Cell Cycle Synchronization of Schizosaccharomyces pombe by Lactose Gradient Centrifugation to Isolate Small Cells

Abstract The prp (pre-mRNA processing) mutants of the fission yeast Schizosaccharomyces pombe have a defect in pre-mRNA splicing and accumulate mRNA precursors at a restrictive temperature. One of the prp mutants, prp1-4, also has a defect in poly(A)+ RNA transport. The prp1 + gene encodes a protein of 906 amino acid residues that contains 19 repeats of 34 amino acids termed tetratrico peptide repeat (TPR) motifs, which were proposed to mediate protein-protein interactions. The amino acid sequence of Prplp shares 29.6% identity and 50.6% similarity with that of the PRP6 protein of Saccharomyces cerevisiae, which is a component of the U4/U6 snRNP required for spliceosome assembly. No functional complementation was observed between S. pombe prp1 + and S. cerevisiae PRP6. We examined synthetic lethality of prp1-4 with the other known prp mutations in S. pombe. The results suggest that Prp1p interacts either physically or functionally with Prp4p, Prp6p and Prp13p. Interestingly, the prp1 + gene was found to be identical with the zer1 + gene that functions in cell cycle control. These results suggest that Prp1p/Zer1p is either directly or indirectly involved in cell cycle progression and/or poly(A)+ RNA nuclear export, in addition to pre-mRNA splicing.

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

Journal of Cell Science ◽

10.1242/jcs.108.2.475 ◽

1995 ◽

Vol 108 (2) ◽

pp. 475-486 ◽

Cited By ~ 9

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.

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Nuclear proteins of the bovine esophageal epithelium. I. Monoclonal antibody W2 specifically reacts with condensed nuclei of differentiated superficial cells

Journal of Cell Science ◽

10.1242/jcs.104.2.237 ◽

1993 ◽

Vol 104 (2) ◽

pp. 237-247 ◽

Cited By ~ 3

Author(s):

T.K. Tang ◽

T.M. Hong ◽

C.Y. Lin ◽

M.L. Lai ◽

C.H. Liu ◽

...

Keyword(s):

Calcium Binding ◽

Metal Ion ◽

Gradient Centrifugation ◽

Basal Layer ◽

Small Cells ◽

Target Antigen ◽

Esophageal Epithelium ◽

Nuclear Condensation ◽

Calcium Binding Site

Cells from three layers of the bovine esophageal epithelium, representing different stages of differentiation, were dissociated and separated by Percoll gradient centrifugation into fractions of small, medium and large sizes. A majority of the large cells possessed condensed nuclei, a characteristic feature of terminal differentiation of the superficial epithelium. The small cells resembled the proliferate cells of the basal layer. In vitro culture of the esophageal epithelial cells resulted in proliferation of the small cells, colony formation, and, in some cases, differentiation into cells with condensed nuclei. Nuclei, or nuclear subfractions derived from cells of the different layers, were used as immunogens for the generation of hybridomas secreting monoclonal antibodies that bound specifically to different regions of the esophageal tissue. One such antibody, designated W2, labeled the condensed nuclei from the superficial layer of stratified esophageal and corneal epithelia in situ, as well as the large cells from esophageal culture in vitro. Thus, the expression of the W2 antigen may be associated with the process of nuclear condensation during epithelial differentiation. Immunoisolation of the target antigen of W2 from extracts of large cells of the bovine esophagus yielded a band of M(r) approximately 33,000 on nonreducing polyacrylamide gels. This band dissociated into two polypeptides, of M(r) approximately 22,000 and approximately 11,000, upon treatment with dithiothreitol. Amino acid sequence analysis of the larger polypeptide showed extensive homology to a group of small calcium-binding proteins, including two helix-turn-helix motifs designated as the EF-hand, characteristic of the configuration of the metal-ion coordinating ligands of the calcium-binding site. Similarly, the sequence at the amino terminus of the polypeptide of approximately 11,000 indicated that it was the light chain counterpart of the same calcium-binding protein complex.

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Intracellular pH homeostasis during cell-cycle progression and growth state transition in Schizosaccharomyces pombe

Journal of Cell Science ◽

10.1242/jcs.114.16.2929 ◽

2001 ◽

Vol 114 (16) ◽

pp. 2929-2941 ◽

Cited By ~ 1

Author(s):

Jim Karagiannis ◽

Paul G. Young

Keyword(s):

Cell Cycle ◽

Schizosaccharomyces Pombe ◽

Intracellular Ph ◽

Cell Cycle Progression ◽

State Transition ◽

Homeostatic Regulation ◽

Ph Homeostasis ◽

Vegetative Cells ◽

Growth State ◽

Internal Ph

Accurate measurement of intracellular pH in unperturbed cells is fraught with difficulty. Nevertheless, using a variety of methods, intracellular pH oscillations have been reported to play a regulatory role in the control of the cell cycle in several eukaryotic systems. Here, we examine pH homeostasis in Schizosaccharomyces pombe using a non-perturbing ratiometric pH sensitive GFP reporter. This method allows for accurate intracellular pH measurements in living, entirely undisturbed, logarithmically growing cells. In addition, the use of a flow cell allows internal pH to be monitored in real time during nutritional, or growth state transition. We can find no evidence for cell-cycle-related changes in intracellular pH. By contrast, all data are consistent with a very tight homeostatic regulation of intracellular pH near 7.3 at all points in the cell cycle. Interestingly, pH set point changes are associated with growth state. Spores, as well as vegetative cells starved of either nitrogen, or a carbon source, show a marked reduction in their internal pH compared with logarithmically growing vegetative cells. However, in both cases, homeostatic regulation is maintained.

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Association of cyclin-bound p34cdc2 with subcellular structures in xenopus eggs

Journal of Cell Science ◽

10.1242/jcs.102.2.285 ◽

1992 ◽

Vol 102 (2) ◽

pp. 285-297 ◽

Cited By ~ 1

Author(s):

D. Leiss ◽

M.A. Felix ◽

E. Karsenti

Keyword(s):

Cell Cycle ◽

Ribosomal Proteins ◽

Cell Cycle Progression ◽

Gradient Centrifugation ◽

Preliminary Evidence ◽

Embryonic Cell ◽

Cytoskeletal Proteins ◽

Cyclin B ◽

Post Translational Modifications ◽

Egg Extracts

Cell cycle progression is controlled by changes in kinase activity of homologs of the fission yeast protein p34cdc2. The p34cdc2 kinase is activated by its association with a cyclin subunit, followed by post-translational modifications. Here, we show that in Xenopus eggs stimulated to enter the early embryonic cell cycle by an electric shock, part of the p34cdc2 becomes associated with subcellular fractions as the eggs progress towards mitosis. This occurs as a result of cyclin accumulation because most of the B-type cyclins and some of the A-type cyclins are found in the particulate fraction. Moreover, as soon as cyclins are degraded, p34cdc2 is released in the soluble fraction. The p34cdc2-cyclin complex can be solubilised by 80 mM beta-glycerophosphate (in the standard MPF extraction buffer) or by high salt concentrations. The post-translational modifications leading to cdc2 kinase activation by cyclin occur in the insoluble form. Following fractionation of egg extracts by sucrose gradient centrifugation, the p34cdc2-cyclin B complex is found in several fractions, but especially in two discrete peaks. We present evidence that in the slow-sedimenting peak the p34cdc2-cyclin B complex is associated with the 60 S subunit of monoribosomes. It could be targeted in this fashion to substrates such as ribosomal proteins and maybe to cytoskeletal proteins, since ribosomes bind to microtubules and are present in the spindle. The p34cdc2-cyclin B complex is also found in a faster-migrating fraction containing various membranous structures, including Golgi stacks. Therefore, as observed by immunofluorescence in other systems, it seems that cyclin subunits target p34cdc2 to specific cellular sites and this is certainly important for its function. In addition, we present preliminary evidence suggesting that some component present in the ribosome-containing fraction is required for activation of the p34cdc2-cyclin B complex.

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