Regulation of DNA chain elongation in SV3T3 cells in relation to growth rate.

Regulation of DNA synthesis was investigated in SV40 transformed 3T3 cells exhibiting variable growth rates and residence times in S phase when cultured in the presence of different serum concentrations. Pulse-labeled DNA was chased into large molecular weight material in vivo much more slowly in slowly growing cells than in cells growing at the normal rate. Consistent with this, the joining of short (less than 10 S) chains to form long (greater than 10 S) chains by whole cell lysate system in vitro was greatly impaired in slowly growing cells compared to controls. Thus the lengthening of S phase in SV3T3 cells growing slowly in low serum is reflected in a reduced rate of DNA chain elongation. The presence of cycloheximide during chase in vivo reduced the rate of conversion of pulse-labeled molecules into large molecular weight DNA in both slowly growing and normally growing cells.     

Replication of DNA in mammalian chromosomes

DNA replication has been studied in cells (CHO) synchronized by mitotic selection from roller cultures. A study of the incorporation of ³H supplied as uridine indicates that cells cannot be blocked precisely at the beginning of the S phase, but DNA synthesis can be stopped in early S by treating with F-dU in G₁. After blockage potential initiation sites continue to increase at a linear rate for atleast 13 hours after division. Incorporation of ³H-thymidine begins at most of these sites within seconds after thymidine is supplied in the medium and incorporation continues at a linear rate for 20–24 minutes. There appears to be a pause after this interval before synthesis is resumed at about two times the initial rate. ³H-bromodeoxyuridine can be substituted for thymidine without affecting the kinetic pattern over a similar period. The increased rate is probably an increase in sites of chain growth rather than a change in rate of chain growth. A study of the labeled DNA segments by band sedimentation in a preformed NaClO₄ isokinetic gradient shows that two distinctly different sized segments can be released from the chromosomes by lysis at submelting conditions. One is the previously reported single chain segments averaging about one-half micron in length, but the other is a much larger segment (26S) which is native DNA with perhaps small regions of single chains presumably at the ends. Primarily single chain DNA is released after 1–2 minute pulse labeling, but after 2 minutes the larger segments (26S) contain most of the newly formed DNA except that attached to the chains of the major part of the template DNA which exhibits a discontinuous distribution, sedimenting far faster than either newly replicated segment. A consideration of the kinetics of formation of the 26S component indicates that is may contain the replicating fork. If this proves to be the correct interpretation the template chains would both have non-adjacent nicks preceeding the fork and also in a post-fork site at a mean distance of about 2 microns in both directions. The isolation of the growing points of DNA replication in chromosomes is now possible and the study of properties of the newly replicated regions should be greatly facilitated.     

Initiation of DNA replication in chromosomes of Chinese hamster ovary cells

The initiation of DNA replication and the subsequent chain elongation were studied using Chinese hamster ovary cells synchronized at the beginning of S phase. The cells were synchronized by a combination of mitotic selection and treatment with 5-fluorodeoxyuridine (FdU). The use of this drug at a concentration of 10^–5 M was found to effectively prevent the leakage of cells into S phase. Reversal of the FdU block by supplying thymidine resulted in the synchronous onset of initiation at multiple sites in each cell. The length of the nascent chains, as determined by autoradiography and velocity sedimentation in alkaline gradients, increased linearly with time during the first twenty minutes of S phase after release. — We applied these procedures to study the effects of the length of an FdU block on the number of functional origins per cell, the rate of chain growth, and the rate of DNA synthesis per cell following reversal of the block. Although no change was noted in the rate of DNA synthesis in cells held at the beginning of S phase from 10.5 to 24 h after division, the rate of chain growth decreased from 0.94 to 0.28 microns per min. This decrease indicated that the number of functional origins increased markedly with length of FdU block. The calculated number of utilized origins per cell increased from 1,900 to 5,700. We also presented arguments that 1,900 origins per cell represents the approximate number of origins utilized by any cell held at the beginning of S phase for less than 10.5 h after division.     

Effects of 3-aminobenzamide on DNA synthesis and cell cycle progression in Chinese hamster ovary cells

3-Aminobenzamide (3AB), an inhibitor of poly(ADP-ribose) polymerase, is a potent inducer of sister chromatid exchanges (SCEs). Because of the possible relation between SCEs and DNA synthesis, the effects of 3AB on DNA synthesis and cell cycle progression in Chinese hamster ovary (CHO) cells were examined. Unlike all other SCE-inducing agents whose effects on DNA synthesis have been studied, short term exposures (30–120 min) of 3AB did not inhibit the overall rate of DNA synthesis and this result was independent of the amount of bromodeoxyuridine (BrdU) in the DNA. Longer exposure times (>24 h) did result in an extended S phase, but this was not due to an effect on the rate of DNA chain elongation. 3AB also delayed the entry of cells into S phase. The overall cell cycle delay was dose dependent, approaching 9 h after a 54 h exposure to 10 mM 3AB. Earlier reports that 3AB is neither mutagenic nor cytotoxic were confirmed. Thus 3AB acts to increase SCE frequency by a mechanism distinct from that which causes cytotoxicity and mutagenicity, and does not involve any inhibition in the rate of DNA chain growth.     

Dna Synthesis Rate Changes During the S Phase In Mouse Epidermis

The in vivo DNA synthesis rate throughout the S phase of mouse epidermal cells was investigated. Epidermal basal cells were isolated at various times of the day from normal animals injected with [³H]TdR 30 min before sacrifice, and from pulse-labelled animals with regenerating and growth-inhibited epidermis. the cells were analysed by DNA flow cytometry combined with cell sorting. Cells from successive fractions of the S phase were sorted on glass slides and subjected to quantitative [³H]TdR autoradiography. The results confirmed the presence of unlabelled (slowly replicating) cells in the S phase, the proportion of which was circadian stage-dependent with minimum values at midnight and in the early morning. the DNA synthesis rate throughout the S phase showed a general trend with high values in the mid-fractions, a pattern which was similar in normal and in growth perturbed epidermis. In the early morning the DNA synthesis rate pattern was bimodal with maxima both in the first and second half of the S phase, with a corresponding trough in mid-S. At this time of day the cell progression rate through S is at its maximum, indicating a relationship between the overall DNA synthesis rate and the rate distribution pattern through S.     

DNA replication in asynchronous and synchronous ehrlich ascites cells in different conditions of growth

Ehrlich ascites tumour cells were labelled for DNA fibre autoradiography within the peritoneal cavity of a tumour-bearing mouse. The generation and the evaluation of the autoradiographic patterns is described and discussed. To study possible changes of the autoradiographic patterns during a natural S phase the labelling was performed in the mouse or in culture with asynchronous cells which were afterwards separated into synchronous subpopulations by zonal centrifugation. The subpopulations obtained were characterized by flow cytofluorometry in connection with the thymidine labelling index. We compared the DNA fibre autoradiographic patterns of several synchronous and asynchronous cell populations growing in the mouse or under different conditions in culture: The replicon size distributions of all populations examined were virtually the same. The fork movement rate was found to depend mainly on the metabolic condition of the cells. In culture it was significantly slower than in the mouse although a shortened S phase and therewith an increased DNA synthesis rate occurred. During a natural S phase it increased slightly, at most, while the DNA synthesis rate was considerably enhanced at the end of S. The changes in the rate of total DNA synthesis cannot account for the changes in the rate of chain growth. We conclude that the DNA synthesis rate is regulated almost exclusively by changing the replicon initiation frequency, while the fork movement rate is limited by the actual metabolic condition of the cells.     

Histone protein and DNA synthesis in HeLa cells after thermal shock

Exposure of suspension-cultured HeLa cells to a 45° thermal shock resulted in cell inactivation and inhibition of both protein and DNA synthesis. DNA synthesis was inhibited in a biphasic manner with a more sensitive (D₀ = 7 min) and a less sensitive (D₀ = 20 min) phase. The less sensitive process was demonstrated to be DNA chain elongation. Transport of thymidine into intracellular pools was significantly less sensitive to thermal shock (D₀ in excess of 200 min). When HeLa cells were heated at 45° for 15 min there was an 80% inhibition of incorporation of precursors into both DNA and protein with little effect on precursor transport into cellular pools. While the rate of synthesis of whole cell and histone protein (H2a, H2b, H3, and H4) and DNA chain elongation recovered by 6 h after cell heating, total precursor incorporation into DNA was only 0.4 of control levels. The long-term depression of the DNA synthetic rate could not be explained by a cell cycle redistribution, a depression in the total fraction of S phase cells synthesizing DNA, or by a depression in the rate of DNA chain elongation. We conclude that thermal shock results in a long-term depression in the fraction of cell replicons involved in DNA replication.     

The abortive synthesis of new DNA chains in Ehrlich ascites tumour cells treated with nitrogen mustard (HN2)

Nitrogen mustard (HN2)-sensitive Ehrlich ascites tumour cells, exposed to HN2 in vivo, showed an inhibition of DNA synthesis which increased with dosage. The effects of alkylation involved at least three distinct components: (1) interference with new 9S DNA chain formation, (2) slowing of the rate of chain growth and (3) loss of newly formed short chains. The dominant effect seemed to be abortive synthesis of new 9S chains; this effect could account for most of the inhibition of DNA synthesis if an initial rapid synthesis of 9S DNA were accompanied by an initial rapid rate of destruction of these chains. By relating the known frequencies of guaninyl alkylations to the postulated ‘replicon’ size observed in control experiments, it appears that only difunctional alkylation frequencies can be directly correlated with the inhibition of discontinuous DNA synthesis by HN2, Mechanistically, this could reflect interference of di-guaninyl alkylations with the integration of 9S chains into 30S, 44S and higher molecular weight species of DNA by ligases. The resulting obstructed short chains, ≦ 9S, might be exposed and so be vulnerable to destruction by the increased nuclease activities expected after alkylation.     

DNA replication in methotrexate-treated human lymphoblasts.

The rate of DNA synthesis in cultures of human lymphoblasts decreased more than 80% within 30 min after the cells were exposed to methotrexate, a potent inhibitor of dihydrofolate reductase. Despite this rapid initial inhibition, DNA continued to be synthesized for at least an additional 6 h. The mode of this subsequent replication appeared to be semiconservative, as indicated by the buoyant density of 5-bromodeoxyuridine-substituted DNA in alkaline CsCl gradients. The growth rates of DNA chains in cells exposed to methotrexate were determined by sedimentation rate analysis in alkaline sucrose gradients. DNA synthesized during 2-min or 10-min pulses with labeled deoxycitidine in the presence of methotrexate had about the same sedimentation coefficient, 35 S, as controls. When methotrexate-treated cultures were pulse-labeled for 10 min and then chased for various times, DNA fragments of about 80 S accumulated. DNA synthesized in the presence of methotrexate was stable and elongated to bulk-size DNA after methotrexate inhibition of growth was removed by addition of thymidine and deoxycytidine. The data suggest that methotrexate reduces the rate of DNA replication by inhibiting chain initiation independently of chain elongation.     

The comparative effects of short-term DNA inhibition on replicon synthesis in mammalian cells

The inter-replicon distance (ID) and rate (R) of DNA chain growth along the replicon were investigated with a [³H]TdR pulse-chase protocol in DNA autoradiographs of cells from seven different cultures of mammalian cells from various species. Asynchronous cultures were labelled with or without a 4 h pretreatment with the DNA inhibitor 5-fluorodeoxyuridine (FUdR). DNA inhibition was found to reduce both the mean ID and R by different amounts in the different cultures. This reduction appeared to correlate with the effectiveness of the inhibition in reducing cell viability. These findings generally could account for the considerable variability found in published data where FUdR pretreatment has been used. When individual values of ID and R in units of μm are plotted against each other, their relationship is given by the mean linear regressions: R = 0.26 ± 0.04 + (0.88 ± 0.05) 10⁻²ID for control, and R = 0.16 ± 0.04 + (1.04 ± 0.06) 10⁻²ID for FUdR-pretreated cultures.The relationship between ID and R in both sets of cultures suggests the presence of a regulating mechanism within a cell which maintains a relatively constant overall rate of chain growth over long stretches of DNA. A mechanism involving changes in the levels of various DNA replication complexes is suggested as one explanation for this relationship.     

Study of Genetic Variability Among Monopycnidial and Monopycnidiospore Isolates Derived from Single Pycnidia of Stagonospora ssp. and Septoria tritici with the use of RAPD-PCR, MP-PCR and rep-PCR Techniques

Thirty-six isolates of Stagonospora avenae f. sp. triticea, S. nodorum and Septoria tritici recovered from asexual fruiting bodies - pycnidia and their spores were assessed for DNA polymorphism with the use of three molecular techniques: microsatellite-primed polymerase chain reaction (MP-PCR), primers correspond to dispersed repetitive elements (rep-PCR) and random amplified polymorphic DNA (RAPD-PCR). These polymerase chain reaction (PCR)-based techniques were simultaneously evaluated for their capacity to detect genetic variation at DNA level. The most polymorphic DNA profiles of monopycnidial and monopycnidiospore isolates were detected with diverse microsatellite motifs used for PCR priming. The lowest similarity values 0.86, 0.76 and 0.84 were identified among monopycnidiospore isolates derived from the same pycnidium of S. avenae f. sp. triticea, S. nodorum and S. tritici, respectively. The above, rather low similarities, found for isolates recovered from single pycnidia, supported a hypothesis that heterokaryosis resulted from high mutation rate of microsatellites and transposons activity. This would have fundamental consequences for the genetic status of asexual populations of Stagonospora spp. and S. tritici. The data produced by this study indicate that more attention should be paid to asexual reproduction as a possible source of genetic variability among populations of the pathogens.     

Rapid detection of viable bacteria by nested polymerase chain reaction via long DNA amplification after ethidium monoazide treatment

In assays to determine whether viable cells of Enterobacteriaceae are present in pasteurized milk, the typical ethidium monoazide (EMA) polymerase chain reaction (PCR) targets a short stretch of DNA. This process often triggers false-positive results owing to the high level of dead cells of Enterobacteriaceae that had initially contaminated the sample. We have developed a novel, direct, real-time PCR that does not require DNA isolation (DQ-PCR) to detect low levels of cells of Enterobacteriaceae regardless of live and dead cells first. We confirmed that the DQ-PCR targeting a long DNA (the 16S ribosomal RNA [rRNA] gene, amplified length of 1514 bp) following EMA treatment is a promising tool to detect live bacteria of all genera owing to the complete suppression of background signal from high levels of dead bacteria in pasteurized milk. However, when identifying viable bacteria in pasteurized milk, commercial PCR primers designed for detecting long stretches of DNA are generally not available. Thus, we treated samples with EMA and then carried out an initial round of PCR of a long stretch of DNA (16S gene, 1514 bp). We then performed another round of PCR, a novel nested PCR to generate short products using commercial primers. This procedure resulted in the rapid detection of low levels of viable cells of Enterobacteriaceae.     

DNA replication in synchronized cultured mammalian cells : VI. The temporal replication of ribosomal cistrons in synchronized cell lines

The time of synthesis of ribosomal genes was studied in a haploid (Rana pipiens), and a pseudodiploid (Chinese hamster) cell line. R. pipiens cells were synchronized by amethopterin block. Chinese hamster cells were synchronized by isoleucine starvation followed by hydroxyurea treatment. DNA replicated during three or four selected intervals of the S period was separated from the remainder of the DNA by bromodeoxyuridine density labeling. Purified bromodeoxyuridine substituted DNA was annealed with radioactive-labeled 28S ribosomal RNA (rRNA) to determine when, during different intervals of S, the nuclear DNA homologous to rRNA was replicated. In the R. pipiens and Chinese hamster cell lines, the percent of nuclear DNA homologous to 28S rRNA is highest in the DNA replicated during the first half of the S period.     

Methionine biosynthesis in Saccharomyces cerevisiae: mutations at the regulatory locus ETH2. I. Genetic data

Summary Evidence has been obtained that sodium azide is an inhibitor of cell division in wild-type and aziA strains of Salmonella typhimurium. The bacteria grown in media containing sodium azide and glucose formed long filaments. It has been found that sodium azide had a stronger inhibitory effect on DNA synthesis than on cell mass increase. When filaments produced by azide action were transferred to azide-free medium very rapid increase in DNA content was observed during the first 45 min. After this time, when relative DNA content was increased the rate of DNA synthesis was reduced and cell divisions reappeared.Inhibitory effect of azide on DNA biosynthesis in vitro was observed with toluenized cells of S. typhimurium. Only ATP-dependent radioactive dTMP incorporation into DNA was affected by sodium azide. It had no effect on the incorporation in the absence of ATP.Mutant aziC was isolated in S. typhimurium by scoring for clones with normal cell division in the presence of sodium azide. Azide had much less effect on DNA biosynthesis in vivo and in vitro in aziC cells as compared with isogenic controls.This work was supported by the Polish Academy of Sciences within the project 09.3.1., and by the U.S. Public Health Service, grant No. 05-032-1.     

First molecular identification of Sarcocystis miescheriana (Protozoa, Apicomplexa) from wild boar (Sus scrofa) in Iran

Sarcocystis isolate obtained from the thigh muscle of a wild boar (Sus scrofa), captured from Gilan Province, northern Iran, was subjected to molecular analysis. Genomic DNA was obtained using a DNA extraction tissue kit and Polymerase chain reaction (PCR) for amplification of the 18S ribosomal DNA region yielded an 842 bp DNA band on agarose gel. Analysis of DNA sequencing by BLAST confirmed the isolate as Sarcocystis miescheriana and the sequence was deposited in GenBank by Accession No. GU395554. This is the first molecular identification of an isolate of S. miescheriana in Iran.     

Involvement of Hus1 in the chain elongation step of DNA replication after exposure to camptothecin or ionizing radiation

DNA damage-induced S phase (S) checkpoint includes inhibition of both replicon initiation and chain elongation. The precise mechanism for controlling the two processes remains unclear. In this study, we showed that Hus1-deficient mouse cells had an impaired S checkpoint after exposure to DNA strand break-inducing agents such as camptothecin (CPT) (≥1.0 µM), or ionizing radiation (IR) (≥15 Gy). The Hus1-dependent S checkpoint contributes to cell resistance to CPT. This impaired S checkpoint induced by CPT or IR in Hus1-deficient cells reflected mainly the chain elongation step of DNA replication and was correlated with the reduction of dissociation of PCNA from DNA replication foci. Although Hus1 is required for Rad9 phosphorylation following exposure of cells to CPT or IR, Hus1-deficient cells showed normal activation of ATR/CHK1 and ATM kinases at doses where the checkpoint defects were manifested, suggesting that Hus1 is not a component of the sensor system for activating these pathways in S checkpoint induced by CPT or IR.     

RADIOAUTOGRAPHIC STUDIES IN SYNCHRONIZED CULTURES OF OEDOGONIUM CARDIACUM

Synthesis of deoxyribonucleic acid (DNA) in synchronized cultures of Oedogonium cardiacum has been studied by radioautography. The germinated zoospores are pulse-labelled for 15 min with thymidine-2-C¹⁴. Use of penicillin in the medium reduces the background in the radioautographs by suppressing the growth of bacteria on Oedogonium cells. Incorporation of labelled precursor is greatly enhanced by growing the cells in a conditioned inorganic medium. The length of the DNA synthesis period (S), as estimated from the curve of percentage of labelled cells versus age during the first cell cycle is about 5-7 hr. The rate of labelling in the nucleus is non-uniform showing a dip during the mid S period. Concomitant with the doubling of DNA in the nucleus a fourfold increase in the nuclear volume is observed.     

Replication of chromosomal DNA in diploid Drosophila melanogaster cells cultured in vitro

Replication rate and replicon sizes in chromosomal DNA of in vitro cultured diploid D. melanogaster cells were determined using autoradiography of ³H-thymidine labeled DNA. Synthesis of DNA in euchromatic and heterochromatic regions of Drosophila diploid cells occurs at different periods of the S phase which lasts 10 h. During the first 4 h the synthesis is observed only in euchromatic regions. The heterochromatic synthesis starts shortly before the synthesis in euchromatic regions is completed and lasts for 6 h until the end of the S phase. The cells were synchronized by 5fluorodeoxyuridine which blocked the diploid cell DNA synthesis. Synthesis was found to start simultaneously in most euchromatic replicons. In the majority of the replicons the synthesis started at a single point and proceeded bidirectionally. The average rate of DNA synthesis per fork was 12.5 m/h (38 kb). The mean distance between the middle points of adjacent labeled regions was 70 m (210 kb). The size of most replicons ranged from 40 to 120 m. — These estimates do not apply to the heterochromatic portions of the D. melanogaster genome since the measurements have been carried out on DNA preparations obtained during the first 2 h of the S phase. — On the average, a replicon can consist of 7 chromomeres since the size of a replicon in diploid cell chromosomal DNA and DNA length of a polytene chromomere average 210 and 30 kb, respectively.     

A single‐molecule approach to DNA replication in Escherichia coli cells demonstrated that DNA polymerase III is a major determinant of fork speed

The replisome catalyses DNA synthesis at a DNA replication fork. The molecular behaviour of the individual replisomes, and therefore the dynamics of replication fork movements, in growing Escherichia coli cells remains unknown. DNA combing enables a single‐molecule approach to measuring the speed of replication fork progression in cells pulse‐labelled with thymidine analogues. We constructed a new thymidine‐requiring strain, eCOMB (E. coli for combing), that rapidly and sufficiently incorporates the analogues into newly synthesized DNA chains for the DNA‐combing method. In combing experiments with eCOMB, we found the speed of most replication forks in the cells to be within the narrow range of 550–750 nt s^?1 and the average speed to be 653 ± 9 nt s^?1 (± SEM). We also found the average speed of the replication fork to be only 264 ± 9 nt s^?1 in a dnaE173‐eCOMB strain producing a mutant‐type of the replicative DNA polymerase III (Pol III) with a chain elongation rate (300 nt s^?1) much lower than that of the wild‐type Pol III (900 nt s^?1). This indicates that the speed of chain elongation by Pol III is a major determinant of replication fork speed in E. coli cells.