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...

Rat embryo cell cultures were synchronized by a double thymidine block. The DNA replication phase (S) was divided into an early, middle, and late period. Cell cultures in the early, middle, or late S phase were pulsed with 0.1 JM 5-bromo[3H]deoxyuridine (BrdU) or equimolar [3HldT. DNA The thymidine analog 5-bromodeoxyuridine (BrdU) induces the synthesis of a latent type C virus in secondary cultures of normal rat embryo cells (1). Release of type C particles into the culture fluids was first detected 2 days after BrdU removal, peaked 8-10 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] (2,3). The specific activity of each DNA sample was determined. Aliquots of radiolabeled DNA were centrifuged to equilibrium in gradients of neutral CsCl (2, 3). Gradient fractions were assayed for radioactivity, absorbance, and refractive index as described (2, 3).Renaturation Kinetics. 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). After thermal denaturation, each ampoule was transferred to a 680 water bath and incubated until the product of initial DNA concentration (mol of 1829 Abbreviations: S, DNA replication phase of cell cycle; p30, group-specific antigen of Friend leukemia virus; BrdU, 5-bromodeoxyuridine; Cot, initial DNA concentration time.

Secondary cultures of normal rat embryo cells were synchronized by a double thymidine block and pulsed with 10(-7) M 5-[3H]bromodeoxyuridine (BrdUrd) OR 10(-7) M[3H]thymidine during an entire S phase (7.5 h). To examine the pattern of [3H]thymidine, DNA was immediately extracted and purified at the completion of the S phase, CsCl density gradient centrifugation revealed that substitution for thymine by bromouracil was less than 7%. Single-strand specific nucleases obtained from Aspergillus oryzae and Neurospora crassa were allowed to react with native and partially depurinated (24-29%) [3H]BrdUrd-labeled rat DNA samples, and the products were assayed by hydroxylapatite column chromatography. Approximately 4-6% of the native, nondepurinated rat DNA was hydrolyzed by both nucleases. However, 24-28% of the partially depurinated, [3H] thymidine-labeled rat DNA was hydrolyzed by both enzymes as determined by loss of mass as well as radioactivity. Whereas comparable levels of depurinated, [3H]BrdUrd-labeled DNA were physically hydrolyzed by both nucleases, nearly 65% of the radioactivity was not recovered. Native, as well as depurinated, enzyme-treated DNA samples were sequentially and preparatively reassociated into highly repetitive, middle repetitive, and nonrepetitive nucleotide sequence components. The absolute and relative specific activities of each subfraction of native [3H]thymidine-labeled DNA were comparable. [3H]BrdUrd was differentially concentrated in the middle repetitive sequences as compared to other reiteration frequency types. When depurinated, nuclease-treated DNA samples were similarly fractionated, [3H]thymine moieties were uniformly distributed thoughout all sequences. However, a differential loss of [3H]BrdUrd moieties was detected predominantly from the middle repetitive nucleotide fraction. Melting profiles of the renatured DNA samples were characteristic of each respective DNA subfraction regardless of isotopic precursor. These results suggest that [3H]BrdUrd may be differentially incorporated into A + T rich clusters of rat DNA, especially in the moderately repeated chromosomal elements.

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