Mapping eukaryotic replication origins in vivo by size analysis of purified nascent DNA strands.

A simple and efficient method for the mapping of eukaryotic replication origins was tested. The method is based on differential labeling of newly synthesized DNA with BrdUrd and subsequent separation of heavy nascent strands from parental DNA by conventional alkaline sucrose and neutral CsCl isopycnic gradient centrifugation. Purified nascent DNA is then size-fractionated on alkaline agarose gels and analyzed by sequential hybridization to specific probes of known location on the DNA segment of interest. Evaluation of the hybridization results allows: (i) determination of the direction of replication fork movement and (ii) location of the initiation site of DNA synthesis. Taking SV40 and polyoma virus as model systems, we demonstrate the feasibility of this procedure. It applicability to the location of chromosomal replication origins is discussed.     

Effect of 5-fluoro-2'-deoxyuridine (FdUrd) on 5-bromo-2'-deoxyuridine (BrdUrd) incorporation into DNA measured with a monoclonal BrdUrd antibody and by the BrdUrd/Hoechst quenching effect

The purpose of this study was to improve the application of bromodeoxyuridine (BrdUrd) for the flow cytometric analysis of cell kinetics. In order to obtain a quantitative measure of the DNA synthesis rate (or the number of divided cells), BrdUrd should replace thymidine (dThd) completely in the newly synthesized DNA strands. The de novo synthesis of dThd monophosphate competing with BrdUrd incorporation was stopped by fluorodeoxyuridine (FdUrd). Cells of a human leukemic cell line (REH) were exposed to BrdUrd for either 20 min, 8 h, or 24 h. Bromodeoxyuridine incorporation was determined by a monoclonal antibody as well as by the BrdUrd/Hoechst (H) technique. Counterstaining of the DNA was performed with propidium iodide or ethidium bromide. DNA fluorescence was measured in both techniques with a two-parameter flow cytometer, the histograms being analyzed by computer. It was found that FdUrd is required in the BrdUrd/H technique for replacement of dThd at low BrdUrd concentrations and long incubation times. With short incubation periods, as used for detection by the monoclonal anti-BrdUrd antibody, FdUrd increases the incorporated BrdUrd amount when BrdUrd concentrations of 10 microM or less are applied.     

Mechanism of conjugation and recombination in bacteria. XVII. Further evidence for single-stranded regions in recipient DNA during conjugation in Escherichia coli K-12.

The secondary structure of recipient DNA mated with Hfr strain was investigated by CsCl density gradient fractionation. After 45 min of HfrH64 X 3h-f-ab1157 mating one-fourth of the radioactive recipient DNA was recovered as a single-strand but only after shearing of cell lysates prior to centrifugation. This heavier than native DNA fraction of radioactive material (obtained after the first centrifugation) was degraded by single-strand specific nuclease S from Aspergillus oryzae. These findings thus confirm the authors' earlier results suggesting that in the course of mating are generated local single-stranded regions in recipient DNA.     

Autoradiographic studies of the replication of satellite DNA in the kangaroo rat. Autoradiographs of satellite DNA

Cells of the Kangaroo rat Dipidomys ordii contain large quantities of satellite DNA (Bostock et al., 1972). Radioactive DNA from such cells has been examined by autoradiography. After a long period of radioactive labeling, long tandem arrays of short radioactive pieces are suddenly observed which are attributed to the replication of satellite DNA. Autoradiograms of DNA isolated by CsCl density centrifugation from such lysates are in agreement with this hypothesis.This material is non-randomly distributed in lysates of single cells. The length of these pieces and their spacings are not remarkably different from DNA made earlier (presumably non-satellite DNA).     

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.     

DNA synthesis in L-cells in the presence of 5-fluorodeoxyuridine.

After 16 h of incubation with 10-minus6 M FdUrd, the rate of (32P) orthophosphate uptake into DNA isolated from L-cells amounted to 15% of that of an untreated culture, although cell division had stopped several hours earlier. All 4 deoxynucleotides were present in this DNA but its nucleotide composition, as measured by enzymatic digestion and chromatography, reflected a decreased thymidine precursor pool in FdUrd-treated cells. Sedimentation analysis in alkaline sucrose gradients revealed that the DNA formed in the presence of FdUrd had a sedimentation coefficient of 10 S which corresponded to a single-stranded molecular weight of 5.5.105. This DNA could be "chased" into a high molecular weight DNA if the FdUrd block was bypassed with added dThd or BrdUrd. Other analyses failed to detect RNA covalently linked to the DNA fragments at a level of more than 5% RNA or about 90 ribonucleotides. The accumulation of these DNA fragments could be explained by assuming that in the presence of limiting precursor pool the rate of DNA chain initiation is greater than the rate of chain elongation.     

Isolation and characterization of satellite DNA from mustard seedlings

The DNA from mustard (Sinapis alba L.) seedlings was examined by neutral CsCl and Ag⁺/Cs₂SO₄ density gradient centrifugation. Different satellite fractions were revealed by these two methods. The satellite fractions obtained from the Ag⁺/Cs₂SO₄ density gradient could not be generally correlated with satellite DNA fractions observed in CsCl. In CsCl density gradient centrifugation, a main band at density 1,695 g/cm³ and a heavy shoulder at density 1,703 g/cm³ are found. By preparative CsCl gradient centrifugation the heavy shoulder can be enriched but not completely separated from the main band DNA.—Gradient centrifugation by complexing the DNA with Ag⁺ rf. 0.25 to DNA phosphate reveals three distinct fractions which are further characterized: The heavy satelite DNA fraction revealed by Ag⁺/Cs₂SO₄ gradient centrifugation has the same density in a CsCl gradient and the same Tm value as the main band, but differs from main band DNA in the details of its melting profile and in its renaturation kinetics. The light Ag⁺/Cs₂SO₄ satellite DNA fraction had a higher melting temperature corresponding to a GC-rich base composition. Differences between these 3 fractions are observed in thermal denaturation and renaturation profiles, hybridization in situ with ribosomal RNA, and their response to restriction endonuclease digestion. The light satellite fraction from the Ag⁺/Cs₂SO₄ gradient, rich in ribosomal cistrons corresponds to the heavy shoulder DNA of neutral CsCl gradients which also is rich in ribosomal cistrons. The heavy satellite fraction from Ag⁺/Cs₂SO₄ gradient which contains highly repetitive short nucleotide sequences could not be revealed by the classical CsCl gradient centrifugation technique.     

DNA synthesis in cytokinin-autotrophic tobacco cells

DNA isolated from various Nicotiana tabacum cell types, differing in their degree of hormone autotrophy and incubated in the presence of bromodeoxyuridine (BrdUrd), was analyzed by isopycnic CsCl gradient centrifugation. All cell types incorporate BrdUrd into DNA in such a way that hybrid DNA is formed with 60–80% of thymine (Thy) residues replaced by bromouracil (BrUra) in the newly synthesized strand. This DNA is not replicated further under ordinary culture conditions. Whereas in normal hormone-dependent cells this state is final and cells necrotize, in tumor (cytokinin-auxin autotrophic) and cytokinin-autotrophic cells a mechanism is induced leading to the reduction of BrUra content in DNA. As a result a decrease in the buoyant density (in CsCl) of BrUra DNA can be observed. In the case of cytokinin-autotrophic cells supplemented with kinetin, the buoyant density of the whole DNA decreases gradually to the value of that of unsubstituted DNA, but specific radioactivities of different DNA fractions reflect the retention of the pyrimidine ring of BrUra in DNA. This is interpreted as debromination of DNA in situ. The process can be inhibited by fluorodeoxyuridine (FdUrd) and deoxycytidine (dCyd). Moreover, FdUrd (but not dCyd) allows replication of hybrid DNA in tumor cells in such a way that HH DNA with all Thy residues replaced by BrUra is formed. For cytokinin-autotrophic cells FdUrd and kinetin are required. In hormone-dependent cells replication of hybrid DNA cannot be induced under any conditions. Most of these conclusions complement our previous findings that BrdUrd tolerance in hormone-autotrophic tobacco cells in hormone controlled. It is postulated that a modulation of thymidylate synthetase specificity is one factor affecting the level of BrUra substitution in DNA. The possibility of cytokinins being involved in the control of DNA synthesis is discussed.Abbreviations BrdUrd 5-bromo-2-deoxyuridine - BrUra 5-bromouracil - dCyd 2-deoxycytidine - FdUrd 5-fluoro-2-deoxyuridine - dThd thymidine - Thy thymine - EDTA Na₂-ethylenedia-minotetraacetate - IAA idole-3-acetic acid (auxin) - SDS Na-dodecylsulphate - LL, HL, HH DNA light-light (unsubstituted), heavy-light (unifilarly BrUra substituted), heavy-heavy (bifilarly BrUra substituted) DNAs, respectively     

Hepatitis B core antigen. Detection of antibody by radioimmunoprecipitation.

Radioactive cores were prepared from concentrated Dane particles by DNA polymerase reaction followed by CsCl density gradient centrifugation. Two radioactive peaks were obtained: one peak with an average density of 1.36 g/cm-3 in CsCl contained cores that possessed full serologic reactivity; the other peak, with a density of 1.28 to 1.32 g/cm-3, contained cores associated with globulin. A double antibody immunoprecipitation test was developed, using the radioactive heavy cores as a source of antigen. The test was at least 300 times as sensitive as complement fixation for detecting antibody to core.     

DNA fork displacement rates in synchronous aneuploid and diploid mammalian cells.

DNA fork displacement rates were measured in Chinese hamster ovary cells (CHO), human HeLa cells and human diploid fibroblasts. For CHO cells two independent techniques were used: one based on CsCl equilibrium density gradients and the other on 313 nm photolysis of incorporated bromodeoxyuridine (BrdUrd). Both methods indicated that there was no significant variation in fork displacement rates in CHO cells as they progressed through S phase. Asynchronous CHO cultures displayed the same average value (1.0 micron/min) and range of values as found in synchronous cells. In contrast, the rate of DNA fork displacement in HeLa cells, measured by the BrdUrd-313 nm method, increased continuously from 0.8 micron/min in early S to 2.5 micron/min in late S. For human diploid fibroblasts, in early S, the rate was approximately 0.7 micron/min and decreased to a minimum of 0.5 micron/min in mid S. The replication fork displacement rate then increased to a maximum of 0.9 micron/min in late S and declined again before the end of S phase. This pattern of DNA fork displacement rates roughly paralleled the overall thymidine incorporation rate and appears quite different from the patterns found for HeLa and CHO cells.     

Structure of the replication fork in ultraviolet light-irradiated human cells.

The DNA extracted from xeroderma pigmentosum human fibroblasts previously irradiated with 12.5 J/m2 of UV light and pulse-labeled for 45 min with radioactive and (or) heavy precursors, was used to determine the structural characteristics of the replication fork. Density equilibrium centrifugation experiments showed that a fork moved 6 micrometer in 45 min and bypassed 3 pyrimidine dimers in both strands. The same length was covered in 15-20 min in control cells. The delay in irradiated cells was apparently due to pyrimidine dimers acting as temporary blocks to the fork movement. Evidence for this interpretation comes from kinetics of incorporation of [3H]thymidine into DNA, which show that the time necessary to attain a new stable level of DNA synthesis in irradiated cells is equivalent to that required for the replication fork to cover the interdimer distance in one strand. On the other hand, the action of S1 nuclease on DNA synthesized soon after irradiation gives rise to a bimodal distribution in neutral sucrose gradients, one peak corresponding to 43 X 10(6) daltons and the other to 3 X 10(6) daltons. These two DNA species are generated by the attack of the S1 nuclease on single-stranded regions associated with the replication fork. A possible explanation for these results is given by a model according to which there is a delayed bypass of the dimer in the leading strand and the appearance of gaps opposite pyrimidine dimers in the lagging strand, as a direct consequence of the discontinuous mode of DNA replication. In terms of the model, the DNA of 43 X 10(6) daltons corresponds to the leading strand, linked to the unreplicated branch of the forks, whereas the piece of 3 X 10(6) daltons is the intergap DNA coming from the lagging strand. Pulse and chase experiments reveal that the low molecular weight DNA grows in a pattern that suggests that more than one gap may be formed per replication fork.     

Reversal of 5-fluorodeoxyuridine-caused growth inhibition in intact and excised etiolated hypocotyls of Sinapis alba L. by thymidine

Summary The elongation growth in etiolated, intact seedlings and excised hypocotyl segments of Sinapis alba is inhibited by FdUrd in the same fashion, and in either case there is a direct correlation between FdUrd concentration and inhibition of elongation growth. Removal of the roots reduced elongation; however, the percentage inhibition by FdUrd remained the same. Therefore, the growth inhibition by FdUrd is not a consequence of root growth inhibition.The growth inhibition in excised hypocotyls cultured on synthetic media is inversely proportional to the size of the segments, and of the seedlings from which they are taken. Elongation of the smaller segments is more sensitive to FdUrd than that of the larger ones. Anatomical observations showed that the inhibition of growth elongation by FdUrd in the hypocotyl segments is due to inhibition of cell elongation, and not of cell division. Root formation is inhibited in all isolated segments.The growth inhibition by FdUrd could be reversed by dThd but not by uridine, and this reversibility depended upon the FdUrd concentration. When FdUrd inhibition is partial (up to 10^-7M) relatively high dThd concentration (up to 100 fold) are required for complete reversal; when inhibition is maximal relatively lower dThd concentrations effect a complete or near-complete reversal. Irreversible, unspecific effects of FdUrd were not found.These experiments confirm that DNA synthesis is involved in cell elongation of the hypocotyls even when the apical meristem and roots are removed, and that the growth inhibition by FdUrd is not a nonspecific, toxic effect.Abbreviations FdUrd 5-fluorodeoxyuridine - dThd thymidine     

MINOR COMPONENTS OF THE DNA OF PHYSARUM POLYCEPHALUM : Cellular Location and Metabolism

DNA metabolism in the slime mold Physarum polycephalum was studied by centrifugation in CsCl of lysates of cultures labeled with radioactive thymidine at various times in the cell cycle. During the G₂ (premitotic) phase of the cell cycle, two components of the DNA are labeled. One component is lighter (buoyant density 1.686 g/cc) than the mean of the principal DNA (1.700 g/cc), and one is heavier (approximately 1.706 g/cc). The labeled light DNA was identified chemically by its denaturability, its susceptibility to DNase, and the recovery of its radioactivity in thymine. Cell fractionation studies showed that the heavy and the principal DNA components are located in the nucleus and that the light DNA is in the cytoplasm. The light DNA comprises approximately 10% of the DNA. About ⅓–½ of the light DNA is synthesized during the S period, and the remainder is synthesized throughout G₂ (there is no G₁ in Physarum). The light DNA is metabolically stable. A low, variable level of incorporation of radioactive thymidine into the principal, nuclear DNA component was observed during G₂.     

DNA synthesis rate and unlabeled cells with S-phase DNA content in P388 leukemic cells in vivo studied by flow cytometry and cell sorting

DNA synthesis kinetics of P388 leukemic cells growing in ascites form in BDF1 hybrid mice were investigated during the periods of exponential growth and growth restriction. Incorporation of tritiated thymidine, and in some instances tritiated uridine, was studied by autoradiography in cells sorted from S-phase fractions during DNA flow cytometry. During exponential growth continuous labeling with tritiated thymidine indicated a growth fraction of unity, whereas the growth fraction was about 30% during growth restriction. At this growth phase the majority of cells with S phase DNA content remained unlabeled after pulse labeling with tritiated thymidine or uridine, indicating that both the "salvage" and the "de novo" DNA synthesis pathways were blocked in most S-phase cells. After pulse labeling with tritiated thymidine the DNA synthesis rate pattern was investigated by sorting of consecutive fractions of cells throughout the S phase followed by quantitative autoradiography. With exception of a reduced rate in the middle of S phase, the DNA synthesis rate increased as the cells progressed through S phase during exponential growth. In contrast, the DNA synthesis rate pattern had a relative peak in the middle of S phase during growth restriction, which is otherwise characterized by a low mean DNA synthesis rate.     

Flow cytometric estimation of cell cycle parameters using a monoclonal antibody to bromodeoxyuridine

An estimation of cell kinetic parameters was made by simultaneous flow cytometric measurements of DNA and bromodeoxyuridine (BrdUrd) contents of cells. The procedure described in this paper involves the incorporation of BrdUrd by S phase cells, labeling the BrdUrd with an indirect immunofluorescent technique using a monoclonal anti-BrdUrd antibody, and staining DNA with propidium iodide (PI). The amount of incorporated BrdUrd in HeLa cells was proportional to that of synthesized DNA through S phase. For all cell lines examined, the pattern of BrdUrd incorporation was essentially the same and the rate of DNA synthesis during S phase was not constant. The bivariate BrdUrd/DNA distributions showed a horse-shoe pattern, maximum in the mid S phase and minimum in the early and late S phases. Furthermore, the durations of cell cycle (Tc) and S phase (Ts) were estimated from a FLSm (fraction of labeled cells in mid S phase) curve that was generated by plotting the percentage of BrdUrd pulse-labeled cells in a narrow window defined in the mid S phase of the DNA histogram. The values of these parameters in NIH 3T3, HeLa S3, and HL-60 cells were in good accordance with the reported data. This FCM method using the monoclonal anti-BrdUrd antibody allows rapid determination of both cell cycle compartments and also Ts and Tc without the use of radioactive DNA precursors.     

Approximation of baseline and BrdU-induced SCE frequencies

In the present paper we have developed a new rationale and an experimental schedule to approximate the frequency of SCEs which occur independently of BrdU incorporation, namely, the baseline frequency of SCEs. The method used includes the analysis of SCE yields in second and third division chromosomes after BrdU-substitution for 1, 2, and/or 3 successive replication rounds in the presence of this thymidine analogue, leading to a set of ten different experimental results. As a result of formulating various mathematical equations and applying them to the data, an accurate estimation of the frequency of baseline (BrdU-independent) and BrdU-induced SCEs, can be made, thus avoiding the difficulties inherent in the current extrapolation methods. The conclusions are that 1) SCEs seem to be formed after DNA synthesis (by exchanging post-replicative DNA portions), but, obviously, very near to the replication fork and 2) that under our experimental conditions about 0.065 SCEs per picogram of DNA per cell cycle occur as a consequence of chromosome replication, this frequency being increased by BrdU-substitution. The methodology seems to be reliable enough to be used in other species and systems in order to compare baseline SCE frequencies.Abbreviations SCEs sister-chromatid exchanges - BrdU(BrdUrd) 5-bromodeoxyuridine - dTh(dThd) thymidine - ³H-dTh(³H-dThd) tritiated thymidine - FdU(FdUrd) 5-fluorodeoxyuridine - Urd uridine - FPG fluorescent plus Giemsa     

Effect of exogenous thymidine on sister-chromatid exchange frequency in Chinese hamster ovary cells with bromodeoxyuridine- and chlorodeoxyuridine-substituted chromosomes

There are conflicting reports on the effect of exogenous thymidine (dThd) on the frequency of sister-chromatid exchanges (SCEs) in Chinese hamster ovary (CHO) cells. Thymidine has been reported either to increase or to have no effect on SCE frequency under similar experimental conditions. To resolve this controversy, we have carried out a series of experiments to examine the effect of dThd on CHO cells cultured with 5-bromodeoxyuridine (BrdUrd). In addition, we have examined the effect of dThd on CHO cells cultured with 5-chlorodeoxyuridine (CldUrd), a much more potent inducer of SCEs than BrdUrd. The addition of 100 microM dThd to the culture medium caused a consistent decrease in the yield of SCEs in cells grown in BrdUrd for two cell cycles. The decrease was even greater when cells were grown in dThd and CldUrd. Analysis of twin and single SCEs indicated that dThd must be present during the first cell cycle to reduce the frequency of SCEs. Because excess dThd is thought to have an effect when DNA replicates on a template substituted with a halogenated nucleoside, dThd at concentrations from 100 microM to 9 mM was added to cultures for the second cell cycle after a first cell cycle in BrdUrd. In this experiment, the presence of dThd increased SCE frequency in a dose-dependent manner. The results suggest that if dThd competes with halogenated nucleosides and thus decreases their incorporation into DNA, SCEs are suppressed in the subsequent cell cycle, whereas if excess dThd creates a dNTP pool imbalance, SCEs can be increased.     

Heterogeneity in the replicating population of cultured human epidermal keratinocytes

We studied the replication of keratinocytes in stratified squamous epithelia. Other studies have revealed functional and morphological heterogeneity in the replicating population of such cells. To examine possible kinetic heterogeneity, we determined the cell-cycle lengths of replicating cells in cultures of human epidermal keratinocytes. A double-label assay was developed, which measures the time between two successive cycles of DNA synthesis. The first cycle of DNA synthesis was marked by pulse labeling cultures for a brief period with 14C-thymidine (dThd), and the second cycle was detected by labeling at a later time with bromodeoxyuridine (BrdUrd). The time taken for the 14C-labeled DNA to become doubly labeled with BrdUrd was shown to correspond to the length of the cell cycle. In subconfluent cultures in which the cell number increased at an exponential rate, the average cell-cycle time was 21.5 h. In confluent cultures in which desquamation was balanced by cell renewal, the average cell cycle was 31.5 h. However, in confluent cultures, three populations of replicating cells were evident, these having cycle times of 22, 33, and 40 h. In subconfluent cultures, there was no clear evidence for cell-cycle heterogeneity of the replicating cells, although the most rapidly cycling cells in these cultures had a cycle time (16 h) considerably less than the most rapidly cycling cells in the confluent cultures (21 h). It is possible that the rapidly cycling cells seen in the subconfluent cultures were stem cells.(ABSTRACT TRUNCATED AT 250 WORDS)     

Analysis of the genome of plants. II. Characterization of repetitive DNA in barley (Hordeum vulgare) and wheat (Triticum aestivum).

Barley and wheat DNAs have been characterized by studying their kinetics of reassociation, melting properties and sedimentation behaviour in neutral CsCl gradients as well as in Cs2SO4 gradients containing Ag+ or Hg2+. In both species, reassociation kinetics have revealed the presence of approx. 76% redundant nucleotide sequences which have been grouped into very rapidly reassociating (Cot 0-0.01), rapidly reassociating (Cot 0.01-1.0) and slowly reassociating (Cot 1-100) fractions. The barley Cot 0-0.01 and Cot 0.01-1.0 fractions as well as the wheat Cot 0.01-1.0 fraction form narrow bands upon centrifugation in CsCl gradients. Under similar experimental conditions both Cot 0.01 and Cot 1.0-100 wheat fractions and the barley Cot 1.0-100 fraction form broad bands each having several shoulders. Thermal denaturation studies of most of the above reassociated fractions have shown a considerable degree of order in their duplexes with an average hyperchromicity of 21.5%. When native, high molecular weight barley DNA is centrifuged in Ag+/CS2SO4 density gradients (RF = 0.2), two satellites appear on the heavier side of the main band, as against one in the case of wheat. The two minor peaks, designated as satellites I and II, have buoyant densities of 1.702 and 1.698 g/cm3, respectively, in neutral CsCl gradients and together represent about 8-9% of total barley DNA. Upon centrifugation in Hg2+/CS2SO4 density gradients, one satellite is observed in both barley and wheat and it accounts for 1-2% of their genomes.