Autoradiographic evidence for bidirectional DNA replication in Escherichia coli

Autoradiographic evidence is presented that demonstrates bidirectional DNA replication during a synchronous round of DNA synthesis in a culture of a reversible temperature sensitive DNA initiation mutant of Escherichia coli K12. High specific activity [³H]thymine was incorporated into the origins and termini of chromosomes which were otherwise uniformly labeled with low specific activity [³H]thymine. Autoradiographs of such differentially labeled chromosomes show two regions of high grain density symmetrically disposed on the circular chromosomes. This demonstrates that the origins and termini of replication are not contiguous; therefore replication must have proceeded in two directions.     

Families of replicating units in cultured hamster fibroblasts

An examination of the patterns of DNA replication in pseudodiploid Don C and diploid Don cell lines in culture has been made. Pulse-chase labelling experiments with ³H-thymidine in both synchronized and log-phase cells indicate that the newly replicated DNA can be divided into two and three large temporally distinct fractions in Don C and Don cells, respectively. This is shown radiochemically by fluctuations in the incorporation of ³H-thymidine into the DNA of synchronized cells and autoradiographically by fluctuations in counts of labelled metaphases and grain over mitotic figures. Pulse-chase experiments and fluorometric determinations indicate that the periodic incorporation of ³H-thymidine can be accounted for by discontinuous synthesis and turnover of DNA during the cell cycle.A survey of the literature reveals that fluctuations in DNA synthetic activity during the S phase are to be found in a large number of published graphs of cell population kinetics. The phenomenon is observable in both diploid and non-diploid cells. A change in the timing of DNA replicon synthesis during the S phase according to the developmental stage and age of the cell is proposed.     

Interchromosomal asynchrony of DNA replication in polytene chromosomes of Drosophila pseudoobscura

Analysis of ³H-thymidine autoradiograms of late third instar larval salivary glands of Drosophila pseudoobscura revealed a unique example of asynchrony of replication in the autosome complement. The two autosomal arms, 2 and 3, show similar labeling pattern during the initial phases, DD to 3C, and thereafter, the chromosome 3 has fewer labeled sites than chromosome 2 until the most terminal pattern, 1D. Detailed sitewise analysis of ³H-thymidine labeling shows that while nearly 54% of the sites examined in chromosome 2 have a labeling frequency greater than 50%, only 13% of all sites in chromosome 3 have labeling frequency at that range. The number of labeled sites on chromosome 3 plotted against that on chromosome 2 shows a hyperbolic profile rather than a linear relationship. The silver grain ratio of the 2nd to 3rd increases from 1.5 to 3.1 through different stages of the cycle. These results suggest that both chromosomes start replication simultaneously but the third chromosome appears to complete the replication earlier than the second. These data open up the possibility of separate control mechanisms for the initiation and termination of DNA replication in polytene chromosomes.This paper is dedicated to the memory of the late Prof. H. D. Berendes.     

Direction of DNA replication in mammalian cells

We have re-examined the direction of DNA synthesis in mammalian cells by means of pulse-labeling with [³H]thymidine and DNA autoradiography. Our results show that, whether or not the cells are treated with 5-fluoro-deoxyuridine, and whether they are labeled first with high specific activity [³H]thymidine and then with low, or vice versa, most (? 90%) of the unambiguous autoradiographic patterns can be explained by bidirectional replication but not by unidirectional replication.We also find that in autoradiographic experiments using two different specific activities of [³H]thymidine, obvious differences in grain density are obtained only when the difference in specific activity is threefold or more. Thus, the apparently contradictory findings of Lark et al. (1971) can be explained by the low difference in specific activity used by those authors.     

Effect of thiopyrimidine ribonucleosides on DNA and RNA synthesis in synchronized mammalian cell cultures

The uptake of ³H-uridine into RNA and of ³H-thymidine into DNA was investigated in synchronized Chinese hamster cells which had been exposed to thiopyrimidine ribonucleosides. The cells were synchronized at metaphase by reversal of colcemid inhibition; these cells were then labeled with either ³H-thymidine or ³H-uridine at selected times, and analyzed in autoradiographs. Incorporation of ³H-thymidine into DNA was not inhibited by administration to the cells of 2-thiouridine or 4-thiouridine (4 × 10⁻³ M). Exposure of the cells to the anti-metabolites for over 15 h significantly reduced the incorporation of ³H-uridine into nuclear RNA and completely blocked the labeling of cytoplasmic RNA. This finding is interpreted as an indication that RNA synthesis was inhibited in cells which continued to synthesize DNA. The inhibition of RNA synthesis hindered cell division and decreased cell viability. This lethal effect is similar to the “unbalanced growth” induced by inhibitors of DNA synthesis. The thiopyrimidine ribonucleosides, however, killed mammalian cells without inhibiting DNA synthesis.     

Histones H1 and H4 are present near the replication fork

The presence of histones H1 and H4 at the sites of actual DNA synthesis has been studied with Ehrlich ascites tumour cells, pulse labeled for different times with ³H-thymidine and then treated with formaldehyde to crosslink histones to DNA. The fixed chromatin fragments were sonicated to reduce the size of DNA, purified in a CsCl gradient and immunoprecipitated with antibodies to histones H1 and H4. Determination of specific radioactivity in precipitated probes showed that both histones have been associated with nascent DNA even upon 1 min pulse with ³H-thymidine, thus indicating their presence near the replication fork.     

Effects of reovirus infection on the spatial and temporal organization of DNA replication in L cells

DNA fiber autoradiography was used to analyze the spatial and temporal organization of activated initiation sites for DNA replication in mouse L929 cells infected with reovirus type 3 (Dearing strain) and in uninfected control cells. Cells were labeled for 10 min with³H-thymidine at high specific activity followed by 3 h of low specific activity labeling. Reovirus infection causes no change in the rate of replication fork progression, but increases both the mean distance between activated initiation sites by 30% and the nonrandomness in the spatial distribution of the sites along the DNA fibers. Significant synchronization of initiation in adjacent activated sites was detected on DNA fibers from uninfected cells and from reovirusinfected cells. The mean relative initiation time for pairs of initiation events which had occurred prior to high specific activity labeling did not differ significantly between the infected and uninfected cells. The data are consistent with the interpretation that reovirus infection shuts off initiation sites in a coordinated fashion, possibly by preventing activation of entire clusters of potential initiation sites.     

Detection of replication initiation by a replicon family in DNA of synchronized pea (Pisum sativum) root cells using benzoylated naphthoylated DEAE-cellulose chromatography

Fractionated replicating DNA from pea was obtained from both synchronized cells just starting replication and from carbohydrate-starved cells ending replication. Benzoylated naphthoylated DEAE-cellulose chromatography of pulse-labeled DNA digested with EcoR I gave evidence that a family of replicons initiated replication 45 to 60 min after synchronized cells were released from the G₁/S phase boundary. DNA from cells labeled in late S phase, on the other hand, showed no signs of additional replication initiations before entering G₂ phase. Results with DNA from both early and late S phase cells comply with a model based on the premise that with short pulses of [³H]-thymidine the isotope is localized at replication forks and that longer pulses label both replication forks and recently replicated segments of double-stranded DNA. The model applies only to DNA subjected to fragmentation before chromatography.The results also suggest that benzoylated naphthoylated DEAE-cellulose chromatography is a useful means to isolate origins and replication forks from synchronized plant cells.     

H-thymidine incorporation into nuclear DNA of leaf cells

The incorporation of ³H-thymidine into nuclear DNA of leaf cells of Nanthium pennsylvanicum was studied as a function of concentration and specific activity of the radioisotope. From the assessment of the average number of grains per nucleus and the percent of labeled nuclei, it was concluded that the incorporation was a linear function of concentration of the exogenous radioisotopic solution and a logarithmic function of the incubation time. Ten microcuries per milliliter on the average yielded 20% of labeled nuclei with 18 grains per nucleus. Seven-fold increase in concentration only doubled the amount of ³H-thymidine incorporated. The lamina regions near the vein incorporated a significantly greater amount of the radioisotope than the lamina region at some distance from the vein. The specific activities of 2, 3.35, 6.7 and 15.3 c/mmole had no effect upon the amount of ³H-thymidine incorporated, if the amount of microcuries of the incubation solution was the same in each activity. Considering the total number of molecules, the estimated rates of incorporation indicated that at the activity of 2 c/mmole, the system operated with about 7 times higher rates as compared with the activity of 15.3 c/mmole.     

Responses of mammalian metaphase chromosomes to endonuclease digestion

Digestion of fixed metaphase chromosomes by endonucleases (micrococcal nuclease and DNase II) under optimal digestion conditions followed by Giemsa staining produces sharp banding patterns identical to G-bands. In ³H-thymidine labeled, synchronized metaphase cells of the chinese hamster (CHO line), the band induction is accompanied by the removal of DNA. The single strand specific nuclease S1 and DNase I do not produce such banding patterns.     

Correspondence of banding patterns to 3H-thymidine labeling patterns in polytene chromosomes

³H-thymidine labeling frequencies over X chromosomal region 1A-4E of Drosophila melanogaster, were analysed with reference to chromosome sections with and without prominent bands. A correspondence was found between band sections and late start of silver grain labeling at the initial stage in combination with late labeling at the end stage of replication. A complementary situation is always to be found over puff/interband sections, where an early start of labeling at the initial stage is generally combined with early labeling completion at the end stage of replication.     

Isolation and Partial Characterization of DNA in Capsular Preparations of Rhizobium trifolii

The capsular polysaccharides from thymidine-(methyl-³H) labeled cultures of Rhizobium trifolii; strain 162S7 (Nitragin Co.) were centrifuged from bacterial cells and collected by ethanol precipitation. Following the addition of unlabeled carrier nucleic acids, labeled DNA, termed cap-DNA, was isolated from the capsular polysaccharides. Cap-DNA absorbed maximally at H-260 nm and was DNase sensitive. Approximately 11 μg of ³H-cap-DNA were consistently isolated per liter of 48 h cultures. Cap-DNA production was generally synchronized with the synthesis of the capsular polysaccharide and bacterial growth, attaining maximum recoverable amounts in 48 h cultures. By five days of culture growth, significant decreases in the amount of recoverable cap-DNA were noted. The presence of label in the cap-DNA demonstrated that the cap-DNA originated via de novo synthesis by the Rhizobium cells rather than from an anomalous source. The cap-DNA and intracellular Rhizobium DNA had similar buoyant densities of p= 1.719, indicating that cap-DNA arose specifically from the intracellular DNA. In 48-h cultures the specific activity of the cap-DNA was about one-third that of the intracellular DNA. This implies intracellular DNA was released during early growth with a relatively low specific activity which diluted the isotopic label of DNA released later. The evidence suggests lysogeny was the principal release mechanism.     

Incorporation of Radioactive Pyrimidine Nucleosides into DNA and RNA of Eimeria tenella (Coccidia) Cultured In Vitro*

Autoradiographic methods were used to study the incorporation of tritiated cytidine, thymidine, and uridine into asexual stages of Eimeria tenella cultured in embryonic chick kidney cells. Developing parasites did not incorporate ³H-thymidine either when host cells were labeled prior to infection or when the cultures were labeled for 30 min, 48–72 hr after infection. Continuous exposure of infected cultures to ³H-thymidine for up to 18 hr resulted in light labeling of cell cytoplasm and schizonts. ³H-cytidine and ³H-uridine were incorporated into parasites developing in cultures that were labeled before infection. When the cultures were labeled for 30 min, 48–72 hr postinfection and fixed immediately, schizonts were labeled lightly with ³H-cytidine but contained dense accumulations of ³H-uridine.     

DNA SYNTHESIS,CELL DIVISION,AND CELL DIFFERENTIATION DURING LEAF DEVELOPMENT OF XANTHIUM PENNSYLVANICUM

Leaf growth consists of two basic processes, cell division and cell enlargement. DNA synthesis is an integral part of cell division and can be studied with autoradiographic techniques and incorporation of some labeled precursor. Studies were made on the synthesis of nuclear DNA through incorporation of ³H-thymidine in various parts of the lamina during the entire course of leaf development of Xanthium pennsylvanicum. The time course analysis of DNA synthesis was correlated with cell division and rates of cell enlargement. Significant differences in ³H-thymidine incorporation were found in various parts of the lamina. Cell division and DNA synthesis were highest in the early stages of development. Since no ³H-thymidine was incorporated after cessation of cell division (LPI 2.8) in the leaf lamina, it appears that DNA synthesis is not needed for enlargement and differentiation of Xanthium cells. Rates of cell enlargement were negligible in the early development and reached their maximum after cessation of mitoses, between plastochron ages (LPI) 3 and 4. Cells matured between LPI's 5 and 6. Enzymatic activity was correlated with cell division and cell differentiation at various stages of leaf development.     

The Role of Cytokinin in the Regulation of Growth, DNA Synthesis and Cell Proliferation in Cultured Soybean Tissues (Glycine max var. Biloxi)

Changes in protein content and cell proliferative activity were followed after a cytokinin-requiring strain of cultured Glycine max tissue was transferred to freshly prepared media which either contained or lacked cytokinin. Cell numbers doubled within the first two days after transfer, both in the presence and absence of cytokinin. However, after the second day no further increase in cell number was observed in the absence of cytokinin, while cell numbers continued to increase logarithmically in the presence of cytokinin. The size of the cell population attained after the first six days of growth was a function of the cytokinin concentration of the culture medium. However, the amount of ³H-thymidine incorporated into nuclear DNA bore no relation to the rate of cell proliferation. Tissues cultured on medium lacking cytokinin incorporated the greatest amount of ³H-thymidine per microgram of DNA, while the actively dividing tissues incorporated somewhat less. Using autoradiography and isopycnic CsCl gradient centrifugation, it was shown that the radioactivity derived from ³H-thymidine was associated with nuclear DNA in the cytokinin-deprived cells. Biochemical measurements demonstrated that cells cultured for six days without cytokinin had approximately twice the DNA content of the actively proliferating cells cultured on cytokinin-containing medium. Furthermore, in autoradiographs labeled cells were found to average nearly three times as many silver grains per nucleus in tissues cultured without cytokinin as the cytokinin-grown tissues. This suggests that the ³H-thymidine incorporation in the non-proliferating soybean cells results from nuclear DNA synthesis and that some of the cells became polypoid in the absence of cytokinin. These findings would be consistent with the idea that cytokinin acts as a specific trigger for cytokinesis.     

Periodic acid-Schiff reaction combined with quantitative autoradiography of 3H-thymidine- or 85S-sulfate-Labeled epithelial cells of colon

Summary The influence of periodic acid-Schiff staining on grain counts was examined using autoradiography of guinea pig colon labeled with either ³H-thymidine or ³⁵S-sulfate. Prestaining decreased the grain count, an effect due to the Schiff reagent but not to periodic acid. Poststaining altered the grains which were partly or completely lost, an effect due to periodic acid but not to the Schiff reagent. The suggested procedure is to pretreat the sections with periodic acid, process for autoradiography, and poststain with the Schiff reagent. No silver grain is then lost.     

STUDIES ON THE GERMINATION OF SEEDS OF THE ROOT PARASITES,ALECTRA VOGELII AND STRIGA GESNERIOIDES. II. DNA SYNTHESIS AND DEVELOPMENT OF THE QUIESCENT CENTER IN THE RADICLE

DNA synthetic activity in the radicle meristem of embryos of germinating seeds of the obligate root parasites, Alectra vogelii and Striga gesnerioides was followed by autoradiography of ³H-thymidine incorporation. Incorporation of ³H-thymidine occurred in the nuclei of cells destined to form the vascular tissues, ground meristem and epidermis. An analysis of the distribution of labeled nuclei demonstrated the presence of a quiescent center of 2-4 cells in the radicle at the beginning of seed germination, becoming more prominent at later stages of germination. During continued growth of the radicle which resulted in a reduction in size of the meristem, cells of the original quiescent center were activated to undergo DNA synthesis.     

The replicative organization of DNA in polytene chromosomes ofDrosophila hydei

Salivary-gland nuclei ofDrosophila hydei were pulse-labeledin vitro with³H-thymidine and studied autoradiographically in squash preparations. The distribution of radioactive label over the length of the polytene chromosomes was discontinuous in most of the labeled nuclei; in some nuclei the pattern of incorporation was continuous. Comparison of the various labeling patterns of homologous chromosome regions in different nuclei showed that specific replicating units are replicated in a specific order. By combining autoradiography with cytophotometry of Feulgen-stained chromosomes, it was possible to correlate thymidine labeling of specific bands with their DNA content. The resulting data indicate that during the S-period many or perhaps all of the replicating units in a salivary-gland nucleus start DNA synthesis simultaneously but complete it at different times. Furthermore, the data support the hypothesis that the chromomere is a unit of replication or replicon. The DNA content of haploid chromomeres was found to be about 5×10^-4 pg for the largest bands inDrosophila hydei. From the results of H³-thymidine autoradiography and Feulgen-cytophotometry on neuroblast and anlage nuclei it was concluded that during growth of the polytenic nucleus heterochromatin is for the most part excluded from duplication. The results of DNA measurements in interbands of polytene chromosomes do not agree with a multistrand structure for the haploid chromatid. A chromosome model is proposed which is in accordance with the reported results and with current views concerning the replicative organization of chromosomes.     

Biosynthetic events of hydrid regeneration

Summary The results of a combined morphological and biochemical study of the role of DNA synthesis during distal regeneration inHydra oligactis revealed that a burst of³H-thymidine incorporation into DNA preceded the elaboration of each of the initial three tentacles. In addition, the relative level of each burst of precursor incorporation relfected the number of tentacles formed at that time. Cytological localization of concentrated amounts of labeled material in nuclei of the hypostome and tentacle regions provided corroborative evidence for the biochemical findings.Evidence that the increased DNA specific activity levels described above are associated with tentacle initiation derived from studies in which regenerating hydra were cultured in hydroxyurea and studies in which hydra regenerated proximally rather than distally. Hydra regenerating in 8 mg/ml (0.105 M) hydroxyurea developed morphologically recognizable hypostomes but no tentacles, and incorporated³H-thymidine into DNA at a level distinctly below that exhibited by uncut, untreated animals. Similarly, hydra regenerated a normal, functional basal disc in the absence of any increased DNA specific activity. Therefore, it is suggested that tentacle initiation inH. oligactis requires concomitant DNA synthesis and, as such, represents an epimorphic phenomenon.     

Replication in Drosophila chromosomes XIII. Comparison of late replicating sites in two polytene cell types in D. hydei

We have compared the temporal order of completion of replication of specific sites of X and 2nd chromosomes in two polytene cell types of D. hydei by examining the patterns of autoradiographic labelling in ³H-thymidine pulse (10 min) labelled salivary glands and gastric ceaca of mid 3rd instar larvae. Present results are in agreement with our earlier finding in D. nasuta (Lakhotia & Tiwari, 1984, Chromosoma, 89: 212–217 that in spites of a general similarity in the cytological identity of independently replicating sites in the two polytene cell types, their temporal programme of replication varies in different tissues. This may be related to differential gene activity patterns and polytene organization in the different cell types.