Germination and outgrowth of <i>Schizosaccharomyces pombe</i> ascospores isolated by Urografin density gradient centrifugation

Nishi, Kayoko; Shimoda, Chikashi; Hayashibe, Masaya

doi:10.1139/m78-149

Cited by 23 publications

(19 citation statements)

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“…In agreement with previous investigations, we f0ur.d that the spores have a haploid complement of DNA, about 1.36 x pg spore-1 (Nishi et al, 1978;Bostock, 1970). More importantly, and in analogy to the cell cycle of eukaryotic cells, S. pombe spores have a protracted 'G1' period during which there is a well-defined sequence of events to achieve the transition to vegetative growth and cell division.…”

Section: Discussionsupporting

confidence: 90%

“…Therefore, a 2 1 culture yielded about 4 x 1O1O spores. This quantity and yield was far greater than obtained by zonal centrifugation through sucrose (Padilla et al, 1974) or Urografin gradients (Nishi et al, 1978). Figure 1 (a) summarizes the time course of germination and outgrowth.…”

Section: R E S U L T Smentioning

confidence: 81%

“…The budding yeast Saccharomyces cerevisiae has been used to study the processes underlying this transition (Rousseau & Halvorson, 1973a, b, c ;Steele & Miller, 1974, 1977Tingle et al, 1974), but the fission yeast Schizosaccharomyces pombe would seem preferable for at least two reasons : the spores are spontaneously released from the ascus, and mating and conjugation do not occur prior to completion of the first round of cell division. Unfortunately, until recently (Padilla et al, 1974(Padilla et al, , 1975Nishi et al, 1978), it was difficult to obtain spores free of vegetative cells. Density gradient centrifugation through sucrose (Padilla et al, 1974) or Urografin (Nishi et al, 1978) was employed in these studies, but the yield of spores was very low and the separation procedures were costly and time consuming.…”

Section: Introductionmentioning

confidence: 99%

“…Unfortunately, until recently (Padilla et al, 1974(Padilla et al, , 1975Nishi et al, 1978), it was difficult to obtain spores free of vegetative cells. Density gradient centrifugation through sucrose (Padilla et al, 1974) or Urografin (Nishi et al, 1978) was employed in these studies, but the yield of spores was very low and the separation procedures were costly and time consuming. As S. pombe has been reported to possess sucrase activity (Mitchison & Creanor, 1969), exposure of the ascospores to sucrose may also lead to premature induction of germination.…”

Section: Introductionmentioning

confidence: 99%

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Germination and Outgrowth of Schizosaccharomyces pombe Spores Isolated by a Simple Batch Centrifugation Technique

Johnke

Padilla

1979

Journal of General Microbiology

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Spores of the fission yeast Schizosaccharomyces pombe have been separated from vegetative cells by a simple and rapid centrifugation (800 g for 20 min) through a 35 % Hypaque solution to a purity > 95 %. Approximately 35 yo of the spores were recovered. Regrowth in EMM2 plus glucose showed that over 97 yo of the spores germinated within the first 2 h and outgrowth continued between 5 and 10 h. Sucrose induced germination in > 95 % of the spores with a 1 h delay and outgrowth in 50 % of the spores with a 3 h delay. There was little protein synthesis during germination but the protein content increased linearly coincident with outgrowth. The RNA content increased slightly during germination, but increased linearly 1 h before the onset of outgrowth and protein synthesis. After 8 h of regrowth, coincident with the onset of DNA synthesis, the rate of RNA synthesis was accelerated. The DNA content had increased l.7-fold after 10 h of regrowth from a haploid level of 1.36 x ,ug spore-l.

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Section: Discussionsupporting

confidence: 90%

Section: R E S U L T Smentioning

confidence: 81%

Section: Introductionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

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Germination and Outgrowth of Schizosaccharomyces pombe Spores Isolated by a Simple Batch Centrifugation Technique

Johnke

Padilla

1979

Journal of General Microbiology

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“…Ascal walls were spontaneously dissolved, and single spores were liberated. Spores were isolated by linear 25 to 55% Urografin (Schering Co.) density gradient centrifugation (34). After centrifugation at 25,000 ϫ g for 30 min in an ultracentrifuge (Hitachi CP70G), the cells formed two layers in the gradient.…”

Section: Vol 3 2004mentioning

confidence: 99%

ADAM Family Protein Mde10 Is Essential for Development of Spore Envelopes in the Fission Yeast Schizosaccharomyces pombe

et al. 2004

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We report the identification of Schizosaccharomyces pombe mde10؉ as a gene possessing a FLEX element, which forms a binding site for the meiosis-specific transcription factor Mei4. In fact, mde10؉ is transcribed only in diploid cells that are induced to meiosis in a Mei4-dependent manner. Western blot analysis indicated that the epitope-tagged Mde10 protein accumulates transiently during meiosis and then rapidly decreases. Mde10 is a multidomain protein containing a metalloprotease catalytic domain, a disintegrin domain, a cysteine-rich domain, and membrane-spanning regions, all of which are shared by members of the mammalian ADAM family. A fusion protein of Mde10 and green fluorescent protein localized to the endoplasmic reticulum during meiosis and was located at the peripheral region of spores at the end of meiosis. An mde10⌬ deletion mutant showed no apparent defects in meiosis, sporulation, or spore germination. However, the mutant spores exhibited an aberrant surface appearance, in which the ragged outer spore wall was lost to a large extent. Furthermore, mde10⌬ spores were found to be less tolerant to ethanol and diethyl ether than were wild-type spores. The mutagenic replacement of the conserved glutamic acid in the putative protease active site with an alanine residue did not affect the surface morphology or the resistance of spores to environmental stress. Our observations indicate that Mde10 is important in the development of the spore envelope, although this function of Mde10 seems to be independent of its metalloprotease activity.Gametes in multicellular organisms differentiate into morphologically and functionally specialized cells-most typically, into sperms and eggs. Sporulation in single-celled eukaryotes such as yeasts is a morphogenetic process that is equivalent to gametogenesis because an ascospore is a highly specialized cell and its formation is preceded by meiosis. In morphogenesis and cell differentiation in multicellular organisms, the control of gene expression is extremely important; in addition, DNA microarray analyses have demonstrated that transcriptional regulation occurs during gametogenesis in yeast (5, 40).The forkhead DNA-binding protein Mei4 of the fission yeast Schizosaccharomyces pombe has been identified as a meiosisspecific transcription factor that plays an important role in the progression of meiosis and sporulation (17). To date, approximately 30 genes whose transcription is dependent on Mei4 have been found, including spo4 ϩ and spo6 ϩ , which encode the Cdc7/Dbf4-related protein kinase complex (30)(31)(32)46). An in vitro binding assay with recombinant Mei4 proteins and the 5Ј upstream region of spo6 ϩ has demonstrated that a short DNA stretch of 14 nucleotides, designated the FLEX element, constitutes the recognition site for Mei4 (17). The FLEX element contains a heptamer core sequence, GTAAACA, which is highly conserved among the recognition sequences of mammalian transcription factors with a forkhead DNA-binding domain, such as human FREAC proteins (37).A search ...

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The cyclic AMP/PKA signal pathway is required for initiation of spore germination inSchizosaccharomyces pombe

Hatanaka

Shimoda

2001

Yeast

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Spore germination, a transition from the quiescent G0 phase to the proliferation cycle, is triggered by glucose in Schizosaccharomyces pombe. The role of cAMP/protein kinase A (PKA) signalling in germination is investigated. Gene disruption of cyr1+, pka1+ and gpa2+ encoding adenylate cyclase, PKA and the α‐subunit of a trimeric GTP‐binding protein, respectively, reduced the colony‐forming efficiency of spores in minimal medium. Isolated spores of these null mutants did not germinate in minimal medium for up to 12 h, at which time wild‐type spores had completed germination and formed germ projections. In wild‐type spores, cortical actin patches randomly distributed in the early stage of outgrowth and then localized to one side of spores before the formation of projections. In contrast, the mutant spores exhibited no actin patches, but the cell surface was predominantly stained, like ungerminated spores of wild‐type. Flow fluorocytometric analysis of propidium iodide‐stained spores revealed a distinct 1C DNA peak after germination was completed. The fluorescent profile of the mutant spores, however, did not change during 12 h incubation in the minimal medium. These observations indicate that spores harbouring either cyr1Δ, pka1Δ or gpa2Δ are hardly triggered to germination. When wild‐type spores were exposed to glucose, the intracellular cAMP level transiently increased in a few minutes, but gpa2Δ spores did not respond to glucose. We conclude that S. pombe spores initiate germination in response to glucose through the cyclic AMP–PKA pathway. Copyright © 2000 John Wiley & Sons, Ltd.

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Germination and outgrowth of Schizosaccharomyces pombe ascospores isolated by Urografin density gradient centrifugation

Cited by 23 publications

References 0 publications

Germination and Outgrowth of Schizosaccharomyces pombe Spores Isolated by a Simple Batch Centrifugation Technique

Germination and Outgrowth of Schizosaccharomyces pombe Spores Isolated by a Simple Batch Centrifugation Technique

ADAM Family Protein Mde10 Is Essential for Development of Spore Envelopes in the Fission Yeast Schizosaccharomyces pombe

The cyclic AMP/PKA signal pathway is required for initiation of spore germination inSchizosaccharomyces pombe

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