49 results on '"Sazer S"'
Search Results
2. Disruption of the YRB2 Gene Retards Nuclear Protein Export, Causing a Profound Mitotic Delay, and Can Be Rescued by Overexpression of XPO1/CRM1
- Author
-
Noguchi, E., primary, Saitoh, Y.-h., additional, Sazer, S., additional, and Nishimoto, T., additional
- Published
- 1999
- Full Text
- View/download PDF
3. Mph1, a member of the Mps1-like family of dual specificity protein kinases, is required for the spindle checkpoint in S. pombe
- Author
-
He, X., primary, Jones, M.H., additional, Winey, M., additional, and Sazer, S., additional
- Published
- 1998
- Full Text
- View/download PDF
4. Dis3, implicated in mitotic control, binds directly to Ran and enhances the GEF activity of RCC1.
- Author
-
Noguchi, E., primary, Hayashi, N., additional, Azuma, Y., additional, Seki, T., additional, Nakamura, M., additional, Nakashima, N., additional, Yanagida, M., additional, He, X., additional, Mueller, U., additional, Sazer, S., additional, and Nishimoto, T., additional
- Published
- 1996
- Full Text
- View/download PDF
5. A mutation in the RCC1-related protein pim1 results in nuclear envelope fragmentation in fission yeast.
- Author
-
Demeter, J, primary, Morphew, M, additional, and Sazer, S, additional
- Published
- 1995
- Full Text
- View/download PDF
6. A fission yeast RCC1-related protein is required for the mitosis to interphase transition.
- Author
-
Sazer, S., primary and Nurse, P., additional
- Published
- 1994
- Full Text
- View/download PDF
7. Phosphorylation at Thr167 is required for Schizosaccharomyces pombe p34cdc2 function.
- Author
-
Gould, K.L., primary, Moreno, S., additional, Owen, D.J., additional, Sazer, S., additional, and Nurse, P., additional
- Published
- 1991
- Full Text
- View/download PDF
8. Mitochondrial growth and DNA synthesis occur in the absence of nuclear DNA replication in fission yeast
- Author
-
Sazer, S., primary and Sherwood, S.W., additional
- Published
- 1990
- Full Text
- View/download PDF
9. Characterization of a nuclear protein conferring brefeldin A resistance in Schizosaccharomyces pombe.
- Author
-
Turi, T G, Mueller, U W, Sazer, S, and Rose, J K
- Abstract
The fungal metabolite brefeldin A disrupts protein secretion and causes the redistribution of the Golgi complex to the endoplasmic reticulum. Previously we isolated six genes that, when present in multiple copies, confer brefeldin A resistance to wild type Schizosaccharomyces pombe. Here we describe the characterization of one of these genes, hba1. This gene encodes an essential protein that shares homology with the mammalian protein RanBP1 and the protein encoded by the Saccharomyces cerevisiae gene YRB1 and contains a peptide motif present in several proteins found within the nuclear pore complex. The protein encoded by hba1 is localized to the nucleus, and it was determined that this protein is phosphorylated in vivo. The characterization of hba1 thus demonstrates a novel mechanism of drug resistance in S. pombe.
- Published
- 1996
10. Stage-specific activity of the Leishmania major CRK3 kinase and functional rescue of a Schizosaccharomyces pombe cdc2 mutant
- Author
-
Wang, Y., Dimitrov, K., Garrity, L. K., Sazer, S., and Beverley, S. M.
- Published
- 1998
- Full Text
- View/download PDF
11. The search for the primary function of the Ran GTPase continues
- Author
-
Sazer, S.
- Published
- 1996
- Full Text
- View/download PDF
12. Perturbations in the spi1p GTPase cycle of Schizosaccharomyces pombe through its GTPase-activating protein and guanine nucleotide exchange factor components result in similar phenotypic consequences
- Author
-
Matynia, A, Dimitrov, K, Mueller, U, He, X, and Sazer, S
- Abstract
spi1p of Schizosaccharomyces pombe is a structural homolog of the mammalian GTPase Ran. The distribution between the GTP- and GDP-bound forms of the protein is regulated by evolutionarily conserved gene products, rna1p and pim1p, functioning as GTPase-activating protein (GAP) and guanine nucleotide exchange factor (GEF), respectively. Antibodies to spi1p, pim1p, and rna1p were generated and used to demonstrate that pim1p is exclusively nuclear, while rna1p is cytoplasmic. A loss of pim1p GEF activity or an increase in the rna1p GAP activity correlates with a change in the localization of the GTPase from predominantly nuclear to uniformly distributed, suggesting that the two forms are topologically segregated and that the nucleotide-bound state of spi1p may dictate its intracellular localization. We demonstrate that the phenotype of cells overproducing the GAP resembles the previously reported phenotype of mutants with alterations in the GEF: the cells are arrested in the cell cycle as septated, binucleated cells with highly condensed chromatin, fragmented nuclear envelopes, and abnormally wide septa. Consistent with the expectation that either an increased dosage of the GAP or a mutation in the GEF would lead to an increase of the spi1p-GDP/spi1p-GTP ratio relative to that of wild-type cells, overexpression of the GAP together with a mutation in the GEF is synthetically lethal. The similar phenotypic consequences of altering the functioning of the nuclear GEF or the cytoplasmic GAP suggest that there is a single pool of the spi1p GTPase that shuttles between the nucleus and the cytoplasm. Phenotypically, rna1 null mutants, in which spi1p-GTP would be expected to accumulate, resemble pim1(ts) and rna1p-overproducing cells, in which spi1p-GDP would be expected to accumulate. Taken together, these results support the hypothesis that the balance between the GDP- and GTP-bound forms of spi1p mediates the host of nuclear processes that are adversely affected when the functioning of different components of this system is perturbed in various organisms.
- Published
- 1996
- Full Text
- View/download PDF
13. Ran-binding protein-1 is an essential component of the Ran/RCC1 molecular switch system in budding yeast.
- Author
-
Ouspenski, I I, Mueller, U W, Matynia, A, Sazer, S, Elledge, S J, and Brinkley, B R
- Abstract
We have performed a screen for genes that affect chromosome stability when overexpressed in the budding yeast Saccharomyces cerevisiae. Two of the genes recovered in the screen, CST17 and CST20, share a number of phenotypic properties, suggesting their involvement in the same cellular process. DNA sequence analysis of these genes revealed that they encode components of the Ran/RCC1 molecular switch system: CST17 is Ran itself (Ras-like nuclear protein) and CST20 is a novel yeast protein with a high degree of similarity to mammalian RanBP1, which is known to interact with Ran-GTP in vitro. We demonstrate that the CST20 protein can interact with Ran-GTP in vitro under similar conditions, indicating that it is the functional yeast homolog of mammalian RanBP1. The results of immunoprecipitation experiments show that the two yeast proteins form a complex in vivo. Deletion of the gene encoding RanBP1 revealed that it is essential for viability, as are Ran and RCC1. Similar phenotypic consequences of overproduction of either Ran or RanBP1 indicate that the latter protein is a functional component of the Ran/RCC1 molecular switch system, which is implicated in the control of a number of nuclear functions. Our finding that overproduction of two components of this system results in mitotic chromosome nondisjunction and sensitivity to an anti-microtubule drug benomyl suggest their involvement in mitosis as well. Thus RanBP1 is a functional component of a highly conserved molecular system that affects diverse cellular processes. The availability of this gene in S. cerevisiae provides a genetic system for the analysis of RanBP1 function in vivo.
- Published
- 1995
14. A re-examination of the 5' termini of mouse dihydrofolate reductase RNA.
- Author
-
Sazer, S and Schimke, R T
- Abstract
Using primer extension and nuclease S1-mapping techniques we have re-examined the 5' termini of RNA transcribed from the mouse dihydrofolate reductase gene. We characterize a previously undescribed transcription initiation site at position -55 relative to the AUG codon, in addition to the previously identified start site at position -115. Differences in the 5' noncoding regions of these two transcripts with respect to their length and relative G + C content result in their differential ability to form stable hybrids with the DNA probe used in previous analyses of these transcripts and thus precluded the detection of transcripts initiated at -55. We show that changes in the temperature of the hybridization reaction result in the ability to detect the RNA having a shorter noncoding region and a lower G + C content. That position -55 represents an authentic transcription start site is confirmed by use of a DNA probe with which the two transcripts can form S1-resistant hybrids of equal stability and by primer extension analysis using an oligonucleotide primer that hybridizes near the AUG codon. These analyses also demonstrate that the transcript with a 5' end mapping near position -55 accounts for the majority of cellular dihydrofolate reductase RNA.
- Published
- 1986
- Full Text
- View/download PDF
15. Isolation and characterization of new fission yeast cytokinesis mutants
- Author
-
Balasubramanian, M. K., Mccollum, D., Chang, L., Wong, K. C. Y., Naqvi, N. I., He, X., Sazer, S., and Kathleen Gould
16. The biology and polymer physics underlying large-scale chromosome organization.
- Author
-
Sazer S and Schiessel H
- Subjects
- Animals, Humans, Models, Biological, Chromatin genetics, Chromosomes genetics, DNA, Genome genetics, Polymers
- Abstract
Chromosome large-scale organization is a beautiful example of the interplay between physics and biology. DNA molecules are polymers and thus belong to the class of molecules for which physicists have developed models and formulated testable hypotheses to understand their arrangement and dynamic properties in solution, based on the principles of polymer physics. Biologists documented and discovered the biochemical basis for the structure, function and dynamic spatial organization of chromosomes in cells. The underlying principles of chromosome organization have recently been revealed in unprecedented detail using high-resolution chromosome capture technology that can simultaneously detect chromosome contact sites throughout the genome. These independent lines of investigation have now converged on a model in which DNA loops, generated by the loop extrusion mechanism, are the basic organizational and functional units of the chromosome., (© 2017 The Authors. Traffic published by John Wiley & Sons Ltd.)
- Published
- 2018
- Full Text
- View/download PDF
17. Evolutionary cell biology: two origins, one objective.
- Author
-
Lynch M, Field MC, Goodson HV, Malik HS, Pereira-Leal JB, Roos DS, Turkewitz AP, and Sazer S
- Subjects
- Animals, Archaea chemistry, Archaea cytology, Archaea metabolism, Bacteria chemistry, Bacteria cytology, Bacteria metabolism, Eukaryota chemistry, Eukaryota cytology, Eukaryota metabolism, Humans, Biological Evolution, Cell Biology, Cells chemistry, Cells metabolism
- Abstract
All aspects of biological diversification ultimately trace to evolutionary modifications at the cellular level. This central role of cells frames the basic questions as to how cells work and how cells come to be the way they are. Although these two lines of inquiry lie respectively within the traditional provenance of cell biology and evolutionary biology, a comprehensive synthesis of evolutionary and cell-biological thinking is lacking. We define evolutionary cell biology as the fusion of these two eponymous fields with the theoretical and quantitative branches of biochemistry, biophysics, and population genetics. The key goals are to develop a mechanistic understanding of general evolutionary processes, while specifically infusing cell biology with an evolutionary perspective. The full development of this interdisciplinary field has the potential to solve numerous problems in diverse areas of biology, including the degree to which selection, effectively neutral processes, historical contingencies, and/or constraints at the chemical and biophysical levels dictate patterns of variation for intracellular features. These problems can now be examined at both the within- and among-species levels, with single-cell methodologies even allowing quantification of variation within genotypes. Some results from this emerging field have already had a substantial impact on cell biology, and future findings will significantly influence applications in agriculture, medicine, environmental science, and synthetic biology.
- Published
- 2014
- Full Text
- View/download PDF
18. Deciphering the evolutionary history of open and closed mitosis.
- Author
-
Sazer S, Lynch M, and Needleman D
- Subjects
- Animals, Cell Nucleus metabolism, Cell Nucleus physiology, Cell Nucleus ultrastructure, Models, Biological, Nuclear Envelope metabolism, Nuclear Envelope physiology, Biological Evolution, Mitosis physiology
- Abstract
The origin of the nucleus at the prokaryote-to-eukaryote transition represents one of the most important events in the evolution of cellular organization. The nuclear envelope encircles the chromosomes in interphase and is a selectively permeable barrier between the nucleoplasm and cytoplasm and an organizational scaffold for the nucleus. It remains intact in the 'closed' mitosis of some yeasts, but loses its integrity in the 'open' mitosis of mammals. Instances of both types of mitosis within two evolutionary clades indicate multiple evolutionary transitions between open and closed mitosis, although the underlying genetic changes that influenced these transitions remain unknown. A survey of the diversity of mitotic nuclei that fall between these extremes is the starting point from which to determine the physiologically relevant characteristics distinguishing open from closed mitosis and to understand how they evolved and why they are retained in present-day organisms. The field is now poised to begin addressing these issues by defining and documenting patterns of mitotic nuclear variation within and among species and mapping them onto a phylogenic tree. Deciphering the evolutionary history of open and closed mitosis will complement cell biological and genetic approaches aimed at deciphering the fundamental organizational principles of the nucleus., (Copyright © 2014 Elsevier Ltd. All rights reserved.)
- Published
- 2014
- Full Text
- View/download PDF
19. Transcriptional regulation at the yeast nuclear envelope.
- Author
-
Steglich B, Sazer S, and Ekwall K
- Subjects
- Animals, Genome, Fungal genetics, Humans, Telomere genetics, Gene Expression Regulation, Fungal, Nuclear Envelope genetics, Transcription, Genetic genetics, Yeasts cytology, Yeasts genetics
- Abstract
The spatial organization of the genome inside the nucleus affects many nuclear processes, such as DNA replication, DNA repair, and gene transcription. In metazoans, the nuclear periphery harbors mainly repressed genes that associate with the nuclear lamina. This review discusses how peripheral positioning is connected to transcriptional regulation in yeasts. Tethering of reporter genes to the nuclear envelope was found to result in transcriptional silencing. Similarly, repression of the silent mating type loci and subtelomeric genes is influenced by their position close to the nuclear envelope. In contrast, active genes are bound by nucleoporins and inducible genes associate with the nuclear pore complex upon activation. Taken together, these results portray the nuclear envelope as a platform for transcriptional regulation, both through activation at nuclear pores and silencing at the nuclear envelope.
- Published
- 2013
- Full Text
- View/download PDF
20. Fission yeast Lem2 and Man1 perform fundamental functions of the animal cell nuclear lamina.
- Author
-
Gonzalez Y, Saito A, and Sazer S
- Subjects
- Animals, Microtubules metabolism, Nuclear Envelope metabolism, Protein Transport, Schizosaccharomyces cytology, Nuclear Lamina metabolism, Schizosaccharomyces metabolism, Schizosaccharomyces pombe Proteins metabolism
- Abstract
In animal cells the nuclear lamina, which consists of lamins and lamin-associated proteins, serves several functions: it provides a structural scaffold for the nuclear envelope and tethers proteins and heterochromatin to the nuclear periphery. In yeast, proteins and large heterochromatic domains including telomeres are also peripherally localized, but there is no evidence that yeast have lamins or a fibrous nuclear envelope scaffold. Nonetheless, we found that the Lem2 and Man1 proteins of the fission yeast Schizosaccharomyces pombe, evolutionarily distant relatives of the Lap2/Emerin/Man1 (LEM) sub-family of animal cell lamin-associated proteins, perform fundamental functions of the animal cell lamina. These integral inner nuclear membrane localized proteins, with nuclear localized DNA binding Helix-Extension-Helix (HEH) domains, impact nuclear envelope structure and integrity, are essential for the enrichment of telomeres at the nuclear periphery and by means of their HEH domains anchor chromatin, most likely transcriptionally repressed heterochromatin, to the nuclear periphery. These data indicate that the core functions of the nuclear lamina are conserved between fungi and animal cells and can be performed in fission yeast, without lamins or other intermediate filament proteins.
- Published
- 2012
- Full Text
- View/download PDF
21. Nuclear membrane: nuclear envelope PORosity in fission yeast meiosis.
- Author
-
Sazer S
- Subjects
- Cytoplasm metabolism, Nuclear Envelope physiology, Nuclear Envelope ultrastructure, Permeability, Schizosaccharomyces cytology, Schizosaccharomyces ultrastructure, Schizosaccharomyces pombe Proteins analysis, Anaphase physiology, Meiosis physiology, Nuclear Envelope metabolism, Schizosaccharomyces metabolism, Schizosaccharomyces pombe Proteins metabolism
- Abstract
The fission yeast Schizosaccharomyces pombe undergoes closed mitosis but 'virtual nuclear envelope breakdown' at anaphase of meiosis II, in which the nuclear envelope is structurally closed but functionally open., (Copyright © 2010 Elsevier Ltd. All rights reserved.)
- Published
- 2010
- Full Text
- View/download PDF
22. Nuclear shape, growth and integrity in the closed mitosis of fission yeast depend on the Ran-GTPase system, the spindle pole body and the endoplasmic reticulum.
- Author
-
Gonzalez Y, Meerbrey K, Chong J, Torii Y, Padte NN, and Sazer S
- Subjects
- ATP-Dependent Proteases metabolism, Intracellular Membranes enzymology, Membrane Lipids metabolism, Mitochondrial Proteins metabolism, Mutation, Recombinant Fusion Proteins metabolism, Saccharomyces cerevisiae Proteins metabolism, Schizosaccharomyces genetics, Schizosaccharomyces growth & development, Schizosaccharomyces pombe Proteins genetics, Serine Endopeptidases metabolism, Temperature, Time Factors, ran GTP-Binding Protein genetics, Cell Nucleus Shape, Endoplasmic Reticulum enzymology, Mitosis physiology, Nuclear Envelope enzymology, Schizosaccharomyces enzymology, Schizosaccharomyces pombe Proteins metabolism, Spindle Apparatus enzymology, ran GTP-Binding Protein metabolism
- Abstract
The double lipid bilayer of the nuclear envelope (NE) remains intact during closed mitosis. In the fission yeast Schizosaccharomyces pombe, the intranuclear mitotic spindle has envelope-embedded spindle pole bodies (SPB) at its ends. As the spindle elongates and the nucleus divides symmetrically, nuclear volume remains constant but nuclear area rapidly increases by 26%. When Ran-GTPase function is compromised in S. pombe, nuclear division is strikingly asymmetrical and the newly synthesized SPB is preferentially associated with the smaller nucleus, indicative of a Ran-dependent SPB defect that interferes with symmetrical nuclear division. A second defect, which specifically influences the NE, results in breakage of the NE upon spindle elongation. This defect, but not asymmetric nuclear division, is partially rescued by slowing spindle elongation, stimulating endoplasmic reticulum (ER) proliferation or changing conformation of the ER membrane. We propose that redistribution of lipid within the ER-NE network is crucial for mitosis-specific NE changes in both open and closed mitosis.
- Published
- 2009
- Full Text
- View/download PDF
23. Zebrafish cdc25a is expressed during early development and limiting for post-blastoderm cell cycle progression.
- Author
-
Nogare DE, Arguello A, Sazer S, and Lane ME
- Subjects
- Amino Acid Sequence, Animals, Base Sequence, Cell Cycle genetics, Cloning, Molecular, DNA Primers genetics, Gastrulation genetics, Gene Expression Regulation, Developmental, Genes, Fungal, Genetic Complementation Test, Molecular Sequence Data, Phylogeny, Schizosaccharomyces cytology, Schizosaccharomyces enzymology, Schizosaccharomyces genetics, Sequence Homology, Amino Acid, Zebrafish embryology, Zebrafish genetics, cdc25 Phosphatases genetics
- Abstract
Cdc25 phosphatases are required for eukaryotic cell cycle progression. To investigate mechanisms governing spatiotemporal dynamics of cell cycle progression during vertebrate development, we isolated two cdc25 genes from the zebrafish, Danio rerio, cdc25a, and cdc25d. We propose that Zebrafish cdc25a is the zebrafish orthologue of the tetrapod Cdc25A genes, while cdc25d is of indeterminate origin. We show that both genes have proliferation promoting activity, but that only cdc25d can complement a Schizosaccharomyces pombe loss of function cdc25 mutation. We present expression data demonstrating that cdc25d expression is very limited during early development, while cdc25a is widely expressed and consistent with the mitotic activity in previously identified mitotic domains of the post-blastoderm zebrafish embryo. Finally, we show that cdc25a can accelerate the entry of post-blastoderm cells into mitosis, suggesting that levels of cdc25a are rate limiting for cell cycle progression during gastrulation., (2007 Wiley-Liss, Inc)
- Published
- 2007
- Full Text
- View/download PDF
24. Vesicle-like biomechanics governs important aspects of nuclear geometry in fission yeast.
- Author
-
Lim H W G, Huber G, Torii Y, Hirata A, Miller J, and Sazer S
- Subjects
- Cell Cycle physiology, Cell Nucleus ultrastructure, Cell Nucleus Division physiology, Microscopy, Electron, Mitosis physiology, Models, Biological, Nuclear Envelope metabolism, Nuclear Envelope ultrastructure, Schizosaccharomyces cytology, Schizosaccharomyces metabolism, Cell Nucleus metabolism, Cytoplasmic Vesicles physiology, Schizosaccharomyces physiology
- Abstract
It has long been known that during the closed mitosis of many unicellular eukaryotes, including the fission yeast (Schizosaccharomyces pombe), the nuclear envelope remains intact while the nucleus undergoes a remarkable sequence of shape transformations driven by elongation of an intranuclear mitotic spindle whose ends are capped by spindle pole bodies embedded in the nuclear envelope. However, the mechanical basis of these normal cell cycle transformations, and abnormal nuclear shapes caused by intranuclear elongation of microtubules lacking spindle pole bodies, remain unknown. Although there are models describing the shapes of lipid vesicles deformed by elongation of microtubule bundles, there are no models describing normal or abnormal shape changes in the nucleus. We describe here a novel biophysical model of interphase nuclear geometry in fission yeast that accounts for critical aspects of the mechanics of the fission yeast nucleus, including the biophysical properties of lipid bilayers, forces exerted on the nuclear envelope by elongating microtubules, and access to a lipid reservoir, essential for the large increase in nuclear surface area during the cell cycle. We present experimental confirmation of the novel and non-trivial geometries predicted by our model, which has no free parameters. We also use the model to provide insight into the mechanical basis of previously described defects in nuclear division, including abnormal nuclear shapes and loss of nuclear envelope integrity. The model predicts that (i) despite differences in structure and composition, fission yeast nuclei and vesicles with fluid lipid bilayers have common mechanical properties; (ii) the S. pombe nucleus is not lined with any structure with shear resistance, comparable to the nuclear lamina of higher eukaryotes. We validate the model and its predictions by analyzing wild type cells in which ned1 gene overexpression causes elongation of an intranuclear microtubule bundle that deforms the nucleus of interphase cells.
- Published
- 2007
- Full Text
- View/download PDF
25. Mitosis: ran scales the alps of spindle formation.
- Author
-
Clarke PR and Sazer S
- Subjects
- Schizosaccharomyces metabolism, Microtubule-Associated Proteins metabolism, Mitosis, Schizosaccharomyces cytology, Schizosaccharomyces pombe Proteins metabolism, Spindle Apparatus metabolism, ran GTP-Binding Protein metabolism
- Abstract
Alp7/TACC has been identified as an important target for Ran GTPase in spindle formation in fission yeast. This discovery underlines a general role for Ran in orchestrating mitosis in all eukaryotes.
- Published
- 2007
- Full Text
- View/download PDF
26. New 'omics tools for fission yeast.
- Author
-
Sazer S
- Subjects
- Genes, Fungal, Internet, Saccharomyces cerevisiae Proteins genetics, Gene Library, Open Reading Frames genetics, Schizosaccharomyces genetics, Schizosaccharomyces pombe Proteins genetics
- Published
- 2006
- Full Text
- View/download PDF
27. The view from Awaji island: past, present, and future of RCC1 and the Ran GTPase system.
- Author
-
Sazer S
- Subjects
- Biological Transport, Humans, Nuclear Envelope, Nuclear Pore, Cell Cycle Proteins metabolism, Cell Nucleus metabolism, Guanine Nucleotide Exchange Factors metabolism, Guanosine Triphosphate metabolism, Nuclear Proteins metabolism, ran GTP-Binding Protein metabolism
- Abstract
The International Symposium on Ran and the Cell Cycle was held on October 1-4, 2005, at the Awaji Island Resort near Osaka, to celebrate the career and scientific achievements of Professor Takeharu Nishimoto. One hundred of his former lab members, collaborators and other scientific colleagues from around the world attended the symposium organized by Mary Dasso (National Institutes of Health) and Yoshihiro Yoneda (Osaka University). The program was divided into sessions on cell cycle and chromosomes, nuclear import and export of proteins and RNA, nuclear envelope and the nuclear pore complex, and RCC1 and chromatin. Dr. Nishimoto's retirement from Kyushu University is a perfect time to look back at the history of Ran and RCC1, assess the current state of the field, and discuss the challenges that remain in order to unravel the complexities of the Ran GTPase system.
- Published
- 2005
- Full Text
- View/download PDF
28. The fission yeast Schizosaccharomyces pombe has two importin-alpha proteins, Imp1p and Cut15p, which have common and unique functions in nucleocytoplasmic transport and cell cycle progression.
- Author
-
Umeda M, Izaddoost S, Cushman I, Moore MS, and Sazer S
- Subjects
- Active Transport, Cell Nucleus physiology, Amino Acid Sequence, Basic-Leucine Zipper Transcription Factors genetics, Basic-Leucine Zipper Transcription Factors metabolism, Gene Expression Regulation, Fungal, Genes, Lethal genetics, Genetic Complementation Test, Green Fluorescent Proteins genetics, Green Fluorescent Proteins metabolism, HeLa Cells, Humans, Molecular Sequence Data, Mutation, Nuclear Localization Signals genetics, Nuclear Localization Signals metabolism, Nuclear Proteins metabolism, Oxidative Stress, Phenotype, Protein Binding, Recombinant Fusion Proteins genetics, Recombinant Fusion Proteins metabolism, Schizosaccharomyces growth & development, Schizosaccharomyces metabolism, Schizosaccharomyces pombe Proteins metabolism, Sequence Alignment, Sequence Homology, Amino Acid, alpha Karyopherins metabolism, Cell Cycle physiology, Cell Nucleus metabolism, Schizosaccharomyces genetics, Schizosaccharomyces pombe Proteins genetics, alpha Karyopherins genetics
- Abstract
The nuclear import of classical nuclear localization signal-containing proteins depends on importin-alpha transport receptors. In budding yeast there is a single importin-alpha gene and in higher eukaryotes there are multiple importin-alpha-like genes, but in fission yeast there are two: the previously characterized cut15 and the more recently identified imp1. Like other importin-alpha family members, Imp1p supports nuclear protein import in vitro. In contrast to cut15, imp1 is not essential for viability, but imp1delta mutant cells exhibit a telophase delay and mild temperature-sensitive lethality. Differences in the cellular functions that depend on Imp1p and Cut15p indicate that they each have unique physiological roles. They also have common roles because the imp1delta and the cut15-85 temperature-sensitive mutations are synthetically lethal; overexpression of cut15 partially suppresses the temperature sensitivity, but not the mitotic delay in imp1delta cells; and overexpression of imp1 partially suppresses the mitotic defect in cut15-85 cells but not the loss of viability. Both Imp1p and Cut15p are required for the efficient nuclear import of both an SV40 nuclear localization signal-containing reporter protein and the Pap1p component of the stress response MAP kinase pathway. Imp1p and Cut15p are essential for efficient nuclear protein import in S. pombe.
- Published
- 2005
- Full Text
- View/download PDF
29. SAC-ing mitotic errors: how the spindle assembly checkpoint (SAC) plays defense against chromosome mis-segregation.
- Author
-
Kadura S and Sazer S
- Subjects
- Animals, Cell Cycle Proteins physiology, Cell Transformation, Neoplastic genetics, Chromosomes, Fungal physiology, Fungal Proteins, Kinetochores physiology, Models, Genetic, Mutation, Nuclear Proteins, Phosphorylation, Protein Kinases metabolism, Saccharomyces cerevisiae cytology, Saccharomyces cerevisiae genetics, Saccharomyces cerevisiae Proteins, Schizosaccharomyces cytology, Schizosaccharomyces genetics, Schizosaccharomyces pombe Proteins, Chromosome Segregation, Spindle Apparatus physiology
- Published
- 2005
- Full Text
- View/download PDF
30. Nuclear envelope: nuclear pore complexity.
- Author
-
Sazer S
- Subjects
- Nuclear Pore physiology, Permeability, Aspergillus nidulans cytology, GTP Phosphohydrolases physiology, Mitosis physiology, Nuclear Pore metabolism, ran GTP-Binding Protein physiology
- Abstract
A new study shows that the filamentous fungus, Aspergillus nidulans, which has a closed mitosis, does not maintain a continuous permeability barrier during mitosis. This work challenges current views of the differences between closed and open mitosis and has implications for understanding mitotic specific changes in the nuclear pore complex and Ran GTPase system in lower eukaryotes.
- Published
- 2005
- Full Text
- View/download PDF
31. The A78V mutation in the Mad3-like domain of Schizosaccharomyces pombe Bub1p perturbs nuclear accumulation and kinetochore targeting of Bub1p, Bub3p, and Mad3p and spindle assembly checkpoint function.
- Author
-
Kadura S, He X, Vanoosthuyse V, Hardwick KG, and Sazer S
- Subjects
- Alanine chemistry, Alleles, Cell Cycle Proteins metabolism, Cell Cycle Proteins physiology, Cell Nucleus metabolism, Fungal Proteins, Genotype, Green Fluorescent Proteins metabolism, Interphase, Kinetochores metabolism, Metaphase, Microscopy, Fluorescence, Mitosis, Models, Genetic, Mutagenesis, Site-Directed, Nuclear Proteins, Point Mutation, Protein Serine-Threonine Kinases, Protein Structure, Tertiary, Saccharomyces cerevisiae metabolism, Saccharomyces cerevisiae Proteins metabolism, Schizosaccharomyces metabolism, Schizosaccharomyces pombe Proteins, Cell Cycle Proteins genetics, Mutation, Protein Kinases genetics, Schizosaccharomyces genetics, Spindle Apparatus
- Abstract
During mitosis, the spindle assembly checkpoint (SAC) responds to faulty attachments between kinetochores and the mitotic spindle by imposing a metaphase arrest until the defect is corrected, thereby preventing chromosome missegregation. A genetic screen to isolate SAC mutants in fission yeast yielded point mutations in three fission yeast SAC genes: mad1, bub3, and bub1. The bub1-A78V mutant is of particular interest because it produces a wild-type amount of protein that is mutated in the conserved but uncharacterized Mad3-like region of Bub1p. Characterization of mutant cells demonstrates that the alanine at position 78 in the Mad3-like domain of Bub1p is required for: 1) cell cycle arrest induced by SAC activation; 2) kinetochore accumulation of Bub1p in checkpoint-activated cells; 3) recruitment of Bub3p and Mad3p, but not Mad1p, to kinetochores in checkpoint-activated cells; and 4) nuclear accumulation of Bub1p, Bub3p, and Mad3p, but not Mad1p, in cycling cells. Increased targeting of Bub1p-A78V to the nucleus by an exogenous nuclear localization signal does not significantly increase kinetochore localization or SAC function, but GFP fused to the isolated Bub1p Mad 3-like accumulates in the nucleus. These data indicate that Bub1p-A78V is defective in both nuclear accumulation and kinetochore targeting and that a threshold level of nuclear Bub1p is necessary for the nuclear accumulation of Bub3p and Mad3p.
- Published
- 2005
- Full Text
- View/download PDF
32. Nucleocytoplasmic transport and nuclear envelope integrity in the fission yeast Schizosaccharomyces pombe.
- Author
-
Yoshida M and Sazer S
- Subjects
- Active Transport, Cell Nucleus physiology, Cell Nucleus metabolism, Nuclear Envelope metabolism, Schizosaccharomyces metabolism
- Abstract
The nuclear envelope is essential for compartmentalizing the nucleus from the cytoplasm in all eukaryotic cells. There is a tremendous flux of both RNA and proteins across the nuclear envelope, which is intact throughout the entire cell cycle of yeasts but breaks down during mitosis of animal cells. Transport across the nuclear envelope requires the recognition of cargo molecules by receptors, docking at the nuclear pore, transit through the nuclear pore, and then dissociation of the cargo from the receptor. This process depends on the RanGTPase system, transport receptors, and the nuclear pore complex. We provide an overview of the nuclear transport process, with particular emphasis on the fission yeast Schizosaccharomyces pombe, including strategies for predicting and experimentally verifying the signals that determine the sub-cellular localization of a protein of interest. We also describe a variety of reagents and experimental strategies, including the use of mutants and chemical inhibitors, to study nuclear protein import, nuclear protein export, nucleocytoplasmic protein shuttling, and mRNA export in fission yeast. The RanGTPase and its regulators also play an essential transport independent role in nuclear envelope re-assembly after mitosis in animal cells and in the maintenance of nuclear envelope integrity at mitosis in S. pombe. Several experimental strategies and reagents for studying nuclear size, nuclear shape, the localization of nuclear pores, and the integrity of the nuclear envelope in living fission yeast cells are described.
- Published
- 2004
- Full Text
- View/download PDF
33. The fission yeast Nup107-120 complex functionally interacts with the small GTPase Ran/Spi1 and is required for mRNA export, nuclear pore distribution, and proper cell division.
- Author
-
Baï SW, Rouquette J, Umeda M, Faigle W, Loew D, Sazer S, and Doye V
- Subjects
- Active Transport, Cell Nucleus physiology, Animals, Cell Survival, Chromosomes, Fungal, Humans, Macromolecular Substances, Mutation, Open Reading Frames, Recombinant Fusion Proteins genetics, Recombinant Fusion Proteins metabolism, Saccharomyces cerevisiae Proteins genetics, Schizosaccharomyces cytology, Schizosaccharomyces genetics, Schizosaccharomyces pombe Proteins genetics, Schizosaccharomyces pombe Proteins metabolism, Temperature, ran GTP-Binding Protein genetics, Cell Division physiology, Nuclear Pore metabolism, Nuclear Pore Complex Proteins metabolism, RNA, Messenger metabolism, Saccharomyces cerevisiae Proteins metabolism, Schizosaccharomyces physiology, ran GTP-Binding Protein metabolism
- Abstract
We have characterized Schizosaccharomyces pombe open reading frames encoding potential orthologues of constituents of the evolutionarily conserved Saccharomyces cerevisiae Nup84 vertebrate Nup107-160 nuclear pore subcomplex, namely Nup133a, Nup133b, Nup120, Nup107, Nup85, and Seh1. In spite of rather weak sequence conservation, in vivo analyses demonstrated that these S. pombe proteins are localized at the nuclear envelope. Biochemical data confirmed the organization of these nucleoporins within conserved complexes. Although examination of the S. cerevisiae and S. pombe deletion mutants revealed different viability phenotypes, functional studies indicated that the involvement of this complex in nuclear pore distribution and mRNA export has been conserved between these highly divergent yeasts. Unexpectedly, microscopic analyses of some of the S. pombe mutants revealed cell division defects at the restrictive temperature (abnormal septa and mitotic spindles and chromosome missegregation) that were reminiscent of defects occurring in several S. pombe GTPase Ran (Ran(Sp))/Spi1 cycle mutants. Furthermore, deletion of nup120 moderately altered the nuclear location of Ran(Sp)/Spi1, whereas overexpression of a nonfunctional Ran(Sp)/Spi1-GFP allele was specifically toxic in the Deltanup120 and Deltanup133b mutant strains, indicating a functional and genetic link between constituents of the S. pombe Nup107-120 complex and of the Ran(Sp)/Spi1 pathway.
- Published
- 2004
- Full Text
- View/download PDF
34. HIV-1 Vpr induces defects in mitosis, cytokinesis, nuclear structure, and centrosomes.
- Author
-
Chang F, Re F, Sebastian S, Sazer S, and Luban J
- Subjects
- Cell Cycle, Cell Cycle Proteins metabolism, Cell Division, Cells, Cultured, Checkpoint Kinase 2, Drosophila Proteins metabolism, G2 Phase, HeLa Cells, Humans, Mutation, Plasmids metabolism, Protein Serine-Threonine Kinases metabolism, Saccharomyces cerevisiae Proteins metabolism, Schizosaccharomyces, Spindle Apparatus, Time Factors, Transfection, Umbilical Veins cytology, Cell Nucleus metabolism, Centrosome metabolism, Gene Products, vpr physiology, Mitosis
- Abstract
Human immunodeficiency virus type 1 (HIV-1) Vpr is a 15-kDa accessory protein that contributes to several steps in the viral replication cycle and promotes virus-associated pathology. Previous studies demonstrated that Vpr inhibits G2/M cell cycle progression in both human cells and in the fission yeast Schizosaccharomyces pombe. Here, we report that, upon induction of vpr expression, fission yeast exhibited numerous defects in the assembly and function of the mitotic spindle. In particular, two spindle pole body proteins, sad1p and the polo kinase plo1p, were delocalized in vpr-expressing yeast cells, suggesting that spindle pole body integrity was perturbed. In addition, nuclear envelope structure, contractile actin ring formation, and cytokinesis were also disrupted. Similar Vpr-induced defects in mitosis and cytokinesis were observed in human cells, including aberrant mitotic spindles, multiple centrosomes, and multinucleate cells. These defects in cell division and centrosomes might account for some of the pathological effects associated with HIV-1 infection.
- Published
- 2004
- Full Text
- View/download PDF
35. The Ran GTPase system in fission yeast affects microtubules and cytokinesis in cells that are competent for nucleocytoplasmic protein transport.
- Author
-
Salus SS, Demeter J, and Sazer S
- Subjects
- Cell Cycle physiology, Cell Nucleus metabolism, Cytoskeleton drug effects, Cytoskeleton metabolism, Endopeptidases metabolism, Genes, Fungal, Genes, cdc, Microtubules drug effects, Mutation, Phenotype, Recombinant Fusion Proteins genetics, Recombinant Fusion Proteins metabolism, Schizosaccharomyces cytology, Schizosaccharomyces drug effects, Schizosaccharomyces genetics, Schizosaccharomyces pombe Proteins genetics, Spindle Apparatus metabolism, Thiabendazole pharmacology, ran GTP-Binding Protein genetics, Active Transport, Cell Nucleus physiology, Cell Division physiology, Microtubules metabolism, Saccharomyces cerevisiae Proteins, Schizosaccharomyces physiology, Schizosaccharomyces pombe Proteins metabolism, ran GTP-Binding Protein metabolism
- Abstract
Misregulation of the evolutionarily conserved GTPase Ran in fission yeast results in defects in several cellular processes in cells that are competent for nucleocytoplasmic protein transport. These results suggest that transport is neither the only nor the primary Ran-dependent process in living cells. The ability of Ran to independently regulate multiple cellular processes in vivo is demonstrated by showing that (i) eight different transport-competent RanGEF (guanine nucleotide exchange factor) mutants have defects in mitotic spindle formation; (ii) the RanGEF temperature-sensitive mutant pim1-d1 has abnormal actin ring structures at the septum. Overexpression of Imp2p, which specifically destabilizes these structures, restores viability. (iii) Ran-dependent processes differ in their requirements for active Ran in vivo. Microtubule function, cytokinesis, and nuclear envelope structure are the Ran-dependent processes most sensitive to the amount of Ran protein in the cell, whereas nucleocytoplasmic protein transport is the most robust. Therefore, the ability of Ran from Schizosaccharomyces pombe to independently regulate multiple cellular processes may reflect differences in its interactions with the binding proteins that mediate these functions and explain the complex phenotypic consequences of its misregulation in vivo.
- Published
- 2002
- Full Text
- View/download PDF
36. Three proteins required for early steps in the protein secretory pathway also affect nuclear envelope structure and cell cycle progression in fission yeast.
- Author
-
Matynia A, Salus SS, and Sazer S
- Subjects
- Carrier Proteins genetics, Carrier Proteins metabolism, Endoplasmic Reticulum metabolism, Gene Expression Regulation, Fungal genetics, Intracellular Membranes metabolism, Intracellular Membranes ultrastructure, Molecular Sequence Data, Monomeric GTP-Binding Proteins genetics, Monomeric GTP-Binding Proteins metabolism, Mutation genetics, Nuclear Envelope metabolism, Phosphoproteins genetics, Phosphoproteins metabolism, Phosphotransferases (Phosphomutases) genetics, Phosphotransferases (Phosphomutases) metabolism, Schizosaccharomyces ultrastructure, Sequence Homology, Amino Acid, Vesicular Transport Proteins, Active Transport, Cell Nucleus physiology, Cell Cycle physiology, Endoplasmic Reticulum genetics, Fungal Proteins genetics, Fungal Proteins metabolism, Nuclear Envelope genetics, Saccharomyces cerevisiae Proteins, Schizosaccharomyces genetics, Schizosaccharomyces metabolism
- Abstract
The Ran GTPase is an essential protein that has multiple functions in eukaryotic cells. Fission yeast cells in which Ran is misregulated arrest after mitosis with condensed, unreplicated chromosomes and abnormal nuclear envelopes. The fission yeast sns mutants arrest with a similar cell cycle block and interact genetically with the Ran system. sns-A10, sns-B2 and sns-B9 have mutations in the fission yeast homologues of S. cerevisiae Sar1p, Sec31p and Sec53p, respectively, which are required for the early steps of the protein secretory pathway. The three sns mutants accumulate a normally secreted protein in the endoplasmic reticulum (ER), have an increased amount of ER membrane, and the ER/nuclear envelope lumen is dilated. Neither a post-ER block in the secretory pathway, nor ER proliferation caused by overexpression of an integral ER membrane protein, results in a cell cycle-specific defect. Therefore, the arrest seen in sns-A10, sns-B2 and sns-B9 is most likely due to nuclear envelope defects that render the cells unable to re-establish the interphase organization of the nucleus after mitosis. As a consequence, these mutants are unable to decondense their chromosomes or to initiate of the next round of DNA replication.
- Published
- 2002
- Full Text
- View/download PDF
37. Redundant control of rereplication in fission yeast.
- Author
-
Gopalakrishnan V, Simancek P, Houchens C, Snaith HA, Frattini MG, Sazer S, and Kelly TJ
- Subjects
- Cell Cycle Proteins metabolism, DNA-Binding Proteins metabolism, Fluorescent Antibody Technique, Fungal Proteins metabolism, Phosphorylation, Precipitin Tests, Schizosaccharomyces pombe Proteins, Subcellular Fractions metabolism, Cell Cycle Proteins physiology, DNA Replication, DNA, Fungal biosynthesis, DNA-Binding Proteins physiology, Fungal Proteins physiology, Schizosaccharomyces genetics
- Abstract
The initiation of DNA replication at replication origins in eukaryotic cells is tightly controlled to ensure that the genome is duplicated only once each cell cycle. We present evidence that in fission yeast, independent regulation of two essential components of the initiation complex, Cdc18 and Cdt1, contributes to the prevention of reinitiation of DNA replication. Cdc18 is negatively controlled by cyclin-dependent kinase (CDK) phosphorylation, but low level expression of a mutant form of Cdc18 lacking CDK phosphorylation sites (Cdc18(CDK)) is not sufficient to induce rereplication. Similar to Cdc18, Cdt1 is expressed periodically in the cell cycle, accumulating in the nucleus in G(1) and declining in G(2). When Cdt1 is expressed constitutively from an ectopic promoter, it accumulates in the nucleus throughout the cell cycle but does not promote reinitiation. However, constitutive expression of Cdt1, together with Cdc18(CDK), is sufficient to induce extra rounds of DNA replication in the absence of mitosis. Significantly greater levels of rereplication can be induced by coexpression of Cdc18(CDK) and a Cdt1 mutant lacking a conserved C-terminal motif. In contrast, uncontrolled DNA replication does not occur when either mutant protein is expressed in the absence of the other. Constitutive expression of wild-type or mutant Cdt1 also leads to an increase in the levels of Cdc18(CDK), possibly as a result of increased protein stability. Our data are consistent with the hypothesis that control of rereplication depends on a redundant mechanism in which negative regulation of Cdt1 functions in parallel with the negative regulation of Cdc18.
- Published
- 2001
- Full Text
- View/download PDF
38. The fission yeast ran GTPase is required for microtubule integrity.
- Author
-
Fleig U, Salus SS, Karig I, and Sazer S
- Subjects
- Active Transport, Cell Nucleus physiology, Alleles, Cell Cycle Proteins genetics, Cell Cycle Proteins metabolism, Cell Nucleus metabolism, Chromosomes, Fungal metabolism, Cytoplasm metabolism, Genes, Lethal physiology, Interphase physiology, Microtubule-Associated Proteins genetics, Microtubule-Associated Proteins metabolism, Mitosis physiology, Mutagenesis physiology, Nucleotides metabolism, Phenotype, Schizosaccharomyces cytology, Schizosaccharomyces genetics, Spindle Apparatus metabolism, ran GTP-Binding Protein genetics, Microtubules enzymology, Schizosaccharomyces enzymology, Schizosaccharomyces pombe Proteins, ran GTP-Binding Protein metabolism
- Abstract
The microtubule cytoskeleton plays a pivotal role in cytoplasmic organization, cell division, and the correct transmission of genetic information. In a screen designed to identify fission yeast genes required for chromosome segregation, we identified a strain that carries a point mutation in the SpRan GTPase. Ran is an evolutionarily conserved eukaryotic GTPase that directly participates in nucleocytoplasmic transport and whose loss affects many biological processes. Recently a transport-independent effect of Ran on spindle formation in vitro was demonstrated, but the in vivo relevance of these findings was unclear. Here, we report the characterization of a Schizosaccharomyces pombe Ran GTPase partial loss of function mutant in which nucleocytoplasmic protein transport is normal, but the microtubule cytoskeleton is defective, resulting in chromosome missegregation and abnormal cell shape. These abnormalities are exacerbated by microtubule destabilizing drugs, by loss of the spindle checkpoint protein Mph1p, and by mutations in the spindle pole body component Cut11p, indicating that SpRan influences microtubule integrity. As the SpRan mutant phenotype can be partially suppressed by the presence of extra Mal3p, we suggest that SpRan plays a role in microtubule stability.
- Published
- 2000
- Full Text
- View/download PDF
39. Effects of genome position and the DNA damage checkpoint on the structure and frequency of sod2 gene amplification in fission yeast.
- Author
-
Patterson TE, Albrecht EB, Nurse P, Sazer S, and Stark GR
- Subjects
- Cell Cycle genetics, Chromosome Mapping, Gene Amplification, Gene Frequency, Genome, Fungal, Mutation, Telomere genetics, DNA Damage genetics, Genes, Fungal, Schizosaccharomyces genetics, Sodium-Hydrogen Exchangers genetics
- Abstract
The Schizosaccharomyces pombe sod2 gene, located near the telomere on the long arm of chromosome I, encodes a Na+ (or Li+)/H+ antiporter. Amplification of sod2 has previously been shown to confer resistance to LiCl. We analyzed 20 independent LiCl-resistant strains and found that the only observed mechanism of resistance is amplification of sod2. The amplicons are linear, extrachromosomal elements either 225 or 180 kb long, containing both sod2 and telomere sequences. To determine whether proximity to a telomere is necessary for sod2 amplification, a strain was constructed in which the gene was moved to the middle of the same chromosomal arm. Selection of LiCl-resistant strains in this genetic background also yielded amplifications of sod2, but in this case the amplified DNA was exclusively chromosomal. Thus, proximity to a telomere is not a prerequisite for gene amplification in S. pombe but does affect the mechanism. Relative to wild-type cells, mutants with defects in the DNA damage aspect of the rad checkpoint control pathway had an increased frequency of sod2 amplification, whereas mutants defective in the S-phase completion checkpoint did not. Two models for generating the amplified DNA are presented.
- Published
- 1999
- Full Text
- View/download PDF
40. imp2, a new component of the actin ring in the fission yeast Schizosaccharomyces pombe.
- Author
-
Demeter J and Sazer S
- Subjects
- Cell Compartmentation physiology, Cell Cycle physiology, Cell Cycle Proteins genetics, Cell Cycle Proteins metabolism, Chromosome Mapping, Cytoskeletal Proteins, Endopeptidases isolation & purification, F-Box-WD Repeat-Containing Protein 7, GTP-Binding Proteins genetics, GTP-Binding Proteins metabolism, Gene Deletion, Gene Expression Regulation, Fungal, Mitochondrial Proteins, Molecular Sequence Data, Mutagenesis physiology, Schizosaccharomyces cytology, Schizosaccharomyces pombe Proteins, Sequence Homology, Amino Acid, Actins metabolism, Endopeptidases genetics, Endopeptidases metabolism, F-Box Proteins, Fungal Proteins metabolism, Saccharomyces cerevisiae Proteins, Schizosaccharomyces enzymology, Ubiquitin-Protein Ligases
- Abstract
Cytokinesis is the part of the cell cycle in which the cell is cleaved to form two daughter cells. The unicellular yeast, Schizosaccharomyces pombe is an excellent model organism in which to study cell division, since it shows the general features of eukaryotic cell division and is amenable to genetic analysis. In this manuscript we describe the isolation and characterization of a new protein, imp2, which is required for normal septation in fission yeast. imp2, which colocalizes with the medial ring during septation, is structurally similar to a group of proteins including the S. pombe cdc15 and the mouse PSTPIP that are localized to, and thought to be involved in actin ring organization. Cells in which the imp2 gene is deleted or overexpressed have septation and cell separation defects. An analysis of the actin cytoskeleton shows the lack of a medial ring in septating cells that overexpress imp2, and the appearance of abnormal medial ring structures in septated cells that lack imp2. These observations suggest that imp2 destabilizes the medial ring during septation. imp2 also shows genetic interactions with several, previously characterized septation genes, strengthening the conclusion that it plays a role in normal fission yeast septation.
- Published
- 1998
- Full Text
- View/download PDF
41. The role of fnx1, a fission yeast multidrug resistance protein, in the transition of cells to a quiescent G0 state.
- Author
-
Dimitrov K and Sazer S
- Subjects
- Amino Acid Sequence, Fungal Proteins chemistry, Fungal Proteins genetics, Gene Expression Regulation, Fungal, Models, Molecular, Molecular Sequence Data, Nitrogen metabolism, Open Reading Frames, Promoter Regions, Genetic, Protein Structure, Secondary, Resting Phase, Cell Cycle, Saccharomyces cerevisiae genetics, Schizosaccharomyces drug effects, Schizosaccharomyces genetics, Sequence Alignment, Sequence Homology, Amino Acid, Drug Resistance, Multiple genetics, Fungal Proteins biosynthesis, Schizosaccharomyces physiology, Schizosaccharomyces pombe Proteins, Transcription, Genetic
- Abstract
Most microorganisms live in conditions of nutrient limitation in their natural habitats. When exposed to these conditions they respond with physiological and morphological changes that enable them to survive. To obtain insights into the molecular mechanisms of this response a systematic genetic screen was performed to identify genes that when overexpressed can induce a starvation-like response in the yeast species Schizosaccharomyces pombe. One gene that meets these criteria, fnx1(+), induces, transcriptionally correlates with, and is required for the entry into the quiescent G0 state that is normally induced by nitrogen starvation. fnx1(+) encodes a protein with sequence similarity to the proton-driven plasma membrane transporters from the multidrug resistance group of the major facilitator superfamily of proteins. We propose that fnx1(+) plays a role in the entry into G0, possibly by facilitating the release of a signaling substance into the environment as a means of cell-to-cell communication.
- Published
- 1998
- Full Text
- View/download PDF
42. Isolation and characterization of new fission yeast cytokinesis mutants.
- Author
-
Balasubramanian MK, McCollum D, Chang L, Wong KC, Naqvi NI, He X, Sazer S, and Gould KL
- Subjects
- Actins biosynthesis, Actins genetics, Amino Acid Sequence, Cloning, Molecular, DNA Primers, Genes, Fungal, Genotype, Molecular Sequence Data, Mutagenesis, Polymerase Chain Reaction, Protein Kinases biosynthesis, Protein Kinases chemistry, Schizosaccharomyces genetics, Sequence Alignment, Sequence Homology, Amino Acid, Actins physiology, Cell Cycle Proteins, Cell Division genetics, DNA-Binding Proteins, Protein Kinases genetics, Schizosaccharomyces cytology, Schizosaccharomyces pombe Proteins
- Abstract
Schizosaccharomyces pombe is an excellent organism in which to study cytokinesis as it divides by medial fission using an F-actin contractile ring. To enhance our understanding of the cell division process, a large genetic screen was carried out in which 17 genetic loci essential for cytokinesis were identified, 5 of which are novel. Mutants identifying three genes, rng3(+), rng4(+), and rng5(+), were defective in organizing an actin contractile ring. Four mutants defective in septum deposition, septum initiation defective (sid)1, sid2, sid3, and sid4, were also identified and characterized. Genetic analyses revealed that the sid mutants display strong negative interactions with the previously described septation mutants cdc7-24, cdc11-123, and cdc14-118. The rng5(+), sid2(+), and sid3(+) genes were cloned and shown to encode Myo2p (a myosin heavy chain), a protein kinase related to budding yeast Dbf2p, and Spg1p, a GTP binding protein that is a member of the ras superfamily of GTPases, respectively. The ability of Spg1p to promote septum formation from any point in the cell cycle depends on the activity of Sid4p. In addition, we have characterized a phenotype that has not been described previously in cytokinesis mutants, namely the failure to reorganize actin patches to the medial region of the cell in preparation for septum formation.
- Published
- 1998
- Full Text
- View/download PDF
43. Isolation and characterization of fission yeast sns mutants defective at the mitosis-to-interphase transition.
- Author
-
Matynia A, Mueller U, Ong N, Demeter J, Granger AL, Hinata K, and Sazer S
- Subjects
- Alleles, Amino Acid Sequence, Epistasis, Genetic, GTP Phosphohydrolases genetics, Gene Expression, Genetic Complementation Test, Genetic Linkage, Guanine Nucleotide Exchange Factors genetics, Molecular Sequence Data, Phenotype, Schizosaccharomyces genetics, Schizosaccharomyces isolation & purification, Interphase genetics, Mitosis genetics, Mutation, Schizosaccharomyces cytology
- Abstract
pim1-d1ts was previously identified in a visual screen for fission yeast mutants unable to complete the mitosis-to-interphase transition. pim1+ encodes the guanine nucleotide exchange factor (GEF) for the spi1 GTPase. Perturbations of this GTPase system by either mutation or overproduction of its regulatory proteins cause cells to arrest with postmitotic condensed chromosomes, an unreplicated genome, and a wide medial septum. The septation phenotype of pim1-d1ts was used as the basis for a more extensive screen for this novel class of sns (septated, not in S-phase) mutants. Seventeen mutants representing 14 complementation groups were isolated. Three strains, sns-A3, sns-A5, and sns-A6, representing two different alleles, are mutated in the pim1+ gene. Of the 13 non-pim1ts sns complementation groups, 11 showed genetic interactions with the spi1 GTPase system. The genes mutated in 10 sns strains were synthetically lethal with pim1-d1, and six sns strains were hypersensitive to overexpression of one or more of the known components of the spil GTPase system. Epistasis analysis places the action of the genes mutated in nine of these strains downstream of pim1+ and the action of one gene upstream of pim1+. Three strains, sns-A2, sns-B1, and sns-B9, showed genetic interaction with the spil GTPase system in every test performed. sns-B1 and sns-B9 are likely to identify downstream targets, whereas sns-A2 is likely to identify upstream regulators of the spi1 GTPase system that are required for the mitosis-to-interphase transition.
- Published
- 1998
- Full Text
- View/download PDF
44. The identification of cDNAs that affect the mitosis-to-interphase transition in Schizosaccharomyces pombe, including sbp1, which encodes a spi1p-GTP-binding protein.
- Author
-
He X, Hayashi N, Walcott NG, Azuma Y, Patterson TE, Bischoff FR, Nishimoto T, and Sazer S
- Subjects
- Amino Acid Sequence, Base Sequence, Cell Cycle physiology, Cell Survival genetics, Cloning, Molecular, Conserved Sequence genetics, DNA, Complementary chemistry, Evolution, Molecular, Flow Cytometry, GTP Phosphohydrolases genetics, Gene Expression Regulation, Fungal genetics, Molecular Sequence Data, Nuclear Proteins chemistry, Sequence Analysis, DNA, Sequence Homology, Amino Acid, Fungal Proteins chemistry, GTP-Binding Proteins chemistry, Mitosis genetics, Monomeric GTP-Binding Proteins, Schizosaccharomyces chemistry, Schizosaccharomyces pombe Proteins, ran GTP-Binding Protein
- Abstract
Perturbations of the spi1p GTPase system in fission yeast, caused by mutation or overexpression of several regulatory proteins, result in a unique terminal phenotype that includes condensed chromosomes, a wide medial septum, and a fragmented nuclear envelope. To identify potential regulators or targets of the spi1p GTPase system, a screen for cDNAs whose overexpression results in this terminal phenotype was conducted, and seven clones that represent three genes, named med1, med2, and med3 (mitotic exit defect), were identified. Their genetic interaction with the spi1p GTPase system was established by showing that the spi1p guanine nucleotide exchange factor mutant pim1-d1ts was hypersensitive to their overexpression. med1 encodes a homologue of the human Ran-binding protein, RanBP1, and has been renamed sbp1 (spi1-binding protein). sbp1p binds to spi1p-GTP and costimulates the GTPase-activating protein (GAP)-catalyzed GTPase activity. Cells in which sbp1p is depleted or overproduced phenocopy cells in which the balance between spi1p-GTP and spi1p-GDP is perturbed by other means. Therefore, sbp1p mediates and/or regulates the essential functions of the spi1p GTPase system. med2 and med3 encode novel fission yeast proteins that, based on our phenotypic analyses, are likely to identify additional regulators or effectors of the spi1p GTPase system.
- Published
- 1998
- Full Text
- View/download PDF
45. The Schizosaccharomyces pombe spindle checkpoint protein mad2p blocks anaphase and genetically interacts with the anaphase-promoting complex.
- Author
-
He X, Patterson TE, and Sazer S
- Subjects
- Amino Acid Sequence, Anaphase-Promoting Complex-Cyclosome, Calcium-Binding Proteins genetics, Cell Cycle Proteins, DNA, Complementary, Fungal Proteins genetics, Ligases genetics, Mad2 Proteins, Maturation-Promoting Factor antagonists & inhibitors, Metaphase genetics, Molecular Sequence Data, Nuclear Proteins, Protein Binding, Schizosaccharomyces cytology, Schizosaccharomyces metabolism, Schizosaccharomyces pombe Proteins, Sequence Homology, Amino Acid, Spindle Apparatus, Ubiquitin-Protein Ligases, Anaphase genetics, Calcium-Binding Proteins metabolism, Carrier Proteins, Fungal Proteins metabolism, Ligases metabolism, Schizosaccharomyces genetics, Ubiquitin-Protein Ligase Complexes
- Abstract
The spindle checkpoint monitors mitotic spindle integrity and the attachment of kinetochores to the spindle. Upon sensing a defect the checkpoint blocks cell cycle progression and thereby prevents chromosome missegregation. Previous studies in budding yeast show that the activated spindle checkpoint inhibits the onset of anaphase by an unknown mechanism. One possible target of the spindle checkpoint is anaphase promoting complex (APC), which controls all postmetaphase events that are blocked by spindle checkpoint activation. We have isolated mad2, a spindle checkpoint component in fission yeast, and shown that mad2 overexpression activates the checkpoint and causes a cell cycle arrest at the metaphase-to-anaphase transition. In addition to the observation that mad2-induced arrest can be partially relieved by mitosis-promoting factor inactivation, we present genetic evidence consistent with the hypothesis that the spindle checkpoint imposes a cell cycle arrest by inhibiting APC-dependent proteolysis.
- Published
- 1997
- Full Text
- View/download PDF
46. Mutations in the glutathione synthetase gene cause 5-oxoprolinuria.
- Author
-
Shi ZZ, Habib GM, Rhead WJ, Gahl WA, He X, Sazer S, and Lieberman MW
- Subjects
- Amino Acid Metabolism, Inborn Errors complications, Anemia complications, Anemia genetics, Binding Sites, Erythrocytes pathology, Escherichia coli genetics, Escherichia coli metabolism, Female, Genetic Complementation Test, Glutathione Synthase metabolism, Heterozygote, Humans, Male, Molecular Sequence Data, Pedigree, Polymerase Chain Reaction, RNA Splicing, Recombinant Proteins genetics, Recombinant Proteins metabolism, Schizosaccharomyces genetics, Schizosaccharomyces metabolism, Sequence Analysis, DNA, Amino Acid Metabolism, Inborn Errors genetics, Glutathione Synthase genetics, Mutation, Pyrrolidonecarboxylic Acid metabolism
- Abstract
5-Oxoprolinuria (pyroglutamic aciduria) resulting from glutathione synthetase (GSS) deficiency is an inherited autosomal recessive disorder characterized, in its severe form, by massive urinary excretion of 5-oxoproline, metabolic acidosis, haemolytic anaemia and central nervous system damage. The metabolic defect results in low GSH levels presumably with feedback over-stimulation of gamma-glutamylcysteine synthesis and its subsequent conversion to 5-oxoproline. In this study, we cloned and characterized the human GSS gene and examined three families with four cases of well-documented 5-oxoprolinuria. We identified seven mutations at the GSS locus on six alleles: one splice site mutation, two deletions and four missense mutations. Bacterial expression and yeast complementation assays of the cDNAs encoded by these alleles demonstrated their functional defects. We also characterized a fifth case, an homozygous missense mutation in the gene in an individual affected by a milder-form of the GSS deficiency, which is apparently restricted to erythrocytes and only associated with haemolytic anaemia. Our data provide the first molecular genetic analysis of 5-oxoprolinuria and demonstrate that GSS deficiency with oxoprolinuria and GSS deficiency without 5-oxoprolinuria are caused by mutations in the same gene.
- Published
- 1996
- Full Text
- View/download PDF
47. A single mouse glutathione synthetase gene encodes six mRNAs with different 5' ends.
- Author
-
Shi ZZ, Carter BZ, Habib GM, He X, Sazer S, Lebovitz RM, and Lieberman MW
- Subjects
- Alternative Splicing, Amino Acid Sequence, Animals, Base Sequence, Cloning, Molecular, DNA Primers chemistry, Exons, Gene Expression, Genes, Genetic Complementation Test, Mice, Molecular Sequence Data, Rats, Restriction Mapping, Schizosaccharomyces genetics, Sequence Alignment, Sequence Homology, Amino Acid, Tissue Distribution, Xenopus laevis, Glutathione Synthase genetics, RNA, Messenger genetics
- Abstract
To understand more about the role of glutathione (GSH) in metabolism, we have cloned both cDNA and genomic sequences for mouse glutathione synthetase (GSH syn), the enzyme that catalyzes the last step in the synthesis of glutathione. The mouse cDNA contains an open reading frame (ORF) of 474 aa and shares 64 and 95% deduced amino acid sequence identity with Xenopus cDNA and rat cDNA, respectively. The cDNA complements Schizosaccaromyces pombe strains deficient in GSH syn. The gene is a single-copy gene spanning approximately 30 kb and is composed of at least 15 exons. Steady-state RNA levels and enzyme activity levels are highest in kidney, about 3-fold lower in liver, and 8- to 10-fold lower in lung and brain. We have identified six different GSH syn RNAs: three, termed types A1, A2, and A3, have different 5' ends that localize to different sites in the gene, but appear to encode the same protein (474 aa). Types B, C1, and C2 all have unique 5' ends and type-specific ORFs, which are shorter than that for types A1, A2, and A3. In liver only type A1 GSH syn RNA is detectable, while in kidney 90% of GSH syn RNA is type A1 and types B and C account for about 10%.
- Published
- 1996
- Full Text
- View/download PDF
48. A strategy for quickly identifying all unique two-hybrid or library plasmids within a pool of yeast transformants.
- Author
-
Patterson TE, Stark GR, and Sazer S
- Subjects
- ATP-Dependent Proteases, Gene Dosage, Genes, Fungal genetics, Genes, Suppressor genetics, Leucine, Mitochondrial Proteins, Molecular Probe Techniques, Nucleic Acid Hybridization, Serine Endopeptidases genetics, Cloning, Molecular methods, Genomic Library, Guanine Nucleotide Exchange Factors, Plasmids genetics, Saccharomyces cerevisiae genetics, Saccharomyces cerevisiae Proteins, Schizosaccharomyces genetics, Schizosaccharomyces pombe Proteins
- Published
- 1995
- Full Text
- View/download PDF
49. Transcription factor Sp1 recognizes a DNA sequence in the mouse dihydrofolate reductase promoter.
- Author
-
Dynan WS, Sazer S, Tjian R, and Schimke RT
- Subjects
- Animals, Base Sequence, Binding Sites, Humans, Mice, Transcription, Genetic, Promoter Regions, Genetic, Tetrahydrofolate Dehydrogenase genetics, Transcription Factors
- Abstract
Human (HeLa) cells contain a host-cell-encoded transcription factor, Sp1, which is required for transcription of simian virus 40 (SV40) promoters. Since the discovery of Sp1 we have been interested in learning what role this factor plays in uninfected cells. A monkey cellular gene promoter interacts with Sp1, but no gene products linked to this promoter have yet been identified. The finding that the sequence of the 5'-flanking DNA of the mouse dihydrofolate reductase (DHFR) gene contains several regions showing strong homology to the Sp1 binding region of simian virus 40 (SV40) prompted us to undertake experiments with dhfr. We report here that Sp1 binds to these regions in the dhfr promoter, and that Sp1-containing preparations stimulate transcription from the dhfr promoter in an in vitro reaction. Our results suggest that in addition to its interactions with the SV40 viral promoter, one function of Sp1 is to direct the expression of the cellular DHFR gene.
- Published
- 1986
- Full Text
- View/download PDF
Catalog
Discovery Service for Jio Institute Digital Library
For full access to our library's resources, please sign in.