OBSERVATIONS ON THE UPTAKE OF TRITIATED THYMIDINE IN THE PRONUCLEI OF FERTILIZED SAND DOLLAR EMBRYOS

Following fertilization of the egg of the sand dollar Echinarachnius parma, tritiated thymidine (H³TDR) was taken up independently by the male and female pronuclei beginning within about 15 to 20 minutes, and the labeled pronuclei fused at about 30 to 40 minutes. At cleavage 90 minutes later the labeled nuclear material was distributed to both daughter cells. Unfertilized eggs and sperm exposed to H³TDR did not show nuclear localization of thymidine. DNA replication, thus, is initiated in the haploid pronuclei shortly after fertilization and prior to fusion. The major portion of DNA synthesis, as evidenced by thymidine uptake, appears to be during a 20 to 30 minute period after fertilization. Fertilization is associated with the activation of a mechanism which initiates early and independent replication of DNA in both the male and female pronuclei.     

Simian virus 40 DNA replication in nuclear monolayers.

Simian virus 40 DNA replication has been studied in nuclear monolayers prepared by treatment of monolayers of BSC-1 monkey kidney cells with Nonidet P-40. These nuclear monolayers incorporated [3H]TTP into two types of viral replicative intermediates that sediment as 25-26S and 22-23S species, respectively, in neutral sucrose gradients. The 22-23S species behaves, in dye buoyant density equilibrium gradients, as a late replicative intermediate. Examination of both species in alkaline sucrose gradients revealed the presence of two types of newly synthesized strands: (i) 4-7S strands and (ii) full-length, or nearly full-length, 10-16S strands. At low TTP concentrations (less than 0.5 muM), the two size classes were found in approximately equal amounts. However, at 10 to 50 muM TTP, the proportion of the longer strands increased, with a corresponding decrease in the relative amount of the 4-7S species. Thus, the joining of small, Okazaki-like fragments to the growing chain appears to require a much higher concentration of TTP than the synthesis of the fragments themselves. Replicating simian virus 40 DNA synthesized in the nuclear monolayers is is associated with "M bands", as previously demonstrated for replicating simian virus 40 DNA in cultured whole cells.     

Replication of ColE1 plasmid deoxyribonucleic acid in a thermosensitive dnaA mutant of Escherichia coli.

The replication of ColE1 deoxyribonucleic acid (DNA) took place at the restrictive temperature in a dnaA mutant, dnaA167(Ts). It proceeded at a constant rate at 42 degrees C for at least 3 h. The replication was insensitive to rifampin, which blocked replication at the permissive temperature or in the presence of chloramphenicol, even at the restrictive temperature. A linear DNA strand of ColE1 longer than unit genome size was synthesized. The structure of the replicating molecules observed by electron microscopy was mostly sigma shaped, composed of a circle of a unit genome length with a double-stranded tail. These observations suggest that the replication of ColE1 DNA proceeds via a rolling-circle type of structure in the absence of dnaA function.     

Synthesis and turnover of Euglena gracilis mitochondrial DNA

Replication of mitochondrial DNA was investigated by a density transfer experiment in a strain of Euglena gracilis lacking chloroplast DNA. DNA was uniformly labeled in a medium containing ³²P-labeled inorganic phosphate and [³H]adenine in the presence of the heavy-density label and transferred to a medium containing ³²P-labeled inorganic phosphate but no [³H]adenine following removal of the heavy-density label. Replication of nuclear DNA within these cells was used as an internal control. The densities and ratios of the peaks of nuclear DNA were those expected for a strict semiconservative replication. In contrast, replication of mitochondrial DNA was dispersive, as illustrated by the following results: (1) both native and denatured mitochondrial DNA exhibited a single density peak at 1.1 and 2.2 cell doublings after the density transfer. (2) The specific activity of ³H-labeled DNA varied across the peak of native or denatured DNA, indicating a heterogeneous population of molecules exhibiting different degrees of density and radioisotope labeling. This dispersive replication could involve either multiple recombination events or extensive turnover of the DNA or a mixture of both. Extensive dispersion of the sample obtained at 1.1 cell doublings after the density transfer is shown by the persistence of the same peak density for duplex DNA reduced to a molecular weight of 6 × 10⁵ by shearing.Two measures of the rate of replication of mitochondrial DNA were obtained from the densities of native duplex DNA and the rate of decrease in ³H-specific activities of duplex DNA during the experiment. The average of these rates indicates that mitochondrial DNA replicates at least 1.5 times as fast as nuclear DNA. Since there is a constant ratio of mitochondrial DNA:nuclear DNA in a logarithmic culture, mitochondrial DNA was calculated to have a half-life of 1.8 cell doublings.     

In vitro HeLa cell DNA synthesis similarity to in vivo replication

An in vitro DNA synthesizing system, consisting of a HeLa cell lysate which incorporated dNTPs into an acid-insoluble, DNase-labile product, was optimized for incorporation per nucleus. Synthesis depended on the presence of all four dNTPs and was linear for about 15 minutes, then slowed and finally stopped after one to two hours at 37 °C. The DNA synthesized in vitro was found to be preferentially attached by covalent linkage to sites which had just been replicated in vivo. DNA fiber autoradiography of DNA labeled in vitro suggests that synthesis occurs by the replicon mechanism proposed for in vivo replication, but at a fork movement rate 50 to 60% of that in vivo.When analyzed on alkaline sucrose gradients, dNTPs appeared to be incorporated by a semidiscontinuous mechanism, with label after brief pulses (10 to 20 s) distributed about equally between a peak of Okazaki fragments and a very heterogeneous distribution of longer DNA strands. Okazaki fragments, which can be initiated in vitro, sedimented in a broad peak averaging 180 nucleotides in length.     

Process of infection with bacteriophage phi X 174 XXXVIII. Replication of phi chi 174 replicative form in vivo.

The replication of bacteriophage phi X 174 replicative-form DNA has been studied by structural analysis of pulse-labeled replicative-intermediate molecules. Such intermediates were identified by pulse-labeling with [13H]thymidine and separated into four major fractions (A, B, C, and D) in a propidium diiodide-cesium chloride buoyand density gradient. Sedimentation analysis of each of these fractions suggests the following features of phi X replicative-form DNA replication in vivo. (i) At the end of one cycle of replication, one daughter replicative form (RFII) contains a nascent plus (+) strand of the unit viral length, and the other daughter RFII contains small fragments of nascent minus (-) strand. (ii) Asymmetry is also associated with production of the first supercoiled RFI after addition of pulse label in that only the minus strand becomes radioactive. (iii) A supercoiled DNA (RFI') seems to occur in vivo. This DNA is observed at a position of greater density in a propidium diiodide-cesium chloride buoyant density gradient than normal RFI. (iv) A novel DNA component is observed, at a density greater than RFI, which releases, in alkali, a plus strand longer (1.5 to 1.7 times) than the unit viral length. These results are discussed in terms of the possible sequence of events in phi X 174 replicative-form replication in vivo.     

Inhibition of DNA replication by ultraviolet light.

DNA replication in ultraviolet-irradiated HeLa cells was studied by two different techniques: measurements of the kinetics of semiconservative DNA synthesis, and DNA fiber autoradiography. In examining the kinetics of semiconservative DNA synthesis, density label was used to avoid measuring the incorporation due to repair replication. The extent of inhibition varied with time. After doses of less than 10J/m2 the rate was initially depressed but later showed some recovery. After higher doses, a constant, low rate of synthesis was seen for at least the initial 6 h. An analysis of these data indicated that the inhibition of DNA synthesis could be explained by replication forks halting at pyrimidine dimers. DNA fiber autoradiography was used to further characterize replication after ultraviolet irradiation. The average length of labeled segments in irradiated cells increased in the time immediately after irradiation, and then leveled off. This is the predicted pattern if DNA synthesis in each replicon continued at its previous rate until a lesion is reached, and then halted. The frequency of lesions that block synthesis is approximately the same as the frequency of pyrimidine dimers.     

Replicon size and rate of DNA replication fork movement are correlated in grasses

In eukaryotes, most nuclear DNA replication proceeds bidirectionally from multiple origins of replication. A unit of DNA, replicated by two replication forks from a single origin, is termed a replicon. Using results from DNA fiber autoradiography we show a novel positive correlation between replicon size and the rate of replication fork movement in root meristem nuclei of 13 grass species. Although there is interspecific variation in replicon size, it is balanced by similar variation in the rate of replication fork progression.     

Establishment of exponential growth after a nutritional shift-up in Escherichia coli B/r: accumulation of deoxyribonucleic acid, ribonucleic acid, and protein.

The accumulation of deoxyribonucleic acid (DNA), ribonucleic acid (RNA), and protein was followed in cultures of Escherichia coli B/r during exponential growth in different media and for 2 h after a nutritional shift-up from succinate minimal medium (growth rate [mu1] = 0.67 doublings per h) to glucose plus amino acids medium (mu2 = 3.14 doublings per h). During postshift growth of the culture, the amounts of RNA (R), DNA (D), and protein (P) increased such that the ratios of the increments (delta R/delta P; delta D/delta P) were constants (k1, k2). This implies that the rates of accumulation of nuclei1:k2:1. These constants change from their preshift value to their final postshift value (i.e., k1 and k2) within a few minutes after the shift. k1 is a function of the activity of ribosomes, whereas k2 is related to the initiation of rounds of DNA replication. These parameters and the observed change in the doubling time of RNA (= mu2/mu1) were used to derive kinetic equations that describe the accumulation of DNA, RNA, protein, and cell mass during the 2- to 3-h transition period after a shift-up. The calculated kinetics agree closely with the observed kinetics.     

Cellular localization of oriC during the cell cycle of Escherichia coli as analyzed by fluorescent in situ hybridization

The origin of replication of Escherichia coli, oriC, has been labeled by fluorescent in situ hybridization (FISH). The E. coli K12 strain was grown under steady state conditions with a doubling time of 79 min at 28 degrees C. Under these growth conditions DNA replication starts in the previous cell cycle at -33 min. At birth cells possess two origins which are visible as two separated foci in fully labeled cells. The number of foci increased with cell length. The distance of foci from the nearest cell pole has been measured in various length classes. The data suggest: i) that the two most outwardly located foci keep a constant distance to the cell pole and they therefore move apart gradually in line with cell elongation; and ii) that at the initiation of DNA replication the labeled origins occur near the center of prospective daughter cells.     

Growth of bacteriophage Mu in Escherichia coli dnaA mutants.

In one-step growth experiments we found that bacteriophage Mu grew less efficiently in nonreplicating dnaA mutants than in dnaA+ strains of Escherichia coli. Phage development in dnaA hosts was characterized by latent periods that were 15 to 30 min longer and an average burst size that was reduced by 1.5- to 4-fold. The differences in phage Mu development in dnaA and dnaA+ strains were most pronounced in cells infected at a low multiplicity and became less pronounced in cells infected at a high multiplicity. Many of these differences could be eliminated by allowing the arrested dnaA cells to restart chromosome replication just before infection. In continuous labeling experiments we found that infected dnaA strains incorporated 5 to 40 times more [methyl-3H]thymidine than did uninfected cells, depending on the multiplicity of infection. DNA-DNA hybridization assays showed that greater than 90% of this label was contained in phage Mu DNA sequences and that only small amounts of the label appeared in E. coli sequences. In contrast, substantial amounts of label were incorporated into both host and viral DNA sequences in infected dnaA+ cells. Although our results indicated that phage Mu development is not absolutely dependent on concurrent host chromosomal DNA replication, they did strongly suggest that host replication is necessary for optimal growth of this phage.     

Intermediate in adenovirus type 2 replication.

Replicating chromosomes, called intermediate DNA, have been extracted from the adenovirus replication complex. Compared to mature molecules, intermediate DNA had a greater buoyant density in CsCl gradients and ethidium bromide-cesium chloride gradients. Digestion of intermediate DNA with S1 endonuclease, but not with RNase, abolished the difference in densities. These properties suggest that replicating molecules contain extensive regions of parental single strands. Although intermediate DNA sedimented faster than marker viral DNA in neutral sucrose gradients, single strands longer than unit length could not be detected after alkaline denaturation. Integral size classes of nascent chains in intermediate DNA suggest a relationship between units of replication and the nucleoprotein structure of the virus chromosome. Adenovirus DNA was replicated at a rate of 0.7 x 10-6 daltons/min. Although newly synthesized molecules had the same sedimentation coefficient and buoyant density as mature chromosomes, they still contained single-strand interruptions. Complete joining of daughter strands required an additional 15 to 20 min.     

Replication of colicinogenic factor E 1 DNA: evidence for a discontinuous replication mechanism

The mechanism of Col E 1 DNA replication was investigated in a plasmolysed cell system prepared from chloramphenicoltreated E. coli JC 411 (Col E 1). After pulse-labelling with (3)H-dTTP a considerable fraction of the newly synthesized DNA was recovered as single-stranded fragments. Upon alkali denaturation the pulse label was found in DNA chains sedimenting slower than unit length Col E 1 strands with a prominent peak at 5 S. During a chase with unlabeled precursors the label is transferred nearly completely into supercoiled Col E 1 DNA. DNA ligase appears to be required for the joining of the 5 S pieces since in the absence of NAD an accumulation of short fragments is observed.     

In vitro replication directed by a cloned adenovirus origin

A 5.7-kb recombinant plasmid, called XD-7, contains the terminal XbaI-E fragment from the left end of type 2 adenovirus cloned into the EcoRI site of pBR322. An average of 9% +/- 1% of input supercoiled, protein-free XD-7 DNA replicated as rolling circles with single-stranded tails ranging up to unit length and longer in reaction mixtures containing nuclear and cytoplasmic extracts from adenovirus-infected, but not uninfected, HeLa cells. The adenovirus origin was mapped on XD-7 by electron microscopy at the left boundary of the cloned adenovirus segment. Since replication proceeded rightwards, we conclude that the adenovirus l strand was displaced during replication. No origin was located at or near the EcoRI site on pBR322. Reversing the orientation of the adenovirus origin reversed the direction of replication, and deletion of the adenovirus origin abolished replication.     

Deoxyribonucleotide metabolism in cycling and resting human fibroblasts with a missense mutation in p53R2, a subunit of ribonucleotide reductase

Ribonucleotide reduction provides deoxynucleotides for nuclear and mitochondrial (mt) DNA replication and DNA repair. In cycling mammalian cells the reaction is catalyzed by two proteins, R1 and R2. A third protein, p53R2, with the same function as R2, occurs in minute amounts. In quiescent cells, p53R2 replaces the absent R2. In humans, genetic inactivation of p53R2 causes early death with mtDNA depletion, especially in muscle. We found that cycling fibroblasts from a patient with a lethal mutation in p53R2 contained a normal amount of mtDNA and showed normal growth, ribonucleotide reduction, and deoxynucleoside triphosphate (dNTP) pools. However, when made quiescent by prolonged serum starvation the mutant cells strongly down-regulated ribonucleotide reduction, decreased their dCTP and dGTP pools, and virtually abolished the catabolism of dCTP in substrate cycles. mtDNA was not affected. Also, nuclear DNA synthesis and the cell cycle-regulated enzymes R2 and thymidine kinase 1 decreased strongly, but the mutant cell populations retained unexpectedly larger amounts of the two enzymes than the controls. This difference was probably due to their slightly larger fraction of S phase cells and therefore not induced by the absence of p53R2 activity. We conclude that loss of p53R2 affects ribonucleotide reduction only in resting cells and leads to a decrease of dNTP catabolism by substrate cycles that counterweigh the loss of anabolic activity. We speculate that this compensatory mechanism suffices to maintain mtDNA in fibroblasts but not in muscle cells with a larger content of mtDNA necessary for their high energy requirements.     

A detailed analysis of cyclin A accumulation at the G(1)/S border in normal and transformed cells

The temporal relationship between cyclin A accumulation and the onset of DNA replication was analyzed in detail. Five untransformed and nine transformed asynchronously growing cell cultures were investigated using a triple immunofluorescence staining protocol combined with computerized evaluation of staining intensities in individual cells. The simultaneous staining of BrdU, cyclin A, and cyclin E made it possible to determine the cell cycle position of each cell investigated. Cells at the G(1)/S border were identified on the basis of cyclin E content and were further analyzed with respect to cyclin A and BrdU content. A method was developed to calculate objective thresholds defining the highest staining intensity found in the negative cells in the population. Using the thresholds we could distinguish cells with minute amounts of cyclin A and BrdU from truly negative cells. We show that the onset of cyclin A accumulation and the start of DNA replication occurs at the same time, or deviating by a few minutes at the most. We also show that cyclin A accumulates continuously during S. This study clearly demonstrates that nuclear cyclin A can be used as a reliable marker for the S and G(2) phases in both normal and transformed interphase cells.     

THE REPLICATION TIME AND PATTERN OF CARCINOGEN-INDUCED HEPATOMA CELLS

The replication time and pattern have been investigated in hepatoma cells induced by feeding 3'Me-DAB to male rats for 5 months. With the use of tritiated thymidine as a DNA label along with autoradiography, mitotic nuclear labeling has been studied 0.5 to 72 hours after the administration of the label. The following time intervals have been estimated: replication time, 31 hours; DNA synthesis, 17 hours; G₂ plus Mitosis, 2 hours; G₁, 12 hours. Only about 8 per cent of the tumor cell (interphase) population is "flash" labeled, following a single dose of 50 µC of H³TDR. This group of cells has been followed through three cycles of division. The repeated rhythmic passage of tumor cells through cell division is similar to that previously reported for normal liver cells in the growing rat. However, tumor cells have longer replication and DNA synthesis times. In addition, the several time intervals studied vary more in the tumor cell population than they do in the growing normal cell population.     

Correlations of geographic distribution and temperature of embryonic development with the nuclear DNA content in the Salamandridae (Urodela, Amphibia).

We used flow cytometry to measure the nuclear DNA content in erythrocytes of 27 salamandrid species. Across these species, diploid genome size varied more than 2 fold (51.3-104.4 pg). According to genome size and geographic distribution, 3 groups of newt species were recognized: West Palearctics with smaller amounts of nuclear DNA; Nearctic, with intermediate values; and East Asiatic, with higher genome sizes. Viviparous West Palearctic salamanders differed from most of the oviparous West Palearctic newts in possessing larger genome sizes. The nuclear DNA content strongly correlates with species range limits. At the same temperature, embryos of salamandrid species with larger genome sizes have a markedly longer developmental time than those with smaller genomes. We present an analysis of the relationships between the amount of nuclear DNA and water temperature at the breeding sites.