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

Abstract

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.

Tyrosine aminotransferase sensitivity to bromodeoxyuridine during restricted intervals of S phase in hepatoma cells.

“…Although in some systems the order in which genes replicate appears to be highly temporal (3, l9), this is not always the case (24,25). The period of TAT sensitivity to BUdR reported here, though broad, is similar in extent to that reported for the effect of the analogue on other systems (4,9,17,22).…”

“…The plating efficiency of HeLa cells (9), various enzymes in THE JOURNAL of CELL BIOLOGY -VOLUME 87 DECEMBER 1980 629-632 L cells (10), viral antigen production in rat embryo cultures (17), and mutagenesis in various hamster cell lines (4,22) have been found to respond to the incorporation of BUR into specific intervals of S phase . In HTC cells, however, TAT decreased whenever BUdR was incorporated into DNA during S phase (19) .…”

Nonrandom Correlation between Selective Distribution of Virogenic 5-Bromodeoxyuridine and DNA-Binding Nonhistone Proteins in Rat DNA

“…We have extensively examined the nature of BrdU activation of latent type C virus from normal rat embryo cells (21,22,(24)(25)(26). Many other workers have similarly studied BrdU-mediated transcriptional alterations in several dif ferent types of eukaryotic cells (19).…”

“…specific activity of unfractionated DNA sample 24,25). The majority of rat DNA repetitive sequences are believed to average 250 400 nucleotides in length, and are predominantly interspersed between the nonrepeated elements (5).…”

“…On the basis of all these findings, a generalized observa tion has been made that DNA-binding proteins bind more tightly to bromouracil-containing nucleotide sequences, and thereby constitute a more stable complex. Our previous reports have described studies concerning the elucidation of the molecular mechanism responsible for the BrdU-mediated activation of a latent endogenous type C RNA virus from normal rat embryo cells in vitro (21,22,(24)(25)(26). In this particular system, minimal replacement (<7%) of bromouracil for thymine into finite nucleotide sequence elements was sufficient for substantial virogenic induction to occur.…”