Yona Kassir - Academia.edu (original) (raw)
Papers by Yona Kassir
Methods in molecular biology, 2017
We describe a simple computational approach that can be used for the study and simulation of regu... more We describe a simple computational approach that can be used for the study and simulation of regulatory networks. The advantage of this approach is that it requires neither computational background nor exact quantitative data about the biological system under study. Moreover, it is suitable for examining alternative hypotheses about the structure of a biological network. We used a tool called BioNSi (Biological Network Simulator) that is based on a simple computational model, which can be easily integrated as part of the lab routine, in parallel to experimental work. One benefit of this approach is that it enables the identification of regulatory proteins, which are missing from the experimental work. We describe the general methodology for modeling a network's dynamics in the tool, and then give a point by point example for a specific known network, entry into meiosis in budding yeast.
Current Drug Targets, Nov 1, 2006
Molecular Genetics And Genomics, Mar 1, 1993
The IME1 gene of Saccharomyces cerevisiae is required for initiation of meiosis. Transcription of... more The IME1 gene of Saccharomyces cerevisiae is required for initiation of meiosis. Transcription of IME1 is detected under conditions which are known to induce initiation of meiosis, namely starvation for nitrogen and glucose, and the presence of MA Tal and MA Te2 gene products. In this paper we show that IME1 is also subject to translational regulation. Translation of IME1 mRNA is achieved either upon nitrogen starvation, or upon G1 arrest. In the presence of nutrients, constitutively elevated transcription of IME1 is also sufficient for the translation of IME1 RNA. Four different conditions were found to cause expression of Imel protein in vegetative cultures: elevated transcription levels due to the presence of 1ME1 on a multicopy plasmid; elevated transcription provided by a Gal-IME1 construct; G1 arrest due to a-factor treatment; G1 arrest following mild heat-shock treatment of cdc28 diploids. Using these conditions, we obtained evidence that starvation is required not only for transcription and efficient translation of IME1, but also for either the activation of Imel protein or for the induction/activation of another factor that, either alone or in combination with Imel, induces meiosis.
Methods in molecular biology, 2017
The budding yeast Saccharomyces cerevisiae has a long history as a model organism for studies of ... more The budding yeast Saccharomyces cerevisiae has a long history as a model organism for studies of meiosis and the cell cycle. The popularity of this yeast as a model is in large part due to the variety of genetic and cytological approaches that can be effectively performed with the cells. Cultures of the cells can be induced to synchronously progress through meiosis and sporulation allowing large-scale gene expression and biochemical studies to be performed. Additionally, the spore tetrads resulting from meiosis make it possible to characterize the haploid products of meiosis allowing investigation of meiotic recombination and chromosome segregation. Here we describe genetic methods for analysis progression of S. cerevisiae through meiosis and sporulation with an emphasis on strategies for the genetic analysis of regulators of meiosis-specific genes.
PLOS ONE, Dec 17, 2013
In budding yeasts, the histone deacetylase Rpd3 resides in two different complexes called Rpd3L (... more In budding yeasts, the histone deacetylase Rpd3 resides in two different complexes called Rpd3L (large) and Rpd3S (small) that exert opposing effects on the transcription of meiosis-specific genes. By introducing mutations that disrupt the integrity and function of either Rpd3L or Rpd3S, we show here that Rpd3 function is determined by its association with either of these complexes. Specifically, the catalytic activity of Rpd3S activates the transcription of the two major positive regulators of meiosis, IME1 and IME2, under all growth conditions and activates the transcription of NDT80 only during vegetative growth. In contrast, the effects of Rpd3L depends on nutrients; it represses or activates transcription in the presence or absence of a nitrogen source, respectively. Further, we show that transcriptional activation does not correlate with histone H4 deacetylation, suggesting an effect on a nonhistone protein. Comparison of rpd3-null and catalytic-site point mutants revealed an inhibitory activity that is independent of either the catalytic activity of Rpd3 or the integrity of Rpd3L and Rpd3S.
Cell Cycle, Feb 15, 2009
Progression through the cell cycle depends on sequential activation of Cyclin-Dependent Kinase(s)... more Progression through the cell cycle depends on sequential activation of Cyclin-Dependent Kinase(s). In this report we use budding-yeast meiosis as a tool to elucidate the specific functions of mammalian Cdks. Yeast meiosis is regulated by both Cdc28 (yCdk1) and Ime2 (a meiosis-specific Cdk-like kinase). We show that human Cdk2 is a functional homolog for most of Ime2 functions. It promotes efficient and timely entry into premeiotic DNA replication and the first nuclear division, as well as the regulated transcription of IME1 and the early meiosis-specific genes. We show that this effect is specific, and that neither mice Cdk1, nor mice Cdk4 can suppress ime2. We show that Cdk1 is a functional homolog of Cdc28 that also suppresses one of its meiotic functions, namely inhibiting the transcription of IME1. Cdk2, on the other hand, show dominant negative effects on entry into the cell cycle, most probably by inhibiting the function of Cdc28. Finally, we show that in the meiotic pathway Cdk4 functions as a transcriptional activator.
Molecular and Cellular Biology, Aug 1, 2004
Phosphorylation is the main mode by which signals are transmitted to key regulators of developmen... more Phosphorylation is the main mode by which signals are transmitted to key regulators of developmental pathways. The glycogen synthase kinase 3 family plays pivotal roles in the development and well-being of all eukaryotic organisms. Similarly, the budding yeast homolog Rim11 is essential for the exit of diploid cells from the cell cycle and for entry into the meiotic developmental pathway. In this report we show that in vivo, in cells grown in a medium promoting vegetative growth with acetate as the sole carbon source (SA medium), Rim11 phosphorylates Ime1, the master transcriptional activator required for entry into the meiotic cycle and for the transcription of early meiosis-specific genes. We demonstrate that in the presence of glucose, the kinase activity of Rim11 is inhibited. This inhibition could be due to phosphorylation on Ser-5, Ser-8, and/or Ser-12 because in the rim11S5AS8AS12A mutant, Ime1 is incorrectly phosphorylated in the presence of glucose and cells undergo sporulation. We further show that this nutrient signal is transmitted to Rim11 and consequently to Ime1 by the cyclic AMP/protein kinase A signal transduction pathway. Ime1 is phosphorylated in SA medium on at least two residues, Tyr-359 and Ser-302 and/or Ser-306. Ser-302 and Ser-306 are part of a consensus site for the mammalian homolog of Rim11, glycogen synthase kinase 3-. Phosphorylation on Tyr-359 but not Ser-302 or Ser-306 is essential for the transcription of early meiosis-specific genes and sporulation. We show that Tyr-359 is phosphorylated by Rim11.
Genetics, Feb 20, 1976
A supposed sporulation-deficient mutation of Saccharomyces cerevisiae is found to affect mating i... more A supposed sporulation-deficient mutation of Saccharomyces cerevisiae is found to affect mating in haploids and in diploids, and to be inseparable from the mating-type locus by recombination. The mutation is regarded as a defective a allele and is designated a*. This is confirmed by its dominance relations in diploids, triploids, and tetraploids. Tetrad analysis of tetraploids and of their sporulating diploid progeny suggests the existence of an additional locus, RIME, which regulates sporulation in yeast strains that can mate. Thus the recessive homozygous constitution rme/rme enables the diploids a*/a, a/a*, and (u/a to go through meiosis. Haploids carrying rme show apparent premeiotic DNA replication in sporulation conditions. This new regulatory locus is linked to the centromere of the mating-type chromosome, and its two alleles, rme and RME, are found among standard laboratory strains. HE mating-type locus i n yeast regulates the alternation between the haplo-Tphase and the diplophase. Mating between two cells is accomplished only if they are of different mating types (a x a; aa x aa x e, etc). The ploidy of the cells does not play a role in mating (ROMAN and SANDS 1953). The meiotic process can start when the cell is heterozygous for the mating-type locus, i.e. a/a. Normal a/, diploids will accomplish meiosis and spore formation, while a/a disomic haploids will enter meiosis but will not form viable ascospores (ROTH and FOGEL 1971). One of the main approaches to the study of the function and structure of the mating-type locus is to obtain mutations which affect mating and/or meiosis. HAWTHORNE (1963) selected for a haploid that was able to mate with other haploids from the same mating type. He obtained a mutation from the a allele to the a allele. This mutation was associated with a recessive lethal deletion in the same region. MACKAY and MANNEY (1974a, b) selected for non-mating haploids. They characterized at least five loci. The loci were defined through complementation and genetic mapping, and the mutants differed in their ability to produce sex hormone, to respond to the hormone of the opposite mating type, to mate at low frequency and to sporulate. GERLACH (1974) obtained a recessive mutation that was unlinked to the mating-type locus and enabled aa or aa diploids to sporulate. HOPPER and HALL (1975) also selected for mutants that enabled aa or ea diploids to sporulate. In this way they could identify a mutation
Current Genetics, Mar 1, 1989
The yeast Saccharomyces cerevisiae is a simple unicellular eukaryotic organism that shares certai... more The yeast Saccharomyces cerevisiae is a simple unicellular eukaryotic organism that shares certain common features with higher organisms (plants and animals), namely the basic developmental processes of the cell the vegetative cell cycle and the meiotic process. Cell differentiation is initiated following both external and internal signals. Upon reaching the G1 stage, yeast cells probe the environment; if they find it rich in nutrients, they proceed into the mitotic cell cycle. Under starvation conditions, the cells remain arrested in G1 or initiate meiosis, depending on the cell type. Meiosis, like other developmental pathways, is initiated from G1.
Molecular and Cellular Biology, May 1, 1983
Molecular Genetics And Genomics, Nov 1, 1998
The S. cerevisiae CDC40 gene was originally identified as a cell-division-specific gene that is e... more The S. cerevisiae CDC40 gene was originally identified as a cell-division-specific gene that is essential only at elevated temperatures. Cells carrying mutations in this gene arrest with a large bud and a single nucleus with duplicated DNA content. Cdc40p is also required for spindle establishment or maintenance. Sequence analysis reveals that CDC40 is identical to PRP17, a gene involved in pre-mRNA splicing. In this paper, we show that Cdc40p is required at all temperatures for efficient entry into S-phase and that cell cycle arrest associated with cdc40 mutations is independent of all the known checkpoint mechanisms. Using immunofluorescence, we show that Cdc40p is localized to the nuclear membrane, weakly associated with the nuclear pore. Our results point to a link between cell cycle progression, pre-mRNA splicing, and mRNA export.
Developmental Genetics, 1995
The IME1 gene of Saccharomyces cerevisiae encodes a transcription factor that is required for the... more The IME1 gene of Saccharomyces cerevisiae encodes a transcription factor that is required for the expression of meiosis-specific genes. Like many of the genes it regulates, IME1 itself is expressed according to the following complex pattern: barely detectable levels during vegetative growth, and high induced levels under starvation conditions, followed by a subsequent decline in the course of meiosis. This report examines the influence of Ime1 protein on its own expression, demonstrating feedback regulation. Disruption of either IME1 or IME2 leads to constantly increasing levels of Ime1-lacZ expression, under meiotic conditions. This apparent negative regulation is due to cis elements in the IME1 upstream region, which confer transient meiotic expression to heterologous promoter-less genes. A specific DNA/protein complex, whose level is transiently increased under meiotic conditions, is detected on this element. In ime1- diploids, the level of this DNA/protein complex increases, without any decline. These results indicate that the transient expression of IME1 is apparently due to transcriptional regulation. This report also presents evidence suggesting that Ime1p is directly responsible for regulating its own transcription. Positive feedback regulation in mitotic conditions is suggested by the observation that overexpression of Ime1p leads to increased levels of IME1-lacZ. Negative autoregulation in meiotic cultures is demonstrated by the observation that a specific point mutation in IME1, ime1-3, permits expression of meiosis-specific genes, as well as induction of meiosis, but is defective in negative-feedback regulation of IME1.
Genetics, Dec 1, 2001
In the budding yeast Saccharomyces cerevisiae initiation and progression through the mitotic cell... more In the budding yeast Saccharomyces cerevisiae initiation and progression through the mitotic cell cycle are determined by the sequential activity of the cyclin-dependent kinase Cdc28. The role of this kinase in entry and progression through the meiotic cycle is unclear, since all cdc28 temperature-sensitive alleles are leaky for meiosis. We used a "heat-inducible Degron system" to construct a diploid strain homozygous for a temperature-degradable cdc28-deg allele. We show that this allele is nonleaky, giving no asci at the nonpermissive temperature. We also show, using this allele, that Cdc28 is not required for premeiotic DNA replication and commitment to meiotic recombination. IME2 encodes a meiosis-specific hCDK2 homolog that is required for the correct timing of premeiotic DNA replication, nuclear divisions, and asci formation. Moreover, in ime2⌬ diploids additional rounds of DNA replication and nuclear divisions are observed. We show that the delayed premeiotic DNA replication observed in ime2⌬ diploids depends on a functional Cdc28. Ime2⌬ cdc28-4 diploids arrest prior to initiation of premeiotic DNA replication and meiotic recombination. Ectopic overexpression of Clb1 at early meiotic times advances premeiotic DNA replication, meiotic recombination, and nuclear division, but the coupling between these events is lost. The role of Ime2 and Cdc28 in initiating the meiotic pathway is discussed.
Genetics, Sep 20, 1978
Vegetative cells carrying the new temperature-sensitive mutation cdc40 arrest at the restrictive ... more Vegetative cells carrying the new temperature-sensitive mutation cdc40 arrest at the restrictive temperature with a medial nuclear division phenotype. DNA replication is observed under these conditions, but most cells remain sensitive to hydroxyurea and do not complete the ongoing cell cycle if the drug is present during release from the temperature block. It is suggested that the cdc40 lesion affects an essential function in DNA synthesis. Normal meiosis is observed at the permissive temperature in cdc40 homozygotes. At the restrictive temperature, a full round of premeiotic DNA replication is observed, but neither commitment to recombination nor later meiotic events occur. Meiotic cells that are already committed to the recombination process at the permissive temperature do not complete it if transferred to the restrictive temperature before recombination is realized. These temperature shift-up experiments demonstrate that the CDC40 function is required for the completion of recombination events, as well as for the earlier stage of recombination commitment. Temperature shift-down experiments with cdc40 homozygotes suggest that meiotic segregation depends on the final events of recombination rather than on commitment to recombination.
Methods in Enzymology, 1991
Publisher Summary This chapter provides methods used for monitoring meiosis and sporulation in Sa... more Publisher Summary This chapter provides methods used for monitoring meiosis and sporulation in Saccharomyces cerevisiae . To study the effect of a mutation on meiosis, one must first determine the terminal phenotype at which the mutant arrests. When the mutation is conditional, one applies the restrictive conditions at the time of transfer to medium, or after 1–2 hr in SPM to allow the completion of the mitotic division. Important parameters to examine are whether the mutant cells are able to complete the premeiotic DNA replication, commitment to recombination, recombination, and commitment to haploidization. Electron microscopy can reveal= ultrastructures such as the spindle plaque body and the presence or absence of the synaptonemal complex. Analysis of ts mutations gives more information. Temperature shift-up experiments reveal whether the gene product is required more than once in meiosis as well as the time at which it is required. Shift-down experiments may give some clues as to the functions that do take place at the restrictive temperature and the reversibility of the specific meiotic defect.
Molecular and Cellular Biology, May 1, 1996
The transcription of meiosis-specific genes, as well as the initiation of meiosis, in the budding... more The transcription of meiosis-specific genes, as well as the initiation of meiosis, in the budding yeast Saccharomyces cerevisiae depends on IME1. IME1 encodes a transcriptional activator which lacks known DNA binding motifs. In this study we have determined the mode by which Ime1 specifically activates the transcription of meiotic genes. We demonstrate that Ime1 is recruited to the promoters of meiotic genes by interacting with a DNA-binding protein, Ume6. This association between Ime1 and Ume6 depends on both starvation and the activity of a protein kinase, encoded by RIM11. In the absence of Ime1, Ume6 represses the transcription of meiotic genes. However, in the presence of Ime1, or when Ume6 is fused in frame to the Gal4 activation domain, Ume6 is converted from a repressor to an activator, resulting in the transcription of meiosis-specific genes and the formation of asci.
International review of cytology, 2003
Initiation of meiosis in Saccharomyces cerevisiae is regulated by mating type and nutritional con... more Initiation of meiosis in Saccharomyces cerevisiae is regulated by mating type and nutritional conditions that restrict meiosis to diploid cells grown under starvation conditions. Specifically, meiosis occurs in MATa/MATalpha cells shifted to nitrogen depletion media in the absence of glucose and the presence of a nonfermentable carbon source. These conditions lead to the expression and activation of Ime 1, the master regulator of meiosis. IME1 encodes a transcriptional activator recruited to promoters of early meiosis-specific genes by association with the DNA-binding protein, Ume6. Under vegetative growth conditions these genes are silent due to recruitment of the Sin3/Rpd3 histone deacetylase and Isw2 chromatin remodeling complexes by Ume6. Transcription of these meiotic genes occurs following histone acetylation by Gcn5. Expression of the early genes promote entry into the meiotic cycle, as they include genes required for premeiotic DNA synthesis, synapsis of homologous chromosomes, and meiotic recombination. Two of the early meiosis specific genes, a transcriptional activator, Ndt80, and a CDK2 homologue, Ime2, are required for the transcription of middle meiosis-specific genes that are involved with nuclear division and spore formation. Spore maturation depends on late genes whose expression is indirectly dependent on Ime1, Ime2, and Ndt80. Finally, phosphorylation of Imel by Ime2 leads to its degradation, and consequently to shutting down of the meiotic transcriptional cascade. This review is focusing on the regulation of gene expression governing initiation and progression through meiosis.
Nucleic Acids Research, 1992
Cell, Mar 1, 1988
/ME1 (Inducer of MEiosis) was cloned due to its high copy number effect: it enabled MAT insuffici... more /ME1 (Inducer of MEiosis) was cloned due to its high copy number effect: it enabled MAT insufficient strains to undergo meiosis. Disruption of /ME1 results in a recessive Spa-phenotype. Diploids homozygous for the two mutations imel-0, rmel-7 are also meiosis deficient. We conclude that /ME1 is a positive regulator of meiosis that normally is repressed by RME7. RME1 is repressed by a complex of MATal and MATa gene products. /ME7 is also regulated by the environment: no transcripts could be detected in glucose growing cells, in contrast to acetate growing cells. Starvation for nitrogen further induced (6-to 8-fold) transcription of IME7, but, as expected, the induction was found only in MATaIMATu or rmel-llrmel-7 diploids. Furthermore, the /ME7 multicopy plasmids promoted sporulation in rich media.
BMC Research Notes, Aug 6, 2013
Background: Bench biologists often do not take part in the development of computational models fo... more Background: Bench biologists often do not take part in the development of computational models for their systems, and therefore, they frequently employ them as "black-boxes". Our aim was to construct and test a model that does not depend on the availability of quantitative data, and can be directly used without a need for intensive computational background. Results: We present a discrete transition model. We used cell-cycle in budding yeast as a paradigm for a complex network, demonstrating phenomena such as sequential protein expression and activity, and cell-cycle oscillation. The structure of the network was validated by its response to computational perturbations such as mutations, and its response to mating-pheromone or nitrogen depletion. The model has a strong predicative capability, demonstrating how the activity of a specific transcription factor, Hcm1, is regulated, and what determines commitment of cells to enter and complete the cell-cycle. Conclusion: The model presented herein is intuitive, yet is expressive enough to elucidate the intrinsic structure and qualitative behavior of large and complex regulatory networks. Moreover our model allowed us to examine multiple hypotheses in a simple and intuitive manner, giving rise to testable predictions. This methodology can be easily integrated as a useful approach for the study of networks, enriching experimental biology with computational insights.
Methods in molecular biology, 2017
We describe a simple computational approach that can be used for the study and simulation of regu... more We describe a simple computational approach that can be used for the study and simulation of regulatory networks. The advantage of this approach is that it requires neither computational background nor exact quantitative data about the biological system under study. Moreover, it is suitable for examining alternative hypotheses about the structure of a biological network. We used a tool called BioNSi (Biological Network Simulator) that is based on a simple computational model, which can be easily integrated as part of the lab routine, in parallel to experimental work. One benefit of this approach is that it enables the identification of regulatory proteins, which are missing from the experimental work. We describe the general methodology for modeling a network's dynamics in the tool, and then give a point by point example for a specific known network, entry into meiosis in budding yeast.
Current Drug Targets, Nov 1, 2006
Molecular Genetics And Genomics, Mar 1, 1993
The IME1 gene of Saccharomyces cerevisiae is required for initiation of meiosis. Transcription of... more The IME1 gene of Saccharomyces cerevisiae is required for initiation of meiosis. Transcription of IME1 is detected under conditions which are known to induce initiation of meiosis, namely starvation for nitrogen and glucose, and the presence of MA Tal and MA Te2 gene products. In this paper we show that IME1 is also subject to translational regulation. Translation of IME1 mRNA is achieved either upon nitrogen starvation, or upon G1 arrest. In the presence of nutrients, constitutively elevated transcription of IME1 is also sufficient for the translation of IME1 RNA. Four different conditions were found to cause expression of Imel protein in vegetative cultures: elevated transcription levels due to the presence of 1ME1 on a multicopy plasmid; elevated transcription provided by a Gal-IME1 construct; G1 arrest due to a-factor treatment; G1 arrest following mild heat-shock treatment of cdc28 diploids. Using these conditions, we obtained evidence that starvation is required not only for transcription and efficient translation of IME1, but also for either the activation of Imel protein or for the induction/activation of another factor that, either alone or in combination with Imel, induces meiosis.
Methods in molecular biology, 2017
The budding yeast Saccharomyces cerevisiae has a long history as a model organism for studies of ... more The budding yeast Saccharomyces cerevisiae has a long history as a model organism for studies of meiosis and the cell cycle. The popularity of this yeast as a model is in large part due to the variety of genetic and cytological approaches that can be effectively performed with the cells. Cultures of the cells can be induced to synchronously progress through meiosis and sporulation allowing large-scale gene expression and biochemical studies to be performed. Additionally, the spore tetrads resulting from meiosis make it possible to characterize the haploid products of meiosis allowing investigation of meiotic recombination and chromosome segregation. Here we describe genetic methods for analysis progression of S. cerevisiae through meiosis and sporulation with an emphasis on strategies for the genetic analysis of regulators of meiosis-specific genes.
PLOS ONE, Dec 17, 2013
In budding yeasts, the histone deacetylase Rpd3 resides in two different complexes called Rpd3L (... more In budding yeasts, the histone deacetylase Rpd3 resides in two different complexes called Rpd3L (large) and Rpd3S (small) that exert opposing effects on the transcription of meiosis-specific genes. By introducing mutations that disrupt the integrity and function of either Rpd3L or Rpd3S, we show here that Rpd3 function is determined by its association with either of these complexes. Specifically, the catalytic activity of Rpd3S activates the transcription of the two major positive regulators of meiosis, IME1 and IME2, under all growth conditions and activates the transcription of NDT80 only during vegetative growth. In contrast, the effects of Rpd3L depends on nutrients; it represses or activates transcription in the presence or absence of a nitrogen source, respectively. Further, we show that transcriptional activation does not correlate with histone H4 deacetylation, suggesting an effect on a nonhistone protein. Comparison of rpd3-null and catalytic-site point mutants revealed an inhibitory activity that is independent of either the catalytic activity of Rpd3 or the integrity of Rpd3L and Rpd3S.
Cell Cycle, Feb 15, 2009
Progression through the cell cycle depends on sequential activation of Cyclin-Dependent Kinase(s)... more Progression through the cell cycle depends on sequential activation of Cyclin-Dependent Kinase(s). In this report we use budding-yeast meiosis as a tool to elucidate the specific functions of mammalian Cdks. Yeast meiosis is regulated by both Cdc28 (yCdk1) and Ime2 (a meiosis-specific Cdk-like kinase). We show that human Cdk2 is a functional homolog for most of Ime2 functions. It promotes efficient and timely entry into premeiotic DNA replication and the first nuclear division, as well as the regulated transcription of IME1 and the early meiosis-specific genes. We show that this effect is specific, and that neither mice Cdk1, nor mice Cdk4 can suppress ime2. We show that Cdk1 is a functional homolog of Cdc28 that also suppresses one of its meiotic functions, namely inhibiting the transcription of IME1. Cdk2, on the other hand, show dominant negative effects on entry into the cell cycle, most probably by inhibiting the function of Cdc28. Finally, we show that in the meiotic pathway Cdk4 functions as a transcriptional activator.
Molecular and Cellular Biology, Aug 1, 2004
Phosphorylation is the main mode by which signals are transmitted to key regulators of developmen... more Phosphorylation is the main mode by which signals are transmitted to key regulators of developmental pathways. The glycogen synthase kinase 3 family plays pivotal roles in the development and well-being of all eukaryotic organisms. Similarly, the budding yeast homolog Rim11 is essential for the exit of diploid cells from the cell cycle and for entry into the meiotic developmental pathway. In this report we show that in vivo, in cells grown in a medium promoting vegetative growth with acetate as the sole carbon source (SA medium), Rim11 phosphorylates Ime1, the master transcriptional activator required for entry into the meiotic cycle and for the transcription of early meiosis-specific genes. We demonstrate that in the presence of glucose, the kinase activity of Rim11 is inhibited. This inhibition could be due to phosphorylation on Ser-5, Ser-8, and/or Ser-12 because in the rim11S5AS8AS12A mutant, Ime1 is incorrectly phosphorylated in the presence of glucose and cells undergo sporulation. We further show that this nutrient signal is transmitted to Rim11 and consequently to Ime1 by the cyclic AMP/protein kinase A signal transduction pathway. Ime1 is phosphorylated in SA medium on at least two residues, Tyr-359 and Ser-302 and/or Ser-306. Ser-302 and Ser-306 are part of a consensus site for the mammalian homolog of Rim11, glycogen synthase kinase 3-. Phosphorylation on Tyr-359 but not Ser-302 or Ser-306 is essential for the transcription of early meiosis-specific genes and sporulation. We show that Tyr-359 is phosphorylated by Rim11.
Genetics, Feb 20, 1976
A supposed sporulation-deficient mutation of Saccharomyces cerevisiae is found to affect mating i... more A supposed sporulation-deficient mutation of Saccharomyces cerevisiae is found to affect mating in haploids and in diploids, and to be inseparable from the mating-type locus by recombination. The mutation is regarded as a defective a allele and is designated a*. This is confirmed by its dominance relations in diploids, triploids, and tetraploids. Tetrad analysis of tetraploids and of their sporulating diploid progeny suggests the existence of an additional locus, RIME, which regulates sporulation in yeast strains that can mate. Thus the recessive homozygous constitution rme/rme enables the diploids a*/a, a/a*, and (u/a to go through meiosis. Haploids carrying rme show apparent premeiotic DNA replication in sporulation conditions. This new regulatory locus is linked to the centromere of the mating-type chromosome, and its two alleles, rme and RME, are found among standard laboratory strains. HE mating-type locus i n yeast regulates the alternation between the haplo-Tphase and the diplophase. Mating between two cells is accomplished only if they are of different mating types (a x a; aa x aa x e, etc). The ploidy of the cells does not play a role in mating (ROMAN and SANDS 1953). The meiotic process can start when the cell is heterozygous for the mating-type locus, i.e. a/a. Normal a/, diploids will accomplish meiosis and spore formation, while a/a disomic haploids will enter meiosis but will not form viable ascospores (ROTH and FOGEL 1971). One of the main approaches to the study of the function and structure of the mating-type locus is to obtain mutations which affect mating and/or meiosis. HAWTHORNE (1963) selected for a haploid that was able to mate with other haploids from the same mating type. He obtained a mutation from the a allele to the a allele. This mutation was associated with a recessive lethal deletion in the same region. MACKAY and MANNEY (1974a, b) selected for non-mating haploids. They characterized at least five loci. The loci were defined through complementation and genetic mapping, and the mutants differed in their ability to produce sex hormone, to respond to the hormone of the opposite mating type, to mate at low frequency and to sporulate. GERLACH (1974) obtained a recessive mutation that was unlinked to the mating-type locus and enabled aa or aa diploids to sporulate. HOPPER and HALL (1975) also selected for mutants that enabled aa or ea diploids to sporulate. In this way they could identify a mutation
Current Genetics, Mar 1, 1989
The yeast Saccharomyces cerevisiae is a simple unicellular eukaryotic organism that shares certai... more The yeast Saccharomyces cerevisiae is a simple unicellular eukaryotic organism that shares certain common features with higher organisms (plants and animals), namely the basic developmental processes of the cell the vegetative cell cycle and the meiotic process. Cell differentiation is initiated following both external and internal signals. Upon reaching the G1 stage, yeast cells probe the environment; if they find it rich in nutrients, they proceed into the mitotic cell cycle. Under starvation conditions, the cells remain arrested in G1 or initiate meiosis, depending on the cell type. Meiosis, like other developmental pathways, is initiated from G1.
Molecular and Cellular Biology, May 1, 1983
Molecular Genetics And Genomics, Nov 1, 1998
The S. cerevisiae CDC40 gene was originally identified as a cell-division-specific gene that is e... more The S. cerevisiae CDC40 gene was originally identified as a cell-division-specific gene that is essential only at elevated temperatures. Cells carrying mutations in this gene arrest with a large bud and a single nucleus with duplicated DNA content. Cdc40p is also required for spindle establishment or maintenance. Sequence analysis reveals that CDC40 is identical to PRP17, a gene involved in pre-mRNA splicing. In this paper, we show that Cdc40p is required at all temperatures for efficient entry into S-phase and that cell cycle arrest associated with cdc40 mutations is independent of all the known checkpoint mechanisms. Using immunofluorescence, we show that Cdc40p is localized to the nuclear membrane, weakly associated with the nuclear pore. Our results point to a link between cell cycle progression, pre-mRNA splicing, and mRNA export.
Developmental Genetics, 1995
The IME1 gene of Saccharomyces cerevisiae encodes a transcription factor that is required for the... more The IME1 gene of Saccharomyces cerevisiae encodes a transcription factor that is required for the expression of meiosis-specific genes. Like many of the genes it regulates, IME1 itself is expressed according to the following complex pattern: barely detectable levels during vegetative growth, and high induced levels under starvation conditions, followed by a subsequent decline in the course of meiosis. This report examines the influence of Ime1 protein on its own expression, demonstrating feedback regulation. Disruption of either IME1 or IME2 leads to constantly increasing levels of Ime1-lacZ expression, under meiotic conditions. This apparent negative regulation is due to cis elements in the IME1 upstream region, which confer transient meiotic expression to heterologous promoter-less genes. A specific DNA/protein complex, whose level is transiently increased under meiotic conditions, is detected on this element. In ime1- diploids, the level of this DNA/protein complex increases, without any decline. These results indicate that the transient expression of IME1 is apparently due to transcriptional regulation. This report also presents evidence suggesting that Ime1p is directly responsible for regulating its own transcription. Positive feedback regulation in mitotic conditions is suggested by the observation that overexpression of Ime1p leads to increased levels of IME1-lacZ. Negative autoregulation in meiotic cultures is demonstrated by the observation that a specific point mutation in IME1, ime1-3, permits expression of meiosis-specific genes, as well as induction of meiosis, but is defective in negative-feedback regulation of IME1.
Genetics, Dec 1, 2001
In the budding yeast Saccharomyces cerevisiae initiation and progression through the mitotic cell... more In the budding yeast Saccharomyces cerevisiae initiation and progression through the mitotic cell cycle are determined by the sequential activity of the cyclin-dependent kinase Cdc28. The role of this kinase in entry and progression through the meiotic cycle is unclear, since all cdc28 temperature-sensitive alleles are leaky for meiosis. We used a "heat-inducible Degron system" to construct a diploid strain homozygous for a temperature-degradable cdc28-deg allele. We show that this allele is nonleaky, giving no asci at the nonpermissive temperature. We also show, using this allele, that Cdc28 is not required for premeiotic DNA replication and commitment to meiotic recombination. IME2 encodes a meiosis-specific hCDK2 homolog that is required for the correct timing of premeiotic DNA replication, nuclear divisions, and asci formation. Moreover, in ime2⌬ diploids additional rounds of DNA replication and nuclear divisions are observed. We show that the delayed premeiotic DNA replication observed in ime2⌬ diploids depends on a functional Cdc28. Ime2⌬ cdc28-4 diploids arrest prior to initiation of premeiotic DNA replication and meiotic recombination. Ectopic overexpression of Clb1 at early meiotic times advances premeiotic DNA replication, meiotic recombination, and nuclear division, but the coupling between these events is lost. The role of Ime2 and Cdc28 in initiating the meiotic pathway is discussed.
Genetics, Sep 20, 1978
Vegetative cells carrying the new temperature-sensitive mutation cdc40 arrest at the restrictive ... more Vegetative cells carrying the new temperature-sensitive mutation cdc40 arrest at the restrictive temperature with a medial nuclear division phenotype. DNA replication is observed under these conditions, but most cells remain sensitive to hydroxyurea and do not complete the ongoing cell cycle if the drug is present during release from the temperature block. It is suggested that the cdc40 lesion affects an essential function in DNA synthesis. Normal meiosis is observed at the permissive temperature in cdc40 homozygotes. At the restrictive temperature, a full round of premeiotic DNA replication is observed, but neither commitment to recombination nor later meiotic events occur. Meiotic cells that are already committed to the recombination process at the permissive temperature do not complete it if transferred to the restrictive temperature before recombination is realized. These temperature shift-up experiments demonstrate that the CDC40 function is required for the completion of recombination events, as well as for the earlier stage of recombination commitment. Temperature shift-down experiments with cdc40 homozygotes suggest that meiotic segregation depends on the final events of recombination rather than on commitment to recombination.
Methods in Enzymology, 1991
Publisher Summary This chapter provides methods used for monitoring meiosis and sporulation in Sa... more Publisher Summary This chapter provides methods used for monitoring meiosis and sporulation in Saccharomyces cerevisiae . To study the effect of a mutation on meiosis, one must first determine the terminal phenotype at which the mutant arrests. When the mutation is conditional, one applies the restrictive conditions at the time of transfer to medium, or after 1–2 hr in SPM to allow the completion of the mitotic division. Important parameters to examine are whether the mutant cells are able to complete the premeiotic DNA replication, commitment to recombination, recombination, and commitment to haploidization. Electron microscopy can reveal= ultrastructures such as the spindle plaque body and the presence or absence of the synaptonemal complex. Analysis of ts mutations gives more information. Temperature shift-up experiments reveal whether the gene product is required more than once in meiosis as well as the time at which it is required. Shift-down experiments may give some clues as to the functions that do take place at the restrictive temperature and the reversibility of the specific meiotic defect.
Molecular and Cellular Biology, May 1, 1996
The transcription of meiosis-specific genes, as well as the initiation of meiosis, in the budding... more The transcription of meiosis-specific genes, as well as the initiation of meiosis, in the budding yeast Saccharomyces cerevisiae depends on IME1. IME1 encodes a transcriptional activator which lacks known DNA binding motifs. In this study we have determined the mode by which Ime1 specifically activates the transcription of meiotic genes. We demonstrate that Ime1 is recruited to the promoters of meiotic genes by interacting with a DNA-binding protein, Ume6. This association between Ime1 and Ume6 depends on both starvation and the activity of a protein kinase, encoded by RIM11. In the absence of Ime1, Ume6 represses the transcription of meiotic genes. However, in the presence of Ime1, or when Ume6 is fused in frame to the Gal4 activation domain, Ume6 is converted from a repressor to an activator, resulting in the transcription of meiosis-specific genes and the formation of asci.
International review of cytology, 2003
Initiation of meiosis in Saccharomyces cerevisiae is regulated by mating type and nutritional con... more Initiation of meiosis in Saccharomyces cerevisiae is regulated by mating type and nutritional conditions that restrict meiosis to diploid cells grown under starvation conditions. Specifically, meiosis occurs in MATa/MATalpha cells shifted to nitrogen depletion media in the absence of glucose and the presence of a nonfermentable carbon source. These conditions lead to the expression and activation of Ime 1, the master regulator of meiosis. IME1 encodes a transcriptional activator recruited to promoters of early meiosis-specific genes by association with the DNA-binding protein, Ume6. Under vegetative growth conditions these genes are silent due to recruitment of the Sin3/Rpd3 histone deacetylase and Isw2 chromatin remodeling complexes by Ume6. Transcription of these meiotic genes occurs following histone acetylation by Gcn5. Expression of the early genes promote entry into the meiotic cycle, as they include genes required for premeiotic DNA synthesis, synapsis of homologous chromosomes, and meiotic recombination. Two of the early meiosis specific genes, a transcriptional activator, Ndt80, and a CDK2 homologue, Ime2, are required for the transcription of middle meiosis-specific genes that are involved with nuclear division and spore formation. Spore maturation depends on late genes whose expression is indirectly dependent on Ime1, Ime2, and Ndt80. Finally, phosphorylation of Imel by Ime2 leads to its degradation, and consequently to shutting down of the meiotic transcriptional cascade. This review is focusing on the regulation of gene expression governing initiation and progression through meiosis.
Nucleic Acids Research, 1992
Cell, Mar 1, 1988
/ME1 (Inducer of MEiosis) was cloned due to its high copy number effect: it enabled MAT insuffici... more /ME1 (Inducer of MEiosis) was cloned due to its high copy number effect: it enabled MAT insufficient strains to undergo meiosis. Disruption of /ME1 results in a recessive Spa-phenotype. Diploids homozygous for the two mutations imel-0, rmel-7 are also meiosis deficient. We conclude that /ME1 is a positive regulator of meiosis that normally is repressed by RME7. RME1 is repressed by a complex of MATal and MATa gene products. /ME7 is also regulated by the environment: no transcripts could be detected in glucose growing cells, in contrast to acetate growing cells. Starvation for nitrogen further induced (6-to 8-fold) transcription of IME7, but, as expected, the induction was found only in MATaIMATu or rmel-llrmel-7 diploids. Furthermore, the /ME7 multicopy plasmids promoted sporulation in rich media.
BMC Research Notes, Aug 6, 2013
Background: Bench biologists often do not take part in the development of computational models fo... more Background: Bench biologists often do not take part in the development of computational models for their systems, and therefore, they frequently employ them as "black-boxes". Our aim was to construct and test a model that does not depend on the availability of quantitative data, and can be directly used without a need for intensive computational background. Results: We present a discrete transition model. We used cell-cycle in budding yeast as a paradigm for a complex network, demonstrating phenomena such as sequential protein expression and activity, and cell-cycle oscillation. The structure of the network was validated by its response to computational perturbations such as mutations, and its response to mating-pheromone or nitrogen depletion. The model has a strong predicative capability, demonstrating how the activity of a specific transcription factor, Hcm1, is regulated, and what determines commitment of cells to enter and complete the cell-cycle. Conclusion: The model presented herein is intuitive, yet is expressive enough to elucidate the intrinsic structure and qualitative behavior of large and complex regulatory networks. Moreover our model allowed us to examine multiple hypotheses in a simple and intuitive manner, giving rise to testable predictions. This methodology can be easily integrated as a useful approach for the study of networks, enriching experimental biology with computational insights.