro.
Kucherlapati
R S
RS
Spencer
J
J
Moore
P D
PD
D002460:Cell Line; D002474:Cell-Free System; D004269:DNA, Bacterial / Q000235:genetics; D004926:Escherichia coli / Q000235:genetics; D005785:Gene Conversion; D005798:Genes, Bacterial; D006801:Humans; D009355:Neomycin / Q000633:toxicity; D010770:Phosphotransferases / Q000235:genetics; D010957:Plasmids; D011995:Recombination, Genetic
D004269:DNA, Bacterial; D010770:Phosphotransferases; D009355:Neomycin