Nuclear DNA in normal and refed Amoeba proteus

Amoeba proteus synthesizes DNA in G2 phase of the cell cycle upon feeding after starvation. The characteristics of the DNA synthesized in G2 have been studied by microscope photometry of individual Feulgen-stained nuclei and by buoyant density centrifugation of nuclear DNA in CsCl. Amoeba nuclei were found to contain 42.8 pg of DNA. This DNA bands in CsCl at a density of 1.693 g/cm³ with a satellite at 1.714 g/cm³ which makes up 24% of nuclear DNA. DNA from whole cells has an additional non-nuclear satellite at 1.726 g/cm³. When cells are starved and re-fed with food labeled with [³H]thymidine, the DNA synthesized is predominantly the 1.714 satellite. The amount of DNA synthesized in G2 is small since there is no measurable difference in Feulgen dye binding to nuclei of starved vs starved and re-fed cells. The data suggest that refeeding induces a resumption of late S phase DNA synthesis, or the preferential synthesis of specific DNA sequences such as rRNA genes.     

Separate effect of hydroxyurea on the initiation and elongation of DNA synthesis in BHK cells

Summary BHK21/C₁ cells, starved for 30 h in serum deficient medium and treated for 15 h with 1 mm hydroxyurea (HU) in order to obtain a synchronous cell population in the G₁/S-boundary, incorporate a residual proportion of ³H-thymidine (dThd). This residual incorporation is due to semiconservative synthesis and may not be reduced by increasing the drug concentration without affecting the reversion capacity of the cells proportionally. As shown by autoradiographic analysis, the residual DNA synthesis does not correspond to ³H-dThd incorporation within a small number of resistant cells, but is located in the nuclei of a high proportion of cells with reduced density of silver grains. After treatment with 0.05 mm HU, however, the incorporation of ³H-dThd increases considerably over the control values. The determination of the radioactivity incorporated by µg DNA corresponding to nuclei in S phase indicates that this concentration of HU is also able to reduce the rate of DNA polymerization. Kinetic data on the appearance of this increased ³H-dThd incorporation and on the accumulation of labelled nuclei in cells growing at random and labelled continuously with the radioactive DNA precursor indicate that HU stimulates the cells to enter the S phase. The reported results are consistent with a mechanism of action of HU which affects initiation and elongation of DNA chains separately.     

Kinetic Differences Between Fed and Starved Chinese Hamster Ovary Cells

When Chinese hamster (CHO-K1) cells are grown as monolayer cultures, they eventually reach a population-density plateau after which no net increase in cell numbers occurs. the kinetics of aged cells in nutritionally deprived (starved) or density-inhibited (fed) late plateau-phase cultures were studied by four methods: (i) Reproductive integrity and cell viability were monitored daily by clonogenic-cell assay and erythrosin-b dye-exclusion techniques. (ii) Mitotic frequencies of cells from 18 day old cultures were determined during regrowth by analysing time-lapse video microscope records of dividing cells. (iii) Tritiated-thymidine ([³H]TdR) auto-radiography was used to determine the fractions of DNA-synthesizing cells in cultures entering plateau phase and during regrowth after harvest. (iv) the rate of labelled nucleoside uptake and incorporation into DNA was measured using liquid scintillation or sodium iodide crystal counters after labelling with [³H]TdR or [¹²⁵]UdR. Non-cycling cells in starved cultures accumulate primarily as G₁, phase cells. Most cells not in G₁ phase had stopped in G₂, phase. Very few cells (< 2%) were found in S phase. In contrast, about half of the cells in periodically fed cultures were found to be in DNA-synthetic phase, and the percentage of these S phase cells fluctuated in a manner reflecting the frequency of medium replacement. Populations of both types of plateau-phase cultures demonstrate extremely coherent cyclic patterns of DNA synthesis upon harvest and reculturing. They retain this high degree of synchrony for more than three generations after the resumption of growth. From these data it is concluded that nutritionally deprived (starved) late plateau-phase cells generally stop in either G₁, or G₂, phase, whereas periodically fed late plateau-phase cultures contain a very large fraction of cycling cells. Populations of cells from these two types of non-expanding cultures are kinetically dissimilar, and should not be expected to respond to extracellular stimuli in the same manner.     

Site-specific and temporally controlled initiation of DNA replication in a human cell-free system

We have recently established a cell-free system from human cells that initiates semi-conservative DNA replication in nuclei isolated from cells which are synchronised in late G₁ phase of the cell division cycle. We now investigate origin specificity of initiation using this system. New DNA replication foci are established upon incubation of late G₁ phase nuclei in a cytosolic extract from proliferating human cells. The intranuclear sites of replication foci initiated in vitro coincide with the sites of earliest replicating DNA sequences, where DNA replication had been initiated in these nuclei in vivo upon entry into S phase of the previous cell cycle. In contrast, intranuclear sites that replicate later in S phase in vivo do not initiate in vitro. DNA replication initiates in this cell-free system site-specifically at the lamin B2 DNA replication origin, which is also activated in vivo upon release of mimosine-arrested late G₁ phase cells into early S phase. In contrast, in the later replicating ribosomal DNA locus (rDNA) we neither detected replicating rDNA in the human in vitro initiation system nor upon entry of intact mimosine-arrested cells into S phase in vivo. As a control, replicating rDNA was detected in vivo after progression into mid S phase. These data indicate that early origin activity is faithfully recapitulated in the in vitro system and that late origins are not activated under these conditions, suggesting that early and late origins may be subject to different mechanisms of control.     

EXPERIMENTAL CONTROL OF DNA SYNTHESIZING AND DIVIDING CELLS IN EXCISED ROOT TIPS OF PISUM

The number of dividing and DNA-synthesizing cells in excised pea roots can be regulated by eliminating the carbohydrate normally supplied in the culture medium. When the excised roots were allowed to remain for 24 hr in a medium lacking carbohydrate, the number of mitotic figures and tritiated thymidine (H³-T) labeled cells was reduced almost to zero. After an additional 24 hr in the incomplete culture medium, 15% of the interphase cells were H³-T labeled, the percentage of the cells that were dividing never exceeded 1.4, and 30% of these were H³-T labeled. When the roots remained in the deficient medium for 72 hr, neither cell division nor cells synthesizing DNA were observed. Upon addition of 2% sucrose, cell division and DNA synthesis were resumed in the roots that were maintained for 24 or 72 hr without an exogenous carbohydrate supply. It has been hypothesized that some proliferative systems consist of two cellular subpopulations which selectively stop or remain in either the pre-DNA synthetic (G₁) or post-DNA synthetic (G₂) periods of the mitotic cycle. The addition of sucrose, H³-T, and 5-aminouracil to the medium, after the roots had been maintained for 24 hr without a carbohydrate, indicated that most of the proliferative cells in the roots had accumulated in either G₁, a quasi-G₁ condition, i.e., DNA synthesis stopped sometime before completion, or G₂ periods of interphase; the majority, however, were in G₁ or quasi-G₁ conditions. The results suggested that DNA synthesis (S period) and mitosis or the onset of these processes have the highest metabolic requirements in the mitotic cycle and that G₁ and G₂ were the most probable states for proliferative cells in a meristem with a low metabolic level.     

Cessation of Nuclear DNA Synthesis in Differentiating Naegleria*

SYNOPSIS. DNA synthesis during growth and differentiation in Naegleria gruberi strain NEG populations has been studied. Autoradiography of cells labeled with [³H]thymidine revealed that grains are concentrated over the nuclei in logarithmically growing populations of cells, whereas in differentiating cells, grains are scattered over the cytoplasm; i.e. no significant nuclear labeling is detectable. It was established by MAK chromatographic analysis that [³H]thymidine is incorporated into double-stranded DNA in Naegleria and that the actual amount of incorporation in the logarithmically growing populations of cells is 20 times greater than that in differentiating cells. These results suggest that nuclear DNA synthesis is reduced markedly soon after the initiation of differentiation, while cytoplasmic DNA synthesis continues. It was established from cell cycle analysis that the approximate intervals of G₁, S, G₂, and M phases were 180, 183, 90, and 28 min, respectively. Hence, the reduction in the nuclear DNA synthesis in differentiating cells is not due to the inhibition of initiation of DNA replication, but rather to the termination of the DNA replicating process. Thus DNA synthesis is curtailed in the presence of RNA and protein synthesis which are required for differentiation.     

Extra DNA synthesis in the dedifferentiating cells ofVicia faba roots

Summary Cell dedifferentiation was induced inVicia faba root tissues by removing the whole root meristem (decapitation) and the behaviour of the nuclear DNA in the dedifferentiating cells was studied by means of cytophotometric and autoradiographic analyses. Cytophotometric determination after Feulgen-staining showed that: 1. the vast majority of nuclei in differentiated cells were in the DNA postsynthetic phase, but their Feulgen absorption was lower than that of DNA postsynthetic nuclei (G₂, 4 C) in the meristem; 2. such a Feulgen absorption was detected in certain nuclei after root decapitation; 3. all the mitoses in the dedifferentiating tissues were diploid, fully matching the Feulgen absorption of mitoses in the meristem.After³H-thymidine (³H-T) feeding of the decapitated roots and autoradiography, the following results were obtained: 1. two populations of labeled nuclei, characterized by two different levels of scattered labeling occurred in dedifferentiating tissues, slightly labeled nuclei being much more numerous than heavily labeled nuclei; 2. the percentage of labeled nuclei was much greater than that of DNA presynthetic nuclei in the root tissues; 3. almost all the mitoses were labeled after a 16-hour³H-T feeding; 4. the percentage of slightly labeled nuclei paralleled that of dedifferentiating cells; 5. the duration of the DNA synthesis phase and that of the gap between completion of DNA synthesis and mitosis differed in heavily and slightly labeled nuclei; 6. all nuclei which entered DNA synthesis also entered mitosis.These results are interpreted to mean that: 1. after decapitation, two different DNA syntheses occur in the dedifferentiating root tissues ofV. faba: DNA reduplication in cells which dedifferentiate starting from a DNA presynthetic nuclear condition (heavily labeled nuclei) and extra DNA synthesis in cells which dedifferentiate starting from a DNA postsynthetic nuclear condition (slightly labeled nuclei); 2. extra DNA synthesis is required in these dedifferentiating cells for entry into mitosis.     

Utilization of the label from bacterial [3H] DNA by isolated barley roots and embryos under different conditions

[³H] DNA fromEscherichia coli and [³H] thymidine were applied, in sterile conditions, on isolated barley embryos and on roots excised from these embryos, both cultivated in the liquid medium and on halves of barley seeds, through the endosperm bridge. In embryos and roots, the labelled compounds were applied in 1.5% sucrose + 0.2 SSC alone, or together with either unlabelled thymidine or DEAE-dextran. Similar labelling indices were found after [³H] thymidine and [³H] DNA treatment which shows that the activity of [³H] DNA is utilized during the S phase. After application of [³H] thymidine, only cell nuclei in S phase were labelled. After the application of [³H] DNA an extranuclear label, in addition to the labelling of nuclei in the S phase, was observed in some experimental variants. The density of label above labelled nuclei after [³H] DNA treatment sharply decreased when unlabelled thymidine or DEAE-dextran was added, while the density of label above nuclei labelled by [³H] thymidine decreased when unlabelled thymidine but not DEAE-dextran was added. The labelling of nuclei with the label from [³H] DNA is the result of degradation of exogenous DNA reutilization of low molecular weight products. Extranuclear labelling is most probably due to the polymerous or partly degraded DNA.     

SEPARATION OF NUCLEI REPRESENTING DIFFERENT PHASES OF THE GROWTH CYCLE FROM UNSYNCHRONIZED MAMMALIAN CELL CULTURES

Nuclei have been isolated from unsynchronized cultures of Chinese hamster fibroblasts after varying intervals of growth following the incorporation of thymidine ^-3H for 20 min. These nuclei were fractionated by unit gravity sedimentation in a stabilizing density gradient of sucrose, and fractions were analyzed for the concentration of nuclei, DNA, and radioactivity. A more rapidly sedimenting population of nuclei in the G₂ phase of the cell cycle was separated from a group of nuclei in the G₁ phase, and nuclei in progressive stages of DNA synthesis (S phase) were distributed between these two regions. The fractionation of intact cells by sedimentation according to their position in the cell cycle was found to be less satisfactory than the corresponding separation of nuclei. This probably results from the continuous accumulation of mass within individual cells throughout the entire cell cycle, whereas most of the mass of a nucleus is replicated during a relatively narrow interval of the total cell cycle.     

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.     

The effect of lead on the cell cycle in the root meristem ofAllium cepa L.

Summary Allium cepa (L.) adventitious roots were treated with lead (2.5 mg of Pb²⁺ [from Pb(NO₃)₂] per dm³) for 30–72 h. The cell cycle was studied by pulse labeling with [³H]thymidine. Mitotic activity kinetics, occurrence of disturbed mitoses (c-mitoses), and level of DNA synthesis were examined. It was found that lead prolonged the cell cycle and that cells in two phases of the cycle, G₂ and S, differed in their sensitivity to lead. Cells in G₂ were more sensitive; lead lengthened their cycle by 216% and disturbed the course of cell division by causing c-mitoses. Cells in S phase were less sensitive. Their cell cycle was longer by 55%. They went through their G₂ phase without major disturbances, mitosis in these cells was normal. During treatment ofA. cepa with lead, its destructive effects on cells were exerted only during the first few hours (around 6 h) of incubation. That is when the inhibition of mitotic activity, numerous disturbances of cell division, a decline in the number of cells synthesizing DNA, and a lower level of DNA synthesis were observed. As the incubation continued, the above processes were found to return to normal. In the discussion, data are presented supporting the hypothesis that during the initial period of exposure ofA. cepa to lead, this metal enters both the root apoplast and symplast, exerting a destructive effect on cells, while later, lead penetrates only into the root apoplast, and in this way remains harmless to cells.     

Location of heitz's zerstäubungsstadium (dispersion phase) in the mitotic cycle ofPhaseolus coccineus and the concept of Angiosperm endomitosis

Summary DNA microdensitometry and autoradiography after treatment with³H-thymidine were used to study the phase of dispersion of chromocenters (Z phase) in parallel with chromocentric nuclei inPhaseolus coccineus. In all materials studied, two types of chromocentric nuclei were present.In radicle apices of dry seeds, two classes of nuclear DNA contents were measured, 2 C (G₁) and 4 C (G₂). The 2 C DNA class comprised all chromocentric type I nuclei, the 4 C class included Z phases and chromocentric type II nuclei. The 4 C (G₂) condition of Z phases implies that Z phases maintain their nuclear structure for some time after the end of DNA replication. Shoot apices also contain 2 C (G₁) and 4 C (G₂) nuclei but 4 C nuclei (Z phases and chromocentric type II nuclei) are rare.In seedling root apices, Z phases are from 1.02 to 4.08 times as frequent as prophases. This excludes that Z phase is a very early prophase. DNA microdensitometry shows that the chromocentric type I includes 2 C (G₁) nuclei and nuclei in the first part of the S phase, Z phases include 4 C (G₂) nuclei and nuclei in the last stage of the S phase and chromocentric type II includes mainly 4 C (G₂) nuclei and nuclei in the second part of S. After 90 minutes of treatment with³H-thymidine all Z phase nuclei are labeled. This result and the microdensitometric data unequivocally demonstrate that Z phase is located at the end of S.The present results and those of previous authors on Z phase are discussed in relation to Geitler's concept of Angiosperm endomitosis. It is concluded that the term Angiosperm endomitosis must be abandoned and substituted by the term chromosome endoreduplication.     

Regulation of p27Kip1 by intracellular iron levels

Enhanced intracellular iron levels are essential for proliferation of mammalian cells. If cells have entered S phase when iron is limiting, an adequate supply of deoxynucleotides cannot be maintained and the cells arrest with incompletely replicated DNA. In contrast, proliferating cells that are not in S phase, but have low iron pools, arrest in late G1. In this report the mechanism of iron-dependent G1 arrest in normal fibroblasts was investigated. Cells were synchronized in G0 by contact inhibition and serum deprivation. Addition of serum caused the cells to re-enter the cell cycle and enter S phase. However, if the cells were also treated with the iron chelator deferoxamine, S phase entry was blocked. This corresponded to elevated levels of the cyclin dependent kinase inhibitor p27^Kip1 and inhibition of CDK2 activity. Expression of other cell cycle regulatory proteins was not affected, including the induction of cyclins D1 and E. When the quiescent serum starved cells were supplemented with a readily usable form of iron in the absence of serum or any other growth factors, a significant population of the cells entered S phase. This was associated with downregulation of p27^Kip1 and increased CDK2 activity. Using an IPTG-responsive construct to artificially raise p27^Kip1 levels blocked the ability of iron supplementation to promote S phase entry. Thus it appears that p27^Kip1 is a mediator of G1 arrest in iron depleted Swiss 3T3 fibroblasts. We propose that this is part of an iron-sensitive checkpoint that functions to ensure that cells have sufficient iron pools to support DNA synthesis prior to entry into S phase.     

Immunocytochemical localization of callose in root cortical cells parasitized by the ring nematodeCriconemella xenoplax

Summary Under hypoxia (10 and 5% partial oxygen tension) meristematic cells ofAllium cepa L. roots acquired new cycle kinetics, characterized by reduced but constant rates of root growth. Under these conditions, there was preferential lengthening of G₁ and of the last third of the S period, S₃. Since hyperoxygenation shortened S₃ but not G₁ in these cells, the high sensitivity of late replication to environmental oxygen is demonstrated. The preferential depression of the replication rate when those cells replicated the last third of their DNA was not associated with diminished cell size. Rather, the lower the oxygen level the larger the mean size of the cycling cells. Under anoxia (0% oxygen tension) the rate of growth slowed, accompanied by preferential accumulation of cells in G₁. However, steady state kinetics of root growth was not achieved under these extreme conditions.Abbreviations Mean cell length - LI labelling index or frequency of cells with labelled nuclei after [³H]thymidine - G₁, S, G₂ pre-replicative, replicative, and post-replicative periods of the interphase of cycling cells - M mitosis     

Dynamics of association of origins of DNA replication with the nuclear matrix during the cell cycle

DNA of replication foci attached to the nuclear matrix was isolated from Chinese hamster ovary cells and human HeLa cells synchronized at different stages of the G₁ and S phases of the cell cycle. The abundance of sequences from dihydrofolate reductase ori-β and the β-globin replicator was determined in matrix-attached DNA. The results show that matrix-attached DNA isolated from cells in late G₁ phase was enriched in origin sequences in comparison with matrix-attached DNA from early G₁ phase cells. The concentration of the early firing ori-β in DNA attached to the matrix decreased in early S phase, while the late firing β-globin origin remained attached until late S phase. We conclude that replication origins associate with the nuclear matrix in late G₁ phase and dissociate after initiation of DNA replication in S phase.     

Glycogen metabolism in resting and growing cells of Saccharomyces carlsbergensis

Saccharomyces carlsbergensis cells, growing under carbohydrate or nitrogen limitation, initially deplete their glycogen, which is resynthesized only during the late exponential phase. Cells, harvested in the carly exponential phase, are even unable to synthesize glycogen in glucose-containing phosphate buffer. This is in contrast to cells from the stationary phase which rapidly synthesize glycogen under the same conditions. Lack of O₂ slows down glycogen synthesis.Contrary to cells suspended in complete medium, addition of ammonia alone to nitrogen free-media induced neither breakdown of glycogen, nor complete cessation of glycogen synthesis. Ammonia slowed down glycogen synthesis (both aerobic and anaerobic), only, in cells grown either under carbohydrate or under nitrogen limitation.Glycogen synthesis was observed 1 min after addition of glucose to a starved cell suspension in phosphate buffer. Removal of the sugar from the buffer resulted in an instantanous decrease of the glycogen level in the cells. The results indicate that glycogen-metabolism is regulated by a variety of endogenous and environmental factors.     

Critical nuclear DNA size and distribution associated with S phase initiation

Using HeLa S-3 cells synchronized by selective detachment, in this paper we report a parallel study of nuclear morphology and autoradiography grain patterns between middle G₁ and middle S phases: Our results show two distinct [³H]-thymidine labeling patterns. The first “peripheral” labeling pattern has a characteristic nuclear size distribution, in contrast to the heterogeneous and varying size distributions of Feulgen-stained nuclei, and apparently is characteristic of very early S phase. The sizes of the second labeling pattern—homogeneous or inhomogeneous grain distribution throughout the nucleus—are equal or larger than the first and vary with S phase progression. Together, the corresponding nuclear sizes of the labeled nuclei represent the larger extreme of nuclear areas, and the labeling index closely parallels the fraction of nuclei with areas larger than the minimum size of the labeled nuclei. These results suggest a characteristic nuclear size (reflecting unique intranuclear DNA distribution) as a necessary, if not sufficient, requirement for S phase initiation. Parallel experimentation with rat liver cells—synchronized in vivo by partial hepatectomy and analyzed by thin section autoradiography—confirms the existence of a peripheral labeling pattern in both the very early part and the very late part of S phase, which reconciles our data with previous results and points to the fact that both initiation and termination sites for DNA replication are near the nuclear periphery.     

The cytoplasmic appearance of three functions expressed during the G0→G1→S transition is nucleus-dependent

tsAF8, ts13, tsHJ-4, and TK^?ts13 cells are G₁-specific temperature-sensitive (ts) mutants of BHK cells that do not enter S phase when serumstimulated from quiescence at nonpermissive temperature (39.6°-40.6°). TK^?ts13 are, in addition, defective in thymidine kinase. Different G₁ functions must be involved in these cells, since the first three cell lines complement each other when forming heterokaryons. We have used these cells to study the role of the nucleus in the cytoplasmic expression of these G₁ functions during the transition of cells from the non-proliferating to the proliferating state. We fused cytoplasts from either serumstarved (G₀) or serum-stimulated (S) tsAF8 cells with G₀-ts13, G₀-tsHJ-4, and G₀-TK^?ts13 recipient cells and determined, after serum stimulation of the fusion products, which type of cytoplasts could complement the defective G₁ functions. Cytoplasts from S-tsAF8 cells complemented all three functions, i.e., cybridoids between S phase cytoplasts and ts13 or tsHJ-4 recipient cells entered S at the nonpermissive temperature, and TK^?ts13 recipient cells incorporated exogenous thymidine. Cytoplasts isolated from G₀-tsAF8 cells (3 days of serum starvation) complemented ts13 cells but not tsHJ-4 and TK^?ts13 cells. Cytoplasts from 6-day starved tsAF8 cells lost the complementing capacity for ts13 cells. However, when the 6-day starved tsAF8 cells were fused with G₀-ts13 cells, the heterokaryons entered S phase at the nonpermissive temperature. Also, cytoplasts isolated from the 6-day starved cells that were serum stimulated for 40 hr before enucleation regained the capacity to complement ts13 cells. These results demonstrate that three functions required in G₁ cannot be detected in the cytoplasm of serum-starved cells, although they are present in the cytoplasm of S-phase cells. These results suggest that a functional nucleus is required for the cytoplasmic appearance of certain G₁ functions in serumstimulated cells.     

Variability of the time at which PHA-stimulated lymphocytes initiate DNA synthesis

A method is presented for the study of the entrance of in vitro stimulated cells into their first poststimulation S phase. PHA-stimulated lymphocytes were incubated continuously with ¹⁴C-TdR. This isotope was then removed at different intervals and the cells were incubated for 8 h in medium containing ³H-TdR. Cells which had incorporated ³H-TdR but not ¹⁴C-TdR were considered to have entered their first post-stimulation S phase during the 8 h incubation with ³H-TdR. These cells were identified by double-layer autoradiography.The majority of PHA-stimulated lymphocytes entered their first period of DNA synthesis between 48 and 72 h after the addition of PHA. However, the variability was pronounced, some cells entering their first S phase at about 24 h and others some 100 h later. Cells entering their first S phase accounted for a considerable part of the population of cells in DNA synthesis still as late as 72 h after the addition of PHA.Calculation of the total number of cells that entered their first S phase during the 6 day culture period showed that DNA synthesis was initiated in some 40 % of the cells of the initial population.     

Enhanced expression of a chromatin associated protein tyrosine phosphatase during G0 to S transition

The non-transmembrane protein tyrosine phosphatase, PTP-S, is located predominantly in the cell nucleus in association with chromatin. Here we have analysed the expression of PTP-S upon mitogenic stimulation and during cell division cycle. During liver regeneration after partial hepatectomy, PTP-S mRNA levels increased 16-fold after 6 h (G₁ phase) and declined thereafter. Upon stimulation of serum starved cells in culture with serum, PTP-S mRNA levels increased reaching a maximum during late G₁ phase and declined thereafter. No significant change in PTP-S RNA levels was observed in growing cells during cell cycle. PTP^-S protein levels were also found to increase upon mitogenic stimulation. Upon serum starvation for 72 h, PTP-S protein disappears from the nucleus and is seen in the cytoplasm; after 96 h of serum starvation the PTP-S protein disappears from the nucleus as well as cytoplasm. Refeeding of starved cells for 6 h results in reappearance of this protein in the nucleus. Our results suggest a role of this phosphatase during cell proliferation.