Isolation of replicating DNA segments from Chinese hamster cells by density equilibrium centrifugation

When DNA is extracted from Chinese hamster cells grown in the presence of 5-bromodeoxyuridine from the beginning of the S (synthesis) phase until the middle of the first replication round, a significant fraction of total replicated DNA bands at intermediate densities between light-light and light-heavy DNA, in a CsCl gradient. Incomplete bromodeoxyuridine substitution compared with light-heavy DNA justifies the displaced banding of these molecules. Since “intermediate DNA” following alkaline or thermal denaturation gives rise to unsubstituted and fully substituted single strands, its particular density in neutral gradients cannot be ascribed to a uniformly reduced degree of bromodeoxyuridine substitution nor to covalently joined light and heavy strands. The segregation of DNA of intermediate densities into light-light and light-heavy components after shearing suggests that it includes at least one junction between replicated and still unreplicated segments, i. e. one replication fork that may or may not have lost one of its prongs. DNA of intermediate density specifically contains one to two sites sensitive to breakage by Neurospora crassa endonuclease.When a two-minute pulse of tritiated bromodeoxyuridine is given during replication in unlabelled heavy medium, the DNA fragments (mol. wt 35 × 10⁶) containing labelled segments band essentially at intermediate positions and are progressively converted to light-heavy molecules, with increasing duration of chase. The half-life of this pulse-labelled intermediate DNA (about 25 min) is consistent with the proportion of total replicated DNA found at displaced densities (10 to 15%) and, together with the distribution of the intermediate radioactivity, is compatible with the existence of adjacent growing replicons.If DNA is labelled and extracted during the second replication round in the presence of bromodeoxyuridine, “intermediate DNA” with similar properties is found between the light-heavy and heavy-heavy peaks.     

Messenger Ribonucleic Acid of Dormant Spores of Bacillus subtilis

Evidence of the presence of messenger ribonucleic acid (mRNA) in dormant spores of Bacillus subtilis has been obtained. The bulk RNA from spores was isolated and labeled in vitro with tritiated dimethyl sulfate. The spore RNA hybridized to 2.4 to 3.2% of the B. subtilis genome. The RNA hybridized to both the complementary heavy and light fractions of deoxyribonucleic acid (DNA). Bulk RNA from log-phase cells competed with virtually all the spore RNA for the heavy DNA fraction and with part of the spore RNA for the light DNA fraction. Bulk RNA from stage IV cells in sporulation also competed with all of the spore RNA for the heavy DNA fraction and with essentially all the spore RNA for the light DNA fraction. These results indicate that dormant spores contain mRNA species present in both log-phase cells and stage IV cells of sporulation. The RNA polymerase in the developing forespore must be able to recognize promotor sites for both log-phase and sporulation genes.     

Multiple Initiation of Bacteriophage T4 DNA Replication: Delaying Effect of Bromodeoxyuridine

Effects of bromodeoxyuridine (BUdR) substitutions in phage T4 DNA on the initial stages of DNA replication were investigated. Electron microscope studies of partially replicated, light (thymidine-containing) T4 DNA revealed the presence of multiple loops and forks. These DNA preparations had no BUdR in either parental or newly synthesized DNA, and the observations thus show that multiple initiation of DNA replication is a normal event in T4 development and is not caused by the presence of BUdR. A comparison of early replicative stages of light and heavy (BUdR-containing) DNA in cells mixedly infected with light and heavy T4 phage showed that early DNA synthesis occurs preferentially on the light template. Heavy and light parental DNA became associated with the protein complex of replicative DNA with equal efficiency, and there was no effect of BUdR on the net rate of DNA synthesis after infection. Newly synthesized DNA from heavy templates sedimented more slowly through alkaline sucrose gradients than did newly synthesized DNA from light templates and appeared to represent fewer replicative regions per molecule. These data indicate that BUdR substitutions in the DNA caused a slight delay in initiation but that replication of heavy DNA proceeded normally once initiated.     

Segregation of nucleosomes in replicated mouse alpha-globin gene

DNA of mouse erythroleukemia cells grown in vitro was labeled with bromodeoxyuridine during cycloheximide-inhibited protein synthesis. Isolated nuclei were digested with micrococcal nuclease to obtain monosomes and monosomal dsDNA. The protection of the heavy and of the light strands of the newly replicated DNA was studied by dot hybridization with the coding and with its complementary noncoding strand of the alpha-globin gene. The results show that both sides of the replication fork contain protected sequences of the gene, thus supporting a bilateral (dispersive) mode of nucleosome segregation during DNA replication.     

Buoyant density heterogeneity in spores of Bacillus subtilis: biochemical and physiological basis

The biochemical and physiological basis of density heterogeneity in Renografin of Bacillus subtilis W23 spores was determined by analysis of metals, macromolecules, and dipicolinic acid in the two density classes of the population. Germination rate and heat resistance were measured in both density classes. Atomic absorption spectrophotometry revealed that heavy spores (density = 1.335 g/ml) have 30% more calcium than light spores (density = 1.290 g/ml). Other metals found in greater amounts in heavy spores were manganese and potassium. However, light spores had more sodium than heavy spores. The amounts of carbohydrates, nucleic acids, and proteins were the same in both types of spores, but light spores contained more lipids, whereas heavy spores had 30% more dipicolinic acid than light spores. Calcium and lipid were excluded as causes of the heterogeneity in density in that alteration of their contents in spores did not detectably affect the density of these spores. Spores of two densities were genetically similar. Furthermore, light density spores arose earlier during sporulation than heavy spores as determined by releasing refractile forespores at various times during sporulation. We concluded that light spores represent an incomplete stage in development because they became heavy when reinoculated into spent sporulation medium. This must involve the additional accretion or synthesis of dipicolinic acid.     

The integration of donor and recipient deoxyribonucleic acid during transformation of Bacillus subtilis

1. DNA labelled with 5-bromo[(3)H]uracil was used to transform auxotrophic strains of Bacillus subtilis. 2. After various times of incubation, DNA was extracted from the transformed culture and subjected to equilibrium sedimentation in caesium chloride gradients. 3. In addition to heavy donor DNA and light recipient DNA, a component with an intermediate density was found and is believed to consist of a biological hybrid of donor and recipient. 4. The component of intermediate density was isolated and found to possess activity in transformation derived from both donor and recipient strains. 5. Denaturation of the component of intermediate density followed by centrifugation gave only one component, indicating that integration had occurred in both strands of the recipient DNA. 6. No integrated band was observed after uptake by competent cells of B. subtilis of heavy DNA prepared from Escherichia coli.     

Validation of a quantitative PCR diagnostic method for detection of the microsporidian Ovipleistophora ovariae in the cyprinid fish Notemigonus crysoleucas

Microsporidian parasites are easily detected by light microscopy when infections are heavy and spores are present. However, early infections without spores, or light infections with low numbers of spores, are easily missed. This limitation has made it difficult to conduct investigations into microsporidian prevalence and transmission. In this study, we developed a quantitative TaqMan polymerase chain reaction assay to assess the presence of Ovipleistophora ovariae in the tissues of the cyprinid fish Notemigonus crysoleucas (golden shiner). The efficiency of the primer set was 100.8%, with a correlation coefficient of threshold position to copy number of 0.997 over 9 logs using a plasmid containing the cloned reaction product. No product was produced from other closely related microsporidian species (Nucleospora salmonis, Pseudoloma neurophila, Glugea stephani, Heterosporis sp., and O. mirandella). The coefficient of variation for replicate assays done on different days was 12.4%. The assay detects O. ovariae reliably at less than 10 genomic copies and 0.14 spores per reaction, but maximum sensitivity is only achieved when sonication is included as part of the DNA purification step. Using the assay, we found 4.44 x 10(1) to 7.91 x 10(6) copies microg(-1) host DNA in female golden shiners, with the spore density increasing during the spawning season. The parasite was also detected for the first time in the testes of male golden shiners at 2.60 x 10(1) to 8.62 x 102 copies microg(-1) host DNA.     

Evidence for the formation of hybrid DNA during mitotic recombination in Chinese hamster cells

Direct evidence is provided for the formation of hybrid DNA during mitotic recombination in CHO cells. The cells were labeled for one round of replication in medium containing BUdR, so that the density of the DNA was heavy light (HL) and then returned to light medium. Further DNA synthesis, during either repair or chromosome replication, can only result in HL or fully light (LL) DNA; however, the formation of hybrid DNA as part of the process of recombinational repair will produce some fully heavy (HH) DNA.A small fraction of DNA containing regions of HH DNA has been detected on neutral CsCl gradients, and the amount of this DNA is increased by treatment of the cells with mitomycin C. Increasing doses of mitomycin C produce similar increases in both the amount of HH DNA and the frequency of sister chromatid exchanges measured cytologically. This correlation provides evidence that the HH DNA is hybrid DNA, formed as an intermediate in recombinational repair.     

Production of DNA bifilarly substituted with bromodeoxyuridine in the first round of synthesis: branch migration during isolation of cellular DNA.

Incubation of human lymphoid cells with bromodeoxyuridine (BrdUrd) for short periods produces three classes of DNA containing analog: DNAHL (hybrid DNA, density approximately equal to 1.75 g/cm3), DNAint (intermediate density DNA, density approximately equal to 1.71 g/cm3), and DNAHH (DNA with both strands containing analog, density approximately equal to 1.80 g/cm3). Preparations of DNAint yield DNAHH after extensive shearing and/or treatment with single strand specific endonuclease. Cross-linking of pulse-labeled (BrdUrd + 3HdT) DNA in cells by treatment with trioxsalen and near UV light before lysis prevents the appearance of DNAHH.Cross-linking after lysis has little effect. A large fraction of DNAHH is obtained after incubation of cells with caffeine. Extraction of DNA at high salt concentration or cross-linking with trioxsalen and near UV light drastically reduced the amount of DNAHH obtained from caffeine-treated cells. We conclude that most DNAHH arises from in vitro branch migration in isolated DNA growing points.     

Size maturation process of nascent DNA intermediates into chromosomal-sized DNA in Tetrahymena pyriformis macronuclear DNA replication

An analysis was made of the size maturation process of nascent DNA intermediates in macronuclear DNA replication of Tetrahymena pyriformis. The first discrete size class of nascent intermediates larger than Okazaki fragments were replicon-sized DNA (about 2 X 10(7) D single-stranded (ss) DNA) and accumulated in cells treated with cycloheximide. On removal of cycloheximide, the replicon-sized intermediates were converted to middle-sized intermediates (about 10 X 10(7) D ssDNA) and then merged into chromosomal-sized DNA. As indicated by either aphidicolin inhibition or the technique of the photolysis of bromodeoxyuridine (BrdU)-substituted DNA with long-wave ultraviolet light, four to eight replicon-sized intermediates were joined together to form a middle-sized intermediate after rapid sealing by DNA synthesis of the late-replicating regions located between adjacent replicon-sized intermediates. The late-replicating regions may represent the short gaps or terminal regions where DNA synthesis was retarded by cycloheximide, since the size of late-replicating regions was suggested to be shorter than the replicon size by DNA fiber autoradiography. Therefore, it is probable that four to eight completed replicons are joined as a group such as a replicon cluster, as has been reported in DNA replication of other eukaryotic cells.     

The degree of ultraviolet light damage to DNA containing iododeoxyuridine or bromodeoxyuridine is dependent on the DNA sequence.

The sequence selectivity of 300 nm ultraviolet light damage to DNA containing bromodeoxyuridine or iododeoxyuridine was examined on DNA sequencing gels. This was accomplished using a system where an M13 template was employed to direct synthesis of DNA in which thymidine was fully substituted with bromodeoxyuridine or iododeoxyuridine. The sites of damage corresponded to the positions of analogue incorporation. The extent of damage varied considerably at different sites of cleavage and ranged from the undetectable to over fifteen times the limit of detection (as assessed by laser densitometer scans). Strong damage sites had the "consensus" sequence CTT while sites of no detectable damage had the "consensus" sequence GTR. Bromodeoxyuridine and iododeoxyuridine had the same sites of damage although the extent of damage varied at different sites and bromodeoxyuridine damage was slightly greater than iododeoxyuridine. DNA containing thymidine was not damaged to any detectable level in this system with 300 nm ultraviolet light. The use of three closely related DNA sequences as targets for damage confirmed that (1) the sites of analogue incorporation are the cause of ultraviolet damage; and (2) that the neighbouring DNA sequence is an important parameter in determining the extent of damage. It is proposed that the microstructure of DNA--in particular the distance between the 5-carbon of the pyrimidine base (which is attached to the halogen) and hydrogen on the 2' carbon of the 5'-deoxyribose--ultimately determines the degree of cleavage with large distances giving a small degree of damage and smaller distances a large degree of damage.     

Action of FdUrD and dCyd on the incorporation of BrdUrd in chinese hamster somatic cell DNA and the isolation of auxotrophic mutants.

Chinese hamster somatic cells grown in the presence of bromodeoxyuridine, deoxycytidine and fluorodeoxyuridine incorporate more bromodeoxyuridine in the DNA than cells grown in the presence of bromodeoxyuridine alone. Thus they become more sensitive to light irradiation. Our data suggest that 0.05 mM--0.2 mM bromodeoxyuridine, 0.05 mM deoxyctidine and 10 mmug/ml fluorodeoxyuridine is one of the best possible combinations for the selection of Chinese hamster somatic cells mutants. Auxotrophs for proline, inositol or both were thus isolated at high frequency.     

Differences in nucleotide and base DNA excision repair observed during mitogenic stimulation of bovine lymphocytes.

The bromodeoxyuridine density-shift technique was used to examine nucleotide and base DNA excision repair in quiescent and lectin stimulated bovine lymphocytes damaged with either ultraviolet light or dimethyl sulfate (DMS). Compared to a number of human cell lines, quiescent lymphocytes were less proficient in the repair of both types of damage. Repair replication was enhanced upon mitogenic stimulation, but both the amount and time course of the increase in repair depended upon the damaging agent used. A 2-3-fold increase in UV light induced repair replication occurred early during stimulation and subsided only gradually as stimulation proceeded. However, the profile of DMS induced repair increased 7-fold and then decreased, in parallel with measurements of lectin-stimulated DNA replication. Estimates of average repair patch sizes showed that quiescent lymphocytes produced smaller patches of 7 nucleotides in response to DMS damage while UV light irradiation resulted in repair patches of 20 nucleotides. During stimulation, patch sizes appeared to increase to maximum values of 45 and 33 nucleotides in response to UV light and DMS, respectively, one day prior to the peak of DNA replication. These increases in patch size were followed by a gradual decrease towards unstimulated levels. However, the appearance of a DNA species of intermediate density in the gradient profiles made the interpretation of repair patch sizes in stimulated cells difficult. These results are discussed as evidence not only for differences in the mechanisms of nucleotide and base excision repair but also for changes in repair as the cell progresses through the cell cycle.     

Density Transfer Studies of DNA Isolated from BACILLUS SUBTILIS after Exposure to Phenethyl Alcohol

The aim of this study was to determine whether there are specific weak points in the Bacillus subtilis chromosome and if so whether the replication point is the site of breakage. To answer these questions, B. subtilis chromosomes were partially labeled with 5-bromodeoxyuridine (5-BUdR). Sheared or unsheared preparations of partially labeled chromosomes which may or may not contain replication forks were analyzed for the distribution of genetic markers in a CsCl density gradient. Two sets of experiments based upon the density transfer experiments of Yoshikawa and Sueoka (1963) were performed: (1) experiments in which the origin of the chromosome was labeled and (2) experiments in which the terminus of the chromosome was labeled. In the first experiment, strain 23 (thy(-), his(-)) spores were germinated in the presence of 5-BUdR for various lengths of time and then transferred to fresh medium containing phenethyl alcohol (PEA) and thymidine (TdR). The DNA was isolated before and after transfer to PEA and TdR. In the second experiment strain 23 (thy(-), his(-)) spores were germinated in the presence of TdR and then PEA was added. After various lengths of time transfer was made to fresh medium containing PEA and 5-BUdR. The DNA was extracted by an extremely gentle technique to avoid breakage and centrifuged in a CsCl density gradient. PEA was added to the germinated spores to prevent dichotomous replication, but PEA did not prevent dichotomous replication in any of these experiments. This contradicts the conclusion of others that PEA prevents the chromosome from entering a new round of replication, but allows the chromosome to complete the round of replication already begun. The following observations offer support for the hypothesis that the replication point is a weak point in the chromosome: (1) when conditions were created to obtain partially labeled chromosomes with replication points: (a) labeled markers appeared at the hybrid density, (b) unlabeled markers appeared at the light density, (c) shearing of the DNA had little effect on the CsCl density gradient, except on a small proportion of labeled markers which had not appeared at the hybrid density prior to shearing; (2) when conditions were created to obtain partially labeled chromosomes with no replication points: (a) the majority of DNA molecules appeared at an intermediate density between the hybrid and the light densities, (b) the labeled and unlabeled markers appeared in the intermediate peak with approximately the same ratio as in the DNA preparations, (c) the labeled markers were found in the intermediate peak except where dichotomous replication had occurred, (d) after shearing, the labeled markers appeared at the hybrid density and the unlabeled markers appeared at the light density. Thus it is concluded that the replication point is a weak point in the B. subtilis chromosome where breakage easily occurs.     

GROWTH REGULATION BY ETHYLENE IN FERN GAMETOPHYTES. V. ETHYLENE AND THE EARLY EVENTS OF SPORE GERMINATION

Early events during the germination of spores of the fern Onoclea sensibilis were studied to determine the time during germination when ethylene had its greatest inhibiting effect. Water imbibition by dry spores was rapid and did not appear to be inhibited by ethylene. During normal germination DNA synthesis occurred about four hours before the nucleus moved from a central position to the spore periphery. Following nuclear movement, mitosis and cell division occurred, partitioning the spore into a small rhizoid cell and a large protonemal cell. Cell division was complete approximately six hours after nuclear movement. Ethylene treatment of the spores blocked DNA synthesis, nuclear movement, and cell division. The earliest DNA replication in uninhibited spores was observed after 14 hours of germination, and the maximal rate of spore labeling with ³H-thymidine was between 16 and 20 hours. Spores were most sensitive to ethylene, however, during the stages of germination prior to DNA synthesis, and it was concluded that ethylene did not directly inhibit DNA replication but blocked germination at some earlier fundamental step. The effects of ethylene were reversible. since complete recovery from inhibition of germination was possible if ethylene was released and the spores were kept in light. Recovery was much slower in darkness. It was hypothesized that light acted photosynthetically to overcome the ethylene inhibition of germination. Consistent with this, it was shown that spores exhibit net photosynthesis after only two hours of germination.     

Bromodeoxyuridine combined with UV light and gamma irradiation promotes the production of asymmetric somatic hybrid calli

Summary The degree of gamma- or X-ray-induced donor chromosome elimination in asymmetric somatic hybrids is highly variable. Here the beneficial use of bromodeoxyuridine and UV light as additional chromosome destabilizing agents is described. Protoplasts of Nicotiana tabacum were fused with protoplasts of Nicotiana plumbaginifolia (Np) that carried the kanamycin-resistance and glucuronidase (GUS) genes on separate chromosomes. Prior to fusion, the Np donor protoplasts were pretreated with bromodeoxyuridine and then were inactivated by treatment with iodoacetate ± UV light ± 200 Gy gamma irradiation. Hybrids were selected on medium containing kanamycin. The elimination of Np DNA was assessed by scoring of the fraction of hybrid calli that expressed GUS and by dot-blot analysis using a Np-specific probe. Gamma irradiation alone resulted in elimination of 50% of Np DNA. Pretreatment with bromodeoxyuridine (10 M) followed by 2.5 to 5 min UV light resulted in the elimination of 35–45% of the donor genome, but incorporation of bromodeoxyuridine (10 M) followed by 2.5 to 5 min UV light and 200 Gy gamma irradiation resulted in 85 to 90% elimination of Np DNA.Abbreviations BrdU bromodeoxyuridine (5-bromo-2-deoxyuridine) - GUS glucuronidase - HPT hygromycin B phosphotransferase - KmR kanamycin resistant - Np Nicotiana plumbaginifolia - NPT neomycin phosphotransferase II - Nt Nicotiana tabacum - UV ultraviolet     

Influence of cellular differentiation on ultraviolet induced DNA damage and its repair mechanisms in B. cereus

Susceptibility to UV irradiation of B. cereus BIS-59 spores undergoing germination at various stages-dormant spores to vegetative cell stage and their ability to recover from radiation damage were studied. For a given dose of radiation, the number of spore photoproducts (SPP) formed in the DNA of dormant spores was about 5-times greater than that of thymine dimers (TT) formed in the DNA of vegetative cells. At intermediate stages of the germination cycle, there was a rapid decline in the UV radiation-induced SPP formed in DNA with a concomitant increase in the UV radiation-induced TT formed in DNA. Bacterial spores undergoing germination (up to 3 hr) in the low nutrient medium (0.3% yeast extract) displayed much higher resistance to UV radiation than those germinating in the rich nutrient medium, even though there was no discernible difference under the two incubation conditions in respect of the extent of germination and the time at which the outgrowth stage appeared (3 hr). This was due to the formation TT in the DNA of spores germinating in the low nutrient as compared to that of spores germinating in the rich-nutrient medium. In UV-irradiated dormant spores, SPP formed in the spore DNA did not disappear even after prolonged incubation in the non-germinating medium. However, when the UV-irradiated dormant spores were germinated in low or rich nutrient medium, a significant proportion of SPP in DNA was eliminated. The dormant spores incubated in either of the germinating media for 15 min and then UV-irradiated were capable of eliminating SPP (presumably by monomerization) even by incubation in a non-germinating medium and in the complete absence of protein synthesis (buffer holding recovery), thereby implying that spore-repair enzymes were activated in response to initial's germination. The acquisition of photo-reactivation ability appeared in spores subjected to germination only in the rich-nutrient medium at the outgrowth stage and required de novo synthesis of the required enzymes.     

Inhibition and recovery of the rate of DNA synthesis in V79 Chinese hamster cells following ultraviolet light irradiation. Variation in the rate of movement of the replication fork

Chinese hamster fibroblasts (V79 cell line) exhibit the phenomenon of recovery of DNA synthesis from the initial inhibition observed after ultraviolet light irradiation, in the absence of significant excision of pyrimidine dimers. In an attempt to determine whether the initial inhibition and subsequent recovery can be accounted for by parallel variations in the rate of movement of the replication fork, the cells were pulse-labeled with radioactive bromodeoxyuridine at different times following irradiation and their DNA centrifuged in neutral CsCl density gradients. When DNA synthesis inhibition was at a maximum, an accumulation of DNA, of density intermediate between hybrid and nonsubstituted DNA, was noticed in the density-distribution profiles. This intermediate-density DNA has been previously shown to correspond to fork structures, and thus it seems that inhibition of DNA synthesis after irradiation is to a great extent caused by the forks pausing at the lesions. Later on, when recovery in the rate of DNA synthesis occurs, the accumulation of intermediate-density DNA is no longer observed. The density distribution of DNA along the gradient can thus provide an estimate of the rate of movement of the replication fork, and the results indicate that most of the variation in the overall rate of DNA synthesis can be accounted for by a parallel variation in the rate of fork movement.     

Effects of mutant small, acid-soluble spore proteins from Bacillus subtilis on DNA in vivo and in vitro.

alpha/beta-type small, acid-soluble spore proteins (SASP) of Bacillus subtilis bind to DNA and alter its conformation, topology, and photochemistry, and thereby spore resistance to UV light. Three mutations have been introduced into the B. subtilis sspC gene, which codes for the alpha/beta-type wild-type SASP, SspCwt. One mutation (SspCTyr) was a conservative change, as residue 29 (Leu) was changed to Tyr, an amino acid found at this position in other alpha/beta-type SASP. The other mutations changed residues conserved in all alpha/beta-type SASP. In one (SspCAla), residue 52 (Gly) was changed to Ala; in the second (SspCGln), residue 57 (Lys) was changed to Gln. The effects of the wild-type and mutant SspC on DNA properties were examined in vivo in B. subtilis spores and Escherichia coli as well as in vitro with use of purified protein. Both SspCwt and SspCTyr interacted similarly with DNA in vivo and in vitro, restoring much UV resistance to spores lacking major alpha/beta-type SASP, causing a large increase in plasmid negative supercoiling, and altering DNA UV photochemistry from cell type to spore type. In contrast, SspCAla had no detectable effect on DNA properties in vivo or in vitro, while SspCGln had effects intermediate between those of SspCAla and SspCwt. Strikingly, neither SspCAla nor SspCGln bound well to DNA in vitro. These results confirm the importance of the conserved primary sequence of alpha/beta-type SASP in the ability of these proteins to bind to spore DNA and cause spore UV resistance.     

5-Methylcytosine content of rat hepatoma DNA substituted with bromodeoxyuridine.

The aim of these experiments was to test whether incorporation of bromodeoxyuridine into DNA affects DNA methylation. Rat hepatoma (HTC) cells in culture were labeled for two generations with [14C]bromodeoxyuridine and [3H]thymidine to yield DNA which was 2.1, 20.6, 52.6, and 95.0% bromodeoxyuridine-substituted in the newly made strands. The DNA then was fractionated into highly repetitive, moderately repetitive, and single copy sequences. As determined by a comparison of 14C and 3H counts per min, the percentage of substitution with bromodeoxyuridine was found to be the same in each repetition class. The 5-methylcytosine content of each fraction was determined using high pressure liquid chromatography. It was found that bromodeoxyuridine, even at a level of substitution into newly mad DNA of 95%, has no effect on the 5-methylcytosine content of DNA. At all levels of bromodeoxyuridine substitution, highly repetitive DNA has slightly more 5-methylcytosine (3.0% of total cytosine) than does single copy DNA or moderately repetitive DNA (2.3%). The 5-methylcytosine content of whole HTC DNA is the same as that of rat liver DNA (2.4%).