The effects of incorporation of bromodeoxyuridine into mammalian DNA on the migration patterns of DNA fragments subjected to pulsed-field gel electrophoresis after X irradiation or cutting with a restriction enzyme.
Incorporation of bromodeoxyuridine (BrdU) into DNA in both Chinese hamster ovary (CHO) cells and human melanoma (U1) cells reduced the rate of DNA migration in transverse alternating-field electrophoresis (TAFE) agarose gel optimized for separating molecules larger than 2 Mb. This "BrdU migration effect" was independent of the method of damaging the DNA; i.e., the effect was observed after irradiation of the cells or treatment of the plugs containing DNA with the restriction enzyme, Mlu-1, and similar results were found for CHO and U1 cells. However, when the amount of cutting of DNA was estimated from the amount of DNA migrating from the plugs, a difference between U1 cells and CHO cells was observed in that incorporation of BrdU enhanced the cutting of DNA by either irradiation or Mlu-1 digestion for U1 cells but not for CHO cells. Therefore, the "BrdU migration effect" could not be attributed to an increase in large molecules because of reduced cutting of the BrdU-labeled DNA. The decrease in migration rate during pulsed-field gel electrophoresis when BrdU replaces thymidine in the DNA is hypothesized to result from the increase in negative charge on BrdU-labeled DNA. An increase in electron charge density is expected to increase the reorientation time of BrdU-labeled DNA fragments during each voltage pulse due to an increase in the elongation length of the DNA caused by an increase in electronegativity.
Latz
D L
DL
Radiation Oncology Research Laboratory, University of California San Francisco 94103.
Trinh
M M
MM
Thompson
L L
LL
Gardiner
K
K
Zhu
Y
Y
Bodell
W J
WJ
Dewey
W C
WC
D000818:Animals; D001426:Bacterial Proteins; D001483:Base Sequence; D015139:Blotting, Southern; D001973:Bromodeoxyuridine / Q000378:metabolism* / Q000633:toxicity; D016466:CHO Cells; D002460:Cell Line; D002470:Cell Survival / Q000187:drug effects / Q000528:radiation effects; D006224:Cricetinae; D004247:DNA / Q000302:isolation & purification* / Q000378:metabolism / Q000528:radiation effects; D015342:DNA Probes; D004273:DNA, Neoplasm / Q000302:isolation & purification* / Q000378:metabolism / Q000528:radiation effects; D015252:Deoxyribonucleases, Type II Site-Specific / Q000378:metabolism*; D004587:Electrophoresis, Agar Gel / Q000379:methods; D006801:Humans; D008545:Melanoma; D008969:Molecular Sequence Data; D014407:Tumor Cells, Cultured; D014965:X-Rays
D001426:Bacterial Proteins; D015342:DNA Probes; D004273:DNA, Neoplasm; D004247:DNA; C035041:endodeoxyribonuclease MluI; D015252:Deoxyribonucleases, Type II Site-Specific; D001973:Bromodeoxyuridine