The cell cycle of Bacillus subtilis as studied by electron microscopy

Bacillus subtilis strain Marburg was grown exponentially with a doubling time of 65 min. To follow the time course of various cell cycle events, cells were collected by agar filtration and were then classified according to length. The DNA replication cycle was determined by a quantitative analysis of radioautograms of tritiated thymidine pulse labeled cells. The DNA replication period was found to be 45 min. This period is preceded and followed by periods without DNA synthesis of about 10 min.The morphology and segregation of nucleoplasmic bodies was studied in thin sections. B. subtilis contains two sets of genomes. DNA replication and DNA segregation seem to go hand in hand and DNA segregation is completed shortly after termination of DNA replication.Cell division and cell separation were investigated in whole mount preparations (agar filtration) and in thin sections. Cell division starts about 20 min after cell birth; cell separation starts at about 45 min and before completion of the septum.     

Perturbation of DNA replication and cell cycle progression by commonly used [3H]thymidine labeling protocols.

The effect of tritiated thymidine incorporation on DNA replication was studied in Chinese hamster ovary cells. Rapidly eluting (small) DNA from cells labeled with 2 microCi of [3H]thymidine per ml (200 microCi/mmol) for 60 min matured to a large nonelutable size within approximately 2 to 4 h, as measured by the alkaline elution technique. However, DNA from cells exposed to 10 microCi of [3H]thymidine per ml (66 microCi/mmol) was more rapidly eluting initially and did not mature to a nonelutable size during subsequent incubation. Semiconservative DNA replication measured by cesium chloride gradient analysis of bromodeoxyuridine-substituted DNA was also found to be affected by the final specific activity of the [3H]thymidine used in the labeling protocol. Dramatic cell cycle perturbations accompanied these effects on DNA replication, suggesting that labeling protocols commonly used to study DNA metabolism produce aberrant DNA replication and subsequent cell cycle perturbations.     

Relationship between tracheary element differentiation and the cell cycle in single cells isolated from the mesophyll of Zinnia elegans

A relationship between tracheary element differentiation and the cell cycle was studied in single cells isolated from the mesophyll of Zinnia elegans L. cv. Canary bird. Almost all nuclei of isolated mesophyll cells were at the 2 C level of DNA, indicating that almost all cells were initially in the G₁ phase and that somatic polyploidy was absent. Cultured cells underwent partially synchronous DNA replication at 42 h and mitosis at 54 h of culture, and the first cell cycle time was approximately 58 h.
The occurrence and timing of DNA replication and mitosis during cytodifferentiation to tracheary elements were investigated using microspectrophotometry, microfluorometry, tritiated thymidine autoradiography, and serial observation. More than 55% of the nuclei of the immature tracheary elements were at the 2 C level of DNA and were not labeled by continuous feeding with tritiated thymidine, providing clear evidence that these cells differentiated without interventing DNA replication. Some tracheary elements (approximately 30%) were formed after one round of the cell cycle, and others (less than 5%) were formed after passing through the S phase, but without intervening mitosis. All types of tracheary elements appeared simultaneously after 58 h of culture, and their patterns of increase in number were similar. From the results, we propose a hypothesis concerning the relationship between cytodifferentiation and the cell cycle.     

Cell Division During Inhibition of Deoxyribonucleic Acid Synthesis in Escherichia coli

When cultures of Escherichia coli B/r growing at various rates were exposed to ultraviolet light, mitomycin C, or nalidixic acid, deoxyribonucleic acid (DNA) synthesis stopped but cell division continued for at least 20 min. The chromosome configurations in the cells which divided were estimated by determining the rate of DNA synthesis during the division cycle. The cultures were pulse-labeled with (14)C-thymidine, and the amount of label incorporated into cells of different ages was found by measuring the radioactivity in cells born subsequent to the labeling period. The cells which divided in the absence of DNA synthesis were those which had completed a round of chromosome replication prior to the treatments. It was concluded that completion of a round of replication is a necessary and sufficient condition of DNA synthesis for cell division.     

Escherichia coli contains a DNA replication compartment in the cell center

The active replication forks of E. coli B/r K cells growing with a doubling time of 210 min have been pulse-labeled with [(3)H] thymidine for 10 min. By electron-microscopic autoradiography the silver grains have been localized in the various length classes. From the known pattern of the DNA replication period in the cell cycle at slow growth and from the average position of grains per length class it was deduced that DNA replication starts in the cell center and that it remains there for a substantial part of the DNA replication period. This suggests the occurrence of a centrally located DNA replication compartment.     

Kinetics of Incorporation of DNA Precursors from Ingested Bacteria into Macronuclear DNA of Paramecium aurelia12

SYNOPSIS. The kinetics of transfer of tritium-labeled material from the DNA of ingested bacteria into macronuclear DNA of Paramecium was examined by autoradiography. Bacteria labeled with tritiated thymidine were almost immediately incorporated into food vacuoles, thus becoming available for digestion and a potential source of labeled DNA precursors. Soluble label derived from food vacuoles appeared in low concentrations in the cytoplasm soon after cells were transferred to medium with labeled bacteria; incorporation of labeled precursors into macronuclear DNA began within 5 min. Labeled food vacuoles remained as potential sources of tritiated DNA precursors for a long and variable period after removal of labeled cells to non-labeled medium. The activity of the soluble cytoplasmic DNA precursors decreased parallel to the loss of labeled food vacuoles and no soluble DNA precursors were carried over from one macronuclear DNA synthetic period to the next. Labeling experiments were designed, using this information, which allowed determination of the pattern of macronuclear DNA synthesis and nuclear mass increase during the cell cycle. Macronuclear DNA synthesis began 25–30% of the way thru the cell cycle, continued at a constant rate during the middle half, and decreased in rate during the last quarter. Macronuclear mass increased in an approximately linear fashion, beginning with the onset of DNA synthesis and doubling by the time of karyokinesis.     

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.     

Mechanism of killing Chinese hamster ovary cells heated in G1: effects on DNA synthesis and blocking in G2

To determine where in the cell cycle Chinese hamster ovary cells die following heating in G1, a mild hyperthermia treatment, i.e., 10 or 11.5 min at 45.5 degrees C, resulting in 40-50% cell kill was used. After a 7-14-h delay in G1, the cells heated in G1 eventually entered S phase and replicated all their DNA. Both an autoradiographic analysis with tritiated thymidine and a bromodeoxyuridine-propidium iodide bivariate analysis by flow cytometry revealed that both clonogenic and nonclonogenic cells were delayed in progression through S phase for at least 4 h. Then they completed replication of all their DNA and entered G2. Alkaline sucrose gradient sedimentation analysis revealed that these heated cells could complete replicon elongation into cluster-sized molecules of 120-160 S which persisted for 2-12 h after heating. However, further replicon elongation into multicluster-sized molecules greater than 160 S required an additional 12 h in heated cells compared to the 4 h needed in unheated control cells. Our results when compared with the literature suggest that when G1 cells are heated to a survival level of about 50%, the nonclonogenic cells recover from a long delay in G1, traverse S at a reduced rate, and then die either in G2 or as multinucleated cells after an aberrant division.     

The cell cycle of glial cells grown in vitro: an immunocytochemical method of analysis.

Studies of cell cycles have traditionally employed [3H]- and [14C]-thymidine to label the DNA of proliferating cells and autoradiography to reveal the thymidine label. The development of antibodies to the thymidine analogue 5-bromodeoxyuridine (BrdU) has allowed the development of an immunocytochemical method analogous to the thymidine autoradiographic technique. In direct comparisons, we found that the immunocytochemical method consistently detected a larger number of proliferating cells. This suggests that it may be a more sensitive index of proliferation than thymidine autoradiography in some systems. We used the BrdU method to analyze the cycle of astroglia cultured from neonatal mouse cerebral cortex. Cells were exposed to BrdU for 1 hr to label a discrete subpopulation of proliferating cells. At 2-36 hr after the pulse, a combination of anti-BrdU immunocytochemistry and counterstaining with propidium iodide was used to identify proliferating cells. The length of the cell cycle was determined by charting the percent of BrdU-labeled mitotic cells vs time after the pulse. We found the average length of the cell cycle of astrocytes grown in vitro to be 20.5 hr. The combined G2 + M phases were 2-3 hr. These values are virtually identical with those found for glial cells in vivo, suggesting that the culture environment does not interfere with the normal control of cell cycle length.     

Replication of Deoxyribonucleic Acid During the Division Cycle of Salmonella typhimurium

The rate of thymidine incorporation into cells of Salmonella typhimurium growing in different media has been measured. In glucose-minimal medium, deoxyribonucleic acid (DNA) replication occurs during the first two-thirds of the division cycle; the final one-third of the division cycle was devoid of DNA replication. The measured doubling time of S. typhimurium in this medium is approximately 48 min, indicating that C (the time for a round of replication) and D (the time between termination and cell division) are approximately 32 and 16 min, respectively. At slower growth rates the pattern of replication is the same as glucose minimal medium. At faster growth rates the "gap" in DNA synthesis disappears. At rapid growth rates evidence for multiple forks is obtained.     

The Schaechter-Bentzon-Maaløe experiment and the analysis of cell cycle events in eukaryotic cells

The Schaechter-Bentzon-Maal?e (SBM) experiment, performed more than 40 years ago, provides an important lesson for the analysis of the eukaryotic cell cycle. Before this experiment, temperature shifts had been used to synchronize bacteria and determine the pattern of DNA synthesis during the bacterial division cycle. These experiments indicated that DNA replication occurred during a fraction of the division cycle with gaps before and after DNA synthesis, a pattern similar to the eukaryotic division cycle. The SBM experiment studied DNA replication during the division cycle by labeling an unperturbed culture with a short pulse of tritiated thymidine. All cells were found to be labeled, indicating that unperturbed cells synthesize DNA throughout the division cycle. Thus, the SBM experiment was a control experiment demonstrating that artifacts can be introduced by synchronization methods. The idea of an control experiment under unperturbed conditions is proposed for the analysis of data on cell-cycle-specific gene expression in yeast and mammalian cells.     

Does capacity of DNA replication change during in vitro ageing?

We described elsewhere how a lack of change in the rate of DNA chain elongation occurred during in vitro ageing of human diploid fibroblasts. Here we further examined the rate of actual incorporation of tritiated thymidine, the center-to-center distance of replicons and the length of each phase of the cell cycle in order to extend our previous results to the other aspects of DNA replication. The results obtained showed that the rate of net DNA synthesis, the replicon size and the duration of S phase did not change during in vitro ageing. Our findings indicated that the reason why the greater part of the cell population at high population doubling levels becomes incapable of proliferating might not be the gradual decline in the ability of DNA replication. The regulation system(s) of DNA replication may alter during the period of culturing without any change in the capacities of the DNA replication machinery and, consequently, the non-cycling cells increase.     

DNA replication and the nuclear membrane

To investigate the relationship between the nuclear membrane and DNA replication, Chinese hamster cells were labeled with tritiated thymidine and examined by electron microscope autoradiography. Unsynchronized cells were labeled for periods ranging from 0.5 to 20 minutes. There was no relative increase in the frequency of membrane-associated grains with the shorter labeling times, indicating that the replication point is not necessarily close to the nuclear membrane. When cells were synchronized to the beginning of the S period with mitotic selection and hydroxyurea, the percentage of membrane-associated grains was very low, indicating that DNA synthesis is not initiated at the nuclear membrane. When cells synchronized by mitotic selection were labeled at various times throughout the cell cycle, the percentage of peripheral grains was low in early S period and became progressively higher toward late S period as heterochromatin began to replicate. The labeling of Unsynchronized Microtus agrestis cells indicated that much of the peripheral labeling is due to the replication of intercallary heterochromatin. The results indicate that there is no association between the nuclear membrane and DNA replication.     

Tritiated thymidine effects on DNA, RNA, and protein synthetic rates in synchronized L-cells

Exponentially growing L -cells were synchronized by the double thymidine-block method and exposed to high specific activities of tritiated thymidine. DNA, RNA, and protein synthetic rates were measured through one cell cycle with 1-hour pulses of the appropriate C¹⁴-labelled precursors. Equivalent doses of tritiated water were substituted for tritiated thymidine in some experiments. Total amounts of DNA and histones per nucleus were determined photometrically in Feulgen and fast-green stained cells. It was observed that incorporated tritiated thymidine has an effect distinct from that of tritiated water and that it enhances the incorporation of the precursors at specific stages of the cell cycle, to a degree roughly proportional to the dose. Photometric data indicated an increase in DNA net synthesis and a metabolic instability of histones in the H³-thymidine-treated cells, resulting in higher DNA:histone ratios.     

Autoradiographic studies of chromosome replication during the cell cycle of Streptococcus faecium.

Analysis of the distribution of autoradiographic grains around cells of Streptococcus faecium which had been either continuously or pulse-labeled with tritiated thymidine (mass doubling time, 90 min) showed a non-Poisson distribution even when the distribution of cell sizes in the populations studied was taken into account. These non-Poisson distributions of grains were assumed to reflect the discontinuous nature of chromosome replication. To study this discontinuous process further, we fitted an equation to the grain distribution observed for the pulse-labeled cells that assumed that in any population of cells there were subpopulations in which there were zero, one, or two replicating chromosomes. This analysis predicted an average time for chromosome replication and for the period between completion of rounds of chromosome replication and division of 55 and 43 min, respectively, which were in excellent agreement with estimates made by other techniques. The present investigation extended past studies in indicating that the initiation and completion of rounds of chromosome replication are poorly phased with increases in cell volume and that the amount of chromosome replication may be different in different cell halves.     

The Application of Autoradiography to Developmental Studies in the Hydrozoa

The uptake of isotopically labelled nucleotides and amino acidshas been studied in five species of hydrozoans. In three speciesthe label was introduced both through immersion in a mediumcontaining the labeled compound and by injecting the labeledcompound into the gastrovascular cavity. In the remaining twospecies the label was introduced by immersion only. The comparisonof soaked and injected specimens clearly indicates that injectionis the method of choice whenever the injection of compoundsinto the gastrovascular cavity is possible. The relative easewith which labeled compounds were absorbed can be correlatedwith the ultrastructure of the epidermal and gastrodermal cellsurfaces and their associated extracellular coats. The use ofthese autoradiographic techniques is illustrated by the useof injected tritiated thymidine and tritiated uiidine to followthe replacement cycle of the zymogenic secretory cell in Hydra,and the use of immersion to introduce tritiated thymidine intothe planula larva of Pennaria.     

A Comparison of the Killing of Cultured Mammalian Cells Induced by Decay of Incorporated Tritiated Molecules at -196°C

The killing efficiency of tritium disintegrations in frozen mammalian cells labeled with tritiated uridine, histidine, and lysine was compared with the killing efficiency of incorporated tritiated thymidine. In each case, the distribution of tritium in the cells was determined by chemical fractionation as well as by radio-autography. Of all tritium disintegrations, by far the most effective were those occurring in DNA molecules within frozen cells; such incorporated tritium has a killing efficiency of 0.006. When cells were incubated with tritiated uridine for 10 min to label nuclear RNA, the killing efficiency was 0.0015. When the cells were pulse labeled with tritiated uridine and permitted to grow in nonradioactive media for 10 hr before freezing in order to incorporate tritium into cytoplasmic RNA, the killing efficiency was reduced to 0.0010. The results suggest that decay of tritium in nuclear RNA is more effective than that in cytoplasmic RNA. When the cells were labeled with tritiated histidine or lysine for 30 min, tritium atoms were found mainly in the acid soluble rather than in the protein fraction and the killing efficiency in each case was approximately 0.0007. The results of these suicide experiments indicate that the killing efficiency of tritium disintegrations depends on where tritium is located within the cells. Tritium disintegrations in the nucleus are more effective in killing the cell than that in cytoplasm; and tritium disintegrations on DNA in the nucleus is more effective in killing the cell than that of nuclear RNA.     

The action of 5-amino uracil on log growth and division-synchronized Tetrahymena

The influence of 5-amino uracil (5-AU) was investigated on the cell cycle of log growth and division-synchronized Tetrahymena pyriformis GL. The division index of log growth phase Tetrahymena was suppressed by 50% after 40 min in 8 mM 5-AU. Cells division-synthronized by one heat shock per generation were also treated with 5-AU. Cells treated either prior to the first synchronous division (80 min EH) or up to 25 min prior to the second synchronous division (after 160 min EH) were not delayed in their progress through the cell cycle. Cells treated during the S phase of the first free running cell cycle, however, were delayed 5-30 min from reaching the second synchronous division. The effect of 5-AU on DNA and RNA synthesis was also examined. Incorporation of [3H]thymidine into acid-precipitable material was reduced in the presence of 5-AU; the rate of DNA synthesis was also reduced. The depression in the rate of DNA synthesis was greater at the beginning of S than at the end of S. The size of the thymidine pool (nucleosides + nucleotides) did not change during 5-AU treatment; however, an accumulation of thymidine tri-phosphate and a decrease in the amount of thymidine nucleoside was observed. A suppression of [14C]uridine incorporation resulting from 5-AU treatment was observed throughout the cell cycle. The rate of RNA synthesis as monitored by [14C]uridine incorporation into acid precipitable material was also reduced during 5-AU treatment. No change in either the size or the composition of the pool of uridine (nucleoside + nucleotide) was detected in 5-AU treated cells as compared to controls.     

Electron Microscopy of Adenovirus 12 Replication II. High-Resolution Autoradiography of Infected KB Cells Labeled with Tritiated Thymidine

Incorporation of tritiated thymidine by KB cells infected with oncogenic adenovirus 12 was studied by means of high-resolution electron microscopic autoradiography. After a 1-hr pulse with tritiated thymidine, infected and control cultures were fixed at 8, 16, 24, 30, and 36 hr. Infected cultures showed a higher percentage of labeled cells. During early stages, the frequency of silver grains in the nucleus and in the nucleolus was higher in infected material. From 24 hr on, there was an inhibition of nuclear and nucleolar deoxyribonucleic acid (DNA) synthesis. At late stages, one-third of the label was located over nuclear inclusions, type II and IV, previously shown to be composed of DNA and protein, while a large majority of the remaining grains were located over the nucleoplasm. The possibility is considered, that the early increase in nuclear and nucleolar DNA synthesis produced by adeno 12 replication could in part be due to newly synthesized cellular DNA, as has been reported by others with respect to other oncogenic DNA viruses.     

The initiation of SV40 DNA synthesis is not unique to the replication origin

Replicative intermediates of SV40 were isolated, digested with the restriction endonuclease Bgl I and examined by electron microscopy. Over 98% of the replicative intermediates isolated following infection with wild-type virions at 33 degrees, 37 degrees or 40 degrees C or with tsA209 at 33 degrees C had initiated replication about 35 nucleotides to one side of the Bgl I site. Approximately 1% of the molecules had initiated replication about 2400 nucleotides from the Bgl I site. The remaining molecules may have initiated at other sites. When tsA209 virion-infected cultures were shifted to 40.5 degrees C for 90 min, the relative rate of thymidine incorporation into superhelical viral DNA dropped by more than 97%. The remaining incorporation was not due to "leakiness." The label incorporated into mature superhelical molecules during brief pulses was not preferentially incorporated near the terminus of replication as it was at 33 degrees C. Approximately 33% of the incorporated label represented repair synthesis. Electron microscopy revealed that half of the replicative intermediates formed under these conditions appear to have been initiated randomly around the SV40 genome. Rolling circle molecules contaminated all the preparations of replicative intermediates.