Functional Characterization of the Fission Yeast Phosphatidylserine Synthase Gene, pps1, Reveals Novel Cellular Functions for Phosphatidylserine (original) (raw)
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Functional Characterization of Fission Yeast Transcription Factors by Overexpression Analysis
Genetics, 2013
In Schizosaccharomyces pombe, over 90% of transcription factor genes are nonessential. Moreover, the majority do not exhibit significant growth defects under optimal conditions when deleted, complicating their functional characterization and target gene identification. Here, we systematically overexpressed 99 transcription factor genes with the nmt1 promoter and found that 64 transcription factor genes exhibited reduced fitness when ectopically expressed. Cell cycle defects were also often observed. We further investigated three uncharacterized transcription factor genes (toe1 + -toe3 + ) that displayed cell elongation when overexpressed. Ectopic expression of toe1 + resulted in a G1 delay while toe2 + and toe3 + overexpression produced an accumulation of septated cells with abnormalities in septum formation and nuclear segregation, respectively. Transcriptome profiling and ChIP-chip analysis of the transcription factor overexpression strains indicated that Toe1 activates target genes of the pyrimidine-salvage pathway, while Toe3 regulates target genes involved in polyamine synthesis. We also found that ectopic expression of the putative target genes SPBC3H7.05c, and dad5 + and SPAC11D3.06 could recapitulate the cell cycle phenotypes of toe2 + and toe3 + overexpression, respectively. Furthermore, single deletions of the putative target genes urg2 + and SPAC1399.04c, and SPBC3H7.05c, SPACUNK4.15, and rds1 + , could suppress the phenotypes of toe1 + and toe2 + overexpression, respectively. This study implicates new transcription factors and metabolism genes in cell cycle regulation and demonstrates the potential of systematic overexpression analysis to elucidate the function and target genes of transcription factors in S. pombe. Available freely online through the author-supported open access option. Supporting information is available online at http://www.genetics.org/lookup/suppl/
We previously determined the copy number limits of overexpression for cell division cycle (cdc) regulatory genes in the fission yeast Schizosaccharomyces pombe using the ''genetic tug-of-war'' (gTOW) method. In this study, we measured the levels of tandem affinity purification (TAP)-tagged target proteins when their copy numbers are increased in gTOW. Twenty analyzed genes showed roughly linear correlations between increased protein levels and gene copy numbers, which suggested a general lack of compensation for gene dosage in S. pombe. Cdc16 and Sid2 protein levels but not their mRNA levels were much lower than that expected by their copy numbers, which suggested the existence of a post-transcriptional down regulation of these genes. The cyclin Cig1 protein level and its mRNA level were much higher than that expected by its copy numbers, which suggested a positive feedback mechanism for its expression. A higher Cdc10 protein level and its mRNA level, probably due to cloning its gene into a plasmid, indicated that Cdc10 regulation was more robust than that previously predicted.
Yeast, 2002
Genetic studies in yeasts enable an in vivo analysis of gene functions required for the cell division cycle (cdc genes) in eukaryotes. In order to characterize new functions involved in cell cycle regulation, we searched for genes causing cell division defects by overexpression in the fission yeast Schizosaccharomyces pombe. By using this dominant genetic strategy, 26 independent clones were isolated from a Sz. pombe cDNA library. The cloned cDNAs were partially sequenced and identified by computer analysis. The 26 clones isolated corresponded to 21 different genes. Among them, six were genes previously characterized in Sz. pombe, 11 were homologues to genes identified and characterized in other organisms, and four represented genes with unknown functions. In addition to known cell cycle regulators encoding inhibitory protein kinases (wee1, pka1 ) and DNA checkpoint proteins (Pcna, rad24 ), we have identified genes that are involved in a number of cellular processes. This includes protein synthesis (ribosomal proteins L7, L10, L29, L41, S6, S11, S17 and the PolyA-Binding Protein PABP), protein degradation (UBI3 ), nucleolar rRNA expression (fib, imp1, dbp2 ), cell cytoskeleton (act1 ) and glycolysis (pfk1 ). The interference caused in the cell cycle by overexpression of these genes may elucidate novel mechanisms coupling different cellular processes with the control of the cell division. The effect caused by some of them is described in more detail.
Transcriptional Regulation in the Yeast Life Cycle
Science, 1987
120. I dedicate this article to Viktor Hamburger, who promoted and took part in this search, and to whom I am forever indebted for invaluable suggestions and generosity. Without him, the nerve growth factor would never have come to our attention. To my dear friends, Pietro Calissano and Luigi Aloe, I wish to express my deepest gratitude for their fundamental contributions. In this 35-year-long investigation, a large number of colleagues, research associates, and graduate students took part in this scientific adventure. I am particularly indebted and I very gratefully acknowledge the most important work performed by two ofthem: Drs.
Genetics, 2007
Cdc25 phosphatase primes entry to mitosis by removing the inhibitory phosphate that is transferred to mitosis promoting factor (MPF) by Wee1 related kinases. A positive feedback loop then boosts Cdc25 and represses Wee1 activities to drive full-scale MPF activation and commitment to mitosis. Dominant mutations in the Schizosaccharomyces pombe spindle pole body (SPB) component Cut12 enable cdc25.22 mutants to overcome a G2 arrest at 36° and enter mitosis. The recessive temperature-sensitive cut12.1 mutation results in the formation of monopolar spindles in which the spindle pole marker Sad1 is enriched on the nonfunctional SPB at 36°. We identified mutations at five loci that suppressed the lethality of the recessive cut12.1 mutation at 36° and conferred lethality at 20°. Three of the five mutations led to the formation of monopolar spindles at restrictive temperatures, affected cell size at commitment to mitosis, and generated multiple Sad1 foci at nuclear periphery. The five loci, ...
Identification of Cell Cycle-regulated Genes in Fission Yeast
Molecular Biology of the Cell, 2005
Cell cycle progression is both regulated and accompanied by periodic changes in the expression levels of a large number of genes. To investigate cell cycle-regulated transcriptional programs in the fission yeast Schizosaccharomyces pombe, we developed a whole-genome oligonucleotide-based DNA microarray. Microarray analysis of both wild-type and cdc25 mutant cell cultures was performed to identify transcripts whose levels oscillated during the cell cycle. Using an unsupervised algorithm, we identified 747 genes that met the criteria for cell cycle-regulated expression. Peaks of gene expression were found to be distributed throughout the entire cell cycle. Furthermore, we found that four promoter motifs exhibited strong association with cell cycle phase-specific expression. Examination of the regulation of MCB motif-containing genes through the perturbation of DNA synthesis control/MCB-binding factor (DSC/MBF)-mediated transcription in arrested synchronous cdc10 mutant cell cultures r...
Journal of Biological Chemistry, 1993
The mammalian transcription factor E2F binds to several cellular proteins including Rb, p107, cyclin A, cyclin E, and p33cdk2 protein kinase in a stage-specific manner during cell cycle. Its recognition sequence, TTTCGCGC, is present in two of the human adenovirns early promoters and in several promoters of cellular genes whose products are implicated in the control of cell proliferation. These observations suggest that E2F may play an important role in cell-cycle regulation and prompted us to ask whether E2F-like activities are present in yeast. We found that the E2F motif can function as an activating sequence in Schizosaccharomyces pombe when cloned upstream of a reporter gene. Consistent with this, the expression of adenovirus E2 promoter in S. pombe was dependent on both E2F motifs of this promoter. A protein, spESF, that binds to the E2F site was partially purified from S. pombe using DNA-affinity chromatography. The binding specificity of this protein was compared to that of human E2F using a number of mutant E2F sites as competitors. These studies showed that spE2F recognizes a sequence closely related to the E2F site. Ultraviolet cross-linking and Southwestern blot studies indicated that the molecular size of spE2F is 30 kDa. Previous studies have shown that a cis-acting element, ACGCGTNA, also called M u 1 cell cycle box, or MCB, is critical for the regulated expression of cell cycle related genes both in fission and budding yeast. In S. pombe, the cdclO gene product binds to this element and controls the cell cycle related genes. Electrophoretic mobility shift assays and molecular size determination studies indicated that spE2F is different from that encoded by cdcl0. Thus, our studies suggest that spE2F is a novel transcription factor. We discuss these results in light of recent observations about the periodically expressed genes involved in the cell cycle progression in yeast.
Quantitative Analysis of a Dynamic Cell Cycle Regulatory Model of Schizosaccharomyces pombe
Current Synthetic and Systems Biology, 2013
Cell cycle is the central process that regulates growth and division in all eukaryotes. Based on the environmental condition sensed, the cell lies in a resting phase G0 or proceeds through the cyclic cell division process (G1->S->G2->M). These series of events and the irreversible phase transitions are governed mainly by the highly conserved Cyclin dependent kinases (Cdks) and its positive and negative regulators which results in a highly interconnected network. The dynamics of the cell cycle regulation is due to this underlying complex network that governs this process. In in silico models it is the parameter set that directly reflects the characteristics of the system. Synthesis rate constants indirectly represent the source of complexity. Therefore, a recently developed model for fission yeast Schizosaccharomyces pombe cell cycle regulation was utilized to investigate the influence of synthesis level regulation on the overall cell cycle period. A systematic local and the global perturbation of sixteen synthesis rate constants of the model were performed to study the synthesis level influence of these regulators on (i) viability, (ii) cell cycle period and (iii) robustness. The results of sensitivity analysis indicates that the cell cycle time is robust to perturbation in the synthesis rate constant of single regulators but fragile to simultaneous perturbation of the multiple regulators. In addition, a perspective on emergence of robustness with respect to multiple layers of complex regulators over a fragile core network is demonstrated based on a systematic regulator deletion and addition analysis. Some of the key predictions that emerge from this study includes, that (i) seven regulatory components Slp1, Cdc2, Cdc13, PP1, APC, and Cdc25 along with Mik1 or Wee1 are sufficient to drive cell cycle regulation. This can be verified by designing appropriate synthetic biology experiments; (ii) either one of the G2 regulatory kinases Wee1 or Mik1 could have emerged through whole chromosome duplication events during evolution which can be tested experimentally to arrive with a conclusive proof.
Periodic gene expression program of the fission yeast cell cycle
Nature Genetics, 2004
Cell-cycle control of transcription seems to be universal, but little is known about its global conservation and biological significance. We report on the genome-wide transcriptional program of the Schizosaccharomyces pombe cell cycle, identifying 407 periodically expressed genes of which 136 show high-amplitude changes. These genes cluster in four major waves of expression. The forkhead protein Sep1p regulates mitotic genes in the first cluster, including Ace2p, which activates transcription in the second cluster during the M-G1 transition and cytokinesis. Other genes in the second cluster, which are required for G1-S progression, are regulated by the MBF complex independently of Sep1p and Ace2p. The third cluster coincides with S phase and a fourth cluster contains genes weakly regulated during G2 phase. Despite conserved cell-cycle transcription factors, differences in regulatory circuits between fission and budding yeasts are evident, revealing evolutionary plasticity of transcriptional control. Periodic transcription of most genes is not conserved between the two yeasts, except for a core set of ∼40 genes that seem to be universally regulated during the eukaryotic cell cycle and may have key roles in cell-cycle progression.
Current Genetics, 1991
Cell division cycle mutants defective in G 1, DNA replication or nuclear division were tested for sporulation at semi-restrictive temperatures. In cdcl-7, cdc5-120, cdcl7-L16 and cdc18-46 no abnormalities were observed; cdc10-129, cdc20-MlO, cdc21-M6B, cdc23-M36 and cdc24-M38 formed four-spored asci but with a low efficiency; cdc22-M45 was completely defective in meiosis, but could conjugate and formed zygotes with a single nucleus. Mutants defective in the mitotic initiation genes cdc2, cdc25 and cdcl3 were blocked in meiosis II. None of the weel-50, adh.niml + and winl + alleles had any affect on sporulation, suggesting that their interactions with cdc25 and cdc2 are specific to mitosis. The meiotic function of cdcl3 is TBZ-sensitive and probably exerted downstream of cdc2. Single mutants in cutl or cut2 did not effect sporulation, whereas the double mutant cutl cut2 formed two-spored asci. The results demonstrate that the cell division cycle and the meiotic developmental pathway share common genes and regulatory cascades.