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

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.

1991 ◽  
Vol 99 (1) ◽  
pp. 193-199
Author(s):  
J. VICENTE-SOLER ◽  
J. CREANOR ◽  
Y. BISSET ◽  
J. M. MITCHISON

The activity of alcohol dehydrogenase (ADHase) was followed in synchronous cultures of Schizosaccharomyces pombe. In selection synchronised cultures of wild-type cells, it followed a linear pattern in which there was a constant rate of increase of activity followed by a doubling of this rate at the end of the cycle. The same pattern was also found in selection synchronised cells of wee mutants except that the point of rate change was shifted to 0.27 of the cycle. A similar linear pattern was also found in the shortened cell cycles produced by induction synchrony (block and release of the mutant cdc2.33) but the rate change point was at about 0.75 of the cycle. In the mutant cdcl3.117, there was a marked fall in the rate of activity increase at 35°C but not at 37°C. In all these situations, the ADHase activity closely paralleled in pattern and in timing the rate of production of CO2 established in earlier papers. This suggests a coordinate control of the flux through glycolysis and the activity of the last enzyme in the glycolytic pathway in yeast. However, an interesting difference indicating a loss of the coordinate control occurred in ‘synchronous’ cultures of cdc2.33 in which small cells had been selected but in which the DNAdivision cycle had been blocked by a shift-up to the restrictive temperature. Rate changes both in CO2 production and in ADHase activity continued in these blocked synchronous cultures but the timing was different. With ADHase activity the timing was 15% greater than that in a normal cell cycle whereas with CO2 production it was 15% less. We suggest that these and other periodic events are subject to independent oscillatory controls in these blocked cultures with timings that differ from each other and from the normal cycle but in the normal cycle the oscillators are all entrained by one or more events of the DNA-division cycle.


1987 ◽  
Vol 87 (2) ◽  
pp. 323-325
Author(s):  
B. Novak ◽  
J.M. Mitchison

CO2 production has been followed by manometry in synchronous and asynchronous control cultures of Schizosaccharomyces pombe prepared by elutriation from the same initial culture. Earlier results showed a periodic change in the rate of production, which took place once per cell cycle. These changes were most clearly shown as oscillations in the difference between values of the second differential (acceleration) for the synchronous and asynchronous cultures. This paper shows that the oscillations continue for at least three cycles in the presence of cycloheximide (with and without chloramphenicol). Protein synthesis is virtually absent and there is no cell division. The control of this metabolic oscillation is therefore not directly dependent on translation. The period of the oscillation under these conditions is about 60% of the normal cycle time.


2002 ◽  
Vol 22 (10) ◽  
pp. 3537-3548 ◽  
Author(s):  
Takashi Morishita ◽  
Yasuhiro Tsutsui ◽  
Hiroshi Iwasaki ◽  
Hideo Shinagawa

ABSTRACT To identify novel genes involved in DNA double-strand break (DSB) repair, we previously isolated Schizosaccharomyces pombe mutants which are hypersensitive to methyl methanesulfonate (MMS) and synthetic lethals with rad2. This study characterizes one of these mutants, rad60-1. The gene that complements the MMS sensitivity of this mutant was cloned and designated rad60. rad60 encodes a protein with 406 amino acids which has the conserved ubiquitin-2 motif found in ubiquitin family proteins. rad60-1 is hypersensitive to UV and γ rays, epistatic to rhp51, and defective in the repair of DSBs caused by γ-irradiation. The rad60-1 mutant is also temperature sensitive for growth. At the restrictive temperature (37°C), rad60-1 cells grow for several divisions and then arrest with 2C DNA content; the arrested cells accumulate DSBs and have a diffuse and often aberrantly shaped nuclear chromosomal domain. The rad60-1 mutant is a synthetic lethal with rad18-X, and expression of wild-type rad60 from a multicopy plasmid partially suppresses the MMS sensitivity of rad18-X cells. rad18 encodes a conserved protein of the structural maintenance of chromosomes (SMC) family (A. R. Lehmann, M. Walicka, D. J. Griffiths, J. M. Murray, F. Z. Watts, S. McCready, and A. M. Carr, Mol. Cell. Biol. 15:7067-7080, 1995). These results suggest that S. pombe Rad60 is required to repair DSBs, which accumulate during replication, by recombination between sister chromatids. Rad60 may perform this function in concert with the SMC protein Rad18.


1992 ◽  
Vol 70 (10-11) ◽  
pp. 1088-1096 ◽  
Author(s):  
Maleki Daya-Makin ◽  
Philippe Szankasi ◽  
Liren Tang ◽  
Diana MacRae ◽  
Steven L. Pelech

Temperature-sensitive pat1 mutants of the fission yeast Schizosaccharomyces pombe can be induced to undergo meiosis at the restrictive temperature, irrespective of the mat1 configuration and the nutritional conditions. Using a combination of exit from stationary phase and thermal inactivation of the 52-kilodalton protein kinase that is encoded by the pat1 (also called ran1) gene, highly synchronous meiotic cultures were obtained. Synthesis and tyrosyl phosphorylation of p34cdc2 was evident during meiotic G1 and S phases. During this period there was increased expression of p105wee1, a protein kinase implicated in the tyrosyl phosphorylation of p34cdc2. Following a relatively brief G2 period, during which a reduction in the steady-state level of p105wee1 occurred, there was an approximately 19-fold increase in the histone H1 phosphotransferase activity of p34cdc2. Only a single peak of histone H1 kinase activation was observed, which implies that unlike meiosis in amphibians and echinoderms, p34cdc2 is functional only during one of the meiotic divisions in S. pombe, presumably meiosis II. Stimulation of the kinase activity of p34cdc2 was associated with its tyrosyl dephosphorylation. This is analogous to mitotic M phase and suggests parallels in the mechanism of activation of p34cdc2 during mitosis and one of the meiotic divisions in S. pombe.Key words: wee1, cdc2, ran1, cell cycle, meiosis.


1999 ◽  
Vol 19 (4) ◽  
pp. 2535-2546 ◽  
Author(s):  
Lynne D. Berry ◽  
Anna Feoktistova ◽  
Melanie D. Wright ◽  
Kathleen L. Gould

ABSTRACT The Schizosaccharomyces pombe dim1 + gene is required for entry into mitosis and for chromosome segregation during mitosis. To further understand dim1p function, we undertook a synthetic lethal screen with the temperature-sensitive dim1-35 mutant and isolated lid (for lethal in dim1-35) mutants. Here, we describe the temperature-sensitive lid1-6mutant. At the restrictive temperature of 36°C, lid1-6mutant cells arrest with a “cut” phenotype similar to that ofcut4 and cut9 mutants. An epitope-tagged version of lid1p is a component of a multiprotein ∼20S complex; the presence of lid1p in this complex depends upon functionalcut9 +. lid1p-myc coimmunoprecipitates with several other proteins, including cut9p and nuc2p, and the presence of cut9p in a 20S complex depends upon the activity oflid1 +. Further, lid1 +function is required for the multiubiquitination of cut2p, an anaphase-promoting complex or cyclosome (APC/C) target. Thus, lid1p is a component of the S. pombe APC/C. In dim1mutants, the abundances of lid1p and the APC/C complex decline significantly, and the ubiquitination of an APC/C target is abolished. These data suggest that at least one role of dim1p is to maintain or establish the steady-state level of the APC/C.


1982 ◽  
Vol 28 (2) ◽  
pp. 261-264 ◽  
Author(s):  
Stephen M. King ◽  
Jeremy S. Hyams

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.


1988 ◽  
Vol 106 (4) ◽  
pp. 1171-1183 ◽  
Author(s):  
T Hirano ◽  
Y Hiraoka ◽  
M Yanagida

A temperature-sensitive mutant nuc2-663 of the fission yeast Schizosaccharomyces pombe specifically blocks mitotic spindle elongation at restrictive temperature so that nuclei in arrested cells contain a short uniform spindle (approximately 3-micron long), which runs through a metaphase plate-like structure consisting of three condensed chromosomes. In the wild-type or in the mutant cells at permissive temperature, the spindle is fully extended approximately 15-micron long in anaphase. The nuc2' gene was cloned in a 2.4-kb genomic DNA fragment by transformation, and its complete nucleotide sequence was determined. Its coding region predicts a 665-residues internally repeating protein (76.250 mol wt). By immunoblots using anti-sera raised against lacZ-nuc2+ fused proteins, a polypeptide (designated p67; 67,000 mol wt) encoded by nuc2+ is detected in the wild-type S. pombe extracts; the amount of p67 is greatly increased when multi-copy or high-expression plasmids carrying the nuc2+ gene are introduced into the S. pombe cells. Cellular fractionation and Percoll gradient centrifugation combined with immunoblotting show that p67 cofractionates with nuclei and is enriched in resistant structure that is insoluble in 2 M NaCl, 25 mM lithium 3,5'-diiodosalicylate, and 1% Triton but is soluble in 8 M urea. In nuc2 mutant cells, however, soluble p76, perhaps an unprocessed precursor, accumulates in addition to insoluble p67. The role of nuc2+ gene may be to interconnect nuclear and cytoskeletal functions in chromosome separation.


1996 ◽  
Vol 109 (6) ◽  
pp. 1297-1310 ◽  
Author(s):  
D.A. Stirling ◽  
T.F. Rayner ◽  
A.R. Prescott ◽  
M.J. Stark

We have generated three temperature-sensitive alleles of SPC110, which encodes the 110 kDa component of the yeast spindle pole body (SPB). Each of these alleles carries point mutations within the calmodulin (CaM) binding site of Spc110p which affect CaM binding in vitro; two of the mutant proteins fail to bind CaM detectably (spc110-111, spc110-118) while binding to the third (spc110-124) is temperature-sensitive. All three alleles are suppressed to a greater or lesser extent by elevated dosage of the CaM gene (CMD1), suggesting that disruption of CaM binding is the primary defect in each instance. To determine the consequences on Spc110p function of loss of effective CaM binding, we have therefore examined in detail the progression of synchronous cultures through the cell division cycle at the restrictive temperature. In each case, cells replicate their DNA but then lose viability. In spc110-124, most cells duplicate and partially separate the SPBs but fail to generate a functional mitotic spindle, a phenotype which we term ‘abnormal metaphase’. Conversely, spc110-111 cells initially produce nuclear microtubules which appear well-organised but on entry into mitosis accumulate cells with ‘broken spindles’, where one SPB has become completely detached from the nuclear DNA. In both cases, the bulk of the cells suffer a lethal failure to segregate the DNA.


1969 ◽  
Vol 5 (2) ◽  
pp. 373-391
Author(s):  
J. M. MITCHISON ◽  
J. CREANOR ◽  
D. A. WILLAMS

The synthesis of sucrase, acid phosphatase and alkaline phosphatase has been followed in synchronous cultures of the fission yeast Schizosaccharomyces pombe prepared by gradient sedimentation. These three enzymes follow a linear pattern of synthesis through the cell cycle, with a doubling in rate at a ‘critical point’ about one-fifth of the way through the cycle. Sucrase can be rapidly derepressed by lowering the glucose concentration in the medium. This has been used to measure the sucrase ‘potential’ or capacity to synthesize sucrase on derepression. The potential exists at all times in the cycle, and follows a stepwise pattern with a sharp rise at the critical point. These results suggest that the functional genome doubles at the critical point. Since, however, the period of DNA synthesis is nearly one-third of a cycle before this point, there must be an appreciable delay between chemical replication and functional replication of the genome. In this respect S. pombe, a eukaryotic cell, differs markedly from bacteria. Other physiological events take place near the critical point, and a tentative model is suggested of what may be happening at the chromosomal level. Experiments with cycloheximide indicate that there is a delay between the synthesis and the appearance of the active enzyme in the case of sucrase and alkaline phosphatase.


1984 ◽  
Vol 69 (1) ◽  
pp. 199-210
Author(s):  
J. Creanor ◽  
J.M. Mitchison

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.


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