Distribution of 5-Bromodeoxyuridine in the DNA of Rat Embryo Cells

Abstract

Rat embryo cells were treated with 3H-labeled 5-bromodeoxyuridine (BrdU) in order to determine the nature of substitution and distribution of the analog in DNA. At optimal oncornavirus-inducing doses of BrdU (10-4 M), density gradient centrifugation and DNA-DNA reassociation experiments revealed extensive (>90%) and uniform base substitution in The thymidine analog 5-bromodeoxyuridine (BrdU) selectively suppresses differentiated functions as well as activates specific synthetic processes in animal cells (1-10). It is generally assumed that the analog is incorporated into DNA, and that bromouracil substitutes exclusively for thymine (1,4,5,8,11,12 U/ml of penicillin (10,15). Cells were dispersed into 670-cm2 glass roller vessels (Bellco) and maintained in a 95% air, 5% CO2 atmosphere at 37°. Replicate roller bottle cultures were grown to subconfluency. Half of the cultures in logarithmic phase were treated with 50 ml of medium 199 containing 1o-4 M BrdU (Sigma) and 10 uCi/ml of [3H]BrdU (12.7 Ci/mmol, New England Nuclear Corp.) (8,10). The remaining cultures were exposed only to 50 ml of medium 199 containing 10 /ACi/ml of [3H]dT (18.3 Ci/mmol, New England Nuclear Corp.). Culture fluids were removed 24 hr later; cells were harvested with 0.25% (w/v) trypsin and concentrated by low-speed centrifugation.Preparation of DNA. Concentrated cell pellets were suspended in 0.15 M NaCl-0.015 M trisodium citrate (pH 7.0) and made 1% in sodium dodecyl sulfate, 1% in 2-mercaptoethanol, and incubated at 600 for 10 min (16). An equal volume of cold chloroform-isoamyl alcohol (24:1, w/v) was added, and the suspension was extracted three times at room temperature. The aqueous phases were again extracted with equal volumes of phenol (equilibrated with NaCl-trisodium citrate) until no material remained at the interface. Three volumes of ice-cold ethanol were layered onto the aqueous phase, and DNA was spooled out of solution. The fibers were immediately dissolved in 80 mM sodium phosphate buffer (pH 6.8). The DNA solution was extensively dialyzed against 80 mM phosphate buffer, and adsorbed to a column of hydroxyapatite (HTP-DNA Grade, Bio Rad) equilibrated at 600 with 80 mM phosphate buffer (17, 18). Contaminating RNA was eluted with several column volumes of 0.18 M phosphate buffer, and the double-stranded DNA was subsequently eluted with 0.40 M phosphate buffer (17, 18). The column-purified DNA was diluted 3fold, made lmM in EDTA, and mechanically sheared in a Ribi cell fractionator (Sorvall) at 50,000 lbs./inch2 (17,18). The procedure consistently prepared DNA fragments of uniform size (300-400 nucleotides in length) with a sedimentation coefficient of 5.4 S, according to the alkaline band method of Studier (19). Sheared, characterized DNA preparations were again purified over hydroxyapatite, assayed for A216, frozen, and stored at -40 until use. Unlabeled, bulk DNA from W/Fu cell cultures was extracted, purified, sheared, and stored in the same manner.Isopycnic CsCl Centrifugationm. Aliquots of radioactive sheared DN...

Distribution and virogenic effects of 5-bromodeoxyuridine in synchronized rat embryo cells.

“…Cells were rinsed and 2 mM dT was added for an additional 16 hr. Cultures were again rinsed and released into synchrony with 10,MM dG, dA, and dC as before (5,6 (2,3). The specific activity of each DNA sample was determined.…”

“…Secondary cultures of 16-day old Wistar/ Furth (W/Fu) rat embryo cells were maintained as described (1)(2)(3) (4)(5)(6). Subconfluent roller cultures were treated with 2 mM dT (Calbiochem).…”

“…Release of type C particles into the culture fluids was first detected 2 days after BrdU removal, peaked [8][9][10] days later, and disappeared 14-16 days after removal of the analog. Density gradient centrifugation and DNA -DNA reassociation experiments revealed extensive and uniform base substitutions throughout the DNA sequences of rat embryo cells at the optimal virogenic BrdU dose (0.1 mM) (2). However, [3H]BrdU was incorporated predominantly into the repetitive and intermediate repetitive DNA sequences when the concentration of the analog was reduced to 0.1 pAM (2,3).…”

“…Each DNA sample was made 0.40 M in sodium phosphate buffer, pH 6.8, and 3 mM in EDTA (2,3,7,8). The initial A260 of the reaction mixture was determined, and the sample was heat sealed in 5 ml glass ampoules and immersed completely in boiling water at 1000 for 10-15 min (2, 3, 7).…”

“…Density gradient centrifugation and DNA -DNA reassociation experiments revealed extensive and uniform base substitutions throughout the DNA sequences of rat embryo cells at the optimal virogenic BrdU dose (0.1 mM) (2). However, [3H] (2,3). The specific activity of each DNA sample was determined.…”