Intermolecular plasmid recombination in fibroblasts from humans with DNA damage-processing defects.
We have evaluated the ability of immortalized human fibroblasts to recombine transfected plasmid DNA. A number of cell lines from normal individuals and from patients with DNA damage-processing defects were examined. Two plasmid recombination substrates were derived from pSV2neo and contained nonoverlapping deletions in the aminoglycoside phosphotransferase II gene. Intermolecular recombination was assessed by two methods after cotransfection. In a short-term, extrachromosomal recombination assay, low molecular weight DNA was extracted from the human cells 48 h after transfection, and recombinant plasmids were detected by transformation into appropriate indicator bacteria. In a long-term stable recombination assay the fibroblasts were cotransfected and G418-resistant colonies allowed to form. By the former assay all but two cultures were recombination-proficient, whereas all were recombination-proficient by the latter assay. The efficiency of transfection of human cells with plasmids appears to be a major variable affecting recombination. Recombination can be stimulated by uv irradiation of plasmid DNA prior to transfection. Cells from patients with Fanconi anemia, ataxia telangiectasia, and xeroderma pigmentosum complementation groups A, C, D, E, and G are not defective at intermolecular plasmid recombination.
Timme
T L
TL
Department of Cell Biology, Baylor College of Medicine, Houston, Texas 77030.
Wood
C M
CM
Moses
R E
RE
D002460:Cell Line; D002872:Chromosome Deletion; D004247:DNA / Q000235:genetics*; D004249:DNA Damage; D004926:Escherichia coli / Q000235:genetics; D005347:Fibroblasts; D006801:Humans; D019868:Kanamycin Kinase; D010770:Phosphotransferases / Q000235:genetics; D010957:Plasmids; D011995:Recombination, Genetic; D015183:Restriction Mapping; D014162:Transfection; D014169:Transformation, Bacterial
D004247:DNA; D010770:Phosphotransferases; D019868:Kanamycin Kinase