Ultrastructure of meristematic cells of dormant and released buds in Tradescantia paludosa

The lateral bud meristems of Tradescantia paludosa show a characteristic cytohistological zonation during dormancy. The cells comprising this so called ‘zone of inhibition’, which is located at the extreme tip of the bud apex, rarely synthesize nuclear DNA or undergo mitotic division. These nuclei are as large as prophase nuclei, yet contain only telophase (2C) amounts of DNA and significantly lower amounts of histone as compared to the 2C nuclei of the actively dividing cells.Ultrastructural observations of the nuclei in the ‘zone of inhibition’ show that a large proportion of the chromatin is organized as less condensed, diffuse, euchromatin fibrils; however, the chromatin of the actively dividing nuclei of the cells outside the ‘zone of inhibition’ or in the released bud meristems is organized to a greater extent as condensed clumps of heterochromatin. When the dormancy is released, the nuclei in the ‘zone of inhibition’ synthesize DNA and histone and undergo cell division in approx. 4 days. Striking changes in the organization of chromatin fibrils take place during this transition period. The diffuse chromatin fibrils of the nuclei in the ‘zone of inhibition’ progressively become more and more condensed as the cell prepares to undergo the first mitotic division after the release of dormancy. This change which is coupled with the synthesis of histones in the nuclei of the ‘zone of inhibition’ suggests a prominent structural role of these basic proteins in the organization of the chromatin. The large volume of 2C nuclei of the ‘zone of inhibition’ seems, therefore, to result not from a great nuclear mass, but probably from a relatively small degree of condensation of chromatin.     

Early effects of floral induction on cell division in the shoot apex of Silene

The changes in cell number, the relative proportions of interphase nuclei with different amounts of DNA, mitotic index and labelling index have been investigated in the shoot apex of Silene coeli-rosa L. (a long-day plant) during the first long day of photoinduction, and compared with the corresponding changes in plants in short days. 3 h after the start of induction the proportion of nuclei in the G2 phase of the cell cycle had increased, the mitotic index tended to be higher, and the labelling index was lower than in plants in short days. 8–9 h later the values for plants in the long day had become similar to those for plants in short days. No evidence was obtained for a synchronisation of cells in one phase of the cell cycle as a result of photoinduction. The results obtained were consistent with a temporary shortening of the cell cycle in the induced apices over the first long day which resulted in a greater increase in cell number by the end of the first day of photoinduction than in plants in short days.Abbreviations LD long day - SD short day     

Cellular responses of leaf explants of Cocos nucifera L. in vitro

Leaf explants of Cocos nucifera L. (coconut palm) were studied in vitro in order to establish whether or not rapid cellular changes contribute to the well known recalcitrance of coconut cells in tissue culture. Segments from the base of immature leaves were cultured on modified Eeuwens' medium at 30°C in darkness. The mitotic index, nuclear DNA amounts, cell and nuclear size were measured both before and during culture (from 0 to 70 days). There was no basipetal gradient of cell division in immature coconut leaves; the mitotic index never exceeded 2% and showed neither a positional nor temporal relationship with leaf development. Moreover the vast majority of cells were in G1 of the cell cycle. This cell cycle pattern was maintained for most of the period in culture although at 70 days there was an increase in the proportion of cells in S- and G2-phases consistent with low rates of callus formation. The nuclear: cell size ratio was constant in cells within the immature leaf irrespective of developmental age. However upon transfer to culture media, cell size but not nuclear size increased. We suggest that this uncoupling of cell and nuclear size disrupts cell co-ordination and is a key contributor to recalcitrant cellular behaviour of this species in vitro.     

Cyclin A, cyclin B and stringlike are regulated separately in cell cycle arrested trochoblasts of Patella vulgata embryos

 Trochoblasts are the first cells to differentiate during the development of spiralian embryos. Differentiation is accompanied by a cell division arrest. In embryos of the limpet Patella vulgata, the participation of cell cycle-regulating factors in trochoblast arrest was analysed as a first step to unravel its cause. We determined the cell cycle phase in which the trochoblasts are arrested by analysing the subcellular locations of mitotic cyclins. The results show that the trochoblasts are most likely arrested in the G2 phase. This was supported by measurement of the DNA content in trochoblast nuclei after the last division. Trochoblasts complete their final division at the sixth mitotic cycle. This mitotic cycle resembles the first postblastoderm cell cycle of Drosophila, in which mitotic activity is controlled by expression of the string gene. As failure of string expression results in cell cycle arrest in the G2 phase, negative regulation of a Patella string homolog could be responsible for trochoblast arrest. Although Stl messengers disappeared from trochoblasts during their final division, expression was observed again 20 min later. Messengers remained present in all trochoblasts at low levels during further development. Thus, expression of the stringlike gene allows the cell cycle arrest of these cells, whereas in Drosophila cells arrested in division lack string messengers. Received: 10 February 1997 / Accepted: 23 November 1997     

Water deficit in developing endosperm of maize: cell division and nuclear DMA endoreduplication

Water deficit severely decreases maize (Zea mays L.) kernel growth; the effect is most pronounced in apical regions of ears. The capacity for accumulation of storage material in endosperms is thought to he partially determined by the extent of cell division and endoreduplication (post-mitotic nuclear DNA synthesis). To gain a better understanding of the regulatory mechanisms involved, we have examined the effect of water deficit on cellular development during the post-fertilization period. Greenhouse-grown maize was subjected to water-limited treatments during rapid cell division [from 1 to 10days after pollination (DAP)] or rapid endoreduplication (9 to 15 DAP). The number of nuclei and the nuclear DNA content were determined with flow cytometry. Water deficit from 1 to 10 DAP substantially decreased the rate of endosperm cell division in apical-region kernels, but had little effect on middle-region endosperms. Rewatcring did not allow cell division to recover in apical-region endosperms. Water deficit from 9 to 15 DAP also decreased cell division in apical-region endosperms. Endoreduplication was not affected by the late treatment in either region of the car, but was inhibited by the early treatment in the apical region. In particular, the proportion of nuclei entering higher DN A-content size classes was reduced. We conclude that cell division is highly responsive to water deficit, whereas endoreduplication is less so. We also conclude that the reduced proportion of nuclei entering higher DNA-content size classes during endoreduplication is indicative of multiple control points in the mitotic and endoreduplication cycles.     

Characterization of abnormal nuclei in renal proximal tubules after irradiation

The distribution and kinetics of proximal tubular cells with abnormally large nuclei, which were observed in irradiated mouse kidneys before any other obvious histological effects, were investigated. Six months after the administration of 13 or 15 Gy, little histopathological change was noted, in the kidneys of C3H mice; however, proliferation of proximal tubular cells was stimulated, and some of these cells had abnormally large nuclei. The relative DNA content of these large nuclei was measured with a quantitative image analysis system. Most of the large nuclear cells had more than diploid DNA content. The labeling index of the large nuclei was higher than that of unselected proximal tubular nuclei. These cells might be hyperploid cells that are dying after having gone through an abortive mitotic division. Examination and quantitation of these abnormal nuclei should be useful in elucidating the steps involved in cell loss in the proximal tubules after irradiation and as an assay for radiation damage to the kidney.     

SEXUAL REPRODUCTION OF PERIDINIUM WILLEI (DINOPHYCEAE)1

Sexual reproduction was induced in the dinoflagellate Peridinium willei Huitfeld-Kass when exponentially growing cells were inoculated into nitrogen deficient medium. Small, naked vegetative cells produced by division of thecate cells acted as gametes. The zygote remained motile 13–14 days, during which time it enlarged and the theca formed became warty. Fourteen to 15 days following plasmogamy the zygote was nonmotile with the protoplast contracted. A large red oil droplet appeared and the wall thickened, becoming chitinized. Hypnozygotes with 4 nuclei were observed 7–8 wk following formation. Meiosis was inferred. The hypnozygote germinated, within 8 wk producing one post-zygotic cell retaining the red oil droplet. This cell divided within 24 h into 2 daughter cells each with a prominent red oil droplet. These daughter cells divided after 2 to 3 days into ordinary vegetative cells. Attempts to induce sexual reproduction by inoculation of cells into media deficient in a number of basic elements were unsuccessful.     

Image analysis of rat satellite cell proliferation in vitro

Myogenic cells were isolated from adult rat skeletal muscles and cultured in vitro. Cell proliferation was analyzed between days 1 and 14. The cell cycle phases were determined by examining Feulgen-stained cultures with a cell image processor. The nuclei were automatically analyzed by calculating 18 parameters relating to the texture and densitometry of chromatin and the shape of each nucleus. Cell cycle phases were characterized (Moustafa and Brugal, 1984). The recognition methods made it possible to analyse the nuclei of the myogenic cell populations which were either involved in each phase of the mitotic cycle, or left out of the cycle after fusion into myotubes.After 3 hr of culture 10% of the cell population was involved in the cell cycle. In the presence of foetal calf serum, this percentage increased until day 3 after plating. At that time, the DNA content of 28.2% of the cell population was higher than 3C, whereas it is 2C in G1 or G0 nuclei; image analysis showed that 42% of these cells were in S or G2 phase. From day 4, the proliferation rate gradually slowed down until day 8. After day 8, when numerous myotubes differentiated, the percentage of S and G2 phase cells had diminished to between 3 and 8%. The percentage of nuclei in G0 increased when the first myotubes differentiated around day 5. Myotube nuclei were largely in G0. When horse serum was added to the culture medium on day 4 to enhance myotube differentiation, significant cell proliferation was observed before cell fusion.These methods of analysis give the first daily pattern of myogenic cell proliferation and fusion in a cell population isolated from adult muscles.     

Isolation and phenotypic characterization ofChlamydomonas reinhardtii mutants defective in chloroplast DNA segregation

Summary Each wild-typeChlamydomonas reinhardtii cell has one large chloroplast containing several nuclei (nucleoids). We used DNA insertional mutagenesis to isolate Chlamydomonas mutants which contain a single, large chloroplast (cp) nucleus and which we namedmoc (monokaryotic chloroplast). DAPI-fluorescence microscopy and microphotometry observations revealed thatmoc mutant cells only contain one cp-nucleus throughout the cell division cycle, and that unequal segregation of cpDNA occurred during cell division in themoc mutant. One cell with a large amount of cpDNA and another with a small amount of cpDNA were produced after the first cell division. Unequal segregation also occurred in the second cell division, producing one cell with a large amount (about 70 copies) of cpDNA and three other cells with a small amount (only 2–8 copies) of cpDNA. However, most individualmoc cells contained several dozen cpDNA copies 12 h after the completion of cell division, suggesting that cpDNA synthesis was activated immediately after chloroplast division. In contrast to the cpDNA, the mitochondrial (mt) DNA of themoc mutants was observed as tiny granules scattered throughout the entire cell. These segregated to each daughter cell equally during cell division. Electron-microscopic observation of the ultrastructure ofmoc mutants showed that a low-electron-density area, which was identified as the cp-nucleus by immunoelectron microscopy with anti-DNA antibody, existed near the pyrenoid. However, there were no other structural differences between the chloroplasts of wild-type cells andmoc mutants. The thylakoid membranes and pyrenoid were identical. Therefore, we propose that the novelmoc mutants are only defective in the dispersion and segregation of cpDNA. This strain should be useful to elucidate the mechanism for the segregation of cpDNA.Abbreviations DAPI 4,6-diamidino-2-phenylindole - VIMPCS video-intensified microscope photon-counting system     

Hypoploidy and hyperplasia in the developing brain exposed to alcohol in utero

Prenatal effects of acute maternal alcohol ingestion on chromosome segregation and mitotic frequency in the brain cells of the fetus were evaluated in mice by direct chromosome and mitotic counts and by flow cytometry. Fetuses were exposed to acute transplacental doses of alcohol for 4 days and killed on the fifth day. Those litters in which the fetuses were developed to the equivalent of normal 16th-17th-day gestation age were analyzed. A marked increase in the number of hypoploid metaphases was observed in direct proportion to the dose ingested by the mother. An over 30% increase in hypoploidy over controls was measured in the fetuses exposed to the highest dose. Counts of mitotic cells showed an over tenfold increase in the mitotic index of the fetal brain exposed to alcohol. Flow cytometric measurements of DNA content in isolated fetal brain cell nuclei showed a shift from a single G0/G1 peak in controls to a bimodal G0/G1-G2 + M distribution in alcohol-exposed fetuses of the same developmental age.     

Histopathogenesis of Galls Induced by Meloidogyne naasi in Wheat Roots

Histopathogenesis of galls induced by Meloidogyne naasi in wheat roots was studied. Large numbers of larvae penetrated wheat root tips within 24 hr; larvae migrated both inter- and intracellularly, causing cortical hypertrophy. Giant cells were formed in the stele around the head of each nematode within 4 to 5 days. Initial pathological alterations in giant cell formation consisted of hypertrophy of protophloem and protoxylem cells, their nuclei and nucleoli. Giant ceils contained 2 to 8 agglomerated multinucleolate nuclei. Synchronous mitotic divisions were first observed 9 days after inoculation. After 21 days, giant cells became highly vacuolate. Observations 40 days after inoculation revealed a complete degeneration of cell contents in many giant cells but their thick walls remained intact. Abnormal xylem completely surrounded the degenerated or partially degenerated giant cells.     

Occurrence of amitotic division of trophoblast cell nuclei in blastocysts of the western spotted skunk (Spilogale putorius latifrons)

A cytogenetic examination of spreaded cells of diapausing and early activated blastocysts obtained from 7 female western spotted skunks was performed. Mitosis was not observed in 1626 cells obtained from 9 diapausing blastocysts; however, 12 (1.5%) figures of diploid mitosis were seen in 851 cells from 5 early activated embryos. Diameter of the cell nuclei varied from 4 to 29 microm during diapause, and from 5 to 40 microm in activated blastocyst, and the heterogeneity in nuclear size was significantly different between diapausing and activated embryos (P<0.01). About 80% of nuclei from diapausing blastocysts measured 9 to 16 microm, whereas a similar percentage of nuclei from activated blastocysts ranged from 15 to 27 microm. Many enlarged nuclei exhibited morphological features characteristic of mammalian polytene (i.e. endopolyploid with polytenic organization of chromosomes) trophoblast cells. The number of silver stained nucleoli in all the nuclei did not exceed 2, which corresponds to the number of nucleolus organizers in the diploid karyotype in this species of skunk and suggests the polytene organization of chromosomes in enlarged nuclei. About 10% of large interphase nuclei were observed to undergo amitosis, i.e. direct division by constriction. The resulting nuclear fragments in diapausing blastocysts usually had normal morphology and active nucleoli. In activated embryos, nearly 15% of amitotically divided nuclei appeared to be dividing into fragments of unequal size, one of which had normal cell nuclear morphology and extremely large silver positive nucleoli, and the other fragment exhibited signs of cell death. We interpret these data as indicating that 1) amitotic division of trophoblast endopolyploid cell nuclei in the skunk blastocysts may generate new trophoblast cells which contribute to increased cell number during both diapause and activation stages, and 2) activation of blastocysts after diapause is related to the production of trophoblast cells with enhanced synthetic capabilities.     

Light-induced synchrony of cell division in the protonema of the fern,Pteris vittata

Summary In protonemata of Pteris vittata grown for 6 days under red light, which brings about a marked depression of mitotic activity, the first division of the cells was synchronously induced by irradiation with blue light, and subsequent cell divisions were also promoted. The peak of the mitotic index reached a maximum of about 70% at 11.5 hrs, and 90% of all protonemata divided between the 11th and 13th hour after exposure to blue light. When the protonemata were continuously irradiated with blue light, synchronism of the next cell division in the apical cells decreased to a mitotic index of about 30%, and further divisions occurred randomly.The synchronization of cell division was found to be a combined effect of red and blue light. Red light maintained the cells in the early G₁ phase of the cell cycle; blue light caused the cells to progress synchronously through the cell cycle, with an average duration of 12 hr. By using ³H-thymidine, the average duration of the G₁, S, G₂ and M phases was determined to be about 3.5, 5, 2.5 and 1 hr, respectively.Synchronous cell division could be induced in older protonemata grown for 6 to 12 days in red light and even in protonemata having two cells. It could be repeated in the same protonema by reexposure to red light for 24 hrs or more before another irradiation with blue light.     

Regulation of cell division in the subapical shoot meristem of dwarf watermelon seedlings by gibberellic acid and polyethylene glycol 4000

The stimulatory effects of gibberellic acid (GA₃) and the inhibitory effects of polyethylene glycol 4000 (PEG) on hypocotyl elongation and cell cycle kinetics in subapical pith cells of dwarf watermelon seedlings (Citrullus lanatus [Thunb.] Matsu and Nakai) were investigated. Mitotic indices (MI) were determined from direct counts of pith cells stained by a modified Feulgen technique. Labeling indices (LI) were determined from direct counts of labeled pith cells sampled 1.5 h after apical applications of³H-thymidine. Root application of 0.32 mM GA₃ at 96, 120, or 144 h after sowing resulted in significant increases in both mitotic and labeling indices within 4.5 to 7.5 h following treatment. A single mitotic peak at 13.5 h occurred in all three treatment periods. Labeling peaks were often less defined than mitotic peaks; however, a relatively high proportion of labeled nuclei were usually observed between 7.5 and 9 h after GA₃ treatment and at 16.5 h, the latter period coinciding with progression of cells into S phase from the peak period of mitosis. The results suggest that GA₃ increases the proportion of rapidly dividing cells in the subapical meristem by increasing the probability that slowly cycling or nonproliferative cells in both 2C and 4C DNA states will enter the proliferative pool. The addition of PEG (200 g/l, = 1.5 mPA) to the rooting medium of dwarf watermelon seedlings inhibited hypocotyl elongation and reduced both mitotic and labeling indices simultaneously within 4.5 h after treatment. Within 24–28 h after PEG treatment, mitotic and labeling indices approached 0. Seedlings transferred from PEG to either water or GA₃ exhibited rapid recovery of cell division and hypocotyl elongation. Mitotic and labeling indices increased within 4.5–7.5 h into the recovery period in either water or GA₃ and reached control values within 10.5 h. GA₃ hastened the recovery from PEG-induced stress. It is concluded that water stress imposed by PEG 4000 causes arrest of cell division in meristematic cells of watermelon seedlings in both G₁ and G₂ periods. PEG and GA treatments resulted in only a partial and transitory synchronization of the cell cycle.     

Cell proliferation in ectodermal explants from Xenopus embryos

Summary Dissociated prospective ectoderm cells from Xenopus laevis embryos divide autonomously up to the 17th division cycle of the embryo. To examine the requirements for the further proliferation of these cells, the continuation of cell division in compact ectodermal explants beyond the 17th division cycle has been studied. Such explants develop into aggregates of epidermal cells, as can be shown immunohistochemically with an anti-serum against Xenopus epidermal cytokeratin. Cell division in these explants is comparable to the in vivo proliferation rate at least during the first 24 h of cultivation, that is, well beyond the 17th division cycle. Thus, epidermal cells are provided with all the factors necessary for continued proliferation, but these can be effective only when the cells form tight aggregates. The long-term changes in cell number are complex. Mitotic figures are present until the explants disintegrate after 3–4 days. However, the total cell number per explant does not increase during later development. The production of cells by mitotic divisions is likely to be countered by the loss of cells due to cell death, which is indicated by the presence of pyknotic nuclei.     

Studies of adult amphibian heart cells in Vitro: DNA synthesis and mitosis

The ventricle of the adult newt heart was excised and cut into several pieces of approximately 0.5 – 1.0 mm. These heart pieces were then cultured for 60 days at 25 °C in a modified Leibovitz medium (L-15). Approximately 37% of the explants were attached to the substrate and more than 33% of the attached explants and approximately 15% of the unattached explants established pulsation rates which ranged 3–67 beats/min. The explants were labeled with 1 μCi/ml of ³H-thymidine for 24 hr at 7, 15, 21, 30, 45 and 60 days of culture initiation, and processed for electron microscopic autoradiography. The examination of the autoradiograms revealed that as the culture continued, the cardiac muscle cells altered their morphology, resembling embryonic cardiac muscle cells. These altered muscle cells were termed dedifferentiated cardiac muscle cells. The number of these dedifferentiated cells increased over the period of culture, showing 10.3–94% dedifferentiated cells after 7–60 days of culture respectively. DNA synthesis and mitosis were observed in the dedifferentiated cardiac muscle cells, apart from the non-muscle cells. The quantitation of the autoradiograms revealed that the number of labeled nuclei in the cardiac muscle cells gradually increased over the period of culture, and a maximum number of labeled cardiac muscle cells (30%) was observed in the third week. The peak was followed by a decline in the eighth week which exhibited 1.5 % labeled cardiac muscle cells. The trend of mitosis was similar to that of DNA synthesis. The maximum number of mitotic figures (9%) was observed in the third week of culture, which was followed by a decline and finally absent in the eighth week. The cardiac non-muscle cells, mostly fibroblasts and endothelial cells, also showed incorporation of ³H-thymidine in their nuclei. The number of labeled non-muscle cells nuclei and the mitotic index were highest (61 and 15% respectively) in the first week of culture, but then they decreased gradually over the eight-week period in culture. This study provides evidence for the first time that the adult amphibian cardiac myocytes can undergo DNA synthesis and mitosis when explanted and cultured. The significance of this cell replication is discussed.     

Cell division during the development ofArtemia salina

Summary Cell division during embryonic development of the brine shrimp,Artemia salina has been studied by counting nuclei and mitotic figures. No cell division was observed during development of the encysted gastrula until about an hour before emergence of the embryo (a pre-nauplius) from the cyst, and even then only a few mitotic figures were observed. Following emergence, and during further development up to the stage II nauplius larva an increase of about 25% in the number of cells occurs. However, when the newly hatched larva is exposed to FUdR (10 g/ml) cell division is largely inhibited, but observable development nevertheless proceeds normally. Evidently all processes involved with the development of the gastrula into a stage II nauplius larva can occur with far fewer cells than normally are present.     

The course of chloroplast DNA replication and its relationship to other reproductive processes in the chloroplast and nucleocytoplasmic compartment during the cell cycle of the algaScenedesmus quadricauda

Summary DNA containing structures (cellular, chloroplast and mitochondrial nuclei) were stained with the fluorochrome DAPI. Fluorescence intensity, as a measure of DNA content, was estimated during the mitotic cycle in synchronized populations of the chlorococcal alga,Scenedesmus quadricauda. In cells yielding eight daughter cells, three consecutive steps in chloroplast DNA increase occurred over one mitotic cycle. The first step was performed shortly after releasing the daughter cells, the second and third steps occurred consecutively during the first half of the mitotic cycle. Commitment to chloroplast DNA replication was chronologically separated from commitment to division of chloroplast nuclei, revealing that these two chloroplast reproductive steps were under different control mechanisms. The replication of chloroplast DNA occurred at a different time to that of cell-nuclear DNA. The coordination of chloroplast reproductive processes and those in the nucleocytoplasmic compartment were governed by the mutual trophic and metabolic dependency of these compartments rather than by any direct or feedback control controlled by either of them.Abbreviations DAPI 46-diamidino-2-phenylindole - ptDNA DNA in chloroplast nuclei - nucDNA DNA in cell nuclei     

DNA endoreduplication in maize endosperm cells: the effect of exposure to short-term high temperature

DNA endoreduplication in Zea mays L. (cv. A619 × W64A) endosperm peaks between 16 and 18 d after pollination (DAP). The physiological function of DNA endoreduplication is not known but it is believed to be important in maize kernel development. In the present study, we investigated how 2, 4 or 6 d of high temperature (35 °C) affected DNA endoreduplication and maize kernel development in comparison with control kernels grown at 25 °C. Data were collected on fresh weight (FW), nuclei number, mitotic index, and DNA endoreduplication. Maize endosperm FW and nuclei number were reduced by exposure to 4 or 6 d of high temperature. At 18 DAP, the 2 d high temperature treatment (HTT) caused a reduction in FW and nuclei number, but had no effect on DNA endoreduplication and average DNA content per endosperm. However, when the exposure to high temperature was increased to 4 or 6 d, FW, nuclei number and the magnitude of DNA endoreduplication were progressively reduced, and the peak mitotic index was delayed compared with the control endosperm. At 18 DAP, the 4 d treatment showed 54·7% of the cells were 3 or 6 C, whereas only 41·2% were 12 C or higher. Six days of high temperature also resulted in a reduction in endosperm FW, nuclei number and a delay in the peak of mitotic index. DNA endoreduplication occurred in the kernels exposed to this treatment, although the magnitude was severely reduced compared with the control kernels. Nuclear DNA content was highly correlated (r = 0·93) with kernel FW, suggesting an important role of DNA endoreduplication in determining endosperm FW. The data suggest that high temperature during endosperm cell division exerted negative effects on DNA endoreduplication by dramatically reducing the nuclei number, leaving fewer nuclei available for DNA endoreduplication. However, the data also suggest that prolonged exposure to high temperature restricts entry of mitotic cells into the endoreduplication phase of the cell cycle.