Isolation of conditional mutations using hypersensitivity to formamide in Schizosaccharomyces pombe

Motivation: Conditional mutations are very powerful tools to study the function of the majority of genes in single-cell eukaryots. High or low temperature are the most commonly used conditions so far. However, some other options such as drug sensitivity, have been proved useful strategies to find new alleles that might not be isolated otherwise. Thus we have searched for new cell cycle mutantions that confer sensitivity to Formamide (HCONH2). This is a small molecule, which presumably enters the cells and is not metabolized. This molecule is widely used as an ionizing solvent in vitro that destabilizes non-covalent bonds. In this study, we have used these properties in vivo to search for mutations in the fission yeast Schizosaccharomyces pombe that lead specifically to chromosome segregation defects in the presence of this molecule.Methods: We performed a traditional mutagenesis using four different mutagens and searched for formamide sensitive mutants (fsm), as a novel conditional phenotype in S. pombe. The sensitivity of fsm strains to other conditions was also tested in a qualitative drop assay. The initial characterization of these mutants was made with a chromatin and cell wall staining. In addition, we have also screened for sensitivity to formamide in a genome-wide S. pombe non-essential gene deletion library . Results: We have isolated 72 conditional mutations that show differential or no sensitivity to other traditional conditions used. Several of these mutants display morphological alterations, cell cycle defects and genomic instability. We have also described that there is a clear enrichment of RNA metabolism deleted genes that are hypersensitive to formamide. In order to confirm these result, we tested sensitivity to formamide of a selected group of splicing mutants (prp: pre-mRNA processing, a key process in RNA metabolism). In agreement with our prediction, all prp mutant strains are hyper-sensitive to formamide.Conclusions: Viable S. pombe deletions which are sensitive to formamide have a special predominance of RNA metabolism related genes. Consistently, all prp alleles tested are hypersensitive to formamide. In vivo effect of formamide can efficiently be used as a condition for the search of new alleles of interest with a special focus on RNA related processes that may have escaped other screenings in fission yeast.

Centrosomal MPF triggers the mitotic and morphogenetic switches of fission yeast

PMCID: PMC3549529.-- et al.Activation of mitosis-promoting factor (MPF) drives mitotic commitment. In human cells active MPF appears first on centrosomes. We show that local activation of MPF on the equivalent organelle of fission yeast, the spindle pole body (SPB), promotes Polo kinase activity at the SPBs long before global MPF activation drives mitotic commitment. Artificially promoting MPF or Polo activity at various locations revealed that this local control of Plo1 activity on G2 phase SPBs dictates the timing of mitotic commitment. Cytokinesis of the rod-shaped fission yeast cell generates a naive, new, cell end. Growth is restricted to the experienced old end until a point in G2 phase called new end take off (NETO) when bipolar growth is triggered. NETO coincided with MPF activation of Plo1 on G2 phase SPBs (ref. 4). Both MPF and Polo activities were required for NETO and both induced NETO when ectopically activated at interphase SPBs. NETO promotion by MPF required polo. Thus, local MPF activation on G2 SPBs directs polo kinase to control at least two distinct and temporally separated, cell-cycle transitions at remote locations.This work was supported by Cancer Research UK (CRUK) grant number C147/A6058. V.S. and A.K. were supported by the Swiss National Science Foundation and EPFL.Peer reviewe

Extending the Schizosaccharomyces pombe molecular genetic toolbox

This is an open-access article distributed under the terms of the Creative Commons Attribution License.-- et al.Targeted alteration of the genome lies at the heart of the exploitation of S. pombe as a model system. The rate of analysis is often determined by the efficiency with which a target locus can be manipulated. For most loci this is not a problem, however for some loci, such as fin1+, rates of gene targeting below 5% can limit the scope and scale of manipulations that are feasible within a reasonable time frame. We now describe a simple modification of transformation procedure for directing integration of genomic sequences that leads to a 5-fold increase in the transformation efficiency when antibiotic based dominant selection markers are used. We also show that removal of the pku70+ and pku80+ genes, which encode DNA end binding proteins required for the non-homologous end joining DNA repair pathway, increases the efficiency of gene targeting at fin1+ to around 75-80% (a 16-fold increase). We describe how a natMX6/rpl42+ cassette can be used for positive and negative selection for integration at a targeted locus. To facilitate the evaluation of the impact of a series of mutations on the function of a gene of interest we have generated three vector series that rely upon different selectable markers to direct the expression of tagged/untagged molecules from distinct genomic integration sites. pINTL and pINTK vectors use ura4+ selection to direct disruptive integration of leu1+ and lys1+ respectively, while pINTH vectors exploit nourseothricin resistance to detect the targeted disruption of a hygromycin B resistance conferring hphMX6 cassette that has been integrated on chromosome III. Finally, we have generated a series of multi-copy expression vectors that use resistance to nourseothricin or kanamycin/G418 to select for propagation in prototrophic hosts. Collectively these protocol modifications and vectors extend the versatility of this key model system.Swiss National Science Foundation (www.snf.ch). École polytechnique fédérale de Lausanne (www.epfl.ch). Cancer Research UK [CRUK] C147/A6058.Peer Reviewe

R-loops and regulatory changes in chronologically ageing fission yeast cells drive non-random patterns of genome rearrangements

Aberrant repair of DNA double-strand breaks can recombine distant chromosomal breakpoints. Chromosomal rearrangements compromise genome function and are a hallmark of ageing. Rearrangements are challenging to detect in non-dividing cell populations, because they reflect individually rare, heterogeneous events. The genomic distribution of de novo rearrangements in non-dividing cells, and their dynamics during ageing, remain therefore poorly characterized. Studies of genomic instability during ageing have focussed on mitochondrial DNA, small genetic variants, or proliferating cells. To characterize genome rearrangements during cellular ageing in non-dividing cells, we interrogated a single diagnostic measure, DNA breakpoint junctions, using Schizosaccharomyces pombe as a model system. Aberrant DNA junctions that accumulated with age were associated with microhomology sequences and R-loops. Global hotspots for age-associated breakpoint formation were evident near telomeric genes and linked to remote breakpoints elsewhere in the genome, including the mitochondrial chromosome. Formation of breakpoint junctions at global hotspots was inhibited by the Sir2 histone deacetylase and might be triggered by an age-dependent de-repression of chromatin silencing. An unexpected mechanism of genomic instability may cause more local hotspots: age-associated reduction in an RNA-binding protein triggering R-loops at target loci. This result suggests that biological processes other than transcription or replication can drive genome rearrangements. Notably, we detected similar signatures of genome rearrangements that accumulated in old brain cells of humans. These findings provide insights into the unique patterns and possible mechanisms of genome rearrangements in non-dividing cells, which can be promoted by ageing-related changes in gene-regulatory proteins

Isolation of a new spliceosome mutant with cell cycle defects by a novel conditional screening in S. pombe

Resumen del póster presentado al 7th International Fission Yeast Meeting (EMBO Conference), celebrado en Londres (UK) del 24 al 29 de junio de 2013.Conditional screenings have so far identified hundreds of mutants in many biological processes that allow the study of loss-of-function under the restrictive condition, but also the propagation and genetic crosses of these strains under the permissive condition. Although drug sensitivity mutants have been isolated, the most spread condition used is temperature. We have searched for formamide sensitive mutants, a novel conditionality in S. pombe, that display cell cycle defects. Among others, we have identified one mutation in the conserved spliceosome component cwf15, that allows growth in normal conditions but it blocks cell cycle progression in the presence of formamide. This mutation (cwf15.32Fs) consists of 13 nucleotides direct duplication that generates a premature stop codon and thus a C-terminal truncated protein. Cwf15 (Complexed with cdc5 protein 15) is an essential and evolutionary conserved splicing factor from yeast to humans. It has been shown to physically interact to other spliceosome components but no further characterization has been done. Here we characterize cwf15.32Fs cell cycle defects and test for genetic interactions with other splicing mutants.Peer Reviewe

Gene dosis and the timing of mitosis

Cell Cycle News & ViewsIn all organisms, cell size is crucial for biological function and is tightly controlled by genetic, environmental and physiological factors. Cell-size homeostasis is mediated by a finely balanced coordination between cell growth and division. At a critical point in the cell cycle, the G2-M transition, cells stop growth and enter mitosis; advancing or delaying this transition leads to smaller or larger cells, respectivelyPeer Reviewe

Controlling mitotic commitment from the G2 spindle pole

Resumen del trabajo presentado al XXXVII Congreso de la Sociedad Española de Genética, celebrado en Torremolinos (Málaga) del 29 de septiembre al 2 de octubre de 2009.The activation of MPF to promote mitotic commitment is regulated by the co-ordination of the signals from a number of signalling cascades. MPF is held in check in interphase cells by phosphorylation in the ATP binding site of the catalytic, Cdk1, subunit. This phosphate is then removed by a phosphatase called Cdc25 to activate the Cdk1 kinase and promote entrance into mitosis. The essential spindle pole body (SPB) component Cut12 appears to play a key role in the activation of MPF in fission yeast. The cut12.1 loss of function mutation leads to insertion defects of the new SPB and blocks spindle formation while the gain of function mutation cut12.s11 enable cells to live without the otherwise essential phosphatase Cdc25. Increasing Cdc25 levels suppresses the conditional lethality and the spindle formation defect of cut12.1, indicating that these cells are unable to form a spindle because of a defect in MPF activation. Thus, this SPB component seems to play a critical role in regulating entrance to mitosis. We think that this function is fulfilled by alteration of the activity of the polo kinase Plo1. Polo has been implicated in a positive feedback loop that boosts Cdc25 and inhibits Wee1 during mitotic entry in a variety of systems. We found that boosting Plo1 activity independently of any change in Cut12 also suppresses cdc25.22 defects. Furthermore the gain of function cut12.s11 mutation boosts global and SPB associated Plo1 activity and the loss of function mutant cut12.1 reduces both. Importantly, at 30°C Plo1 is recruited to the SPB in G2 100 mins before commitment to mitosis. The cdc25 bypassing cut12.s11 mutation promotes this recruitment 25 minutes earlier. Fluorescence Recovery After Photobleaching (FRAP) experiments demonstrate that this premature recruitment is not due to turnover increase. Live imaging experiments with analogue sensitive cdc2.as mutant establish that recruitment of Plo1 to the G2 SPB relies upon the activity of MPF itself. Thus from a point in G2 there is a futile cycle that involves MPF activity on the SPB.Peer reviewe

Aurora B kinase erases monopolar microtubule-kinetochore arrays at the meiosis I-II transition

Summary: During meiosis, faithful chromosome segregation requires monopolar spindle microtubule-kinetochore arrays in MI to segregate homologous chromosomes, but bipolar in MII to segregate sister chromatids. Using fission yeasts, we found that the universal Aurora B kinase localizes to kinetochores in metaphase I and in the mid-spindle during anaphase I, as in mitosis; but in the absence of an intervening S phase, the importin α Imp1 propitiates its release from the spindle midzone to re-localize at kinetochores during meiotic interkinesis. We show that “error-correction” activity of kinetochore re-localized Aurora B becomes essential to erase monopolar arrangements from anaphase I, a prerequisite to satisfy the spindle assembly checkpoint (SAC) and to generate proper bipolar arrays at the onset of MII. This microtubule-kinetochore resetting activity of Aurora B at the MI-MII transition is required to prevent chromosome missegregation in meiosis II, a type of error often associated with birth defects and infertility in humans

Hsp 90 interaction with Cdc2 and Plo1 kinases contributes to actomyosin ring condensation in fission yeast

In Schizosaccharomyces pombe, cytokinesis occurs by ordered recruitment of actomyosin components at the division site, followed by lateral condensation to produce a ring-like structure early in anaphase, which eventually matures and contracts at the end of mitosis. We found that in temperature-sensitive hsp 90-w1 mutant cells, encoding an Hsp 90 mutant protein, ring components were recruited to form a cortical network at the division site, but this network failed to condense into a compact ring, suggesting a role for Hsp 90 in this particular step. hsp 90-w1 mutant shows strong genetic interaction with specific mutant alleles of the fission yeast cdc2, such as cdc2-33. Interestingly, actomyosin ring defects in hsp 90-w1 cdc2-33 mutant cells resembled that of hsp 90-w1 single mutant at restrictive temperature. Noteworthy, similar genetic interaction was found with a mutant allele of polo-like kinase, plo1-ts4, suggesting that Hsp 90 collaborates with Cdc2 and Plo1 cell cycle kinases to condense medial ring components. In vitro analyses suggested that Cdc2 and Plo1 physically interact with Hsp 90. Association of Cdc2 to Hsp 90 was ATP independent, while Plo1 binds to this chaperone in an ATP-dependent manner, indicating that these two kinases interact with different Hsp 90 complexes. Overall, our analyses of hsp 90-w1 reveal a possible role for this chaperone in medial ring condensation in association with Cdc2 and Plo1 kinases.This work was supported by grants from the Ministerio de Ciencia e Innovación of the Spanish Government (BFU2009-13565).Peer Reviewe

Genome-wide search of Schizosaccharomyces pombe genes causing overexpression-mediated cell cycle defects

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 (Pena, 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 (pjk1). 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 ivision. The effect caused by some of them is described in more detail.This work was supported by grants from the Spanish Ministerio de Educación y Cultura (BMC2000-0124) and the EC (BIO4-CT96-0159). C.P. was awarded a Fellowship from the Spanish Ministerio de Educacióon y Ciencia.Peer Reviewe