Regulation of the start of DNA replication in Schizosaccharomyces pombe

1999 ◽  
Vol 112 (6) ◽  
pp. 939-946 ◽  
Author(s):  
C.R. Carlson ◽  
B. Grallert ◽  
T. Stokke ◽  
E. Boye
Keyword(s):  
Cell Cycle ◽  
Flow Cytometry ◽  
Growth Rate ◽  
Schizosaccharomyces Pombe ◽  
Growth Rates ◽  
Cell Separation ◽  
Cell Mass ◽  
Nitrogen Sources ◽  
S Phase ◽  
G1 Phase

Cells of Schizosaccharomyces pombe were grown in minimal medium with different nitrogen sources under steady-state conditions, with doubling times ranging from 2.5 to 14 hours. Flow cytometry and fluorescence microscopy confirmed earlier findings that at rapid growth rates, the G1 phase was short and cell separation occurred at the end of S phase. For some nitrogen sources, the growth rate was greatly decreased, the G1 phase occupied 30–50% of the cell cycle, and cell separation occurred in early G1. In contrast, other nitrogen sources supported low growth rates without any significant increase in G1 duration. The method described allows manipulation of the length of G1 and the relative cell cycle position of S phase in wild-type cells. Cell mass was measured by flow cytometry as scattered light and as protein-associated fluorescence. The extensions of G1 were not related to cell mass at entry into S phase. Our data do not support the hypothesis that the cells must reach a certain fixed, critical mass before entry into S. We suggest that cell mass at the G1/S transition point is variable and determined by a set of molecular parameters. In the present experiments, these parameters were influenced by the different nitrogen sources in a way that was independent of the actual growth rate.

The Effect of 2-Phenyl Ethanol on the DNA Synthesis Cycle of Schizosaccharomyces Pombe

1970 ◽  
Vol 7 (2) ◽  
pp. 523-530
Author(s):  
C. J. BOSTOCK
Keyword(s):  
Cell Cycle ◽  
Dna Synthesis ◽  
Schizosaccharomyces Pombe ◽  
Normal Growth ◽  
S Phase ◽  
G1 Phase ◽  
Synchronous Cultures ◽  
Number Of Cells

The effect of different concentrations of 2-phenyl ethanol (PE) on growth and DNA synthesis of Schizosaccharomyces pombe is described. o.3% PE inhibits the entry of cells into S phase, but allows a doubling in the number of cells in the culture. The effect of o.2% PE on random and synchronous cultures of S. pombe shows that, in the continued presence of the inhibitor, the S phase is moved to a different point in the cell cycle. Cells continue to grow in the presence of o.2% PE with a G1 phase occupying a significant portion of the cell cycle. This differs from normal growth when the G1 phase is absent.

Effects of temperature and nutritional conditions on the mitotic cell cycle of Saccharomyces cerevisiae

1978 ◽  
Vol 31 (1) ◽  
pp. 71-78
Author(s):  
M.N. Jagadish ◽  
B.L. Carter
Keyword(s):  
Cell Cycle ◽  
Growth Rate ◽  
Growth Rates ◽  
Mitotic Cell ◽  
S Phase ◽  
Yeast Cells ◽  
Batch Cultures ◽  
Nutritional Conditions ◽  
Limiting Nutrient ◽  
Effects Of Temperature

Yeast cells were cultivated at different growth rates in a chemostat by alterations in the flow of the limiting nutrient glucose and in batch cultures where variations in growth rate were achieved by alterations in the composition of nutrients. It was observed that the stage in the cycle at which S-phase was completed varied with growth rate. The faster the growth rate, the earlier the stage in the cycle in which completion of S-phase occurred. When stage in the cycle is converted into time before division it was observed that the time from completion of S-phase to cell division varied only slightly with growth rate except at extremely slow growth rates. Expansion of cell cycle transit time as the growth rate was slowed was achieved primarily by an expansion in time of the period from division to the completion of S-phase. In contrast, when cells were grown at different rates by alterations in the temperature of cultivation, completion of S-phase occurred at approximately the same stage in the cell cycle at all growth rates.

scholarly journals Immunochemical detection of cell cycle synchronization in a human erythroleukemia cell line, K562.

1987 ◽  
Vol 35 (10) ◽  
pp. 1143-1148 ◽  
Author(s):  
M M Walker ◽  
P E Wanda
Keyword(s):  
Cell Cycle ◽  
Flow Cytometry ◽  
K562 Cells ◽  
Culture Conditions ◽  
S Phase ◽  
Immunofluorescent Staining ◽  
G1 Phase ◽  
Cell Cycle Synchronization ◽  
Immunochemical Detection ◽  
Erythroleukemia Cell

Cells of the human erythroleukemic line K562 can be induced by manipulation of culture conditions to arrest within the G1 phase of the cell cycle, and subsequently to enter S phase synchronously after release from G1. Cell cultures subjected to serum deprivation and hydroxyurea (HU) treatment demonstrated less than 5% of the cells to be in S phase. Four hours after release from HU, 63% of the cells were in S phase, as detected by immunofluorescent staining. This protocol offers a method for synchronization of K562 cells at the G1/S border and a technique for detection of S-phase cells without the use of radioisotopes or flow cytometry instrumentation.

scholarly journals CLN3 expression is sufficient to restore G1-to-S-phase progression in Saccharomyces cerevisiae mutants defective in translation initiation factor eIF4E

1999 ◽  
Vol 340 (1) ◽  
pp. 135-141 ◽  
Author(s):  
Parisa DANAIE ◽  
Michael ALTMANN ◽  
Michael N. HALL ◽  
Hans TRACHSEL ◽  
Stephen B. HELLIWELL
Keyword(s):  
Cell Cycle ◽  
S Phase ◽  
Translation Initiation Factor ◽  
Yeast Cells ◽  
Initiation Factor ◽  
G1 Phase ◽  
Mrna Levels ◽  
Wild Type ◽  
Phase Progression ◽  
Type Gene

The essential cap-binding protein (eIF4E) of Saccharomycescerevisiae is encoded by the CDC33 (wild-type) gene, originally isolated as a mutant, cdc33-1, which arrests growth in the G1 phase of the cell cycle at 37 °C. We show that other cdc33 mutants also arrest in G1. One of the first events required for G1-to-S-phase progression is the increased expression of cyclin 3. Constructs carrying the 5ʹ-untranslated region of CLN3 fused to lacZ exhibit weak reporter activity, which is significantly decreased in a cdc33-1 mutant, implying that CLN3 mRNA is an inefficiently translated mRNA that is sensitive to perturbations in the translation machinery. A cdc33-1 strain expressing either stable Cln3p (Cln3-1p) or a hybrid UBI4 5ʹ-CLN3 mRNA, whose translation displays decreased dependence on eIF4E, arrested randomly in the cell cycle. In these cells CLN2 mRNA levels remained high, indicating that Cln3p activity is maintained. Induction of a hybrid UBI4 5ʹ-CLN3 message in a cdc33-1 mutant previously arrested in G1 also caused entry into a new cell cycle. We conclude that eIF4E activity in the G1-phase is critical in allowing sufficient Cln3p activity to enable yeast cells to enter a new cell cycle.

Evaluation of genomic sequence-based growth rate methods for synchronized Synechococcus cultures

2021 ◽  
Author(s):  
Julia Carroll ◽  
Nicolas Van Oostende ◽  
Bess B. Ward
Keyword(s):  
Cell Cycle ◽  
Growth Rate ◽  
Dna Replication ◽  
Growth Rates ◽  
Genomic Sequence ◽  
Niche Differentiation ◽  
Trophic Levels ◽  
Individual Species ◽  
Cell Counts

Standard methods for calculating microbial growth rates (μ) through the use of proxies, such as in situ fluorescence, cell cycle, or cell counts, are critical for determining the magnitude of the role bacteria play in marine carbon (C) and nitrogen (N) cycles. Taxon-specific growth rates in mixed assemblages would be useful for attributing biogeochemical processes to individual species and understanding niche differentiation among related clades, such as found in Synechococcus and Prochlorococcus . We tested three novel DNA sequencing-based methods (iRep, bPTR, and GRiD) for evaluating growth of light synchronized Synechococcus cultures under different light intensities and temperatures. In vivo fluorescence and cell cycle analysis were used to obtain standard estimates of growth rate for comparison with the sequence-based methods (SBM). None of the SBM values were correlated with growth rates calculated by standard techniques despite the fact that all three SBM were correlated with percentage of cells in S phase (DNA replication) over the diel cycle. Inaccuracy in determining the time of maximum DNA replication is unlikely to account entirely for the absence of relationship between SBM and growth rate, but the fact that most microbes in the surface ocean exhibit some degree of diel cyclicity is a caution for application of these methods. SBM correlate with DNA replication but cannot be interpreted quantitatively in terms of growth rate. Importance Small but abundant, cyanobacterial strains such as the photosynthetic Synechococcus spp. are essential because they contribute significantly to primary productivity in the ocean. These bacteria generate oxygen and provide biologically-available carbon, which is essential for organisms at higher trophic levels. The small size and diversity of natural microbial assemblages means that taxon-specific activities (e.g., growth rate) are difficult to obtain in the field. It has been suggested that sequence-based methods (SBM) may be able to solve this problem. We find, however, that SBM can detect DNA replication and are correlated with phases of the cell cycle but cannot be interpreted in terms of absolute growth rate for Synechococcus cultures growing under a day-night cycle, like that experienced in the ocean.

The Schizosaccharomyces pombe hus5 gene encodes a ubiquitin conjugating enzyme required for normal mitosis

1995 ◽  
Vol 108 (2) ◽  
pp. 475-486 ◽  
Author(s):  
F. al-Khodairy ◽  
T. Enoch ◽  
I.M. Hagan ◽  
A.M. Carr
Keyword(s):  
Cell Cycle ◽  
Dna Synthesis ◽  
Schizosaccharomyces Pombe ◽  
Cell Cycle Control ◽  
S Phase ◽  
Temperature Sensitive ◽  
Checkpoint Control ◽  
Ubiquitin Conjugating Enzyme ◽  
Efficient Recovery ◽  
Mediate Cell

Normal eukaryotic cells do not enter mitosis unless DNA is fully replicated and repaired. Controls called ‘checkpoints’, mediate cell cycle arrest in response to unreplicated or damaged DNA. Two independent Schizosaccharomyces pombe mutant screens, both of which aimed to isolate new elements involved in checkpoint controls, have identified alleles of the hus5+ gene that are abnormally sensitive to both inhibitors of DNA synthesis and to ionizing radiation. We have cloned and sequenced the hus5+ gene. It is a novel member of the E2 family of ubiquitin conjugating enzymes (UBCs). To understand the role of hus5+ in cell cycle control we have characterized the phenotypes of the hus5 mutants and the hus5 gene disruption. We find that, whilst the mutants are sensitive to inhibitors of DNA synthesis and to irradiation, this is not due to an inability to undergo mitotic arrest. Thus, the hus5+ gene product is not directly involved in checkpoint control. However, in common with a large class of previously characterized checkpoint genes, it is required for efficient recovery from DNA damage or S-phase arrest and manifests a rapid death phenotype in combination with a temperature sensitive S phase and late S/G2 phase cdc mutants. In addition, hus5 deletion mutants are severely impaired in growth and exhibit high levels of abortive mitoses, suggesting a role for hus5+ in chromosome segregation. We conclude that this novel UBC enzyme plays multiple roles and is virtually essential for cell proliferation.

scholarly journals Replication fork rate and origin activation during the S phase of Saccharomyces cerevisiae.

1980 ◽  
Vol 85 (1) ◽  
pp. 108-115 ◽  
Author(s):  
C J Rivin ◽  
W L Fangman
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Cell Cycle ◽  
Cycle Length ◽  
Replication Fork ◽  
Nitrogen Sources ◽  
S Phase ◽  
Phase Duration ◽  
Yeast Saccharomyces Cerevisiae ◽  
Origin Activation ◽  
Cell Cycle Length

When the growth rate of the yeast Saccharomyces cerevisiae is limited with various nitrogen sources, the duration of the S phase is proportional to cell cycle length over a fourfold range of growth rates (C.J. Rivin and W. L. Fangman, 1980, J. Cell Biol. 85:96-107). Molecular parameters of the S phases of these cells were examined by DNA fiber autoradiography. Changes in replication fork rate account completely for the changes in S-phase duration. No changes in origin-to-origin distances were detected. In addition, it was found that while most adjacent replication origins are activated within a few minutes of each other, new activations occur throughout the S phase.

Post-transcriptional control of the onset of DNA synthesis by an insulin-like growth factor

1984 ◽  
Vol 4 (9) ◽  
pp. 1807-1814
Author(s):  
J Campisi ◽  
A B Pardee
Keyword(s):  
Cell Cycle ◽  
Growth Factors ◽  
Growth Factor ◽  
Dna Synthesis ◽  
Transcriptional Control ◽  
S Phase ◽  
G1 Phase ◽  
Insulin Like Growth Factor ◽  
Igf I ◽  
Translational Inhibition

The control of eucaryotic cell proliferation is governed largely by a series of regulatory events which occur in the G1 phase of the cell cycle. When stimulated to proliferate, quiescent (G0) 3T3 fibroblasts require transcription, rapid translation, and three growth factors for the growth state transition. We examined exponentially growing 3T3 cells to relate the requirements for G1 transit to those necessary for the transition from the G0 to the S phase. Cycling cells in the G1 phase required transcription, rapid translation, and a single growth factor (insulin-like growth factor [IGF] I) to initiate DNA synthesis. IGF I acted post-transcriptionally at a late G1 step. All cells in the G1 phase entered the S phase on schedule if either insulin (hyperphysiological concentration) or IGF I (subnanomolar concentration) was provided as the sole growth factor. In medium lacking all growth factors, only cells within 2 to 3 h of the S phase were able to initiate DNA synthesis. Similarly, cells within 2 to 3 h of the S phase were less dependent on transcription and translation for entry into the S phase. Cells responded very differently to inhibited translation than to growth factor deprivation. Cells in the early and mid-G1 phases did not progress toward the S phase during transcriptional or translational inhibition, and during translational inhibition they actually regressed from the S phase. In the absence of growth factors, however, these cells continued progressing toward the S phase, but still required IGF at a terminal step before initiating DNA synthesis. We conclude that a suboptimal condition causes cells to either progress or regress in the cell cycle rather than freezing them at their initial position. By using synchronized cultures, we also show that in contrast to earlier events, this final, IGF-dependent step did not require new transcription. This result is in contrast to findings that other growth factors induce new transcription. We examined the requirements for G1 transit by using a chemically transformed 3T3 cell line (BPA31 cells) which has lost some but not all ability to regulate its growth. Early- and mid-G1-phase BPA31 cells required transcription and translation to initiate DNA synthesis, although they did not regress from the S phase during translational inhibition. However, these cells did not need IGF for entry into the S phase.

Yeast L double-stranded ribonucleic acid is synthesized during the G1 phase but not the S phase of the cell cycle

1981 ◽  
Vol 1 (8) ◽  
pp. 673-679
Author(s):  
V A Zakian ◽  
D W Wagner ◽  
W L Fangman
Keyword(s):  
Cell Cycle ◽  
Deoxyribonucleic Acid ◽  
Cell Cycle Control ◽  
S Phase ◽  
G1 Phase ◽  
Restrictive Temperature ◽  
Permissive Temperature ◽  
Phase Synthesis ◽  
Ribonucleic Acids ◽  
Alpha Factor

The cytoplasm of Saccharomyces cerevisiae contains two major classes of protein-encapsulated double-stranded ribonucleic acids (dsRNA's), L and M. Replication of L and M dsRNA's was examined in cells arrested in the G1 phase by either alpha-factor, a yeast mating pheromone, or the restrictive temperature for a cell cycle mutant (cdc7). [3H]uracil was added during the arrest periods to cells prelabeled with [14C]uracil, and replication was monitored by determining the ratio of 3H/14C for purified dsRNA's. Like mitochondrial deoxyribonucleic acid, both L and M dsRNA's were synthesized in the G1 arrested cells. The replication of L dsRNA was also examined during the S phase, using cells synchronized in two different ways. Cells containing the cdc7 mutation, treated sequentially with alpha-factor and then the restrictive temperature, enter a synchronous S phase when transferred to permissive temperature. When cells entered the S phase, synthesis of L dsRNA ceased, and little or no synthesis was detected throughout the S phase. Synthesis of L dsRNA was also observed in G1 phase cells isolated from asynchronous cultures by velocity centrifugation. Again, synthesis ceased when cells entered the S phase. These results indicate that L dsRNA replication is under cell cycle control. The control differs from that of mitochondrial deoxyribonucleic acid, which replicates in all phases of the cell cycle, and from that of 2-micron DNA, a multiple-copy plasmid whose replication is confined to the S phase.

scholarly journals Expression of p27Kip1 in Osteoblast-Like Cells during Differentiation with Parathyroid Hormone*

Endocrinology ◽  
1997 ◽  
Vol 138 (5) ◽  
pp. 1995-2004 ◽  
Author(s):  
Takehisa Onishi ◽  
Keith Hruska
Keyword(s):  
Cell Cycle ◽  
Cell Proliferation ◽  
Protein Kinase ◽  
Protein Kinase A ◽  
Osteogenic Sarcoma ◽  
S Phase ◽  
G1 Phase ◽  
Osteoblastic Cell ◽  
Cyclin Dependent Kinases ◽  
Kinase A

Abstract PTH is a major systemic regulator of bone metabolism and plays an important role in both bone formation and resorption. PTH either inhibits or stimulates osteoblastic cell proliferation depending on the model that is studied. We analyzed the cell cycle of the UMR-106 cell line, a relatively differentiated osteoblastic osteogenic sarcoma line in which PTH is known to inhibit proliferation but the mechanism of action is unknown. PTH decreased the proportion of cells in S phase and increased the number of G1 phase cells. We examined the effect of PTH on the regulators of the G1 phase cyclin-dependent kinases and found that PTH increased p27Kip1, but not p21Cip1, levels. This effect was mimicked by 8-bromo-cAMP, but not by phorbol 12-myristate 13-acetate. The protein kinase A inhibitor KT5720 abolished the effect of PTH on the increase in p27Kip1 expression. PTH increased CDK2-associated p27Kip1 without affecting the levels of CDK2. CDK2 activity was down-regulated by both PTH and 8-bromo-cAMP treatment. These data suggest that PTH blocks entry of cells into S phase and inhibits cell proliferation as the consequence of an increase in p27Kip1, which is mediated through the protein kinase A pathway. The inhibition of G1 cyclin-dependent kinases by p27Kip1 could cause a reduction of phosphorylation of key substrates and inactivation of transcription factors essential for entry into S phase. The inhibition of cell cycle progression through PKA-mediated p27Kip1 induction might play an important role in PTH-induced differentiation of osteoblasts.

Sign in / Sign up

Export Citation Format

Share Document