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1.
James Pipkin Jeanne Anson William Hinson Jerry Hudson 《Biochimica et Biophysica Acta (BBA) - Gene Structure and Expression》1982,699(2):155-163
The in vivo administration of hydroxyurea for 12 h counteracts DNA synthesis and cell cycling stimulated by 72 h of isoproterenol treatment in rat salivary gland, as determined by fluorescence-activated flow cytometry. Hydroxyurea has little effect on [3H]leucine incorporation (protein synthesis) of the nuclear proteins soluble in 0.35 M NaCl, when examined by polyacrylamide gel chromatography and autoradiography from electro-statically sorted nuclei of (G0+G1) and (G2+M) phases of the in vivo cell cycle. Differential incorporation of [3H]leucine into nuclear proteins was observed during various phases of the cell cycle. Proteins ‘X’ and ‘Z’, observed in stained gel chromatographs of the 0.35 M NaCl-soluble nuclear proteins, were identified by biochemical analyses as ubiquitin and protein A24, respectively. Ubiquitin appeared transiently while A24 increased in gel chromatograms concomitant with progressive quiescence of the salivary gland induced by hydroxyurea. 相似文献
2.
Zainy M. Banjara Robert C. Briggs Lubomir S. Hnilica Janet L. Stein Gary S. Stein 《Molecular and cellular biochemistry》1985,67(2):101-110
Summary Antisera to 0.35 M NaCl extracts and residues of S phase HeLa nuclei were reacted with electrophoretically separated proteins
from the nuclei or nuclear material of HeLa cells synchronized in G1, S, G2 or M phases of the cell cycle. Quantitative evaluation of the peroxidase-antiperoxidase stained nitrocellulose transfers
(Western blots) revealed significant changes in the quantities of nuclear non-histone proteins during the cell cycle. Immunochemical
staining of electrophoretically separated nuclear antigens permits their selective detection in minute quantities and in the
presence of many additional proteins. 相似文献
3.
The distribution of cellular and nuclear volume in synchronous populations of NHIK 3025 cells, which derive from a cervix carcinoma, have been measured by electronic sizing during the first cell cycle after mitotic selection. Cells given an X-ray dose of 580 rad in G1, were also studied. During the entire cell cycle the volume distribution of both cells and nuclei is an approximately Gaussian peak with a relative width at half maximum of about 30%. About half of this width is due to imperfect synchrony whereas the rest is associated with various time invariant factors. During S the mean volume of the cells grows exponentially whereas the nuclear volume increases faster than for exponential kinetics. Hence, although cellular and nuclear volumes are closely correlated, their ratio does not remain constant during the cell cycle. Volume growth during the first half of G1 is negligible especially for nuclei where the growth appears to be closely associated with DNA-synthesis. For unirradiated cells the growth of cellular and nuclear volume is negligible also during G2+ M. In contrast, the X-irradiated cells continue to grow during the 6 hr mitotic delay with a rate that is constant and about half of that observed in late S. Hence, radiation induced mitotic delay does not appear merely as a lengthening of an otherwise normal G2. During G1 and S the irradiated cells were identical to unirradiated ones with respect to all the parameters measured. 相似文献
4.
In the 3-d-old 2-mm root tip of Pisum sativum L. cv. Lincoln the percentage of actively proliferating cells is estimated to be 70%. The remaining cells are non-cycling
and arrested with 2C and 4C DNA content in G0 and in G2Q, respectively. In this work we studied the kinetic significance of these quiescent cells, using the sorting capabilities
of flow cytometry and immunofluorescence techniques to detect the proliferation marker PCNA (proliferating cell nuclear antigen)
inside cells within the different cell-cycle compartments. While in animal cells, PCNA is present at a high level only in
actively proliferating cells, in 3-d-old pea root tips 95% of the cells are PCNA-positive. After flow cytometry and sorting
of pea non-cycling nuclear populations, all G2Q nuclei appeared strongly PCNA-positive, indicating that these cells had recently left the cell cycle. By contrast, most
G0 nuclei showed a low level of PCNA immunofluorescence intensity, as measured by image analysis, with about 25% of the nuclei
being PCNA-negative. This small percentage was found to correspond to root cap cells, as could be observed in the root tip
section. These are the only cells in the root apical region which are fully differentiated and which, therefore, lack the
competence to enter the cell cycle. In contrast, the more or less PCNA-positive G0 nuclei could represent a kinetically heterogeneous population of cells competent to proliferate, but which have either recently
left the cell cycle or are progressing to the G0-G1 transition.
Received: 6 November 1996 / Accepted: 14 January 1997 相似文献
5.
Arlette Nougarède 《Protoplasma》1977,93(2-3):341-356
Summary In pea axillary meristems submitted to or released from apical dominance, basic nuclear proteins and their fractions (lysine or arginine-rich) were localized at the infrastructural level using convergent methods. In the inhibited nuclei, the condensed chromatin and the nucleoli are the most reactive regions to alcoholic solution of phosphotungstic acid and to ammoniacal silver nitrate. It is the same in the reactivated nuclei after the release from dominance, but the increase in diameter of the nucleoli is accompanied by the creation of a granular component which is observed around the nucleoli during the G1 S or G2 phases. This structure is built up essentially by a lysin-rich ribonucleoprotein complex characteristic of active nuclei. 相似文献
6.
Dora Višnjić Josip Ćurić Vladiana Crljen Drago Batinić Stefano Volinia Hrvoje Banfić 《Biochimica et Biophysica Acta (BBA)/Molecular and Cell Biology of Lipids》2003,1631(1):61-71
The activity of nuclear phosphoinositide 3-kinase C2β (PI3K-C2β) was investigated in HL-60 cells blocked by aphidicolin at G1/S boundary and allowed to progress synchronously through the cell cycle. The activity of immunoprecipitated PI3K-C2β in the nuclei and nuclear envelopes showed peak activity at 8 h after release from the G1/S block, which correlates with G2/M phase of the cell cycle. In the nuclei and nuclear envelopes isolated from HL-60 cells at 8 h after release from G1/S block, a significant increase in the level of incorporation of radiolabeled phosphate into phosphatidylinositol 3-phosphate (PtdIns(3)P) was observed with no change in the level of radiolabeled PtdIns(4)P, PtdIns(4,5)P2 and PtdIns(3,4,5)P3. On Western blots, PI3K-C2β revealed a single immunoreactive band of 180 kDa, whereas in the nuclei and nuclear envelopes isolated at 8 h after release, the gel shift of 18 kDa was observed. When nuclear envelopes were treated for 20 min with μ-calpain in vitro, the similar gel shift and increase in PI3K-C2β activity was observed which was completely inhibited by pretreatment with calpain inhibitor calpeptin. The presence of PI3K inhibitor LY 294002 completely abolished the calpain-mediated increase in the activity of PI3K-C2β but did not prevent the gel shift. When HL-60 cells were released from G1/S block in the presence of either calpeptin or LY 294002, the activation of nuclear PI3K-C2β was completely inhibited. These results demonstrate the calpain-mediated activation of the nuclear PI3K-C2β during G2/M phase of the cell cycle in HL-60 cells. 相似文献
7.
Taizo Motomura 《Phycological Research》1996,44(1):11-17
The cell cycle (nuclear division cycle) of a multinucleate green alga, Boergesenia forbesii (Harvey) Feldmann was studied using microspectrophotometry and BrdU incorporation techniques. Mitosis was observed frequently 1-4 h after the beginning of the light period, on a 16:8 h LD cycle at 25°C. Mitotic nuclei formed discrete patches. Other nuclei remained in the G1 period. The DNA synthetic phase (S phase) was estimated to last about 12 h from microspectrophotometric study using aphidicolin inhibition just before the S phase and release from it. The G2 period was estimated to be about 2 h, because a labeled prophase nucleus could be detected when the samples were labeled with BrdU continuously over 3 h. The incorporation pattern of BrdU changed through the S phase nucleus. In early S phase, BrdU staining was detected as many dots in the entire nucleus, while in late S phase, it was detected as several discrete regions along the nuclear membrane. Almost all nuclei in B. forbesii were in the G1 stage after nuclear division, and the nuclei in several patches of the cell simultaneously initiated DNA synthesis. Once the nuclei entered into S phase, these nuclei continued into G2 and mitosis. In other words, the cell cycle regulation of entrance into S phase from G1 is an important factor in the growth and morphogenesis in B. forbesii. 相似文献
8.
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. 相似文献
9.
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. 相似文献
10.
THE SYNTHESIS OF ACIDIC CHROMOSOMAL PROTEINS DURING THE CELL CYCLE OF HELA S-3 CELLS : I. The Accelerated Accumulation of Acidic Residual Nuclear Protein before the Initiation of DNA Replication 总被引:40,自引:4,他引:36
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The synthesis and accumulation of acidic proteins in the tightly bound residual nuclear fraction goes on throughout the cell cycle of continuously dividing populations of HeLa S-3 cells; however, during late G1 there is an increased rate of synthesis and accumulation of these proteins which precedes the onset of DNA synthesis. Unlike that of the histones, whose synthesis is tightly coupled to DNA replication, the synthesis of acidic residual nuclear proteins is insensitive to inhibitors of DNA synthesis. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of acidic residual nuclear proteins shows different profiles during the G1, S, and G2 phases of the cell cycle. These results suggest that, in contrast to histones whose synthesis appears to be highly regulated, the acidic residual proteins may have a regulatory function in the control of cell proliferation in continuously dividing mammalian cells. 相似文献
11.
Cell cycle dependent chromosomal movement in pre-mitotic human T-lymphocyte nuclei 总被引:20,自引:0,他引:20
Fluorescent in situ hybridization with chromosome specific probes was used in conjunction with laser scanning confocal microscopy to assess the three-dimensional distribution of chromosomes in human T-lymphocyte nuclei. Cells in the G1-phase of the cell cycle exhibit a distinctly non-random chromosome organization: centromeric regions of the ten chromosomes examined are localized on the nuclear periphery, often making contact with the nuclear membrane, while telomeric domains are consistently localized within the interior 50% of the nuclear volume. Chromosome homolog pairing is not observed. Transition from the G1 to G2 cell cycle phase is accompanied by extensive chromosome movement, with centromeres assuming a more interior location. Chromosome condensation and chromatin depleted areas are observed in a small subset of G2 nuclei approaching mitosis. These results demonstrate that dynamic chromosome rearrangements occur in non-mitotic nuclei during the cell cycle.by L. Manuelidis 相似文献
12.
Change of chromatin morphology during the cell cycle detected by means of automated image analysis 总被引:2,自引:0,他引:2
Mouse embryo fibroblasts growing asynchronously in vitro stained with Feulgen method and their nuclear chromatin was analysed by means of the image analysing computer Quantimet 720D. Cells with 2C, 3C and 4C content of DNA were considered as being in G1, middle S and G2 phase of cell cycle, respectively. It was found that the projected area of nuclei increases during the cell cycle and that the mean optical density of chromatin increases from G1 through S to G2 phase. The curves showing the areas of chromatin at different optical density thresholds are different for cells in G1, S and G2 phase. The results demonstrate cyclic changes in chromatin morphology in the interphase nuclei during the cell cycle. 相似文献
13.
Summary Although many putative cdk (cyclin-dependent kinase) homologue genes have been identified in higher plants, their function and involvement in cell proliferation are still unclear. In this work we investigated the presence and distribution of cdk-like proteins in root tip meristem nuclei at different germination times (before, during, and after the onset of cell proliferation) and in nuclei of differentiated leaves. Nuclear cdk-like proteins were found in the root meristem throughout seed germination with a higher amount in actively proliferating cells, but were not detected in differentiated leaf. Characterization of the detected pea cdk-like proteins by immunoblotting led to the identification of two specific principal proteins of 33.2 and 34 kDa with the cdk conserved motif PSTAIRE. The p33.2 protein was also recognized by the anti-human p33cdk2 antibody, suggesting that the p33.2 and p34 proteins could be pea homologues of human p33cdk2 and p34cdk1, involved in the G1-S and G2-M transitions, respectively. Additional analysis of pea cdk protein localization has shown partial localization of these proteins at DNA replication sites during the G1 to S transition. These microscopical and biochemical data support the hypothesis that, in pea nuclei as in mammals, many PSTAIRE-cdks are present with different functions related to cell proliferation, one of which is probably involved in the control of the G1-S transition.Abbreviations Cdk
cyclin-dependent kinase
- HU
hydroxyurea
- BrdU
bromodeoxyuridine
- DAPI
4,6-diamidino-2-phenylindole
- SR 101
sulforhodamine 101
- PI
propidium iodide 相似文献
14.
Summary When the red-light grown protonema ofAdiantum capillus-veneris was transferred to the dark, the nucleus ceased its migration ca. 5 hours before cell plate formation (Mineyuki andFuruya 1980). To see whether the nucleus was held by some cytoplasmic structure during nuclear positioning, protonemata were treated with various centrifugal forces at different stages of the cell cycle. Nuclei of G1 phase were easily displaced by centrifugation at 360×g for 15 minutes, but those of G2 or M phase were not displaced by it, suggesting that the nuclei were held by some cytoplasmic elements in G2 or M phase. This nuclear anchoring was not detectable in protonemata that were treated with 5mM colchicine. With this treatment, the nucleus did not stop its migration at late G2 and moved even in prophase. And the retardation of organelle movement which was observed in cytoplasm on the lateral side of the nucleus after the cessation of premitotic nuclear migration (Mineyuki andFuruya 1984) was not observed in the presence of colchicine. Thus the nuclei appear to be held by colchicine-sensitive structure in cytoplasm between the lateral surface of the nucleus and cell wall during the premitotic nuclear positioning. Electron micrographs showing cytoplasmic microtubules were consistent with the idea.Abbreviations PPN
Premitotic positioning of the nucleus
- L region
Cytoplasm between the lateral surface of the nucleus and cell wall (seeMineyuki
et al. 1984) 相似文献
15.
Centrifugal elutriation was used to separate 9L rat brain tumour cells into fractions enriched in the G1, S, or G2/M phases of the cell cycle. Cells enriched in early G1, phase were recultured, grown in synchrony, and harvested periodically for analysis of their DNA distribution and polyamine content. Mathematical analysis of the DNA distributions indicated that excellent synchrony was obtained with low dissersion throughout the cell cycle. Polyamine accumulation began at the time of seeding, and intracellular levels of putrescine, spermidine, and spermine increased continuously during the cell cycle. In cells in the G2/M phase of the cell cycle, putrescine and spermidine levels were twice as high as in cells in the G1, phase. DNA distribution and polyamine levels were also analysed in cells taken directly from the various elutriation fractions enriched in G1, S, or G2/M. Because we did not obtain pure S or G2/M populations by elutriation or by harvesting synchronized cells, a mathematical procedure—which assumed that the measured polyamine levels for any population were linearly related to the fraction of cells in the G1, S, and G2/M phases times the polyamine levels in these phases and that polyamine levels did not vary within these phases—was used to estimate ‘true’ phase-specific polyamine levels (levels to be expected if perfect synchrony were achieved). Estimated ‘true’ phase-specific polyamine levels calculated from the data obtained from cells either sorted by elutriation or obtained from synchronously growing cultures were very similar. 相似文献
16.
Summary Using cytophotometric procedures, we measured the nuclear and nucleolar protein content of successive zones of growth and differentiation in consecutive (1–7 mm) root segments obtained from eight species of the Angiospermae after staining the preparations with Feulgen-Naphthol Yellow S (F-NYS). In meristematic cells the nuclear and nucleolar protein content was found to double during the cell cycle. In species in which differentiation occurs at the same time as nuclear DNA endoreplication, i.e. Vicia faba subsp. minor, V. faba subsp. major, Pisum sativum, Hordeum vulgare and Amaryllis belladonna, the pool of nuclear proteins observed during the G2 phase of the cell cycle was seen in the differentiated zone in nuclei containing 8C DNA. Species in which differentiation is not accompanied by the process of nuclear DNA endoreplication, i.e. Levisticum officinale, Tulipa kaufmanniana and Haemanthus katharinae, exhibited the highest nuclear proteins content during the G2 phase of the cell cycle; comparably high values were not found in the differentiated zone. A decrease in nucleolar protein content was observed during the process of differentiation, this tendency being more evident in the studied species that do not exhibit endoreplication.This work was supported by the Polish Academy of Sciences as a part of project no 09.7.1.4.5 相似文献
17.
When minced rat ventral prostate was incubated with labelled amino acids and cycloheximide or puromycin, the specific radioactivity of proteins associated with Triton X 100-washed nuclei exceeded that of the 105 000 g cytosol. The distribution of radioactive proteins from incubated mince, examined by SDS polyacrylamide gel electrophoresis was also consistent with labelling of some nuclear proteins that was resistant to inhibitors. Highly purified prostate nuclei, washed with detergent, labelled proteins of from 1–6 × 104 D with radioactive amino acids. When these proteins were fractionated according to solubility, NaOH-soluble ‘acidic’ proteins, examined by SDS polyacrylamide gel electrophoresis, were highly labelled, with a distribution of radioactivity that differed from the patterns of 0.4 N H2SO4-soluble basic proteins (including histones), and proteins soluble in Krebs-Ringer-phosphate buffer. Although these results cannot be interpreted unambiguously, they are consistent with the synthesis of certain nuclear proteins at a site(s) sequestered from cycloheximide and puromycin. Nuclei may represent one such site. 相似文献
18.
The lipoglycoproteins of the WNT family act on seven transmembrane-spanning Class Frizzled receptors. Here, we show that WNT-5A evokes a proliferative response in a mouse microglia-like cell line (N13), which is sensitive to pertussis toxin, thus implicating the involvement of heterotrimeric G proteins of the Gi/o family. We continue to show that WNT-5A stimulation of N13 membranes and permeabilized cells evokes the exchange of GDP for GTP of pertussis toxin-sensitive G proteins employing [γ-35S]GTP assay and activity state-specific antibodies to GTP-bound Gi proteins. Our functional analysis of the PTX-sensitivity of WNT-induced G protein activation and PCR analysis of G protein and FZD expression patterns suggest that WNT-5A stimulation leads to the activation of Gi2/3 proteins in N13 cells possibly mediated by FZD5, the predominant FZD expressed. In summary, we provide for the first time molecular proof that WNT-5A stimulation results in the activation of heterotrimeric Gi2/3 proteins in mammalian cells with physiological protein stochiometry. 相似文献
19.
M. G. Taylor I. K. Vasil 《TAG. Theoretical and applied genetics. Theoretische und angewandte Genetik》1987,74(6):681-686
Summary Mesophyll cell nuclei isolated from leaves of Pennisetum purpureum were analysed by flow cytometry to determine the nuclear DNA content and the percentage of cells in different phases of the cell cycle. Samples taken from base, middle and tip regions of leaves 2 to 8 (leaf 1, which was adjacent to the meristem, was too small to sample) showed no significant differences in the amount of DNA per G1 nucleus due to either age or position. The average amount of DNA per G1 nucleus was 5.78 pg. Although the majority of cells for each sample were in G1, samples taken from older leaves had higher percentages of cells in G2 and S phases. More specifically, base and middle regions of older leaves had a higher percentage of cells in G2 than all three positions in younger leaves. Electrophoretic analysis of nuclear DNA from leaves 2 to 7 showed no evidence of degradation or difference in fragment size for any sample or position. This study was compared to previous work on the relationship between leaf age and embryogenic competence in Pennisetum purpureum. The results suggest that changes in the cell cycle, and/or a loss or fragmentation of the nuclear DNA, are not responsible for loss of embryogenic competence in mature leaf tissue. 相似文献
20.
Cell Cycle Activity and β-Tubulin Accumulation During Dormancy Breaking of Acer platanoides L. seeds
Cell cycle events in embryo axes of Norway maple (Acer platanoides L.) seeds were studied during dormancy breaking by flow cytometric analyses of the nuclear DNA content and by immunodetection
of β-tubulin. Most embryonic nuclei of dry, fully matured seeds were arrested in the G2 phase of the cell cycle. In addition, the lowest content of β-tubulin was detected in dry, mature seeds. Imbibition in water
and cold stratification resulted in a decrease in the number of nuclei in G2, and a simultaneous increase in β-tubulin content. In germinated seeds the content of β-tubulin was the highest and the number
of cells in G2 was the lowest. Both cell cycle events preceded cell expansion and division and subsequent growth of the radicle through
the seed coat. The anatomical investigation has proved that the main reason for decrease in the number of nuclei in G2 is mitosis, started with seeds germination (radicle protrusion). The activation of the cell cycle and the β-tubulin accumulation
were associated with embryo dormancy breaking.
This revised version was published online in July 2006 with corrections to the Cover Date. 相似文献