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1.
Replication Protein A (RPA), the replicative single-strand DNA binding protein from eukaryotic cells, is a stable heterotrimeric complex consisting of three polypeptides. Cytological studies have investigated the subcellular distribution and association characteristics of the three RPA subunits during different stages of the cell cycle with varying results. In this study, various HeLa cell fractions were subjected to separation by either immunoprecipitation or velocity sedimentation. These separations were evaluated by immunoblotting for specific RPA subunits to determine whether the RPA in these fractions retains its heterotrimeric association. Immunoprecipitation of either the large (RPA70) or middle-sized (RPA32) subunit of RPA followed by immunoblotting for the other subunits demonstrate that RPA remains complexed throughout the G1, S and G2 phases of the cell cycle. Immunoprecipitation and sedimentation separations of both the nucleosolic and chromatin-bound RPA populations from both cycling and nocodazole-blocked cells showed that the majority of RPA remains complexed under all conditions examined. Consistent with previous reports, hypotonic extracts from 293 cells were shown to contain some RPA32 not complexed with RPA70. These results indicate that in some cell types, extracts may contain small amounts of RPA32 free of RPA70; however, in HeLa cells the majority of RPA clearly remains complexed as a heterotrimer throughout the cell cycle. 相似文献
2.
SEPARATION OF NUCLEI REPRESENTING DIFFERENT PHASES OF THE GROWTH CYCLE FROM UNSYNCHRONIZED MAMMALIAN CELL CULTURES 总被引:5,自引:3,他引:2
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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. 相似文献
3.
Asynchronous 9L cells were separated into relatively homogeneously-sized populations using centrifugal elutriation with both
a conventional collection method and a long collection method. A substantial increase in the homogeneity of the volume distributions
and in the degree of synchrony of the separated fractions was obtained using the long collection method. Autoradiographic
data indicated that fractions containing ≥97% G1 cells, ≥80% S cells, and 70–75% G2 cells could be routinely recovered with this procedure. Recovery in these fractions varied from 5 to 8% of the total number
of cells elutriated. The colony forming efficiency (CFE) of cells from fractions representing each phase of the cell cycle
was a constant 60–70%, which was comparable to the 60–80% usually found for asynchronous 9L cells. The percentage of cells
in the G1, S, and G2 phases in the elutriated fractions was more accurately determined from the volume distribution than from computer fits of
the DNA histogram obtained from flow cytometry. In general, the degree of synchrony was related to the coefficient of variation
(CV) of the volume distributions of the elutriated fractions. The CV was about 14% for all elutriated fractions. When the
≥97% G1 population was allowed to progress to S and G2, the CVs were about 17 and 20.2%, respectively. Thus, the best nonperturbing method for obtaining synchronous 9L cells in
the S or G2 phases was direct elutriation with the long collection method. 相似文献
4.
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
5.
6.
Chang liver cells from exponentially growing suspension cultures have been separated by sedimentation at unit gravity. Determinations of the protein content per cell showed that the fractionation procedure resulted in good separation of cells of different size. On the other hand, the DNA content of individual cells from the fractions, as determined cytofluorimetrically, indicated considerable heterogeneity in the size of cells from the same stage of the division cycle. On the basis of earlier results on intermitotic growth and the variation in the length of the cell cycle in homogeneous cell populations, a mathematical model has been constructed and tested using a computer program. The present results on the size distribution of cells from the different stages of the mitotic cycle are consistent with a regeneration of size heterogeneity in each cell generation, as a result of the dispersion of intermitotic times. The variation in cell cycle times may be related to a probabilistic event in the G1 period. In the mathematical model it was necessary to include a mechanism by which the regeneration of abnormally large cells is prevented. The experimental data are compatible with a gradually increasing inhibition of growth in cells larger than a certain size (circa 400 pg protein per cell). 相似文献
7.
Anna Maria Bolognani Fantin Antonella Franchini Roberta Malgara Barbara Rebecchi Anna Maria Fuhrman Conti 《Biology of the cell / under the auspices of the European Cell Biology Organization》1998,90(2):155-159
The changes in the expression of glycoconjugates and adhesion molecules were studied by selective lectin binding and immunocytochemical reactions in a human embryonic epithelial cell line (EUE cells), synchronized in the cell cycle phases. The results can be summarized as follows: most of the tested lectins display a more diffuse binding for the cytoplasm in G1 than S and G2 phases; in the S and particularly in G2 phases the cytoplasm glycoconjugates are rearranged around the nucleus; cells in mitosis always show a strong binding towards all tested lectins. Cellular fibronectin and its receptor β1 integrin are well expressed in G1, but the strongest reaction is observed in the S phase. The immunoreactions for laminin and uvomorulin (L-CAM) are poorly positive in all cell cycle phases. The immunocytochemical reaction for heparan sulfate is positive, with a stronger reaction in S and G2 than in G1; on the contrary a diffuse staining with the anti-dermatan sulfate proteoglycan antibody appears unchanged during the cell cycle. 相似文献
8.
The technique of velocity sedimentation was employed to fractionate mouse bone marrow CFU-s relative to two different phases of the cell cycle. CFU-s in DNA synthesis which are destroyed by hydroxyurea were separated from other smaller CFU-s which were shown to be insensitive to the drug and are presumably those CFU-s in the G2 phase of cell cycle. 相似文献
9.
S. Papa M. Vitale G. Mazzotti R. Rizzoli M. Falconi A. Bartoletti F. A. Manzoli 《Histochemistry and cell biology》1988,89(3):241-245
Summary A number of techniques are currently employed for the fractionation of heterogeneous cell populations or for the separation of cells in different phases of their cycle. With the development of osmotically inert colloidal silica particles media, density gradient centrifugation became an established method for the separation and purification of cells and subcellular particles. We have applied this technique to the separation of cycling from resting Friend erythroleukemia cells, to obtain purified populations for further biological assays. The flow cytometric analysis of DNA content of the different fractions obtained by the gradient and stained with Propidium lodide (PI), showed the S compartment highly concentrated in the 1.073/77g/ml interface, while the upper levels of the gradient were highly enriched of cells in G1 phase. Moreover, the dual parameter analysis of DNA content by means of Bromodeoxyuridine (BrdUrd) incorporation and PI staining, showed that part of the cells in the 1.067/73 fraction represented the early S phase even if their DNA level, measured on the basis of PI fluorescence was within the diploid cell cluster. This method seems to be suitable to obtain pure cell fractions even when dealing with numerically large populations. 相似文献
10.
To date two inhibitors of epidermal cell proliferation have been characterized: (1) a factor which depresses DNA synthesis, and (2) a factor which depresses mitotic rate. In the absence of experimental proof it has been assumed that the respective targets for these purified inhibitory factors are in G1 and G2 phases of the cell cycle. In the experiments reported here both these fractions were subjected to cell cycle phase specificity tests in order to verify these assumptions. In addition, an epidermally derived “cell line” (the sebaceous gland) and two nonectodermal tissues were examined for a response. The results suggest that the response induced by the inhibitor of DNA synthesis is cell cycle phase-specific, that the target cells are at the G1-S phase boundary, and that only epidermal cells respond. Similarly the factor which depresses the flow of cells from G2 into mitosis had no measurable effect on DNA synthesis by any of the tissues tested. The G2 inhibitor lacks an inhibitory effect on mitosis in the sebaceous gland.The physiological roles which epidermal chalones may play are briefly discussed. It is suggested that a G1–G2 chalone system may have been effective in isolating kinetically cell populations with modified function during the evolutionary development in the vertebrates. 相似文献
11.
《Neurochemistry international》1986,9(1):111-119
The hydrophobic interactions of globular forms of acetylcholinesterase from adult and embryonic chick muscles have been analyzed by sucrose gradient centrifugation and non denaturing polyacrylamide gel electrophoresis. The presence of positively- or negatively-charged detergents influences the electrophoretic migrations of hydrophobic globular forms, whereas the mobility of hydrophilic components is unchanged. We defined an hydrophobicity index (HI) which quantitatively reflects this interaction.Globular forms of acetylcholinesterase were isolated in preparative sucrose gradients of muscle extracts. The G1 form (5 S) appeared as a single band in electrophoresis, the G2 form (7 S) under two and the G4 form (11 S) under three electromorphs. The G1 and the G2 forms interacted with detergents: this resulted in a shift in their sedimentation in sucrose gradients upon removal of detergents, and in a modification of their electrophoretic migrations in the presence of charged detergents (HI = 1.0 for G1, HI = 1.7 for G2). The G4 form was heterogenous: one band (G4f) did not interact with detergent (HI = 0.1). The other variants (G4i and G4s) were clearly hydrophobic (HI = 0.5 and HI = I respectively). The hydrophilic and hydrophobic variants of the G4 form however, were not resolved by sedimentation analysis performed in the presence of Triton X100, but their separation was improved in the presence of 10-oleyl-ether. Therefore, the combination of electrophoretic and sedimentation methods, as described in this paper, can be used successfully for subdividing a single molecular form (size isomer defined by hydrodynamic parameters) into several constituents differing by their hydrophobic interactions. 相似文献
12.
There are few data available on cell cycle events that occur when proliferation of normal cells in culture is curtailed due to “natural aging” of the culture conditions. Stathmokinetic and cytofluorometry studies were performed on PHA-stimulated human lymphocyte cultures for eight consecutive days. Cell proliferation peaked on day 5 and then gradually decreased. Percent labeled mitosis curves performed each day demonstrated that, for those cells which progressed to mitosis, the cell cycle time remained constant at 18 ± 1 hour throughout the entire period of culture. However when the fate of all cells pulse-labeled with 3H-thymidine (S phase cells) was followed daily, only 64 ± 5% of labeled cells reached mitosis on day 3 and <20% on day 6. When the growth fraction was estimated by standard methods (with the labeling index) and used to predict future cell counts in the culture, proliferation was greatly overestimated; but after correcting the growth fraction for labeled cells not reaching mitosis, proliferation was accurately predicted by a newly derived “dividing fraction.” Flow cytofluorometry confirmed accumulation of cells in S and G2 + M phases, and mitotic indices ruled out accumulation in M phase. Assessment of non-viable cells with cytofluorometry demonstrated that death occurred in all phases of the cell cycle. We conclude that with increasing age of culture, an increased fraction of cycling PHA-stimulated lymphocytes fail to progress all the way to mitosis and are arrested in S or G2 phases. These observations provide evidence against the existence of a specific “restriction point” in G1 or at the G1/S interface in aging proliferating human lymphocyte cultures, but it remains to be determined whether cells arrested in S or G2 phases retain the capacity to complete the cell cycle in more favorable culture environments. 相似文献
13.
Summary A new technique of exploitation of the data was proposed after DNA scanning microdensitometry. By using all of the measurements obtained from the seriated sections of a single nucleus, this method made it possible to estimate six characteristic parameters during the different phases of the cell cycle in the various shoot apical cells. The cells whose rate of proliferation was the highest showed the biggest variations of their nuclear and nucleolar volumes during the cell cycle. In the axial zone, where the cells have a slow cell cycle and display the longest duration of the G1 phase, the volume occupied by dispersed DNA was greater than in the cells of the lateral zone and of the rib meristem, where the cell cycle and the G1 phase were short. No matter what the cell type, the proportion of the dispersed and condensed DNA varied little when the G1 and G2 phases were compared. In the Z phase, characterized by a decondensation of the DNA, the mean DNA amount was 3.4 C. The evolution of the nuclear density during the interphase was also estimated. It is demonstrated that the main feature of the shoot apex zonation was the decondensation of the condensed DNA in the axial zone in both the G1 and G2 phases. 相似文献
14.
H.-P. BECK 《Cell proliferation》1978,11(2):139-148
L-cells synchronized by mitotic selection were investigated by flow-cytometry nd the fractions of cells in the various cell cycle compartments were determined as a function of time. A new analytical evaluation procedure was developed, by which the mean transit-times of cells through various cell cycle phases can be calculated from these data. Three examples for application of the method are presented: (1) determination of the duration of G1, S, G2+ M and of the whole cell cycle; (2) calculation of the rate of DNA synthesis in several subcompartments of the S-phase; and (3) evaluation of the degree of synchronization at different stages of the cell cycle. 相似文献
15.
Conservative homologous recombination preferentially repairs DNA double-strand breaks in the S phase of the cell cycle in human cells 总被引:8,自引:2,他引:6
DNA double-strand breaks (DSBs) are repaired by either homologous recombination (HR) or non-homologous end joining (NHEJ) in mammalian cells. Repair with NHEJ or HR using single-strand annealing (SSA) often results in deletions and is generally referred to as non-conservative recombination. Error-free, conservative HR involves strand invasion and requires a homologous DNA template, and therefore it is generally believed that this type of repair occurs preferentially in the late S, G2 and M phases of the cell cycle, when the sister chromatid is available. There are several observations supporting this hypothesis, although it has not been tested directly. Here, we synchronize human SW480SN.3 cells in the G1/G0 (with serum starvation), S (with thymidine block) and M (with nocodazole) phases of the cell cycle and investigate the efficiency of conservative HR repair of an I-SceI-induced DSB. The frequency of HR repair of DSBs was 39 times higher in S-phase cells than in M-phase cells and 24-fold higher than in G1/G0 cells. This low level of conservative HR occurs even though a homologous template is present within the recombination substrate. We propose that this can be explained by an absence of recombination proteins outside the S phase or alternatively that there maybe factors that suppress HR in G1/G0 and M. Furthermore, we found that HR repair of DSBs involves short tract gene conversion in all the phases of the cell cycle. This indicates that the same pathway for conservative HR is employed in the repair of DSBs regardless of phase of the cell cycle and that only the frequency is affected. 相似文献
16.
Masao Nakagaki Ryuzo Takei Eiichi Nagashima Toshinobu Yaginuma 《Development genes and evolution》1991,200(4):223-229
Summary In the silkworm, Bombyx mori, diapause occurs at a specific embryonic stage, i.e. after formation of the germ band with cephalic lobes and telson and sequential mesoderm segmentation. As long as the eggs are incubated at 25° C, cell divisions and morphological development of the embryos cease. To examine changes in percentage of embryonic cells in the G1, S and G2 phases during embryogenesis, nuclear fractions were isolated from embryos, stained with propidium iodide and then subjected to flow cytometric analysis. The percentages of embryonic cells in G1, S and G2 were 10, 35 and 55%, respectively, at the stage of formation of cephalic lobes, whilst 98% of cells were in G2 at diapause stage. After termination of diapause by acclimation at 5° C or by a combination of chilling and HCl, cell division resumed in the embryos. During this period, the cells rapidly entered S phase through G1 from G2, suggesting that their G1 phase was short. In eggs in which diapause was averted by HCl-treatment after incubation at 25° C for 20 h after oviposition, embryonic development proceeded continuously for 9.5 days at 25° C until hatching. Along with this development, the G1 fraction increased to levels of about 90%. These results indicate that embryonic cells are arrested in G2 at diapause and suggest that, concomitant with further embryonic development, cell cycles become slower in proportion to an increasing length of G1. Finally, most of the cells may be arrested in G1, while there is only a small fraction of cells continuously cycling.
Offprint requests to: T. Yaginuma 相似文献
17.
Y. Kowyama T. Kawase H. Yamagata 《TAG. Theoretical and applied genetics. Theoretische und angewandte Genetik》1984,68(4):297-303
Summary In order to examine changes in survival and mutation rates during a cell cycle in higher plant, fertilized egg cells of rice were irradiated with X-rays at 2 h intervals for the first 36 h after pollination, i.e., at different phases of the first and second cell cycles. The most sensitive phase in lethality was late G1 to early S, followed by late G2 to M, which were more sensitive than the other phases. In both M1 and M2 generations, sterile plants appeared most frequently when fertilized egg cells were irradiated at G2 and M phases. Different kinds of mutated characters gave rise to the respective maximum mutation rates at different phases of a cell cycle: namely, albino and viridis were efficiently induced at early G1, xantha at early S, short-culm mutant at mid G2, heading-date mutant at M to early G1. The present study suggests the possibility that the differential mutation spectrums concerning agronomic traits are obtained by selecting the time of irradiation after pollination. 相似文献
18.
George Constantopoulos Khalid Iqbal† Anatole S. Dekaban 《Journal of neurochemistry》1980,34(6):1399-1411
Abstract: Brain cellular fractions were prepared in bulk from four non-neurological patients and from five patients with mucopolysaccharidosis (MPS). Glycosaminoglycans and lipids were isolated and chemically analyzed. Results of the present study: in the normal controls glycosaminoglycans as μg per mg protein (mean) were 2.2 in neuronal perikarya, 2.0 in astroglia, 2.1 in oligodendroglia, 3.3 in neuropile from gray matter and 3.2 in a mixed fraction from white matter. In the partially myelinated axons from gray and white matter of an 8-month-old infant, the concentration was 6.9 and 2.6 μg per mg protein, compared with 2.8 and 0.8 μg per mg protein, respectively, in the adult patients. It was estimated that chondroitin sulfates constituted more than one-half of the total glycosaminoglycan. Hyaluronic acid, heparan sulfate and dermatan sulfate were also present in all cell types and fractions. Cholesterol, phospholipids, cerebrosides, sulfatide and gangliosides were present in all cell types and fractions, but differed widely in concentration. There was a four- to sixfold increase in the concentration of total glycosaminoglycans in the neuronal perikarya of patients with MPS IH, II and IIIA. The increased glycosaminoglycans were heparan sulfate in MPS IIIA and dermatan sulfate plus heparan sulfate in MPS IH and II. Similar changes were found in the astroglia and in the other brain fractions of those patients. The concentration of the gangliosides Gm 2, Gm 3, Gd 3 and ceramide dihexoside was markedly increased in the neurons and other brain fractions of the same patients. The quantities of Gm 3, Gm 2 and Gd 3 together amounted to 65% of the total gangliosides of the neurons, indicating changes of the same magnitude seen in the gangliosidoses. All these patients exhibited mental retardation. The concentration and composition of glycosaminoglycans, gangliosides and neutral hexosyl ceramides in the neuronal perikarya of the patient with MPS IS was normal. There was only a small increase of dermatan sulfate content in the neuropile, mixed fraction and myelinated axons from the white matter and some increase of ceramide dihexoside content in the myelinated axons. This patient was an adult of normal intelligence. 相似文献
19.
Summary Exponentially growing HeLa cells have been separated according to their cell cycle age by sedimenting at unit gravity for
3 hr on a phosphate-buffered sucrose density gradient. Measurements of cell size, cell number, DNA content, and tritiated
thymidine incorporation in consecutive portions of the gradient showed that cells in upper fractions were in G1, cells in middle fractions were in S, and cells in lower fractions were in G2. Basic amino acids were rapidly incorporated into nuclear protein during late G1 and S; some incorporation also took place during G2.
This work is supported by grant A-3458 from the National Research Council of Canada. 相似文献
20.
To better understand how the flow cytometric bromodeoxyuridine (BrdUrd)-pulse-chase method detects perturbed cell kinetics we applied it to measure cell cycle progression delays following exposure to ionizing radiation. Since this method will allow both the use of asynchronous cell populations and the determination of the alterations in cell cycle progression specific to cells irradiated in given cell cycle phases, it has a significant advantage over laborious synchronization methods. Exponentially growing Chinese hamster ovary (CHO) K1 cells were irradiated with graded doses of X-rays and pulse-labelled with BrdUrd immediately thereafter. Cells were subcultured in a BrdUrd-free medium for various time intervals and prepared for flow cytometric analysis. Of five flow cytometric parameters examined, only those that involved cell transit through G2, i.e. the fraction of BrdUrd-negative G2 cells and the fraction of BrdUrd-positive cells that had not divided, showed radiation dose-dependent delays. The magnitude of the effects indicates that the cells irradiated in G2 and in S are equally delayed. S phase transit of cells irradiated in S or in G1 did not appear to be affected. There were apparent changes in flow of cells out of G1, which could be explained by the delayed entry of G2 cells into the compartment because of G2 arrest. Thus, in asynchronous cells the method was able to detect G2 delay in those cells irradiated in S and G2 phases and demonstrate the absence of cell-cycle delays in other phases. 相似文献