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.

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.

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.

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.

Protein synthesis and its relation to the DNA-division cycle in the fission yeast Schizosaccharomyces pombe

1984 ◽  
Vol 69 (1) ◽  
pp. 199-210
Author(s):  
J. Creanor ◽  
J.M. Mitchison
Keyword(s):  
Cell Cycle ◽  
Protein Synthesis ◽  
Schizosaccharomyces Pombe ◽  
Protein Content ◽  
Fission Yeast ◽  
Nuclear Division ◽  
Restrictive Temperature ◽  
Tentative Conclusion ◽  
Synchronous Cultures

The rate of protein synthesis has been measured with pulse labels of [3H]tryptophan in synchronous and asynchronous cultures of cdc mutants of Schizosaccharomyces pombe shifted up to the restrictive temperature. The cell cycle related fluctuations in rate that occur in normal synchronous cultures vanish when nuclear division is blocked in synchronous cultures of cdc2 and cdc10. But they persist in cdc11 where nuclear division continues and cleavage is stopped. We conclude that nuclear division affects the rate of synthesis and that this effect is inhibitory and probably persists for the last 40% of the cycle. When nuclear division has been blocked, the rate of synthesis continues to increase until a plateau is reached where the rate remains constant. Three size mutants of cdc2 reach the plateau at the same average protein content per cell although their initial protein contents vary over a threefold range. Comparison of these results with those from cdc10 leads to the tentative conclusion that the plateau starts when the cells reach a critical protein/DNA ratio.

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.

scholarly journals Separation of phenotypes in mutant alleles of the Schizosaccharomyces pombe cell-cycle checkpoint gene rad1+.

1995 ◽  
Vol 6 (12) ◽  
pp. 1793-1805 ◽  
Author(s):  
G Kanter-Smoler ◽  
K E Knudsen ◽  
G Jimenez ◽  
P Sunnerhagen ◽  
S Subramani
Keyword(s):  
Cell Cycle ◽  
Dna Damage ◽  
Dna Replication ◽  
Schizosaccharomyces Pombe ◽  
Synthetic Lethality ◽  
Cell Cycle Checkpoint ◽  
Site Directed Mutagenesis ◽  
Restrictive Temperature ◽  
Wild Type ◽  
Cycle Checkpoint

The Schizosaccharomyces pombe rad1+ gene is involved in the G2 DNA damage cell-cycle checkpoint and in coupling mitosis to completed DNA replication. It is also required for viability when the cdc17 (DNA ligase) or wee1 proteins are inactivated. We have introduced mutations into the coding regions of rad1+ by site-directed mutagenesis. The effects of these mutations on the DNA damage and DNA replication checkpoints have been analyzed, as well as their associated phenotypes in a cdc17-K42 or a wee1-50 background. For all alleles, the resistance to radiation or hydroxyurea correlates well with the degree of functioning of checkpoint pathways activated by these treatments. One mutation, rad1-S3, completely abolishes the DNA replication checkpoint while partially retaining the DNA damage checkpoint. As single mutants, the rad1-S1, rad1-S2, rad1-S5, and rad1-S6 alleles have a wild-type phenotype with respect to radiation sensitivity and checkpoint functions; however, like the rad1 null allele, the rad1-S1 and rad1-S2 alleles exhibit synthetic lethality at the restrictive temperature with the cdc17-K42 or the wee1-50 mutation. The rad1-S5 and rad1-S6 alleles allow growth at higher temperatures in a cdc17-K42 or wee1-50 background than does wild-type rad1+, and thus behave like "superalleles." In most cases both chromosomal and multi-copy episomal mutant alleles have been investigated, and the agreement between these two states is very good. We provide evidence that the functions of rad1 can be dissociated into three groups by specific mutations. Models for the action of these rad1 alleles are discussed. In addition, a putative negative regulatory domain of rad1 is identified.

Carbon dioxide evolution during the cell cycle of the fission yeast Schizosaccharomyces pombe

1978 ◽  
Vol 33 (1) ◽  
pp. 385-397
Author(s):  
J. Creanor
Keyword(s):  
Cell Cycle ◽  
Dna Synthesis ◽  
Schizosaccharomyces Pombe ◽  
Fission Yeast ◽  
Nuclear Division ◽  
Complete Medium ◽  
Co2 Evolution ◽  
Carbon Dioxide Evolution ◽  
Synchronous Cultures ◽  
Constant Addition

The rate of CO2 evolution was measured in synchronous cultures of the fission yeast Schizosaccharomyces pombe growing in a minimal medium. The rate of CO2 evolution was found to double sharply at about the time of nuclear division (0.75 of the way through the cell cycle). For the remainder of the cell cycle the rate remained constant. Addition of inhibitors of DNA synthesis or nuclear division did not affect the pattern of CO2 evolution in synchronous cultures. Similarly, in an induced synchronous culture, in which DNA synthesis, nuclear division and cell division—but not growth, were synchronized, CO2 evolution showed a continuous pattern and not the step-wise increase associated with the normal synchronous cultures. When S. pombe was grown in a complete medium, the evolution of CO2 in a synchronous cultures was shown to increase in a continuous manner but at a rate faster than the growth of the culture.

scholarly journals Observations on ultracentrifuging wild-type and mutant (cdc2.33) cells of Schizosaccharomyces pombe

1989 ◽  
Vol 92 (3) ◽  
pp. 345-348
Author(s):  
A. Khar ◽  
J.M. Mitchison
Keyword(s):  
Schizosaccharomyces Pombe ◽  
Fission Yeast ◽  
Nuclear Dna ◽  
Restrictive Temperature ◽  
Wild Type ◽  
Intense Fluorescence ◽  
Cytoplasmic Dna ◽  
Dnase Treatment

Ultracentrifuging (400,000 g for 4–6 h at 4 degrees C) living wild-type cells of the fission yeast Schizosaccharomyces pombe moves the nucleus towards the ends of the cells but scarcely affects their viability. However, in the long cells produced by growing the mutant cdc2.33 for 4–6 h at the restrictive temperature (36.5 degrees C), ultracentrifuging (as above) gives an intense fluorescence with DAPI in about half of the cytoplasm in about 80% of the cells. This is probably nuclear DNA that has moved into the cytoplasm, both because of the DAPI stain and because it is removed by DNase treatment. These cells ultimately divide and are viable, and we suggest that the extended cytoplasmic DNA returns to the nucleus.

Identification of temperature-sensitive DNA- mutants of Chinese hamster cells affected in cellular and viral DNA synthesis

1986 ◽  
Vol 6 (12) ◽  
pp. 4594-4601
Author(s):  
J J Dermody ◽  
B E Wojcik ◽  
H Du ◽  
H L Ozer
Keyword(s):  
Dna Replication ◽  
Dna Synthesis ◽  
Chinese Hamster ◽  
Human Adenovirus ◽  
Temperature Sensitive ◽  
Restrictive Temperature ◽  
Permissive Temperature ◽  
Dependent Manner ◽  
Viral Dna ◽  
Cell Functions

We described a strategy which facilitates the identification of cell mutants which are restricted in DNA synthesis in a temperature-dependent manner. A collection of over 200 cell mutants temperature-sensitive for growth was isolated in established Chinese hamster cell lines (CHO and V79) by a variety of selective and nonselective techniques. Approximately 10% of these mutants were identified as ts DNA- based on differential inhibition of macromolecular synthesis at the restrictive temperature (39 degrees C) as assessed by incorporation of [3H]thymidine and [35S]methionine. Nine such mutants, selected for further study, demonstrated rapid shutoff of DNA replication at 39 degrees C. Infections with two classes of DNA viruses extensively dependent on host-cell functions for their replication were used to distinguish defects in DNA synthesis itself from those predominantly affecting other aspects of DNA replication. All cell mutants supported human adenovirus type 2 (Ad2) and mouse polyomavirus DNA synthesis at the permissive temperature. Five of the nine mutants (JB3-B, JB3-O, JB7-K, JB8-D, and JB11-J) restricted polyomavirus DNA replication upon transfection with viral sequences at 33 degrees C and subsequent shift to 39 degrees C either before or after the onset of viral DNA synthesis. Only one of these mutants (JB3-B) also restricted Ad2 DNA synthesis after virion infection under comparable conditions. No mutant was both restrictive for Ad2 and permissive for polyomavirus DNA synthesis at 39 degrees C. The differential effect of these cell mutants on viral DNA synthesis is expected to assist subsequent definition of the biochemical defect responsible.

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