Synchronisation of mitosis in a cell division cycle mutant of Schizosaccharomyces pombe released from temperature arrest

1982 ◽  
Vol 28 (2) ◽  
pp. 261-264 ◽  
Author(s):  
Stephen M. King ◽  
Jeremy S. Hyams
Keyword(s):  
Cell Division ◽  
Schizosaccharomyces Pombe ◽  
Temperature Shift ◽  
Cell Plate ◽  
Restrictive Temperature ◽  
C Cells ◽  
Wild Type ◽  
Ultrastructural Morphology ◽  
Synchronous Cultures ◽  
A Cell

When cultures of Schizosaccharomyces pombe cdc 2.33 were shifted to 25 °C, after 5 h at the restrictive temperature of 35 °C, cells entered cycles of synchronous division as judged by the appearance of peaks in the cell plate index at 1.5, 3, and 4.75 h. The timing and ultrastructural morphology of events occurring in such synchronous cultures were examined. Most cells underwent mitosis between 10 and 50 min after the temperature shift, with a maximal value after approximately 30 min. The ultrastructure of mitosis was consistent with previous descriptions of this process in wild-type cells.

Growth in cell length in the fission yeast Schizosaccharomyces pombe

1985 ◽  
Vol 75 (1) ◽  
pp. 357-376 ◽  
Author(s):  
J.M. Mitchison ◽  
P. Nurse
Keyword(s):  
Schizosaccharomyces Pombe ◽  
Cell Size ◽  
Single Cells ◽  
Temperature Shift ◽  
Time Lapse ◽  
Cell Length ◽  
Wild Type ◽  
Cycle Growth ◽  
A Cell ◽  
Mutant Cells

The cylindrical cells of Schizosaccharomyces pombe grow in length by extension at the ends and not the middle. At the beginning of the cell cycle, growth is restricted to the ‘old end’, which existed in the previous cycle. Later on, the ‘new end’, formed from the septum, starts to grow at a point in the cycle that we have called NETO (‘new end take-off’). Fluorescence microscopy on cells stained with Calcofluor has been used to study NETO in size mutants, in blocked cdc mutants and with different growth temperatures and media. In wild-type cells (strain 972) NETO happens at 0.34 of the cycle with a cell length of 9.5 microns. With size mutants that are smaller at division, NETO takes place at the same size (9.0-9.5 microns) but this is not achieved until later in the cycle. Another control operates in larger size mutants since NETO occurs at the same stage of the cycle (about 0.32) as in wild type but at a larger cell size. This control is probably a requirement to have completed an event in early G2, since most cdc mutant cells blocked before this point in the cycle do not show NETO whereas most of those blocked in late G2 do show it. We conclude that NETO only happens if: (1) the cell length is greater than a critical value of 9.0-9.5 microns; and (2) the cell has traversed the first 0.3-0.35 of the cycle and passed early G2. NETO is delayed in poor media, in which cell size is also reduced. Temperature has little effect on NETO under steady-state conditions, but there is a transient delay for some hours after a temperature shift. NETO is later in another wild-type strain, 132. Time-lapse photomicrography was used to follow the rates of length growth in single cells. Wild-type cells showed two linear segments during the first 75% of the cycle. There was a rate-change point (RCP), coincident with NETO, where the rate of total length extension increased by 35%. This increase was not due simply to the start of new-end growth, since old-end growth slowed down in some cells at the RCP. cdc 11.123 is a mutant in which septation and division is blocked at 35 degrees C but nuclear division continues.(ABSTRACT TRUNCATED AT 400 WORDS)

Continued DNA synthesis after a mitotic block in the double mutant cut1 cdc11 of the fission yeast Schizosaccharomyces pombe

1990 ◽  
Vol 96 (3) ◽  
pp. 435-438
Author(s):  
J. Creanor ◽  
J.M. Mitchison
Keyword(s):  
Dna Synthesis ◽  
Schizosaccharomyces Pombe ◽  
Double Mutant ◽  
Single Cells ◽  
Restrictive Temperature ◽  
Synchronous Cultures ◽  
Fluorescence Measurements ◽  
Dna Assays ◽  
A Cell ◽  
Two Peaks

DNA synthesis is normally dependent on a cell having previously gone through mitosis. Hirano et al. (1986), however, found that DNA synthesis continued at the restrictive temperature in the double mutant cut1 cdc11 of Schizosaccharomyces pombe even though mitosis was blocked in some of the cells. We have confirmed this result with bulk DNA assays of asynchronous cultures. Synchronous cultures of a diploid double mutant at the restrictive temperature showed two peaks of incorporation with an interval between them that was approximately the same as the doubling time in cell length. Flow cytometry showed that the cells had increased their DNA content from 4C (the diploid value) to about 16C after 7h. The cytological appearance at this time was mixed, with uninucleate, binucleate and dead cells, but fluorescence measurements on single cells indicated that about half the population had single nuclei with about the 16C value and had therefore gone through two rounds of DNA synthesis without mitosis.

Arginase and sucrase potential in the fission yeast Schizosaccharomyces pombe

1980 ◽  
Vol 46 (1) ◽  
pp. 399-431
Author(s):  
T. Benitez ◽  
P. Nurse ◽  
J.M. Mitchison
Keyword(s):  
Dna Replication ◽  
Dna Synthesis ◽  
Schizosaccharomyces Pombe ◽  
Fission Yeast ◽  
Gene Dosage ◽  
Critical Ratio ◽  
Restrictive Temperature ◽  
Wild Type ◽  
Synchronous Cultures ◽  
Short Period

The induction potentials of 2 enzymes, sucrase and arginase, have been measured in asynchronous and synchronous cultures of the fission yeast Schizosaccharomyces pombe. The effect on potential of inhibiting DNA synthesis is asynchronous cultures has been studied using 2 temperature-sensitive dcd mutants, one blocked in DNA replication and the other blocked in mitosis. The results show that despite inhibition of DNA synthesis, sucrase and arginase potential both continue to increase exponentially for at least a generation of growth after shifting the cdc mutants from the permissive to the restrictive temperature. A second method of inhibiting DNA synthesis, using deoxyadenosine, has also been tested. Cells treated with deoxyadenosine stop the increase in potential for a short period. However, experiments carried out using a cdc mutant together with deoxyadenosine show that the block to the increase in potential is due to a side effect of the inhibitor. It appears that increase in potential is not dependent upon continued DNA replication, and that gene dosage does not control potential in the normal cell cycle. This conclusion is supported by measurements on mutants of different cell sizes. potential is proportional to size (protein content per cell is asynchronous culture) and not to DNA content. Although potential is not gene limited in normal cells, it does appear to be so in the abnormally large cells produced by a cdc block. If cdc mutants of different sizes are grown asynchronously, and DNA synthesis is inhibited by a shift to the restrictive temperature, there is no increase in potential. This critical ratio is different for the 2 enzymes, but for each enzyme it is similar in all the mutants tested. When large cells (produced by a mutant block for 4.5 h) are shifted down in temperature, there are synchronous rounds of DNA synthesis and division and also step doublings in potential. In synchronous cultures of wild type cells, both enzymes show a stepwise doubling of potential at 0.2 of a cycle after DNA replication. In synchronous cultures of cdc mutants blocked either in replication or in mitosis, the potential steps continue with the normal timing observed in wild type cells. This shows that the steps are not dependent on the events of the DNA-division cycle but are controlled by another mechanism. Attainment of a critical size might be part of this mechanism, but tests with size mutants argue against this.

Schizosaccharomyces pombe mutants affected in their division response to starvation

1987 ◽  
Vol 88 (3) ◽  
pp. 295-304 ◽  
Author(s):  
P.G. Young ◽  
P.A. Fantes
Keyword(s):  
Schizosaccharomyces Pombe ◽  
Protein Content ◽  
Nitrogen Starvation ◽  
Size Control ◽  
Fold Increase ◽  
Cell Plate ◽  
Cell Number ◽  
Restrictive Temperature ◽  
Wild Type ◽  
Altered Response

Schizosaccharomyces pombe mutants have been selected on the basis of an altered response to nutritional stimulation of cell division (changed division response, cdr). Two new loci (cdr1 and cdr2) were identified and characterized. When suspended in nitrogen-free medium wild-type cells underwent stimulated rates of division and became reduced to approximately 30% in protein content with a concomitant 3.6-fold increase in cell number after 24 h starvation. cdr cells had significantly smaller increases in cell number. The ratio of starved/unstarved protein content was higher for the cdr strains than for the wild type. cdr cells were also affected in their response to nitrogen-source shifts from proline to glutamate (or vice versa) or when shifted from serine phosphate to inorganic phosphate, showing that the alteration in division response was not restricted to nitrogen metabolism. Upon nitrogen starvation wild-type cells arrested prior to the cdc10 execution point, whereas cdr cells arrested later in the cell cycle. cdc25-22 cdr1 or cdr2 double mutants grew very slowly and were extremely elongated at all temperatures; the restrictive temperature was reduced to 27 degrees C. wee1 was epistatic to cdr mutations with respect to cell length at the cell plate stage. cdr+ genes are postulated to play a role in the nutritional modulation of the mitotic size control.

Nucleoside diphosphokinase and cell cycle control in the fission yeast Schizosaccharomyces pombe

1983 ◽  
Vol 60 (1) ◽  
pp. 355-365
Author(s):  
J.R. Dickinson
Keyword(s):  
Cell Cycle ◽  
Protein Synthesis ◽  
Enzyme Activity ◽  
Dna Replication ◽  
Schizosaccharomyces Pombe ◽  
S Phase ◽  
Restrictive Temperature ◽  
Wild Type ◽  
Synchronous Cultures ◽  
In The Wild

Centrifugal elutriation was used to prepare synchronous cultures of Schizosaccharomyces pombe. Nucleoside diphosphokinase activity was measured throughout the cell cycle. In the wild-type strain (972) nucleoside diphosphokinase activity doubled in a stepwise fashion. The midpoint of the rise in enzyme activity was at 0.65 of a cycle, 0.29 of a cycle before the next S phase. Synchronous cultures of the mutant wee 1–6 were also prepared. In this strain S phase is delayed, occurring about 0.3 cycle later than in the wild-type. In wee 1–6 the midpoint of the stepwise doubling in nucleoside diphosphokinase activity occurred at 0.084; showing that the rise in enzyme activity is also delayed. Addition of cycloheximide to an exponentially growing culture caused an immediate inhibition of protein synthesis, yet nucleoside diphosphokinase activity continued to increase exponentially for a further 300 min. This indicates that the stepwise doubling of nucleoside diphosphokinase activity during the cell cycle is not achieved by a simple control on protein synthesis. Two temperature-sensitive cdc- mutants were also used: cdc2-33, a mutant whose single genetic lesion results in the twin defects of a loss of mitotic control and a loss of commitment to the cell cycle; and cdc 10–129, which has a defect in DNA replication. In both mutants a temperature shift-up of an asynchronously growing culture from the permissive (25 degrees C) to the restrictive temperature (36.5 degrees C) results in a rapid inhibition of DNA replication. In both mutants nucleoside diphosphokinase continues to increase exponentially. Therefore, although nucleoside diphosphokinase is required for DNA replication, apparently DNA replication is not required for an increase in nucleoside diphosphokinase activity.

Mild temperature shock alters the transcription of a discrete class of Saccharomyces cerevisiae genes

1983 ◽  
Vol 3 (3) ◽  
pp. 457-465
Author(s):  
C H Kim ◽  
J R Warner
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Ribosomal Protein ◽  
Ribosomal Proteins ◽  
Temperature Shift ◽  
Restrictive Temperature ◽  
Ribosomal Protein Genes ◽  
Wild Type ◽  
Temperature Shock ◽  
Mild Temperature ◽  
Mutant Cells

In Saccharomyces cerevisiae the synthesis of ribosomal proteins declines temporarily after a culture has been subjected to a mild temperature shock, i.e., a shift from 23 to 36 degrees C, each of which support growth. Using cloned genes for several S. cerevisiae ribosomal proteins, we found that the changes in the synthesis of ribosomal proteins parallel the changes in the concentration of mRNA of each. The disappearance and reappearance of the mRNA is due to a brief but severe inhibition of the transcription of each of the ribosomal protein genes, although the total transcription of mRNA in the cells is relatively unaffected by the temperature shock. The precisely coordinated response of these genes, which are scattered throughout the genome, suggests that either they or the enzyme which transcribes them has unique properties. In certain S. cerevisiae mutants, the synthesis of ribosomal proteins never recovers from a temperature shift. Yet both the decline and the resumption of transcription of these genes during the 30 min after the temperature shift are indistinguishable from those in wild-type cells. The failure of the mutant cells to grow at the restrictive temperature appears to be due to their inability to process the RNA transcribed from genes which have introns (Rosbash et al., Cell 24:679-686, 1981), a large proportion of which appear to be ribosomal protein genes.

scholarly journals Cell cycle arrest of cdc mutants and specificity of the RAD9 checkpoint.

Genetics ◽  
1993 ◽  
Vol 134 (1) ◽  
pp. 63-80 ◽  
Author(s):  
T A Weinert ◽  
L H Hartwell
Keyword(s):  
Cell Cycle ◽  
Cell Division ◽  
Dna Replication ◽  
Cell Cycle Control ◽  
Dna Lesions ◽  
Temperature Sensitive ◽  
Restrictive Temperature ◽  
A Cell ◽  
G2 Phase ◽  
Rad Mutants

Abstract In eucaryotes a cell cycle control called a checkpoint ensures that mitosis occurs only after chromosomes are completely replicated and any damage is repaired. The function of this checkpoint in budding yeast requires the RAD9 gene. Here we examine the role of the RAD9 gene in the arrest of the 12 cell division cycle (cdc) mutants, temperature-sensitive lethal mutants that arrest in specific phases of the cell cycle at a restrictive temperature. We found that in four cdc mutants the cdc rad9 cells failed to arrest after a shift to the restrictive temperature, rather they continued cell division and died rapidly, whereas the cdc RAD cells arrested and remained viable. The cell cycle and genetic phenotypes of the 12 cdc RAD mutants indicate the function of the RAD9 checkpoint is phase-specific and signal-specific. First, the four cdc RAD mutants that required RAD9 each arrested in the late S/G2 phase after a shift to the restrictive temperature when DNA replication was complete or nearly complete, and second, each leaves DNA lesions when the CDC gene product is limiting for cell division. Three of the four CDC genes are known to encode DNA replication enzymes. We found that the RAD17 gene is also essential for the function of the RAD9 checkpoint because it is required for phase-specific arrest of the same four cdc mutants. We also show that both X- or UV-irradiated cells require the RAD9 and RAD17 genes for delay in the G2 phase. Together, these results indicate that the RAD9 checkpoint is apparently activated only by DNA lesions and arrests cell division only in the late S/G2 phase.

Change in the rate of CO2 production in synchronous cultures of the fission yeast Schizosaccharomyces pombe: a periodic cell cycle event that persists after the DNA-division cycle has been blocked

1986 ◽  
Vol 86 (1) ◽  
pp. 191-206
Author(s):  
B. Novak ◽  
J.M. Mitchison
Keyword(s):  
Schizosaccharomyces Pombe ◽  
Rate Change ◽  
Co2 Production ◽  
Temperature Sensitive ◽  
Restrictive Temperature ◽  
Normal Cycle ◽  
Periodic Cell ◽  
Linear Pattern ◽  
Synchronous Cultures ◽  
Oscillatory Pattern

CO2 production has been followed by manometry in synchronous and asynchronous cultures of Schizosaccharomyces pombe prepared by elutriation from the same initial culture. The rate of production follows a linear pattern in synchronous cultures with a rate change once per cycle at the time of cell division. This pattern is most clearly shown in oscillations of the difference between values of the second differential (acceleration) for the synchronous and asynchronous cultures. The association between the rate change and the time of division is maintained during growth speeded up in rich medium and slowed down in poor medium and at lower temperature. It is also maintained after a shift-up in temperature. Results with wee mutants suggest that the association is with the S period rather than division itself. The rate and acceleration of CO2 production are approximately proportional to cell size (protein content) in asynchronous cultures. When synchronous cultures of the temperature-sensitive mutants cdc2.33 and cdc2.33 wee1.6 are shifted up to the restrictive temperature, the DNA-division cycle is blocked. The oscillatory pattern of CO2 production, however, continues for one to two cycles until the acceleration reaches a constant value, after which the oscillations are undetectable. This point is reached later in the double mutant and there is a phase difference in the oscillations compared to those in the single mutant. With both blocked mutants the ‘free-running’ oscillations are about 15% shorter than the normal cycle time. There are well-known examples of such oscillations in eggs but they are rare in growing systems.

Oxygen uptake during the cell cycle of the fission yeast Schizosaccharomyces pombe

1978 ◽  
Vol 33 (1) ◽  
pp. 399-411
Author(s):  
J. Creanor
Keyword(s):  
Cell Cycle ◽  
Cell Division ◽  
Oxygen Uptake ◽  
Dna Synthesis ◽  
Schizosaccharomyces Pombe ◽  
Fission Yeast ◽  
Nuclear Division ◽  
Synchronous Culture ◽  
Synchronous Cultures ◽  
The Many

Oxygen uptake was measured in synchronous cultures of the fission yeast Schizosaccharomyces pombe. The rate of oxygen uptake was found to increase in a step-wise manner at the beginning of the cycle and again in the middle of the cycle. The increases in rate were such that overall, oxygen uptake doubled in rate once per cell cycle. Addition of inhibitors of DNA synthesis or nuclear division to a synchronous culture did not affect the uptake of oxygen. In an induced synchronous culture, in which DNA synthesis, cell division, and nuclear division, but not ‘growth’ were synchronized, oxygen uptake increased continuously in rate and did not show the step-wise rises which were shown in the selection-synchronized culture. These results were compared with previous measurements of oxygen uptake in yeast and an explanation is suggested for the many different patterns which have been reported.

scholarly journals Analysis of ftsQ Mutant Alleles in Escherichia coli: Complementation, Septal Localization, and Recruitment of Downstream Cell Division Proteins

2002 ◽  
Vol 184 (3) ◽  
pp. 695-705 ◽  
Author(s):  
Joseph C. Chen ◽  
Michael Minev ◽  
Jon Beckwith
Keyword(s):  
Escherichia Coli ◽  
Cell Division ◽  
Random Mutagenesis ◽  
Dominant Negative ◽  
Restrictive Temperature ◽  
Permissive Temperature ◽  
Wild Type ◽  
Negative Effects ◽  
Mutant Proteins ◽  
Division Site

ABSTRACT FtsQ, a 276-amino-acid, bitopic membrane protein, is one of the nine proteins known to be essential for cell division in gram-negative bacterium Escherichia coli. To define residues in FtsQ critical for function, we performed random mutagenesis on the ftsQ gene and identified four alleles (ftsQ2, ftsQ6, ftsQ15, and ftsQ65) that fail to complement the ftsQ1(Ts) mutation at the restrictive temperature. Two of the mutant proteins, FtsQ6 and FtsQ15, are functional at lower temperatures but are unable to localize to the division site unless wild-type FtsQ is depleted, suggesting that they compete poorly with the wild-type protein for septal targeting. The other two mutants, FtsQ2 and FtsQ65, are nonfunctional at all temperatures tested and have dominant-negative effects when expressed in an ftsQ1(Ts) strain at the permissive temperature. FtsQ2 and FtsQ65 localize to the division site in the presence or absence of endogenous FtsQ, but they cannot recruit downstream cell division proteins, such as FtsL, to the septum. These results suggest that FtsQ2 and FtsQ65 compete efficiently for septal targeting but fail to promote the further assembly of the cell division machinery. Thus, we have separated the localization ability of FtsQ from its other functions, including recruitment of downstream cell division proteins, and are beginning to define regions of the protein responsible for these distinct capabilities.

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