Nothing is more fundamental to life than the ability to reproduce and duplicate theinformation cells store in their genomes. The mechanism of duplication of DNA has beenconserved from prokaryotes to eukaryotes. The aim of the study was to quantify whichevolutionary forces could produce the pattern of genome replication architecture observedin present-day organisms. This was achieved using an evolutionary simulation, combiningrandom genome sequence shuffling, mutation, selection and the mathematical modeling of DNAreplication. We have found parameter values which explained evolutionary pressures of DNAreplication in E.coli, P.calidifontis and S.cerevisae. Surprisingly, the results of the evolutionary simulation suggeststhat for a fixed cost per replication origin it is more advantageous for genomes to reducethe number of replication origins under increasing uncertainty in origin activationtiming.

How does DNA, the molecule containing genetic information, change its three-dimensionalshape during the complex cellular processes of replication, recombination and repair? Thisis one of the core questions in molecular biology which cannot be answered without helpfrom mathematical modeling. Basic concepts of topology and geometry can be introduced inundergraduate teaching to help students understand counterintuitive complex structuraltransformations that occur in every living cell. Topoisomerases, a fascinating class ofenzymes involved in replication, recombination and repair, catalyze a change in DNAtopology through a series of highly coordinated mechanistic steps. Undergraduate biologyand mathematics students can visualize and explore the principles of topoisomerase actionby using easily available materials such as Velcro, ribbons, telephone cords, zippers andtubing. These simple toys can be used as powerful teaching tools to engage students inhands-on exploration with the goal of learning about both the mathematics and the biologyof DNA structure.

A plastid-like organelle, the apicoplast, is essential to the majority of medically and veterinary important apicomplexan protozoa including Toxoplasma gondii and Plasmodium. The apicoplast contains multiple copies of a 35 kb genome, the replication of which is dependent upon nuclear-encoded proteins that are imported into the organelle. In P. falciparum an unusual multi-functional gene, pfprex, was previously identified and inferred to encode a protein with DNA primase, DNA helicase and DNA polymerase activities. Herein, we report the presence of a prex orthologue in T. gondii. The protein is predicted to have a bi-partite apicoplast targeting sequence similar to that demonstrated on the PfPREX polypeptide, capable of delivering marker proteins to the apicoplast. Unlike the P. falciparum gene that is devoid of introns, the T. gondii prex gene carries 19 introns, which are spliced to produce a contiguous mRNA. Bacterial expression of the polymerase domain reveals the protein to be active. Consistent with the reported absence of a plastid in Cryptosporidium species, in silico analysis of their genomes failed to demonstrate an orthologue of prex. These studies indicate that prex is conserved across the plastid-bearing apicomplexans and may play an important role in the replication of the plastid genome.

Prototheca richardsi, an unpigmented heterotrophic alga, causes growth inhibition in amphibian larvae and has proved refractory to culture in Vitro. P. richardsi replication is dependent on regular passaging through tadpole digestive systems; uptake of thymidine by free-living Prototheca cells and incorporation into DNA are very low by comparison with leucine uptake and incorporation into protein, but DNA synthesis is detectable in cells isolated from tadpole intestines. DNA replication was elicited 6–8 h after ingestion in protothecans fed to tadpoles and subsequently re-isolated from them, providing that the tadpoles were fed subsequent to the ingestion. It appears that passaging through tadpole intestines provides an essential stimulus to maintaining an active cell division cycle in P. richardsi.

Immature mouse oocytes (germinal vesicle stage, GV), oocytes at different stages during maturation (prometaphase to anaphase I) and matured oocytes (metaphase II arrested) were cultured in 6-dimethylaminopurine (6-DMAP)-supplemented medium also containing bromodeoxyuridine for the assessment of DNA replication in these cells. Immature oocytes remained arrested at the GV stage and DNA replication was never detected in them. On the other hand, oocytes at the prometaphase to anaphase-telophase I stages responded to 6-DMAP treatment by forming nuclei which synthesised DNA. Mature (metaphase II) oocytes did not respond to 6-DMAP and their chromatin remained condensed. DNA synthesis could even be induced in GV-staged oocytes, but only when they were fused to freshly activated oocytes and incubated in 6-DMAP-supplemented medium.

We address the developmental activation, in the zebrafish embryo, of intrinsic cell-cycle checkpoints which monitor the DNA replication process and progression through the cell cycle. Eukaryotic DNA replication is probably carried out by a multiprotein complex containing numerous enzymes and accessory factors that act in concert to effect processive DNA synthesis (Applegren, N. et al. (1995) J. Cell. Biochem. 59, 91–107). We have exposed early zebrafish embryos to three chemical agents which are predicted to specifically inhibit the DNA polymerase α, topoisomerase I and topoisomerase II components of the DNA replication complex. We present four findings: (1) Before mid-blastula transition (MBT) an inhibition of DNA synthesis does not block cells from attempting to proceed through mitosis, implying the lack of functional checkpoints. (2) After MBT, the embryo displays two distinct modes of intrinsic checkpoint operation. One mode is a rapid and complete stop of cell division, and the other is an ‘adaptive’ response in which the cell cycle continues to operate, perhaps in a ‘repair’ mode, to generate daughter nuclei with few visible defects. (3) The embryo does not display a maximal capability for the ‘adaptive’ response until several hours after MBT, which is consistent with a slow rranscriptional control mechanism for checkpoint activation. (4) The slow activation of checkpoints at MBT provides a window of time during which inhibitors of DNA synthesis will induce cytogenetic lesions without killing the embryo. This could be useful in the design of a deletion-mutagenesis strategy.

Freshly harvested tomato (Lycopersicon esculentum Mill, cv. Moneymaker) seeds were osmotically primed for 8 d in −1.0 MPa PEG-6000 solution and dried to about 6% water content for storage. Such so–called ‘fresh PEG priming’ enhanced seed germination and improved seedling performance as compared with the untreated control. Fresh PEG priming neither alleviated seed dormancy nor promoted DNA replication as was the case when seeds were dried upon harvest and subsequently primed in PEG (normal PEG priming). However, the addition of 10 μM GA4+7 to the osmotic priming solution triggered replicative DNA synthesis of fresh-priming seeds and further enhanced the germination process. After 5 months of storage in ambient temperature conditions, fresh PEG-primed seeds maintained more positive effects gained from priming, whereas, normal PEG-primed seeds had lost the promoting effects on germination. Normal PEG-primed seeds were much more susceptible to controlled deterioration than fresh PEG-primed seeds. It is suggested that the advancement of germination is negatively correlated with seed storability. The mechanisms of seed priming in relation to nuclear replication activities and physical changes are discussed.

Links between germination, DNA replication and β-tubulin accumulation were studied with cabbage (Brassica oleracea L.) seeds, by using flow cytometric analysis of nuclear DNA content and immunodetection of β-tubulin levels. The seeds were incubated in water or 0.1–500 mM hydroxyurea solutions. Radicle tips isolated from dry cabbage seeds revealed most 2C (Go or G1 stage) and some 4C (G2 stage) signals of nuclear DNA contents and a constitutive level of β-tubulin. The onset of DNA replication in the radicle tip was observed between 12 and 24 h of imbibition in water and was preceded by an increase of β-tubulin levels. Incubation of the seeds in 1 mM hydroxyurea retarded DNA replication, whereas an arrest of DNA replication occurred upon incubation in 10 mM hydroxyurea or higher concentrations. The arrest of DNA replication and cell division did not block radicle protrusion and allowed some extension of the radicle. This demonstrated that DNA replication is not a prerequisite for radicle protrusion and initial extension. However, further seedling development, including root growth and root hair development, was dependent on DNA replication. Accumulation of β-tubulin was not affected by hydroxyurea. Thus, it can be deduced that both DNA replication and β-tubulin accumulation are two parallel and independent cell cycle events during seed germination.

Using flow cytometry, we determined relative DNA amounts expressed as C-values, of large numbers of nuclei isolated from embryo root tips in three seed lots of pepper (Capsicum annuum) and tomato (Lycopersicon esculentum). The nuclei in embryos of fully matured dry pepper and tomato seeds predominantly revealed 2C signals, which indicates that the cells of maturing seeds have arrested at G1 phase of the cell cycle. Priming of pepper and tomato seeds in −1.1, −1.3 or −1.5 MPa polyethylene glycol (PEG) for 14 days reduced the mean time to germination. In all seed lots an induction of 4C signals was found after priming, indicating that during priming the cells of the embryonic root tip had replicated their DNA and arrested at the G2 phase of the cell cycle. For both tomato and pepper, the frequency of 4C signals was highest at the lowest PEG concentration (osmotic potential of −1.1 MPa). A significant inverse correlation was found between the frequency of root tip cells expressing 4C DNA signals and the mean time to germination for each individual pepper and tomato seed lot. However, the effect of a specific osmotic treatment differed between seed lots of the same cultivar, with regard to both the decrease in the mean time to germination and to the percentage of 4C cells, suggesting specific differences among seed lots in the response to priming. The present results indicate that flow cytometry can be used to follow the progress in nuclear replication activities during priming. The advancement in DNA synthesis can thus be used to measure the efficiency of a priming treatment with regard to enhancement of seed lot germination rate.

The effect of two priming treatments in PEG solutions on nuclear replication stages of pepper (Capsicum annuum L.) embryos were evaluated using flow cytometry. Priming at −1.1 MPa for 10 d induced almost 40% of nuclei in the embryo root tips to enter the synthetic phase of nuclear division, while no DNA synthesis occurred during 6 d at−1.5 MPa and in less than 10% of the nuclei at 10 d. Both priming treatments effectively increased seed performance. A decrease of 2.7 d in mean germination time (MGT) was observed after priming in PEG solution at −1.1 MPa, while the treatment at−1.5 MPa lowered the MGT by 2.1 d. Primed and untreated seeds were subjected to controlled deterioration treatments by exposure to 45°C after equilibration at 75% RH. Both osmotic treatments considerably lowered seed tolerance to adverse storage conditions as compared with untreated seeds. However, seeds in which DNA replication was induced by priming were more sensitive to controlled deterioration than seeds in which priming did not induce nuclei to enter the synthetic phase. This could be a consequence of a higher DNA content, which is a more vulnerable target for mutation inducing factors. Alternatively the increased sensitivity could be a consequence of their more advanced progress in germinative events, making seeds less resistant to deteriorative factors imposed during storage. On the whole, from the present results it appears that the nucleic acid DNA content of the embryo only plays an additive role in influencing seed storability.

Storage of tomato seeds under deteriorating conditions reduced the rate of germination, the uniformity of germination, total germination and the proportion of normal seedlings. Pre-storage humidification and hydropriming of seeds resulted in a significant increase in resistance to deterioration. In contrast, osmoprimed seeds were more deterioration sensitive. Humidification or hydropriming for one day did not allow nuclei to enter the S phase of the cell cycle. Osmopriming strongly increased the percentage of nuclei with replicated DNA in the embryonic root tip, indicating initiation of the cell cycle and progression towards the G2 phase. The interaction between the prestorage treatments, cell cycle progression and deterioration resistance, is discussed. Based on the changes in moisture content equilibrium and cell cycle activity it is hypothesized that the beneficial effects of pre-storage humidification and hydropriming were related to metabolic activities induced by the partial hydration and that the adverse effects of osmopriming were caused by a decrease in DNA repair activity due to progression in the cell cycle.

Cell cycle activity in dry and germinating untreated and treated (soaked in water and subsequently in fungicide) seeds of two sugarbeet cultivars, collected at commercial harvest time (late mature seeds) and about 2 weeks before this (immature seeds), was investigated by flow cytometry, and by immuno-detection of β-tubulin and the B-subunit of the 11 S globulin. Germination capacity and field emergence were tested. With dry seeds of both cultivars, higher G2 / G1 ratios were observed in the radicle tips of late mature seeds, as compared with those from immature seeds. The late mature seeds contained more partly degraded (soluble) B-subunit of 11 S globulin, typical of germinating or primed sugarbeet seeds. Thus events associated with the onset of germination had occurred in the seed lots collected at commercial harvest time. The cytoskeleton protein β-tubulin was not detectable in dry seeds from either harvest. Western blotting revealed an accumulation of β-tubulin during germination and this was faster in the late mature harvested seeds which was correlated with the onset of DNA replication. Soaking enhanced the rate of cell cycle activation during germination as well as vigour, germination capacity, and field emergence. There was positive correlation between the G2 / G1 ratio and the traits examined in laboratory and field tests. It is concluded that a combined analysis of proteins and cell-cycle-related events can be used in understanding and predicting sugarbeet seed quality.

The correlation between physiological markers for seed maturity (acquisition of germinability, definitive morphological traits and desiccation tolerance) and timing of the shutdown of nuclear replication activity and β-tubulin synthesis has been investigated in developing pepper seeds. Nuclear replication activity was analysed by flow cytometry. Expression of β-tubulin, a cell-cycle related structural protein, was detected in western blots of total protein extracts. In pepper embryos, DNA synthesis stopped 42–49 days after anthesis (DAA), at which time seeds started to acquire desiccation tolerance. A clear β-tubulin signal was detected up to 63 DAA, only 1 week before the acquisition of complete desiccation tolerance. Both nuclear replication and β-tubulin synthesis can therefore be used as markers for the physiological stage reached by developing pepper seeds. Detection of β-tubulin signals in embryonic tissues can also be helpful in discriminating immature seeds within a commercial seed lot.

We find explicit analytical formulae for the time dependence of the probability of the number of Okazaki fragments produced during the process of DNA replication. This extends a result of Cowan on the asymptotic probability distribution of these fragments.

It has been demonstrated that in the zygotes of some mammals a unique checkpoint controls the onset of DNA replication. Thus, DNA replication begins in the maternal pronucleus only after the paternal pronucleus is fully formed. In our experiments we have investigated whether this checkpoint also operates in porcine zygotes produced either by in vitro fertilization (IVF) or by intracytoplasmic sperm injection (ICSI). Our results show that the onset of DNA replication occurs in the maternal pronucleus even in the presence of an intact sperm head in zygotes produced by ICSI, as well as in polyspermic eggs where some sperm heads are intact or male pronuclei are not yet fully developed. We conclude that in porcine zygotes there is an absence of the DNA replication checkpoint that is typical for some other mammals.

This study reports that inhibition of Leishmania Topo-I with the minor groove-binding ligands (MGBLs) Hoechst 33342 (Ho342) blocks parasite growth in culture by mechanisms involving DNA breakage. While Ho342 inhibited the replication of several species of Leishmania in a dose- and time-dependent manner, Ho258 was not effective. Cytofluorometric analysis suggested that superior effectiveness of Ho342 over Ho258 was attributed to Leishmania parasites being more permeable toward Ho342. This observation was supported by the ability of both Ho342 and Ho258 to block the relaxation of supercoiled pBR322 DNA by Leishmania Topo-I. The Ho342 specificity toward L. donovani Topo-I was reinforced by the observation that increased Topo-I gene expression and Topo-I activity in Leishmania was paralleled by augmented resistance for this compound. Furthermore, the capacity of NaCl treatment to reverse MGBL-mediated DNA break suggests that Ho342 targetted Topo-I. Moreover, we observed that Ho342-inducible arrest of Leishmania growth was accompanied by G1 arrest and induction of cell death that closely resembles apoptosis. Taken together, our results suggest that MGBL compounds show promise as Topo-I inhibitors against Leishmania infection.

Thymocyte nuclei were microinjected into the cytoplasm of parthenogenetic mouse eggs within 60 min or 3 h after egg activation and DNA replication and RNA synthesis were analysed in remodelled thymocyte nuclei and female pronuclei. We show that thymocyte nuclei which transform into pronucleus-like nuclei (thymocytes injected not later than 60 min after activation) enter S-phase 1 h earlier than the female pronuclei. At the beginning of the first cell cycle they remain transcriptionally silent, but in G2 undertake transcription earlier than the female pronuclei. Partly remodelled thymocyte nuclei (injected 3 h after activation) start to replicate DNA at the same time as the female pronuclei. They reinitiate RNA synthesis within 2 h after transfer and continue to transcribe irrespective of the transcriptional activity of the female pronucleus. We show that the observed transcription is only nuclear, i.e. RNA polymerase II-dependent.

In the course of the cell cycles of synchronous cultures of the chlorococcal alga Scenedesmus quadricauda, the following were monitored: total protein and RNA accumulation as a measure of growth processes, the timing of the commitment points at which the cells trigger the sequence of reproductive processes (DNA replication, nuclear and cellular division) and the course of the reproductive processes. The synchronous cultures were grown either under various lighting regimes, or in the temporary presence of specific inhibitors of either proteosynthesis (cycloheximide) or DNA replication (5-fluorodeoxyuridine). By adjusting the length of the light period, the cell cycle could be manipulated. Cell cycle patterns could be altered to give different numbers of sequences of reproductive processes. The extent of their mutual overlap could be influenced and the number of daughter cells produced could be altered. Schematic illustrations of various cell cycle patterns and comparisons with those of higher plants and other algal species are presented.

During DNA replication, small fragments of DNA are formed. These have been observed experimentally and the mechanism of their formation modelled mathematically. Using the stochastic model of Cowan and Chiu (1992), (1994), we find the probability distribution of the number of fragments. A new discrete distribution arises. The work has interest as an application of the recent theory on quasirenewal equations in Piau (2000).