WorldWideScience

Sample records for budding yeast chromosomes

  1. Recruiting a microtubule-binding complex to DNA directs chromosome segregation in budding yeast

    OpenAIRE

    Murray, Andrew W.; Lacefield, Soni; Lau, Tsz Cham Derek

    2009-01-01

    Accurate chromosome segregation depends on the kinetochore, the complex of proteins that link microtubules to centromeric DNA1. The budding yeast kinetochore consists of more than 80 proteins assembled on a 125bp region of DNA1. We studied the assembly and function of kinetochore components by fusing individual kinetochore proteins to the lactose repressor (LacI) and testing their ability to improve the segregation of a plasmid carrying tandem repeats of the lactose operator (LacO). Targeting...

  2. Chromosome Conformation Capture Carbon Copy (5C) in Budding Yeast.

    Science.gov (United States)

    Belton, Jon-Matthew; Dekker, Job

    2015-06-01

    Chromosome conformation capture carbon copy (5C) is a high-throughput method for detecting ligation products of interest in a chromosome conformation capture (3C) library. 5C uses ligation-mediated amplification (LMA) to generate carbon copies of 3C ligation product junctions using single-stranded oligonucleotide probes. This procedure produces a 5C library of short DNA molecules which represent the interactions between the corresponding restriction fragments. The 5C library can be amplified using universal primers containing the Illumina paired-end adaptor sequences for subsequent high-throughput sequencing.

  3. Spindle checkpoint proteins and chromosome-microtubule attachment in budding yeast.

    Science.gov (United States)

    Gillett, Emily S; Espelin, Christopher W; Sorger, Peter K

    2004-02-16

    Accurate chromosome segregation depends on precise regulation of mitosis by the spindle checkpoint. This checkpoint monitors the status of kinetochore-microtubule attachment and delays the metaphase to anaphase transition until all kinetochores have formed stable bipolar connections to the mitotic spindle. Components of the spindle checkpoint include the mitotic arrest defective (MAD) genes MAD1-3, and the budding uninhibited by benzimidazole (BUB) genes BUB1 and BUB3. In animal cells, all known spindle checkpoint proteins are recruited to kinetochores during normal mitoses. In contrast, we show that whereas Saccharomyces cerevisiae Bub1p and Bub3p are bound to kinetochores early in mitosis as part of the normal cell cycle, Mad1p and Mad2p are kinetochore bound only in the presence of spindle damage or kinetochore lesions that interfere with chromosome-microtubule attachment. Moreover, although Mad1p and Mad2p perform essential mitotic functions during every division cycle in mammalian cells, they are required in budding yeast only when mitosis goes awry. We propose that differences in the behavior of spindle checkpoint proteins in animal cells and budding yeast result primarily from evolutionary divergence in spindle assembly pathways. Copyright The Rockefeller University Press

  4. Ontogeny of Unstable Chromosomes Generated by Telomere Error in Budding Yeast

    Science.gov (United States)

    Weinert, Ted

    2016-01-01

    DNA replication errors at certain sites in the genome initiate chromosome instability that ultimately leads to stable genomic rearrangements. Where instability begins is often unclear. And, early instability may form unstable chromosome intermediates whose transient nature also hinders mechanistic understanding. We report here a budding yeast model that reveals the genetic ontogeny of genome rearrangements, from initial replication error to unstable chromosome formation to their resolution. Remarkably, the initial error often arises in or near the telomere, and frequently forms unstable chromosomes. Early unstable chromosomes may then resolve to an internal "collection site" where a dicentric forms and resolves to an isochromosome (other outcomes are possible at each step). The initial telomere-proximal unstable chromosome is increased in mutants in telomerase subunits, Tel1, and even Rad9, with no known telomere-specific function. Defects in Tel1 and in Rrm3, a checkpoint protein kinase with a role in telomere maintenance and a DNA helicase, respectively, synergize dramatically to generate unstable chromosomes, further illustrating the consequence of replication error in the telomere. Collectively, our results suggest telomeric replication errors may be a common cause of seemingly unrelated genomic rearrangements located hundreds of kilobases away. PMID:27716774

  5. Ontogeny of Unstable Chromosomes Generated by Telomere Error in Budding Yeast.

    Science.gov (United States)

    Beyer, Tracey; Weinert, Ted

    2016-10-01

    DNA replication errors at certain sites in the genome initiate chromosome instability that ultimately leads to stable genomic rearrangements. Where instability begins is often unclear. And, early instability may form unstable chromosome intermediates whose transient nature also hinders mechanistic understanding. We report here a budding yeast model that reveals the genetic ontogeny of genome rearrangements, from initial replication error to unstable chromosome formation to their resolution. Remarkably, the initial error often arises in or near the telomere, and frequently forms unstable chromosomes. Early unstable chromosomes may then resolve to an internal "collection site" where a dicentric forms and resolves to an isochromosome (other outcomes are possible at each step). The initial telomere-proximal unstable chromosome is increased in mutants in telomerase subunits, Tel1, and even Rad9, with no known telomere-specific function. Defects in Tel1 and in Rrm3, a checkpoint protein kinase with a role in telomere maintenance and a DNA helicase, respectively, synergize dramatically to generate unstable chromosomes, further illustrating the consequence of replication error in the telomere. Collectively, our results suggest telomeric replication errors may be a common cause of seemingly unrelated genomic rearrangements located hundreds of kilobases away.

  6. Dicentric chromosome stretching during anaphase reveals roles of Sir2/Ku in chromatin compaction in budding yeast.

    Science.gov (United States)

    Thrower, D A; Bloom, K

    2001-09-01

    We have used mitotic spindle forces to examine the role of Sir2 and Ku in chromatin compaction. Escherichia coli lac operator DNA was placed between two centromeres on a conditional dicentric chromosome in budding yeast cells and made visible by expression of a lac repressor-green fluorescent fusion protein. Centromeres on the same chromatid of a dicentric chromosome attach to opposite poles approximately 50% of the time, resulting in chromosome bridges during anaphase. In cells deleted for yKU70, yKU80, or SIR2, a 10-kb region of the dicentric chromosome stretched along the spindle axis to a length of 6 microm during anaphase. On spindle disassembly, stretched chromatin recoiled to the bud neck and was partitioned to mother and daughter cells after cytokinesis and cell separation. Chromatin immunoprecipitation revealed that Sir2 localizes to the lacO region in response to activation of the dicentric chromosome. These findings indicate that Ku and Sir proteins are required for proper chromatin compaction within regions of a chromosome experiencing tension or DNA damage. The association of Sir2 with the affected region suggests a direct role in this process, which may include the formation of heterochromatic DNA.

  7. Kinetochore function and chromosome segregation rely on critical residues in histones H3 and H4 in budding yeast.

    Science.gov (United States)

    Ng, Tessie M; Lenstra, Tineke L; Duggan, Nicole; Jiang, Shuangying; Ceto, Steven; Holstege, Frank C P; Dai, Junbiao; Boeke, Jef D; Biggins, Sue

    2013-11-01

    Accurate chromosome segregation requires that sister kinetochores biorient and attach to microtubules from opposite poles. Kinetochore biorientation relies on the underlying centromeric chromatin, which provides a platform to assemble the kinetochore and to recruit the regulatory factors that ensure the high fidelity of this process. To identify the centromeric chromatin determinants that contribute to chromosome segregation, we performed two complementary unbiased genetic screens using a library of budding yeast mutants in every residue of histone H3 and H4. In one screen, we identified mutants that lead to increased loss of a nonessential chromosome. In the second screen, we isolated mutants whose viability depends on a key regulator of biorientation, the Aurora B protein kinase. Nine mutants were common to both screens and exhibited kinetochore biorientation defects. Four of the mutants map near the unstructured nucleosome entry site, and their genetic interaction with reduced IPL1 can be suppressed by increasing the dosage of SGO1, a key regulator of biorientation. In addition, the composition of purified kinetochores was altered in six of the mutants. Together, this work identifies previously unknown histone residues involved in chromosome segregation and lays the foundation for future studies on the role of the underlying chromatin structure in chromosome segregation.

  8. Three Different Pathways Prevent Chromosome Segregation in the Presence of DNA Damage or Replication Stress in Budding Yeast.

    Directory of Open Access Journals (Sweden)

    Gloria Palou

    2015-09-01

    Full Text Available A surveillance mechanism, the S phase checkpoint, blocks progression into mitosis in response to DNA damage and replication stress. Segregation of damaged or incompletely replicated chromosomes results in genomic instability. In humans, the S phase checkpoint has been shown to constitute an anti-cancer barrier. Inhibition of mitotic cyclin dependent kinase (M-CDK activity by Wee1 kinases is critical to block mitosis in some organisms. However, such mechanism is dispensable in the response to genotoxic stress in the model eukaryotic organism Saccharomyces cerevisiae. We show here that the Wee1 ortholog Swe1 does indeed inhibit M-CDK activity and chromosome segregation in response to genotoxic insults. Swe1 dispensability in budding yeast is the result of a redundant control of M-CDK activity by the checkpoint kinase Rad53. In addition, our results indicate that Swe1 is an effector of the checkpoint central kinase Mec1. When checkpoint control on M-CDK and on Pds1/securin stabilization are abrogated, cells undergo aberrant chromosome segregation.

  9. Spatial organization of the budding yeast genome in the cell nucleus and identification of specific chromatin interactions from multi-chromosome constrained chromatin model

    Science.gov (United States)

    Gürsoy, Gamze; Xu, Yun

    2017-01-01

    Nuclear landmarks and biochemical factors play important roles in the organization of the yeast genome. The interaction pattern of budding yeast as measured from genome-wide 3C studies are largely recapitulated by model polymer genomes subject to landmark constraints. However, the origin of inter-chromosomal interactions, specific roles of individual landmarks, and the roles of biochemical factors in yeast genome organization remain unclear. Here we describe a multi-chromosome constrained self-avoiding chromatin model (mC-SAC) to gain understanding of the budding yeast genome organization. With significantly improved sampling of genome structures, both intra- and inter-chromosomal interaction patterns from genome-wide 3C studies are accurately captured in our model at higher resolution than previous studies. We show that nuclear confinement is a key determinant of the intra-chromosomal interactions, and centromere tethering is responsible for the inter-chromosomal interactions. In addition, important genomic elements such as fragile sites and tRNA genes are found to be clustered spatially, largely due to centromere tethering. We uncovered previously unknown interactions that were not captured by genome-wide 3C studies, which are found to be enriched with tRNA genes, RNAPIII and TFIIS binding. Moreover, we identified specific high-frequency genome-wide 3C interactions that are unaccounted for by polymer effects under landmark constraints. These interactions are enriched with important genes and likely play biological roles. PMID:28704374

  10. The role of replication bypass pathways in dicentric chromosome formation in budding yeast.

    Science.gov (United States)

    Paek, Andrew L; Jones, Hope; Kaochar, Salma; Weinert, Ted

    2010-12-01

    Gross chromosomal rearrangements (GCRs) are large scale changes to chromosome structure and can lead to human disease. We previously showed in Saccharomyces cerevisiae that nearby inverted repeat sequences (∼20-200 bp of homology, separated by ∼1-5 kb) frequently fuse to form unstable dicentric and acentric chromosomes. Here we analyzed inverted repeat fusion in mutants of three sets of genes. First, we show that genes in the error-free postreplication repair (PRR) pathway prevent fusion of inverted repeats, while genes in the translesion branch have no detectable role. Second, we found that siz1 mutants, which are defective for Srs2 recruitment to replication forks, and srs2 mutants had opposite effects on instability. This may reflect separate roles for Srs2 in different phases of the cell cycle. Third, we provide evidence for a faulty template switch model by studying mutants of DNA polymerases; defects in DNA pol delta (lagging strand polymerase) and Mgs1 (a pol delta interacting protein) lead to a defect in fusion events as well as allelic recombination. Pol delta and Mgs1 may collaborate either in strand annealing and/or DNA replication involved in fusion and allelic recombination events. Fourth, by studying genes implicated in suppression of GCRs in other studies, we found that inverted repeat fusion has a profile of genetic regulation distinct from these other major forms of GCR formation.

  11. Condensin suppresses recombination and regulates double-strand break processing at the repetitive ribosomal DNA array to ensure proper chromosome segregation during meiosis in budding yeast

    Science.gov (United States)

    Li, Ping; Jin, Hui; Yu, Hong-Guo

    2014-01-01

    During meiosis, homologues are linked by crossover, which is required for bipolar chromosome orientation before chromosome segregation at anaphase I. The repetitive ribosomal DNA (rDNA) array, however, undergoes little or no meiotic recombination. Hyperrecombination can cause chromosome missegregation and rDNA copy number instability. We report here that condensin, a conserved protein complex required for chromosome organization, regulates double-strand break (DSB) formation and repair at the rDNA gene cluster during meiosis in budding yeast. Condensin is highly enriched at the rDNA region during prophase I, released at the prophase I/metaphase I transition, and reassociates with rDNA before anaphase I onset. We show that condensin plays a dual role in maintaining rDNA stability: it suppresses the formation of Spo11-mediated rDNA breaks, and it promotes DSB processing to ensure proper chromosome segregation. Condensin is unnecessary for the export of rDNA breaks outside the nucleolus but required for timely repair of meiotic DSBs. Our work reveals that condensin coordinates meiotic recombination with chromosome segregation at the repetitive rDNA sequence, thereby maintaining genome integrity. PMID:25103240

  12. Fusion of nearby inverted repeats by a replication-based mechanism leads to formation of dicentric and acentric chromosomes that cause genome instability in budding yeast.

    Science.gov (United States)

    Paek, Andrew L; Kaochar, Salma; Jones, Hope; Elezaby, Aly; Shanks, Lisa; Weinert, Ted

    2009-12-15

    Large-scale changes (gross chromosomal rearrangements [GCRs]) are common in genomes, and are often associated with pathological disorders. We report here that a specific pair of nearby inverted repeats in budding yeast fuse to form a dicentric chromosome intermediate, which then rearranges to form a translocation and other GCRs. We next show that fusion of nearby inverted repeats is general; we found that many nearby inverted repeats that are present in the yeast genome also fuse, as does a pair of synthetically constructed inverted repeats. Fusion occurs between inverted repeats that are separated by several kilobases of DNA and share >20 base pairs of homology. Finally, we show that fusion of inverted repeats, surprisingly, does not require genes involved in double-strand break (DSB) repair or genes involved in other repeat recombination events. We therefore propose that fusion may occur by a DSB-independent, DNA replication-based mechanism (which we term "faulty template switching"). Fusion of nearby inverted repeats to form dicentrics may be a major cause of instability in yeast and in other organisms.

  13. Measuring mitotic spindle dynamics in budding yeast

    Science.gov (United States)

    Plumb, Kemp

    In order to carry out its life cycle and produce viable progeny through cell division, a cell must successfully coordinate and execute a number of complex processes with high fidelity, in an environment dominated by thermal noise. One important example of such a process is the assembly and positioning of the mitotic spindle prior to chromosome segregation. The mitotic spindle is a modular structure composed of two spindle pole bodies, separated in space and spanned by filamentous proteins called microtubules, along which the genetic material of the cell is held. The spindle is responsible for alignment and subsequent segregation of chromosomes into two equal parts; proper spindle positioning and timing ensure that genetic material is appropriately divided amongst mother and daughter cells. In this thesis, I describe fluorescence confocal microscopy and automated image analysis algorithms, which I have used to observe and analyze the real space dynamics of the mitotic spindle in budding yeast. The software can locate structures in three spatial dimensions and track their movement in time. By selecting fluorescent proteins which specifically label the spindle poles and cell periphery, mitotic spindle dynamics have been measured in a coordinate system relevant to the cell division. I describe how I have characterised the accuracy and precision of the algorithms by simulating fluorescence data for both spindle poles and the budding yeast cell surface. In this thesis I also describe the construction of a microfluidic apparatus that allows for the measurement of long time-scale dynamics of individual cells and the development of a cell population. The tools developed in this thesis work will facilitate in-depth quantitative analysis of the non-equilibrium processes in living cells.

  14. How to halve ploidy: lessons from budding yeast meiosis.

    Science.gov (United States)

    Kerr, Gary William; Sarkar, Sourav; Arumugam, Prakash

    2012-09-01

    Maintenance of ploidy in sexually reproducing organisms requires a specialized form of cell division called meiosis that generates genetically diverse haploid gametes from diploid germ cells. Meiotic cells halve their ploidy by undergoing two rounds of nuclear division (meiosis I and II) after a single round of DNA replication. Research in Saccharomyces cerevisiae (budding yeast) has shown that four major deviations from the mitotic cell cycle during meiosis are essential for halving ploidy. The deviations are (1) formation of a link between homologous chromosomes by crossover, (2) monopolar attachment of sister kinetochores during meiosis I, (3) protection of centromeric cohesion during meiosis I, and (4) suppression of DNA replication following exit from meiosis I. In this review we present the current understanding of the above four processes in budding yeast and examine the possible conservation of molecular mechanisms from yeast to humans.

  15. Sociobiology of the budding yeast

    Indian Academy of Sciences (India)

    Dominika M Wloch-Salamon

    2014-04-01

    Social theory has provided a useful framework for research with microorganisms. Here I describe the advantages and possible risks of using a well-known model organism, the unicellular yeast Saccharomyces cerevisiae, for sociobiological research. I discuss the problems connected with clear classification of yeast behaviour based on the fitness-based Hamilton paradigm. Relevant traits include different types of communities, production of flocculins, invertase and toxins, and the presence of apoptosis.

  16. Bipolar budding in yeasts - an electron microscope study

    NARCIS (Netherlands)

    Kreger-van Rij, N.J.W.; Veenhuis, M.

    1971-01-01

    Bud formation in yeasts with bipolar budding was studied by electron microscopy of thin sections. Budding in yeasts of the species Saccharomycodes ludwigii, Hanseniaspora valbyensis and Wickerhamia fluorescens resulted in concentric rings of scar ridges on the wall of the mother cell. The wall

  17. Bipolar budding in yeasts - an electron microscope study

    NARCIS (Netherlands)

    Kreger-van Rij, N.J.W.; Veenhuis, M.

    1971-01-01

    Bud formation in yeasts with bipolar budding was studied by electron microscopy of thin sections. Budding in yeasts of the species Saccharomycodes ludwigii, Hanseniaspora valbyensis and Wickerhamia fluorescens resulted in concentric rings of scar ridges on the wall of the mother cell. The wall betwe

  18. Taxonomy Icon Data: Budding yeast [Taxonomy Icon

    Lifescience Database Archive (English)

    Full Text Available Budding yeast Saccharomyces cerevisiae Saccharomyces_cerevisiae_L.png Saccharomyces..._cerevisiae_NL.png Saccharomyces_cerevisiae_S.png Saccharomyces_cerevisiae_NS.png http://biosciencedbc.jp/taxonomy..._icon/icon.cgi?i=Saccharomyces+cerevisiae&t=L http://biosciencedbc.jp/taxonomy_icon/icon.cgi?i=Saccharomy...ces+cerevisiae&t=NL http://biosciencedbc.jp/taxonomy_icon/icon.cgi?i=Saccharomy...ces+cerevisiae&t=S http://biosciencedbc.jp/taxonomy_icon/icon.cgi?i=Saccharomyces+cerevisiae&t=NS http://togodb.biosciencedbc.jp/togodb/view/taxonomy_icon_comment_en?species_id=216 ...

  19. DNA Replication Forks Pause at Silent Origins near the HML Locus in Budding Yeast

    OpenAIRE

    Wang, Yangzhou; Vujcic, Marija; Kowalski, David

    2001-01-01

    Chromosomal replicators in budding yeast contain an autonomously replicating sequence (ARS) that functions in a plasmid, but certain ARSs are silent as replication origins in their natural chromosomal context. In chromosome III, the HML ARS cluster (ARS302-ARS303-ARS320) and ARS301 flank the transcriptionally silent mating-type locus HML, and all of these ARSs are silent as replication origins. ARS301 and ARS302 function in transcriptional silencing mediated by the origin recognition complex ...

  20. Electrochemical regulation of budding yeast polarity.

    Directory of Open Access Journals (Sweden)

    Armin Haupt

    2014-12-01

    Full Text Available Cells are naturally surrounded by organized electrical signals in the form of local ion fluxes, membrane potential, and electric fields (EFs at their surface. Although the contribution of electrochemical elements to cell polarity and migration is beginning to be appreciated, underlying mechanisms are not known. Here we show that an exogenous EF can orient cell polarization in budding yeast (Saccharomyces cerevisiae cells, directing the growth of mating projections towards sites of hyperpolarized membrane potential, while directing bud emergence in the opposite direction, towards sites of depolarized potential. Using an optogenetic approach, we demonstrate that a local change in membrane potential triggered by light is sufficient to direct cell polarization. Screens for mutants with altered EF responses identify genes involved in transducing electrochemical signals to the polarity machinery. Membrane potential, which is regulated by the potassium transporter Trk1p, is required for polarity orientation during mating and EF response. Membrane potential may regulate membrane charges through negatively charged phosphatidylserines (PSs, which act to position the Cdc42p-based polarity machinery. These studies thus define an electrochemical pathway that directs the orientation of cell polarization.

  1. Tolerance of budding yeast Saccharomyces cerevisiae to ultra high pressure

    Science.gov (United States)

    Shibata, M.; Torigoe, M.; Matsumoto, Y.; Yamamoto, M.; Takizawa, N.; Hada, Y.; Mori, Y.; Takarabe, K.; Ono, F.

    2014-05-01

    Our studies on the tolerance of plants and animals against very high pressure of several GPa have been extended to a smaller sized fungus, the budding yeast Saccharomyces cerevisiae. Several pieces of budding yeast (dry yeast) were sealed in a small teflon capsule with a liquid pressure medium fluorinate, and exposed to 7.5 GPa by using a cubic anvil press. The pressure was kept constant for various duration of time from 2 to 24 h. After the pressure was released, the specimens were brought out from the teflon capsule, and they were cultivated on a potato dextrose agar. It was found that the budding yeast exposed to 7.5 GPa for up to 6 h showed multiplication. However, those exposed to 7.5 GPa for longer than 12 h were found dead. The high pressure tolerance of budding yeast is a little weaker than that of tardigrades.

  2. Characterization of Septin Ultrastructure in Budding Yeast Using Electron Tomography

    Science.gov (United States)

    Bertin, Aurélie; Nogales, Eva

    2015-01-01

    Summary Septins are essential for the completion of cytokinesis. In budding yeast, Saccharomyces cerevisiae, septins are located at the bud neck during mitosis and are closely connected to the inner plasma membrane. In vitro, yeast septins have been shown to self-assemble into a variety of filamentous structures, including rods, paired filaments, bundles and rings [1–3]. Using electron tomography of freeze-substituted section and cryo-electron tomography of frozen sections, we determined the three dimensional organization of the septin cytoskeleton in dividing budding yeast with molecular resolution [4,5]. Here we describe the detailed procedures used for our characterization of the septin cellular ultrastructure. PMID:26519309

  3. Mechanical feedback stabilizes budding yeast morphogenesis

    Science.gov (United States)

    Banavar, Samhita; Trogdon, Michael; Petzold, Linda; Campas, Otger

    Walled cells have the ability to remodel their shape while sustaining an internal turgor pressure that can reach values up to 10 atmospheres. This requires a tight and simultaneous regulation of cell wall assembly and mechanochemistry, but the underlying mechanisms by which this is achieved remain unclear. Using the growth of mating projections in budding yeast (S. cerevisiae) as a motivating example, we have developed a theoretical description that couples the mechanics of cell wall expansion and assembly via a mechanical feedback. In the absence of a mechanical feedback, cell morphogenesis is inherently unstable. The presence of a mechanical feedback stabilizes changes in cell shape and growth, and provides a mechanism to prevent cell lysis in a wide range of conditions. We solve for the dynamics of the system and obtain the different dynamical regimes. In particular, we show that several parameters affect the stability of growth, including the strength of mechanical feedback in the system. Finally, we compare our results to existing experimental data.

  4. Regulation of homologous recombination at telomeres in budding yeast

    DEFF Research Database (Denmark)

    Eckert-Boulet, Nadine; Lisby, Michael

    2010-01-01

    Homologous recombination is suppressed at normal length telomere sequences. In contrast, telomere recombination is allowed when telomeres erode in the absence of telomerase activity or as a consequence of nucleolytic degradation or incomplete replication. Here, we review the mechanisms...... that contribute to regulating mitotic homologous recombination at telomeres and the role of these mechanisms in signalling short telomeres in the budding yeast Saccharomyces cerevisiae....

  5. Apoptosis at inflection point in liquid culture of budding yeasts.

    Directory of Open Access Journals (Sweden)

    Toshiyuki Hagiwara

    Full Text Available Budding yeasts are highly suitable for aging studies, because the number of bud scars (stage proportionally correlates with age. Its maximum stages are known to reach at 20-30 stages on an isolated agar medium. However, their stage dynamics in a liquid culture is virtually unknown. We investigate the population dynamics by counting scars in each cell. Here one cell division produces one new cell and one bud scar. This simple rule leads to a conservation law: "The total number of bud scars is equal to the total number of cells." We find a large discrepancy: extremely fewer cells with over 5 scars than expected. Almost all cells with 6 or more scars disappear within a short period of time in the late log phase (corresponds to the inflection point. This discrepancy is confirmed directly by the microscopic observations of broken cells. This finding implies apoptosis in older cells (6 scars or more.

  6. Hi-C in Budding Yeast.

    Science.gov (United States)

    Belton, Jon-Matthew; Dekker, Job

    2015-07-01

    Hi-C enables simultaneous detection of interaction frequencies between all possible pairs of restriction fragments in the genome. The Hi-C method is based on chromosome conformation capture (3C), which uses formaldehyde cross-linking to fix chromatin regions that interact in three-dimensional space, irrespective of their genomic locations. In the Hi-C protocol described here, cross-linked chromatin is digested with HindIII and the ends are filled in with a nucleotide mix containing biotinylated dCTP. These fragments are ligated together, and the resulting chimeric molecules are purified and sheared to reduce length. Finally, biotinylated ligation junctions are pulled down with streptavidin-coated beads, linked to high-throughput sequencing adaptors, and amplified via polymerase chain reaction (PCR). The resolution of the Hi-C data set will depend on the depth of sequencing and choice of restriction enzyme. When sufficient sequence reads are obtained, information on chromatin interactions and chromosome conformation can be derived at single restriction fragment resolution for complete genomes.

  7. The Malleable Nature of the Budding Yeast Nuclear Envelope: Flares, Fusion, and Fenestrations.

    Science.gov (United States)

    Meseroll, Rebecca A; Cohen-Fix, Orna

    2016-11-01

    In eukaryotes, the nuclear envelope (NE) physically separates nuclear components and activities from rest of the cell. The NE also provides rigidity to the nucleus and contributes to chromosome organization. At the same time, the NE is highly dynamic; it must change shape and rearrange its components during development and throughout the cell cycle, and its morphology can be altered in response to mutation and disease. Here we focus on the NE of budding yeast, Saccharomyces cerevisiae, which has several unique features: it remains intact throughout the cell cycle, expands symmetrically during interphase, elongates during mitosis and, expands asymmetrically during mitotic delay. Moreover, its NE is safely breached during mating and when large structures, such as nuclear pore complexes and the spindle pole body, are embedded into its double membrane. The budding yeast NE lacks lamins and yet the nucleus is capable of maintaining a spherical shape throughout interphase. Despite these eccentricities, studies of the budding yeast NE have uncovered interesting, and likely conserved, processes that contribute to NE dynamics. In particular, we discuss the processes that drive and enable NE expansion and the dramatic changes in the NE that lead to extensions and fenestrations. J. Cell. Physiol. 231: 2353-2360, 2016. Published 2016. This article is a U.S. Government work and is in the public domain in the USA.

  8. Assembly of eukaryotic algal chromosomes in yeast

    OpenAIRE

    Karas, Bogumil J.; Molparia, Bhuvan; Jablanovic, Jelena; Hermann, Wolfgang J; Lin, Ying-Chi; Dupont, Christopher L.; Tagwerker, Christian; Yonemoto, Isaac T.; Noskov, Vladimir N.; Chuang, Ray-Yuan; Allen, Andrew E; Glass, John I.; Hutchison, Clyde A; Smith, Hamilton O; Venter, J Craig

    2013-01-01

    Background Synthetic genomic approaches offer unique opportunities to use powerful yeast and Escherichia coli genetic systems to assemble and modify chromosome-sized molecules before returning the modified DNA to the target host. For example, the entire 1 Mb Mycoplasma mycoides chromosome can be stably maintained and manipulated in yeast before being transplanted back into recipient cells. We have previously demonstrated that cloning in yeast of large (> ~ 150 kb), high G + C (55%) prokaryoti...

  9. Dynamical Analysis of Protein Regulatory Network in Budding Yeast Nucleus

    Institute of Scientific and Technical Information of China (English)

    LI Fang-Ting; JIA Xun

    2006-01-01

    @@ Recent progresses in the protein regulatory network of budding yeast Saccharomyces cerevisiae have provided a global picture of its protein network for further dynamical research. We simplify and modularize the protein regulatory networks in yeast nucleus, and study the dynamical properties of the core 37-node network by a Boolean network model, especially the evolution steps and final fixed points. Our simulation results show that the number of fixed points N(k) for a given size of the attraction basin k obeys a power-law distribution N(k)∝k-2.024. The yeast network is more similar to a scale-free network than a random network in the above dynamical properties.

  10. The coordination of centromere replication, spindle formation, and kinetochore-microtubule interaction in budding yeast.

    Directory of Open Access Journals (Sweden)

    Hong Liu

    2008-11-01

    Full Text Available The kinetochore is a protein complex that assembles on centromeric DNA to mediate chromosome-microtubule interaction. Most eukaryotic cells form the spindle and establish kinetochore-microtubule interaction during mitosis, but budding yeast cells finish these processes in S-phase. It has long been noticed that the S-phase spindle in budding yeast is shorter than that in metaphase, but the biological significance of this short S-phase spindle structure remains unclear. We addressed this issue by using ask1-3, a temperature-sensitive kinetochore mutant that exhibits partially elongated spindles at permissive temperature in the presence of hydroxyurea (HU, a DNA synthesis inhibitor. After exposure to and removal of HU, ask1-3 cells show a delayed anaphase entry. This delay depends on the spindle checkpoint, which monitors kinetochore-microtubule interaction defects. Overproduction of microtubule-associated protein Ase1 or Cin8 also induces spindle elongation in HU-arrested cells. The spindle checkpoint-dependent anaphase entry delay is also observed after ASE1 or CIN8 overexpression in HU-arrested cells. Therefore, the shorter spindle in S-phase cells is likely to facilitate proper chromosome-microtubule interaction.

  11. EdU Incorporation for FACS and Microscopy Analysis of DNA Replication in Budding Yeast.

    Science.gov (United States)

    Talarek, Nicolas; Petit, Julie; Gueydon, Elisabeth; Schwob, Etienne

    2015-01-01

    DNA replication is a key determinant of chromosome segregation and stability in eukaryotes. The yeast Saccharomyces cerevisiae has been extensively used for cell cycle studies, yet simple but key parameters such as the fraction of cells in S phase in a population or the subnuclear localization of DNA synthesis have been difficult to gather for this organism. 5-ethynyl-2'-deoxyuridine (EdU) is a thymidine analogue that can be incorporated in vivo and later detected using copper-catalyzed azide alkyne cycloaddition (Click reaction) without prior DNA denaturation. This chapter describes a budding yeast strain and conditions that allow rapid EdU incorporation at moderate extracellular concentrations, followed by its efficient detection for the analysis of DNA replication in single cells by flow cytometry and fluorescence microscopy.

  12. MEN, destruction and separation: mechanistic links between mitotic exit and cytokinesis in budding yeast.

    Science.gov (United States)

    Yeong, Foong May; Lim, Hong Hwa; Surana, Uttam

    2002-07-01

    Cellular events must be executed in a certain sequence during the cell division in order to maintain genome integrity and hence ensure a cell's survival. In M phase, for instance, chromosome segregation always precedes mitotic exit (characterized by mitotic kinase inactivation via cyclin destruction); this is then followed by cytokinesis. How do cells impose this strict order? Recent findings in budding yeast have suggested a mechanism whereby partitioning of chromosomes into the daughter cell is a prerequisite for the activation of mitotic exit network (MEN). So far, however, a regulatory scheme that would temporally link the initiation of cytokinesis to the execution of mitotic exit has not been determined. We propose that the requirement of MEN components for cytokinesis, their translocation to the mother-daughter neck and triggering of this translocation by inactivation of the mitotic kinase may be the three crucial elements that render initiation of cytokinesis dependent on mitotic exit.

  13. Signal transduction by MAP kinase cascades in budding yeast.

    Science.gov (United States)

    Posas, F; Takekawa, M; Saito, H

    1998-04-01

    Budding yeast contain at least four distinct MAPK (mitogen activated protein kinase) cascades that transduce a variety of intracellular signals: mating-pheromone response, pseudohyphal/invasive growth, cell wall integrity, and high osmolarity adaptation. Although each MAPK cascade contains a conserved set of three protein kinases, the upstream activation mechanisms for these cascades are diverse, including a trimeric G protein, monomeric small G proteins, and a prokaryotic-like two-component system. Recently, it became apparent that there is extensive sharing of signaling elements among the MAPK pathways; however, little undesirable cross-talk occurs between various cascades. The formation of multi-protein signaling complexes is probably centrally important for this insulation of individual MAPK cascades.

  14. The cellular robustness by genetic redundancy in budding yeast.

    Directory of Open Access Journals (Sweden)

    Jingjing Li

    2010-11-01

    Full Text Available The frequent dispensability of duplicated genes in budding yeast is heralded as a hallmark of genetic robustness contributed by genetic redundancy. However, theoretical predictions suggest such backup by redundancy is evolutionarily unstable, and the extent of genetic robustness contributed from redundancy remains controversial. It is anticipated that, to achieve mutual buffering, the duplicated paralogs must at least share some functional overlap. However, counter-intuitively, several recent studies reported little functional redundancy between these buffering duplicates. The large yeast genetic interactions released recently allowed us to address these issues on a genome-wide scale. We herein characterized the synthetic genetic interactions for ∼500 pairs of yeast duplicated genes originated from either whole-genome duplication (WGD or small-scale duplication (SSD events. We established that functional redundancy between duplicates is a pre-requisite and thus is highly predictive of their backup capacity. This observation was particularly pronounced with the use of a newly introduced metric in scoring functional overlap between paralogs on the basis of gene ontology annotations. Even though mutual buffering was observed to be prevalent among duplicated genes, we showed that the observed backup capacity is largely an evolutionarily transient state. The loss of backup capacity generally follows a neutral mode, with the buffering strength decreasing in proportion to divergence time, and the vast majority of the paralogs have already lost their backup capacity. These observations validated previous theoretic predictions about instability of genetic redundancy. However, departing from the general neutral mode, intriguingly, our analysis revealed the presence of natural selection in stabilizing functional overlap between SSD pairs. These selected pairs, both WGD and SSD, tend to have decelerated functional evolution, have higher propensities of co

  15. Genome-wide studies of telomere biology in budding yeast

    Directory of Open Access Journals (Sweden)

    Yaniv Harari

    2014-03-01

    Full Text Available Telomeres are specialized DNA-protein structures at the ends of eukaryotic chromosomes. Telomeres are essential for chromosomal stability and integrity, as they prevent chromosome ends from being recognized as double strand breaks. In rapidly proliferating cells, telomeric DNA is synthesized by the enzyme telomerase, which copies a short template sequence within its own RNA moiety, thus helping to solve the “end-replication problem”, in which information is lost at the ends of chromosomes with each DNA replication cycle. The basic mechanisms of telomere length, structure and function maintenance are conserved among eukaryotes. Studies in the yeast Saccharomyces cerevisiae have been instrumental in deciphering the basic aspects of telomere biology. In the last decade, technical advances, such as the availability of mutant collections, have allowed carrying out systematic genome-wide screens for mutants affecting various aspects of telomere biology. In this review we summarize these efforts, and the insights that this Systems Biology approach has produced so far.

  16. Characterization of dependencies between growth and division in budding yeast.

    Science.gov (United States)

    Mayhew, Michael B; Iversen, Edwin S; Hartemink, Alexander J

    2017-02-01

    Cell growth and division are processes vital to the proliferation and development of life. Coordination between these two processes has been recognized for decades in a variety of organisms. In the budding yeast Saccharomyces cerevisiae, this coordination or 'size control' appears as an inverse correlation between cell size and the rate of cell-cycle progression, routinely observed in G1 prior to cell division commitment. Beyond this point, cells are presumed to complete S/G2/M at similar rates and in a size-independent manner. As such, studies of dependence between growth and division have focused on G1 Moreover, in unicellular organisms, coordination between growth and division has commonly been analysed within the cycle of a single cell without accounting for correlations in growth and division characteristics between cycles of related cells. In a comprehensive analysis of three published time-lapse microscopy datasets, we analyse both intra- and inter-cycle dependencies between growth and division, revisiting assumptions about the coordination between these two processes. Interestingly, we find evidence (i) that S/G2/M durations are systematically longer in daughters than in mothers, (ii) of dependencies between S/G2/M and size at budding that echo the classical G1 dependencies, and (iii) in contrast with recent bacterial studies, of negative dependencies between size at birth and size accumulated during the cell cycle. In addition, we develop a novel hierarchical model to uncover inter-cycle dependencies, and we find evidence for such dependencies in cells growing in sugar-poor environments. Our analysis highlights the need for experimentalists and modellers to account for new sources of cell-to-cell variation in growth and division, and our model provides a formal statistical framework for the continued study of dependencies between biological processes.

  17. Interorganelle interactions and inheritance patterns of nuclei and vacuoles in budding yeast meiosis.

    Science.gov (United States)

    Tsai, I-Ting; Lin, Jyun-Liang; Chiang, Yi-Hsuan; Chuang, Yu-Chien; Liang, Shu-Shan; Chuang, Chi-Ning; Huang, Tzyy-Nan; Wang, Ting-Fang

    2014-02-01

    Many of the mechanisms by which organelles are inherited by spores during meiosis are not well understood. Dramatic chromosome motion and bouquet formation are evolutionarily conserved characteristics of meiotic chromosomes. The budding yeast bouquet genes (NDJ1, MPS3, CSM4) mediate these movements via telomere attachment to the nuclear envelope (NE). Here, we report that during meiosis the NE is in direct contact with vacuoles via nucleus-vacuole junctions (NVJs). We show that in meiosis NVJs are assembled through the interaction of the outer NE-protein Nvj1 and the vacuolar membrane protein Vac8. Notably, NVJs function as diffusion barriers that exclude the nuclear pore complexes, the bouquet protein Mps3 and NE-tethered telomeres from the outer nuclear membrane and nuclear ER, resulting in distorted NEs during early meiosis. An increase in NVJ area resulting from Nvj1-GFP overexpression produced a moderate bouquet mutant-like phenotype in wild-type cells. NVJs, as the vacuolar contact sites of the nucleus, were found to undergo scission alongside the NE during meiotic nuclear division. The zygotic NE and NVJs were partly segregated into 4 spores. Lastly, new NVJs were also revealed to be synthesized de novo to rejoin the zygotic NE with the newly synthesized vacuoles in the mature spores. In conclusion, our results revealed that budding yeast nuclei and vacuoles exhibit dynamic interorganelle interactions and different inheritance patterns in meiosis, and also suggested that nvj1Δ mutant cells may be useful to resolve the technical challenges pertaining to the isolation of intact nuclei for the biochemical study of meiotic nuclear proteins.

  18. 5'-end sequences of budding yeast full-length cDNA clones and quality scores - Budding yeast cDNA sequencing project | LSDB Archive [Life Science Database Archive metadata

    Lifescience Database Archive (English)

    Full Text Available List Contact us Budding yeast cDNA sequencing project 5'-end sequences of budding yeast full-length cDNA clones and quality score...s Data detail Data name 5'-end sequences of budding yeast full-length cDNA clones and quality score...or-capping method, the sequence quality score generated by the Phred software, and links to SGD, dbEST and U...es. FASTA format. Quality Phred's quality score About This Database Database Desc...g yeast full-length cDNA clones and quality scores - Budding yeast cDNA sequencing project | LSDB Archive ...

  19. Identification of an amphipathic helix important for the formation of ectopic septin spirals and axial budding in yeast axial landmark protein Bud3p.

    Science.gov (United States)

    Guo, Jia; Gong, Ting; Gao, Xiang-Dong

    2011-03-08

    Correct positioning of polarity axis in response to internal or external cues is central to cellular morphogenesis and cell fate determination. In the budding yeast Saccharomyces cerevisiae, Bud3p plays a key role in the axial bud-site selection (axial budding) process in which cells assemble the new bud next to the preceding cell division site. Bud3p is thought to act as a component of a spatial landmark. However, it is not clear how Bud3p interacts with other components of the landmark, such as the septins, to control axial budding. Here, we report that overexpression of Bud3p causes the formation of small septin rings (∼1 µm in diameter) and arcs aside from previously reported spiral-like septin structures. Bud3p closely associates with the septins in vivo as Bud3p colocalizes with these aberrant septin structures and forms a complex with two septins, Cdc10p and Cdc11p. The interaction of Bud3p with the septins may involve multiple regions of Bud3p including 1-858, 850-1220, and 1221-1636 a.a. since they all target to the bud neck but exhibit different effects on septin organization when overexpressed. In addition, our study reveals that the axial budding function of Bud3p is mediated by the N-terminal region 1-858. This region shares an amphipathic helix (850-858) crucial for bud neck targeting with the middle portion 850-1103 involved in the formation of ectopic septin spirals and rings. Interestingly, the Dbl-homology domain located in 1-858 is dispensable for axial bud-site selection. Our findings suggest that multiple regions of Bud3p ensure efficient targeting of Bud3p to the bud neck in the assembly of the axial landmark and distinct domains of Bud3p are involved in axial bud-site selection and other cellular processes.

  20. Timing robustness in the budding and fission yeast cell cycles.

    KAUST Repository

    Mangla, Karan

    2010-02-01

    Robustness of biological models has emerged as an important principle in systems biology. Many past analyses of Boolean models update all pending changes in signals simultaneously (i.e., synchronously), making it impossible to consider robustness to variations in timing that result from noise and different environmental conditions. We checked previously published mathematical models of the cell cycles of budding and fission yeast for robustness to timing variations by constructing Boolean models and analyzing them using model-checking software for the property of speed independence. Surprisingly, the models are nearly, but not totally, speed-independent. In some cases, examination of timing problems discovered in the analysis exposes apparent inaccuracies in the model. Biologically justified revisions to the model eliminate the timing problems. Furthermore, in silico random mutations in the regulatory interactions of a speed-independent Boolean model are shown to be unlikely to preserve speed independence, even in models that are otherwise functional, providing evidence for selection pressure to maintain timing robustness. Multiple cell cycle models exhibit strong robustness to timing variation, apparently due to evolutionary pressure. Thus, timing robustness can be a basis for generating testable hypotheses and can focus attention on aspects of a model that may need refinement.

  1. Consider the workhorse: Nonhomologous end-joining in budding yeast.

    Science.gov (United States)

    Emerson, Charlene H; Bertuch, Alison A

    2016-10-01

    DNA double strand breaks (DSBs) are dangerous sources of genome instability and must be repaired by the cell. Nonhomologous end-joining (NHEJ) is an evolutionarily conserved pathway to repair DSBs by direct ligation of the ends, with no requirement for a homologous template. While NHEJ is the primary DSB repair pathway in mammalian cells, conservation of the core NHEJ factors throughout eukaryotes makes the pathway attractive for study in model organisms. The budding yeast, Saccharomyces cerevisiae, has been used extensively to develop a functional picture of NHEJ. In this review, we will discuss the current understanding of NHEJ in S. cerevisiae. Topics include canonical end-joining, alternative end-joining, and pathway regulation. Particular attention will be paid to the NHEJ mechanism involving core factors, including Yku70/80, Dnl4, Lif1, and Nej1, as well as the various factors implicated in the processing of the broken ends. The relevance of chromatin dynamics to NHEJ will also be discussed. This review illustrates the use of S. cerevisiae as a powerful system to understand the principles of NHEJ, as well as in pioneering the direction of the field.

  2. Consider the workhorse: Nonhomologous end joining in budding yeast

    Science.gov (United States)

    Emerson, Charlene H.; Bertuch, Alison A.

    2017-01-01

    DNA double strand breaks (DSBs) are dangerous sources of genome instability and must be repaired by the cell. Nonhomologous end joining (NHEJ) is an evolutionarily conserved pathway to repair DSBs by direct ligation of the ends, with no requirement for a homologous template. While NHEJ is the primary DSB repair pathway in mammalian cells, conservation of the core NHEJ factors throughout eukaryotes make the pathway attractive for study in model organisms. The budding yeast, Saccharomyces cerevisiae, has been used extensively to develop a functional picture of NHEJ. In this review, we will discuss the current understanding of NHEJ in S. cerevisiae. Topics include canonical end-joining, alternative end-joining, and pathway regulation. Particular attention will be paid to the NHEJ mechanism involving core factors, including Yku70/80, Dnl4, Lif1, and Nej1, as well as the various factors implicated in the processing of the broken ends. The relevance of chromatin dynamics to NHEJ will also be discussed. This review illustrates the use of S. cerevisiae as a powerful system to understand the principles of NHEJ, as well as in pioneering the direction of the field. PMID:27240172

  3. Asymmetric nucleosomes flank promoters in the budding yeast genome.

    Science.gov (United States)

    Ramachandran, Srinivas; Zentner, Gabriel E; Henikoff, Steven

    2015-03-01

    Nucleosomes in active chromatin are dynamic, but whether they have distinct structural conformations is unknown. To identify nucleosomes with alternative structures genome-wide, we used H4S47C-anchored cleavage mapping, which revealed that 5% of budding yeast (Saccharomyces cerevisiae) nucleosome positions have asymmetric histone-DNA interactions. These asymmetric interactions are enriched at nucleosome positions that flank promoters. Micrococcal nuclease (MNase) sequence-based profiles of asymmetric nucleosome positions revealed a corresponding asymmetry in MNase protection near the dyad axis, suggesting that the loss of DNA contacts around H4S47 is accompanied by protection of the DNA from MNase. Chromatin immunoprecipitation mapping of selected nucleosome remodelers indicated that asymmetric nucleosomes are bound by the RSC chromatin remodeling complex, which is required for maintaining nucleosomes at asymmetric positions. These results imply that the asymmetric nucleosome-RSC complex is a metastable intermediate representing partial unwrapping and protection of nucleosomal DNA on one side of the dyad axis during chromatin remodeling.

  4. Measurement of spatial proximity and accessibility of chromosomal loci in yeast using Cre/loxP site-specific recombination

    OpenAIRE

    Lui, Doris; Burgess, Sean M.

    2009-01-01

    Several methods have been developed to measure interactions between homologous chromosomes during meiosis in budding yeast. These include cytological analysis of fixed, spread nuclei using fluorescence in situ Hybridization (FISH) (1, 2), visualization of GFP-labeled chromosomal loci in living cells (3), and Chromosome-Conformation Capture (3C) (4). Here we describe a quantitative genetic assay that uses exogenous site-specific recombination to monitor the level of homolog associations betwee...

  5. Origin of irreversibility of cell cycle start in budding yeast.

    Directory of Open Access Journals (Sweden)

    Gilles Charvin

    2010-01-01

    Full Text Available Budding yeast cells irreversibly commit to a new division cycle at a regulatory transition called Start. This essential decision-making step involves the activation of the SBF/MBF transcription factors. SBF/MBF promote expression of the G1 cyclins encoded by CLN1 and CLN2. Cln1,2 can activate their own expression by inactivating the Whi5 repressor of SBF/MBF. The resulting transcriptional positive feedback provides an appealing, but as yet unproven, candidate for generating irreversibility of Start. Here, we investigate the logic of the Start regulatory module by quantitative single-cell time-lapse microscopy, using strains in which expression of key regulators is efficiently controlled by changes of inducers in a microfluidic chamber. We show that Start activation is ultrasensitive to G1 cyclin. In the absence of CLN1,2-dependent positive feedback, we observe that Start transit is reversible, due to reactivation of the Whi5 transcriptional repressor. Introduction of the positive feedback loop makes Whi5 inactivation and Start activation irreversible, which therefore guarantees unidirectional entry into S phase. A simple mathematical model to describe G1 cyclin turn on at Start, entirely constrained by empirically measured parameters, shows that the experimentally measured ultrasensitivity and transcriptional positive feedback are necessary and sufficient dynamical characteristics to make the Start transition a bistable and irreversible switch. Our study thus demonstrates that Start irreversibility is a property that arises from the architecture of the system (Whi5/SBF/Cln2 loop, rather than the consequence of the regulation of a single component (e.g., irreversible protein degradation.

  6. Origin of irreversibility of cell cycle start in budding yeast.

    Science.gov (United States)

    Charvin, Gilles; Oikonomou, Catherine; Siggia, Eric D; Cross, Frederick R

    2010-01-19

    Budding yeast cells irreversibly commit to a new division cycle at a regulatory transition called Start. This essential decision-making step involves the activation of the SBF/MBF transcription factors. SBF/MBF promote expression of the G1 cyclins encoded by CLN1 and CLN2. Cln1,2 can activate their own expression by inactivating the Whi5 repressor of SBF/MBF. The resulting transcriptional positive feedback provides an appealing, but as yet unproven, candidate for generating irreversibility of Start. Here, we investigate the logic of the Start regulatory module by quantitative single-cell time-lapse microscopy, using strains in which expression of key regulators is efficiently controlled by changes of inducers in a microfluidic chamber. We show that Start activation is ultrasensitive to G1 cyclin. In the absence of CLN1,2-dependent positive feedback, we observe that Start transit is reversible, due to reactivation of the Whi5 transcriptional repressor. Introduction of the positive feedback loop makes Whi5 inactivation and Start activation irreversible, which therefore guarantees unidirectional entry into S phase. A simple mathematical model to describe G1 cyclin turn on at Start, entirely constrained by empirically measured parameters, shows that the experimentally measured ultrasensitivity and transcriptional positive feedback are necessary and sufficient dynamical characteristics to make the Start transition a bistable and irreversible switch. Our study thus demonstrates that Start irreversibility is a property that arises from the architecture of the system (Whi5/SBF/Cln2 loop), rather than the consequence of the regulation of a single component (e.g., irreversible protein degradation).

  7. Download - Budding yeast cDNA sequencing project | LSDB Archive [Life Science Database Archive metadata

    Lifescience Database Archive (English)

    Full Text Available Budding yeast cDNA sequencing project Download First of all, please read the license of this database. Data ...names and data descriptions are about the downloadable data in this page. They might not correspond to the c...f the data. # Data name File Simple search and download 1 README README_e.html - 2 5'-end sequences of buddi...ng yeast full-length cDNA clones and quality scores yeast_seq_qual.zip (59.9MB) Simple search and download 3...Downlaod via FTP Joomla SEF URLs by Artio About This Database Database Description Download License Update H

  8. Ndc10 is a platform for inner kinetochore assembly in budding yeast

    Energy Technology Data Exchange (ETDEWEB)

    Cho, Uhn-Soo; Harrison, Stephen C. (Harvard-Med)

    2012-01-10

    Kinetochores link centromeric DNA to spindle microtubules and ensure faithful chromosome segregation during mitosis. In point-centromere yeasts, the CBF3 complex Skp1-Ctf13-(Cep3){sub 2}-(Ndc10){sub 2} recognizes a conserved centromeric DNA element through contacts made by Cep3 and Ndc10. We describe here the five-domain organization of Kluyveromyces lactis Ndc10 and the structure at 2.8 {angstrom} resolution of domains I-II (residues 1-402) bound to DNA. The structure resembles tyrosine DNA recombinases, although it lacks both endonuclease and ligase activities. Structural and biochemical data demonstrate that each subunit of the Ndc10 dimer binds a separate fragment of DNA, suggesting that Ndc10 stabilizes a DNA loop at the centromere. We describe in vitro association experiments showing that specific domains of Ndc10 interact with each of the known inner-kinetochore proteins or protein complexes in budding yeast. We propose that Ndc10 provides a central platform for inner-kinetochore assembly.

  9. The budding yeast Dbf2 protein kinase localises to the centrosome and moves to the bud neck in late mitosis.

    Science.gov (United States)

    Frenz, L M; Lee, S E; Fesquet, D; Johnston, L H

    2000-10-01

    Dbf2 is a multifunctional protein kinase in Saccharomyces cerevisiae that functions in transcription, the stress response and as part of a network of genes in exit from mitosis. By analogy with fission yeast it seemed likely that these mitotic exit genes would be involved in cytokinesis. As a preliminary investigation of this we have used Dbf2 tagged with GFP to examine intracellular localisation of the protein in living cells. Dbf2 is found on the centrosomes/spindle pole bodies (SPBs) and also at the bud neck where it forms a double ring. The localisation of Dbf2 is cell cycle regulated. It is on the SPBs for much of the cell cycle and migrates from there to the bud neck in late mitosis, consistent with a role in cytokinesis. Dbf2 partly co-localises with septins at the bud neck. A temperature-sensitive mutant of dbf2 also blocks progression of cytokinesis at 37 degrees C. Following cytokinesis some Dbf2 moves into the nascent bud. Localisation to the bud neck depends upon the septins and also the mitotic exit network proteins Mob1, Cdc5, Cdc14 and Cdc15. The above data are consistent with Dbf2 acting downstream in a pathway controlling cytokinesis.

  10. High Throughput Analyses of Budding Yeast ARSs Reveal New DNA Elements Capable of Conferring Centromere-Independent Plasmid Propagation.

    Science.gov (United States)

    Hoggard, Timothy; Liachko, Ivan; Burt, Cassaundra; Meikle, Troy; Jiang, Katherine; Craciun, Gheorghe; Dunham, Maitreya J; Fox, Catherine A

    2016-04-07

    The ability of plasmids to propagate in Saccharomyces cerevisiae has been instrumental in defining eukaryotic chromosomal control elements. Stable propagation demands both plasmid replication, which requires a chromosomal replication origin (i.e., an ARS), and plasmid distribution to dividing cells, which requires either a chromosomal centromere for segregation or a plasmid-partitioning element. While our knowledge of yeast ARSs and centromeres is relatively advanced, we know less about chromosomal regions that can function as plasmid partitioning elements. The Rap1 protein-binding site (RAP1) present in transcriptional silencers and telomeres of budding yeast is a known plasmid-partitioning element that functions to anchor a plasmid to the inner nuclear membrane (INM), which in turn facilitates plasmid distribution to daughter cells. This Rap1-dependent INM-anchoring also has an important chromosomal role in higher-order chromosomal structures that enhance transcriptional silencing and telomere stability. Thus, plasmid partitioning can reflect fundamental features of chromosome structure and biology, yet a systematic screen for plasmid partitioning elements has not been reported. Here, we couple deep sequencing with competitive growth experiments of a plasmid library containing thousands of short ARS fragments to identify new plasmid partitioning elements. Competitive growth experiments were performed with libraries that differed only in terms of the presence or absence of a centromere. Comparisons of the behavior of ARS fragments in the two experiments allowed us to identify sequences that were likely to drive plasmid partitioning. In addition to the silencer RAP1 site, we identified 74 new putative plasmid-partitioning motifs predicted to act as binding sites for DNA binding proteins enriched for roles in negative regulation of gene expression and G2/M-phase associated biology. These data expand our knowledge of chromosomal elements that may function in plasmid

  11. From START to FINISH: computational analysis of cell cycle control in budding yeast.

    Science.gov (United States)

    Kraikivski, Pavel; Chen, Katherine C; Laomettachit, Teeraphan; Murali, T M; Tyson, John J

    2015-01-01

    In the cell division cycle of budding yeast, START refers to a set of tightly linked events that prepare a cell for budding and DNA replication, and FINISH denotes the interrelated events by which the cell exits from mitosis and divides into mother and daughter cells. On the basis of recent progress made by molecular biologists in characterizing the genes and proteins that control START and FINISH, we crafted a new mathematical model of cell cycle progression in yeast. Our model exploits a natural separation of time scales in the cell cycle control network to construct a system of differential-algebraic equations for protein synthesis and degradation, post-translational modifications, and rapid formation and dissociation of multimeric complexes. The model provides a unified account of the observed phenotypes of 257 mutant yeast strains (98% of the 263 strains in the data set used to constrain the model). We then use the model to predict the phenotypes of 30 novel combinations of mutant alleles. Our comprehensive model of the molecular events controlling cell cycle progression in budding yeast has both explanatory and predictive power. Future experimental tests of the model's predictions will be useful to refine the underlying molecular mechanism, to constrain the adjustable parameters of the model, and to provide new insights into how the cell division cycle is regulated in budding yeast.

  12. History of chromosome rearrangements reflects the spatial organization of yeast chromosomes.

    Science.gov (United States)

    Khrameeva, Ekaterina E; Fudenberg, Geoffrey; Gelfand, Mikhail S; Mirny, Leonid A

    2016-04-01

    Three-dimensional (3D) organization of genomes affects critical cellular processes such as transcription, replication, and deoxyribo nucleic acid (DNA) repair. While previous studies have investigated the natural role, the 3D organization plays in limiting a possible set of genomic rearrangements following DNA repair, the influence of specific organizational principles on this process, particularly over longer evolutionary time scales, remains relatively unexplored. In budding yeast S.cerevisiae, chromosomes are organized into a Rabl-like configuration, with clustered centromeres and telomeres tethered to the nuclear periphery. Hi-C data for S.cerevisiae show that a consequence of this Rabl-like organization is that regions equally distant from centromeres are more frequently in contact with each other, between arms of both the same and different chromosomes. Here, we detect rearrangement events in Saccharomyces species using an automatic approach, and observe increased rearrangement frequency between regions with higher contact frequencies. Together, our results underscore how specific principles of 3D chromosomal organization can influence evolutionary events.

  13. Nur1 dephosphorylation confers positive feedback to mitotic exit phosphatase activation in budding yeast.

    Directory of Open Access Journals (Sweden)

    Molly Godfrey

    2015-01-01

    Full Text Available Substrate dephosphorylation by the cyclin-dependent kinase (Cdk-opposing phosphatase, Cdc14, is vital for many events during budding yeast mitotic exit. Cdc14 is sequestered in the nucleolus through inhibitory binding to Net1, from which it is released in anaphase following Net1 phosphorylation. Initial Net1 phosphorylation depends on Cdk itself, in conjunction with proteins of the Cdc14 Early Anaphase Release (FEAR network. Later on, the Mitotic Exit Network (MEN signaling cascade maintains Cdc14 release. An important unresolved question is how Cdc14 activity can increase in early anaphase, while Cdk activity, that is required for Net1 phosphorylation, decreases and the MEN is not yet active. Here we show that the nuclear rim protein Nur1 interacts with Net1 and, in its Cdk phosphorylated form, inhibits Cdc14 release. Nur1 is dephosphorylated by Cdc14 in early anaphase, relieving the inhibition and promoting further Cdc14 release. Nur1 dephosphorylation thus describes a positive feedback loop in Cdc14 phosphatase activation during mitotic exit, required for faithful chromosome segregation and completion of the cell division cycle.

  14. Study of budding yeast colony formation and its characterizations by using circular granular cell

    Science.gov (United States)

    Aprianti, D.; Haryanto, F.; Purqon, A.; Khotimah, S. N.; Viridi, S.

    2016-03-01

    Budding yeast can exhibit colony formation in solid substrate. The colony of pathogenic budding yeast can colonize various surfaces of the human body and medical devices. Furthermore, it can form biofilm that resists drug effective therapy. The formation of the colony is affected by the interaction between cells and with its growth media. The cell budding pattern holds an important role in colony expansion. To study this colony growth, the molecular dynamic method was chosen to simulate the interaction between budding yeast cells. Every cell was modelled by circular granular cells, which can grow and produce buds. Cohesion force, contact force, and Stokes force govern this model to mimic the interaction between cells and with the growth substrate. Characterization was determined by the maximum (L max) and minimum (L min) distances between two cells within the colony and whether two lines that connect the two cells in the maximum and minimum distances intersect each other. Therefore, it can be recognized the colony shape in circular, oval, and irregular shapes. Simulation resulted that colony formation are mostly in oval shape with little branch. It also shows that greater cohesion strength obtains more compact colony formation.

  15. Continuous High-resolution Microscopic Observation of Replicative Aging in Budding Yeast

    NARCIS (Netherlands)

    Huberts, Daphne H. E. W.; Janssens, Georges E.; Lee, Sung Sik; Vizcarra, Ima Avalos; Heinemann, Matthias

    We demonstrate the use of a simple microfluidic setup, in which single budding yeast cells can be tracked throughout their entire lifespan. The microfluidic chip exploits the size difference between mother and daughter cells using an array of micropads. Upon loading, cells are trapped underneath

  16. Whole lifespan microscopic observation of budding yeast aging through a microfluidic dissection platform

    NARCIS (Netherlands)

    Lee, Sung Sik; Avalos Vizcarra, Ima; Huberts, Daphne H E W; Lee, Luke P; Heinemann, Matthias

    2012-01-01

    Important insights into aging have been generated with the genetically tractable and short-lived budding yeast. However, it is still impossible today to continuously track cells by high-resolution microscopic imaging (e.g., fluorescent imaging) throughout their entire lifespan. Instead, the field

  17. Whole lifespan microscopic observation of budding yeast aging through a microfluidic dissection platform

    NARCIS (Netherlands)

    Lee, Sung Sik; Avalos Vizcarra, Ima; Huberts, Daphne H E W; Lee, Luke P; Heinemann, Matthias

    2012-01-01

    Important insights into aging have been generated with the genetically tractable and short-lived budding yeast. However, it is still impossible today to continuously track cells by high-resolution microscopic imaging (e.g., fluorescent imaging) throughout their entire lifespan. Instead, the field st

  18. Vector sequences - Budding yeast cDNA sequencing project | LSDB Archive [Life Science Database Archive metadata

    Lifescience Database Archive (English)

    Full Text Available [ Credits ] BLAST Search Image Search Home About Archive Update History Contact us ...od - Number of data entries 7 entries - Joomla SEF URLs by Artio About This Database Database Description Download License Update His...tory of This Database Site Policy | Contact Us Vector sequences - Budding yeast cDNA sequencing project | LSDB Archive ...

  19. Healing of Broken Linear Dicentric Chromosomes in Yeast

    OpenAIRE

    Haber, James E; Thorburn, Patricia C.

    1984-01-01

    In yeast, meiotic recombination between a linear chromosome III and a haploid-viable circular chromosome will yield a dicentric, tandemly duplicated chromosome. Spores containing apparently intact dicentric chromosomes were recovered from tetrads with three viable spores. The spore containing the dicentric inherited URA3 (part of the recombinant DNA used to join regions near the ends of the chromosome into a circle) as well as HML, HMR and MAL2 (located near the two ends of a linear but dele...

  20. Role of transcription at centromeres in budding yeast.

    Science.gov (United States)

    Ohkuni, Kentaro; Kitagawa, Katsumi

    2012-01-01

    Centromeres are specialized chromosomal loci that are essential for proper chromosome segregation. Recent data show that a certain level of active transcription, regulated by transcription factors Cbf1 and Ste12, makes a direct contribution to centromere function in Saccharomyces cerevisiae. Here, we discuss the requirement and function of transcription at centromeres.

  1. Genome Diversity and Evolution in the Budding Yeasts (Saccharomycotina).

    Science.gov (United States)

    Dujon, Bernard A; Louis, Edward J

    2017-06-01

    Considerable progress in our understanding of yeast genomes and their evolution has been made over the last decade with the sequencing, analysis, and comparisons of numerous species, strains, or isolates of diverse origins. The role played by yeasts in natural environments as well as in artificial manufactures, combined with the importance of some species as model experimental systems sustained this effort. At the same time, their enormous evolutionary diversity (there are yeast species in every subphylum of Dikarya) sparked curiosity but necessitated further efforts to obtain appropriate reference genomes. Today, yeast genomes have been very informative about basic mechanisms of evolution, speciation, hybridization, domestication, as well as about the molecular machineries underlying them. They are also irreplaceable to investigate in detail the complex relationship between genotypes and phenotypes with both theoretical and practical implications. This review examines these questions at two distinct levels offered by the broad evolutionary range of yeasts: inside the best-studied Saccharomyces species complex, and across the entire and diversified subphylum of Saccharomycotina. While obviously revealing evolutionary histories at different scales, data converge to a remarkably coherent picture in which one can estimate the relative importance of intrinsic genome dynamics, including gene birth and loss, vs. horizontal genetic accidents in the making of populations. The facility with which novel yeast genomes can now be studied, combined with the already numerous available reference genomes, offer privileged perspectives to further examine these fundamental biological questions using yeasts both as eukaryotic models and as fungi of practical importance. Copyright © 2017 by the Genetics Society of America.

  2. The fascinating and secret wild life of the budding yeast S. cerevisiae.

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    Liti, Gianni

    2015-03-25

    The budding yeast Saccharomyces cerevisiae has been used in laboratory experiments for over a century and has been instrumental in understanding virtually every aspect of molecular biology and genetics. However, it wasn't until a decade ago that the scientific community started to realise how little was known about this yeast's ecology and natural history, and how this information was vitally important for interpreting its biology. Recent large-scale population genomics studies coupled with intensive field surveys have revealed a previously unappreciated wild lifestyle of S. cerevisiae outside the restrictions of human environments and laboratories. The recent discovery that Chinese isolates harbour almost twice as much genetic variation as isolates from the rest of the world combined suggests that Asia is the likely origin of the modern budding yeast.

  3. Chromosome-refolding model of mating-type switching in yeast.

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    Avşaroğlu, Barış; Bronk, Gabriel; Li, Kevin; Haber, James E; Kondev, Jane

    2016-10-24

    Chromosomes are folded into cells in a nonrandom fashion, with particular genetic loci occupying distinct spatial regions. This observation raises the question of whether the spatial organization of a chromosome governs its functions, such as recombination or transcription. We consider this general question in the specific context of mating-type switching in budding yeast, which is a model system for homologous recombination. Mating-type switching is induced by a DNA double-strand break (DSB) at the MAT locus on chromosome III, followed by homologous recombination between the cut MAT locus and one of two donor loci (HMLα and HMRa), located on the same chromosome. Previous studies have suggested that in MATa cells after the DSB is induced chromosome III undergoes refolding, which directs the MAT locus to recombine with HMLα. Here, we propose a quantitative model of mating-type switching predicated on the assumption of DSB-induced chromosome refolding, which also takes into account the previously measured stochastic dynamics and polymer nature of yeast chromosomes. Using quantitative fluorescence microscopy, we measure changes in the distance between the donor (HMLα) and MAT loci after the DSB and find agreement with the theory. Predictions of the theory also agree with measurements of changes in the use of HMLα as the donor, when we perturb the refolding of chromosome III. These results establish refolding of yeast chromosome III as a key driving force in MAT switching and provide an example of a cell regulating the spatial organization of its chromosome so as to direct homology search during recombination.

  4. Regulation of Budding Yeast CENP-A levels Prevents Misincorporation at Promoter Nucleosomes and Transcriptional Defects.

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    Erica M Hildebrand

    2016-03-01

    Full Text Available The exclusive localization of the histone H3 variant CENP-A to centromeres is essential for accurate chromosome segregation. Ubiquitin-mediated proteolysis helps to ensure that CENP-A does not mislocalize to euchromatin, which can lead to genomic instability. Consistent with this, overexpression of the budding yeast CENP-A(Cse4 is lethal in cells lacking Psh1, the E3 ubiquitin ligase that targets CENP-A(Cse4 for degradation. To identify additional mechanisms that prevent CENP-A(Cse4 misincorporation and lethality, we analyzed the genome-wide mislocalization pattern of overexpressed CENP-A(Cse4 in the presence and absence of Psh1 by chromatin immunoprecipitation followed by high throughput sequencing. We found that ectopic CENP-A(Cse4 is enriched at promoters that contain histone H2A.Z(Htz1 nucleosomes, but that H2A.Z(Htz1 is not required for CENP-A(Cse4 mislocalization. Instead, the INO80 complex, which removes H2A.Z(Htz1 from nucleosomes, promotes the ectopic deposition of CENP-A(Cse4. Transcriptional profiling revealed gene expression changes in the psh1Δ cells overexpressing CENP-A(Cse4. The down-regulated genes are enriched for CENP-A(Cse4 mislocalization to promoters, while the up-regulated genes correlate with those that are also transcriptionally up-regulated in an htz1Δ strain. Together, these data show that regulating centromeric nucleosome localization is not only critical for maintaining centromere function, but also for ensuring accurate promoter function and transcriptional regulation.

  5. Regulation of Budding Yeast CENP-A levels Prevents Misincorporation at Promoter Nucleosomes and Transcriptional Defects.

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    Hildebrand, Erica M; Biggins, Sue

    2016-03-01

    The exclusive localization of the histone H3 variant CENP-A to centromeres is essential for accurate chromosome segregation. Ubiquitin-mediated proteolysis helps to ensure that CENP-A does not mislocalize to euchromatin, which can lead to genomic instability. Consistent with this, overexpression of the budding yeast CENP-A(Cse4) is lethal in cells lacking Psh1, the E3 ubiquitin ligase that targets CENP-A(Cse4) for degradation. To identify additional mechanisms that prevent CENP-A(Cse4) misincorporation and lethality, we analyzed the genome-wide mislocalization pattern of overexpressed CENP-A(Cse4) in the presence and absence of Psh1 by chromatin immunoprecipitation followed by high throughput sequencing. We found that ectopic CENP-A(Cse4) is enriched at promoters that contain histone H2A.Z(Htz1) nucleosomes, but that H2A.Z(Htz1) is not required for CENP-A(Cse4) mislocalization. Instead, the INO80 complex, which removes H2A.Z(Htz1) from nucleosomes, promotes the ectopic deposition of CENP-A(Cse4). Transcriptional profiling revealed gene expression changes in the psh1Δ cells overexpressing CENP-A(Cse4). The down-regulated genes are enriched for CENP-A(Cse4) mislocalization to promoters, while the up-regulated genes correlate with those that are also transcriptionally up-regulated in an htz1Δ strain. Together, these data show that regulating centromeric nucleosome localization is not only critical for maintaining centromere function, but also for ensuring accurate promoter function and transcriptional regulation.

  6. Fimbrin phosphorylation by metaphase Cdk1 regulates actin cable dynamics in budding yeast.

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    Miao, Yansong; Han, Xuemei; Zheng, Liangzhen; Xie, Ying; Mu, Yuguang; Yates, John R; Drubin, David G

    2016-01-01

    Actin cables, composed of actin filament bundles nucleated by formins, mediate intracellular transport for cell polarity establishment and maintenance. We previously observed that metaphase cells preferentially promote actin cable assembly through cyclin-dependent kinase 1 (Cdk1) activity. However, the relevant metaphase Cdk1 targets were not known. Here we show that the highly conserved actin filament crosslinking protein fimbrin is a critical Cdk1 target for actin cable assembly regulation in budding yeast. Fimbrin is specifically phosphorylated on threonine 103 by the metaphase cyclin-Cdk1 complex, in vivo and in vitro. On the basis of conformational simulations, we suggest that this phosphorylation stabilizes fimbrin's N-terminal domain, and modulates actin filament binding to regulate actin cable assembly and stability in cells. Overall, this work identifies fimbrin as a key target for cell cycle regulation of actin cable assembly in budding yeast, and suggests an underlying mechanism.

  7. Strategy of transcription regulation in the budding yeast.

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    Sagi Levy

    Full Text Available Cells must adjust their gene expression in order to compete in a constantly changing environment. Two alternative strategies could in principle ensure optimal coordination of gene expression with physiological requirements. First, characters of the internal physiological state, such as growth rate, metabolite levels, or energy availability, could be feedback to tune gene expression. Second, internal needs could be inferred from the external environment, using evolutionary-tuned signaling pathways. Coordination of ribosomal biogenesis with the requirement for protein synthesis is of particular importance, since cells devote a large fraction of their biosynthetic capacity for ribosomal biogenesis. To define the relative contribution of internal vs. external sensing to the regulation of ribosomal biogenesis gene expression in yeast, we subjected S. cerevisiae cells to conditions which decoupled the actual vs. environmentally-expected growth rate. Gene expression followed the environmental signal according to the expected, but not the actual, growth rate. Simultaneous monitoring of gene expression and growth rate in continuous cultures further confirmed that ribosome biogenesis genes responded rapidly to changes in the environments but were oblivious to longer-term changes in growth rate. Our results suggest that the capacity to anticipate and prepare for environmentally-mediated changes in cell growth presented a major selection force during yeast evolution.

  8. cDNA sequence quality data - Budding yeast cDNA sequencing project | LSDB Archive [Life Science Database Archive metadata

    Lifescience Database Archive (English)

    Full Text Available Budding yeast cDNA sequencing project cDNA sequence quality data Data detail Data name cDNA sequence quality... data Description of data contents Phred's quality score. PHD format, one file to a single cDNA data, and co...ription Download License Update History of This Database Site Policy | Contact Us cDNA sequence quality data - Budding yeast cDNA sequencing project | LSDB Archive ...

  9. A nutrient dependant switch explains mutually exclusive existence of meiosis and mitosis initiation in budding yeast.

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    Wannige, C T; Kulasiri, D; Samarasinghe, S

    2014-01-21

    Nutrients from living environment are vital for the survival and growth of any organism. Budding yeast diploid cells decide to grow by mitosis type cell division or decide to create unique, stress resistant spores by meiosis type cell division depending on the available nutrient conditions. To gain a molecular systems level understanding of the nutrient dependant switching between meiosis and mitosis initiation in diploid cells of budding yeast, we develop a theoretical model based on ordinary differential equations (ODEs) including the mitosis initiator and its relations to budding yeast meiosis initiation network. Our model accurately and qualitatively predicts the experimentally revealed temporal variations of related proteins under different nutrient conditions as well as the diverse mutant studies related to meiosis and mitosis initiation. Using this model, we show how the meiosis and mitosis initiators form an all-or-none type bistable switch in response to available nutrient level (mainly nitrogen). The transitions to and from meiosis or mitosis initiation states occur via saddle node bifurcation. This bidirectional switch helps the optimal usage of available nutrients and explains the mutually exclusive existence of meiosis and mitosis pathways.

  10. Distinct roles of the polarity factors Boi1 and Boi2 in the control of exocytosis and abscission in budding yeast.

    Science.gov (United States)

    Masgrau, Aina; Battola, Andrea; Sanmartin, Trinidad; Pryszcz, Leszek P; Gabaldón, Toni; Mendoza, Manuel

    2017-09-13

    Boi1 and Boi2 (Boi1/2) are budding yeast plasma membrane proteins that function in polarized growth, and in cytokinesis inhibition in response to chromosome bridges via the NoCut abscission checkpoint. How Boi1/2 act in these two distinct processes is not understood. We demonstrate that Boi1/2 are required for a late step in the fusion of secretory vesicles with the plasma membrane of the growing bud. Cells lacking Boi1/2 accumulate secretory vesicles and are defective in bud growth. In contrast, Boi2 is specifically required for abscission inhibition in cells with chromatin bridges. The SH3 domain of Boi2, which is dispensable for bud growth and targets Boi2 to the site of abscission, is necessary and sufficient for abscission inhibition. Gain of function of the exocyst, a conserved protein complex involved in tethering of exocytic vesicles to the plasma membrane, rescued secretion and bud growth defects in boi mutant cells, and abrogated NoCut checkpoint function. Thus, Boi2 functions redundantly with Boi1 to promote the fusion of secretory vesicles with the plasma membrane at sites of polarized growth, and acts as an abscission inhibitor during cytokinesis in response to chromatin bridges. © 2017 by The American Society for Cell Biology.

  11. Direct and indirect control of the initiation of meiotic recombination by DNA damage checkpoint mechanisms in budding yeast.

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    Bilge Argunhan

    Full Text Available Meiotic recombination plays an essential role in the proper segregation of chromosomes at meiosis I in many sexually reproducing organisms. Meiotic recombination is initiated by the scheduled formation of genome-wide DNA double-strand breaks (DSBs. The timing of DSB formation is strictly controlled because unscheduled DSB formation is detrimental to genome integrity. Here, we investigated the role of DNA damage checkpoint mechanisms in the control of meiotic DSB formation using budding yeast. By using recombination defective mutants in which meiotic DSBs are not repaired, the effect of DNA damage checkpoint mutations on DSB formation was evaluated. The Tel1 (ATM pathway mainly responds to unresected DSB ends, thus the sae2 mutant background in which DSB ends remain intact was employed. On the other hand, the Mec1 (ATR pathway is primarily used when DSB ends are resected, thus the rad51 dmc1 double mutant background was employed in which highly resected DSBs accumulate. In order to separate the effect caused by unscheduled cell cycle progression, which is often associated with DNA damage checkpoint defects, we also employed the ndt80 mutation which permanently arrests the meiotic cell cycle at prophase I. In the absence of Tel1, DSB formation was reduced in larger chromosomes (IV, VII, II and XI whereas no significant reduction was found in smaller chromosomes (III and VI. On the other hand, the absence of Rad17 (a critical component of the ATR pathway lead to an increase in DSB formation (chromosomes VII and II were tested. We propose that, within prophase I, the Tel1 pathway facilitates DSB formation, especially in bigger chromosomes, while the Mec1 pathway negatively regulates DSB formation. We also identified prophase I exit, which is under the control of the DNA damage checkpoint machinery, to be a critical event associated with down-regulating meiotic DSB formation.

  12. Partial purification of histone H3 proteolytic activity from the budding yeast Saccharomyces cerevisiae.

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    Azad, Gajendra Kumar; Tomar, Raghuvir Singh

    2016-06-01

    The proteolytic clipping of histone tails has recently emerged as a novel form of irreversible post-translational modification (PTM) of histones. Histone clipping has been implicated as a regulatory process leading to the permanent removal of PTMs from histone proteins. However, there is scarcity of literature that describes the identification and characterization of histone-specific proteases. Here, we employed various biochemical methods to report histone H3-specific proteolytic activity from budding yeast. Our results demonstrate that H3 proteolytic activity was associated with sepharose bead matrices and activity was not affected by a variety of stress conditions. We have also identified the existence of an unknown protein that acts as a physiological inhibitor of the H3-clipping activity of yeast H3 protease. Moreover, through protease inhibition assays, we have also characterized yeast H3 protease as a serine protease. Interestingly, unlike glutamate dehydrogenase (GDH), yeast H3 proteolytic activity was not inhibited by Stefin B. Together, our findings suggest the existence of a novel H3 protease in yeast that is different from other reported histone H3 proteases. The presence of histone H3 proteolytic activity, along with the physiological inhibitor in yeast, suggests an interesting molecular mechanism that regulates the activity of histone proteases. Copyright © 2016 John Wiley & Sons, Ltd.

  13. Focal accumulation of preribosomes outside the nucleolus during metaphase-anaphase in budding yeast.

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    Moriggi, Giulia; Gaspar, Sonia G; Nieto, Blanca; Bustelo, Xosé R; Dosil, Mercedes

    2017-09-01

    Saccharomyces cerevisiae contains one nucleolus that remains intact in the mother-cell side of the nucleus throughout most of mitosis. Based on this, it is assumed that the bulk of ribosome production during cell division occurs in the mother cell. Here, we show that the ribosome synthesis machinery localizes not only in the nucleolus but also at a center that is present in the bud side of the nucleus after the initiation of mitosis. This center can be visualized by live microscopy as a punctate body located in close proximity to the nuclear envelope and opposite to the nucleolus. It contains ribosomal DNA (rDNA) and precursors of both 40S and 60S ribosomal subunits. Proteins that actively participate in ribosome synthesis, but not functionally defective variants, accumulate in that site. The formation of this body occurs in the metaphase-to-anaphase transition when discrete regions of rDNA occasionally exit the nucleolus and move into the bud. Collectively, our data unveil the existence of a previously unknown mechanism for preribosome accumulation at the nuclear periphery in budding yeast. We propose that this might be a strategy to expedite the delivery of ribosomes to the growing bud. © 2017 Moriggi et al.; Published by Cold Spring Harbor Laboratory Press for the RNA Society.

  14. Frequent and efficient use of the sister chromatid for DNA double-strand break repair during budding yeast meiosis.

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    Tamara Goldfarb

    Full Text Available Recombination between homologous chromosomes of different parental origin (homologs is necessary for their accurate segregation during meiosis. It has been suggested that meiotic inter-homolog recombination is promoted by a barrier to inter-sister-chromatid recombination, imposed by meiosis-specific components of the chromosome axis. Consistent with this, measures of Holliday junction-containing recombination intermediates (joint molecules [JMs] show a strong bias towards inter-homolog and against inter-sister JMs. However, recombination between sister chromatids also has an important role in meiosis. The genomes of diploid organisms in natural populations are highly polymorphic for insertions and deletions, and meiotic double-strand breaks (DSBs that form within such polymorphic regions must be repaired by inter-sister recombination. Efforts to study inter-sister recombination during meiosis, in particular to determine recombination frequencies and mechanisms, have been constrained by the inability to monitor the products of inter-sister recombination. We present here molecular-level studies of inter-sister recombination during budding yeast meiosis. We examined events initiated by DSBs in regions that lack corresponding sequences on the homolog, and show that these DSBs are efficiently repaired by inter-sister recombination. This occurs with the same timing as inter-homolog recombination, but with reduced (2- to 3-fold yields of JMs. Loss of the meiotic-chromosome-axis-associated kinase Mek1 accelerates inter-sister DSB repair and markedly increases inter-sister JM frequencies. Furthermore, inter-sister JMs formed in mek1Δ mutants are preferentially lost, while inter-homolog JMs are maintained. These findings indicate that inter-sister recombination occurs frequently during budding yeast meiosis, with the possibility that up to one-third of all recombination events occur between sister chromatids. We suggest that a Mek1-dependent reduction in

  15. Affected chromosome homeostasis and genomic instability of clonal yeast cultures.

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    Adamczyk, Jagoda; Deregowska, Anna; Panek, Anita; Golec, Ewelina; Lewinska, Anna; Wnuk, Maciej

    2016-05-01

    Yeast cells originating from one single colony are considered genotypically and phenotypically identical. However, taking into account the cellular heterogeneity, it seems also important to monitor cell-to-cell variations within a clone population. In the present study, a comprehensive yeast karyotype screening was conducted using single chromosome comet assay. Chromosome-dependent and mutation-dependent changes in DNA (DNA with breaks or with abnormal replication intermediates) were studied using both single-gene deletion haploid mutants (bub1, bub2, mad1, tel1, rad1 and tor1) and diploid cells lacking one active gene of interest, namely BUB1/bub1, BUB2/bub2, MAD1/mad1, TEL1/tel1, RAD1/rad1 and TOR1/tor1 involved in the control of cell cycle progression, DNA repair and the regulation of longevity. Increased chromosome fragility and replication stress-mediated chromosome abnormalities were correlated with elevated incidence of genomic instability, namely aneuploid events-disomies, monosomies and to a lesser extent trisomies as judged by in situ comparative genomic hybridization (CGH). The tor1 longevity mutant with relatively balanced chromosome homeostasis was found the most genomically stable among analyzed mutants. During clonal yeast culture, spontaneously formed abnormal chromosome structures may stimulate changes in the ploidy state and, in turn, promote genomic heterogeneity. These alterations may be more accented in selected mutated genetic backgrounds, namely in yeast cells deficient in proper cell cycle regulation and DNA repair.

  16. Effect of chromosome tethering on nuclear organization in yeast.

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    Barış Avşaroğlu

    Full Text Available Interphase chromosomes in Saccharomyces cerevisiae are tethered to the nuclear envelope at their telomeres and to the spindle pole body (SPB at their centromeres. Using a polymer model of yeast chromosomes that includes these interactions, we show theoretically that telomere attachment to the nuclear envelope is a major determinant of gene positioning within the nucleus only for genes within 10 kb of the telomeres. We test this prediction by measuring the distance between the SPB and the silent mating locus (HML on chromosome III in wild-type and mutant yeast strains that contain altered chromosome-tethering interactions. In wild-type yeast cells we find that disruption of the telomere tether does not dramatically change the position of HML with respect to the SPB, in agreement with theoretical predictions. Alternatively, using a mutant strain with a synthetic tether that localizes an HML-proximal site to the nuclear envelope, we find a significant change in the SPB-HML distance, again as predicted by theory. Our study quantifies the importance of tethering at telomeres on the organization of interphase chromosomes in yeast, which has been shown to play a significant role in determining chromosome function such as gene expression and recombination.

  17. Profiling DNA damage-induced phosphorylation in budding yeast reveals diverse signaling networks.

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    Zhou, Chunshui; Elia, Andrew E H; Naylor, Maria L; Dephoure, Noah; Ballif, Bryan A; Goel, Gautam; Xu, Qikai; Ng, Aylwin; Chou, Danny M; Xavier, Ramnik J; Gygi, Steven P; Elledge, Stephen J

    2016-06-28

    The DNA damage response (DDR) is regulated by a protein kinase signaling cascade that orchestrates DNA repair and other processes. Identifying the substrate effectors of these kinases is critical for understanding the underlying physiology and mechanism of the response. We have used quantitative mass spectrometry to profile DDR-dependent phosphorylation in budding yeast and genetically explored the dependency of these phosphorylation events on the DDR kinases MEC1, RAD53, CHK1, and DUN1. Based on these screens, a database containing many novel DDR-regulated phosphorylation events has been established. Phosphorylation of many of these proteins has been validated by quantitative peptide phospho-immunoprecipitation and examined for functional relevance to the DDR through large-scale analysis of sensitivity to DNA damage in yeast deletion strains. We reveal a link between DDR signaling and the metabolic pathways of inositol phosphate and phosphatidyl inositol synthesis, which are required for resistance to DNA damage. We also uncover links between the DDR and TOR signaling as well as translation regulation. Taken together, these data shed new light on the organization of DDR signaling in budding yeast.

  18. Intersection between the regulators of sister chromatid cohesion establishment and maintenance in budding yeast indicates a multi-step mechanism.

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    Noble, Daniel; Kenna, Margaret A; Dix, Melissa; Skibbens, Robert V; Unal, Elçin; Guacci, Vincent

    2006-11-01

    Sister chromatid cohesion is established during S phase and maintained until anaphase. The cohesin complex (Mcd1p/Scc1p, Smc1p, Smc3p Irr1p/Scc3p in budding yeast) serves a structural role as it is required at all times when cohesion exists. Pds5p colocalizes temporally and spatially with cohesin on chromosomes but is thought to serve as a regulator of cohesion maintenance during mitosis. In contrast, Ctf7p/Eco1p is required during S phase for establishment but is not required during mitosis. Here we provide genetic and biochemical evidence that the pathways of cohesion establishment and maintenance are intimately linked. Our results show that mutants in ctf7 and pds5 are synthetically lethal. Moreover, over-expression of either CTF7 or PDS5 exhibits reciprocal suppression of the other mutant's temperature sensitivity. The suppression by CTF7 is specific for pds5 mutants as CTF7 over-expression increases the temperature sensitivity of an mcd1 mutant but has no effect on smc1 or smc3 mutants. Three additional findings provide new insights into the process of cohesion establishment. First, over-expression of ctf7 alleles deficient in acetylase activity exhibit significantly reduced suppression of the pds5 mutant but exacerbated toxicity to the mcd1 mutant. Second, using chromosome spreads and chromatin immuno-precipitation, we find either cohesin complex or Pds5p chromosomal localization is altered in ctf7 mutants. Finally, biochemical analysis reveals that Ctf7p and Pds5p coimmunoprecipitate, which physically links these regulators of cohesion establishment and maintenance. We propose a model whereby Ctf7p and Pds5p cooperate to facilitate efficient establishment by mediating changes in cohesin complex on chromosomes after its deposition.

  19. Budding Yeast Rif1 Controls Genome Integrity by Inhibiting rDNA Replication.

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    Shyian, Maksym; Mattarocci, Stefano; Albert, Benjamin; Hafner, Lukas; Lezaja, Aleksandra; Costanzo, Michael; Boone, Charlie; Shore, David

    2016-11-01

    The Rif1 protein is a negative regulator of DNA replication initiation in eukaryotes. Here we show that budding yeast Rif1 inhibits DNA replication initiation at the rDNA locus. Absence of Rif1, or disruption of its interaction with PP1/Glc7 phosphatase, leads to more intensive rDNA replication. The effect of Rif1-Glc7 on rDNA replication is similar to that of the Sir2 deacetylase, and the two would appear to act in the same pathway, since the rif1Δ sir2Δ double mutant shows no further increase in rDNA replication. Loss of Rif1-Glc7 activity is also accompanied by an increase in rDNA repeat instability that again is not additive with the effect of sir2Δ. We find, in addition, that the viability of rif1Δ cells is severely compromised in combination with disruption of the MRX or Ctf4-Mms22 complexes, both of which are implicated in stabilization of stalled replication forks. Significantly, we show that removal of the rDNA replication fork barrier (RFB) protein Fob1, alleviation of replisome pausing by deletion of the Tof1/Csm3 complex, or a large deletion of the rDNA repeat array all rescue this synthetic growth defect of rif1Δ cells lacking in addition either MRX or Ctf4-Mms22 activity. These data suggest that the repression of origin activation by Rif1-Glc7 is important to avoid the deleterious accumulation of stalled replication forks at the rDNA RFB, which become lethal when fork stability is compromised. Finally, we show that Rif1-Glc7, unlike Sir2, has an important effect on origin firing outside of the rDNA locus that serves to prevent activation of the DNA replication checkpoint. Our results thus provide insights into a mechanism of replication control within a large repetitive chromosomal domain and its importance for the maintenance of genome stability. These findings may have important implications for metazoans, where large blocks of repetitive sequences are much more common.

  20. Budding Yeast Rif1 Controls Genome Integrity by Inhibiting rDNA Replication.

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    Maksym Shyian

    2016-11-01

    Full Text Available The Rif1 protein is a negative regulator of DNA replication initiation in eukaryotes. Here we show that budding yeast Rif1 inhibits DNA replication initiation at the rDNA locus. Absence of Rif1, or disruption of its interaction with PP1/Glc7 phosphatase, leads to more intensive rDNA replication. The effect of Rif1-Glc7 on rDNA replication is similar to that of the Sir2 deacetylase, and the two would appear to act in the same pathway, since the rif1Δ sir2Δ double mutant shows no further increase in rDNA replication. Loss of Rif1-Glc7 activity is also accompanied by an increase in rDNA repeat instability that again is not additive with the effect of sir2Δ. We find, in addition, that the viability of rif1Δ cells is severely compromised in combination with disruption of the MRX or Ctf4-Mms22 complexes, both of which are implicated in stabilization of stalled replication forks. Significantly, we show that removal of the rDNA replication fork barrier (RFB protein Fob1, alleviation of replisome pausing by deletion of the Tof1/Csm3 complex, or a large deletion of the rDNA repeat array all rescue this synthetic growth defect of rif1Δ cells lacking in addition either MRX or Ctf4-Mms22 activity. These data suggest that the repression of origin activation by Rif1-Glc7 is important to avoid the deleterious accumulation of stalled replication forks at the rDNA RFB, which become lethal when fork stability is compromised. Finally, we show that Rif1-Glc7, unlike Sir2, has an important effect on origin firing outside of the rDNA locus that serves to prevent activation of the DNA replication checkpoint. Our results thus provide insights into a mechanism of replication control within a large repetitive chromosomal domain and its importance for the maintenance of genome stability. These findings may have important implications for metazoans, where large blocks of repetitive sequences are much more common.

  1. Screening the budding yeast genome reveals unique factors affecting K2 toxin susceptibility.

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    Elena Servienė

    Full Text Available BACKGROUND: Understanding how biotoxins kill cells is of prime importance in biomedicine and the food industry. The budding yeast (S. cerevisiae killers serve as a convenient model to study the activity of biotoxins consistently supplying with significant insights into the basic mechanisms of virus-host cell interactions and toxin entry into eukaryotic target cells. K1 and K2 toxins are active at the cell wall, leading to the disruption of the plasma membrane and subsequent cell death by ion leakage. K28 toxin is active in the cell nucleus, blocking DNA synthesis and cell cycle progression, thereby triggering apoptosis. Genome-wide screens in the budding yeast S. cerevisiae identified several hundred effectors of K1 and K28 toxins. Surprisingly, no such screen had been performed for K2 toxin, the most frequent killer toxin among industrial budding yeasts. PRINCIPAL FINDINGS: We conducted several concurrent genome-wide screens in S. cerevisiae and identified 332 novel K2 toxin effectors. The effectors involved in K2 resistance and hypersensitivity largely map in distinct cellular pathways, including cell wall and plasma membrane structure/biogenesis and mitochondrial function for K2 resistance, and cell wall stress signaling and ion/pH homeostasis for K2 hypersensitivity. 70% of K2 effectors are different from those involved in K1 or K28 susceptibility. SIGNIFICANCE: Our work demonstrates that despite the fact that K1 and K2 toxins share some aspects of their killing strategies, they largely rely on different sets of effectors. Since the vast majority of the host factors identified here is exclusively active towards K2, we conclude that cells have acquired a specific K2 toxin effectors set. Our work thus indicates that K1 and K2 have elaborated different biological pathways and provides a first step towards the detailed characterization of K2 mode of action.

  2. Aneuploidy as a cause of impaired chromatin silencing and mating-type specification in budding yeast.

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    Mulla, Wahid A; Seidel, Chris W; Zhu, Jin; Tsai, Hung-Ji; Smith, Sarah E; Singh, Pushpendra; Bradford, William D; McCroskey, Scott; Nelliat, Anjali R; Conkright, Juliana; Peak, Allison; Malanowski, Kathryn E; Perera, Anoja G; Li, Rong

    2017-08-25

    Aneuploidy and epigenetic alterations have long been associated with carcinogenesis, but it was unknown whether aneuploidy could disrupt the epigenetic states required for cellular differentiation. In this study, we found that ~3% of random aneuploid karyotypes in yeast disrupt the stable inheritance of silenced chromatin during cell proliferation. Karyotype analysis revealed that this phenotype was significantly correlated with gains of chromosomes III and X. Chromosome X disomy alone was sufficient to disrupt chromatin silencing and yeast mating-type identity as indicated by a lack of growth response to pheromone. The silencing defect was not limited to cryptic mating type loci and was associated with broad changes in histone modifications and chromatin localization of Sir2 histone deacetylase. The chromatin-silencing defect of disome X can be partially recapitulated by an extra copy of several genes on chromosome X. These results suggest that aneuploidy can directly cause epigenetic instability and disrupt cellular differentiation.

  3. Measuring dynamic changes in histone modifications and nucleosome density during activated transcription in budding yeast.

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    Govind, Chhabi K; Ginsburg, Daniel; Hinnebusch, Alan G

    2012-01-01

    Chromatin immunoprecipitation is widely utilized to determine the in vivo binding of factors that regulate transcription. This procedure entails formaldehyde-mediated cross-linking of proteins and isolation of soluble chromatin followed by shearing. The fragmented chromatin is subjected to immunoprecipitation using antibodies against the protein of interest and the associated DNA is identified using quantitative PCR. Since histones are posttranslationally modified during transcription, this technique can be effectively used to determine the changes in histone modifications that occur during transcription. In this paper, we describe a detailed methodology to determine changes in histone modifications in budding yeast that takes into account reductions in nucleosome.

  4. Budding yeast dma proteins control septin dynamics and the spindle position checkpoint by promoting the recruitment of the Elm1 kinase to the bud neck.

    Science.gov (United States)

    Merlini, Laura; Fraschini, Roberta; Boettcher, Barbara; Barral, Yves; Lucchini, Giovanna; Piatti, Simonetta

    2012-01-01

    The first step towards cytokinesis in budding yeast is the assembly of a septin ring at the future site of bud emergence. Integrity of this ring is crucial for cytokinesis, proper spindle positioning, and the spindle position checkpoint (SPOC). This checkpoint delays mitotic exit and cytokinesis as long as the anaphase spindle does not properly align with the division axis. SPOC signalling requires the Kin4 protein kinase and the Kin4-regulating Elm1 kinase, which also controls septin dynamics. Here, we show that the two redundant ubiquitin-ligases Dma1 and Dma2 control septin dynamics and the SPOC by promoting the efficient recruitment of Elm1 to the bud neck. Indeed, dma1 dma2 mutant cells show reduced levels of Elm1 at the bud neck and Elm1-dependent activation of Kin4. Artificial recruitment of Elm1 to the bud neck of the same cells is sufficient to re-establish a normal septin ring, proper spindle positioning, and a proficient SPOC response in dma1 dma2 cells. Altogether, our data indicate that septin dynamics and SPOC function are intimately linked and support the idea that integrity of the bud neck is crucial for SPOC signalling.

  5. Budding yeast dma proteins control septin dynamics and the spindle position checkpoint by promoting the recruitment of the Elm1 kinase to the bud neck.

    Directory of Open Access Journals (Sweden)

    Laura Merlini

    Full Text Available The first step towards cytokinesis in budding yeast is the assembly of a septin ring at the future site of bud emergence. Integrity of this ring is crucial for cytokinesis, proper spindle positioning, and the spindle position checkpoint (SPOC. This checkpoint delays mitotic exit and cytokinesis as long as the anaphase spindle does not properly align with the division axis. SPOC signalling requires the Kin4 protein kinase and the Kin4-regulating Elm1 kinase, which also controls septin dynamics. Here, we show that the two redundant ubiquitin-ligases Dma1 and Dma2 control septin dynamics and the SPOC by promoting the efficient recruitment of Elm1 to the bud neck. Indeed, dma1 dma2 mutant cells show reduced levels of Elm1 at the bud neck and Elm1-dependent activation of Kin4. Artificial recruitment of Elm1 to the bud neck of the same cells is sufficient to re-establish a normal septin ring, proper spindle positioning, and a proficient SPOC response in dma1 dma2 cells. Altogether, our data indicate that septin dynamics and SPOC function are intimately linked and support the idea that integrity of the bud neck is crucial for SPOC signalling.

  6. Cleavage of the SUN-domain protein Mps3 at its N-terminus regulates centrosome disjunction in budding yeast meiosis

    Science.gov (United States)

    Koch, Bailey A.; Han, Xuemei

    2017-01-01

    Centrosomes organize microtubules and are essential for spindle formation and chromosome segregation during cell division. Duplicated centrosomes are physically linked, but how this linkage is dissolved remains unclear. Yeast centrosomes are tethered by a nuclear-envelope-attached structure called the half-bridge, whose components have mammalian homologues. We report here that cleavage of the half-bridge protein Mps3 promotes accurate centrosome disjunction in budding yeast. Mps3 is a single-pass SUN-domain protein anchored at the inner nuclear membrane and concentrated at the nuclear side of the half-bridge. Using the unique feature in yeast meiosis that centrosomes are linked for hours before their separation, we have revealed that Mps3 is cleaved at its nucleus-localized N-terminal domain, the process of which is regulated by its phosphorylation at serine 70. Cleavage of Mps3 takes place at the yeast centrosome and requires proteasome activity. We show that noncleavable Mps3 (Mps3-nc) inhibits centrosome separation during yeast meiosis. In addition, overexpression of mps3-nc in vegetative yeast cells also inhibits centrosome separation and is lethal. Our findings provide a genetic mechanism for the regulation of SUN-domain protein-mediated activities, including centrosome separation, by irreversible protein cleavage at the nuclear periphery. PMID:28609436

  7. Cleavage of the SUN-domain protein Mps3 at its N-terminus regulates centrosome disjunction in budding yeast meiosis.

    Directory of Open Access Journals (Sweden)

    Ping Li

    2017-06-01

    Full Text Available Centrosomes organize microtubules and are essential for spindle formation and chromosome segregation during cell division. Duplicated centrosomes are physically linked, but how this linkage is dissolved remains unclear. Yeast centrosomes are tethered by a nuclear-envelope-attached structure called the half-bridge, whose components have mammalian homologues. We report here that cleavage of the half-bridge protein Mps3 promotes accurate centrosome disjunction in budding yeast. Mps3 is a single-pass SUN-domain protein anchored at the inner nuclear membrane and concentrated at the nuclear side of the half-bridge. Using the unique feature in yeast meiosis that centrosomes are linked for hours before their separation, we have revealed that Mps3 is cleaved at its nucleus-localized N-terminal domain, the process of which is regulated by its phosphorylation at serine 70. Cleavage of Mps3 takes place at the yeast centrosome and requires proteasome activity. We show that noncleavable Mps3 (Mps3-nc inhibits centrosome separation during yeast meiosis. In addition, overexpression of mps3-nc in vegetative yeast cells also inhibits centrosome separation and is lethal. Our findings provide a genetic mechanism for the regulation of SUN-domain protein-mediated activities, including centrosome separation, by irreversible protein cleavage at the nuclear periphery.

  8. Effect of Chromosome Tethering on Nuclear Organization in Yeast

    OpenAIRE

    Barış Avşaroğlu; Gabriel Bronk; Susannah Gordon-Messer; Jungoh Ham; Debra A Bressan; Haber, James E; Jane Kondev

    2014-01-01

    Interphase chromosomes in Saccharomyces cerevisiae are tethered to the nuclear envelope at their telomeres and to the spindle pole body (SPB) at their centromeres. Using a polymer model of yeast chromosomes that includes these interactions, we show theoretically that telomere attachment to the nuclear envelope is a major determinant of gene positioning within the nucleus only for genes within 10 kb of the telomeres. We test this prediction by measuring the distance between the SPB and the sil...

  9. Confinement to Organelle-Associated Inclusion Structures Mediates Asymmetric Inheritance of Aggregated Protein in Budding Yeast

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    Rachel Spokoini

    2012-10-01

    Full Text Available The division of the S. cerevisiae budding yeast, which produces one mother cell and one daughter cell, is asymmetric with respect to aging. Remarkably, the asymmetry of yeast aging coincides with asymmetric inheritance of damaged and aggregated proteins by the mother cell. Here, we show that misfolded proteins are retained in the mother cell by being sequestered in juxtanuclear quality control compartment (JUNQ and insoluble protein deposit (IPOD inclusions, which are attached to organelles. Upon exposure to stress, misfolded proteins accumulate in stress foci that must be disaggregated by Hsp104 in order to be degraded or processed to JUNQ and IPOD. Cells that fail to deliver aggregates to an inclusion pass on aggregates to subsequent generations.

  10. The yeast prefoldin-like URI-orthologue Bud27 associates with the RSC nucleosome remodeler and modulates transcription.

    Science.gov (United States)

    Mirón-García, María Carmen; Garrido-Godino, Ana Isabel; Martínez-Fernández, Verónica; Fernández-Pevida, Antonio; Cuevas-Bermúdez, Abel; Martín-Expósito, Manuel; Chávez, Sebastián; de la Cruz, Jesús; Navarro, Francisco

    2014-09-01

    Bud27, the yeast orthologue of human URI/RMP, is a member of the prefoldin-like family of ATP-independent molecular chaperones. It has recently been shown to mediate the assembly of the three RNA polymerases in an Rpb5-dependent manner. In this work, we present evidence of Bud27 modulating RNA pol II transcription elongation. We show that Bud27 associates with RNA pol II phosphorylated forms (CTD-Ser5P and CTD-Ser2P), and that its absence affects RNA pol II occupancy of transcribed genes. We also reveal that Bud27 associates in vivo with the Sth1 component of the chromatin remodeling complex RSC and mediates its association with RNA pol II. Our data suggest that Bud27, in addition of contributing to Rpb5 folding within the RNA polymerases, also participates in the correct assembly of other chromatin-associated protein complexes, such as RSC, thereby modulating their activity.

  11. Ingression Progression Complexes Control Extracellular Matrix Remodelling during Cytokinesis in Budding Yeast

    Science.gov (United States)

    Foltman, Magdalena; Molist, Iago; Arcones, Irene; Sacristan, Carlos; Filali-Mouncef, Yasmina; Roncero, Cesar; Sanchez-Diaz, Alberto

    2016-01-01

    Eukaryotic cells must coordinate contraction of the actomyosin ring at the division site together with ingression of the plasma membrane and remodelling of the extracellular matrix (ECM) to support cytokinesis, but the underlying mechanisms are still poorly understood. In eukaryotes, glycosyltransferases that synthesise ECM polysaccharides are emerging as key factors during cytokinesis. The budding yeast chitin synthase Chs2 makes the primary septum, a special layer of the ECM, which is an essential process during cell division. Here we isolated a group of actomyosin ring components that form complexes together with Chs2 at the cleavage site at the end of the cell cycle, which we named ‘ingression progression complexes’ (IPCs). In addition to type II myosin, the IQGAP protein Iqg1 and Chs2, IPCs contain the F-BAR protein Hof1, and the cytokinesis regulators Inn1 and Cyk3. We describe the molecular mechanism by which chitin synthase is activated by direct association of the C2 domain of Inn1, and the transglutaminase-like domain of Cyk3, with the catalytic domain of Chs2. We used an experimental system to find a previously unanticipated role for the C-terminus of Inn1 in preventing the untimely activation of Chs2 at the cleavage site until Cyk3 releases the block on Chs2 activity during late mitosis. These findings support a model for the co-ordinated regulation of cell division in budding yeast, in which IPCs play a central role. PMID:26891268

  12. Ingression Progression Complexes Control Extracellular Matrix Remodelling during Cytokinesis in Budding Yeast.

    Directory of Open Access Journals (Sweden)

    Magdalena Foltman

    2016-02-01

    Full Text Available Eukaryotic cells must coordinate contraction of the actomyosin ring at the division site together with ingression of the plasma membrane and remodelling of the extracellular matrix (ECM to support cytokinesis, but the underlying mechanisms are still poorly understood. In eukaryotes, glycosyltransferases that synthesise ECM polysaccharides are emerging as key factors during cytokinesis. The budding yeast chitin synthase Chs2 makes the primary septum, a special layer of the ECM, which is an essential process during cell division. Here we isolated a group of actomyosin ring components that form complexes together with Chs2 at the cleavage site at the end of the cell cycle, which we named 'ingression progression complexes' (IPCs. In addition to type II myosin, the IQGAP protein Iqg1 and Chs2, IPCs contain the F-BAR protein Hof1, and the cytokinesis regulators Inn1 and Cyk3. We describe the molecular mechanism by which chitin synthase is activated by direct association of the C2 domain of Inn1, and the transglutaminase-like domain of Cyk3, with the catalytic domain of Chs2. We used an experimental system to find a previously unanticipated role for the C-terminus of Inn1 in preventing the untimely activation of Chs2 at the cleavage site until Cyk3 releases the block on Chs2 activity during late mitosis. These findings support a model for the co-ordinated regulation of cell division in budding yeast, in which IPCs play a central role.

  13. Cdc14 phosphatase directs centrosome re-duplication at the meiosis I to meiosis II transition in budding yeast

    Science.gov (United States)

    2017-01-01

    Background Gametes are generated through a specialized cell division called meiosis, in which ploidy is reduced by half because two consecutive rounds of chromosome segregation, meiosis I and meiosis II, occur without intervening DNA replication. This contrasts with the mitotic cell cycle where DNA replication and chromosome segregation alternate to maintain the same ploidy. At the end of mitosis, CDKs are inactivated. This low CDK state in late mitosis/G1 allows for critical preparatory events for DNA replication and centrosome/spindle pole body (SPB) duplication. However, their execution is inhibited until S phase, where further preparatory events are also prevented. This “licensing” ensures that both the chromosomes and the centrosomes/SPBs replicate exactly once per cell cycle, thereby maintaining constant ploidy. Crucially, between meiosis I and meiosis II, centrosomes/SPBs must be re-licensed, but DNA re-replication must be avoided. In budding yeast, the Cdc14 protein phosphatase triggers CDK down regulation to promote exit from mitosis. Cdc14 also regulates the meiosis I to meiosis II transition, though its mode of action has remained unclear. Methods Fluorescence and electron microscopy was combined with proteomics to probe SPB duplication in cells with inactive or hyperactive Cdc14. Results We demonstrate that Cdc14 ensures two successive nuclear divisions by re-licensing SPBs at the meiosis I to meiosis II transition. We show that Cdc14 is asymmetrically enriched on a single SPB during anaphase I and provide evidence that this enrichment promotes SPB re-duplication. Cells with impaired Cdc14 activity fail to promote extension of the SPB half-bridge, the initial step in morphogenesis of a new SPB. Conversely, cells with hyper-active Cdc14 duplicate SPBs, but fail to induce their separation. Conclusion Our findings implicate reversal of key CDK-dependent phosphorylations in the differential licensing of cyclical events at the meiosis I to meiosis I

  14. An insight into the complex prion-prion interaction network in the budding yeast Saccharomyces cerevisiae.

    Science.gov (United States)

    Du, Zhiqiang; Valtierra, Stephanie; Li, Liming

    2014-01-01

    The budding yeast Saccharomyces cerevisiae is a valuable model system for studying prion-prion interactions as it contains multiple prion proteins. A recent study from our laboratory showed that the existence of Swi1 prion ([SWI(+)]) and overproduction of Swi1 can have strong impacts on the formation of 2 other extensively studied yeast prions, [PSI(+)] and [PIN(+)] ([RNQ(+)]) (Genetics, Vol. 197, 685-700). We showed that a single yeast cell is capable of harboring at least 3 heterologous prion elements and these prions can influence each other's appearance positively and/or negatively. We also showed that during the de novo [PSI(+)] formation process upon Sup35 overproduction, the aggregation patterns of a preexisting inducer ([RNQ(+)] or [SWI(+)]) can undergo significant remodeling from stably transmitted dot-shaped aggregates to aggregates that co-localize with the newly formed Sup35 aggregates that are ring/ribbon/rod- shaped. Such co-localization disappears once the newly formed [PSI(+)] prion stabilizes. Our finding provides strong evidence supporting the "cross-seeding" model for prion-prion interactions and confirms earlier reports that the interactions among different prions and their prion proteins mostly occur at the initiation stages of prionogenesis. Our results also highlight a complex prion interaction network in yeast. We believe that elucidating the mechanism underlying the yeast prion-prion interaction network will not only provide insight into the process of prion de novo generation and propagation in yeast but also shed light on the mechanisms that govern protein misfolding, aggregation, and amyloidogenesis in higher eukaryotes.

  15. Quantitative Analysis of Pac1/LIS1-mediated Dynein Targeting: Implications for Regulation of Dynein Activity in Budding Yeast

    OpenAIRE

    Markus, Steven M.; Plevock, Karen M.; St. Germain, Bryan J.; Punch, Jesse J.; Meaden, Christopher W.; Lee, Wei-Lih

    2011-01-01

    LIS1 is a critical regulator of dynein function during mitosis and organelle transport. Here, we investigated how Pac1, the budding yeast LIS1 homologue, regulates dynein targeting and activity during nuclear migration. We show that Pac1 and Dyn1 (dynein heavy chain) are dependent upon each other and upon Bik1 (budding yeast CLIP-170 homologue) for plus end localization, whereas Bik1 is independent of either. Dyn1, Pac1 and Bik1 interact in vivo at the plus ends, where an excess amount of Bik...

  16. Microtubule dynamics from mating through the first zygotic division in the budding yeast Saccharomyces cerevisiae.

    Science.gov (United States)

    Maddox, P; Chin, E; Mallavarapu, A; Yeh, E; Salmon, E D; Bloom, K

    1999-03-08

    We have used time-lapse digital imaging microscopy to examine cytoplasmic astral microtubules (Mts) and spindle dynamics during the mating pathway in budding yeast Saccharomyces cerevisiae. Mating begins when two cells of opposite mating type come into proximity. The cells arrest in the G1 phase of the cell cycle and grow a projection towards one another forming a shmoo projection. Imaging of microtubule dynamics with green fluorescent protein (GFP) fusions to dynein or tubulin revealed that the nucleus and spindle pole body (SPB) became oriented and tethered to the shmoo tip by a Mt-dependent search and capture mechanism. Dynamically unstable astral Mts were captured at the shmoo tip forming a bundle of three or four astral Mts. This bundle changed length as the tethered nucleus and SPB oscillated toward and away from the shmoo tip at growth and shortening velocities typical of free plus end astral Mts (approximately 0.5 micrometer/min). Fluorescent fiduciary marks in Mt bundles showed that Mt growth and shortening occurred primarily at the shmoo tip, not the SPB. This indicates that Mt plus end assembly/disassembly was coupled to pushing and pulling of the nucleus. Upon cell fusion, a fluorescent bar of Mts was formed between the two shmoo tip bundles, which slowly shortened (0.23 +/- 0.07 micrometer/min) as the two nuclei and their SPBs came together and fused (karyogamy). Bud emergence occurred adjacent to the fused SPB approximately 30 min after SPB fusion. During the first mitosis, the SPBs separated as the spindle elongated at a constant velocity (0.75 micrometer/min) into the zygotic bud. There was no indication of a temporal delay at the 2-micrometer stage of spindle morphogenesis or a lag in Mt nucleation by replicated SPBs as occurs in vegetative mitosis implying a lack of normal checkpoints. Thus, the shmoo tip appears to be a new model system for studying Mt plus end dynamic attachments and much like higher eukaryotes, the first mitosis after haploid

  17. The budding yeast nuclear envelope adjacent to the nucleolus serves as a membrane sink during mitotic delay.

    Science.gov (United States)

    Witkin, Keren L; Chong, Yolanda; Shao, Sichen; Webster, Micah T; Lahiri, Sujoy; Walters, Alison D; Lee, Brandon; Koh, Judice L Y; Prinz, William A; Andrews, Brenda J; Cohen-Fix, Orna

    2012-06-19

    The mechanisms that dictate nuclear shape are largely unknown. Here we screened the budding yeast deletion collection for mutants with abnormal nuclear shape. A common phenotype was the appearance of a nuclear extension, particularly in mutants in DNA repair and chromosome segregation genes. Our data suggest that these mutations led to the abnormal nuclear morphology indirectly, by causing a checkpoint-induced cell-cycle delay. Indeed, delaying cells in mitosis by other means also led to the appearance of nuclear extensions, whereas inactivating the DNA damage checkpoint pathway in a DNA repair mutant reduced the fraction of cells with nuclear extensions. Formation of a nuclear extension was specific to a mitotic delay, because cells arrested in S or G2 had round nuclei. Moreover, the nuclear extension always coincided with the nucleolus, while the morphology of the DNA mass remained largely unchanged. Finally, we found that phospholipid synthesis continued unperturbed when cells delayed in mitosis, and inhibiting phospholipid synthesis abolished the formation of nuclear extensions. Our data suggest a mechanism that promotes nuclear envelope expansion during mitosis. When mitotic progression is delayed, cells sequester the added membrane to the nuclear envelope associated with the nucleolus, possibly to avoid disruption of intranuclear organization.

  18. Specificity of mutations induced by carbon ions in budding yeast Saccharomyces cerevisiae

    Energy Technology Data Exchange (ETDEWEB)

    Matuo, Youichirou [Graduate School of Engineering, Osaka University, Yamada-oka 2-1, Suita, Osaka 565-0871 (Japan); Nishijima, Shigehiro [Graduate School of Engineering, Osaka University, Yamada-oka 2-1, Suita, Osaka 565-0871 (Japan); Hase, Yoshihiro [Radiation-Applied Biology Division, Quantum Beam Science Directorate, Japan Atomic Energy Agency (JAEA), Watanuki-machi 1233, Takasaki, Gunma 370-1292 (Japan); Sakamoto, Ayako [Radiation-Applied Biology Division, Quantum Beam Science Directorate, Japan Atomic Energy Agency (JAEA), Watanuki-machi 1233, Takasaki, Gunma 370-1292 (Japan); Tanaka, Atsushi [Radiation-Applied Biology Division, Quantum Beam Science Directorate, Japan Atomic Energy Agency (JAEA), Watanuki-machi 1233, Takasaki, Gunma 370-1292 (Japan); Shimizu, Kikuo [Radioisotope Research Center, Osaka University, Yamada-oka 2-4, Suita, Osaka 565-0871 (Japan)]. E-mail: shimizu@rirc.osaka-u.ac.jp

    2006-12-01

    To investigate the nature of mutations induced by accelerated ions in eukaryotic cells, the effects of carbon-ion irradiation were compared with those of {gamma}-ray irradiation in the budding yeast Saccharomyces cerevisiae. The mutational effect and specificity of carbon-ion beams were studied in the URA3 gene of the yeast. Our experiments showed that the carbon ions generated more than 10 times the number of mutations induced by {gamma}-rays, and that the types of base changes induced by carbon ions include transversions (68.7%), transitions (13.7%) and deletions/insertions (17.6%). The transversions were mainly G:C {sup {yields}} T:A, and all the transitions were G:C {sup {yields}} A:T. In comparison with the surrounding sequence context of mutational base sites, the C residues in the 5'-AC(A/T)-3' sequence were found to be easily changed. Large deletions and duplications were not observed, whereas ion-induced mutations in Arabidopsis thaliana were mainly short deletions and rearrangements. The remarkable feature of yeast mutations induced by carbon ions was that the mutation sites were localized near the linker regions of nucleosomes, whereas mutations induced by {gamma}-ray irradiation were located uniformly throughout the gene.

  19. Complete DNA sequence of Kuraishia capsulata illustrates novel genomic features among budding yeasts (Saccharomycotina).

    Science.gov (United States)

    Morales, Lucia; Noel, Benjamin; Porcel, Betina; Marcet-Houben, Marina; Hullo, Marie-Francoise; Sacerdot, Christine; Tekaia, Fredj; Leh-Louis, Véronique; Despons, Laurence; Khanna, Varun; Aury, Jean-Marc; Barbe, Valérie; Couloux, Arnaud; Labadie, Karen; Pelletier, Eric; Souciet, Jean-Luc; Boekhout, Teun; Gabaldon, Toni; Wincker, Patrick; Dujon, Bernard

    2013-01-01

    The numerous yeast genome sequences presently available provide a rich source of information for functional as well as evolutionary genomics but unequally cover the large phylogenetic diversity of extant yeasts. We present here the complete sequence of the nuclear genome of the haploid-type strain of Kuraishia capsulata (CBS1993(T)), a nitrate-assimilating Saccharomycetales of uncertain taxonomy, isolated from tunnels of insect larvae underneath coniferous barks and characterized by its copious production of extracellular polysaccharides. The sequence is composed of seven scaffolds, one per chromosome, totaling 11.4 Mb and containing 6,029 protein-coding genes, ~13.5% of which being interrupted by introns. This GC-rich yeast genome (45.7%) appears phylogenetically related with the few other nitrate-assimilating yeasts sequenced so far, Ogataea polymorpha, O. parapolymorpha, and Dekkera bruxellensis, with which it shares a very reduced number of tRNA genes, a novel tRNA sparing strategy, and a common nitrate assimilation cluster, three specific features to this group of yeasts. Centromeres were recognized in GC-poor troughs of each scaffold. The strain bears MAT alpha genes at a single MAT locus and presents a significant degree of conservation with Saccharomyces cerevisiae genes, suggesting that it can perform sexual cycles in nature, although genes involved in meiosis were not all recognized. The complete absence of conservation of synteny between K. capsulata and any other yeast genome described so far, including the three other nitrate-assimilating species, validates the interest of this species for long-range evolutionary genomic studies among Saccharomycotina yeasts.

  20. Complete DNA Sequence of Kuraishia capsulata Illustrates Novel Genomic Features among Budding Yeasts (Saccharomycotina)

    Science.gov (United States)

    Morales, Lucia; Noel, Benjamin; Porcel, Betina; Marcet-Houben, Marina; Hullo, Marie-Francoise; Sacerdot, Christine; Tekaia, Fredj; Leh-Louis, Véronique; Despons, Laurence; Khanna, Varun; Aury, Jean-Marc; Barbe, Valérie; Couloux, Arnaud; Labadie, Karen; Pelletier, Eric; Souciet, Jean-Luc; Boekhout, Teun; Gabaldon, Toni; Wincker, Patrick; Dujon, Bernard

    2013-01-01

    The numerous yeast genome sequences presently available provide a rich source of information for functional as well as evolutionary genomics but unequally cover the large phylogenetic diversity of extant yeasts. We present here the complete sequence of the nuclear genome of the haploid-type strain of Kuraishia capsulata (CBS1993T), a nitrate-assimilating Saccharomycetales of uncertain taxonomy, isolated from tunnels of insect larvae underneath coniferous barks and characterized by its copious production of extracellular polysaccharides. The sequence is composed of seven scaffolds, one per chromosome, totaling 11.4 Mb and containing 6,029 protein-coding genes, ∼13.5% of which being interrupted by introns. This GC-rich yeast genome (45.7%) appears phylogenetically related with the few other nitrate-assimilating yeasts sequenced so far, Ogataea polymorpha, O. parapolymorpha, and Dekkera bruxellensis, with which it shares a very reduced number of tRNA genes, a novel tRNA sparing strategy, and a common nitrate assimilation cluster, three specific features to this group of yeasts. Centromeres were recognized in GC-poor troughs of each scaffold. The strain bears MAT alpha genes at a single MAT locus and presents a significant degree of conservation with Saccharomyces cerevisiae genes, suggesting that it can perform sexual cycles in nature, although genes involved in meiosis were not all recognized. The complete absence of conservation of synteny between K. capsulata and any other yeast genome described so far, including the three other nitrate-assimilating species, validates the interest of this species for long-range evolutionary genomic studies among Saccharomycotina yeasts. PMID:24317973

  1. Update History of This Database - Budding yeast cDNA sequencing project | LSDB Archive [Life Science Database Archive metadata

    Lifescience Database Archive (English)

    Full Text Available [ Credits ] BLAST Search Image Search Home About Archive Update History Contact us ...Budding yeast cDNA sequencing project Update History of This Database Date Update contents 2010/03/29 Buddin...tio About This Database Database Description Download License Update History of This Database Site Policy | Contact Us Update History

  2. Sequential Feedback Induction Stabilizes the Phosphate Starvation Response in Budding Yeast

    Directory of Open Access Journals (Sweden)

    Noam Vardi

    2014-11-01

    Full Text Available Depletion of essential nutrients triggers regulatory programs that prolong cell growth and survival. Starvation-induced processes increase nutrient transport, mobilize nutrient storage, and recycle nutrients between cellular components. This leads to an effective increase in intracellular nutrients, which may act as a negative feedback that downregulates the starvation program. To examine how cells overcome this potential instability, we followed the transcription response of budding yeast transferred to medium lacking phosphate. Genes were induced in two temporal waves. The first wave was stably maintained and persisted even upon phosphate replenishment, indicating a positive feedback loop. This commitment was abolished after 2 hr with the induction of the second expression wave, coinciding with the reduction in cell growth rate. We show that the overall temporal stability of the expression response depends on the sequential pattern of gene induction. Our results emphasize the key role of gene expression dynamics in optimizing cellular adaptation.

  3. Commitment to meiosis: what determines the mode of division in budding yeast?

    Science.gov (United States)

    Simchen, Giora

    2009-02-01

    In budding yeast, commitment to meiosis is attained when meiotic cells cannot return to the mitotic cell cycle even if the triggering cue (nutrients deprivation) is withdrawn. Commitment is arrived at gradually, and different aspects of meiosis may be committed at different times. Cells become fully committed to meiosis at the end of Prophase I, long after DNA replication and just before the first meiotic division (M(I)). Whole-genome gene expression analysis has shown that committed cells have a distinct and rapid response to nutrients, and are not simply insulated from environmental signals. Thus becoming committed to meiosis is an active process. The cellular event most likely to be associated with commitment to meiosis is the separation of the duplicated spindle-pole bodies (SPBs) and the formation of the spindle. Commitment to the mitotic cell cycle is also associated with the separation of SPBs, although it occurs in G1, before DNA replication.

  4. Yeast pol4 promotes tel1-regulated chromosomal translocations.

    Directory of Open Access Journals (Sweden)

    Jose F Ruiz

    Full Text Available DNA double-strand breaks (DSBs are one of the most dangerous DNA lesions, since their erroneous repair by nonhomologous end-joining (NHEJ can generate harmful chromosomal rearrangements. PolX DNA polymerases are well suited to extend DSB ends that cannot be directly ligated due to their particular ability to bind to and insert nucleotides at the imperfect template-primer structures formed during NHEJ. Herein, we have devised genetic assays in yeast to induce simultaneous DSBs in different chromosomes in vivo. The repair of these breaks in trans could result in reciprocal chromosomal translocations that were dependent on classical Ku-dependent NHEJ. End-joining events leading to translocations were mainly based on the formation of short base pairing between 3'-overhanging DNA ends coupled to gap-filling DNA synthesis. A major proportion of these events were specifically dependent on yeast DNA polymerase Pol4 activity. In addition, we have discovered that Pol4-Thr(540 amino acid residue can be phosphorylated by Tel1/ATM kinase, which could modulate Pol4 activity during NHEJ. Our data suggest that the role of Tel1 in preventing break-induced chromosomal translocations can, to some extent, be due to its stimulating effect on gap-filling activity of Pol4 to repair DSBs in cis. Overall, this work provides further insight to the molecular mechanisms of DSB repair by NHEJ and presents a new perspective to the understanding of how chromosomal translocations are formed in eukaryotic cells.

  5. Unconventional genomic architecture in the budding yeast saccharomyces cerevisiae masks the nested antisense gene NAG1.

    Science.gov (United States)

    Ma, Jun; Dobry, Craig J; Krysan, Damian J; Kumar, Anuj

    2008-08-01

    The genomic architecture of the budding yeast Saccharomyces cerevisiae is typical of other eukaryotes in that genes are spatially organized into discrete and nonoverlapping units. Inherent in this organizational model is the assumption that protein-coding sequences do not overlap completely. Here, we present evidence to the contrary, defining a previously overlooked yeast gene, NAG1 (for nested antisense gene) nested entirely within the coding sequence of the YGR031W open reading frame in an antisense orientation on the opposite strand. NAG1 encodes a 19-kDa protein, detected by Western blotting of hemagglutinin (HA)-tagged Nag1p with anti-HA antibodies and by beta-galactosidase analysis of a NAG1-lacZ fusion. NAG1 is evolutionarily conserved as a unit with YGR031W in bacteria and fungi. Unlike the YGR031WP protein product, however, which localizes to the mitochondria, Nag1p localizes to the cell periphery, exhibiting properties consistent with those of a plasma membrane protein. Phenotypic analysis of a site-directed mutant (nag1-1) disruptive for NAG1 but silent with respect to YGR031W, defines a role for NAG1 in yeast cell wall biogenesis; microarray profiling of nag1-1 indicates decreased expression of genes contributing to cell wall organization, and the nag1-1 mutant is hypersensitive to the cell wall-perturbing agent calcofluor white. Furthermore, production of Nag1p is dependent upon the presence of the cell wall integrity pathway mitogen-activated protein kinase Slt2p and its downstream transcription factor Rlm1p. Thus, NAG1 is important for two reasons. First, it contributes to yeast cell wall biogenesis. Second, its genomic context is novel, raising the possibility that other nested protein-coding genes may exist in eukaryotic genomes.

  6. Analysis of Recombination and Chromosome Structure during Yeast Meiosis.

    Science.gov (United States)

    Börner, G Valentin; Cha, Rita S

    2015-11-02

    Meiosis is a diploid-specific differentiation program that consists of a single round of genome duplication followed by two rounds of chromosome segregation. These events result in halving of the genetic complement, which is a requirement for formation of haploid reproductive cells (i.e., spores in yeast and gametes in animals and plants). During meiosis I, homologous maternal and paternal chromosomes (homologs) pair and separate, whereas sister chromatids remain connected at the centromeres and separate during the second meiotic division. In most organisms, accurate homolog disjunction requires crossovers, which are formed as products of meiotic recombination. For the past two decades, studies of yeast meiosis have provided invaluable insights into evolutionarily conserved mechanisms of meiosis.

  7. Maintenance of cellular ATP level by caloric restriction correlates chronological survival of budding yeast

    Energy Technology Data Exchange (ETDEWEB)

    Choi, Joon-Seok; Lee, Cheol-Koo, E-mail: cklee2005@korea.ac.kr

    2013-09-13

    Highlights: •CR decreases total ROS and mitochondrial superoxide during the chronological aging. •CR does not affect the levels of oxidative damage on protein and DNA. •CR contributes extension of chronological lifespan by maintenance of ATP level -- Abstract: The free radical theory of aging emphasizes cumulative oxidative damage in the genome and intracellular proteins due to reactive oxygen species (ROS), which is a major cause for aging. Caloric restriction (CR) has been known as a representative treatment that prevents aging; however, its mechanism of action remains elusive. Here, we show that CR extends the chronological lifespan (CLS) of budding yeast by maintaining cellular energy levels. CR reduced the generation of total ROS and mitochondrial superoxide; however, CR did not reduce the oxidative damage in proteins and DNA. Subsequently, calorie-restricted yeast had higher mitochondrial membrane potential (MMP), and it sustained consistent ATP levels during the process of chronological aging. Our results suggest that CR extends the survival of the chronologically aged cells by improving the efficiency of energy metabolism for the maintenance of the ATP level rather than reducing the global oxidative damage of proteins and DNA.

  8. A pathway of targeted autophagy is induced by DNA damage in budding yeast

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    Eapen, Vinay V.; Waterman, David P.; Bernard, Amélie; Schiffmann, Nathan; Sayas, Enrich; Kamber, Roarke; Lemos, Brenda; Memisoglu, Gonen; Ang, Jessie; Mazella, Allison; Chuartzman, Silvia G.; Loewith, Robbie J.; Schuldiner, Maya; Denic, Vladimir; Klionsky, Daniel J.; Haber, James E.

    2017-01-01

    Autophagy plays a central role in the DNA damage response (DDR) by controlling the levels of various DNA repair and checkpoint proteins; however, how the DDR communicates with the autophagy pathway remains unknown. Using budding yeast, we demonstrate that global genotoxic damage or even a single unrepaired double-strand break (DSB) initiates a previously undescribed and selective pathway of autophagy that we term genotoxin-induced targeted autophagy (GTA). GTA requires the action primarily of Mec1/ATR and Rad53/CHEK2 checkpoint kinases, in part via transcriptional up-regulation of central autophagy proteins. GTA is distinct from starvation-induced autophagy. GTA requires Atg11, a central component of the selective autophagy machinery, but is different from previously described autophagy pathways. By screening a collection of ∼6,000 yeast mutants, we identified genes that control GTA but do not significantly affect rapamycin-induced autophagy. Overall, our findings establish a pathway of autophagy specific to the DNA damage response. PMID:28154131

  9. Evidence for widespread adaptive evolution of gene expression in budding yeast.

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    Fraser, Hunter B; Moses, Alan M; Schadt, Eric E

    2010-02-16

    Changes in gene expression have been proposed to underlie many, or even most, adaptive differences between species. Despite the increasing acceptance of this view, only a handful of cases of adaptive gene expression evolution have been demonstrated. To address this discrepancy, we introduce a simple test for lineage-specific selection on gene expression. Applying the test to genome-wide gene expression data from the budding yeast Saccharomyces cerevisiae, we find that hundreds of gene expression levels have been subject to lineage-specific selection. Comparing these findings with independent population genetic evidence of selective sweeps suggests that this lineage-specific selection has resulted in recent sweeps at over a hundred genes, most of which led to increased transcript levels. Examination of the implicated genes revealed a specific biochemical pathway--ergosterol biosynthesis--where the expression of multiple genes has been subject to selection for reduced levels. In sum, these results suggest that adaptive evolution of gene expression is common in yeast, that regulatory adaptation can occur at the level of entire pathways, and that similar genome-wide scans may be possible in other species, including humans.

  10. Formation of complex and unstable chromosomal translocations in yeast.

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    Kristina H Schmidt

    Full Text Available Genome instability, associated with chromosome breakage syndromes and most human cancers, is still poorly understood. In the yeast Saccharomyces cerevisiae, numerous genes with roles in the preservation of genome integrity have been identified. DNA-damage-checkpoint-deficient yeast cells that lack Sgs1, a RecQ-like DNA helicase related to the human Bloom's-syndrome-associated helicase BLM, show an increased rate of genome instability, and we have previously shown that they accumulate recurring chromosomal translocations between three similar genes, CAN1, LYP1 and ALP1. Here, the chromosomal location, copy number and sequence similarity of the translocation targets ALP1 and LYP1 were altered to gain insight into the formation of complex translocations. Among 844 clones with chromosomal rearrangements, 93 with various types of simple and complex translocations involving CAN1, LYP1 and ALP1 were identified. Breakpoint sequencing and mapping showed that the formation of complex translocation types is strictly dependent on the location of the initiating DNA break and revealed that complex translocations arise via a combination of interchromosomal translocation and template-switching, as well as from unstable dicentric intermediates. Template-switching occurred between sequences on the same chromosome, but was inhibited if the genes were transferred to different chromosomes. Unstable dicentric translocations continuously gave rise to clones with multiple translocations in various combinations, reminiscent of intratumor heterogeneity in human cancers. Base substitutions and evidence of DNA slippage near rearrangement breakpoints revealed that translocation formation can be accompanied by point mutations, and their presence in different translocation types within the same clone provides evidence that some of the different translocation types are derived from each other rather than being formed de novo. These findings provide insight into eukaryotic

  11. [CHL15--a new gene controlling the replication of chromosomes in saccharomycetes yeast: cloning, physical mapping, sequencing, and sequence analysis].

    Science.gov (United States)

    Kuprina, N Iu; Krol', E S; Koriabin, M Iu; Shestopalov, B V; Bliskovskiĭ, V V; Bannikov, V M; Gizatullin, R Z; Kirillov, A V; Kravtsov, V Iu; Zakhar'ev, V M

    1993-01-01

    We have analyzed the CHL15 gene, earlier identified in a screen for yeast mutants with increased loss of chromosome III and artificial circular and linear chromosomes in mitosis. Mutations in the CHL15 gene lead to a 100-fold increase in the rate of chromosome III loss per cell division and a 200-fold increase in the rate of marker homozygosis on this chromosome by mitotic recombination. Analysis of segregation of artificial circular minichromosome and artificially generated nonessential marker chromosome fragment indicated that sister chromatid loss (1:0 segregation) is a main reason of chromosome destabilization in the chl15-1 mutant. A genomic clone of CHL15 was isolated and used to map its physical position on chromosome XVI. Nucleotide sequence analysis of CHL15 revealed a 2.8-kb open reading frame with a 105-kD predicted protein sequence. At the N-terminal region of the protein sequences potentially able to form DNA-binding domains defined as zinc-fingers were found. The C-terminal region of the predicted protein displayed a similarity to sequence of regulatory proteins known as the helix-loop-helix (HLH) proteins. Data on partial deletion analysis suggest that the HLH domain is essential for the function of the CHL15 gene product. Analysis of the upstream untranslated region of CHL15 revealed the presence of the hexamer element, ACGCGT (an MluI restriction site) controlling both the periodic expression and coordinate regulation of the DNA synthesis genes in budding yeast. Deletion in the RAD52 gene, the product of which is involved in double-strand break/recombination repair and replication, leads to a considerable decrease in the growth rate of the chl15 mutant. We suggest that CHL15 is a new DNA synthesis gene in the yeast Saccharomyces cerevisiae.

  12. Genetic dissection of ethanol tolerance in the budding yeast Saccharomyces cerevisiae.

    Science.gov (United States)

    Hu, X H; Wang, M H; Tan, T; Li, J R; Yang, H; Leach, L; Zhang, R M; Luo, Z W

    2007-03-01

    Uncovering genetic control of variation in ethanol tolerance in natural populations of yeast Saccharomyces cerevisiae is essential for understanding the evolution of fermentation, the dominant lifestyle of the species, and for improving efficiency of selection for strains with high ethanol tolerance, a character of great economic value for the brewing and biofuel industries. To date, as many as 251 genes have been predicted to be involved in influencing this character. Candidacy of these genes was determined from a tested phenotypic effect following gene knockout, from an induced change in gene function under an ethanol stress condition, or by mutagenesis. This article represents the first genomics approach for dissecting genetic variation in ethanol tolerance between two yeast strains with a highly divergent trait phenotype. We developed a simple but reliable experimental protocol for scoring the phenotype and a set of STR/SNP markers evenly covering the whole genome. We created a mapping population comprising 319 segregants from crossing the parental strains. On the basis of the data sets, we find that the tolerance trait has a high heritability and that additive genetic variance dominates genetic variation of the trait. Segregation at five QTL detected has explained approximately 50% of phenotypic variation; in particular, the major QTL mapped on yeast chromosome 9 has accounted for a quarter of the phenotypic variation. We integrated the QTL analysis with the predicted candidacy of ethanol resistance genes and found that only a few of these candidates fall in the QTL regions.

  13. Time scale and dimension analysis of a budding yeast cell cycle model

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    Novák Béla

    2006-11-01

    Full Text Available Abstract Background The progress through the eukaryotic cell division cycle is driven by an underlying molecular regulatory network. Cell cycle progression can be considered as a series of irreversible transitions from one steady state to another in the correct order. Although this view has been put forward some time ago, it has not been quantitatively proven yet. Bifurcation analysis of a model for the budding yeast cell cycle has identified only two different steady states (one for G1 and one for mitosis using cell mass as a bifurcation parameter. By analyzing the same model, using different methods of dynamical systems theory, we provide evidence for transitions among several different steady states during the budding yeast cell cycle. Results By calculating the eigenvalues of the Jacobian of kinetic differential equations we have determined the stability of the cell cycle trajectories of the Chen model. Based on the sign of the real part of the eigenvalues, the cell cycle can be divided into excitation and relaxation periods. During an excitation period, the cell cycle control system leaves a formerly stable steady state and, accordingly, excitation periods can be associated with irreversible cell cycle transitions like START, entry into mitosis and exit from mitosis. During relaxation periods, the control system asymptotically approaches the new steady state. We also show that the dynamical dimension of the Chen's model fluctuates by increasing during excitation periods followed by decrease during relaxation periods. In each relaxation period the dynamical dimension of the model drops to one, indicating a period where kinetic processes are in steady state and all concentration changes are driven by the increase of cytoplasmic growth. Conclusion We apply two numerical methods, which have not been used to analyze biological control systems. These methods are more sensitive than the bifurcation analysis used before because they identify those

  14. Bridge-induced chromosome translocation in yeast relies upon a Rad54/Rdh54-dependent, Pol32-independent pathway.

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    Valentina Tosato

    Full Text Available While in mammalian cells the genetic determinism of chromosomal translocation remains unclear, the yeast Saccharomyces cerevisiae has become an ideal model system to generate ad hoc translocations and analyze their cellular and molecular outcome. A linear DNA cassette carrying a selectable marker flanked by perfect homologies to two chromosomes triggers a bridge-induced translocation (BIT in budding yeast, with variable efficiency. A postulated two-step process to produce BIT translocants is based on the cooperation between the Homologous Recombination System (HRS and Break-Induced Replication (BIR; however, a clear indication of the molecular factors underlying the genetic mechanism is still missing. In this work we provide evidence that BIT translocation is elicited by the Rad54 helicase and completed by a Pol32-independent replication pathway. Our results demonstrate also that Rdh54 is involved in the stability of the translocants, suggesting a mitotic role in chromosome pairing and segregation. Moreover, when RAD54 is over-expressed, an ensemble of secondary rearrangements between repeated DNA tracts arise after the initial translocation event, leading to severe aneuploidy with loss of genetic material, which prompts the identification of fragile sites within the yeast genome.

  15. Single-Cell Analysis of Growth in Budding Yeast and Bacteria Reveals a Common Size Regulation Strategy.

    Science.gov (United States)

    Soifer, Ilya; Robert, Lydia; Amir, Ariel

    2016-02-08

    To maintain a constant cell size, dividing cells have to coordinate cell-cycle events with cell growth. This coordination has long been supposed to rely on the existence of size thresholds determining cell-cycle progression [1]. In budding yeast, size is controlled at the G1/S transition [2]. In agreement with this hypothesis, the size at birth influences the time spent in G1: smaller cells have a longer G1 period [3]. Nevertheless, even though cells born smaller have a longer G1, the compensation is imperfect and they still bud at smaller cell sizes. In bacteria, several recent studies have shown that the incremental model of size control, in which size is controlled by addition of a constant volume (in contrast to a size threshold), is able to quantitatively explain the experimental data on four different bacterial species [4-7]. Here, we report on experimental results for the budding yeast Saccharomyces cerevisiae, finding, surprisingly, that cell size control in this organism is very well described by the incremental model, suggesting a common strategy for cell size control with bacteria. Additionally, we argue that for S. cerevisiae the "volume increment" is not added from birth to division, but rather between two budding events. Copyright © 2016 Elsevier Ltd. All rights reserved.

  16. Characterization of the minimum domain required for targeting budding yeast myosin II to the site of cell division

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    Tolliday Nicola J

    2006-06-01

    Full Text Available Abstract Background All eukaryotes with the exception of plants use an actomyosin ring to generate a constriction force at the site of cell division (cleavage furrow during mitosis and meiosis. The structure and filament forming abilities located in the C-terminal or tail region of one of the main components, myosin II, are important for localising the molecule to the contractile ring (CR during cytokinesis. However, it remains poorly understood how myosin II is recruited to the site of cell division and how this recruitment relates to myosin filament assembly. Significant conservation between species of the components involved in cytokinesis, including those of the CR, allows the use of easily genetically manipulated organisms, such as budding yeast (Saccharomyces cerevisiae, in the study of cytokinesis. Budding yeast has a single myosin II protein, named Myo1. Unlike most other class II myosins, the tail of Myo1 has an irregular coiled coil. In this report we use molecular genetics, biochemistry and live cell imaging to characterize the minimum localisation domain (MLD of budding yeast Myo1. Results We show that the MLD is a small region in the centre of the tail of Myo1 and that it is both necessary and sufficient for localisation of Myo1 to the yeast bud neck, the pre-determined site of cell division. Hydrodynamic measurements of the MLD, purified from bacteria or yeast, show that it is likely to exist as a trimer. We also examine the importance of a small region of low coiled coil forming probability within the MLD, which we call the hinge region. Removal of the hinge region prevents contraction of the CR. Using fluorescence recovery after photobleaching (FRAP, we show that GFP-tagged MLD is slightly more dynamic than the GFP-tagged full length molecule but less dynamic than the GFP-tagged Myo1 construct lacking the hinge region. Conclusion Our results define the intrinsic determinant for the localization of budding yeast myosin II and show

  17. Proteomics analysis for asymmetric inheritance of preexisting proteins between mother and daughter cells in budding yeast.

    Science.gov (United States)

    Okada, Mitsuhiro; Kusunoki, Shunta; Ishibashi, Yuko; Kito, Keiji

    2017-06-01

    In budding yeast, a mother cell can produce a finite number of daughter cells over its life. The accumulation of a variety of types of damaged components has an impact on the aging process. Asymmetrical inheritance during cell division causes these aberrant intracellular constituents to be retained in mother cells and prevents them from segregating to daughter cells. However, the understanding of asymmetrical inheritance of individual proteins that are damaged or old age, and their relevance to the aging process, has been limited. The aim of this study is to propose a proteomics strategy for asymmetrical inheritance of preexisting proteins between mother and daughter cells. During synchronous culture for one generation, newly synthesized proteins were labeled with stable isotope amino acids to discriminate preexisting proteins originally expressed in mother cells, followed by separation of mother and daughter cells using a conventional method based on biotin labeling. Isotope incorporation ratios for individual proteins were quantified using mass spectrometry. We successfully identified 21 proteins whose preexisting versions were asymmetrically inherited in mother cells, including plasma membrane transporter involved in the aging process and organelle-anchoring proteins related to the stress response to misfolded proteins. Thus, our approach would be useful for making catalog of asymmetrically inherited proteins. © 2017 Molecular Biology Society of Japan and John Wiley & Sons Australia, Ltd.

  18. Photobleaching of the "Raman spectroscopic signature of life" and mitochondrial activity in rho- budding yeast cells.

    Science.gov (United States)

    Onogi, Chikao; Hamaguchi, Hiro-O

    2009-08-06

    Raman spectra of mitochondria in single living budding yeast (zygote of Saccharomyces cerevisiae and Saccharomyces bayanus) cells have been measured and compared for two types of tetraploid strains, the wild-type strain and the rho- strain. The "Raman spectroscopic signature of life", a strong Raman band at 1602 cm(-1) that sharply reflects the mitochondrial activity, has been found in both of the two strains with similar intensities. This signature showed photobleaching under irradiation of the 632.8 nm line of a He-Ne laser, only for the rho- strain. The photobleaching recovered by keeping the cell in the dark for two hours without laser irradiation. These findings indicate that the molecular species that gives rise to the 1602 cm(-1) band is an intermediate formed and accumulated during the metabolic cycle and that it absorbs at 632.8 nm to undergo photodecomposition. We also found a number of weak Raman bands in the 400-1200 cm(-1) region that show the same temporal behaviors as that of the 1602 cm(-1) band by an SVD analysis of time-resolved Raman spectra. Several new pieces of information on the origin of the "Raman spectroscopic signature of life" have thus been obtained.

  19. A conserved signaling network monitors delivery of sphingolipids to the plasma membrane in budding yeast.

    Science.gov (United States)

    Clarke, Jesse; Dephoure, Noah; Horecka, Ira; Gygi, Steven; Kellogg, Douglas

    2017-08-09

    In budding yeast, cell cycle progression and ribosome biogenesis are dependent upon plasma membrane growth, which ensures that events of cell growth are coordinated with each other and with the cell cycle. However, the signals that link the cell cycle and ribosome biogenesis to membrane growth are poorly understood. Here, we used proteome-wide mass spectrometry to systematically discover signals associated with membrane growth. The results suggest that membrane trafficking events required for membrane growth generate sphingolipid-dependent signals. A conserved signaling network appears to play an essential role in signaling by responding to delivery of sphingolipids to the plasma membrane. In addition, sphingolipid-dependent signals control phosphorylation of protein kinase C (Pkc1), which plays an essential role in the pathways that link the cell cycle and ribosome biogenesis to membrane growth. Together, these discoveries provide new clues to how growth-dependent signals control cell growth and the cell cycle. © 2017 by The American Society for Cell Biology.

  20. Sequestration of mRNAs Modulates the Timing of Translation during Meiosis in Budding Yeast.

    Science.gov (United States)

    Jin, Liang; Zhang, Kai; Xu, Yifeng; Sternglanz, Rolf; Neiman, Aaron M

    2015-10-01

    Starvation of diploid cells of the budding yeast Saccharomyces cerevisiae induces them to enter meiosis and differentiate into haploid spores. During meiosis, the precise timing of gene expression is controlled at the level of transcription, and also translation. If cells are returned to rich medium after they have committed to meiosis, the transcript levels of most meiotically upregulated genes decrease rapidly. However, for a subset of transcripts whose translation is delayed until the end of meiosis II, termed protected transcripts, the transcript levels remain stable even after nutrients are reintroduced. The Ime2-Rim4 regulatory circuit controls both the delayed translation and the stability of protected transcripts. These protected mRNAs localize in discrete foci, which are not seen for transcripts of genes with different translational timing and are regulated by Ime2. These results suggest that Ime2 and Rim4 broadly regulate translational delay but that additional factors, such as mRNA localization, modulate this delay to tune the timing of gene expression to developmental transitions during sporulation.

  1. The nuclear exosome is active and important during budding yeast meiosis.

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    Stephen Frenk

    Full Text Available Nuclear RNA degradation pathways are highly conserved across eukaryotes and play important roles in RNA quality control. Key substrates for exosomal degradation include aberrant functional RNAs and cryptic unstable transcripts (CUTs. It has recently been reported that the nuclear exosome is inactivated during meiosis in budding yeast through degradation of the subunit Rrp6, leading to the stabilisation of a subset of meiotic unannotated transcripts (MUTs of unknown function. We have analysed the activity of the nuclear exosome during meiosis by deletion of TRF4, which encodes a key component of the exosome targeting complex TRAMP. We find that TRAMP mutants produce high levels of CUTs during meiosis that are undetectable in wild-type cells, showing that the nuclear exosome remains functional for CUT degradation, and we further report that the meiotic exosome complex contains Rrp6. Indeed Rrp6 over-expression is insufficient to suppress MUT transcripts, showing that the reduced amount of Rrp6 in meiotic cells does not directly cause MUT accumulation. Lack of TRAMP activity stabilises ∼ 1600 CUTs in meiotic cells, which occupy 40% of the binding capacity of the nuclear cap binding complex (CBC. CBC mutants display defects in the formation of meiotic double strand breaks (DSBs, and we see similar defects in TRAMP mutants, suggesting that a key function of the nuclear exosome is to prevent saturation of the CBC complex by CUTs. Together, our results show that the nuclear exosome remains active in meiosis and has an important role in facilitating meiotic recombination.

  2. The nuclear exosome is active and important during budding yeast meiosis.

    Science.gov (United States)

    Frenk, Stephen; Oxley, David; Houseley, Jonathan

    2014-01-01

    Nuclear RNA degradation pathways are highly conserved across eukaryotes and play important roles in RNA quality control. Key substrates for exosomal degradation include aberrant functional RNAs and cryptic unstable transcripts (CUTs). It has recently been reported that the nuclear exosome is inactivated during meiosis in budding yeast through degradation of the subunit Rrp6, leading to the stabilisation of a subset of meiotic unannotated transcripts (MUTs) of unknown function. We have analysed the activity of the nuclear exosome during meiosis by deletion of TRF4, which encodes a key component of the exosome targeting complex TRAMP. We find that TRAMP mutants produce high levels of CUTs during meiosis that are undetectable in wild-type cells, showing that the nuclear exosome remains functional for CUT degradation, and we further report that the meiotic exosome complex contains Rrp6. Indeed Rrp6 over-expression is insufficient to suppress MUT transcripts, showing that the reduced amount of Rrp6 in meiotic cells does not directly cause MUT accumulation. Lack of TRAMP activity stabilises ∼ 1600 CUTs in meiotic cells, which occupy 40% of the binding capacity of the nuclear cap binding complex (CBC). CBC mutants display defects in the formation of meiotic double strand breaks (DSBs), and we see similar defects in TRAMP mutants, suggesting that a key function of the nuclear exosome is to prevent saturation of the CBC complex by CUTs. Together, our results show that the nuclear exosome remains active in meiosis and has an important role in facilitating meiotic recombination.

  3. Coordinate responses to alkaline pH stress in budding yeast

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    Albert Serra-Cardona

    2015-05-01

    Full Text Available Alkalinization of the medium represents a stress condition for the budding yeast Saccharomyces cerevisiae to which this organism responds with profound remodeling of gene expression. This is the result of the modulation of a substantial number of signaling pathways whose participation in the alkaline response has been elucidated within the last ten years. These regulatory inputs involve not only the conserved Rim101/PacC pathway, but also the calcium-activated phosphatase calcineurin, the Wsc1-Pkc1-Slt2 MAP kinase, the Snf1 and PKA kinases and oxidative stress-response pathways. The uptake of many nutrients is perturbed by alkalinization of the environment and, consequently, an impact on phosphate, iron/copper and glucose homeostatic mechanisms can also be observed. The analysis of available data highlights cases in which diverse signaling pathways are integrated in the gene promoter to shape the appropriate response pattern. Thus, the expression of different genes sharing the same signaling network can be coordinated, allowing functional coupling of their gene products.

  4. Csm4, in collaboration with Ndj1, mediates telomere-led chromosome dynamics and recombination during yeast meiosis.

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    Jennifer J Wanat

    2008-09-01

    Full Text Available Chromosome movements are a general feature of mid-prophase of meiosis. In budding yeast, meiotic chromosomes exhibit dynamic movements, led by nuclear envelope (NE-associated telomeres, throughout the zygotene and pachytene stages. Zygotene motion underlies the global tendency for colocalization of NE-associated chromosome ends in a "bouquet." In this study, we identify Csm4 as a new molecular participant in these processes and show that, unlike the two previously identified components, Ndj1 and Mps3, Csm4 is not required for meiosis-specific telomere/NE association. Instead, it acts to couple telomere/NE ensembles to a force generation mechanism. Mutants lacking Csm4 and/or Ndj1 display the following closely related phenotypes: (i elevated crossover (CO frequencies and decreased CO interference without abrogation of normal pathways; (ii delayed progression of recombination, and recombination-coupled chromosome morphogenesis, with resulting delays in the MI division; and (iii nondisjunction of homologs at the MI division for some reason other than absence of (the obligatory CO(s. The recombination effects are discussed in the context of a model where the underlying defect is chromosome movement, the absence of which results in persistence of inappropriate chromosome relationships that, in turn, results in the observed mutant phenotypes.

  5. YODA: Software to facilitate high-throughput analysis of chronological life span, growth rate, and survival in budding yeast

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    Murakami Christopher J

    2010-03-01

    Full Text Available Abstract Background The budding yeast Saccharomyces cerevisiae is one of the most widely studied model organisms in aging-related science. Although several genetic modifiers of yeast longevity have been identified, the utility of this system for longevity studies has been limited by a lack of high-throughput assays for quantitatively measuring survival of individual yeast cells during aging. Results Here we describe the Yeast Outgrowth Data Analyzer (YODA, an automated system for analyzing population survival of yeast cells based on the kinetics of outgrowth measured by optical density over time. YODA has been designed specifically for quantification of yeast chronological life span, but can also be used to quantify growth rate and survival of yeast cells in response to a variety of different conditions, including temperature, nutritional composition of the growth media, and chemical treatments. YODA is optimized for use with a Bioscreen C MBR shaker/incubator/plate reader, but is also amenable to use with any standard plate reader or spectrophotometer. Conclusions We estimate that use of YODA as described here reduces the effort and resources required to measure chronological life span and analyze the resulting data by at least 15-fold.

  6. CHL12, a gene essential for the fidelity of chromosome transmission in the yeast Saccharomyces cerevisiae.

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    Kouprina, N; Kroll, E; Kirillov, A; Bannikov, V; Zakharyev, V; Larionov, V

    1994-12-01

    We have analyzed the CHL12 gene, earlier identified in a screen for yeast mutants with increased rates of mitotic loss of chromosome III and circular centromeric plasmids. A genomic clone of CHL12 was isolated and used to map its physical position on the right arm of chromosome XIII near the ADH3 locus. Nucleotide sequence analysis of CHL12 revealed a 2.2-kb open reading frame with a 84-kD predicted protein sequence. Analysis of the sequence upstream of the CHL12 open reading frame revealed the presence of two imperfect copies of MluI motif, ACGCGT, a sequence associated with many DNA metabolism genes in yeast. Analysis of the amino acid sequence revealed that the protein contains a NTP-binding domain and shares a low degree of homology with subunits of replication factor C (RF-C). A strain containing a null allele of CHL12 was viable under standard growth conditions, and as well as original mutants exhibited an increase in the level of spontaneous mitotic recombination, slow growth and cold-sensitive phenotypes. Most of cells carrying the null chl12 mutation arrested as large budded cells with the nucleus in the neck at nonpermissive temperature that typical for cell division cycle (cdc) mutants that arrest in the cell cycle at a point either immediately preceding M phase or during S phase. Cell cycle arrest of the chl12 mutant requires the RAD9 gene. We conclude that the CHL12 gene product has critical role in DNA metabolism.

  7. Novel E3 ubiquitin ligases that regulate histone protein levels in the budding yeast Saccharomyces cerevisiae.

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    Rakesh Kumar Singh

    Full Text Available Core histone proteins are essential for packaging the genomic DNA into chromatin in all eukaryotes. Since multiple genes encode these histone proteins, there is potential for generating more histones than what is required for chromatin assembly. The positively charged histones have a very high affinity for negatively charged molecules such as DNA, and any excess of histone proteins results in deleterious effects on genomic stability and cell viability. Hence, histone levels are known to be tightly regulated via transcriptional, posttranscriptional and posttranslational mechanisms. We have previously elucidated the posttranslational regulation of histone protein levels by the ubiquitin-proteasome pathway involving the E2 ubiquitin conjugating enzymes Ubc4/5 and the HECT (Homologous to E6-AP C-Terminus domain containing E3 ligase Tom1 in the budding yeast. Here we report the identification of four additional E3 ligases containing the RING (Really Interesting New Gene finger domains that are involved in the ubiquitylation and subsequent degradation of excess histones in yeast. These E3 ligases are Pep5, Snt2 as well as two previously uncharacterized Open Reading Frames (ORFs YKR017C and YDR266C that we have named Hel1 and Hel2 (for Histone E3 Ligases respectively. Mutants lacking these E3 ligases are sensitive to histone overexpression as they fail to degrade excess histones and accumulate high levels of endogenous histones on histone chaperones. Co-immunoprecipitation assays showed that these E3 ligases interact with the major E2 enzyme Ubc4 that is involved in the degradation related ubiquitylation of histones. Using mutagenesis we further demonstrate that the RING domains of Hel1, Hel2 and Snt2 are required for histone regulation. Lastly, mutants corresponding to Hel1, Hel2 and Pep5 are sensitive to replication inhibitors. Overall, our results highlight the importance of posttranslational histone regulatory mechanisms that employ multiple E3

  8. The Budding Yeast “Saccharomyces cerevisiae” as a Drug Discovery Tool to Identify Plant-Derived Natural Products with Anti-Proliferative Properties

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    Qaddouri, Bouchra; Guaadaoui, Abdelkarim; Bellirou, Ahmed; Hamal, Abdellah; Melhaoui, Ahmed; Brown, Grant W.; Bellaoui, Mohammed

    2011-01-01

    The budding yeast Saccharomyces cerevisiae is a valuable system to study cell-cycle regulation, which is defective in cancer cells. Due to the highly conserved nature of the cell-cycle machinery between yeast and humans, yeast studies are directly relevant to anticancer-drug discovery. The budding yeast is also an excellent model system for identifying and studying antifungal compounds because of the functional conservation of fungal genes. Moreover, yeast studies have also contributed greatly to our understanding of the biological targets and modes of action of bioactive compounds. Understanding the mechanism of action of clinically relevant compounds is essential for the design of improved second-generation molecules. Here we describe our methodology for screening a library of plant-derived natural products in yeast in order to identify and characterize new compounds with anti-proliferative properties. PMID:19596744

  9. The Budding Yeast “Saccharomyces cerevisiae” as a Drug Discovery Tool to Identify Plant-Derived Natural Products with Anti-Proliferative Properties

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    Bouchra Qaddouri

    2011-01-01

    Full Text Available The budding yeast Saccharomyces cerevisiae is a valuable system to study cell-cycle regulation, which is defective in cancer cells. Due to the highly conserved nature of the cell-cycle machinery between yeast and humans, yeast studies are directly relevant to anticancer-drug discovery. The budding yeast is also an excellent model system for identifying and studying antifungal compounds because of the functional conservation of fungal genes. Moreover, yeast studies have also contributed greatly to our understanding of the biological targets and modes of action of bioactive compounds. Understanding the mechanism of action of clinically relevant compounds is essential for the design of improved second-generation molecules. Here we describe our methodology for screening a library of plant-derived natural products in yeast in order to identify and characterize new compounds with anti-proliferative properties.

  10. Role of endocytosis in localization and maintenance of the spatial markers for bud-site selection in yeast.

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    Tuo, Shanshan; Nakashima, Kenichi; Pringle, John R

    2013-01-01

    The yeast Saccharomyces cerevisiae normally selects bud sites (and hence axes of cell polarization) in one of two distinct patterns, the axial pattern of haploid cells and the bipolar pattern of diploid cells. These patterns depend on distinct sets of cortical-marker proteins that transmit positional information through a common signaling pathway based on a Ras-type GTPase. It has been reported previously that various proteins of the endocytic pathway may be involved in determining the bipolar pattern but not the axial pattern. To explore this question systematically, we constructed and analyzed congenic haploid and diploid deletion mutants for 14 genes encoding proteins that are involved in endocytosis. The mutants displayed a wide range of severities in their overall endocytosis defects, as judged by their growth rates and abilities to take up the lipophilic dye FM 4-64. Consistent with the previous reports, none of the mutants displayed a significant defect in axial budding, but they displayed defects in bipolar budding that were roughly correlated with the severities of their overall endocytosis defects. Both the details of the mutant budding patterns and direct examination of GFP-tagged marker proteins suggested that both initial formation and maintenance of the normally persistent bipolar marks depend on endocytosis, as well as polarized exocytosis, in actively growing cells. Interestingly, maintenance of the bipolar marks in non-growing cells did not appear to require normal levels of endocytosis. In some cases, there was a striking lack of correlation between the overall severities of the general-endocytosis defect and the bud-site selection defect, suggesting that various endocytosis proteins may differ in their importance for the uptake of various plasma-membrane targets.

  11. Four linked genes participate in controlling sporulation efficiency in budding yeast.

    Directory of Open Access Journals (Sweden)

    Giora Ben-Ari

    2006-11-01

    Full Text Available Quantitative traits are conditioned by several genetic determinants. Since such genes influence many important complex traits in various organisms, the identification of quantitative trait loci (QTLs is of major interest, but still encounters serious difficulties. We detected four linked genes within one QTL, which participate in controlling sporulation efficiency in Saccharomyces cerevisiae. Following the identification of single nucleotide polymorphisms by comparing the sequences of 145 genes between the parental strains SK1 and S288c, we analyzed the segregating progeny of the cross between them. Through reciprocal hemizygosity analysis, four genes, RAS2, PMS1, SWS2, and FKH2, located in a region of 60 kilobases on Chromosome 14, were found to be associated with sporulation efficiency. Three of the four "high" sporulation alleles are derived from the "low" sporulating strain. Two of these sporulation-related genes were verified through allele replacements. For RAS2, the causative variation was suggested to be a single nucleotide difference in the upstream region of the gene. This quantitative trait nucleotide accounts for sporulation variability among a set of ten closely related winery yeast strains. Our results provide a detailed view of genetic complexity in one "QTL region" that controls a quantitative trait and reports a single nucleotide polymorphism-trait association in wild strains. Moreover, these findings have implications on QTL identification in higher eukaryotes.

  12. Cdc14 phosphatase directs centrosome re-duplication at the meiosis I to meiosis II transition in budding yeast [version 2; referees: 3 approved, 1 approved with reservations

    Directory of Open Access Journals (Sweden)

    Colette Fox

    2017-02-01

    Full Text Available Background Gametes are generated through a specialized cell division called meiosis, in which ploidy is reduced by half because two consecutive rounds of chromosome segregation, meiosis I and meiosis II, occur without intervening DNA replication. This contrasts with the mitotic cell cycle where DNA replication and chromosome segregation alternate to maintain the same ploidy. At the end of mitosis, cyclin-dependent kinases (CDKs are inactivated. This low CDK state in late mitosis/G1 allows for critical preparatory events for DNA replication and centrosome/spindle pole body (SPB duplication. However, their execution is inhibited until S phase, where further preparatory events are also prevented. This “licensing” ensures that both the chromosomes and the centrosomes/SPBs replicate exactly once per cell cycle, thereby maintaining constant ploidy. Crucially, between meiosis I and meiosis II, centrosomes/SPBs must be re-licensed, but DNA re-replication must be avoided. In budding yeast, the Cdc14 protein phosphatase triggers CDK down regulation to promote exit from mitosis. Cdc14 also regulates the meiosis I to meiosis II transition, though its mode of action has remained unclear. Methods Fluorescence and electron microscopy was combined with proteomics to probe SPB duplication in cells with inactive or hyperactive Cdc14. Results We demonstrate that Cdc14 ensures two successive nuclear divisions by re-licensing SPBs at the meiosis I to meiosis II transition. We show that Cdc14 is asymmetrically enriched on a single SPB during anaphase I and provide evidence that this enrichment promotes SPB re-duplication. Cells with impaired Cdc14 activity fail to promote extension of the SPB half-bridge, the initial step in morphogenesis of a new SPB. Conversely, cells with hyper-active Cdc14 duplicate SPBs, but fail to induce their separation. Conclusion Our findings implicate reversal of key CDK-dependent phosphorylations in the differential licensing of

  13. Complete DNA sequence of Kuraishia capsulata illustrates novel genomic features among budding yeasts (Saccharomycotina)

    NARCIS (Netherlands)

    Morales, L.; Noel, B.; Porcel, B.; Marcet-Houben, M.; Hullo, M.F.; Sacerdot, C.; Tekaia, F.; Leh-Louis, V.; Despons, L.; Khanna, V.; Aury, J.M.; Barbe, V.; Couloux, A.; Labadie, K.; Pelletier, E.; Souciet, J.L.; Boekhout, T.; Gabaldon, T.; Wincker, P.; Dujon, B.

    2013-01-01

    The numerous yeast genome sequences presently available provide a rich source of information for functional as well as evolutionary genomics, but unequally cover the large phylogenetic diversity of extant yeasts. We present here the complete sequence of the nuclear genome of the haploid type strain

  14. Complete DNA sequence of Kuraishia capsulata illustrates novel genomic features among budding yeasts (Saccharomycotina)

    NARCIS (Netherlands)

    Morales, L.; Noel, B.; Porcel, B.; Marcet-Houben, M.; Hullo, M.F.; Sacerdot, C.; Tekaia, F.; Leh-Louis, V.; Despons, L.; Khanna, V.; Aury, J.M.; Barbe, V.; Couloux, A.; Labadie, K.; Pelletier, E.; Souciet, J.L.; Boekhout, T.; Gabaldon, T.; Wincker, P.; Dujon, B.

    2013-01-01

    The numerous yeast genome sequences presently available provide a rich source of information for functional as well as evolutionary genomics, but unequally cover the large phylogenetic diversity of extant yeasts. We present here the complete sequence of the nuclear genome of the haploid type strain

  15. Dual control by Cdk1 phosphorylation of the budding yeast APC/C ubiquitin ligase activator Cdh1.

    Science.gov (United States)

    Höckner, Sebastian; Neumann-Arnold, Lea; Seufert, Wolfgang

    2016-07-15

    The antagonism between cyclin-dependent kinases (Cdks) and the ubiquitin ligase APC/C-Cdh1 is central to eukaryotic cell cycle control. APC/C-Cdh1 targets cyclin B and other regulatory proteins for degradation, whereas Cdks disable APC/C-Cdh1 through phosphorylation of the Cdh1 activator protein at multiple sites. Budding yeast Cdh1 carries nine Cdk phosphorylation sites in its N-terminal regulatory domain, most or all of which contribute to inhibition. However, the precise role of individual sites has remained unclear. Here, we report that the Cdk phosphorylation sites of yeast Cdh1 are organized into autonomous subgroups and act through separate mechanisms. Cdk sites 1-3 had no direct effect on the APC/C binding of Cdh1 but inactivated a bipartite nuclear localization sequence (NLS) and thereby controlled the partitioning of Cdh1 between cytoplasm and nucleus. In contrast, Cdk sites 4-9 did not influence the cell cycle-regulated localization of Cdh1 but prevented its binding to the APC/C. Cdk sites 4-9 reside near two recently identified APC/C interaction motifs in a pattern conserved with the human Cdh1 orthologue. Thus a Cdk-inhibited NLS goes along with Cdk-inhibited APC/C binding sites in yeast Cdh1 to relay the negative control by Cdk1 phosphorylation of the ubiquitin ligase APC/C-Cdh1.

  16. Isolation of a cdc28 mutation that abrogates the dependence of S phase on completion of M phase of the budding yeast cell cycle

    Indian Academy of Sciences (India)

    Santanu Kumar Ghosh; Pratima Sinha

    2000-01-01

    We have isolated a mutation in the budding yeast Saccharomyces cerevisisae CDC28 gene that allows cdc13 cells, carrying damaged DNA, to continue with the cell division cycle. While cdc13 mutant cells are arrested as large-budded cells at the nonpermissive temperature 37°C, the cdc13 cdc28 double mutant culture showed cells with one or more buds, most of which showed apical growth. The additional buds emerged without the intervening steps of nuclear division and cell separation. We suggest that the cdc28 mutation abrogates a checkpoint function and allows cells with damaged or incompletely replicated DNA an entry to another round of cell cycle and bypasses the mitotic phase of the cell cycle.

  17. Budding yeast Wee1 distinguishes spindle pole bodies to guide their pattern of age-dependent segregation.

    Science.gov (United States)

    Lengefeld, Jette; Hotz, Manuel; Rollins, Meaghen; Baetz, Kristin; Barral, Yves

    2017-08-01

    Many asymmetrically dividing cells unequally partition cellular structures according to age. Yet, it is unclear how cells differentiate pre-existing from newly synthesized material. Yeast cells segregate the spindle pole body (SPB, centrosome equivalent) inherited from the previous mitosis to the bud, while keeping the new one in the mother cell. Here, we show that the SPB inheritance network (SPIN), comprising the kinases Swe1 (also known as Wee1) and Kin3 (also known as Nek2) and the acetyltransferase NuA4 (also known as Tip60), distinguishes pre-existing from new SPBs. Swe1 phosphorylated Nud1 (orthologous to Centriolin) on young SPBs as they turned into pre-existing ones. The subsequent inactivation of Swe1 protected newly assembling SPBs from being marked. Kin3 and NuA4 maintained age marks on SPBs through following divisions. Downstream of SPIN, the Hippo regulator Bfa1-Bub2 bound the marked SPB, directed the spindle-positioning protein Kar9 towards it and drove its partition to the bud. Thus, coordination of SPIN activity and SPB assembly encodes age onto SPBs to enable their age-dependent segregation.

  18. Detection of QTL for Cold Tolerance at Bud Bursting Stage Using Chromosome Segment Substitution Lines in Rice (Oryza sativa)

    Institute of Scientific and Technical Information of China (English)

    LIN Jing; WANG Yan-ping; WANG Cai-lin; ZHU Wen-yin; ZHANG Ya-dong; ZHU Zhen; ZHAO Ling; CHEN Tao; ZHAO Qing-yong; ZHOU Li-hui; FANG Xian-wen

    2011-01-01

    The cold tolerance at the bud bursting stage (CTB) was evaluated at 5℃ by using a set of 95 chromosome segment substitution lines (CSSLs) derived from an indica rice 9311 and a japonica rice Nipponbare with a genetic background of 9311.The result showed that six CSSLs had slightly stronger effect on CTB than 9311.Total four quantitative trait loci (QTLs) for CTB were preliminary mapped on chromosomes 5 and 7 by substitution mapping.qCTB-5-1,qCTB-5-2 and qCTB-5-3 were mapped in the region of RM267-RM1237,RM2422-RM6054 and RM3321-RM1054,which were 21.3 cM,27.4 cM and 12.7 cM in genetic distance on rice chromosome 5,respectively.qCTB-7 was mapped in a 6.8-cM region of RM11-RM2752 on rice chromosome 7.

  19. Protein Kinase C Controls Binding of Igo/ENSA Proteins to Protein Phosphatase 2A in Budding Yeast.

    Science.gov (United States)

    Thai, Vu; Dephoure, Noah; Weiss, Amit; Ferguson, Jacqueline; Leitao, Ricardo; Gygi, Steven P; Kellogg, Douglas R

    2017-03-24

    Protein phosphatase 2A (PP2A) plays important roles in controlling mitosis in all eukaryotic cells. The form of PP2A that controls mitosis is associated with a conserved regulatory subunit that is called B55 in vertebrates and Cdc55 in budding yeast. The activity of this form of PP2A can be inhibited by binding of conserved Igo/ENSA proteins. Although the mechanisms that activate Igo/ENSA to bind and inhibit PP2A are well understood, little is known about how Igo/Ensa are inactivated. Here, we have analyzed regulation of Igo/ENSA in the context of a checkpoint pathway that links mitotic entry to membrane growth in budding yeast. Protein kinase C (Pkc1) relays signals in the pathway by activating PP2A(Cdc55) We discovered that constitutively active Pkc1 can drive cells through a mitotic checkpoint arrest, which suggests that Pkc1-dependent activation of PP2A(Cdc55) plays a critical role in checkpoint signaling. We therefore used mass spectrometry to determine how Pkc1 modifies the PP2A(Cdc55) complex. This revealed that Pkc1 induces changes in the phosphorylation of multiple subunits of the complex, as well as dissociation of Igo/ENSA. Pkc1 directly phosphorylates Cdc55 and Igo/ENSA, and phosphorylation site mapping and mutagenesis indicate that phosphorylation of Cdc55 contributes to Igo/ENSA dissociation. Association of Igo2 with PP2A(Cdc55) is regulated during the cell cycle, yet mutation of Pkc1-dependent phosphorylation sites on Cdc55 and Igo2 did not cause defects in mitotic progression. Together, the data suggest that Pkc1 controls PP2A(Cdc55) by multiple overlapping mechanisms. © 2017 by The American Society for Biochemistry and Molecular Biology, Inc.

  20. Hormesis of Glyceollin I, an Induced Phytoalexin from Soybean, on Budding Yeast Chronological Lifespan Extension

    Directory of Open Access Journals (Sweden)

    Yuancai Liu

    2014-01-01

    Full Text Available Glyceollin I, an induced phytoalexin isolated from soybean, has been reported to have various bioactivities, including anti-bacterial, anti-nematode, anti-fungal, anti-estrogenic and anti-cancer, anti-oxidant, anti-inflammatory, insulin sensitivity enhancing, and attenuation of vascular contractions. Here we show that glyceollin I has hormesis and extends yeast life span at low (nM doses in a calorie restriction (CR-dependent manner, while it reduces life span and inhibits yeast cell proliferation at higher (μM doses. In contrast, the other two isomers (glyceollin II and III cannot extend yeast life span and only show life span reduction and antiproliferation at higher doses. Our results in anti-aging activity indicate that glyceollin I might be a promising calorie restriction mimetic candidate, and the high content of glyceollins could improve the bioactivity of soybean as functional food ingredients.

  1. alpha-Synuclein budding yeast model: toxicity enhanced by impaired proteasome and oxidative stress.

    Science.gov (United States)

    Sharma, Nijee; Brandis, Katrina A; Herrera, Sara K; Johnson, Brandon E; Vaidya, Tulaza; Shrestha, Ruja; Debburman, Shubhik K

    2006-01-01

    Parkinson's disease (PD) is a common neurodegenerative disorder that results from the selective loss of midbrain dopaminergic neurons. Misfolding and aggregation of the protein alpha-synuclein, oxidative damage, and proteasomal impairment are all hypotheses for the molecular cause of this selective neurotoxicity. Here, we describe a Saccharomyces cerevisiae model to evaluate the misfolding, aggregation, and toxicity-inducing ability of wild-type alpha-synuclein and three mutants (A30P, A53T, and A30P/A53T), and we compare regulation of these properties by dysfunctional proteasomes and by oxidative stress. We found prominent localization of wild-type and A53T alpha-synuclein near the plasma membrane, supporting known in vitro lipid-binding ability. In contrast, A30P was mostly cytoplasmic, whereas A30P/A53T displayed both types of fluorescence. Surprisingly, alpha-synuclein was not toxic to several yeast strains tested. When yeast mutants for the proteasomal barrel (doa3-1) were evaluated, delayed alpha-synuclein synthesis and membrane association were observed; yeast mutant for the proteasomal cap (sen3-1) exhibited increased accumulation and aggregation of alpha-synuclein. Both sen3-1and doa3-1 mutants exhibited synthetic lethality with alpha-synuclein. When yeasts were challenged with an oxidant (hydrogen peroxide), alpha-synuclein was extremely lethal to cells that lacked manganese superoxide dismutase Mn-SOD (sod2Delta) but not to cells that lacked copper, zinc superoxide dismutase Cu,Zn-SOD (sod1Delta). Despite the toxicity, sod2Delta cells never displayed intracellular aggregates of alpha-synuclein. We suggest that the toxic alpha-synuclein species in yeast are smaller than the visible aggregates, and toxicity might involve alpha-synuclein membrane association. Thus, yeasts have emerged effective organisms for characterizing factors and mechanisms that regulate alpha-synuclein toxicity.

  2. A stochastic model of kinetochore-microtubule attachment accurately describes fission yeast chromosome segregation.

    Science.gov (United States)

    Gay, Guillaume; Courtheoux, Thibault; Reyes, Céline; Tournier, Sylvie; Gachet, Yannick

    2012-03-19

    In fission yeast, erroneous attachments of spindle microtubules to kinetochores are frequent in early mitosis. Most are corrected before anaphase onset by a mechanism involving the protein kinase Aurora B, which destabilizes kinetochore microtubules (ktMTs) in the absence of tension between sister chromatids. In this paper, we describe a minimal mathematical model of fission yeast chromosome segregation based on the stochastic attachment and detachment of ktMTs. The model accurately reproduces the timing of correct chromosome biorientation and segregation seen in fission yeast. Prevention of attachment defects requires both appropriate kinetochore orientation and an Aurora B-like activity. The model also reproduces abnormal chromosome segregation behavior (caused by, for example, inhibition of Aurora B). It predicts that, in metaphase, merotelic attachment is prevented by a kinetochore orientation effect and corrected by an Aurora B-like activity, whereas in anaphase, it is corrected through unbalanced forces applied to the kinetochore. These unbalanced forces are sufficient to prevent aneuploidy.

  3. A stochastic model of kinetochore–microtubule attachment accurately describes fission yeast chromosome segregation

    OpenAIRE

    Gay, Guillaume; Courtheoux, Thibault; Reyes, Céline; Tournier, Sylvie; Gachet, Yannick

    2012-01-01

    In fission yeast, erroneous attachments of spindle microtubules to kinetochores are frequent in early mitosis. Most are corrected before anaphase onset by a mechanism involving the protein kinase Aurora B, which destabilizes kinetochore microtubules (ktMTs) in the absence of tension between sister chromatids. In this paper, we describe a minimal mathematical model of fission yeast chromosome segregation based on the stochastic attachment and detachment of ktMTs. The model accurately reproduce...

  4. Improved statistical analysis of budding yeast TAG microarrays revealed by defined spike-in pools.

    Science.gov (United States)

    Peyser, Brian D; Irizarry, Rafael A; Tiffany, Carol W; Chen, Ou; Yuan, Daniel S; Boeke, Jef D; Spencer, Forrest A

    2005-09-15

    Saccharomyces cerevisiae knockout collection TAG microarrays are an emergent platform for rapid, genome-wide functional characterization of yeast genes. TAG arrays report abundance of unique oligonucleotide 'TAG' sequences incorporated into each deletion mutation of the yeast knockout collection, allowing measurement of relative strain representation across experimental conditions for all knockout mutants simultaneously. One application of TAG arrays is to perform genome-wide synthetic lethality screens, known as synthetic lethality analyzed by microarray (SLAM). We designed a fully defined spike-in pool to resemble typical SLAM experiments and performed TAG microarray hybridizations. We describe a method for analyzing two-color array data to efficiently measure the differential knockout strain representation across two experimental conditions, and use the spike-in pool to show that the sensitivity and specificity of this method exceed typical current approaches.

  5. The yeast I-Sce I meganuclease induces site-directed chromosomal recombination in mammalian cells.

    Science.gov (United States)

    Choulika, A; Perrin, A; Dujon, B; Nicolas, J F

    1994-11-01

    Double-strand breaks in genomic DNA stimulate recombination. Until now it was not possible to induce in vivo site-directed double-strand breaks in a mammalian chromosomal target. In this article we describe the use of I-Sce I meganuclease, a very rare cutter yeast endonuclease, to induce site-directed double-strand breaks mediated recombination. The results demonstrate the potential of the I-Sce I system for chromosome manipulation in mammalian cells.

  6. Whole-cell imaging of the budding yeast Saccharomyces cerevisiae by high-voltage scanning transmission electron tomography

    Energy Technology Data Exchange (ETDEWEB)

    Murata, Kazuyoshi, E-mail: kazum@nips.ac.jp [National Institute for Physiological Sciences, Okazaki, Aichi 444-8585 (Japan); Esaki, Masatoshi; Ogura, Teru [Institute of Molecular Embryology and Genetics, Kumamoto University, Kumamoto 860-0811 (Japan); Arai, Shigeo; Yamamoto, Yuta; Tanaka, Nobuo [Ecotopia Science Institute, Nagoya University, Nagoya, Aichi 464-8603 (Japan)

    2014-11-15

    Electron tomography using a high-voltage electron microscope (HVEM) provides three-dimensional information about cellular components in sections thicker than 1 μm, although in bright-field mode image degradation caused by multiple inelastic scattering of transmitted electrons limit the attainable resolution. Scanning transmission electron microscopy (STEM) is believed to give enhanced contrast and resolution compared to conventional transmission electron microscopy (CTEM). Samples up to 1 μm in thickness have been analyzed with an intermediate-voltage electron microscope because inelastic scattering is not a critical limitation, and probe broadening can be minimized. Here, we employed STEM at 1 MeV high-voltage to extend the useful specimen thickness for electron tomography, which we demonstrate by a seamless tomographic reconstruction of a whole, budding Saccharomyces cerevisiae yeast cell, which is ∼3 μm in thickness. High-voltage STEM tomography, especially in the bright-field mode, demonstrated sufficiently enhanced contrast and intensity, compared to CTEM tomography, to permit segmentation of major organelles in the whole cell. STEM imaging also reduced specimen shrinkage during tilt-series acquisition. The fidelity of structural preservation was limited by cytoplasmic extraction, and the spatial resolution was limited by the relatively large convergence angle of the scanning probe. However, the new technique has potential to solve longstanding problems of image blurring in biological specimens beyond 1 μm in thickness, and may facilitate new research in cellular structural biology. - Highlights: • High voltage TEM and STEM tomography were compared to visualize whole yeast cells. • 1-MeV STEM-BF tomography had significant improvements in image contrast and SNR. • 1-MeV STEM tomography showed less specimen shrinkage than the TEM tomography. • KMnO{sub 4} post-treatment permitted segmenting the major cellular components.

  7. Ndj1, a telomere-associated protein, regulates centrosome separation in budding yeast meiosis

    Science.gov (United States)

    Li, Ping; Shao, Yize; Jin, Hui

    2015-01-01

    Yeast centrosomes (called spindle pole bodies [SPBs]) remain cohesive for hours during meiotic G2 when recombination takes place. In contrast, SPBs separate within minutes after duplication in vegetative cells. We report here that Ndj1, a previously known meiosis-specific telomere-associated protein, is required for protecting SPB cohesion. Ndj1 localizes to the SPB but dissociates from it ∼16 min before SPB separation. Without Ndj1, meiotic SPBs lost cohesion prematurely, whereas overproduction of Ndj1 delayed SPB separation. When produced ectopically in vegetative cells, Ndj1 caused SPB separation defects and cell lethality. Localization of Ndj1 to the SPB depended on the SUN domain protein Mps3, and removal of the N terminus of Mps3 allowed SPB separation and suppressed the lethality of NDJ1-expressing vegetative cells. Finally, we show that Ndj1 forms oligomeric complexes with Mps3, and that the Polo-like kinase Cdc5 regulates Ndj1 protein stability and SPB separation. These findings reveal the underlying mechanism that coordinates yeast centrosome dynamics with meiotic telomere movement and cell cycle progression. PMID:25897084

  8. Transcription factor genes essential for cell proliferation and replicative lifespan in budding yeast

    Energy Technology Data Exchange (ETDEWEB)

    Kamei, Yuka; Tai, Akiko; Dakeyama, Shota; Yamamoto, Kaori; Inoue, Yamato; Kishimoto, Yoshifumi; Ohara, Hiroya; Mukai, Yukio, E-mail: y_mukai@nagahama-i-bio.ac.jp

    2015-07-31

    Many of the lifespan-related genes have been identified in eukaryotes ranging from the yeast to human. However, there is limited information available on the longevity genes that are essential for cell proliferation. Here, we investigated whether the essential genes encoding DNA-binding transcription factors modulated the replicative lifespan of Saccharomyces cerevisiae. Heterozygous diploid knockout strains for FHL1, RAP1, REB1, and MCM1 genes showed significantly short lifespan. {sup 1}H-nuclear magnetic resonance analysis indicated a characteristic metabolic profile in the Δfhl1/FHL1 mutant. These results strongly suggest that FHL1 regulates the transcription of lifespan related metabolic genes. Thus, heterozygous knockout strains could be the potential materials for discovering further novel lifespan genes. - Highlights: • Involvement of yeast TF genes essential for cell growth in lifespan was evaluated. • The essential TF genes, FHL1, RAP1, REB1, and MCM1, regulate replicative lifespan. • Heterozygous deletion of FHL1 changes cellular metabolism related to lifespan.

  9. The third exon of the budding yeast meiotic recombination gene HOP2 is required for calcium-dependent and recombinase Dmc1-specific stimulation of homologous strand assimilation.

    Science.gov (United States)

    Chan, Yuen-Ling; Brown, M Scott; Qin, Daoming; Handa, Naofumi; Bishop, Douglas K

    2014-06-27

    During meiosis in Saccharomyces cerevisiae, the HOP2 and MND1 genes are essential for recombination. A previous biochemical study has shown that budding yeast Hop2-Mnd1 stimulates the activity of the meiosis-specific strand exchange protein ScDmc1 only 3-fold, whereas analogous studies using mammalian homologs show >30-fold stimulation. The HOP2 gene was recently discovered to contain a second intron that lies near the 3'-end. We show that both HOP2 introns are efficiently spliced during meiosis, forming a predominant transcript that codes for a protein with a C-terminal sequence different from that of the previously studied version of the protein. Using the newly identified HOP2 open reading frame to direct synthesis of wild type Hop2 protein, we show that the Hop2-Mnd1 heterodimer stimulated Dmc1 D-loop activity up to 30-fold, similar to the activity of mammalian Hop2-Mnd1. ScHop2-Mnd1 stimulated ScDmc1 activity in the presence of physiological (micromolar) concentrations of Ca(2+) ions, as long as Mg(2+) was also present at physiological concentrations, leading us to hypothesize that ScDmc1 protomers bind both cations in the active Dmc1 filament. Co-factor requirements and order-of-addition experiments suggested that Hop2-Mnd1-mediated stimulation of Dmc1 involves a process that follows the formation of functional Dmc1-ssDNA filaments. In dramatic contrast to mammalian orthologs, the stimulatory activity of budding yeast Hop2-Mnd1 appeared to be specific to Dmc1; we observed no Hop2-Mnd1-mediated stimulation of the other budding yeast strand exchange protein Rad51. Together, these results support previous genetic experiments indicating that Hop2-Mnd1 specifically stimulates Dmc1 during meiotic recombination in budding yeast.

  10. Misregulation of Scm3p/HJURP Causes Chromosome Instability in Saccharomyces cerevisiae and Human Cells

    OpenAIRE

    Prashant K. Mishra; Wei-Chun Au; John S. Choy; P Henning Kuich; Baker, Richard E.; Foltz, Daniel R.; Basrai, Munira A.

    2011-01-01

    The kinetochore (centromeric DNA and associated proteins) is a key determinant for high fidelity chromosome transmission. Evolutionarily conserved Scm3p is an essential component of centromeric chromatin and is required for assembly and function of kinetochores in humans, fission yeast, and budding yeast. Overexpression of HJURP, the mammalian homolog of budding yeast Scm3p, has been observed in lung and breast cancers and is associated with poor prognosis; however, the physiological relevanc...

  11. Positive feedback promotes mitotic exit via the APC/C-Cdh1-separase-Cdc14 axis in budding yeast.

    Science.gov (United States)

    Hatano, Yuhki; Naoki, Koike; Suzuki, Asuka; Ushimaru, Takashi

    2016-10-01

    The mitotic inhibitor securin is degraded via the ubiquitin ligase anaphase-promoting complex/cyclosome (APC/C)-Cdc20 after anaphase onset. This triggers activation of the mitotic protease separase and thereby sister chromatid separation. However, only a proportion of securin molecules are degraded at metaphase-anaphase transition and the remaining molecules are still present in anaphase. The roles of securin and separase in late mitosis remain elusive. Here, we show that securin still inhibits separase to repress mitotic exit in anaphase in budding yeast. APC/C-Cdh1-mediated securin degradation at telophase further liberated separase, which promotes Cdc14 release and mitotic exit. Separase executed these events via its proteolytic action and that in the Cdc14 early release (FEAR) network. Cdc14 release further activated APC/C-Cdh1 in the manner of a positive feedback loop. Thus, the positive feedback promotes mitotic exit via the APC/C-Cdh1-separase-Cdc14 axis. This study shows the importance of the two-step degradation mode of securin and the role of separase in mitotic exit.

  12. Anoxia-Induced Suspended Animation in Budding Yeast as an Experimental Paradigm for Studying Oxygen-Regulated Gene Expression▿

    Science.gov (United States)

    Chan, Kin; Roth, Mark B.

    2008-01-01

    A lack of oxygen can force many organisms to enter into recoverable hypometabolic states. To better understand how organisms cope with oxygen deprivation, our laboratory previously had shown that when challenged with anoxia, both the nematode Caenorhabditis elegans and embryos of the zebrafish Danio rerio enter into suspended animation, in which all life processes that can be observed by light microscopy reversibly halt pending the restoration of oxygen (P. A. Padilla and M. B. Roth, Proc. Natl. Acad. Sci. USA 98:7331-7335, 2001, and P. A. Padilla, T. G. Nystul, R. A. Zager, A. C. Johnson, and M. B. Roth, Mol. Biol. Cell 13:1473-1483, 2002). Here, we show that both sporulating and vegetative cells of the budding yeast Saccharomyces cerevisiae also enter into a similar state of suspended animation when made anoxic on a nonfermentable carbon source. Transcriptional profiling using cDNA microarrays and follow-on quantitative real-time PCR analysis revealed a relative derepression of aerobic metabolism genes in carbon monoxide (CO)-induced anoxia when compared to nitrogen (N2) gas-induced anoxia, which is consistent with the known oxygen-mimetic effects of CO. We also found that mutants deleted for components of the mitochondrial retrograde signaling pathway can tolerate prolonged exposure to CO but not to N2. We conclude that the cellular response to anoxia is dependent on whether the anoxic gas is an oxygen mimetic and that the mitochondrial retrograde signaling pathway is functionally important for mediating this response. PMID:18708563

  13. Formation of new chromosomes as a virulence mechanism in yeast Candida glabrata

    DEFF Research Database (Denmark)

    Poláková, S.; Blume, C.; Zárate, J. A.

    2009-01-01

    , Candida glabrata, for their genome structure and stability. This organism has recently become the second most prevalent yeast pathogen in humans. Although the gene sequences were well conserved among different strains, their chromosome structures differed drastically. The most frequent events reshaping...

  14. Simultaneous measurement of the frequencies of intrachromosomal recombination and chromosome gain using the yeast DEL assay.

    Science.gov (United States)

    Howlett, N G; Schiestl, R H

    2000-11-06

    The yeast DEL assay measures the frequency of intrachromosomal recombination between two partially-deleted his3 alleles on chromosome XV. The his3Delta alleles share approximately 400bp of overlapping homology, and are separated by an intervening LEU2 sequence. Homologous recombination between the his3Delta alleles results in deletion of the intervening LEU2 sequence (DEL), and reversion to histidine prototrophy. In this study we have attempted to further extend the use of the yeast DEL assay to measure the frequency of chromosome XV gain events. Reversion to His(+)Leu(+) in the haploid yeast DEL tester strain RSY6 occurs upon non-disjunction of chromosome XV sister chromatids, coupled with a subsequent DEL event. Here we have tested the ability of the yeast DEL assay to accurately predict the aneugenic potential of the diversely-acting, known or suspected aneugens actinomycin D, benomyl, chloral hydrate, ethyl methanesulfonate (EMS), methyl methanesulfonate (MMS), and methotrexate. Actinomycin D and benomyl strongly induced aneuploidy. EMS and methotrexate modestly induced aneuploidy, while chloral hydrate and MMS failed to illicit any significant induction. In addition, by FACS-analysis of DNA content it was shown that the majority of both spontaneous- and chemically-induced His(+)Leu(+) revertants were heterodiploid. Thus, our results indicate endoreduplication of almost entire chromosome sets as a major mechanism of aneuploidy induction in haploid Saccharomyces cerevisiae.

  15. A strategy for constructing aneuploid yeast strains by transient nondisjunction of a target chromosome

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    Peck Anders T

    2009-07-01

    Full Text Available Abstract Background Most methods for constructing aneuploid yeast strains that have gained a specific chromosome rely on spontaneous failures of cell division fidelity. In Saccharomyces cerevisiae, extra chromosomes can be obtained when errors in meiosis or mitosis lead to nondisjunction, or when nuclear breakdown occurs in heterokaryons. We describe a strategy for constructing N+1 disomes that does not require such spontaneous failures. The method combines two well-characterized genetic tools: a conditional centromere that transiently blocks disjunction of one specific chromosome, and a duplication marker assay that identifies disomes among daughter cells. To test the strategy, we targeted chromosomes III, IV, and VI for duplication. Results The centromere of each chromosome was replaced by a centromere that can be blocked by growth in galactose, and ura3::HIS3, a duplication marker. Transient exposure to galactose induced the appearance of colonies carrying duplicated markers for chromosomes III or IV, but not VI. Microarray-based comparative genomic hybridization (CGH confirmed that disomic strains carrying extra chromosome III or IV were generated. Chromosome VI contains several genes that are known to be deleterious when overexpressed, including the beta-tubulin gene TUB2. To test whether a tubulin stoichiometry imbalance is necessary for the apparent lethality caused by an extra chromosome VI, we supplied the parent strain with extra copies of the alpha-tubulin gene TUB1, then induced nondisjunction. Galactose-dependent chromosome VI disomes were produced, as revealed by CGH. Some chromosome VI disomes also carried extra, unselected copies of additional chromosomes. Conclusion This method causes efficient nondisjunction of a targeted chromosome and allows resulting disomic cells to be identified and maintained. We used the method to test the role of tubulin imbalance in the apparent lethality of disomic chromosome VI. Our results indicate

  16. The dynamics of homologous pairing during mating type interconversion in budding yeast.

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    Peter L Houston

    2006-06-01

    Full Text Available Cells repair most double-strand breaks (DSBs that arise during replication or by environmental insults through homologous recombination, a high-fidelity process critical for maintenance of genomic integrity. However, neither the detailed mechanism of homologous recombination nor the specific roles of critical components of the recombination machinery-such as Bloom and Werner syndrome proteins-have been resolved. We have taken a novel approach to examining the mechanism of homologous recombination by tracking both a DSB and the template from which it is repaired during the repair process in individual yeast cells. The two loci were labeled with arrays of DNA binding sites and visualized in live cells expressing green fluorescent protein-DNA binding protein chimeras. Following induction of an endonuclease that introduces a DSB next to one of the marked loci, live cells were imaged repeatedly to determine the relative positions of the DSB and the template locus. We found a significant increase in persistent associations between donor and recipient loci following formation of the DSB, demonstrating DSB-induced pairing between donor and template. However, such associations were transient and occurred repeatedly in every cell, a result not predicted from previous studies on populations of cells. Moreover, these associations were absent in sgs1 or srs2 mutants, yeast homologs of the Bloom and Werner syndrome genes, but were enhanced in a rad54 mutant, whose protein product promotes efficient strand exchange in vitro. Our results indicate that a DSB makes multiple and reversible contacts with a template during the repair process, suggesting that repair could involve interactions with multiple templates, potentially creating novel combinations of sequences at the repair site. Our results further suggest that both Sgs1 and Srs2 are required for efficient completion of recombination and that Rad54 may serve to dissociate such interactions. Finally, these

  17. A 6. 5-Mb yeast artificial chromosome contig incorporating 33 DNA markers on the human X chromosome at Xq22

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    Vetrie, D.; Kendall, E.; Coffey, A.; Hassock, S.; Collins, J.; Todd, C.; Bobrow, M.; Bentley, D.R. (Paediatric Research Unit, London (United Kingdom)); Lehrach, H. (Imperial Cancer Research Fund, London (United Kingdom)); Harris, A. (John Radcliffe Hospital, Oxford (United Kingdom))

    1994-01-01

    The Xq22 region of the human X chromosome contains genes for a number of inherited disorders. Sixty-nine yeast artificial chromosome clones have been isolated and assembled into a 6.5-Mb contig that contains 33 DNA markers localized to this region. This contig extends distally from DXS366 to beyond DXS87 and includes the genes involved in X-linked agammaglobulinemia (btk), Fabry disease (GLA), and Pelizaeus-Merzbacher disease (PLP). The order of markers in this contig is consistent with the known genetic and physical mapping information of Xq22. This cloned material provides a source from which to isolate other genes located in this part of the X chromosome. 45 refs., 2 figs., 2 tabs.

  18. Distinct differences in chromatin structure at subtelomeric X and Y' elements in budding yeast.

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    Xuefeng Zhu

    Full Text Available In Saccharomyces cerevisiae, all ends of telomeric DNA contain telomeric repeats of (TG(1-3, but the number and position of subtelomeric X and Y' repeat elements vary. Using chromatin immunoprecipitation and genome-wide analyses, we here demonstrate that the subtelomeric X and Y' elements have distinct structural and functional properties. Y' elements are transcriptionally active and highly enriched in nucleosomes, whereas X elements are repressed and devoid of nucleosomes. In contrast to X elements, the Y' elements also lack the classical hallmarks of heterochromatin, such as high Sir3 and Rap1 occupancy as well as low levels of histone H4 lysine 16 acetylation. Our analyses suggest that the presence of X and Y' elements govern chromatin structure and transcription activity at individual chromosome ends.

  19. Identification of She3 as an SCF(Grr1 substrate in budding yeast.

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    Ruiwen Wang

    Full Text Available The highly orchestrated progression of the cell cycle depends on the degradation of many regulatory proteins at different cell cycle stages. One of the key cell cycle ubiquitin ligases is the Skp1-cullin-F-box (SCF complex. Acting in concert with the substrate-binding F-box protein Grr1, SCF(Grr1 promotes the degradation of cell cycle regulators as well as various metabolic enzymes. Using a yeast two-hybrid assay with a Grr1 derivative as the bait, we identified She3, which is an adaptor protein in the asymmetric mRNA transport system, as a novel Grr1 substrate. We generated stabilized She3 mutants, which no longer bound to Grr1, and found that the degradation of She3 is not required for regulating asymmetric mRNA transport. However, She3 stabilization leads to slower growth compared to wild-type cells in a co-culture assay, demonstrating that the degradation of She3 by Grr1 is required for optimal cell growth.

  20. Organelle Size Scaling of the Budding Yeast Vacuole by Relative Growth and Inheritance.

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    Chan, Yee-Hung M; Reyes, Lorena; Sohail, Saba M; Tran, Nancy K; Marshall, Wallace F

    2016-05-09

    It has long been noted that larger animals have larger organs compared to smaller animals of the same species, a phenomenon termed scaling [1]. Julian Huxley proposed an appealingly simple model of "relative growth"-in which an organ and the whole body grow with their own intrinsic rates [2]-that was invoked to explain scaling in organs from fiddler crab claws to human brains. Because organ size is regulated by complex, unpredictable pathways [3], it remains unclear whether scaling requires feedback mechanisms to regulate organ growth in response to organ or body size. The molecular pathways governing organelle biogenesis are simpler than organogenesis, and therefore organelle size scaling in the cell provides a more tractable case for testing Huxley's model. We ask the question: is it possible for organelle size scaling to arise if organelle growth is independent of organelle or cell size? Using the yeast vacuole as a model, we tested whether mutants defective in vacuole inheritance, vac8Δ and vac17Δ, tune vacuole biogenesis in response to perturbations in vacuole size. In vac8Δ/vac17Δ, vacuole scaling increases with the replicative age of the cell. Furthermore, vac8Δ/vac17Δ cells continued generating vacuole at roughly constant rates even when they had significantly larger vacuoles compared to wild-type. With support from computational modeling, these results suggest there is no feedback between vacuole biogenesis rates and vacuole or cell size. Rather, size scaling is determined by the relative growth rates of the vacuole and the cell, thus representing a cellular version of Huxley's model.

  1. The budding yeast amphiphysin complex is required for contractile actin ring (CAR assembly and post-contraction GEF-independent accumulation of Rho1-GTP.

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    Michael John Cundell

    Full Text Available The late events of the budding yeast cell division cycle, cytokinesis and cell separation, require the assembly of a contractile actomyosin ring (CAR, primary and secondary septum formation followed by enzymatic degradation of the primary septum. Here we present evidence that demonstrates a role for the budding yeast amphiphysin complex, a heterodimer comprising Rvs167 and Rvs161, in CAR assembly and cell separation. The iqg1-1 allele is synthetically lethal with both rvs167 and rvs161 null mutations. We show that both Iqg1 and the amphiphysin complex are required for CAR assembly in early anaphase but cells are able to complete assembly in late anaphase when these activities are, respectively, either compromised or absent. Amphiphysin dependent CAR assembly is dependent upon the Rvs167 SH3 domain, but this function is insufficient to explain the observed synthetic lethality. Dosage suppression of the iqg1-1 allele demonstrates that endocytosis is required for the default cell separation pathway in the absence of CAR contraction but is unlikely to be required to maintain viability. The amphiphysin complex is required for normal, post-mitotic, localization of Chs3 and the Rho1 GEF, Rom2, which are responsible for secondary septum deposition and the accumulation of GTP bound Rho1 at the bud neck. It is concluded that a failure of polarity establishment in the absence of CAR contraction and amphiphysin function leads to loss of viability as a result of the consequent cell separation defect.

  2. IQGAP and mitotic exit network (MEN) proteins are required for cytokinesis and re-polarization of the actin cytoskeleton in the budding yeast, Saccharomyces cerevisiae.

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    Corbett, Mark; Xiong, Yulan; Boyne, James R; Wright, Daniel J; Munro, Ewen; Price, Clive

    2006-11-01

    In budding yeast the final stages of the cell division cycle, cytokinesis and cell separation, are distinct events that require to be coupled, both together and with mitotic exit. Here we demonstrate that mutations in genes of the mitotic exit network (MEN) prevent cell separation and are synthetically lethal in combination with both cytokinesis and septation defective mutations. Analysis of the synthetic lethal phenotypes reveals that Iqg1p functions in combination with the MEN components, Tem1p, Cdc15p Dbf20p and Dbf2p to govern the re-polarization of the actin cytoskeleton to either side of the bud neck. In addition phosphorylation of the conserved PCH protein, Hof1p, is dependent upon these activities and requires actin ring assembly. Recruitment of Dbf2p to the bud neck is dependent upon actin ring assembly and correlates with Hof1p phosphorylation. Failure to phosphorylate Hof1p results in the increased stability of the protein and its persistence at the bud neck. These data establish a mechanistic dependency of cell separation upon an intermediate step requiring actomyosin ring assembly.

  3. Transcript analysis of 250 novel yeast genes from chromosome XIV.

    Science.gov (United States)

    Planta, R J; Brown, A J; Cadahia, J L; Cerdan, M E; de Jonge, M; Gent, M E; Hayes, A; Kolen, C P; Lombardia, L J; Sefton, M; Oliver, S G; Thevelein, J; Tournu, H; van Delft, Y J; Verbart, D J; Winderickx, J

    1999-03-15

    The European Functional Analysis Network (EUROFAN) is systematically analysing the function of novel Saccharomyces cerevisiae genes revealed by genome sequencing. As part of this effort our consortium has performed a detailed transcript analysis for 250 novel ORFs on chromosome XIV. All transcripts were quantified by Northern analysis under three quasi-steady-state conditions (exponential growth on rich fermentative, rich non-fermentative, and minimal fermentative media) and eight transient conditions (glucose derepression, glucose upshift, stationary phase, nitrogen starvation, osmo-stress, heat-shock, and two control conditions). Transcripts were detected for 82% of the 250 ORFs, and only one ORF did not yield a transcript of the expected length (YNL285w). Transcripts ranged from low (62%), moderate (16%) to high abundance (2%) relative to the ACT1 mRNA. The levels of 73% of the 206 chromosome XIV transcripts detected fluctuated in response to the transient states tested. However, only a small number responded strongly to the transients: eight ORFs were induced upon glucose upshift; five were repressed by glucose; six were induced in response to nitrogen starvation; three were induced in stationary phase; five were induced by osmo-stress; four were induced by heat-shock. These data provide useful clues about the general function of these ORFs and add to our understanding of gene regulation on a genome-wide basis.

  4. Autophagy is required for efficient meiosis progression and proper meiotic chromosome segregation in fission yeast.

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    Matsuhara, Hirotada; Yamamoto, Ayumu

    2016-01-01

    Autophagy is a conserved intracellular degradation system, which contributes to development and differentiation of various organisms. Yeast cells undergo meiosis under nitrogen-starved conditions and require autophagy for meiosis initiation. However, the precise roles of autophagy in meiosis remain unclear. Here, we show that autophagy is required for efficient meiosis progression and proper meiotic chromosome segregation in fission yeast. Autophagy-defective strains bearing a mutation in the autophagy core factor gene atg1, atg7, or atg14 exhibit deformed nuclear structures during meiosis. These mutant cells require an extracellular nitrogen supply for meiosis progression following their entry into meiosis and show delayed meiosis progression even with a nitrogen supply. In addition, they show frequent chromosome dissociation from the spindle together with spindle overextension, forming extra nuclei. Furthermore, Aurora kinase, which regulates chromosome segregation and spindle elongation, is significantly increased at the centromere and spindle in the mutant cells. Aurora kinase down-regulation eliminated delayed initiation of meiosis I and II, chromosome dissociation, and spindle overextension, indicating that increased Aurora kinase activity may cause these aberrances in the mutant cells. Our findings show a hitherto unrecognized relationship of autophagy with the nuclear structure, regulation of cell cycle progression, and chromosome segregation in meiosis. © 2015 The Molecular Biology Society of Japan and Wiley Publishing Asia Pty Ltd.

  5. Discovery of a small molecule targeting IRA2 deletion in budding yeast and neurofibromin loss in malignant peripheral nerve sheath tumor cells

    Science.gov (United States)

    Wood, Matthew; Rawe, Melissa; Johansson, Gunnar; Pang, Shu; Soderquist, Ryan S.; Patel, Ami V.; Nelson, Sandra; Seibel, William; Ratner, Nancy; Sanchez, Yolanda

    2011-01-01

    Malignant peripheral nerve sheath tumor (MPNST) is a life-threatening complication of neurofibromatosis type 1 (NF1). NF1 is caused by mutation in the gene encoding neurofibromin, a negative regulator of Ras signaling. There are no effective pharmacologic therapies for MPNST. To identify new therapeutic approaches targeting this dangerous malignancy, we developed assays in NF1+/+ and NF1−/ − MPNST cell lines and in budding yeast lacking the NF1 homologue IRA2 (ira2Δ). Here we describe UC1, a small molecule that targets NF1−/− cell lines and ira2Δ budding yeast. Using yeast genetics we identified NAB3 as a high-copy suppressor of UC1 sensitivity. NAB3 encodes an RNA binding protein that associates with the C-terminal domain of RNA Pol II and plays a role in the termination of non-polyadenylated RNA transcripts. Strains with deletion of IRA2 are sensitive to genetic inactivation of NAB3, suggesting an interaction between Ras signaling and Nab3-dependent transcript termination. This work identifies a lead compound and a possible target pathway for NF1-associated MPNST, and demonstrates a novel model system approach to identify and validate target pathways for cancer cells in which NF1 loss drives tumor formation. PMID:21697395

  6. ADA1 and NET1 Genes of Yeast Mediate Both Chromosome Maintenance and Mitochondrial $\\rho^{-}$ Mutagenesis

    CERN Document Server

    Koltovaya, N A; Tchekhouta, I A; Devin, A B

    2002-01-01

    An increase in the mitochondrial (mt) rho^- mutagenesis is a well-known respose of yeast cells to mutations in the numerous nuclear genes as well as to various kinds of stress. Notwithstanding the extensive studies during several decades the biological significance of this response is not yet fully understood. The genetic approach to solution of this subject includes the study of genes that are required for the high incidence of spontaneous rho^- mutants. Previously we found that mutations in certain nuclear genes including CDC28, the central cell-cycle regulation gene, may decrease the spontaneous rho^- mutability and simultaneously affect maintenance of the yeast chromosomes and plasmids. The present work provides data on identification of two more genes, resembling CDC28 in this respect. These genes NET1 and ADA1 mediate important regulatory protein-protein interactions in the yeast cell. The effects of net1 and ada1 mutations on the maintenance of yeast mt genome, chromosomes and plasmids as well as on ce...

  7. Spt10 and Swi4 Control the Timing of Histone H2A/H2B Gene Activation in Budding Yeast

    OpenAIRE

    Eriksson, Peter R.; Ganguli, Dwaipayan; Clark, David J.

    2010-01-01

    The expression of the histone genes is regulated during the cell cycle to provide histones for nucleosome assembly during DNA replication. In budding yeast, histones H2A and H2B are expressed from divergent promoters at the HTA1-HTB1 and HTA2-HTB2 loci. Here, we show that the major activator of HTA1-HTB1 is Spt10, a sequence-specific DNA binding protein with a putative histone acetyltransferase (HAT) domain. Spt10 binds to two pairs of upstream activation sequence (UAS) elements in the HTA1-H...

  8. Discovery of a small molecule targeting IRA2 deletion in budding yeast and neurofibromin loss in malignant peripheral nerve sheath tumor cells

    OpenAIRE

    Wood, Matthew; Rawe, Melissa; Johansson, Gunnar; Pang, Shu; Soderquist, Ryan S.; Patel, Ami V.; Nelson, Sandra; Seibel, William; Ratner, Nancy; Sanchez, Yolanda

    2011-01-01

    Malignant peripheral nerve sheath tumor (MPNST) is a life-threatening complication of neurofibromatosis type 1 (NF1). NF1 is caused by mutation in the gene encoding neurofibromin, a negative regulator of Ras signaling. There are no effective pharmacologic therapies for MPNST. To identify new therapeutic approaches targeting this dangerous malignancy, we developed assays in NF1+/+ and NF1−/ − MPNST cell lines and in budding yeast lacking the NF1 homologue IRA2 (ira2Δ). Here we describe UC1, a ...

  9. Timely activation of budding yeast APCCdh1 involves degradation of its inhibitor, Acm1, by an unconventional proteolytic mechanism.

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    Michael Melesse

    Full Text Available Regulated proteolysis mediated by the ubiquitin proteasome system is a fundamental and essential feature of the eukaryotic cell division cycle. Most proteins with cell cycle-regulated stability are targeted for degradation by one of two related ubiquitin ligases, the Skp1-cullin-F box protein (SCF complex or the anaphase-promoting complex (APC. Here we describe an unconventional cell cycle-regulated proteolytic mechanism that acts on the Acm1 protein, an inhibitor of the APC activator Cdh1 in budding yeast. Although Acm1 can be recognized as a substrate by the Cdc20-activated APC (APCCdc20 in anaphase, APCCdc20 is neither necessary nor sufficient for complete Acm1 degradation at the end of mitosis. An APC-independent, but 26S proteasome-dependent, mechanism is sufficient for complete Acm1 clearance from late mitotic and G1 cells. Surprisingly, this mechanism appears distinct from the canonical ubiquitin targeting pathway, exhibiting several features of ubiquitin-independent proteasomal degradation. For example, Acm1 degradation in G1 requires neither lysine residues in Acm1 nor assembly of polyubiquitin chains. Acm1 was stabilized though by conditional inactivation of the ubiquitin activating enzyme Uba1, implying some requirement for the ubiquitin pathway, either direct or indirect. We identified an amino terminal predicted disordered region in Acm1 that contributes to its proteolysis in G1. Although ubiquitin-independent proteasome substrates have been described, Acm1 appears unique in that its sensitivity to this mechanism is strictly cell cycle-regulated via cyclin-dependent kinase (Cdk phosphorylation. As a result, Acm1 expression is limited to the cell cycle window in which Cdk is active. We provide evidence that failure to eliminate Acm1 impairs activation of APCCdh1 at mitotic exit, justifying its strict regulation by cell cycle-dependent transcription and proteolytic mechanisms. Importantly, our results reveal that strict cell

  10. Budding yeast greatwall and endosulfines control activity and spatial regulation of PP2A(Cdc55 for timely mitotic progression.

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    Maria Angeles Juanes

    Full Text Available Entry into mitosis is triggered by cyclinB/Cdk1, whose activity is abruptly raised by a positive feedback loop. The Greatwall kinase phosphorylates proteins of the endosulfine family and allows them to bind and inhibit the main Cdk1-counteracting PP2A-B55 phosphatase, thereby promoting mitotic entry. In contrast to most eukaryotic systems, Cdc14 is the main Cdk1-antagonizing phosphatase in budding yeast, while the PP2A(Cdc55 phosphatase promotes, instead of preventing, mitotic entry by participating to the positive feedback loop of Cdk1 activation. Here we show that budding yeast endosulfines (Igo1 and Igo2 bind to PP2A(Cdc55 in a cell cycle-regulated manner upon Greatwall (Rim15-dependent phosphorylation. Phosphorylated Igo1 inhibits PP2A(Cdc55 activity in vitro and induces mitotic entry in Xenopus egg extracts, indicating that it bears a conserved PP2A-binding and -inhibitory activity. Surprisingly, deletion of IGO1 and IGO2 in yeast cells leads to a decrease in PP2A phosphatase activity, suggesting that endosulfines act also as positive regulators of PP2A in yeast. Consistently, RIM15 and IGO1/2 promote, like PP2A(Cdc55, timely entry into mitosis under temperature-stress, owing to the accumulation of Tyr-phosphorylated Cdk1. In addition, they contribute to the nuclear export of PP2A(Cdc55, which has recently been proposed to promote mitotic entry. Altogether, our data indicate that Igo proteins participate in the positive feedback loop for Cdk1 activation. We conclude that Greatwall, endosulfines, and PP2A are part of a regulatory module that has been conserved during evolution irrespective of PP2A function in the control of mitosis. However, this conserved module is adapted to account for differences in the regulation of mitotic entry in different organisms.

  11. Two separate pathways regulate protein stability of ATM/ATR-related protein kinases Mec1 and Tel1 in budding yeast.

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    Goto, Greicy H; Ogi, Hiroo; Biswas, Himadri; Ghosh, Avik; Tanaka, Seiji; Sugimoto, Katsunori

    2017-08-01

    Checkpoint signaling requires two conserved phosphatidylinositol 3-kinase-related protein kinases (PIKKs): ATM and ATR. In budding yeast, Tel1 and Mec1 correspond to ATM and ATR, respectively. The Tel2-Tti1-Tti2 (TTT) complex connects to the Rvb1-Rvb2-Tah1-Pih1 (R2TP) complex for the protein stability of PIKKs; however, TTT-R2TP interaction only partially mediates ATM and ATR protein stabilization. How TTT controls protein stability of ATM and ATR remains to be precisely determined. Here we show that Asa1, like Tel2, plays a major role in stabilization of newly synthesized Mec1 and Tel1 proteins whereas Pih1 contributes to Mec1 and Tel1 stability at high temperatures. Although Asa1 and Pih1 both interact with Tel2, no Asa1-Pih1 interaction is detected. Pih1 is distributed in both the cytoplasm and nucleus wheres Asa1 localizes largely in the cytoplasm. Asa1 and Pih1 are required for proper DNA damage checkpoint signaling. Our findings provide a model in which two different Tel2 pathways promote protein stabilization of Mec1 and Tel1 in budding yeast.

  12. A role for the rap GTPase YlRsr1 in cellular morphogenesis and the involvement of YlRsr1 and the ras GTPase YlRas2 in bud site selection in the dimorphic yeast Yarrowia lipolytica.

    Science.gov (United States)

    Li, Yun-Qing; Li, Min; Zhao, Xiao-Feng; Gao, Xiang-Dong

    2014-05-01

    Yarrowia lipolytica is a dimorphic yeast species that can grow in the ovoid yeast form or in the elongated pseudohyphal or hyphal form depending on the growth conditions. Here, we show that the Rap GTPase Rsr1 of Y. lipolytica (YlRsr1) plays an important role in cellular morphogenesis in this microorganism. Cells deleted for YlRSR1 exhibited impaired polarized growth during yeast-form growth. Pseudohyphal and hyphal development were also abnormal. YlRsr1 is also important for cell growth, since the deletion of YlRSR1 in cells lacking the Ras GTPase YlRas2 caused lethality. Y. lipolytica cells bud in a bipolar pattern in which the cells produce the new buds at the two poles. YlRsr1 plays a prominent role in this bud site selection process. YlRsr1's function in bud site selection absolutely requires the cycling of YlRsr1 between the GTP- and GDP-bound states but its function in cellular morphogenesis does not, suggesting that the two processes are differentially regulated. Interestingly, the Ras GTPase YlRas2 is also involved in the control of bud site selection, as Ylras2Δ cells were severely impaired in bipolar bud site selection. The GTP/GDP cycling and the plasma membrane localization of YlRas2 are important for YlRas2's function in bud site selection. However, they are not essential for this process, suggesting that the mechanism by which YlRas2 acts is different from that of YlRsr1. Our results suggest that YlRsr1 is regulated by the GTPase-activating protein (GAP) YlBud2 and partially by YlCdc25, the potential guanine nucleotide exchange factor (GEF) for YlRas2.

  13. Dot1-dependent histone H3K79 methylation promotes the formation of meiotic double-strand breaks in the absence of histone H3K4 methylation in budding yeast.

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    Mohammad Bani Ismail

    Full Text Available Epigenetic marks such as histone modifications play roles in various chromosome dynamics in mitosis and meiosis. Methylation of histones H3 at positions K4 and K79 is involved in the initiation of recombination and the recombination checkpoint, respectively, during meiosis in the budding yeast. Set1 promotes H3K4 methylation while Dot1 promotes H3K79 methylation. In this study, we carried out detailed analyses of meiosis in mutants of the SET1 and DOT1 genes as well as methylation-defective mutants of histone H3. We confirmed the role of Set1-dependent H3K4 methylation in the formation of double-strand breaks (DSBs in meiosis for the initiation of meiotic recombination, and we showed the involvement of Dot1 (H3K79 methylation in DSB formation in the absence of Set1-dependent H3K4 methylation. In addition, we showed that the histone H3K4 methylation-defective mutants are defective in SC elongation, although they seem to have moderate reduction of DSBs. This suggests that high levels of DSBs mediated by histone H3K4 methylation promote SC elongation.

  14. Massive Amplification at an Unselected Locus Accompanies Complex Chromosomal Rearrangements in Yeast

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    Agnès Thierry

    2016-05-01

    Full Text Available Gene amplification has been observed in different organisms in response to environmental constraints, such as limited nutrients or exposure to a variety of toxic compounds, conferring them with specific phenotypic adaptations via increased expression levels. However, the presence of multiple gene copies in natural genomes has generally not been found in the absence of specific functional selection. Here, we show that the massive amplification of a chromosomal locus (up to 880 copies per cell occurs in the absence of any direct selection, and is associated with low-order amplifications of flanking segments in complex chromosomal alterations. These results were obtained from mutants with restored phenotypes that spontaneously appeared from genetically engineered strains of the yeast Saccharomyces cerevisiae suffering from severe fitness reduction. Grossly extended chromosomes (macrotene were formed, with complex structural alterations but sufficient stability to propagate unchanged over successive generations. Their detailed molecular analysis, including complete genome sequencing, identification of sequence breakpoints, and comparisons between mutants, revealed novel mechanisms causing their formation, whose combined action underlies the astonishing dynamics of eukaryotic chromosomes and their consequences.

  15. Identification and cloning of the CHL4 gene controlling chromosome segregation in yeast.

    Science.gov (United States)

    Kouprina, N; Kirillov, A; Kroll, E; Koryabin, M; Shestopalov, B; Bannikov, V; Zakharyev, V; Larionov, V

    1993-10-01

    A collection of chl mutants characterized by decreased fidelity of chromosome transmission and by minichromosome nondisjunction in mitosis was examined for the ability to maintain nonessential dicentric plasmids. In one of the seven mutants analyzed, chl4, dicentric plasmids did not depress cell division. Moreover, nonessential dicentric plasmids were maintained stably without any rearrangements during many generations in the chl4 mutant. The rate of mitotic heteroallelic recombination in the chl4 mutant was not increased compared to that in an isogenic wild-type strain. Analysis of the segregation of a marked chromosome indicated that sister chromatid nondisjunction and sister chromatid loss contributed equally to chromosome malsegregation in the chl4 mutant. A genomic clone of CHL4 was isolated by complementation of the chl4-1 mutation and was physically mapped to the right arm of chromosome IV near the SUP2 gene. Nucleotide sequence analysis of CHL4 clone revealed a 1.4-kb open reading frame coding for a 53-kD predicted protein which does not have homology to published proteins. A strain containing a null allele of CHL4 is viable under standard growth conditions but has a temperature-sensitive phenotype (conditional lethality at 36 degrees). We suggest that the CHL4 gene is required for kinetochore function in the yeast Saccharomyces cerevisiae.

  16. Asymmetry of the budding yeast Tem1 GTPase at spindle poles is required for spindle positioning but not for mitotic exit.

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    Ilaria Scarfone

    2015-02-01

    Full Text Available The asymmetrically dividing yeast S. cerevisiae assembles a bipolar spindle well after establishing the future site of cell division (i.e., the bud neck and the division axis (i.e., the mother-bud axis. A surveillance mechanism called spindle position checkpoint (SPOC delays mitotic exit and cytokinesis until the spindle is properly positioned relative to the mother-bud axis, thereby ensuring the correct ploidy of the progeny. SPOC relies on the heterodimeric GTPase-activating protein Bub2/Bfa1 that inhibits the small GTPase Tem1, in turn essential for activating the mitotic exit network (MEN kinase cascade and cytokinesis. The Bub2/Bfa1 GAP and the Tem1 GTPase form a complex at spindle poles that undergoes a remarkable asymmetry during mitosis when the spindle is properly positioned, with the complex accumulating on the bud-directed old spindle pole. In contrast, the complex remains symmetrically localized on both poles of misaligned spindles. The mechanism driving asymmetry of Bub2/Bfa1/Tem1 in mitosis is unclear. Furthermore, whether asymmetry is involved in timely mitotic exit is controversial. We investigated the mechanism by which the GAP Bub2/Bfa1 controls GTP hydrolysis on Tem1 and generated a series of mutants leading to constitutive Tem1 activation. These mutants are SPOC-defective and invariably lead to symmetrical localization of Bub2/Bfa1/Tem1 at spindle poles, indicating that GTP hydrolysis is essential for asymmetry. Constitutive tethering of Bub2 or Bfa1 to both spindle poles impairs SPOC response but does not impair mitotic exit. Rather, it facilitates mitotic exit of MEN mutants, likely by increasing the residence time of Tem1 at spindle poles where it gets active. Surprisingly, all mutant or chimeric proteins leading to symmetrical localization of Bub2/Bfa1/Tem1 lead to increased symmetry at spindle poles of the Kar9 protein that mediates spindle positioning and cause spindle misalignment. Thus, asymmetry of the Bub2/Bfa1/Tem1

  17. [Stable maintenance of dicentric mini-chromosomes in CHL4 mutants in yeast].

    Science.gov (United States)

    Kuprina, N Iu; Krol', E S; Koriabin, M Iu; Bannikov, V M; Kirillov, A V; Zakhar'ev, V M; Larionov, V L

    1993-01-01

    Earlier we have identified the chl4-1 mutation in a screen for yeast mutants with increased loss of chromosome III and circular artificial minichromosome in mitosis. Mutation in the CHL4 gene leads to a 50-100-fold promotion in the rate of chromosome loss per cell division compared to the isogenic wild type strain. Detailed analysis of behaviour of the circular minichromosome marked by the CUP1 gene has shown that minichromosome nondisjunction (2:0 segregation) leading to an increase in the copy number of minichromosome in part of a cell population is the main reason of minichromosome instability in the mutant. The unique peculiarity of chl4-1 mutation is the ability of the strains carrying this mutation to stably maintain circular dicentric minichromosomes without any rearrangement during many generations. (In the wild type strains dicentric minichromosomes are extremely unstable. As a consequence of that there is a strong selection for cells harboring monocentric derivatives in a population of cells derived from a cell containing a dicentric plasmid). Introduction of the second centromere into one of the natural chromosomes (chromosomes II or III) in the chl4-1 mutant leads to the same dramatic consequences as that in the wild type strain (mitotic lag of cells harboring dicentric chromosomes and, as a result of that, selective pressure for cells harboring monocentric derivatives of dicentric chromosome). A genomic clone of CHL4 was isolated by complementation of the chl4-1 mutation. Nucleotide sequence analysis of CHL4 revealed a 1.4-kb open reading frame with a predicted 53-kDa protein sequence. Analyzing the sequence of the CHL4 protein we have found a region meeting the necessary requirements for the helix-turn-helix (HTH) structure. This region of the CHL4 protein has about 40% homology with the repressor of tryptophane operon (TrpR) of E. coli. A strain containing a null allele of CHL4 was viable under standard growth conditions, but had temperature

  18. Exposure of ELF-EMF and RF-EMF Increase the Rate of Glucose Transport and TCA Cycle in Budding Yeast

    Science.gov (United States)

    Lin, Kang-Wei; Yang, Chuan-Jun; Lian, Hui-Yong; Cai, Peng

    2016-01-01

    In this study, we investigated the transcriptional response to 50 Hz extremely low frequency electromagnetic field (ELF-EMF) and 2.0 GHz radio frequency electromagnetic field (RF-EMF) exposure by Illumina sequencing technology using budding yeast as the model organism. The transcription levels of 28 genes were upregulated and those of four genes were downregulated under ELF-EMF exposure, while the transcription levels of 29 genes were upregulated and those of 24 genes were downregulated under RF-EMF exposure. After validation by reverse transcription quantitative polymerase chain reaction (RT-qPCR), a concordant direction of change both in differential gene expression (DGE) and RT-qPCR was demonstrated for nine genes under ELF-EMF exposure and for 10 genes under RF-EMF exposure. The RT-qPCR results revealed that ELF-EMF and RF-EMF exposure can upregulate the expression of genes involved in glucose transportation and the tricarboxylic acid (TCA) cycle, but not the glycolysis pathway. Energy metabolism is closely related with the cell response to environmental stress including EMF exposure. Our findings may throw light on the mechanism underlying the biological effects of EMF. PMID:27630630

  19. The Role of the CRL4Cdt2 Target Spd1 in Chromosome Segregation in Fission Yeast

    DEFF Research Database (Denmark)

    Landvad, Katrine

    Ddb1, a component of the E3 ubiquitin ligase CRL4Cdt2, is needed for proper chromosome segregation in fission yeast as ddb1 deleted cells show unequal distribution of DNA to daughter cells and sensitivity to the microtubule destabilising drug TBZ. In this study we show that Δddb1 cells have....... Concomitant deletion of spd1, a known target of CRL4Cdt2, substantially reduces the observed defects of the ddb1 single mutant, indicating that degradation of Spd1 is important to ensure proper chromosome segregation. Spd1 is degraded on proliferating cell nuclear antigen (PCNA) and we propose...

  20. H2B ubiquitylation is part of chromatin architecture that marks exon-intron structure in budding yeast

    LENUS (Irish Health Repository)

    Shieh, Grace S.

    2011-12-22

    Abstract Background The packaging of DNA into chromatin regulates transcription from initiation through 3\\' end processing. One aspect of transcription in which chromatin plays a poorly understood role is the co-transcriptional splicing of pre-mRNA. Results Here we provide evidence that H2B monoubiquitylation (H2BK123ub1) marks introns in Saccharomyces cerevisiae. A genome-wide map of H2BK123ub1 in this organism reveals that this modification is enriched in coding regions and that its levels peak at the transcribed regions of two characteristic subgroups of genes. First, long genes are more likely to have higher levels of H2BK123ub1, correlating with the postulated role of this modification in preventing cryptic transcription initiation in ORFs. Second, genes that are highly transcribed also have high levels of H2BK123ub1, including the ribosomal protein genes, which comprise the majority of intron-containing genes in yeast. H2BK123ub1 is also a feature of introns in the yeast genome, and the disruption of this modification alters the intragenic distribution of H3 trimethylation on lysine 36 (H3K36me3), which functionally correlates with alternative RNA splicing in humans. In addition, the deletion of genes encoding the U2 snRNP subunits, Lea1 or Msl1, in combination with an htb-K123R mutation, leads to synthetic lethality. Conclusion These data suggest that H2BK123ub1 facilitates cross talk between chromatin and pre-mRNA splicing by modulating the distribution of intronic and exonic histone modifications.

  1. Divergent Evolution of the Transcriptional Network Controlled by Snf1-Interacting Protein Sip4 in Budding Yeasts.

    Science.gov (United States)

    Mehlgarten, Constance; Krijger, Jorrit-Jan; Lemnian, Ioana; Gohr, André; Kasper, Lydia; Diesing, Anne-Kathrin; Grosse, Ivo; Breunig, Karin D

    2015-01-01

    Cellular responses to starvation are of ancient origin since nutrient limitation has always been a common challenge to the stability of living systems. Hence, signaling molecules involved in sensing or transducing information about limiting metabolites are highly conserved, whereas transcription factors and the genes they regulate have diverged. In eukaryotes the AMP-activated protein kinase (AMPK) functions as a central regulator of cellular energy homeostasis. The yeast AMPK ortholog SNF1 controls the transcriptional network that counteracts carbon starvation conditions by regulating a set of transcription factors. Among those Cat8 and Sip4 have overlapping DNA-binding specificity for so-called carbon source responsive elements and induce target genes upon SNF1 activation. To analyze the evolution of the Cat8-Sip4 controlled transcriptional network we have compared the response to carbon limitation of Saccharomyces cerevisiae to that of Kluyveromyces lactis. In high glucose, S. cerevisiae displays tumor cell-like aerobic fermentation and repression of respiration (Crabtree-positive) while K. lactis has a respiratory-fermentative life-style, respiration being regulated by oxygen availability (Crabtree-negative), which is typical for many yeasts and for differentiated higher cells. We demonstrate divergent evolution of the Cat8-Sip4 network and present evidence that a role of Sip4 in controlling anabolic metabolism has been lost in the Saccharomyces lineage. We find that in K. lactis, but not in S. cerevisiae, the Sip4 protein plays an essential role in C2 carbon assimilation including induction of the glyoxylate cycle and the carnitine shuttle genes. Induction of KlSIP4 gene expression by KlCat8 is essential under these growth conditions and a primary function of KlCat8. Both KlCat8 and KlSip4 are involved in the regulation of lactose metabolism in K. lactis. In chromatin-immunoprecipitation experiments we demonstrate binding of both, KlSip4 and KlCat8, to

  2. Divergent Evolution of the Transcriptional Network Controlled by Snf1-Interacting Protein Sip4 in Budding Yeasts.

    Directory of Open Access Journals (Sweden)

    Constance Mehlgarten

    Full Text Available Cellular responses to starvation are of ancient origin since nutrient limitation has always been a common challenge to the stability of living systems. Hence, signaling molecules involved in sensing or transducing information about limiting metabolites are highly conserved, whereas transcription factors and the genes they regulate have diverged. In eukaryotes the AMP-activated protein kinase (AMPK functions as a central regulator of cellular energy homeostasis. The yeast AMPK ortholog SNF1 controls the transcriptional network that counteracts carbon starvation conditions by regulating a set of transcription factors. Among those Cat8 and Sip4 have overlapping DNA-binding specificity for so-called carbon source responsive elements and induce target genes upon SNF1 activation. To analyze the evolution of the Cat8-Sip4 controlled transcriptional network we have compared the response to carbon limitation of Saccharomyces cerevisiae to that of Kluyveromyces lactis. In high glucose, S. cerevisiae displays tumor cell-like aerobic fermentation and repression of respiration (Crabtree-positive while K. lactis has a respiratory-fermentative life-style, respiration being regulated by oxygen availability (Crabtree-negative, which is typical for many yeasts and for differentiated higher cells. We demonstrate divergent evolution of the Cat8-Sip4 network and present evidence that a role of Sip4 in controlling anabolic metabolism has been lost in the Saccharomyces lineage. We find that in K. lactis, but not in S. cerevisiae, the Sip4 protein plays an essential role in C2 carbon assimilation including induction of the glyoxylate cycle and the carnitine shuttle genes. Induction of KlSIP4 gene expression by KlCat8 is essential under these growth conditions and a primary function of KlCat8. Both KlCat8 and KlSip4 are involved in the regulation of lactose metabolism in K. lactis. In chromatin-immunoprecipitation experiments we demonstrate binding of both, KlSip4 and

  3. Mapping autonomously replicating sequence elements in a 73-kb region of chromosome II of the fission yeast, Schizosaccharomyces pombe

    Indian Academy of Sciences (India)

    Vinay Kumar Srivastava; Dharani Dhar Dubey

    2007-08-01

    Autonomously replicating sequence (ARS) elements are the genetic determinants of replication origin function in yeasts. They can be easily identified as the plasmids containing them transform yeast cells at a high frequency. As the first step towards identifying all potential replication origins in a 73-kb region of the long arm of fission yeast chromosome II, we have mapped five new ARS elements using systematic subcloning and transformation assay. 2D analysis of one of the ARS plasmids that showed highest transformation frequency localized the replication origin activity within the cloned genomic DNA. All the new ARS elements are localized in two clusters in centromere proximal 40 kb of the region. The presence of at least six ARS elements, including the previously reported ars727, is suggestive of a higher origin density in this region than that predicted earlier using a computer based search.

  4. Delineating Rearrangements in Single Yeast Artificial Chromosomes by Quantitative DNA Fiber Mapping

    Energy Technology Data Exchange (ETDEWEB)

    Weier, Heinz-Ulrich G.; Greulich-Bode, Karin M.; Wu, Jenny; Duell, Thomas

    2009-09-18

    Cloning of large chunks of human genomic DNA in recombinant systems such as yeast or bacterial artificial chromosomes has greatly facilitated the construction of physical maps, the positional cloning of disease genes or the preparation of patient-specific DNA probes for diagnostic purposes. For this process to work efficiently, the DNA cloning process and subsequent clone propagation need to maintain stable inserts that are neither deleted nor otherwise rearranged. Some regions of the human genome; however, appear to have a higher propensity than others to rearrange in any host system. Thus, techniques to detect and accurately characterize such rearrangements need to be developed. We developed a technique termed 'Quantitative DNA Fiber Mapping (QDFM)' that allows accurate tagging of sequence elements of interest with near kilobase accuracy and optimized it for delineation of rearrangements in recombinant DNA clones. This paper demonstrates the power of this microscopic approach by investigating YAC rearrangements. In our examples, high-resolution physical maps for regions within the immunoglobulin lambda variant gene cluster were constructed for three different YAC clones carrying deletions of 95 kb and more. Rearrangements within YACs could be demonstrated unambiguously by pairwise mapping of cosmids along YAC DNA molecules. When coverage by YAC clones was not available, distances between cosmid clones were estimated by hybridization of cosmids onto DNA fibers prepared from human genomic DNA. In addition, the QDFM technology provides essential information about clone stability facilitating closure of the maps of the human genome as well as those of model organisms.

  5. RIM15 antagonistic pleiotropy is responsible for differences in fermentation and stress response kinetics in budding yeast.

    Science.gov (United States)

    Kessi-Pérez, Eduardo I; Araos, Sebastián; García, Verónica; Salinas, Francisco; Abarca, Valentina; Larrondo, Luis F; Martínez, Claudio; Cubillos, Francisco A

    2016-05-01

    Different natural yeast populations have faced dissimilar selective pressures due to the heterogeneous fermentation substrates available around the world; this increases the genetic and phenotypic diversity in Saccharomyces cerevisiae In this context, we expect prominent differences between isolates when exposed to a particular condition, such as wine or sake musts. To better comprehend the mechanisms underlying niche adaptation between two S. cerevisiae isolates obtained from wine and sake fermentation processes, we evaluated fermentative and fungicide resistance phenotypes and identify the molecular origin of such adaptive variation. Multiple regions were associated with fermentation rate under different nitrogen conditions and fungicide resistance, with a single QTL co-localizing in all traits. Analysis around this region identified RIM15 as the causative locus driving fungicide sensitivity, together with efficient nitrogen utilization and glycerol production in the wine strain. A null RIM15 variant confers a greater fermentation rate through the utilization of available glucose instead of its storage. However, this variant has a detrimental effect on fungicide resistance since complex sugars are not synthesized and transported into the membrane. Together, our results reveal the antagonist pleiotropic nature of a RIM15 null variant, positively affecting a series of fermentation related phenotypes, but apparently detrimental in the wild.

  6. Important role of catalase in the cellular response of the budding yeast Saccharomyces cerevisiae exposed to ionizing radiation.

    Science.gov (United States)

    Nishimoto, Takuto; Furuta, Masakazu; Kataoka, Michihiko; Kishida, Masao

    2015-03-01

    Ionizing radiation indirectly causes oxidative stress in cells via reactive oxygen species (ROS), such as hydroxyl radicals (OH(-)) generated by the radiolysis of water. We investigated how the catalase function was affected by ionizing radiation and analyzed the phenotype of mutants with a disrupted catalase gene in Saccharomyces cerevisiae exposed to radiation. The wild-type yeast strain and isogenic mutants with disrupted catalase genes were exposed to various doses of (60)Co gamma-rays. There was no difference between the wild-type strain and the cta1 disruption mutant following exposure to gamma-ray irradiation. In contrast, there was a significant decrease in the ctt1 disruption mutant, suggesting that this strain exhibited decreased survival on gamma-ray exposure compared with other strains. In all three strains, stationary phase cells were more tolerant to the exposure of gamma-rays than exponential phase cells, whereas the catalase activity in the wild-type strain and cta1 disruption mutant was higher in the stationary phase than in the exponential phase. These data suggest a correlation between catalase activity and survival following gamma-ray exposure. However, this correlation was not clear in the ctt1 disruption mutant, suggesting that other factors are involved in the tolerance to ROS induced by irradiation.

  7. Osh proteins regulate COPII-mediated vesicular transport of ceramide from the endoplasmic reticulum in budding yeast.

    Science.gov (United States)

    Kajiwara, Kentaro; Ikeda, Atsuko; Aguilera-Romero, Auxiliadora; Castillon, Guillaume A; Kagiwada, Satoshi; Hanada, Kentaro; Riezman, Howard; Muñiz, Manuel; Funato, Kouichi

    2014-01-15

    Lipids synthesized at the endoplasmic reticulum (ER) are delivered to the Golgi by vesicular and non-vesicular pathways. ER-to-Golgi transport is crucial for maintaining the different membrane lipid composition and identities of organelles. Despite their importance, mechanisms regulating transport remain elusive. Here we report that in yeast coat protein complex II (COPII) vesicle-mediated transport of ceramide from the ER to the Golgi requires oxysterol-binding protein homologs, Osh proteins, which have been implicated in lipid homeostasis. Because Osh proteins are not required to transport proteins to the Golgi, these results indicate a specific requirement for the Osh proteins in the transport of ceramide. In addition, we provide evidence that Osh proteins play a negative role in COPII vesicle biogenesis. Together, our data suggest that ceramide transport and sphingolipid levels between the ER and Golgi are maintained by two distinct functions of Osh proteins, which negatively regulate COPII vesicle formation and positively control a later stage, presumably fusion of ceramide-enriched vesicles with Golgi compartments.

  8. Rif2 promotes a telomere fold-back structure through Rpd3L recruitment in budding yeast.

    Directory of Open Access Journals (Sweden)

    Heiko Poschke

    2012-09-01

    Full Text Available Using a genome-wide screening approach, we have established the genetic requirements for proper telomere structure in Saccharomyces cerevisiae. We uncovered 112 genes, many of which have not previously been implicated in telomere function, that are required to form a fold-back structure at chromosome ends. Among other biological processes, lysine deacetylation, through the Rpd3L, Rpd3S, and Hda1 complexes, emerged as being a critical regulator of telomere structure. The telomeric-bound protein, Rif2, was also found to promote a telomere fold-back through the recruitment of Rpd3L to telomeres. In the absence of Rpd3 function, telomeres have an increased susceptibility to nucleolytic degradation, telomere loss, and the initiation of premature senescence, suggesting that an Rpd3-mediated structure may have protective functions. Together these data reveal that multiple genetic pathways may directly or indirectly impinge on telomere structure, thus broadening the potential targets available to manipulate telomere function.

  9. The stress-regulatory transcription factors Msn2 and Msn4 regulate fatty acid oxidation in budding yeast.

    Science.gov (United States)

    Rajvanshi, Praveen Kumar; Arya, Madhuri; Rajasekharan, Ram

    2017-09-18

    The transcription factors multicopy suppressor of SNF1 mutation (Msn) Msn2 and Msn4 bind the stress-response element in gene promoters in the yeast Saccharomyces cerevisiae. However, the roles of Msn2/4 in primary metabolic pathways such as fatty acid β-oxidation are unclear. Here, in silico analysis revealed that the promoters of most genes involved in the biogenesis, function, and regulation of the peroxisome contain Msn2/4 binding sites. We also found that transcript levels of MSN2/MSN4 are increased in glucose-depletion conditions and that during growth in nonpreferred carbon sources, Msn2 is constantly localized to the nucleus in wildtype cells. Of note, the double mutant msn2Δmsn4Δ exhibited a severe growth defect when grown with oleic acid as the sole carbon source and had reduced transcript levels of major β-oxidation genes. ChIP indicated that Msn2 has increased occupancy on the promoters of β-oxidation genes in glucose-depleted conditions, and in vivo reporter genes analysis indicated reduced expression of these genes in msn2Δmsn4Δ cells. Moreover, mobility shift assays revealed that Msn4 binds β-oxidation gene promoters. Immunofluorescence microscopy with anti-peroxisome membrane protein antibodies disclosed that the msn2Δmsn4Δ strain had fewer peroxisomes than the wildtype, and lipid analysis indicated that the msn2Δmsn4Δ strain had increased triacylglycerol and steryl ester levels. Collectively, our data suggest that Msn2/Msn4 transcription factors activate expression of the genes involved in fatty acid oxidation. As glucose sensing, signaling, and fatty acid β-oxidation pathways are evolutionarily conserved throughout eukaryotes, the msn2Δmsn4Δ strain could therefore be a good model system for further study of these critical processes. Copyright © 2017, The American Society for Biochemistry and Molecular Biology.

  10. Reconstruction of ancestral chromosome architecture and gene repertoire reveals principles of genome evolution in a model yeast genus.

    Science.gov (United States)

    Vakirlis, Nikolaos; Sarilar, Véronique; Drillon, Guénola; Fleiss, Aubin; Agier, Nicolas; Meyniel, Jean-Philippe; Blanpain, Lou; Carbone, Alessandra; Devillers, Hugo; Dubois, Kenny; Gillet-Markowska, Alexandre; Graziani, Stéphane; Huu-Vang, Nguyen; Poirel, Marion; Reisser, Cyrielle; Schott, Jonathan; Schacherer, Joseph; Lafontaine, Ingrid; Llorente, Bertrand; Neuvéglise, Cécile; Fischer, Gilles

    2016-07-01

    Reconstructing genome history is complex but necessary to reveal quantitative principles governing genome evolution. Such reconstruction requires recapitulating into a single evolutionary framework the evolution of genome architecture and gene repertoire. Here, we reconstructed the genome history of the genus Lachancea that appeared to cover a continuous evolutionary range from closely related to more diverged yeast species. Our approach integrated the generation of a high-quality genome data set; the development of AnChro, a new algorithm for reconstructing ancestral genome architecture; and a comprehensive analysis of gene repertoire evolution. We found that the ancestral genome of the genus Lachancea contained eight chromosomes and about 5173 protein-coding genes. Moreover, we characterized 24 horizontal gene transfers and 159 putative gene creation events that punctuated species diversification. We retraced all chromosomal rearrangements, including gene losses, gene duplications, chromosomal inversions and translocations at single gene resolution. Gene duplications outnumbered losses and balanced rearrangements with 1503, 929, and 423 events, respectively. Gene content variations between extant species are mainly driven by differential gene losses, while gene duplications remained globally constant in all lineages. Remarkably, we discovered that balanced chromosomal rearrangements could be responsible for up to 14% of all gene losses by disrupting genes at their breakpoints. Finally, we found that nonsynonymous substitutions reached fixation at a coordinated pace with chromosomal inversions, translocations, and duplications, but not deletions. Overall, we provide a granular view of genome evolution within an entire eukaryotic genus, linking gene content, chromosome rearrangements, and protein divergence into a single evolutionary framework.

  11. Adaptive response to chronic mild ethanol stress involves ROS, sirtuins and changes in chromosome dosage in wine yeasts.

    Science.gov (United States)

    Adamczyk, Jagoda; Deregowska, Anna; Skoneczny, Marek; Skoneczna, Adrianna; Kwiatkowska, Aleksandra; Potocki, Leszek; Rawska, Ewa; Pabian, Sylwia; Kaplan, Jakub; Lewinska, Anna; Wnuk, Maciej

    2016-05-24

    Industrial yeast strains of economic importance used in winemaking and beer production are genomically diverse and subjected to harsh environmental conditions during fermentation. In the present study, we investigated wine yeast adaptation to chronic mild alcohol stress when cells were cultured for 100 generations in the presence of non-cytotoxic ethanol concentration. Ethanol-induced reactive oxygen species (ROS) and superoxide signals promoted growth rate during passages that was accompanied by increased expression of sirtuin proteins, Sir1, Sir2 and Sir3, and DNA-binding transcription regulator Rap1. Genome-wide array-CGH analysis revealed that yeast genome was shaped during passages. The gains of chromosomes I, III and VI and significant changes in the gene copy number in nine functional gene categories involved in metabolic processes and stress responses were observed. Ethanol-mediated gains of YRF1 and CUP1 genes were the most accented. Ethanol also induced nucleolus fragmentation that confirms that nucleolus is a stress sensor in yeasts. Taken together, we postulate that wine yeasts of different origin may adapt to mild alcohol stress by shifts in intracellular redox state promoting growth capacity, upregulation of key regulators of longevity, namely sirtuins and changes in the dosage of genes involved in the telomere maintenance and ion detoxification.

  12. Systematic yeast synthetic lethal and synthetic dosage lethal screens identify genes required for chromosome segregation

    OpenAIRE

    Measday, Vivien; Baetz, Kristin; Guzzo, Julie; Yuen, Karen; Kwok, Teresa; Sheikh, Bilal; Ding, Huiming; Ueta, Ryo; Hoac, Trinh; Cheng, Benjamin; Pot, Isabelle; Tong, Amy; Yamaguchi-Iwai, Yuko; Boone, Charles; Hieter, Phil

    2005-01-01

    Accurate chromosome segregation requires the execution and coordination of many processes during mitosis, including DNA replication, sister chromatid cohesion, and attachment of chromosomes to spindle microtubules via the kinetochore complex. Additional pathways are likely involved because faithful chromosome segregation also requires proteins that are not physically associated with the chromosome. Using kinetochore mutants as a starting point, we have identified genes with roles in chromosom...

  13. The budding yeast Cdc48(Shp1 complex promotes cell cycle progression by positive regulation of protein phosphatase 1 (Glc7.

    Directory of Open Access Journals (Sweden)

    Stefanie Böhm

    Full Text Available The conserved, ubiquitin-selective AAA ATPase Cdc48 regulates numerous cellular processes including protein quality control, DNA repair and the cell cycle. Cdc48 function is tightly controlled by a multitude of cofactors mediating substrate specificity and processing. The UBX domain protein Shp1 is a bona fide substrate-recruiting cofactor of Cdc48 in the budding yeast S. cerevisiae. Even though Shp1 has been proposed to be a positive regulator of Glc7, the catalytic subunit of protein phosphatase 1 in S. cerevisiae, its cellular functions in complex with Cdc48 remain largely unknown. Here we show that deletion of the SHP1 gene results in severe growth defects and a cell cycle delay at the metaphase to anaphase transition caused by reduced Glc7 activity. Using an engineered Cdc48 binding-deficient variant of Shp1, we establish the Cdc48(Shp1 complex as a critical regulator of mitotic Glc7 activity. We demonstrate that shp1 mutants possess a perturbed balance of Glc7 phosphatase and Ipl1 (Aurora B kinase activities and show that hyper-phosphorylation of the kinetochore protein Dam1, a key mitotic substrate of Glc7 and Ipl1, is a critical defect in shp1. We also show for the first time a physical interaction between Glc7 and Shp1 in vivo. Whereas loss of Shp1 does not significantly affect Glc7 protein levels or localization, it causes reduced binding of the activator protein Glc8 to Glc7. Our data suggest that the Cdc48(Shp1 complex controls Glc7 activity by regulating its interaction with Glc8 and possibly further regulatory subunits.

  14. Budding yeast ATM/ATR control meiotic double-strand break (DSB) levels by down-regulating Rec114, an essential component of the DSB-machinery.

    Science.gov (United States)

    Carballo, Jesús A; Panizza, Silvia; Serrentino, Maria Elisabetta; Johnson, Anthony L; Geymonat, Marco; Borde, Valérie; Klein, Franz; Cha, Rita S

    2013-06-01

    An essential feature of meiosis is Spo11 catalysis of programmed DNA double strand breaks (DSBs). Evidence suggests that the number of DSBs generated per meiosis is genetically determined and that this ability to maintain a pre-determined DSB level, or "DSB homeostasis", might be a property of the meiotic program. Here, we present direct evidence that Rec114, an evolutionarily conserved essential component of the meiotic DSB-machinery, interacts with DSB hotspot DNA, and that Tel1 and Mec1, the budding yeast ATM and ATR, respectively, down-regulate Rec114 upon meiotic DSB formation through phosphorylation. Mimicking constitutive phosphorylation reduces the interaction between Rec114 and DSB hotspot DNA, resulting in a reduction and/or delay in DSB formation. Conversely, a non-phosphorylatable rec114 allele confers a genome-wide increase in both DSB levels and in the interaction between Rec114 and the DSB hotspot DNA. These observations strongly suggest that Tel1 and/or Mec1 phosphorylation of Rec114 following Spo11 catalysis down-regulates DSB formation by limiting the interaction between Rec114 and DSB hotspots. We also present evidence that Ndt80, a meiosis specific transcription factor, contributes to Rec114 degradation, consistent with its requirement for complete cessation of DSB formation. Loss of Rec114 foci from chromatin is associated with homolog synapsis but independent of Ndt80 or Tel1/Mec1 phosphorylation. Taken together, we present evidence for three independent ways of regulating Rec114 activity, which likely contribute to meiotic DSBs-homeostasis in maintaining genetically determined levels of breaks.

  15. Budding yeast ATM/ATR control meiotic double-strand break (DSB levels by down-regulating Rec114, an essential component of the DSB-machinery.

    Directory of Open Access Journals (Sweden)

    Jesús A Carballo

    2013-06-01

    Full Text Available An essential feature of meiosis is Spo11 catalysis of programmed DNA double strand breaks (DSBs. Evidence suggests that the number of DSBs generated per meiosis is genetically determined and that this ability to maintain a pre-determined DSB level, or "DSB homeostasis", might be a property of the meiotic program. Here, we present direct evidence that Rec114, an evolutionarily conserved essential component of the meiotic DSB-machinery, interacts with DSB hotspot DNA, and that Tel1 and Mec1, the budding yeast ATM and ATR, respectively, down-regulate Rec114 upon meiotic DSB formation through phosphorylation. Mimicking constitutive phosphorylation reduces the interaction between Rec114 and DSB hotspot DNA, resulting in a reduction and/or delay in DSB formation. Conversely, a non-phosphorylatable rec114 allele confers a genome-wide increase in both DSB levels and in the interaction between Rec114 and the DSB hotspot DNA. These observations strongly suggest that Tel1 and/or Mec1 phosphorylation of Rec114 following Spo11 catalysis down-regulates DSB formation by limiting the interaction between Rec114 and DSB hotspots. We also present evidence that Ndt80, a meiosis specific transcription factor, contributes to Rec114 degradation, consistent with its requirement for complete cessation of DSB formation. Loss of Rec114 foci from chromatin is associated with homolog synapsis but independent of Ndt80 or Tel1/Mec1 phosphorylation. Taken together, we present evidence for three independent ways of regulating Rec114 activity, which likely contribute to meiotic DSBs-homeostasis in maintaining genetically determined levels of breaks.

  16. Regulation of budding yeast mating-type switching donor preference by the FHA domain of Fkh1.

    Science.gov (United States)

    Li, Jin; Coïc, Eric; Lee, Kihoon; Lee, Cheng-Sheng; Kim, Jung-Ae; Wu, Qiuqin; Haber, James E

    2012-01-01

    During Saccharomyces cerevisiae mating-type switching, an HO endonuclease-induced double-strand break (DSB) at MAT is repaired by recombining with one of two donors, HMLα or HMRa, located at opposite ends of chromosome III. MATa cells preferentially recombine with HMLα; this decision depends on the Recombination Enhancer (RE), located about 17 kb to the right of HML. In MATα cells, HML is rarely used and RE is bound by the MATα2-Mcm1 corepressor, which prevents the binding of other proteins to RE. In contrast, in MATa cells, RE is bound by multiple copies of Fkh1 and a single copy of Swi4/Swi6. We report here that, when RE is replaced with four LexA operators in MATa cells, 95% of cells use HMR for repair, but expression of a LexA-Fkh1 fusion protein strongly increases HML usage. A LexA-Fkh1 truncation, containing only Fkh1's phosphothreonine-binding FHA domain, restores HML usage to 90%. A LexA-FHA-R80A mutant lacking phosphothreonine binding fails to increase HML usage. The LexA-FHA fusion protein associates with chromatin in a 10-kb interval surrounding the HO cleavage site at MAT, but only after DSB induction. This association occurs even in a donorless strain lacking HML. We propose that the FHA domain of Fkh1 regulates donor preference by physically interacting with phosphorylated threonine residues created on proteins bound near the DSB, thus positioning HML close to the DSB at MAT. Donor preference is independent of Mec1/ATR and Tel1/ATM checkpoint protein kinases but partially depends on casein kinase II. RE stimulates the strand invasion step of interchromosomal recombination even for non-MAT sequences. We also find that when RE binds to the region near the DSB at MATa then Mec1 and Tel1 checkpoint kinases are not only able to phosphorylate histone H2A (γ-H2AX) around the DSB but can also promote γ-H2AX spreading around the RE region.

  17. Regulation of budding yeast mating-type switching donor preference by the FHA domain of Fkh1.

    Directory of Open Access Journals (Sweden)

    Jin Li

    Full Text Available During Saccharomyces cerevisiae mating-type switching, an HO endonuclease-induced double-strand break (DSB at MAT is repaired by recombining with one of two donors, HMLα or HMRa, located at opposite ends of chromosome III. MATa cells preferentially recombine with HMLα; this decision depends on the Recombination Enhancer (RE, located about 17 kb to the right of HML. In MATα cells, HML is rarely used and RE is bound by the MATα2-Mcm1 corepressor, which prevents the binding of other proteins to RE. In contrast, in MATa cells, RE is bound by multiple copies of Fkh1 and a single copy of Swi4/Swi6. We report here that, when RE is replaced with four LexA operators in MATa cells, 95% of cells use HMR for repair, but expression of a LexA-Fkh1 fusion protein strongly increases HML usage. A LexA-Fkh1 truncation, containing only Fkh1's phosphothreonine-binding FHA domain, restores HML usage to 90%. A LexA-FHA-R80A mutant lacking phosphothreonine binding fails to increase HML usage. The LexA-FHA fusion protein associates with chromatin in a 10-kb interval surrounding the HO cleavage site at MAT, but only after DSB induction. This association occurs even in a donorless strain lacking HML. We propose that the FHA domain of Fkh1 regulates donor preference by physically interacting with phosphorylated threonine residues created on proteins bound near the DSB, thus positioning HML close to the DSB at MAT. Donor preference is independent of Mec1/ATR and Tel1/ATM checkpoint protein kinases but partially depends on casein kinase II. RE stimulates the strand invasion step of interchromosomal recombination even for non-MAT sequences. We also find that when RE binds to the region near the DSB at MATa then Mec1 and Tel1 checkpoint kinases are not only able to phosphorylate histone H2A (γ-H2AX around the DSB but can also promote γ-H2AX spreading around the RE region.

  18. Yeast X-chromosome-associated protein 5 (Xap5) functions with H2A.Z to suppress aberrant transcripts.

    Science.gov (United States)

    Anver, Shajahan; Roguev, Assen; Zofall, Martin; Krogan, Nevan J; Grewal, Shiv I S; Harmer, Stacey L

    2014-08-01

    Chromatin regulatory proteins affect diverse developmental and environmental response pathways via their influence on nuclear processes such as the regulation of gene expression. Through a genome-wide genetic screen, we implicate a novel protein called X-chromosome-associated protein 5 (Xap5) in chromatin regulation. We show that Xap5 is a chromatin-associated protein acting in a similar manner as the histone variant H2A.Z to suppress expression of antisense and repeat element transcripts throughout the fission yeast genome. Xap5 is highly conserved across eukaryotes, and a plant homolog rescues xap5 mutant yeast. We propose that Xap5 likely functions as a chromatin regulator in diverse organisms.

  19. CHARACTERIZATION AND CHROMOSOMAL ASSIGNMENT OF YEAST ARTIFICIAL CHROMOSOMES CONTAINING HUMAN 3P13-P21-SPECIFIC SEQUENCE-TAGGED SITES

    NARCIS (Netherlands)

    MICHAELIS, SC; BARDENHEUER, W; LUX, A; SCHRAMM, A; GOCKEL, A; SIEBERT, R; WILLERS, C; SCHMIDTKE, K; TODT, B; VANDERHOUT, AH; BUYS, CHCM; HEPPELLPARTON, AC; RABBITTS, PH; UNGAR, S; SMITH, D; LEPASLIER, D; COHEN, D; OPALKA, B; SCHUTTE, J

    1995-01-01

    Human chromosomal region 3p12-p23 is proposed to harbor at least three tumor suppressor genes involved in the development of lung cancer, renal cell carcinoma, and other neoplasias. In order to identify one of these genes we defined sequence tagged sites (STSs) specific for 3p13-p24.2 by analyzing a

  20. Arenavirus Budding

    Directory of Open Access Journals (Sweden)

    Shuzo Urata

    2011-01-01

    Full Text Available Several arenaviruses cause hemorrhagic fever disease in humans and pose a significant public health concern in their endemic regions. On the other hand, the prototypic arenavirus LCMV is a superb workhorse for the investigation of virus-host interactions and associated disease. The arenavirus small RING finger protein called Z has been shown to be the main driving force of virus budding. The budding activity of Z is mediated by late (L domain motifs, PT/SAP, and PPXY, located at the C-terminus of Z. This paper will present the current knowledge on arenavirus budding including the diversity of L domain motifs used by different arenaviruses. We will also discuss how improved knowledge of arenavirus budding may facilitate the development of novel antiviral strategies to combat human pathogenic arenaviruses.

  1. BUD SUCKERS

    African Journals Online (AJOL)

    consisted in making sets of suckers of ... Figure 2 : Experimental device for the hydroponic culture of the banana plantain cv Horn 1 bud suckers. ... ning microscope were treated as follow : fixation of ..... determining step in the growth process,.

  2. Mitotic chromosome biorientation in fission yeast is enhanced by dynein and a minus-end-directed, kinesin-like protein.

    Science.gov (United States)

    Grishchuk, Ekaterina L; Spiridonov, Ilia S; McIntosh, J Richard

    2007-06-01

    Chromosome biorientation, the attachment of sister kinetochores to sister spindle poles, is vitally important for accurate chromosome segregation. We have studied this process by following the congression of pole-proximal kinetochores and their subsequent anaphase segregation in fission yeast cells that carry deletions in any or all of this organism's minus end-directed, microtubule-dependent motors: two related kinesin 14s (Pkl1p and Klp2p) and dynein. None of these deletions abolished biorientation, but fewer chromosomes segregated normally without Pkl1p, and to a lesser degree without dynein, than in wild-type cells. In the absence of Pkl1p, which normally localizes to the spindle and its poles, the checkpoint that monitors chromosome biorientation was defective, leading to frequent precocious anaphase. Ultrastructural analysis of mutant mitotic spindles suggests that Pkl1p contributes to error-free biorientation by promoting normal spindle pole organization, whereas dynein helps to anchor a focused bundle of spindle microtubules at the pole.

  3. Chromosome

    Science.gov (United States)

    Chromosomes are structures found in the center (nucleus) of cells that carry long pieces of DNA. DNA ... is the building block of the human body. Chromosomes also contain proteins that help DNA exist in ...

  4. Yeast artificial chromosomes employed for random assembly of biosynthetic pathways and production of diverse compounds in Saccharomyces cerevisiae

    Directory of Open Access Journals (Sweden)

    Mitra Partha P

    2009-08-01

    Full Text Available Abstract Background Natural products are an important source of drugs and other commercially interesting compounds, however their isolation and production is often difficult. Metabolic engineering, mainly in bacteria and yeast, has sought to circumvent some of the associated problems but also this approach is impeded by technical limitations. Here we describe a novel strategy for production of diverse natural products, comprising the expression of an unprecedented large number of biosynthetic genes in a heterologous host. Results As an example, genes from different sources, representing enzymes of a seven step flavonoid pathway, were individually cloned into yeast expression cassettes, which were then randomly combined on Yeast Artificial Chromosomes and used, in a single transformation of yeast, to create a variety of flavonoid producing pathways. Randomly picked clones were analysed, and approximately half of them showed production of the flavanone naringenin, and a third of them produced the flavonol kaempferol in various amounts. This reflected the assembly of 5–7 step multi-species pathways converting the yeast metabolites phenylalanine and/or tyrosine into flavonoids, normally only produced by plants. Other flavonoids were also produced that were either direct intermediates or derivatives thereof. Feeding natural and unnatural, halogenated precursors to these recombinant clones demonstrated the potential to further diversify the type of molecules that can be produced with this technology. Conclusion The technology has many potential uses but is particularly suited for generating high numbers of structurally diverse compounds, some of which may not be amenable to chemical synthesis, thus greatly facilitating access to a huge chemical space in the search for new commercially interesting compounds

  5. A conserved checkpoint monitors meiotic chromosome synapsis inCaenorhabditis elegans

    Energy Technology Data Exchange (ETDEWEB)

    Bhalla, Needhi; Dernburg, Abby F.

    2005-07-14

    We report the discovery of a checkpoint that monitorssynapsis between homologous chromosomes to ensure accurate meioticsegregation. Oocytes containing unsynapsed chromosomes selectivelyundergo apoptosis even if agermline DNA damage checkpoint is inactivated.This culling mechanism isspecifically activated by unsynapsed pairingcenters, cis-acting chromosomesites that are also required to promotesynapsis in Caenorhabditis elegans. Apoptosis due to synaptic failurealso requires the C. elegans homolog of PCH2,a budding yeast pachytenecheckpoint gene, which suggests that this surveillance mechanism iswidely conserved.

  6. Study on Optimization of Jiangxi Yanshan Red Bud Taro Plantlet Chromosome Preparation Technique%江西铅山红芽芋试管苗染色制片技术优化研究

    Institute of Scientific and Technical Information of China (English)

    王艾平; 柯维忠; 林国卫; 吴燕芳; 易雪梅; 叶志康; 黄丽; 余琪; 肖淮宾

    2014-01-01

    以江西铅山红芽芋试管苗为材料,探讨不同取材部位、不同预处理剂、不同酸解离时间和不同改良卡宝品红染色时间对江西铅山红芽芋试管苗染色体制片的影响,以期为江西铅山红芽芋的起源、演化及遗传育种提供一定理论依据。结果表明,切取江西铅山红芽芋试管苗根尖,在室温下用0.1%秋水仙素+0.002mol/L 8-羟基喹啉混合溶液预处理4h ,然后在60℃条件下用1 mol/L盐酸解离15 min ,最后用改良卡宝品红染色5 min ,此时染色体制片效果最佳。%In order to better understand the information about cytology of Jiangxi Yanshan red bud taro ( Colocasia esculenta L .Schott var .cormosus cv .Hongyayu) ,provide information support for genetic breeding of red bud taro .The protocol of different sampling places ,pretreatments solutions ,dissociating time and dying time on chromosome observation from red bud taro plantlets was studied . The results showed that the effect of chromosome preparation was best when the root tips of red bud taro plantlets was cut ,pretreated in a mixture of 0 .1% colchicines and 0 .002mol/L 8-hydroxyquinoline at 25 ℃ for 4h ,dissocated in 1 mol/L HCl at 60 ℃ for 15 min and dyed with modified carbol fuchsin for 5 min .

  7. Structure and dynamics of interphase chromosomes.

    Directory of Open Access Journals (Sweden)

    Angelo Rosa

    Full Text Available During interphase chromosomes decondense, but fluorescent in situ hybridization experiments reveal the existence of distinct territories occupied by individual chromosomes inside the nuclei of most eukaryotic cells. We use computer simulations to show that the existence and stability of territories is a kinetic effect that can be explained without invoking an underlying nuclear scaffold or protein-mediated interactions between DNA sequences. In particular, we show that the experimentally observed territory shapes and spatial distances between marked chromosome sites for human, Drosophila, and budding yeast chromosomes can be reproduced by a parameter-free minimal model of decondensing chromosomes. Our results suggest that the observed interphase structure and dynamics are due to generic polymer effects: confined Brownian motion conserving the local topological state of long chain molecules and segregation of mutually unentangled chains due to topological constraints.

  8. Chromosomal Integration and Expression of Two Bacterial alpha-Acetolactate Decarboxylase Genes in Brewer's Yeast.

    Science.gov (United States)

    Blomqvist, K; Suihko, M L; Knowles, J; Penttilä, M

    1991-10-01

    A bacterial gene encoding alpha-acetolactate decarboxylase, isolated from Klebsiella terrigena or Enterobacter aerogenes, was expressed in brewer's yeast. The genes were expressed under either the yeast phosphoglycerokinase (PGK1) or the alcohol dehydrogenase (ADH1) promoter and were integrated by gene replacement by using cotransformation into the PGK1 or ADH1 locus, respectively, of a brewer's yeast. The expression level of the alpha-acetolactate decarboxylase gene of the PGK1 integrant strains was higher than that of the ADH1 integrants. Under pilot-scale brewing conditions, the alpha-acetolactate decarboxylase activity of the PGK1 integrant strains was sufficient to reduce the formation of diacetyl below the taste threshold value, and no lagering was needed. The brewing properties of the recombinant yeast strains were otherwise unaltered, and the quality (most importantly, the flavor) of the trial beers produced was as good as that of the control beer.

  9. Msa1 and Msa2 Modulate G1-Specific Transcription to Promote G1 Arrest and the Transition to Quiescence in Budding Yeast.

    Directory of Open Access Journals (Sweden)

    Shawna Miles

    2016-06-01

    Full Text Available Yeast that naturally exhaust their glucose source can enter a quiescent state that is characterized by reduced cell size, and high cell density, stress tolerance and longevity. The transition to quiescence involves highly asymmetric cell divisions, dramatic reprogramming of transcription and global changes in chromatin structure and chromosome topology. Cells enter quiescence from G1 and we find that there is a positive correlation between the length of G1 and the yield of quiescent cells. The Swi4 and Swi6 transcription factors, which form the SBF transcription complex and promote the G1 to S transition in cycling cells, are also critical for the transition to quiescence. Swi6 forms a second complex with Mbp1 (MBF, which is not required for quiescence. These are the functional analogues of the E2F complexes of higher eukaryotes. Loss of the RB analogue, Whi5, and the related protein Srl3/Whi7, delays G1 arrest, but it also delays recovery from quiescence. Two MBF- and SBF-Associated proteins have been identified that have little effect on SBF or MBF activity in cycling cells. We show that these two related proteins, Msa1 and Msa2, are specifically required for the transition to quiescence. Like the E2F complexes that are quiescence-specific, Msa1 and Msa2 are required to repress the transcription of many SBF target genes, including SWI4, the CLN2 cyclin and histones, specifically after glucose is exhausted from the media. They also activate transcription of many MBF target genes. msa1msa2 cells fail to G1 arrest and rapidly lose viability upon glucose exhaustion. msa1msa2 mutants that survive this transition are very large, but they attain the same thermo-tolerance and longevity of wild type quiescent cells. This indicates that Msa1 and Msa2 are required for successful transition to quiescence, but not for the maintenance of that state.

  10. Narrowing the genetic interval and yeast artificial chromosome map in the branchio-oto-renal region on chromosome 8q

    Energy Technology Data Exchange (ETDEWEB)

    Kumar, Shrawan; Kimberling, W.J.; Pinnt, J. [Boys Town National Research Hospital, Omaha, NE (United States)] [and others

    1996-01-01

    Branchio-oto-renal (BOR) syndrome is an autosomal dominant disorder characterized by branchial abnormality, hearing loss, and renal anomalies. Recently, the disease gene has been localized to chromosome 8q. Here, we report genetic studies that further refine the disease gene region to a smaller interval and identify several YACs from the critical region. We studied two large, clinically well-characterized BOR families with a set of 13 polymorphic markers spanning the D8S165-D8S275 interval from the chromosome 8q region. Based on multipoint analysis, the highest likelihood for the location of the BOR gene is between markers D8S543 and D8S530, a distance of about 2 cM. YACs that map in the BOR critical region have been identified and characterized by fluorescence in situ hybridization and pulsed-field gel electrophoresis. A YAC contig, based on the STS content map, that covers a minimum of 4 Mb of human DNA in the critical region of BOR is assembled. This lays the groundwork for the construction of a transcriptional map of this region and the eventual identification of genes involved in BOR syndrome. 40 refs., 4 figs., 1 tab.

  11. Sgo1 regulates both condensin and Ipl1/Aurora B to promote chromosome biorientation.

    Directory of Open Access Journals (Sweden)

    Karolina Peplowska

    2014-06-01

    Full Text Available Correct chromosome segregation is essential in order to prevent aneuploidy. To segregate sister chromatids equally to daughter cells, the sisters must attach to microtubules emanating from opposite spindle poles. This so-called biorientation manifests itself by increased tension and conformational changes across kinetochores and pericentric chromatin. Tensionless attachments are dissolved by the activity of the conserved mitotic kinase Aurora B/Ipl1, thereby promoting the formation of correctly attached chromosomes. Recruitment of the conserved centromeric protein shugoshin is essential for biorientation, but its exact role has been enigmatic. Here, we identify a novel function of shugoshin (Sgo1 in budding yeast that together with the protein phosphatase PP2A-Rts1 ensures localization of condensin to the centromeric chromatin in yeast Saccharomyces cerevisiae. Failure to recruit condensin results in an abnormal conformation of the pericentric region and impairs the correction of tensionless chromosome attachments. Moreover, we found that shugoshin is required for maintaining Aurora B/Ipl1 localization on kinetochores during metaphase. Thus, shugoshin has a dual function in promoting biorientation in budding yeast: first, by its ability to facilitate condensin recruitment it modulates the conformation of the pericentric chromatin. Second, shugoshin contributes to the maintenance of Aurora B/Ipl1 at the kinetochore during gradual establishment of bipolarity in budding yeast mitosis. Our findings identify shugoshin as a versatile molecular adaptor that governs chromosome biorientation.

  12. Island rescue PCR: a rapid and efficient method for isolating transcribed sequences from yeast artificial chromosomes and cosmids.

    Science.gov (United States)

    Valdes, J M; Tagle, D A; Collins, F S

    1994-06-07

    The identification of transcripts from large genomic regions cloned in yeast artificial chromosomes (YACs) or cosmids continues to be a critical and often rate-limiting step in positional cloning of human disease genes. We have developed a PCR-based method for rapid and efficient generation of probes from YACs or cosmids that can be used for cDNA library screening. The method, which we call island rescue PCR (IRP), is based upon the observation that the 5' ends of many genes are associated with (G+C)-rich regions called CpG islands. In IRP, the YAC of interest is digested with a restriction enzyme that recognizes sequences of high CpG content, and vectorette linkers are ligated to the cleaved ends. The PCR is used to amplify the region extending from the cleaved restriction enzyme site to the nearest SINE (Alu) repeat. In many cases this product contains sequences from the 5' end of the associated gene. cDNA clones isolated with these products are then verified by mapping them back to the original YAC. The method allows rapid screening of > 500 kb of human genomic insert in one experiment, is tolerant of contaminating yeast sequences, and can also be applied to cosmid pools. In a control experiment, the method was able to identify cDNA clones for the neurofibromatosis type 1 (NF1) gene using a probe generated from a YAC in the region. Application of IRP has yielded nine other genes from YACs isolated from chromosome locations 4p16.3 and 17q21.

  13. A Three-Dimensional Model of the Yeast Genome

    Science.gov (United States)

    Noble, William; Duan, Zhi-Jun; Andronescu, Mirela; Schutz, Kevin; McIlwain, Sean; Kim, Yoo Jung; Lee, Choli; Shendure, Jay; Fields, Stanley; Blau, C. Anthony

    Layered on top of information conveyed by DNA sequence and chromatin are higher order structures that encompass portions of chromosomes, entire chromosomes, and even whole genomes. Interphase chromosomes are not positioned randomly within the nucleus, but instead adopt preferred conformations. Disparate DNA elements co-localize into functionally defined aggregates or factories for transcription and DNA replication. In budding yeast, Drosophila and many other eukaryotes, chromosomes adopt a Rabl configuration, with arms extending from centromeres adjacent to the spindle pole body to telomeres that abut the nuclear envelope. Nonetheless, the topologies and spatial relationships of chromosomes remain poorly understood. Here we developed a method to globally capture intra- and inter-chromosomal interactions, and applied it to generate a map at kilobase resolution of the haploid genome of Saccharomyces cerevisiae. The map recapitulates known features of genome organization, thereby validating the method, and identifies new features. Extensive regional and higher order folding of individual chromosomes is observed. Chromosome XII exhibits a striking conformation that implicates the nucleolus as a formidable barrier to interaction between DNA sequences at either end. Inter-chromosomal contacts are anchored by centromeres and include interactions among transfer RNA genes, among origins of early DNA replication and among sites where chromosomal breakpoints occur. Finally, we constructed a three-dimensional model of the yeast genome. Our findings provide a glimpse of the interface between the form and function of a eukaryotic genome.

  14. Process for assembly and transformation into Saccharomyces cerevisiae of a synthetic yeast artificial chromosome containing a multigene cassette to express enzymes that enhance xylose utilization designed for an automated pla

    Science.gov (United States)

    A yeast artificial chromosome (YAC) containing a multigene cassette for expression of enzymes that enhance xylose utilization (xylose isomerase [XI] and xylulokinase [XKS]) was constructed and transformed into Saccharomyces cerevisiae to demonstrate feasibility as a stable protein expression system ...

  15. AID induces double-strand breaks at immunoglobulin switch regions and c-MYC causing chromosomal translocations in yeast THO mutants.

    Science.gov (United States)

    Ruiz, José F; Gómez-González, Belén; Aguilera, Andrés

    2011-02-01

    Transcription of the switch (S) regions of immunoglobulin genes in B cells generates stable R-loops that are targeted by Activation Induced Cytidine Deaminase (AID), triggering class switch recombination (CSR), as well as translocations with c-MYC responsible for Burkitt's lymphomas. In Saccharomyces cerevisiae, stable R-loops are formed co-transcriptionally in mutants of THO, a conserved nuclear complex involved in mRNP biogenesis. Such R-loops trigger genome instability and facilitate deamination by human AID. To understand the mechanisms that generate genome instability mediated by mRNP biogenesis impairment and by AID, we devised a yeast chromosomal system based on different segments of mammalian S regions and c-MYC for the analysis of chromosomal rearrangements in both wild-type and THO mutants. We demonstrate that AID acts in yeast at heterologous S and c-MYC transcribed sequences leading to double-strand breaks (DSBs) which in turn cause chromosomal translocations via Non-Homologous End Joining (NHEJ). AID-induced translocations were strongly enhanced in yeast THO null mutants, consistent with the idea that AID-mediated DSBs depend on R-loop formation. Our study not only provides new clues to understand the role of mRNP biogenesis in preventing genome rearrangements and the mechanism of AID-mediated genome instability, but also shows that, once uracil residues are produced by AID-mediated deamination, these are processed into DSBs and chromosomal rearrangements by the general and conserved DNA repair functions present from yeast to human cells.

  16. AID induces double-strand breaks at immunoglobulin switch regions and c-MYC causing chromosomal translocations in yeast THO mutants.

    Directory of Open Access Journals (Sweden)

    José F Ruiz

    2011-02-01

    Full Text Available Transcription of the switch (S regions of immunoglobulin genes in B cells generates stable R-loops that are targeted by Activation Induced Cytidine Deaminase (AID, triggering class switch recombination (CSR, as well as translocations with c-MYC responsible for Burkitt's lymphomas. In Saccharomyces cerevisiae, stable R-loops are formed co-transcriptionally in mutants of THO, a conserved nuclear complex involved in mRNP biogenesis. Such R-loops trigger genome instability and facilitate deamination by human AID. To understand the mechanisms that generate genome instability mediated by mRNP biogenesis impairment and by AID, we devised a yeast chromosomal system based on different segments of mammalian S regions and c-MYC for the analysis of chromosomal rearrangements in both wild-type and THO mutants. We demonstrate that AID acts in yeast at heterologous S and c-MYC transcribed sequences leading to double-strand breaks (DSBs which in turn cause chromosomal translocations via Non-Homologous End Joining (NHEJ. AID-induced translocations were strongly enhanced in yeast THO null mutants, consistent with the idea that AID-mediated DSBs depend on R-loop formation. Our study not only provides new clues to understand the role of mRNP biogenesis in preventing genome rearrangements and the mechanism of AID-mediated genome instability, but also shows that, once uracil residues are produced by AID-mediated deamination, these are processed into DSBs and chromosomal rearrangements by the general and conserved DNA repair functions present from yeast to human cells.

  17. Construction and characterization of a 10-genome equivalent yeast artificial chromosome library for the laboratory rat, Rattus norvegicus

    Energy Technology Data Exchange (ETDEWEB)

    Cai, L.; Zee, R.Y.L. [Harvard Medical School, Boston, MA (United States); Schalkwyk, L.C. [Max Planck Institute for Molecular Genetics, Berlin (Germany)] [and others

    1997-02-01

    Increasing attention has been focused in recent years on the rat as a model organism for genetic studies, in particular for the investigation of complex traits, but progress has been limited by the lack of availability of large-insert genomic libraries. Here, we report the construction and characterization of an arrayed yeast artificial chromosome (YAC) library for the rat genome containing approximately 40,000 clones in the AB1380 host using the pCGS966 vector. An average size of 736 kb was estimated from 166 randomly chosen clones; thus the library provides 10-fold coverage of the genome, with a 99.99% probability of containing a unique sequence. Eight of 39 YACs analyzed by fluorescence in situ hybridization were found to be chimeric, indicating a proportion of about 20-30% of chimeric clones. The library was spotted on high-density filters to allow the identification of YAC clones by hybridization and was pooled using a 3-dimensional scheme for screening by PCR. Among 48 probes used to screen the library, an average of 9.3 positive clones were found, consistent with the calculated 10-fold genomic coverage of the library. This YAC library represents the first large-insert genomic library for the rat. It will be made available to the research community at large as an important new resource for complex genome analysis in this species. 35 refs., 4 figs.

  18. Inactivation of the budding yeast cohesin loader Scc2 alters gene expression both globally and in response to a single DNA double strand break

    OpenAIRE

    Lindgren, Emma; Hägg, Sara; Giordano, Fosco; Börkegren, Johan; Ström, Lena

    2014-01-01

    Genome integrity is fundamental for cell survival and cell cycle progression. Important mechanisms for keeping the genome intact are proper sister chromatid segregation, correct gene regulation and efficient repair of damaged DNA. Cohesin and its DNA loader, the Scc2/4 complex have been implicated in all these cellular actions. The gene regulation role has been described in several organisms. In yeast it has been suggested that the proteins in the cohesin network would effect transcription ba...

  19. SLC1 and SLC4 encode partially redundant acyl-coenzyme A 1-acylglycerol-3-phosphate O-acyltransferases of budding yeast

    DEFF Research Database (Denmark)

    Benghezal, Mohammed; Roubaty, Carole; Veepuri, Vijayanath

    2007-01-01

    Phosphatidic acid is the intermediate, from which all glycerophospholipids are synthesized. In yeast, it is generated from lysophosphatidic acid, which is acylated by Slc1p, an sn-2-specific, acyl-coenzyme A-dependent 1-acylglycerol-3-phosphate O-acyltransferase. Deletion of SLC1 is not lethal an......-phosphate O-acyltransferases but also be involved in fatty acid exchange at the sn-2-position of mature glycerophospholipids....

  20. SPOC alert--when chromosomes get the wrong direction.

    Science.gov (United States)

    Caydasi, Ayse Koca; Pereira, Gislene

    2012-07-15

    The asymmetrically dividing budding yeast relies upon the alignment of the mitotic spindle along the mother to daughter cell polarity axis for the fidelity of chromosome segregation during mitosis. In the case of spindle misalignment, a surveillance mechanism named the spindle position checkpoint (SPOC) prevents cells from exiting mitosis through the inhibition of the mitotic exit network (MEN). MEN is a signal transduction pathway that mediates mitotic exit through fully activation of the Cdk-counteracting phosphatase Cdc14. In this mini-review, we briefly describe the mechanisms leading to mitotic exit in budding yeast cells focusing on the control of MEN by the SPOC. In addition, we summarize the recent advances in the molecular understanding of SPOC regulation and discuss whether similar checkpoints may exist in higher eukaryotic cells that undergo asymmetric divisions.

  1. Nested chromosomal fragmentation in yeast using the meganuclease I-Sce I: a new method for physical mapping of eukaryotic genomes.

    Science.gov (United States)

    Thierry, A; Dujon, B

    1992-11-11

    We have developed a new method for the physical mapping of genomes and the rapid sorting of genomic libraries which is based on chromosome fragmentation by the meganuclease I-Sce I, the first available member of a new class of endonucleases with very long recognition sequences. I-Sce I allows complete cleavage at a single artificially inserted site in an entire genome. Sites can be inserted by homologous recombination using specific cassettes containing selectable markers or, at random, using transposons. This method has been applied to the physical mapping of chromosome XI (620 kb) of Saccharomyces cerevisi and to the sorting of a cosmid library. Our strategy has potential applications to various genome mapping projects. A set of transgenic yeast strains carrying the I-Sce I sites at various locations along a chromosome defines physical intervals against which new genes, DNA fragments or clones can be mapped directly by simple hybridizations.

  2. Condensin association with histone H2A shapes mitotic chromosomes.

    Science.gov (United States)

    Tada, Kenji; Susumu, Hiroaki; Sakuno, Takeshi; Watanabe, Yoshinori

    2011-06-01

    Chromosome structure is dynamically regulated during cell division, and this regulation is dependent, in part, on condensin. The localization of condensin at chromosome arms is crucial for chromosome partitioning during anaphase. Condensin is also enriched at kinetochores but its precise role and loading machinery remain unclear. Here we show that fission yeast (Schizosaccharomyces pombe) kinetochore proteins Pcs1 and Mde4--homologues of budding yeast (Saccharomyces cerevisiae) monopolin subunits and known to prevent merotelic kinetochore orientation--act as a condensin 'recruiter' at kinetochores, and that condensin itself may act to clamp microtubule binding sites during metaphase. In addition to the regional recruitment factors, overall condensin association with chromatin is governed by the chromosomal passenger kinase Aurora B. Aurora-B-dependent phosphorylation of condensin promotes its association with histone H2A and H2A.Z, which we identify as conserved chromatin 'receptors' of condensin. Condensin phosphorylation and its deposition onto chromosome arms reach a peak during anaphase, when Aurora B kinase relocates from centromeres to the spindle midzone, where the separating chromosome arms are positioned. Our results elucidate the molecular basis for the spatiotemporal regulation of mitotic chromosome architecture, which is crucial for chromosome partitioning.

  3. Inactivation of the budding yeast cohesin loader Scc2 alters gene expression both globally and in response to a single DNA double strand break.

    Science.gov (United States)

    Lindgren, Emma; Hägg, Sara; Giordano, Fosco; Björkegren, Johan; Ström, Lena

    2014-01-01

    Genome integrity is fundamental for cell survival and cell cycle progression. Important mechanisms for keeping the genome intact are proper sister chromatid segregation, correct gene regulation and efficient repair of damaged DNA. Cohesin and its DNA loader, the Scc2/4 complex have been implicated in all these cellular actions. The gene regulation role has been described in several organisms. In yeast it has been suggested that the proteins in the cohesin network would effect transcription based on its role as insulator. More recently, data are emerging indicating direct roles for gene regulation also in yeast. Here we extend these studies by investigating whether the cohesin loader Scc2 is involved in regulation of gene expression. We performed global gene expression profiling in the absence and presence of DNA damage, in wild type and Scc2 deficient G2/M arrested cells, when it is known that Scc2 is important for DNA double strand break repair and formation of damage induced cohesion. We found that not only the DNA damage specific transcriptional response is distorted after inactivation of Scc2 but also the overall transcription profile. Interestingly, these alterations did not correlate with changes in cohesin binding.

  4. Detection of Multiple Budding Yeast Cells and a Partial Sequence of 43-kDa Glycoprotein Coding Gene of Paracoccidioides brasiliensis from a Case of Lacaziosis in a Female Pacific White-Sided Dolphin (Lagenorhynchus obliquidens).

    Science.gov (United States)

    Minakawa, Tomoko; Ueda, Keiichi; Tanaka, Miyuu; Tanaka, Natsuki; Kuwamura, Mitsuru; Izawa, Takeshi; Konno, Toshihiro; Yamate, Jyoji; Itano, Eiko Nakagawa; Sano, Ayako; Wada, Shinpei

    2016-08-01

    Lacaziosis, formerly called as lobomycosis, is a zoonotic mycosis, caused by Lacazia loboi, found in humans and dolphins, and is endemic in the countries on the Atlantic Ocean, Indian Ocean and Pacific Ocean of Japanese coast. Susceptible Cetacean species include the bottlenose dolphin (Tursiops truncatus), the Indian Ocean bottlenose dolphin (T. aduncus), and the estuarine dolphin (Sotalia guianensis); however, no cases have been recorded in other Cetacean species. We diagnosed a case of Lacaziosis in a Pacific white-sided dolphin (Lagenorhynchus obliquidens) nursing in an aquarium in Japan. The dolphin was a female estimated to be more than 14 years old at the end of June 2015 and was captured in a coast of Japan Sea in 2001. Multiple, lobose, and solid granulomatous lesions with or without ulcers appeared on her jaw, back, flipper and fluke skin, in July 2014. The granulomatous skin lesions from the present case were similar to those of our previous cases. Multiple budding and chains of round yeast cells were detected in the biopsied samples. The partial sequence of 43-kDa glycoprotein coding gene confirmed by a nested PCR and sequencing, which revealed a different genotype from both Amazonian and Japanese lacaziosis in bottlenose dolphins, and was 99 % identical to those derived from Paracoccidioides brasiliensis; a sister fungal species to L. loboi. This is the first case of lacaziosis in Pacific white-sided dolphin.

  5. Microhomology directs diverse DNA break repair pathways and chromosomal translocations.

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    Diana D Villarreal

    Full Text Available Chromosomal structural change triggers carcinogenesis and the formation of other genetic diseases. The breakpoint junctions of these rearrangements often contain small overlapping sequences called "microhomology," yet the genetic pathway(s responsible have yet to be defined. We report a simple genetic system to detect microhomology-mediated repair (MHMR events after a DNA double-strand break (DSB in budding yeast cells. MHMR using >15 bp operates as a single-strand annealing variant, requiring the non-essential DNA polymerase subunit Pol32. MHMR is inhibited by sequence mismatches, but independent of extensive DNA synthesis like break-induced replication. However, MHMR using less than 14 bp is genetically distinct from that using longer microhomology and far less efficient for the repair of distant DSBs. MHMR catalyzes chromosomal translocation almost as efficiently as intra-chromosomal repair. The results suggest that the intrinsic annealing propensity between microhomology sequences efficiently leads to chromosomal rearrangements.

  6. Large-scale cloning of human chromosome 2-specific yeast artificial chromosomes (YACs) using an interspersed repetitive sequences (IRS)-PCR approach.

    Science.gov (United States)

    Liu, J; Stanton, V P; Fujiwara, T M; Wang, J X; Rezonzew, R; Crumley, M J; Morgan, K; Gros, P; Housman, D; Schurr, E

    1995-03-20

    We report here an efficient approach to the establishment of extended YAC contigs on human chromosome 2 by using an interspersed repetitive sequences (IRS)-PCR-based screening strategy for YAC DNA pools. Genomic DNA was extracted from 1152 YAC pools comprised of 55,296 YACs mostly derived from the CEPH Mark I library. Alu-element-mediated PCR was performed for each pool, and amplification products were spotted on hybridization membranes (IRS filters). IRS probes for the screening of the IRS filters were obtained by Alu-element-mediated PCR. Of 708 distinct probes obtained from chromosome 2-specific somatic cell hybrids, 85% were successfully used for library screening. Similarly, 80% of 80 YAC walking probes were successfully used for library screening. Each probe detected an average of 6.6 YACs, which is in good agreement with the 7- to 7.5-fold genome coverage provided by the library. In a preliminary analysis, we have identified 188 YAC groups that are the basis for building contigs for chromosome 2. The coverage of the telomeric half of chromosome 2q was considered to be good since 31 of 34 microsatellites and 22 of 23 expressed sequence tags that were chosen from chromosome region 2q13-q37 were contained in a chromosome 2 YAC sublibrary generated by our experiments. We have identified a minimum of 1610 distinct chromosome 2-specific YACs, which will be a valuable asset for the physical mapping of the second largest human chromosome.

  7. Binding of the Fkh1 Forkhead Associated Domain to a Phosphopeptide within the Mph1 DNA Helicase Regulates Mating-Type Switching in Budding Yeast.

    Directory of Open Access Journals (Sweden)

    Antoinette M Dummer

    2016-06-01

    Full Text Available The Saccharomyces cerevisiae Fkh1 protein has roles in cell-cycle regulated transcription as well as a transcription-independent role in recombination donor preference during mating-type switching. The conserved FHA domain of Fkh1 regulates donor preference by juxtaposing two distant regions on chromosome III to promote their recombination. A model posits that this Fkh1-mediated long-range chromosomal juxtaposition requires an interaction between the FHA domain and a partner protein(s, but to date no relevant partner has been described. In this study, we used structural modeling, 2-hybrid assays, and mutational analyses to show that the predicted phosphothreonine-binding FHA domain of Fkh1 interacted with multiple partner proteins. The Fkh1 FHA domain was important for its role in cell-cycle regulation, but no single interaction partner could account for this role. In contrast, Fkh1's interaction with the Mph1 DNA repair helicase regulated donor preference during mating-type switching. Using 2-hybrid assays, co-immunoprecipitation, and fluorescence anisotropy, we mapped a discrete peptide within the regulatory Mph1 C-terminus required for this interaction and identified two threonines that were particularly important. In vitro binding experiments indicated that at least one of these threonines had to be phosphorylated for efficient Fkh1 binding. Substitution of these two threonines with alanines (mph1-2TA specifically abolished the Fkh1-Mph1 interaction in vivo and altered donor preference during mating-type switching to the same degree as mph1Δ. Notably, the mph1-2TA allele maintained other functions of Mph1 in genome stability. Deletion of a second Fkh1-interacting protein encoded by YMR144W also resulted in a change in Fkh1-FHA-dependent donor preference. We have named this gene FDO1 for Forkhead one interacting protein involved in donor preference. We conclude that a phosphothreonine-mediated protein-protein interface between Fkh1-FHA and

  8. Sequencing of a 9.9 kb segment on the right arm of yeast chromosome VII reveals four open reading frames, including PFK1, the gene coding for succinyl-CoA synthetase (beta-chain) and two ORFs sharing homology with ORFs of the yeast chromosome VIII.

    Science.gov (United States)

    Guerreiro, P; Azevedo, D; Barreiros, T; Rodrigues-Pousada, C

    1997-03-15

    A 9.9 kb DNA fragment from the right arm of chromosome VII of Saccharomyces cerevisiae has been sequenced and analysed. The sequence contains four open reading frames (ORFs) longer than 100 amino acids. One gene, PFK1, has already been cloned and sequenced and the other one is the probable yeast gene coding for the beta-subunit of the succinyl-CoA synthetase. The two remaining ORFs share homology with the deduced amino acid sequence (and their physical arrangement is similar to that) of the YHR161c and YHR162w ORFs from chromosome VIII.

  9. Genome-wide characterisation of the Gcn5 histone acetyltransferase in budding yeast during stress adaptation reveals evolutionarily conserved and diverged roles

    Directory of Open Access Journals (Sweden)

    Brodin David

    2010-03-01

    Full Text Available Abstract Background Gcn5 is a transcriptional coactivator with histone acetyltransferase activity that is conserved with regard to structure as well as its histone substrates throughout the eukaryotes. Gene regulatory networks within cells are thought to be evolutionarily diverged. The use of evolutionarily divergent yeast species, such as S. cerevisiae and S. pombe, which can be studied under similar environmental conditions, provides an opportunity to examine the interface between conserved regulatory components and their cellular applications in different organisms. Results We show that Gcn5 is important for a common set of stress responses in evolutionarily diverged yeast species and that the activity of the conserved histone acetyltransferase domain is required. We define a group of KCl stress response genes in S. cerevisiae that are specifically dependent on Gcn5. Gcn5 is localised to many Gcn5-dependent genes including Gcn5 repressed targets such as FLO8. Gcn5 regulates divergent sets of KCl responsive genes in S. cerevisiae and S. pombe. Genome-wide localization studies showed a tendency for redistribution of Gcn5 during KCl stress adaptation in S. cerevisiae from short genes to the transcribed regions of long genes. An analogous redistribution was not observed in S. pombe. Conclusions Gcn5 is required for the regulation of divergent sets of KCl stress-response genes in S. cerevisiae and S. pombe even though it is required a common group of stress responses, including the response to KCl. Genes that are physically associated with Gcn5 require its activity for their repression or activation during stress adaptation, providing support for a role of Gcn5 as a corepressor as well as a coactivator. The tendency of Gcn5 to re-localise to the transcribed regions of long genes during KCl stress adaptation suggests that Gcn5 plays a specific role in the expression of long genes under adaptive conditions, perhaps by regulating transcriptional

  10. The Cln3 cyclin is down-regulated by translational repression and degradation during the G1 arrest caused by nitrogen deprivation in budding yeast.

    Science.gov (United States)

    Gallego, C; Garí, E; Colomina, N; Herrero, E; Aldea, M

    1997-12-01

    Nutrients are among the most important trophic factors in all organisms. When deprived of essential nutrients, yeast cells use accumulated reserves to complete the current cycle and arrest in the following G1 phase. We show here that the Cln3 cyclin, which has a key role in the timely activation of SBF (Swi4-Swi6)- and MBF (Mbp1-Swi6)-dependent promoters in late G1, is down-regulated rapidly at a post-transcriptional level in cells deprived of the nitrogen source. In addition to the fact that Cln3 is degraded faster by ubiquitin-dependent mechanisms, we have found that translation of the CLN3 mRNA is repressed approximately 8-fold under nitrogen deprivation conditions. As a consequence, both SBF- and MBF-dependent expression is strongly down-regulated. Mainly because of their transcriptional dependence on SBF, and perhaps with the contribution of similar post-transcriptional mechanisms to those found for Cln3, the G1 cyclins Cln1 and 2 become undetectable in starved cells. The complete loss of Cln cyclins and the sustained presence of the Clb-cyclin kinase inhibitor Sic1 in starved cells may provide the molecular basis for the G1 arrest caused by nitrogen deprivation.

  11. Complementation of Yeast Genes with Human Genes as an Experimental Platform for Functional Testing of Human Genetic Variants.

    Science.gov (United States)

    Hamza, Akil; Tammpere, Erik; Kofoed, Megan; Keong, Christelle; Chiang, Jennifer; Giaever, Guri; Nislow, Corey; Hieter, Philip

    2015-11-01

    While the pace of discovery of human genetic variants in tumors, patients, and diverse populations has rapidly accelerated, deciphering their functional consequence has become rate-limiting. Using cross-species complementation, model organisms like the budding yeast, Saccharomyces cerevisiae, can be utilized to fill this gap and serve as a platform for testing human genetic variants. To this end, we performed two parallel screens, a one-to-one complementation screen for essential yeast genes implicated in chromosome instability and a pool-to-pool screen that queried all possible essential yeast genes for rescue of lethality by all possible human homologs. Our work identified 65 human cDNAs that can replace the null allele of essential yeast genes, including the nonorthologous pair yRFT1/hSEC61A1. We chose four human cDNAs (hLIG1, hSSRP1, hPPP1CA, and hPPP1CC) for which their yeast gene counterparts function in chromosome stability and assayed in yeast 35 tumor-specific missense mutations for growth defects and sensitivity to DNA-damaging agents. This resulted in a set of human-yeast gene complementation pairs that allow human genetic variants to be readily characterized in yeast, and a prioritized list of somatic mutations that could contribute to chromosome instability in human tumors. These data establish the utility of this cross-species experimental approach. Copyright © 2015 by the Genetics Society of America.

  12. Large-scale cloning of human chromosome 2-specific yeast artificial chromosomes (YACs) using an interspersed repetitive sequences (IRS)-PCR approach

    Energy Technology Data Exchange (ETDEWEB)

    Liu, J.; Rezonzew, R. [McGill Centre for the Study of Host Resistance, Montreal, Quebec (Canada)]|[McGill Univ., Montreal, Quebec (Canada); Stanton, V.P. Jr. [Massachusetts Institute of Technology, Cambridge, MA (United States)] [and others

    1995-03-20

    We report here an efficient approach to the establishment of extended YAC contigs on human chromosome 2 by using an interspersed repetitive sequences (IRS)-PCR-based screening strategy for YAC DNA pools. Genomic DNA was extracted from 1152 YAC pools comprised of 55,296 YACs mostly derived from the CEPH Mark I library. Alu-element-mediated PCR was performed for each pool, and amplification products were spotted on hybridization membranes (IRS filters). IRS probes for the screening of the IRS filters were obtained by Alu-element-mediated PCR. Of 708 distinct probes obtained from chromosome 2-specific somatic cell hybrids, 85% were successfully used for library screening. Similarly, 80% of 80 YAC walking probes were successfully used for library screening. Each probe detected an average of 6.6 YACs, which is in good agreement with the 7- to 7.5-fold genome coverage provided by the library. In a preliminary analysis, we have identified 188 YAC groups that are the basis for building contigs for chromosome 2. The coverage of the telomeric half of chromosome 2q was considered to be good since 31 of 34 microsatellites and 22 of 23 expressed sequence tags that were chosen from chromosome region 2q13-q37 were contained in a chromosome 2 YAC sublibrary generated by our experiments. We have identified a minimum of 1610 distinct chromosome 2-specific YACs, which will be a valuable asset for the physical mapping of the second largest human chromosome. 81 refs., 8 figs., 3 tabs.

  13. The Gcn2 Regulator Yih1 Interacts with the Cyclin Dependent Kinase Cdc28 and Promotes Cell Cycle Progression through G2/M in Budding Yeast.

    Directory of Open Access Journals (Sweden)

    Richard C Silva

    Full Text Available The Saccharomyces cerevisiae protein Yih1, when overexpressed, inhibits the eIF2 alpha kinase Gcn2 by competing for Gcn1 binding. However, deletion of YIH1 has no detectable effect on Gcn2 activity, suggesting that Yih1 is not a general inhibitor of Gcn2, and has no phenotypic defect identified so far. Thus, its physiological role is largely unknown. Here, we show that Yih1 is involved in the cell cycle. Yeast lacking Yih1 displays morphological patterns and DNA content indicative of a delay in the G2/M phases of the cell cycle, and this phenotype is independent of Gcn1 and Gcn2. Accordingly, the levels of phosphorylated eIF2α, which show a cell cycle-dependent fluctuation, are not altered in cells devoid of Yih1. We present several lines of evidence indicating that Yih1 is in a complex with Cdc28. Yih1 pulls down endogenous Cdc28 in vivo and this interaction is enhanced when Cdc28 is active, suggesting that Yih1 modulates the function of Cdc28 in specific stages of the cell cycle. We also demonstrate, by Bimolecular Fluorescence Complementation, that endogenous Yih1 and Cdc28 interact with each other, confirming Yih1 as a bona fide Cdc28 binding partner. Amino acid substitutions within helix H2 of the RWD domain of Yih1 enhance Yih1-Cdc28 association. Overexpression of this mutant, but not of wild type Yih1, leads to a phenotype similar to that of YIH1 deletion, supporting the view that Yih1 is involved through Cdc28 in the regulation of the cell cycle. We further show that IMPACT, the mammalian homologue of Yih1, interacts with CDK1, the mammalian counterpart of Cdc28, indicating that the involvement with the cell cycle is conserved. Together, these data provide insights into the cellular function of Yih1/IMPACT, and provide the basis for future studies on the role of this protein in the cell cycle.

  14. The Emerging Role of the Cytoskeleton in Chromosome Dynamics

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    Maya Spichal

    2017-05-01

    Full Text Available Chromosomes underlie a dynamic organization that fulfills functional roles in processes like transcription, DNA repair, nuclear envelope stability, and cell division. Chromosome dynamics depend on chromosome structure and cannot freely diffuse. Furthermore, chromosomes interact closely with their surrounding nuclear environment, which further constrains chromosome dynamics. Recently, several studies enlighten that cytoskeletal proteins regulate dynamic chromosome organization. Cytoskeletal polymers that include actin filaments, microtubules and intermediate filaments can connect to the nuclear envelope via Linker of the Nucleoskeleton and Cytoskeleton (LINC complexes and transfer forces onto chromosomes inside the nucleus. Monomers of these cytoplasmic polymers and related proteins can also enter the nucleus and play different roles in the interior of the nucleus than they do in the cytoplasm. Nuclear cytoskeletal proteins can act as chromatin remodelers alone or in complexes with other nuclear proteins. They can also act as transcription factors. Many of these mechanisms have been conserved during evolution, indicating that the cytoskeletal regulation of chromosome dynamics is an essential process. In this review, we discuss the different influences of cytoskeletal proteins on chromosome dynamics by focusing on the well-studied model organism budding yeast.

  15. Dispensability of the SAC Depends on the Time Window Required by Aurora B to Ensure Chromosome Biorientation.

    Directory of Open Access Journals (Sweden)

    Marta Muñoz-Barrera

    Full Text Available Aurora B and the spindle assembly checkpoint (SAC collaborate to ensure the proper biorientation of chromosomes during mitosis. However, lack of Aurora B activity and inactivation of the SAC have a very different impact on chromosome segregation. This is most evident in Saccharomyces cerevisiae, since in this organism the lack of Aurora B is lethal and leads to severe aneuploidy problems, while the SAC is dispensable under normal growth conditions and mutants in this checkpoint do not show evident chromosome segregation defects. We demonstrate that the efficient repair of incorrect chromosome attachments by Aurora B during the initial stages of spindle assembly in budding yeast determines the lack of chromosome segregation defects in SAC mutants, and propose that the differential time window that Aurora B kinase requires to establish chromosome biorientation is the key factor that determines why some cells are more dependent on a functional SAC than others.

  16. Dispensability of the SAC Depends on the Time Window Required by Aurora B to Ensure Chromosome Biorientation

    Science.gov (United States)

    Monje-Casas, Fernando

    2015-01-01

    Aurora B and the spindle assembly checkpoint (SAC) collaborate to ensure the proper biorientation of chromosomes during mitosis. However, lack of Aurora B activity and inactivation of the SAC have a very different impact on chromosome segregation. This is most evident in Saccharomyces cerevisiae, since in this organism the lack of Aurora B is lethal and leads to severe aneuploidy problems, while the SAC is dispensable under normal growth conditions and mutants in this checkpoint do not show evident chromosome segregation defects. We demonstrate that the efficient repair of incorrect chromosome attachments by Aurora B during the initial stages of spindle assembly in budding yeast determines the lack of chromosome segregation defects in SAC mutants, and propose that the differential time window that Aurora B kinase requires to establish chromosome biorientation is the key factor that determines why some cells are more dependent on a functional SAC than others. PMID:26661752

  17. Cytokinesis breaks dicentric chromosomes preferentially at pericentromeric regions and telomere fusions.

    Science.gov (United States)

    Lopez, Virginia; Barinova, Natalja; Onishi, Masayuki; Pobiega, Sabrina; Pringle, John R; Dubrana, Karine; Marcand, Stéphane

    2015-02-01

    Dicentric chromosomes are unstable products of erroneous DNA repair events that can lead to further genome rearrangements and extended gene copy number variations. During mitosis, they form anaphase bridges, resulting in chromosome breakage by an unknown mechanism. In budding yeast, dicentrics generated by telomere fusion break at the fusion, a process that restores the parental karyotype and protects cells from rare accidental telomere fusion. Here, we observed that dicentrics lacking telomere fusion preferentially break within a 25- to 30-kb-long region next to the centromeres. In all cases, dicentric breakage requires anaphase exit, ruling out stretching by the elongated mitotic spindle as the cause of breakage. Instead, breakage requires cytokinesis. In the presence of dicentrics, the cytokinetic septa pinch the nucleus, suggesting that dicentrics are severed after actomyosin ring contraction. At this time, centromeres and spindle pole bodies relocate to the bud neck, explaining how cytokinesis can sever dicentrics near centromeres.

  18. Identification of a putative methylenetetrahydrofolate reductase by sequence analysis of a 6.8 kb DNA fragment of yeast chromosome VII.

    Science.gov (United States)

    Tizon, B; Rodríguez-Torres, M; Rodríguez-Belmonte, E; Cadahia, J L; Cerdan, E

    1996-09-01

    We report the sequence analysis of a 6.8 kb DNA fragment from Saccharomyces cerevisiae chromosome VII. This sequence contains five open reading frames (ORFs) greater than 100 amino acids. There is also an incomplete ORF flanking one of the extremes, G2868, which is the 3' end of the SCS3 gene (Hosaka et al., 1994). The translated sequence of ORF G2882 shows similarity to the human methylenetetrahydrofolate reductase (Goyette et al., 1994). ORF G2889 shows no significant homologies with the sequences compiled in databases. ORF G2893 corresponds to the gene SUP44, coding for the yeast ribosomal protein S4 (All-Robin et al., 1990). G2873 and G2896 are internal ORFs.

  19. Yeast artificial chromosome contigs reveal that distal variable-region genes reside at least 3 megabases from the joining regions in the murine immunoglobulin kappa locus.

    Science.gov (United States)

    George, J B; Li, S; Garrard, W T

    1995-01-01

    The immunoglobulin kappa gene locus encodes 95% of the light chains of murine antibody molecules and is thought to contain up to 300 variable (V kappa)-region genes generally considered to comprise 20 families. To delineate the locus we have isolated 29 yeast artificial chromosome genomic clones that form two contigs, span > 3.5 megabases, and contain two known non-immunoglobulin kappa markers. Using PCR primers specific for 19 V kappa gene families and Southern analysis, we have refined the genetically defined order of these V kappa gene families. Of these, V kappa 2 maps at least 3.0 Mb from the joining (J kappa) region and appears to be the most distal V kappa gene segment. Images Fig. 3 Fig. 4 PMID:8618913

  20. Yeast as a model system for mammalian seven-transmembrane segment receptors

    Energy Technology Data Exchange (ETDEWEB)

    Jeansonne, N.E. [East Carolina Univ. Medical School, Greenville, NC (United States)

    1994-05-01

    Investigators have used the budding yeast Saccharomyces cerevisiae as a model system in which to study the {beta}-adrenergic receptor, the T-cell receptor pathway, initiation of mammalian DNA replication, initiation of mammalian transcription, secretion, the CDC2 kinase system, cell cycle control, and aging, as well as the function of oncogenes. This list continues to growth with the discovery of an immunoglobulin heavy-chain binding homologue in yeast, an Rb binding protein homologue, and a possible yeast arrestin. Yeast is relatively easy to maintain, to grow, and to genetically manipulate. A single gene can be overexpressed, selectively mutated or deleted from its chromosomal location. In this way, the in vivo function of a gene can be studied. It has become reasonable to consider yeast as a model system for studying the seven transmembrane segments (7-TMS) receptor family. Currently, subtypes of the {beta}-adrenergic receptor are being studied in yeast. The receptor and its G{sub {alpha}}-G-protein, trigger the mating pheromone receptor pathway. This provides a powerful assay for determining receptor function. Studies expressing the muscarinic cholinergic receptor in yeast are underway. The yeast pheromone receptor belongs to this receptor family, sharing sequences and secondary structure homology. An effective strategy has been to identify a yeast pathway or process which is homologous to a mammalian system. The pathway is delineated in yeast, identifying other genetic components. Then yeast genes are used to screen for human homologues of these components. The putative human homologues are then expressed in yeast and in mammalian cells to determine function. When this type of {open_quotes}mixing and matching{close_quotes} works, yeast genetics can be a powerful tool. 115 refs.

  1. Identification of region-specific yeast artificial chromosomes using pools of Alu element-mediated polymerase chain reaction probes labeled via linear amplification

    Energy Technology Data Exchange (ETDEWEB)

    Cole, C.G.; Bobrow, M.; Bentley, D.R.; Dunham, I. (United Medical and Dental Schools of Guy' s and St. Thomas Hospitals, London Bridge, London, England (United Kingdom)); Patel, K.; Shipley, J.; Sheer, D. (Imperial Cancer Research Fund, London (United Kingdom))

    1992-12-01

    The ability to identify large numbers of yeast artificial chromosomes (YACS) specific to any given genomic region rapidly and efficiently enhances both the construction of clone maps and the isolation of region-specific landmarks (e.g., polymorphic markers). The authors describe a method of preparing region-specific single-stranded hybridization probes from Alu element-mediated polymerase chain reaction (Alu-PCR) products of somatic cell hybrids for YAC library screening. Pools of up to 50 cloned Alu-PCR products from an irradiation-reduced hybrid containing 22q11.2-q13.1 were labeled to high specific activity by linear amplification using a single vector primer. The resulting single-stranded probes were extensively competed to remove repetitive sequences, while retaining the full complexity of the probe. Extensive coverage of the region by YACs using multiple probe pools was demonstrated as many YACs were detected more than once. In situ analysis using chosen YACs confirmed that the clones were specific for the region. Thus, this pooled probe approach constitutes a rapid method to identify large numbers of YACs relevant to a large chromosomal region. 29 refs., 4 figs.

  2. Identification of region-specific yeast artificial chromosomes using pools of Alu element-mediated polymerase chain reaction probes labeled via linear amplification.

    Science.gov (United States)

    Cole, C G; Patel, K; Shipley, J; Sheer, D; Bobrow, M; Bentley, D R; Dunham, I

    1992-12-01

    The ability to identify large numbers of yeast artificial chromosomes (YACs) specific to any given genomic region rapidly and efficiently enhances both the construction of clone maps and the isolation of region-specific landmarks (e.g., polymorphic markers). We describe a method of preparing region-specific single-stranded hybridization probes from Alu element-mediated polymerase chain reaction (Alu-PCR) products of somatic cell hybrids for YAC library screening. Pools of up to 50 cloned Alu-PCR products from an irradiation-reduced hybrid containing 22q11.2-q13.1 were labeled to high specific activity by linear amplification using a single vector primer. The resulting single-stranded probes were extensively competed to remove repetitive sequences, while retaining the full complexity of the probe. Extensive coverage of the region by YACs using multiple probe pools was demonstrated as many YACs were detected more than once. In situ analysis using chosen YACs confirmed that the clones were specific for the region. Thus, this pooled probe approach constitutes a rapid method to identify large numbers of YACs relevant to a large chromosomal region.

  3. Recent advances in the genome-wide study of DNA replication origins in yeast

    Directory of Open Access Journals (Sweden)

    Chong ePeng

    2015-02-01

    Full Text Available DNA replication, one of the central events in the cell cycle, is the basis of biological inheritance. In order to be duplicated, a DNA double helix must be opened at defined sites, which are called DNA replication origins (ORIs. Unlike in bacteria, where replication initiates from a single replication origin, multiple origins are utilized in the eukaryotic genome. Among them, the ORIs in budding yeast Saccharomyces cerevisiae and the fission yeast Schizosaccharomyces pombe have been best characterized. In recent years, advances in DNA microarray and next-generation sequencing technologies have increased the number of yeast species involved in ORIs research dramatically. The ORIs in some nonconventional yeast species such as Kluyveromyces lactis and Pichia pastoris have also been genome-widely identified. Relevant databases of replication origins in yeast were constructed, then the comparative genomic analysis can be carried out. Here, we review several experimental approaches that have been used to map replication origins in yeast and some of the available web resources related to yeast ORIs. We also discuss the sequence characteristics and chromosome structures of ORIs in the four yeast species, which can be utilized to improve the replication origins prediction.

  4. Per aspera ad astra: When harmful chromosomal translocations become a plus value in genetic evolution. Lessons from Saccharomyces cerevisiae

    Directory of Open Access Journals (Sweden)

    Valentina Tosato

    2015-08-01

    Full Text Available In this review we will focus on chromosomal translocations (either spontaneous or induced in budding yeast. Indeed, very few organisms tolerate so well aneuploidy like Saccharomyces, allowing in depth studies on chromosomal numerical aberrations. Many wild type strains naturally develop chromosomal rearrangements while adapting to different environmental conditions. Translocations, in particular, are valuable not only because they naturally drive species evolution, but because they might allow the artificial generation of new strains that can be optimized for industrial purposes. In this area, several methodologies to artificially trigger chromosomal translocations have been conceived in the past years, such as the chromosomal fragmentation vector (CFV technique, the Cre-loxP procedure, the FLP/FRT recombination method and, recently, the bridge – induced translocation (BIT system. An overview of the methodologies to generate chromosomal translocations in yeast will be presented and discussed considering advantages and drawbacks of each technology, focusing in particular on the recent BIT system. Translocants are important for clinical studies because translocated yeast cells resemble cancer cells from morphological and physiological points of view and because the translocation event ensues in a transcriptional deregulation with a subsequent multi-factorial genetic adaptation to new, selective environmental conditions. The phenomenon of post-translocational adaptation (PTA is discussed, providing some new unpublished data and proposing the hypothesis that translocations may drive evolution through adaptive genetic selection.

  5. What Are Taste Buds?

    Science.gov (United States)

    ... your taste buds for letting you appreciate the saltiness of pretzels and the sweetness of ice cream. ... allow you to experience tastes that are sweet, salty, sour, and bitter. How exactly do your taste ...

  6. An actin-binding protein, CAP, is expressed in a subset of rat taste bud cells.

    Science.gov (United States)

    Ishimaru, Y; Yasuoka, A; Asano-Miyoshi, M; Abe, K; Emori, Y

    2001-02-12

    Single cell cDNA libraries were constructed from taste bud cells of rat circumvallate papillae. Using three steps of screening, including differential hybridization, sequence analyses and in situ hybridization, a clone encoding a rat homolog of yeast adenylyl cyclase-associated protein (CAP) was identified to be highly expressed in a subset of taste bud cells.

  7. Chromosomal localization of three repair genes: The xeroderma pigmentosum group C gene and two human homologs of yeast RAD23

    Energy Technology Data Exchange (ETDEWEB)

    Spek, P.J. van der; Smit, E.M.E.; Beverloo, H.B. [Erasmus Univ., Rotterdam (Netherlands)] [and others

    1994-10-01

    The nucleotide excision repair (NER) disorder xeroderma pigmentosum (XP) is characterized by sun (UV) sensitivity, predisposition to skin cancer, and extensive genetic heterogeneity. Recently, we reported the cloning and analysis of three human NER genes, XPC, HHR23A, and HHR23B. The previously cloned XPC gene is involved in the common XP complementation group C, which is defective in excision repair of nontranscribed sequences in the genome. The XPC protein was found to be complexed with the product of HHR23B, one of the two human homologs of the Saccharomyes cerevisiae NER gene RAD23. Here we present the chromosomal localization by in situ hybridization using haptenized probes of all three genes. The HHR23A gene was assigned to chromosome 19p13.2. Interestingly, the HHR23B and XPC genes, the product of which forms a tight complex, were found to colocalize on band 3p25.1. Pulsed-field gel electrophoresis revealed that the HHR23B and XPC genes possibly share a MluI restriction fragment of about 625 kb. Potential involvement of the HHR23 genes in human genetic disorders is discussed. 53 refs., 4 figs., 2 tabs.

  8. Dicentric chromosomes: unique models to study centromere function and inactivation.

    Science.gov (United States)

    Stimpson, Kaitlin M; Matheny, Justyne E; Sullivan, Beth A

    2012-07-01

    Dicentric chromosomes are products of genome rearrangement that place two centromeres on the same chromosome. Depending on the organism, dicentric stability varies after formation. In humans, dicentrics occur naturally in a substantial portion of the population and usually segregate successfully in mitosis and meiosis. Their stability has been attributed to inactivation of one of the two centromeres, creating a functionally monocentric chromosome that can segregate normally during cell division. The molecular basis for centromere inactivation is not well understood, although studies in model organisms and in humans suggest that genomic and epigenetic mechanisms can be involved. Furthermore, constitutional dicentric chromosomes ascertained in patients presumably represent the most stable chromosomes, so the spectrum of dicentric fates, if it exists, is not entirely clear. Studies of engineered or induced dicentrics in budding yeast and plants have provided significant insight into the fate of dicentric chromosomes. And, more recently, studies have shown that dicentrics in humans can also undergo multiple fates after formation. Here, we discuss current experimental evidence from various organisms that has deepened our understanding of dicentric behavior and the intriguingly complex process of centromere inactivation.

  9. Multiple opposing constraints govern chromosome interactions during meiosis.

    Directory of Open Access Journals (Sweden)

    Doris Y Lui

    Full Text Available Homolog pairing and crossing over during meiosis I prophase is required for accurate chromosome segregation to form euploid gametes. The repair of Spo11-induced double-strand breaks (DSB using a homologous chromosome template is a major driver of pairing in many species, including fungi, plants, and mammals. Inappropriate pairing and crossing over at ectopic loci can lead to chromosome rearrangements and aneuploidy. How (or if inappropriate ectopic interactions are disrupted in favor of allelic interactions is not clear. Here we used an in vivo "collision" assay in budding yeast to test the contributions of cohesion and the organization and motion of chromosomes in the nucleus on promoting or antagonizing interactions between allelic and ectopic loci at interstitial chromosome sites. We found that deletion of the cohesin subunit Rec8, but not other chromosome axis proteins (e.g. Red1, Hop1, or Mek1, caused an increase in homolog-nonspecific chromosome interaction, even in the absence of Spo11. This effect was partially suppressed by expression of the mitotic cohesin paralog Scc1/Mdc1, implicating Rec8's role in cohesion rather than axis integrity in preventing nonspecific chromosome interactions. Disruption of telomere-led motion by treating cells with the actin polymerization inhibitor Latrunculin B (Lat B elevated nonspecific collisions in rec8Δ spo11Δ. Next, using a visual homolog-pairing assay, we found that the delay in homolog pairing in mutants defective for telomere-led chromosome motion (ndj1Δ or csm4Δ is enhanced in Lat B-treated cells, implicating actin in more than one process promoting homolog juxtaposition. We suggest that multiple, independent contributions of actin, cohesin, and telomere function are integrated to promote stable homolog-specific interactions and to destabilize weak nonspecific interactions by modulating the elastic spring-like properties of chromosomes.

  10. The yeast Golgi apparatus.

    Science.gov (United States)

    Suda, Yasuyuki; Nakano, Akihiko

    2012-04-01

    The Golgi apparatus is an organelle that has been extensively studied in the model eukaryote, yeast. Its morphology varies among yeast species; the Golgi exists as a system of dispersed cisternae in the case of the budding yeast Saccharomyces cerevisiae, whereas the Golgi cisternae in Pichia pastoris and Schizosaccharomyces pombe are organized into stacks. In spite of the different organization, the mechanism of trafficking through the Golgi apparatus is believed to be similar, involving cisternal maturation, in which the resident Golgi proteins are transported backwards while secretory cargo proteins can stay in the cisternae. Questions remain regarding the organization of the yeast Golgi, the regulatory mechanisms that underlie cisternal maturation of the Golgi and transport machinery of cargo proteins through this organelle. Studies using different yeast species have provided hints to these mechanisms.

  11. Absence of Non-histone Protein Complexes at Natural Chromosomal Pause Sites Results in Reduced Replication Pausing in Aging Yeast Cells

    Directory of Open Access Journals (Sweden)

    Marleny Cabral

    2016-11-01

    Full Text Available There is substantial evidence that genomic instability increases during aging. Replication pausing (and stalling at difficult-to-replicate chromosomal sites may induce genomic instability. Interestingly, in aging yeast cells, we observed reduced replication pausing at various natural replication pause sites (RPSs in ribosomal DNA (rDNA and non-rDNA locations (e.g., silent replication origins and tRNA genes. The reduced pausing occurs independent of the DNA helicase Rrm3p, which facilitates replication past these non-histone protein-complex-bound RPSs, and is independent of the deacetylase Sir2p. Conditions of caloric restriction (CR, which extend life span, also cause reduced replication pausing at the 5S rDNA and at tRNA genes. In aged and CR cells, the RPSs are less occupied by their specific non-histone protein complexes (e.g., the preinitiation complex TFIIIC, likely because members of these complexes have primarily cytosolic localization. These conditions may lead to reduced replication pausing and may lower replication stress at these sites during aging.

  12. The nucleotide sequence of a 39 kb segment of yeast chromosome IV: 12 new open reading frames, nine known genes and one genes for Gly-tRNA.

    Science.gov (United States)

    Bahr, A; Möller-Rieker, S; Hankeln, T; Kraemer, C; Protin, U; Schmidt, E R

    1997-02-01

    The complete nucleotide sequence of a 39,090 bp segment from the left arm of yeast chromosome IV was determined. Twenty-one open reading frames (ORFs) longer than 100 amino acids and a Gly-tRNA gene were discovered. Nine of the 21 ORFs (D0892, D1022, D1037, D1045, D1057, D1204, D1209, D1214, D1219) correspond to the previously sequenced Saccharomyces cerevisiae genes for the NAD-dependent glutamate dehydrogenase (GDH), the secretory component (SHR3), the GABA transport protein (UGA4), the high mobility group-like protein (NHP2), the hydroxymethylbilane synthase (HEM3), the methylated DNA protein-cysteine S-methyltransferase (MGT1), a putative sugar transport protein, the Shm1 protein (SHM1) and the anti-silencing protein (ASF2). The inferred amino acid sequences of 11 ORFs show significant similarity with known proteins from various organisms, whereas the remaining ORF does not share any similarity with known proteins.

  13. Chromosome segregation: Samurai separation of Siamese sisters.

    Science.gov (United States)

    Glotzer, M

    1999-07-15

    How do cells ensure that sister chromatids are precisely partitioned in mitosis? New studies on budding yeast have revealed that sister chromatid separation at anaphase requires endoproteolytic cleavage of a protein that maintains the association between sister chromatids.

  14. Binding of Multiple Rap1 Proteins Stimulates Chromosome Breakage Induction during DNA Replication.

    Directory of Open Access Journals (Sweden)

    Greicy H Goto

    2015-08-01

    Full Text Available Telomeres, the ends of linear eukaryotic chromosomes, have a specialized chromatin structure that provides a stable chromosomal terminus. In budding yeast Rap1 protein binds to telomeric TG repeat and negatively regulates telomere length. Here we show that binding of multiple Rap1 proteins stimulates DNA double-stranded break (DSB induction at both telomeric and non-telomeric regions. Consistent with the role of DSB induction, Rap1 stimulates nearby recombination events in a dosage-dependent manner. Rap1 recruits Rif1 and Rif2 to telomeres, but neither Rif1 nor Rif2 is required for DSB induction. Rap1-mediated DSB induction involves replication fork progression but inactivation of checkpoint kinase Mec1 does not affect DSB induction. Rap1 tethering shortens artificially elongated telomeres in parallel with telomerase inhibition, and this telomere shortening does not require homologous recombination. These results suggest that Rap1 contributes to telomere homeostasis by promoting chromosome breakage.

  15. The Yeast Genomes in Three Dimensions: Mechanisms and Functions.

    Science.gov (United States)

    Noma, Ken-Ichi

    2017-08-30

    The three-dimensional (3D) genome structure is highly ordered by a hierarchy of organizing events ranging from enhancer-promoter or gene-gene contacts to chromosomal territorial arrangement. It is becoming clear that the cohesin and condensin complexes are key molecular machines that organize the 3D genome structure. These complexes are highly conserved from simple systems, e.g., yeast cells, to the much more complex human system. Therefore, knowledge from the budding and fission yeast systems illuminates highly conserved molecular mechanisms of how cohesin and condensin establish the functional 3D genome structures. Here I discuss how these complexes are recruited across the yeast genomes, mediate distinct genome-organizing events such as gene contacts and topological domain formation, and participate in important nuclear activities including transcriptional regulation and chromosomal dynamics. Expected final online publication date for the Annual Review of Genetics Volume 51 is November 23, 2017. Please see http://www.annualreviews.org/page/journal/pubdates for revised estimates.

  16. Tension-dependent regulation of microtubule dynamics at kinetochores can explain metaphase congression in yeast

    National Research Council Canada - National Science Library

    Gardner, Melissa K; Pearson, Chad G; Sprague, Brian L; Zarzar, Ted R; Bloom, Kerry; Salmon, E D; Odde, David J

    2005-01-01

    During metaphase in budding yeast mitosis, sister kinetochores are tethered to opposite poles and separated, stretching their intervening chromatin, by singly attached kinetochore microtubules (kMTs...

  17. Automated quantification of budding Saccharomyces cerevisiae using a novel image cytometry method.

    Science.gov (United States)

    Laverty, Daniel J; Kury, Alexandria L; Kuksin, Dmitry; Pirani, Alnoor; Flanagan, Kevin; Chan, Leo Li-Ying

    2013-06-01

    The measurements of concentration, viability, and budding percentages of Saccharomyces cerevisiae are performed on a routine basis in the brewing and biofuel industries. Generation of these parameters is of great importance in a manufacturing setting, where they can aid in the estimation of product quality, quantity, and fermentation time of the manufacturing process. Specifically, budding percentages can be used to estimate the reproduction rate of yeast populations, which directly correlates with metabolism of polysaccharides and bioethanol production, and can be monitored to maximize production of bioethanol during fermentation. The traditional method involves manual counting using a hemacytometer, but this is time-consuming and prone to human error. In this study, we developed a novel automated method for the quantification of yeast budding percentages using Cellometer image cytometry. The automated method utilizes a dual-fluorescent nucleic acid dye to specifically stain live cells for imaging analysis of unique morphological characteristics of budding yeast. In addition, cell cycle analysis is performed as an alternative method for budding analysis. We were able to show comparable yeast budding percentages between manual and automated counting, as well as cell cycle analysis. The automated image cytometry method is used to analyze and characterize corn mash samples directly from fermenters during standard fermentation. Since concentration, viability, and budding percentages can be obtained simultaneously, the automated method can be integrated into the fermentation quality assurance protocol, which may improve the quality and efficiency of beer and bioethanol production processes.

  18. The construction of a yeast artificial chromosome (YAC) contig in the vicinity of the Usher syndrome type IIa (USH2A) gene in 1q41

    Energy Technology Data Exchange (ETDEWEB)

    Sumegi, Janos; Wang, Ji-Yi; Zhen, Dong-Kai [Univ. of Nebraska Medical Center, Omaha, NE (United States)] [and others

    1996-07-01

    The gene for Usher syndrome type II (USH2A), and autosomal recessive syndromic deafness, has been mapped to a region of 1q41 flanked proximally by D1S217 and distally by D1S439. Using sequence-tagged sites (STSs) within the region, a total of 21 yeast artificial chromosome (YAC) clones were isolated and ordered into a single contig that spans approximately 11.0 Mb. The order of microsatellite and STS markers in this region was established as D1S505-D1S425-DXS217-D1S556-D1S237-D1S474-EB1-KB6-AFM144XF2-KB1-KB4-D1S229-D1S490-D1S227-TGF{beta}2-D1S439. Analysis of newly positioned polymorphic markers in recombinant individuals in two Usher syndrome type IIa families has enabled us to identify DXS474 and AFM144XF2 as two flanking markers for the Usher type IIa locus. The physical distance between the two markers is 1.0 Mb. This region is covered by eight YACs from the CEPH library: 945f7, 867g9, 762a6, 919h3, 794b8, 785h4, 848b9, and 841g2. A long range physical map of the Usher type IIa critical region, using MluI, BssHII, NotI, EagI, and SacII, has been developed. 41 refs., 5 figs.

  19. Initiation of DNA replication from non-canonical sites on an origin-depleted chromosome.

    Directory of Open Access Journals (Sweden)

    Naomi L Bogenschutz

    Full Text Available Eukaryotic DNA replication initiates from multiple sites on each chromosome called replication origins (origins. In the budding yeast Saccharomyces cerevisiae, origins are defined at discrete sites. Regular spacing and diverse firing characteristics of origins are thought to be required for efficient completion of replication, especially in the presence of replication stress. However, a S. cerevisiae chromosome III harboring multiple origin deletions has been reported to replicate relatively normally, and yet how an origin-deficient chromosome could accomplish successful replication remains unknown. To address this issue, we deleted seven well-characterized origins from chromosome VI, and found that these deletions do not cause gross growth defects even in the presence of replication inhibitors. We demonstrated that the origin deletions do cause a strong decrease in the binding of the origin recognition complex. Unexpectedly, replication profiling of this chromosome showed that DNA replication initiates from non-canonical loci around deleted origins in yeast. These results suggest that replication initiation can be unexpectedly flexible in this organism.

  20. Conservation and Rewiring of Functional Modules Revealed by an Epistasis Map in Fission Yeast

    Science.gov (United States)

    Roguev, Assen; Bandyopadhyay, Sourav; Zofall, Martin; Zhang, Ke; Fischer, Tamas; Collins, Sean R.; Qu, Hongjing; Shales, Michael; Park, Han-Oh; Hayles, Jacqueline; Hoe, Kwang-Lae; Kim, Dong-Uk; Ideker, Trey; Grewal, Shiv I.; Weissman, Jonathan S.; Krogan, Nevan J.

    2009-01-01

    An epistasis map (E-MAP) was constructed in the fission yeast, Schizosaccharomyces pombe, by systematically measuring the phenotypes associated with pairs of mutations. This high-density, quantitative genetic interaction map focused on various aspects of chromosome function, including transcription regulation and DNA repair/replication. The E-MAP uncovered a previously unidentified component of the RNA interference (RNAi) machinery (rsh1) and linked the RNAi pathway to several other biological processes. Comparison of the S. pombe E-MAP to an analogous genetic map from the budding yeast revealed that, whereas negative interactions were conserved between genes involved in similar biological processes, positive interactions and overall genetic profiles between pairs of genes coding for physically associated proteins were even more conserved. Hence, conservation occurs at the level of the functional module (protein complex), but the genetic cross talk between modules can differ substantially. PMID:18818364

  1. Ipl1/Aurora kinase suppresses S-CDK-driven spindle formation during prophase I to ensure chromosome integrity during meiosis.

    Directory of Open Access Journals (Sweden)

    Louise Newnham

    Full Text Available Cells coordinate spindle formation with DNA repair and morphological modifications to chromosomes prior to their segregation to prevent cell division with damaged chromosomes. Here we uncover a novel and unexpected role for Aurora kinase in preventing the formation of spindles by Clb5-CDK (S-CDK during meiotic prophase I and when the DDR is active in budding yeast. This is critical since S-CDK is essential for replication during premeiotic S-phase as well as double-strand break induction that facilitates meiotic recombination and, ultimately, chromosome segregation. Furthermore, we find that depletion of Cdc5 polo kinase activity delays spindle formation in DDR-arrested cells and that ectopic expression of Cdc5 in prophase I enhances spindle formation, when Ipl1 is depleted. Our findings establish a new paradigm for Aurora kinase function in both negative and positive regulation of spindle dynamics.

  2. A FIBER APPARATUS IN THE NUCLEUS OF THE YEAST CELL

    Science.gov (United States)

    Robinow, C. F.; Marak, J.

    1966-01-01

    The structure and mode of division of the nucleus of budding yeast cells have been studied by phase-contrast microscopy during life and by ordinary microscopy after Helly fixation. The components of the nucleus were differentially stained by the Feulgen procedure, with Giemsa solution after hydrolysis, and with iron alum haematoxylin. New information was obtained in cells fixed in Helly's by directly staining them with 0.005% acid fuchsin in 1% acetic acid in water. Electron micrographs have been made of sections of cells that were first fixed with 3% glutaraldehyde, then divested of their walls with snail juice, and postfixed with osmium tetroxide. Light and electron microscopy have given concordant information about the organization of the yeast nucleus. A peripheral segment of the nucleus is occupied by relatively dense matter (the "peripheral cluster" of Mundkur) which is Feulgen negative. The greater part of the nucleus is filled with fine-grained Feulgen-positive matter of low density in which chromosomes could not be identified. Chromosomes become visible in this region under the light microscope at meiosis. In the chromatin lies a short fiber with strong affinity for acid fuchsin. The nucleus divides by elongation and constriction, and during this process the fiber becomes long and thin. Electron microscopy has resolved it into a bundle of dark-edged 150 to 180 A filaments which extends between "centriolar plaques" that are attached to the nuclear envelope. PMID:5331666

  3. Biotechnical Microbiology, yeast and bacteria

    DEFF Research Database (Denmark)

    Villadsen, Ingrid Stampe

    1999-01-01

    This section contains the following single lecture notes: Eukaryotic Cell Biology. Kingdom Fungi. Cell Division. Meiosis and Recombination. Genetics of Yeast. Organisation of the Chromosome. Organization and genetics of the mitochondrial Geneme. Regulatio of Gene Expression. Intracellular...

  4. Biotechnical Microbiology, yeast and bacteria

    DEFF Research Database (Denmark)

    Villadsen, Ingrid Stampe

    1999-01-01

    This section contains the following single lecture notes: Eukaryotic Cell Biology. Kingdom Fungi. Cell Division. Meiosis and Recombination. Genetics of Yeast. Organisation of the Chromosome. Organization and genetics of the mitochondrial Geneme. Regulatio of Gene Expression. Intracellular Compart...

  5. Molecular Genetic Tools and Techniques in Fission Yeast.

    Science.gov (United States)

    Murray, Johanne M; Watson, Adam T; Carr, Antony M

    2016-05-02

    The molecular genetic tools used in fission yeast have generally been adapted from methods and approaches developed for use in the budding yeast, Saccharomyces cerevisiae Initially, the molecular genetics of Schizosaccharomyces pombe was developed to aid gene identification, but it is now applied extensively to the analysis of gene function and the manipulation of noncoding sequences that affect chromosome dynamics. Much current research using fission yeast thus relies on the basic processes of introducing DNA into the organism and the extraction of DNA for subsequent analysis. Targeted integration into specific genomic loci is often used to create site-specific mutants or changes to noncoding regulatory elements for subsequent phenotypic analysis. It is also regularly used to introduce additional sequences that generate tagged proteins or to create strains in which the levels of wild-type protein can be manipulated through transcriptional regulation and/or protein degradation. Here, we draw together a collection of core molecular genetic techniques that underpin much of modern research using S. pombe We summarize the most useful methods that are routinely used and provide guidance, learned from experience, for the successful application of these methods.

  6. Identification of a yeast artificial chromosome (YAC) spanning the synovial sarcoma-specific t(X;18)(p11.2;q11.2) breakpoint

    NARCIS (Netherlands)

    de Leeuw, B; Berger, W; Sinke, R J; Suijkerbuijk, R F; Gilgenkrantz, S; Geraghty, M T; Valle, D; Monaco, A P; Lehrach, H; Ropers, H H

    1993-01-01

    A somatic cell hybrid containing the synovial sarcoma-associated t(X;18)(p11.2;q11.2) derivative (der(X)) chromosome was used to characterize the translocation breakpoint region on the X chromosome. By using Southern hybridization of DNA from this der(X) hybrid in conjunction with Xp-region specific

  7. Process for Assembly and Transformation into Saccharomyces cerevisiae of a Synthetic Yeast Artificial Chromosome Containing a Multigene Cassette to Express Enzymes That Enhance Xylose Utilization Designed for an Automated Platform.

    Science.gov (United States)

    Hughes, Stephen R; Cox, Elby J; Bang, Sookie S; Pinkelman, Rebecca J; López-Núñez, Juan Carlos; Saha, Badal C; Qureshi, Nasib; Gibbons, William R; Fry, Michelle R; Moser, Bryan R; Bischoff, Kenneth M; Liu, Siqing; Sterner, David E; Butt, Tauseef R; Riedmuller, Steven B; Jones, Marjorie A; Riaño-Herrera, Néstor M

    2015-12-01

    A yeast artificial chromosome (YAC) containing a multigene cassette for expression of enzymes that enhance xylose utilization (xylose isomerase [XI] and xylulokinase [XKS]) was constructed and transformed into Saccharomyces cerevisiae to demonstrate feasibility as a stable protein expression system in yeast and to design an assembly process suitable for an automated platform. Expression of XI and XKS from the YAC was confirmed by Western blot and PCR analyses. The recombinant and wild-type strains showed similar growth on plates containing hexose sugars, but only recombinant grew on D-xylose and L-arabinose plates. In glucose fermentation, doubling time (4.6 h) and ethanol yield (0.44 g ethanol/g glucose) of recombinant were comparable to wild type (4.9 h and 0.44 g/g). In whole-corn hydrolysate, ethanol yield (0.55 g ethanol/g [glucose + xylose]) and xylose utilization (38%) for recombinant were higher than for wild type (0.47 g/g and 12%). In hydrolysate from spent coffee grounds, yield was 0.46 g ethanol/g (glucose + xylose), and xylose utilization was 93% for recombinant. These results indicate introducing a YAC expressing XI and XKS enhanced xylose utilization without affecting integrity of the host strain, and the process provides a potential platform for automated synthesis of a YAC for expression of multiple optimized genes to improve yeast strains.

  8. The SPR3 gene encodes a sporulation-specific homologue of the yeast CDC3/10/11/12 family of bud neck microfilaments and is regulated by ABFI.

    Science.gov (United States)

    Ozsarac, N; Bhattacharyya, M; Dawes, I W; Clancy, M J

    1995-10-16

    The SPR3 gene is selectively activated only during the sporulation phase of the Saccharomyces cerevisiae (Sc) life cycle. The predicted amino acid (aa) sequence has homology to microfilament proteins that are involved in cytokinesis and other proteins of unknown function. These include the products of Sc cell division cycle (CDC) genes involved in bud formation (Cdc3p, Cdc10p, Cdc11p and Cdc12p), Candida albicans proteins that accumulate in the hyphal phase (CaCdc3p and CaCdc10p), mouse brain-specific (H5p) and lymphocyte (Diff6p) proteins, Drosophila melanogaster (Dm) protein Pnutp (which is localized to the cleavage furrow of dividing cells), a Diff6p homologue (DmDiff6p), and the Sc septin protein (Sep1hp), a homologue of the 10-nm filament proteins of Sc. One strongly conserved region contains a potential ATP-GTP-binding domain. Primer extension analysis revealed six major transcription start points (tsp) beginning at -142 relative to the ATG start codon. The sequence immediately upstream from the tsp contains consensus binding sites for the HAP2/3/4 and ABFI transcription factors, a T-rich sequence and two putative novel elements for mid to late sporulation, termed SPR3 and PAL. Electrophoretic mobility shift assay (EMSA) and footprint analyses demonstrated that the ABFI protein binds to a region containing the putative ABFI site in vitro, and site-directed mutagenesis showed that the ABFI motif is essential for expression of SPR3 at the appropriate stage in sporulating cells.

  9. Selective Entrapment of Extrachromosomally Amplified DNA by Nuclear Budding and Micronucleation during S Phase

    Science.gov (United States)

    Shimizu, Noriaki; Itoh, Nobuo; Utiyama, Hiroyasu; Wahl, Geoffrey M.

    1998-01-01

    Acentric, autonomously replicating extrachromosomal structures called double-minute chromosomes (DMs) frequently mediate oncogene amplification in human tumors. We show that DMs can be removed from the nucleus by a novel micronucleation mechanism that is initiated by budding of the nuclear membrane during S phase. DMs containing c-myc oncogenes in a colon cancer cell line localized to and replicated at the nuclear periphery. Replication inhibitors increased micronucleation; cell synchronization and bromodeoxyuridine–pulse labeling demonstrated de novo formation of buds and micronuclei during S phase. The frequencies of S-phase nuclear budding and micronucleation were increased dramatically in normal human cells by inactivating p53, suggesting that an S-phase function of p53 minimizes the probability of producing the broken chromosome fragments that induce budding and micronucleation. These data have implications for understanding the behavior of acentric DNA in interphase nuclei and for developing chemotherapeutic strategies based on this new mechanism for DM elimination. PMID:9508765

  10. Anti-aging and anti-microbial effects of melleolide on various types of yeast.

    Science.gov (United States)

    Nakaya, Shigeru; Kobori, Hajime; Sekiya, Atsushi; Kawagishi, Hirokazu; Ushimaru, Takashi

    2014-01-01

    The chronological lifespan (CLS) of the budding yeast Saccharomyces cerevisiae is a model for the aging of post-mitotic cells in higher eukaryotes. In this study, we found that the sesquiterpene aryl ester melleolide expands the CLS of budding yeast. In contrast, melleolide compromised the CLS of the fission yeast Schizosaccharomyces pombe. This indicates that melleolide might have a potential anti-aging activity against some types of cell, and that it might be useful as a selective anti-fungal drug.

  11. Poetry in motion: Increased chromosomal mobility after DNA damage.

    Science.gov (United States)

    Smith, Michael J; Rothstein, Rodney

    2017-08-01

    Double-strand breaks (DSBs) are among the most lethal DNA lesions, and a variety of pathways have evolved to manage their repair in a timely fashion. One such pathway is homologous recombination (HR), in which information from an undamaged donor site is used as a template for repair. Although many of the biochemical steps of HR are known, the physical movements of chromosomes that must underlie the pairing of homologous sequence during mitotic DSB repair have remained mysterious. Recently, several groups have begun to use a variety of genetic and cell biological tools to study this important question. These studies reveal that both damaged and undamaged loci increase the volume of the nuclear space that they explore after the formation of DSBs. This DSB-induced increase in chromosomal mobility is regulated by many of the same factors that are important during HR, such as ATR-dependent checkpoint activation and the recombinase Rad51, suggesting that this phenomenon may facilitate the search for homology. In this perspective, we review current research into the mobility of chromosomal loci during HR, as well as possible underlying mechanisms, and discuss the critical questions that remain to be answered. Although we focus primarily on recent studies in the budding yeast, Saccharomyces cerevisiae, examples of experiments performed in higher eukaryotes are also included, which reveal that increased mobility of damaged loci is a process conserved throughout evolution. Copyright © 2017 Elsevier B.V. All rights reserved.

  12. Multiple Pairwise Analysis of Non-homologous Centromere Coupling Reveals Preferential Chromosome Size-Dependent Interactions and a Role for Bouquet Formation in Establishing the Interaction Pattern.

    Science.gov (United States)

    Lefrançois, Philippe; Rockmill, Beth; Xie, Pingxing; Roeder, G Shirleen; Snyder, Michael

    2016-10-01

    During meiosis, chromosomes undergo a homology search in order to locate their homolog to form stable pairs and exchange genetic material. Early in prophase, chromosomes associate in mostly non-homologous pairs, tethered only at their centromeres. This phenomenon, conserved through higher eukaryotes, is termed centromere coupling in budding yeast. Both initiation of recombination and the presence of homologs are dispensable for centromere coupling (occurring in spo11 mutants and haploids induced to undergo meiosis) but the presence of the synaptonemal complex (SC) protein Zip1 is required. The nature and mechanism of coupling have yet to be elucidated. Here we present the first pairwise analysis of centromere coupling in an effort to uncover underlying rules that may exist within these non-homologous interactions. We designed a novel chromosome conformation capture (3C)-based assay to detect all possible interactions between non-homologous yeast centromeres during early meiosis. Using this variant of 3C-qPCR, we found a size-dependent interaction pattern, in which chromosomes assort preferentially with chromosomes of similar sizes, in haploid and diploid spo11 cells, but not in a coupling-defective mutant (spo11 zip1 haploid and diploid yeast). This pattern is also observed in wild-type diploids early in meiosis but disappears as meiosis progresses and homologous chromosomes pair. We found no evidence to support the notion that ancestral centromere homology plays a role in pattern establishment in S. cerevisiae post-genome duplication. Moreover, we found a role for the meiotic bouquet in establishing the size dependence of centromere coupling, as abolishing bouquet (using the bouquet-defective spo11 ndj1 mutant) reduces it. Coupling in spo11 ndj1 rather follows telomere clustering preferences. We propose that a chromosome size preference for centromere coupling helps establish efficient homolog recognition.

  13. A yeast artificial chromosome contig that spans the RB1-D13S31 interval on human chromosome 13 and encompasses the frequently deleted region in B-cell chronic lymphocytic leukemia

    NARCIS (Netherlands)

    Hawthorn, L; Roberts, T; Verlind, E; Kooy, RF; Cowell, JK

    1995-01-01

    Abnormalities involving chromosome 13 have been reported as the only cytogenetic change in B-cell chronic lymphocytic leukemia (BCLL). Deletions are the most common cytogenetic abnormality and always involve 13q14, but when translocations are seen, the consistent breakpoint is always in 13q14. It is

  14. Identification of a yeast artificial chromosome that spans the human papillary renal cell carcinoma-associated t(X;1) breakpoint in Xp11.2

    NARCIS (Netherlands)

    Suijkerbuijk, R F; Meloni, A M; Sinke, R J; de Leeuw, B; Wilbrink, M; Janssen, H A; Geraghty, M T; Monaco, A P; Sandberg, A A; Geurts van Kessel, A

    1993-01-01

    Recently, a specific chromosome abnormality, t(X;1)(p11;q21), was described for a subgroup of human papillary renal cell carcinomas. The translocation breakpoint in Xp11 is located in the same region as that in t(X;18)(p11;q11)-positive synovial sarcoma. We used fluorescence in situ hybridization (F

  15. Essential roles of Snf21, a Swi2/Snf2 family chromatin remodeler, in fission yeast mitosis.

    Science.gov (United States)

    Yamada, Kentaro; Hirota, Kouji; Mizuno, Ken-Ichi; Shibata, Takehiko; Ohta, Kunihiro

    2008-10-01

    ATP-dependent chromatin remodelers (ADCRs) convert local chromatin structure into both transcriptional active and repressive state. Recent studies have revealed that ADCRs play diverse regulatory roles in chromosomal events such as DNA repair and recombination. Here we have newly identified a fission yeast gene encoding a Swi2/Snf2 family ADCR. The amino acid sequence of this gene, snf21(+), implies that Snf21 is a fission yeast orthologue of the budding yeast Sth1, the catalytic core of the RSC chromatin remodeling complex. The snf21(+) gene product is a nuclear protein essential to cell viability: the null mutant cells stop growing after several rounds of cell divisions. A temperature sensitive allele of snf21(+), snf21-36 exhibits at non-permissive temperature (34 degrees C) a cell cycle arrest at G2-M phase and defects in chromosome segregation, thereby causing cell elongation, lack of cell growth, and death of some cell population. snf21-36 shows thiabendazole (TBZ) sensitivity even at permissive temperature (25 degrees C). The TBZ sensitivity becomes severer as snf21-36 is combined with the deletion of a centromere-localized Mad2 spindle checkpoint protein. The cell cycle arrest phenotype at 34 degrees C cannot be rescued by the mad2(+) deletion, although it is substantially alleviated at 30 degrees C in mad2Delta. These data suggest that Snf21 plays an essential role in mitosis possibly functioning in centromeric chromatin.

  16. Dynamic changes in brewing yeast cells in culture revealed by statistical analyses of yeast morphological data.

    Science.gov (United States)

    Ohnuki, Shinsuke; Enomoto, Kenichi; Yoshimoto, Hiroyuki; Ohya, Yoshikazu

    2014-03-01

    The vitality of brewing yeasts has been used to monitor their physiological state during fermentation. To investigate the fermentation process, we used the image processing software, CalMorph, which generates morphological data on yeast mother cells and bud shape, nuclear shape and location, and actin distribution. We found that 248 parameters changed significantly during fermentation. Successive use of principal component analysis (PCA) revealed several important features of yeast, providing insight into the dynamic changes in the yeast population. First, PCA indicated that much of the observed variability in the experiment was summarized in just two components: a change with a peak and a change over time. Second, PCA indicated the independent and important morphological features responsible for dynamic changes: budding ratio, nucleus position, neck position, and actin organization. Thus, the large amount of data provided by imaging analysis can be used to monitor the fermentation processes involved in beer and bioethanol production.

  17. Confinement-Induced Glassy Dynamics in a Model for Chromosome Organization

    Science.gov (United States)

    Kang, Hongsuk; Yoon, Young-Gui; Thirumalai, D.; Hyeon, Changbong

    2015-11-01

    Recent experiments showing scaling of the intrachromosomal contact probability, P (s )˜s-1 with the genomic distance s , are interpreted to mean a self-similar fractal-like chromosome organization. However, scaling of P (s ) varies across organisms, requiring an explanation. We illustrate dynamical arrest in a highly confined space as a discriminating marker for genome organization, by modeling chromosomes inside a nucleus as a homopolymer confined to a sphere of varying sizes. Brownian dynamics simulations show that the chain dynamics slows down as the polymer volume fraction (ϕ ) inside the confinement approaches a critical value ϕc. The universal value of ϕc∞≈0.44 for a sufficiently long polymer (N ≫1 ) allows us to discuss genome dynamics using ϕ as the sole parameter. Our study shows that the onset of glassy dynamics is the reason for the segregated chromosome organization in humans (N ≈3 ×109, ϕ ≳ϕc∞), whereas chromosomes of budding yeast (N ≈108, ϕ <ϕc∞) are equilibrated with no clear signature of such organization.

  18. Synthetic genome engineering forging new frontiers for wine yeast.

    Science.gov (United States)

    Pretorius, Isak S

    2017-02-01

    Over the past 15 years, the seismic shifts caused by the convergence of biomolecular, chemical, physical, mathematical, and computational sciences alongside cutting-edge developments in information technology and engineering have erupted into a new field of scientific endeavor dubbed Synthetic Biology. Recent rapid advances in high-throughput DNA sequencing and DNA synthesis techniques are enabling the design and construction of new biological parts (genes), devices (gene networks) and modules (biosynthetic pathways), and the redesign of biological systems (cells and organisms) for useful purposes. In 2014, the budding yeast Saccharomyces cerevisiae became the first eukaryotic cell to be equipped with a fully functional synthetic chromosome. This was achieved following the synthesis of the first viral (poliovirus in 2002 and bacteriophage Phi-X174 in 2003) and bacterial (Mycoplasma genitalium in 2008 and Mycoplasma mycoides in 2010) genomes, and less than two decades after revealing the full genome sequence of a laboratory (S288c in 1996) and wine (AWRI1631 in 2008) yeast strain. A large international project - the Synthetic Yeast Genome (Sc2.0) Project - is now underway to synthesize all 16 chromosomes (∼12 Mb carrying ∼6000 genes) of the sequenced S288c laboratory strain by 2018. If successful, S. cerevisiae will become the first eukaryote to cross the horizon of in silico design of complex cells through de novo synthesis, reshuffling, and editing of genomes. In the meantime, yeasts are being used as cell factories for the semi-synthetic production of high-value compounds, such as the potent antimalarial artemisinin, and food ingredients, such as resveratrol, vanillin, stevia, nootkatone, and saffron. As a continuum of previously genetically engineered industrially important yeast strains, precision genome engineering is bound to also impact the study and development of wine yeast strains supercharged with synthetic DNA. The first taste of what the future

  19. Construction of a yeast artificial chromosome contig encompassing the human acidic fibroblast growth factor (FGF1) gene: Toward the cloning of the ANLL/MDS tumor-suppressor gene

    Energy Technology Data Exchange (ETDEWEB)

    Chiu, Ing-Ming; Gilmore, E.C.; Liu, Yang; Payson, R.A. (Ohio State Univ., Columbus, OH (United States))

    1994-02-01

    The region surrounding the human acidic fibroblast growth factor (FGF1) locus on chromosome 5q31 is of particular interest since it represents a critical region consistently lost in acute nonlymphocytic leukemia (ANLL) or myelodysplastic syndrome (MDS) patients who have a demonstrable deletion of the distal portion of the long arm of chromosome 5. It is proposed that an ANLL/MDS leukemia suppressor gene resides on 5q31. The authors have previously shown that the gene is most likely localized between FGF1 and PDGFRB/CSF1R loci. The region has also been linked to at least four other genetic diseases, Treacher Collins syndrome, diastrophic dysplasia, limb-girdle muscular dystrophy, and an autosomal dominant deafness, by linkage analysis. Here, they describe yeast artificial chromosomes (YAC) spanning 450 kb around the FGF1 gene. Six YAC clones were isolated from a human YAC library and their restriction enzyme maps were determined. The overlap of the clones with each other and with FGF1 cosmid and phage clones was characterized. Three of the YAC clones were found to contain the entire FGF1 gene, which spans more than 100 kb. Proximal and distal ends of several of these YAC clones were isolated for further overlap cloning. The proximal ends of both Y2 and Y4 were localized to previously isolated FGF1 DNA by sequence analysis. The distal ends of these two clones also hybridized to a human-hamster hybrid containing chromosome 5 as the only human genetic material. These results suggest that these YAC clones represent colinear DNA around the FGF1 locus. None of the YAC clones were found to contain the CD 14 and GRL genes, the closest known proximal and distal markers (relative to the centromere) to the FGF1 gene, respectively. This contig is useful for the overlap cloning of the 5q31 region and for reverse genetic strategies for the isolation of disease genes in the region. 46 refs., 7 figs., 5 tabs.

  20. Sequence analysis of a 10 kb DNA fragment from yeast chromosome VII reveals a novel member of the DnaJ family.

    Science.gov (United States)

    Rodriguez-Belmonte, E; Rodriguez-Torres, A M; Tizon, B; Cadahia, J L; Gonzalez-Siso, I; Ramil, E; Becerra, M; Gonzalez-Dominguez, M; Cerdan, E

    1996-02-01

    We report the sequence analysis of a 10 kb DNA fragment of Saccharomyces cerevisiae chromosome VII. This sequence contains four complete open reading frames (ORFs) of greater than 100 amino acids. There are also two incomplete ORFs flanking the extremes: one of these, G2868, is the 5' part of the SCS3 gene (Hosaka et al., 1994). ORFs G2853 and G2856 correspond to the genes CEG1, coding for the alfa subunit of the mRNA guanylyl transferase and a 3' gene of unknown function previously sequenced (Shibagaki et al., 1992). G2864 is identical to SOH1 also reported (Fan and Klein, 1994).

  1. Telomere dysfunction and chromosome structure modulate the contribution of individual chromosomes in abnormal nuclear morphologies

    Energy Technology Data Exchange (ETDEWEB)

    Pampalona, J.; Soler, D.; Genesca, A. [Department of Cell Biology, Physiology and Immunology, Universitat Autonoma de Barcelona, Bellaterra E-08193 (Spain); Tusell, L., E-mail: laura.tusell@uab.es [Department of Cell Biology, Physiology and Immunology, Universitat Autonoma de Barcelona, Bellaterra E-08193 (Spain)

    2010-01-05

    The cytokinesis-block micronucleus assay has emerged as a biomarker of chromosome damage relevant to cancer. Although it was initially developed to measure micronuclei, it is also useful for measuring nucleoplasmic bridges and nuclear buds. Abnormal nuclear morphologies are frequently observed in malignant tissues and short-term tumour cell cultures. Changes in chromosome structure and number resulting from chromosome instability are important factors in oncogenesis. Telomeres have become key players in the initiation of chromosome instability related to carcinogenesis by means of breakage-fusion-bridge cycles. To better understand the connection between telomere dysfunction and the appearance of abnormal nuclear morphologies, we have characterised the presence of micronuclei, nucleoplasmic bridges and nuclear buds in human mammary primary epithelial cells. These cells can proliferate beyond the Hayflick limit by spontaneously losing expression of the p16{sup INK4a} protein. Progressive telomere shortening leads to the loss of the capping function, and the appearance of end-to-end chromosome fusions that can enter into breakage-fusion-bridge cycles generating massive chromosomal instability. In human mammary epithelial cells, different types of abnormal nuclear morphologies were observed, however only nucleoplasmatic bridges and buds increased significantly with population doublings. Fluorescent in situ hybridisation using centromeric and painting specific probes for chromosomes with eroded telomeres has revealed that these chromosomes are preferentially included in the different types of abnormal nuclear morphologies observed, thus reflecting their common origin. Accordingly, real-time imaging of cell divisions enabled us to determine that anaphase bridge resolution was mainly through chromatin breakage and the formation of symmetric buds in daughter nuclei. Few micronuclei emerged in this cell system thus validating the scoring of nucleoplasmic bridges and

  2. Budding of Walnut ( Juglans regia L.

    Directory of Open Access Journals (Sweden)

    Majlind Kasmi

    2013-09-01

    Full Text Available The walnut is classified as a strategic species for human nutrition and is included in the FAO’s list of priority plants. Walnut, (Juglans regia L. propagation is more difficult, compared to most fruit species. Due to walnut heterozygosity, propagation by seeds does not lead to inheritance of all the characteristics of certain varieties. That is the reason why propagation technologies are being improved worldwide. The purpose of this experiment was to increase the success of inoculation of the walnut budding var. Franquete. Methods such as the patch budding and chip budding have been employed during the experiment. To establish the most appropriate season of inoculation, June budding on 28 June (with buds taken in the current season, autumn budding on 28 August (with buds taken in the current season and spring budding on 28 May (with buds collected from the winter dormant period, were tested. As rootstocks for the June and August budding, the seedlings of Juglans regia L. of the current year's growth have been employed. For the spring inoculation the one year old scions have been used. Patch budding resulted the most successful method for walnuts. However, the success of the method of patch budding depends on the season of inoculation. An 80 % of successful inoculation was achieved by June budding (on 28 June. Furthermore, cutting off the leaf 20 days before the buds being taken for budding, led to even higher results reaching 87% of successful inoculation. According to the results of the present study, the June budding of the patch method seems to be the best solution for the production of grafted young walnut trees.

  3. CTF4 (CHL15) mutants exhibit defective DNA metabolism in the yeast Saccharomyces cerevisiae.

    Science.gov (United States)

    Kouprina, N; Kroll, E; Bannikov, V; Bliskovsky, V; Gizatullin, R; Kirillov, A; Shestopalov, B; Zakharyev, V; Hieter, P; Spencer, F

    1992-12-01

    We have analyzed the CTF4 (CHL15) gene, earlier identified in two screens for yeast mutants with increased rates of mitotic loss of chromosome III and artificial circular and linear chromosomes. Analysis of the segregation properties of circular minichromosomes and chromosome fragments indicated that sister chromatid loss (1:0 segregation) is the predominant mode of chromosome destabilization in ctf4 mutants, though nondisjunction events (2:0 segregation) also occur at an increased rate. Both inter- and intrachromosomal mitotic recombination levels are elevated in ctf4 mutants, whereas spontaneous mutation to canavanine resistance was not elevated. A genomic clone of CTF4 was isolated and used to map its physical and genetic positions on chromosome XVI. Nucleotide sequence analysis of CTF4 revealed a 2.8-kb open reading frame with a 105-kDa predicted protein sequence. The CTF4 DNA sequence is identical to that of POB1, characterized as a gene encoding a protein that associates in vitro with DNA polymerase alpha. At the N-terminal region of the protein sequence, zinc finger motifs which define potential DNA-binding domains were found. The C-terminal region of the predicted protein displayed similarity to sequences of regulatory proteins known as the helix-loop-helix proteins. Data on the effects of a frameshift mutation suggest that the helix-loop-helix domain is essential for CTF4 function. Analysis of sequences upstream of the CTF4 open reading frame revealed the presence of a hexamer element, ACGCGT, a sequence associated with many DNA metabolism genes in budding yeasts. Disruption of the coding sequence of CTF4 did not result in inviability, indicating that the CTF4 gene is nonessential for mitotic cell division. However, ctf4 mutants exhibit an accumulation of large budded cells with the nucleus in the neck. ctf4 rad52 double mutants grew very slowly and produced extremely high levels (50%) of inviable cell division products compared with either single mutant

  4. Cryo‐scanning x‐ray diffraction microscopy of frozen‐hydrated yeast

    National Research Council Canada - National Science Library

    LIMA, E; DIAZ, A; GUIZAR‐SICAIROS, M; GORELICK, S; PERNOT, P; SCHLEIER, T; MENZEL, A

    2013-01-01

    .... We demonstrate these capabilities in two dimensions by imaging unstained frozen‐hydrated budding yeast cells, achieving a spatial resolution of 85 nm with a phase sensitivity of 0.0053 radians...

  5. Bub2 regulation of cytokinesis and septation in budding yeast

    Directory of Open Access Journals (Sweden)

    Park Su Young

    2009-06-01

    Full Text Available Abstract Background The mitotic exit network (MEN is required for events at the end of mitosis such as degradation of mitotic cyclins and cytokinesis. Bub2 and its binding partner Bfa1 act as a GTPase activating protein (GAP to negatively regulate the MEN GTPase Tem1. The Bub2/Bfa1 checkpoint pathway is required to delay the cell cycle in response to mispositioned spindles. In addition to its role in mitotic exit, Tem1 is required for actomyosin ring contraction. Results To test the hypothesis that the Bub2 pathway prevents premature actin ring assembly, we compared the timing of actin ring formation in wild type, bub2Δ, mad2Δ, and bub2Δmad2Δ cells both with and without microtubules. There was no difference in the timing of actin ring formation between wild type and mutant cells in a synchronized cell cycle. In the presence of nocodazole, both bub2Δ and mad2Δ cells formed rings after a delay of the same duration. Double mutant bub2Δmad2Δ and bfa1Δmad2Δ cells formed rings at the same time with and without nocodazole. To determine if Bub2 has an effect on actomyosin ring contraction through its regulation of Tem1, we used live cell imaging of Myo1-GFP in a bub2Δ strain. We found a significant decrease in the total time of contraction and an increase in rate of contraction compared to wild type cells. We also examined myosin contraction using Myo1-GFP in cells overexpressing an epitope tagged Bub2. Surprisingly, overexpression of Bub2 also led to a significant increase in the rate of contraction, as well as morphological defects. The chained cell phenotype caused by Bub2 overexpression could be rescued by co-overexpression of Tem1, and was not rescued by deletion of BFA1. Conclusion Our data indicate that the Bub2 checkpoint pathway does not have a specific role in delaying actin ring formation. The observed increase in the rate of myosin contraction in the bub2Δ strain provides evidence that the MEN regulates actomyosin ring contraction. Our data suggest that the overexpression of the Bub2 fusion protein acts as a dominant negative, leading to septation defects by a mechanism that is Tem1-dependent.

  6. Defining the budding yeast chromatin-associated interactome

    OpenAIRE

    Lambert, Jean-Philippe; Fillingham, Jeffrey; Siahbazi, Mojgan; Greenblatt, Jack; Baetz, Kristin; Figeys, Daniel

    2010-01-01

    The maintenance of cellular fitness requires living organisms to integrate multiple signals into coordinated outputs. Central to this process is the regulation of the expression of the genetic information encoded into DNA. As a result, there are numerous constraints imposed on gene expression. The access to DNA is restricted by the formation of nucleosomes, in which DNA is wrapped around histone octamers to form chromatin wherein the volume of DNA is considerably reduced. As such, nucleosome ...

  7. Database Description - Yeast Interacting Proteins Database | LSDB Archive [Life Science Database Archive metadata

    Lifescience Database Archive (English)

    Full Text Available Yeast Interacting Proteins Database Database Description General information of database Database name Yeast... Interacting Proteins Database Alternative name - Creator Creator Name: Takashi Ito* Creator Affiliation: Di...-4-7136-3989 FAX: +81-4-7136-3979 E-mail : Database classification Metabolic and Signaling Pathways - Protei...n-protein interactions Organism Taxonomy Name: Saccharomyces cerevisiae Taxonomy ID: 4932 Database descripti...ive yeast two-hybrid analysis of budding yeast proteins. Features and manner of utilization of database Prot

  8. Biotechnological Applications of Dimorphic Yeasts

    Science.gov (United States)

    Doiphode, N.; Joshi, C.; Ghormade, V.; Deshpande, M. V.

    The dimorphic yeasts have the equilibrium between spherical growth (budding) and polarized (hyphal or pseudohyphal tip elongation) which can be triggered by change in the environmental conditions. The reversible growth phenomenon has made dimorphic yeasts as an useful model to understand fungal evolution and fungal differentiation, in general. In nature dimorphism is clearly evident in plant and animal fungal pathogens, which survive and most importantly proliferate in the respective hosts. However, number of organisms with no known pathogenic behaviour also show such a transition, which can be exploited for the technological applications due to their different biochemical make up under different morphologies. For instance, chitin and chitosan production using dimorphic Saccharomyces, Mucor, Rhizopus and Benjaminiella, oil degradation and biotransformation with yeast-form of Yarrowia species, bioremediation of organic pollutants, exopolysac-charide production by yeast-phase of Aureobasidium pullulans, to name a few. Myrothecium verrucaria can be used for seed dressing in its yeast form and it produces a mycolytic enzyme complex in its hyphal-form for the biocontrol of fungal pathogens, while Beauveria bassiana and other entomopathogens kill the insect pest by producing yeast- like cells in the insect body. The form-specific expression of protease, chitinase, lipase, ornithine decarboxylase, glutamate dehydrogenases, etc. make Benjaminiella poitrasii, Basidiobolus sp., and Mucor rouxii strains important in bioremediation, nanobiotechnology, fungal evolution and other areas.

  9. The Energy of COPI for Budding Membranes

    Science.gov (United States)

    Thiam, Abdou Rachid; Pincet, Frédéric

    2015-01-01

    As a major actor of cellular trafficking, COPI coat proteins assemble on membranes and locally bend them to bud 60 nm-size coated particles. Budding requires the energy of the coat assembly to overcome the one necessary to deform the membrane which primarily depends on the bending modulus and surface tension, γ. Using a COPI-induced oil nanodroplet formation approach, we modulated the budding of nanodroplets using various amounts and types of surfactant. We found a Heaviside-like dependence between the budding efficiency and γ: budding was only dependent on γ and occurred beneath 1.3 mN/m. With the sole contribution of γ to the membrane deformation energy, we assessed that COPI supplies ~1500 kBT for budding particles from membranes, which is consistent with common membrane deformation energies. Our results highlight how a simple remodeling of the composition of membranes could mechanically modulate budding in cells. PMID:26218078

  10. Budding yeast cDNA sequencing project: S03052-76_F01 [Budding yeast cDNA sequencing project

    Lifescience Database Archive (English)

    Full Text Available EST - Link to UCSC Genome Browser - Sequence >S03052-76_F01.phd NNNNNNNNNNNNNNNNNNNNNNNNNTNTAAAANNNNGANNNGANNNGTGGNTNTNTNTNT TNT...ANTTTNAANAAANAACNNNCCCTNNNNCNCNNNNNNNGAGNAAAAANNGGGTNTNNT NTTTTNNTNNTNTNTNNNNCNNN Qualit

  11. Detection of myosin immunoanalogue in the yeast Candida albicans.

    Science.gov (United States)

    Ghazali, M; Rodier, M H; el Moudni, B; Quellard, N; Jacquemin, J L

    1995-06-01

    Detection and localization of myosin immunoanalogue protein in the yeast Candida albicans were achieved by immunoblotting, indirect immunofluorescence assay, and immunoelectron microscopy. A polypeptide with an M(r) about 110,000, from cytosolic extract and insoluble fraction in the corresponding membrane pellet, was reacted with polyclonal and monoclonal antibodies raised against vertebrate muscle myosin. This protein was located by immunofluorescence and immunoelectron microscopy in the cell cortex along the plasmalemma, in the cytoplasm, and in the septum corresponding to bud scar region situated between the yeast-mother cell and the bud.

  12. Regulation of mitotic spindle asymmetry by SUMO and the spindle-assembly checkpoint in yeast.

    Science.gov (United States)

    Leisner, Christian; Kammerer, Daniel; Denoth, Annina; Britschi, Mirjam; Barral, Yves; Liakopoulos, Dimitris

    2008-08-26

    During mitosis, the kinetochore microtubules capture and segregate chromosomes, and the astral microtubules position the spindle within the cell. Although the spindle is symmetric, proper positioning of the spindle in asymmetrically dividing cells generally correlates with the formation of morphologically and structurally distinct asters [1]. In budding yeast, the spindle-orientation proteins Kar9 and dynein decorate only one aster of the metaphase spindle and direct it toward the bud [2, 3]. The mechanisms controlling the distribution of Kar9 and dynein remain unclear. Here, we show that SUMO regulates astral-microtubule function in at least two ways. First, Kar9 was sumoylated in vivo. Sumoylation and Cdk1-dependent phosphorylation of Kar9 independently promoted Kar9 asymmetry on the spindle. Second, proper regulation of kinetochore function by SUMO was also required for Kar9 asymmetry. Indeed, activation of the spindle-assembly checkpoint (SAC) due to SUMO and kinetochore defects promoted symmetric redistribution of Kar9 in a Mad2-dependent manner. The control of Kar9 distribution by the SAC was independent of Kar9 sumoylation and phosphorylation. Together, our data reveal that three independent mechanisms contribute to Kar9 asymmetry: Cdk1-dependent phosphorylation, sumoylation, and SAC signaling. Hence, the two seemingly independent spindle domains, kinetochores and astral microtubules, function in a tightly coordinated fashion.

  13. Cell Polarity in Yeast.

    Science.gov (United States)

    Chiou, Jian-Geng; Balasubramanian, Mohan K; Lew, Daniel J

    2017-08-07

    A conserved molecular machinery centered on the Cdc42 GTPase regulates cell polarity in diverse organisms. Here we review findings from budding and fission yeasts that reveal both a conserved core polarity circuit and several adaptations that each organism exploits to fulfill the needs of its lifestyle. The core circuit involves positive feedback by local activation of Cdc42 to generate a cluster of concentrated GTP-Cdc42 at the membrane. Speciesspecific pathways regulate the timing of polarization during the cell cycle, as well as the location and number of polarity sites. Expected final online publication date for the Annual Review of Cell and Developmental Biology Volume 33 is October 6, 2017. Please see http://www.annualreviews.org/page/journal/pubdates for revised estimates.

  14. Programmed cell death during terminal bud senescence in a sympodial branching tree,Eucommia ulmoides

    Institute of Scientific and Technical Information of China (English)

    XU Wenjie; Kalima-N'Koma MWANGE; CUI Keming

    2004-01-01

    Eucommia ulmoides Oliv. is a typical sympodial branching tree. The apical bud of the branch ages and dies every year, replaced by the nearby axillary bud in the second year. Structural assays and a series of biochemical analyses were performed to analyze the senescence mechanism in the apical bud. It was revealed that most cells of the apical bud underwent the programmed cell death (PCD) during the senescence: the chromosomes were congregated and the nuclear contents were condensed, as shown by 4′,6-diamidino-2-phenylindole (DAPI) fluorescence. DNA fragmentation was detected during senescence using the terminal deoxynucleotidyl transferase (TdT)-mediated dUTP nick end in situ labeling (TUNEL) method, coincident with the appearance of a DNA ladder. Moreover, a 20 kD DNase related to fragmentation was found. PCD was initiated first in the young leaves, leaf primordia and peripheral zone cells, then in the central mother cells and initial layer cells in the apical meristem. The terminal buds remain in vegetative growth during senescence, in contrast to buds of many annual plants.

  15. Cellular Factors Required for Lassa Virus Budding

    OpenAIRE

    Urata, Shuzo; Noda, Takeshi; Kawaoka, Yoshihiro; Yokosawa, Hideyoshi; Yasuda, Jiro

    2006-01-01

    It is known that Lassa virus Z protein is sufficient for the release of virus-like particles (VLPs) and that it has two L domains, PTAP and PPPY, in its C terminus. However, little is known about the cellular factor for Lassa virus budding. We examined which cellular factors are used in Lassa virus Z budding. We demonstrated that Lassa Z protein efficiently produces VLPs and uses cellular factors, Vps4A, Vps4B, and Tsg101, in budding, suggesting that Lassa virus budding uses the multivesicula...

  16. Whole-Transcriptome Analysis of Differentially Expressed Genes in the Vegetative Buds, Floral Buds and Buds of Chrysanthemum morifolium.

    Directory of Open Access Journals (Sweden)

    Hua Liu

    Full Text Available Chrysanthemum morifolium is an important floral crop that is cultivated worldwide. However, due to a lack of genomic resources, very little information is available concerning the molecular mechanisms of flower development in chrysanthemum.The transcriptomes of chrysanthemum vegetative buds, floral buds and buds were sequenced using Illumina paired-end sequencing technology. A total of 15.4 Gb of reads were assembled into 91,367 unigenes with an average length of 739 bp. A total of 43,137 unigenes showed similarity to known proteins in the Swissprot or NCBI non-redundant protein databases. Additionally, 25,424, 24,321 and 13,704 unigenes were assigned to 56 gene ontology (GO categories, 25 EuKaryotic Orthologous Groups (KOG categories, and 285 Kyoto Encyclopedia of Genes and Genomes (KEGG pathways, respectively. A total of 1,876 differentially expressed genes (DEGs (1,516 up-regulated, 360 down-regulated were identified between vegetative buds and floral buds, and 3,300 DEGs (1,277 up-regulated, 1,706 down-regulated were identified between floral buds and buds. Many genes encoding important transcription factors (e.g., AP2, MYB, MYC, WRKY, NAC and CRT as well as proteins involved in carbohydrate metabolism, protein kinase activity, plant hormone signal transduction, and the defense responses, among others, were considerably up-regulated in floral buds. Genes involved in the photoperiod pathway and flower organ determination were also identified. These genes represent important candidate genes for molecular cloning and functional analysis to study flowering regulation in chrysanthemum.This comparative transcriptome analysis revealed significant differences in gene expression and signaling pathway components between the vegetative buds, floral buds and buds of Chrysanthemum morifolium. A wide range of genes was implicated in regulating the phase transition from vegetative to reproductive growth. These results should aid researchers in the study of

  17. HIV Pol inhibits HIV budding and mediates the severe budding defect of Gag-Pol.

    Directory of Open Access Journals (Sweden)

    Xin Gan

    Full Text Available The prevailing hypothesis of HIV budding posits that the viral Gag protein drives budding, and that the Gag p6 peptide plays an essential role by recruiting host-cell budding factors to sites of HIV assembly. HIV also expresses a second Gag protein, p160 Gag-Pol, which lacks p6 and fails to bud from cells, consistent with the prevailing hypothesis of HIV budding. However, we show here that the severe budding defect of Gag-Pol is not caused by the absence of p6, but rather, by the presence of Pol. Specifically, we show that (i the budding defect of Gag-Pol is unaffected by loss of HIV protease activity and is therefore an intrinsic property of the Gag-Pol polyprotein, (ii the N-terminal 433 amino acids of Gag and Gag-Pol are sufficient to drive virus budding even though they lack p6, (iii the severe budding defect of Gag-Pol is caused by a dominant, cis-acting inhibitor of budding in the HIV Pol domain, and (iv Gag-Pol inhibits Gag and virus budding in trans, even at normal levels of Gag and Gag-Pol expression. These and other data support an alternative hypothesis of HIV budding as a process that is mediated by the normal, non-viral pathway of exosome/microvesicle biogenesis.

  18. Marker chromosomes.

    Science.gov (United States)

    Rao, Kiran Prabhaker; Belogolovkin, Victoria

    2013-04-01

    Marker chromosomes are a morphologically heterogeneous group of structurally abnormal chromosomes that pose a significant challenge in prenatal diagnosis. Phenotypes associated with marker chromosomes are highly variable and range from normal to severely abnormal. Clinical outcomes are very difficult to predict when marker chromosomes are detected prenatally. In this review, we outline the classification, etiology, cytogenetic characterization, and clinical consequences of marker chromosomes, as well as practical approaches to prenatal diagnosis and genetic counseling.

  19. Increased genome instability is not accompanied by sensitivity to DNA damaging agents in aged yeast cells

    NARCIS (Netherlands)

    Novarina, Daniele; Mavrova, Sara N.; Janssens, Georges E.; Rempel, Irina L.; Veenhoff, Liesbeth M.; Chang, Michael

    The budding yeast Saccharomyces cerevisiae divides asymmetrically, producing a new daughter cell from the original mother cell. While daughter cells are born with a full lifespan, a mother cell ages with each cell division and can only generate on average 25 daughter cells before dying. Aged yeast

  20. Repellence of the red bud borer (Resseliella oculiperda) to grafted apple trees by impregnation of budding tape with essential oils

    NARCIS (Netherlands)

    Tol, van R.W.H.M.; Linden, van der A.; Swarts, H.J.; Visser, J.H.

    2007-01-01

    The red bud borer Resseliella oculiperda (Rübs.) is a pest insect of apple trees when rootstocks are grafted with scion buds by shield budding. The female midges are attracted to the wounds of the grafted buds where they lay their eggs. The larvae feed on the cambium and destroy the buds completely

  1. Dissection and design of yeast prions.

    OpenAIRE

    Osherovich, Lev Z.; Cox, Brian S; Tuite, Mick F; Weissman, Jonathan S.

    2004-01-01

    Many proteins can misfold into beta-sheet-rich, self-seeding polymers (amyloids). Prions are exceptional among such aggregates in that they are also infectious. In fungi, prions are not pathogenic but rather act as epigenetic regulators of cell physiology, providing a powerful model for studying the mechanism of prion replication. We used prion-forming domains from two budding yeast proteins (Sup35p and New1p) to examine the requirements for prion formation and inheritance. In both proteins, ...

  2. Spindle alignment regulates the dynamic association of checkpoint proteins with yeast spindle pole bodies.

    Science.gov (United States)

    Caydasi, Ayse Koca; Pereira, Gislene

    2009-01-01

    In many polarized cells, the accuracy of chromosome segregation depends on the correct positioning of the mitotic spindle. In budding yeast, the spindle positioning checkpoint (SPOC) delays mitotic exit when the anaphase spindle fails to extend toward the mother-daughter axis. However it remains to be established how spindle orientation is translated to SPOC components at the yeast spindle pole bodies (SPB). Here, we used photobleaching techniques to show that the dynamics with which Bub2-Bfa1 turned over at SPBs significantly increased upon SPOC activation. A version of Bfa1 that was stably associated with SPBs rendered the cells SPOC deficient without affecting other Bub2-Bfa1 functions, demonstrating the functional importance of regulating the dynamics of Bfa1 SPB association. In addition, we established that the SPOC kinase Kin4 is the major regulator of Bfa1 residence time at SPBs. We suggest that upon SPOC activation Bfa1-Bub2 spreads throughout the cytoplasm, thereby inhibiting mitotic exit.

  3. Off-Target Effects of Psychoactive Drugs Revealed by Genome-Wide Assays in Yeast

    OpenAIRE

    2008-01-01

    To better understand off-target effects of widely prescribed psychoactive drugs, we performed a comprehensive series of chemogenomic screens using the budding yeast Saccharomyces cerevisiae as a model system. Because the known human targets of these drugs do not exist in yeast, we could employ the yeast gene deletion collections and parallel fitness profiling to explore potential off-target effects in a genome-wide manner. Among 214 tested, documented psychoactive drugs, we identified 81 comp...

  4. Engineering of the Saccharomyces cerevisiae yeast strain with multiple chromosome-integrated genes of human alpha-fetoprotein and its high-yield secretory production, purification, structural and functional characterization.

    Science.gov (United States)

    Dudich, Elena; Dudich, Igor; Semenkova, Lidia; Benevolensky, Sergey; Morozkina, Elena; Marchenko, Aleksey; Zatcepin, Sergey; Dudich, Dmitry; Soboleva, Galina; Khromikh, Luidmila; Roslovtceva, Olga; Tatulov, Eduard

    2012-07-01

    Alpha-fetoprotein (AFP) is a biological drug candidate of high medicinal potential in the treatment of autoimmune diseases, cancer, and regenerative medicine. Large-scale production of recombinant human alpha-fetoprotein (rhAFP) is desirable for structural and functional studies and applied research. In this study we cloned and expressed in the secreted form wild-type glycosylated human rhAFP and non-glycosylated mutant rhAFP(0) (N233S) in the yeast strain Saccharomyces cerevisiae with multiple chromosome-integrated synthetic human AFP genes. RhAFP and rhAFP(0) were successfully produced and purified from the culture liquids active naturally folded proteins. Elimination of the glycosylation by mutation reduced rhAFP(0) secretion about threefold as compared to the wild-type protein showing critical role of the N-linked glycan for heterologous protein folding and secretion. Structural similarity of rhAFP and rhAFP(0) with natural embryonic eAFP was confirmed by circular dichroism technique. Functional tests demonstrated similar type of tumor suppressive and immunosuppressive activity for both recombinant species rhAFP and rhAFP(0) as compared to natural eAFP. It was documented that both types of biological activities attributed to rhAFP and rhAFP(0) are due to the fast induction of apoptosis in tumor cells and mitogen-activated lymphocytes. Despite the fact that rhAFP and rhAFP(0) demonstrated slightly less effective tumor suppressive activity as compared to eAFP but rhAFP(0) had produced statistically notable increase in its ability to induce inhibition of in vitro lymphocyte proliferation as compared to the glycosylated rhAFP and eAFP. We conclude that N-linked glycosylation of rhAFP is required for efficient folding and secretion. However the presence of N-linked sugar moiety was shown to be unimportant for tumor suppressive activity but was critically important for its immunoregulative activity which demonstrates that different molecular mechanisms are involved

  5. A screen for suppressors of gross chromosomal rearrangements identifies a conserved role for PLP in preventing DNA lesions.

    Directory of Open Access Journals (Sweden)

    Pamela Kanellis

    2007-08-01

    Full Text Available Genome instability is a hallmark of cancer cells. One class of genome aberrations prevalent in tumor cells is termed gross chromosomal rearrangements (GCRs. GCRs comprise chromosome translocations, amplifications, inversions, deletion of whole chromosome arms, and interstitial deletions. Here, we report the results of a genome-wide screen in Saccharomyces cerevisiae aimed at identifying novel suppressors of GCR formation. The most potent novel GCR suppressor identified is BUD16, the gene coding for yeast pyridoxal kinase (Pdxk, a key enzyme in the metabolism of pyridoxal 5' phosphate (PLP, the biologically active form of vitamin B6. We show that Pdxk potently suppresses GCR events by curtailing the appearance of DNA lesions during the cell cycle. We also show that pharmacological inhibition of Pdxk in human cells leads to the production of DSBs and activation of the DNA damage checkpoint. Finally, our evidence suggests that PLP deficiency threatens genome integrity, most likely via its role in dTMP biosynthesis, as Pdxk-deficient cells accumulate uracil in their nuclear DNA and are sensitive to inhibition of ribonucleotide reductase. Since Pdxk links diet to genome stability, our work supports the hypothesis that dietary micronutrients reduce cancer risk by curtailing the accumulation of DNA damage and suggests that micronutrient depletion could be part of a defense mechanism against hyperproliferation.

  6. Shrinkage of ipsilateral taste buds and hyperplasia of contralateral taste buds following chorda tympani nerve transection

    Directory of Open Access Journals (Sweden)

    Yi-ke Li

    2015-01-01

    Full Text Available The morphological changes that occur in the taste buds after denervation are not well understood in rats, especially in the contralateral tongue epithelium. In this study, we investigated the time course of morphological changes in the taste buds following unilateral nerve transection. The role of the trigeminal component of the lingual nerve in maintaining the structural integrity of the taste buds was also examined. Twenty-four Sprague-Dawley rats were randomly divided into three groups: control, unilateral chorda tympani nerve transection and unilateral chorda tympani nerve transection + lingual nerve transection. Rats were allowed up to 42 days of recovery before being euthanized. The taste buds were visualized using a cytokeratin 8 antibody. Taste bud counts, volumes and taste receptor cell numbers were quantified and compared among groups. No significant difference was detected between the chorda tympani nerve transection and chorda tympani nerve transection + lingual nerve transection groups. Taste bud counts, volumes and taste receptor cell numbers on the ipsilateral side all decreased significantly compared with control. On the contralateral side, the number of taste buds remained unchanged over time, but they were larger, and taste receptor cells were more numerous postoperatively. There was no evidence for a role of the trigeminal branch of the lingual nerve in maintaining the structural integrity of the anterior taste buds.

  7. Shrinkage of ipsilateral taste buds and hyperplasia of contralateral taste buds following chorda tympani nerve transection

    Institute of Scientific and Technical Information of China (English)

    Yi-ke Li; Juan-mei Yang; Yi-bo Huang; Dong-dong Ren; Fang-lu Chi

    2015-01-01

    The morphological changes that occur in the taste buds after denervation are not well under-stood in rats, especially in the contralateral tongue epithelium. In this study, we investigated the time course of morphological changes in the taste buds following unilateral nerve transection. The role of the trigeminal component of the lingual nerve in maintaining the structural integrity of the taste buds was also examined. Twenty-four Sprague-Dawley rats were randomly divided into three groups:control, unilateral chorda tympani nerve transection and unilateral chorda tympani nerve transection+lingual nerve transection. Rats were allowed up to 42 days of re-covery before being euthanized. The taste buds were visualized using a cytokeratin 8 antibody. Taste bud counts, volumes and taste receptor cell numbers were quantiifed and compared among groups. No signiifcant difference was detected between the chorda tympani nerve transection and chorda tympani nerve transection+lingual nerve transection groups. Taste bud counts, vol-umes and taste receptor cell numbers on the ipsilateral side all decreased signiifcantly compared with control. On the contralateral side, the number of taste buds remained unchanged over time, but they were larger, and taste receptor cells were more numerous postoperatively. There was no evidence for a role of the trigeminal branch of the lingual nerve in maintaining the structural integrity of the anterior taste buds.

  8. Functional genomics in the study of yeast cell polarity: moving in the right direction.

    Science.gov (United States)

    Styles, Erin; Youn, Ji-Young; Mattiazzi Usaj, Mojca; Andrews, Brenda

    2013-01-01

    The budding yeast Saccharomyces cerevisiae has been used extensively for the study of cell polarity, owing to both its experimental tractability and the high conservation of cell polarity and other basic biological processes among eukaryotes. The budding yeast has also served as a pioneer model organism for virtually all genome-scale approaches, including functional genomics, which aims to define gene function and biological pathways systematically through the analysis of high-throughput experimental data. Here, we outline the contributions of functional genomics and high-throughput methodologies to the study of cell polarity in the budding yeast. We integrate data from published genetic screens that use a variety of functional genomics approaches to query different aspects of polarity. Our integrated dataset is enriched for polarity processes, as well as some processes that are not intrinsically linked to cell polarity, and may provide new areas for future study.

  9. Modeling Chromosomes

    Science.gov (United States)

    Robertson, Carol

    2016-01-01

    Learning about chromosomes is standard fare in biology classrooms today. However, students may find it difficult to understand the relationships among the "genome", "chromosomes", "genes", a "gene locus", and "alleles". In the simple activity described in this article, which follows the 5E approach…

  10. Comparative live-cell imaging analyses of SPA-2, BUD-6 and BNI-1 in Neurospora crassa reveal novel features of the filamentous fungal polarisome.

    Directory of Open Access Journals (Sweden)

    Alexander Lichius

    Full Text Available A key multiprotein complex involved in regulating the actin cytoskeleton and secretory machinery required for polarized growth in fungi, is the polarisome. Recognized core constituents in budding yeast are the proteins Spa2, Pea2, Aip3/Bud6, and the key effector Bni1. Multicellular fungi display a more complex polarized morphogenesis than yeasts, suggesting that the filamentous fungal polarisome might fulfill additional functions. In this study, we compared the subcellular organization and dynamics of the putative polarisome components BUD-6 and BNI-1 with those of the bona fide polarisome marker SPA-2 at various developmental stages of Neurospora crassa. All three proteins exhibited a yeast-like polarisome configuration during polarized germ tube growth, cell fusion, septal pore plugging and tip repolarization. However, the localization patterns of all three proteins showed spatiotemporally distinct characteristics during the establishment of new polar axes, septum formation and cytokinesis, and maintained hyphal tip growth. Most notably, in vegetative hyphal tips BUD-6 accumulated as a subapical cloud excluded from the Spitzenkörper (Spk, whereas BNI-1 and SPA-2 partially colocalized with the Spk and the tip apex. Novel roles during septal plugging and cytokinesis, connected to the reinitiation of tip growth upon physical injury and conidial maturation, were identified for BUD-6 and BNI-1, respectively. Phenotypic analyses of gene deletion mutants revealed additional functions for BUD-6 and BNI-1 in cell fusion regulation, and the maintenance of Spk integrity. Considered together, our findings reveal novel polarisome-independent functions of BUD-6 and BNI-1 in Neurospora, but also suggest that all three proteins cooperate at plugged septal pores, and their complex arrangement within the apical dome of mature hypha might represent a novel aspect of filamentous fungal polarisome architecture.

  11. Oxytocin signaling in mouse taste buds.

    Directory of Open Access Journals (Sweden)

    Michael S Sinclair

    Full Text Available BACKGROUND: The neuropeptide, oxytocin (OXT, acts on brain circuits to inhibit food intake. Mutant mice lacking OXT (OXT knockout overconsume salty and sweet (i.e. sucrose, saccharin solutions. We asked if OXT might also act on taste buds via its receptor, OXTR. METHODOLOGY/PRINCIPAL FINDINGS: Using RT-PCR, we detected the expression of OXTR in taste buds throughout the oral cavity, but not in adjacent non-taste lingual epithelium. By immunostaining tissues from OXTR-YFP knock-in mice, we found that OXTR is expressed in a subset of Glial-like (Type I taste cells, and also in cells on the periphery of taste buds. Single-cell RT-PCR confirmed this cell-type assignment. Using Ca2+ imaging, we observed that physiologically appropriate concentrations of OXT evoked [Ca2+]i mobilization in a subset of taste cells (EC50 approximately 33 nM. OXT-evoked responses were significantly inhibited by the OXTR antagonist, L-371,257. Isolated OXT-responsive taste cells were neither Receptor (Type II nor Presynaptic (Type III cells, consistent with our immunofluorescence observations. We also investigated the source of OXT peptide that may act on taste cells. Both RT-PCR and immunostaining suggest that the OXT peptide is not produced in taste buds or in their associated nerves. Finally, we also examined the morphology of taste buds from mice that lack OXTR. Taste buds and their constituent cell types appeared very similar in mice with two, one or no copies of the OXTR gene. CONCLUSIONS/SIGNIFICANCE: We conclude that OXT elicits Ca2+ signals via OXTR in murine taste buds. OXT-responsive cells are most likely a subset of Glial-like (Type I taste cells. OXT itself is not produced locally in taste tissue and is likely delivered through the circulation. Loss of OXTR does not grossly alter the morphology of any of the cell types contained in taste buds. Instead, we speculate that OXT-responsive Glial-like (Type I taste bud cells modulate taste signaling and afferent

  12. Yeast Mph1 helicase dissociates Rad51-made D-loops: implications for crossover control in mitotic recombination.

    Science.gov (United States)

    Prakash, Rohit; Satory, Dominik; Dray, Eloïse; Papusha, Almas; Scheller, Jürgen; Kramer, Wilfried; Krejci, Lumir; Klein, Hannah; Haber, James E; Sung, Patrick; Ira, Grzegorz

    2009-01-01

    Eukaryotes possess mechanisms to limit crossing over during homologous recombination, thus avoiding possible chromosomal rearrangements. We show here that budding yeast Mph1, an ortholog of human FancM helicase, utilizes its helicase activity to suppress spontaneous unequal sister chromatid exchanges and DNA double-strand break-induced chromosome crossovers. Since the efficiency and kinetics of break repair are unaffected, Mph1 appears to channel repair intermediates into a noncrossover pathway. Importantly, Mph1 works independently of two other helicases-Srs2 and Sgs1-that also attenuate crossing over. By chromatin immunoprecipitation, we find targeting of Mph1 to double-strand breaks in cells. Purified Mph1 binds D-loop structures and is particularly adept at unwinding these structures. Importantly, Mph1, but not a helicase-defective variant, dissociates Rad51-made D-loops. Overall, the results from our analyses suggest a new role of Mph1 in promoting the noncrossover repair of DNA double-strand breaks.

  13. [Novel bioconversion systems using a yeast molecular display system].

    Science.gov (United States)

    Shibasaki, Seiji

    2010-11-01

    The budding yeast Saccharomyces cerevisiae has been used for the process of fermentation as well as for studies in biochemistry and molecular biology as a eukaryotic model cell or tool for the analysis of gene functions. Thus, yeast is essential in industries and researches. Yeast cells have a cell wall, which is one characteristic that helps distinguish yeast cells from other eukaryotic cells such as mammalian cells. We have developed a molecular display system using the protein of the yeast cell wall as an anchor for foreign proteins. Yeast cells have been designed for use in sensing and metal adsorption, and have been used in vaccines and for screening novel proteins. Currently, yeast is used not only as a tool for analyzing gene or protein function but also in molecular display technology. The phage display system, which is at the forefront of molecular display technologies, is a powerful tool for screening ligands bound to a target molecule and for analyzing protein-protein interactions; however, in some cases, eukaryotic proteins are not easily expressed by this system. On the other hand, yeast cells have the ability to express eukaryotic proteins and proliferate; thus, these cells display various proteins. Yeast cells are more appropriate for white biotechnology. In this review, displays of enzymes that are important in bioconversion, such as lipases and β-glucosidases, are going to be introduced.

  14. Single-particle tracking of quantum dot-conjugated prion proteins inside yeast cells

    Energy Technology Data Exchange (ETDEWEB)

    Tsuji, Toshikazu; Kawai-Noma, Shigeko [Department of Biomolecular Engineering, Graduate School of Biosciences and Biotechnology, Tokyo Institute of Technology, B56, 4259 Nagatsuta, Midori-ku, Yokohama 226-8501 (Japan); Pack, Chan-Gi [Cellular Informatics Laboratory, RIKEN Advanced Science Institute, Wako-shi, Saitama 351-0198 (Japan); Terajima, Hideki [Department of Biomolecular Engineering, Graduate School of Biosciences and Biotechnology, Tokyo Institute of Technology, B56, 4259 Nagatsuta, Midori-ku, Yokohama 226-8501 (Japan); Yajima, Junichiro; Nishizaka, Takayuki [Department of Physics, Gakushuin University, 1-5-1 Mejiro, Toshima-ku, Tokyo 171-8588 (Japan); Kinjo, Masataka [Laboratory of Molecular Cell Dynamics, Graduate School of Life Sciences, Hokkaido University, Sapporo 001-0021 (Japan); Taguchi, Hideki, E-mail: taguchi@bio.titech.ac.jp [Department of Biomolecular Engineering, Graduate School of Biosciences and Biotechnology, Tokyo Institute of Technology, B56, 4259 Nagatsuta, Midori-ku, Yokohama 226-8501 (Japan)

    2011-02-25

    Research highlights: {yields} We develop a method to track a quantum dot-conjugated protein in yeast cells. {yields} We incorporate the conjugated quantum dot proteins into yeast spheroplasts. {yields} We track the motions by conventional or 3D tracking microscopy. -- Abstract: Yeast is a model eukaryote with a variety of biological resources. Here we developed a method to track a quantum dot (QD)-conjugated protein in the budding yeast Saccharomyces cerevisiae. We chemically conjugated QDs with the yeast prion Sup35, incorporated them into yeast spheroplasts, and tracked the motions by conventional two-dimensional or three-dimensional tracking microscopy. The method paves the way toward the individual tracking of proteins of interest inside living yeast cells.

  15. Yeast Lab

    OpenAIRE

    Lewis, Matt; Powell, Jim

    2016-01-01

    Yeast are grown in a small, capped ask, generating carbon dioxide which is trapped in an inverted jar full of colored water. The volume of carbon dioxide produced can either be measured directly or using time-lapse imagery on an iPad or similar. Students are then challenged to model the resulting data. From this exercise students gain greater understand- ing of ODE compartment models, parameter estimation, population dynamics and limiting factors.

  16. Identification and Quality Assessment of Chrysanthemum Buds by CE Fingerprinting

    Directory of Open Access Journals (Sweden)

    Xiaoping Xing

    2015-01-01

    Full Text Available A simple and efficient fingerprinting method for chrysanthemum buds was developed with the aim of establishing a quality control protocol based on biochemical makeup. Chrysanthemum bud samples were successively extracted by water and alcohol. The fingerprints of the chrysanthemum buds samples were obtained using capillary electrophoresis and electrochemical detection (CE-ED employing copper and carbon working electrodes to capture all of the chemical information. 10 batches of chrysanthemum buds were collected from different regions and various factories to establish the baseline fingerprint. The experimental data of 10 batches electropherogram buds by CE were analyzed by correlation coefficient and the included angle cosine methods. A standard chrysanthemum bud fingerprint including 24 common peaks was established, 12 from each electrode, which was successfully applied to identify and distinguish between chrysanthemum buds from 2 other chrysanthemum species. These results demonstrate that fingerprint analysis can be used as an important criterion for chrysanthemum buds quality control.

  17. Sprouting of dormant buds on border trees

    Science.gov (United States)

    G.R., Jr. Trimble; H. Clay Smith; H. Clay Smith

    1970-01-01

    As part of an evaluation of silvicultura1 systems used in managing Appalachian hardwoods, we are studying degrade of border trees surrounding harvest-cut openings made in the patch cutting and group selection systems. One facet of this research dealt with determining what portion of visually evident dormant buds on border tree boles sprouted when the openings were cut...

  18. Mechanisms of frost adaptation and freeze damage in grapevine buds

    OpenAIRE

    Badulescu Valle, Radu Virgil

    2002-01-01

    Mechanisms of frost hardening in compound (latent) buds of the grapevine cultivar ?Bacchus? were tested with different methods during three winters. The investigated parameters were LTE/HTE (low temperature exotherm/high temperature exotherm), water content, starch, sugar- and anions combination and bud histology. Water content from wood and buds was determined regularly every 2 weeks from March 1998 until Mai 2000. The lowest water content in wood and buds (about 40 %) was found ...

  19. An 18.3 kb DNA fragment from yeast chromosome VII carries four unknown open reading frames, the gene for an Asn synthase, remnants of Ty and three tRNA genes.

    Science.gov (United States)

    Van Dyck, L; Tettelin, H; Purnelle, B; Goffeau, A

    1997-02-01

    An 18.3 kb DNA segment from yeast Saccharomyces cerevisiae VII encompasses the previously characterized MEP1, NUP57 and PPT1 genes as well as seven new open reading frames (ORFs) of at least 100 residues. G6358 is an ubiquitous glutamine-dependent asparagine synthase. G6362 is membrane protein highly homologous to a protein of unknown function in the yeast Schizosaccharomyces pombe. Three ORFs (G6324, G6335 and G6365) have no significant homology with previously reported proteins of characteristic motifs. G6321 and G6359, enclosed in longer ORFs, are not likely to be coding. The segment also contains tRNA genes for Asn, Arg and Ile as well as sigma element and two solo deltas. ORFs and genetic elements are named according to a preliminary working nomenclature.

  20. Heterotrimeric G Protein-coupled Receptor Signaling in Yeast Mating Pheromone Response.

    Science.gov (United States)

    Alvaro, Christopher G; Thorner, Jeremy

    2016-04-08

    The DNAs encoding the receptors that respond to the peptide mating pheromones of the budding yeastSaccharomyces cerevisiaewere isolated in 1985, and were the very first genes for agonist-binding heterotrimeric G protein-coupled receptors (GPCRs) to be cloned in any organism. Now, over 30 years later, this yeast and its receptors continue to provide a pathfinding experimental paradigm for investigating GPCR-initiated signaling and its regulation, as described in this retrospective overview.

  1. An Energy-Independent Pro-longevity Function of Triacylglycerol in Yeast

    OpenAIRE

    Witawas Handee; Xiaobo Li; Hall, Kevin W; Xiexiong Deng; Pan Li; Christoph Benning; Williams, Barry L; Min-Hao Kuo

    2016-01-01

    Author Summary Triacylglycerol (TAG) is a ubiquitous lipid species well-known for its roles in storing surplus energy, providing insulation, and maintaining cellular lipid homeostasis. Here we present evidence for a novel pro-longevity function of TAG in the budding yeast, a model organism for aging research. Yeast cells that are genetically engineered to store more TAG live significantly longer without suffering obvious growth defects, whereas those lean cells that are depleted of TAG die ea...

  2. Molecular model of fission yeast centrosome assembly determined by superresolution imaging.

    Science.gov (United States)

    Bestul, Andrew J; Yu, Zulin; Unruh, Jay R; Jaspersen, Sue L

    2017-08-07

    Microtubule-organizing centers (MTOCs), known as centrosomes in animals and spindle pole bodies (SPBs) in fungi, are important for the faithful distribution of chromosomes between daughter cells during mitosis as well as for other cellular functions. The cytoplasmic duplication cycle and regulation of the Schizosaccharomyces pombe SPB is analogous to centrosomes, making it an ideal model to study MTOC assembly. Here, we use superresolution structured illumination microscopy with single-particle averaging to localize 14 S. pombe SPB components and regulators, determining both the relationship of proteins to each other within the SPB and how each protein is assembled into a new structure during SPB duplication. These data enabled us to build the first comprehensive molecular model of the S. pombe SPB, resulting in structural and functional insights not ascertained through investigations of individual subunits, including functional similarities between Ppc89 and the budding yeast SPB scaffold Spc42, distribution of Sad1 to a ring-like structure and multiple modes of Mto1 recruitment. © 2017 Bestul et al.

  3. Microcompartments within the yeast plasma membrane.

    Science.gov (United States)

    Merzendorfer, Hans; Heinisch, Jürgen J

    2013-02-01

    Recent research in cell biology makes it increasingly clear that the classical concept of compartmentation of eukaryotic cells into different organelles performing distinct functions has to be extended by microcompartmentation, i.e., the dynamic interaction of proteins, sugars, and lipids at a suborganellar level, which contributes significantly to a proper physiology. As different membrane compartments (MCs) have been described in the yeast plasma membrane, such as those defined by Can1 and Pma1 (MCCs and MCPs), Saccharomyces cerevisiae can serve as a model organism, which is amenable to genetic, biochemical, and microscopic studies. In this review, we compare the specialized microcompartment of the yeast bud neck with other plasma membrane substructures, focusing on eisosomes, cell wall integrity-sensing units, and chitin-synthesizing complexes. Together, they ensure a proper cell division at the end of mitosis, an intricately regulated process, which is essential for the survival and proliferation not only of fungal, but of all eukaryotic cells.

  4. Survival and growth of yeast without telomere capping by Cdc13 in the absence of Sgs1, Exo1, and Rad9.

    Directory of Open Access Journals (Sweden)

    Hien-Ping Ngo

    2010-08-01

    Full Text Available Maintenance of telomere capping is absolutely essential to the survival of eukaryotic cells. Telomere capping proteins, such as Cdc13 and POT1, are essential for the viability of budding yeast and mammalian cells, respectively. Here we identify, for the first time, three genetic modifications that allow budding yeast cells to survive without telomere capping by Cdc13. We found that simultaneous inactivation of Sgs1, Exo1, and Rad9, three DNA damage response (DDR proteins, is sufficient to allow cell division in the absence of Cdc13. Quantitative amplification of ssDNA (QAOS was used to show that the RecQ helicase Sgs1 plays an important role in the resection of uncapped telomeres, especially in the absence of checkpoint protein Rad9. Strikingly, simultaneous deletion of SGS1 and the nuclease EXO1, further reduces resection at uncapped telomeres and together with deletion of RAD9 permits cell survival without CDC13. Pulsed-field gel electrophoresis studies show that cdc13-1 rad9Delta sgs1Delta exo1Delta strains can maintain linear chromosomes despite the absence of telomere capping by Cdc13. However, with continued passage, the telomeres of such strains eventually become short and are maintained by recombination-based mechanisms. Remarkably, cdc13Delta rad9Delta sgs1Delta exo1Delta strains, lacking any Cdc13 gene product, are viable and can grow indefinitely. Our work has uncovered a critical role for RecQ helicases in limiting the division of cells with uncapped telomeres, and this may provide one explanation for increased tumorigenesis in human diseases associated with mutations of RecQ helicases. Our results reveal the plasticity of the telomere cap and indicate that the essential role of telomere capping is to counteract specific aspects of the DDR.

  5. Synthetic chromosomes.

    Science.gov (United States)

    Schindler, Daniel; Waldminghaus, Torsten

    2015-11-01

    What a living organism looks like and how it works and what are its components-all this is encoded on DNA, the genetic blueprint. Consequently, the way to change an organism is to change its genetic information. Since the first pieces of recombinant DNA have been used to transform cells in the 1970s, this approach has been enormously extended. Bigger and bigger parts of the genetic information have been exchanged or added over the years. Now we are at a point where the construction of entire chromosomes becomes a reachable goal and first examples appear. This development leads to fundamental new questions, for example, about what is possible and desirable to build or what construction rules one needs to follow when building synthetic chromosomes. Here we review the recent progress in the field, discuss current challenges and speculate on the appearance of future synthetic chromosomes.

  6. Chemotyping of yeast mutants using robotics.

    Science.gov (United States)

    Rieger, K J; El-Alama, M; Stein, G; Bradshaw, C; Slonimski, P P; Maundrell, K

    1999-07-01

    By now, the EUROFAN programme for the functional analysis of genes from the yeast genome has attained its cruising speed. Indeed, several hundreds of yeast mutants with no phenotype as tested by growth on standard media and no significant sequence similarity to proteins of known function are available through the efforts of various laboratories. Based on the methodology initiated during the pilot project on yeast chromosome III (Yeast 13, 1547-1562, 1997) we adapted it to High Throughput Screening (HTS), using robotics. The first 100 different gene deletions from EUROSCARF, constructed in an FY1679 strain background, were run against a collection of about 300 inhibitors. Many of these inhibitors have not been reported until now to interfere in vivo with growth of Saccharomyces cerevisiae. In the present paper we provide a list of novel growth conditions and a compilation of 49 yeast deletants (from chromosomes II, IV, VII, X, XIV, XV) corresponding to 58% of the analysed genes, with at least one clear and stringent phenotype. The majority of these deletants are sensitive to one or two compounds (monotropic phenotype) while a distinct subclass of deletants displays a hyper-pleiotropic phenotype with sensitivities to a dozen or more compounds. Therefore, chemotyping of unknown genes with a large spectrum of drugs opens new vistas for a more in-depth functional analysis and a more precise definition of molecular targets.

  7. Deciphering the genic basis of yeast fitness variation by simultaneous forward and reverse genetics.

    Science.gov (United States)

    Maclean, Calum J; Metzger, Brian P H; Yang, Jian-Rong; Ho, Wei-Chin; Moyers, Bryan; Zhang, Jianzhi

    2017-05-04

    The budding yeast Saccharomyces cerevisiae is the best studied eukaryote in molecular and cell biology, but its utility for understanding the genetic basis of phenotypic variation in natural populations is limited by inefficient association mapping due to strong and complex population structure. To overcome this challenge, we generated genome sequences for 85 strains and performed a comprehensive population genomic survey of a total of 190 diverse strains. We identified considerable variation in population structure among chromosomes and identified 181 genes that are absent from the reference genome. Many of these non-reference genes are expressed and we functionally confirmed that two of these genes confer increased resistance to antifungals. Next, we simultaneously measured the growth rates of over 4500 laboratory strains, each of which lacks a nonessential gene, and 81 natural strains across multiple environments using unique DNA barcode present in each strain. By combining the genome-wide reverse genetic information gained from the gene deletion strains with a genome-wide association analysis from the natural strains, we identified genomic regions associated with fitness variation in natural populations. To experimentally validate a subset of these associations, we used reciprocal hemizygosity tests, finding that while the combined forward and reverse genetic approaches can identify a single causal gene, the phenotypic consequences of natural genetic variation often follow a complicated pattern. The resources and approach provided outline an efficient and reliable route to association mapping in yeast and significantly enhance its value as a model for understanding the genetic mechanisms underlying phenotypic variation and evolution in natural populations. © The Author 2017. Published by Oxford University Press on behalf of the Society for Molecular Biology and Evolution. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.

  8. Telomere DNA recognition in Saccharomycotina yeast: potential lessons for the co-evolution of ssDNA and dsDNA-binding proteins and their target sites

    Directory of Open Access Journals (Sweden)

    Neal F. Lue

    2015-05-01

    Full Text Available In principle, alterations in the telomere repeat sequence would be expected to disrupt the protective nucleoprotein complexes that confer stability to chromosome ends, and hence relatively rare events in evolution. Indeed, numerous organisms in diverse phyla share a canonical 6 bp telomere repeat unit (5’-TTAGGG-3’/5’-CCCTAA-3’, suggesting common descent from an ancestor that carries this particular repeat. All the more remarkable, then, are the extraordinarily divergent telomere sequences that populate the Saccharomycotina subphylum of budding yeast. These sequences are distinguished from the canonical telomere repeat in being long, occasionally degenerate, and frequently non-G/C-rich. Despite the divergent telomere repeat sequences, studies to date indicate that the same families of single-strand (ss and double-strand (ds telomere binding proteins (i.e., the Cdc13 and Rap1 families are responsible for telomere protection in Saccharomycotina yeast. The recognition mechanisms of the protein family members therefore offer an informative paradigm for understanding the co-evolution of DNA-binding proteins and the cognate target sequences. Existing data suggest three potential, inter-related solutions to the DNA recognition problem: (i duplication of the recognition protein and functional modification; (ii combinatorial recognition of target site; and (iii flexibility of the recognition surfaces of the DNA-binding proteins to adopt alternative conformations. Evidence in support of these solutions and the relevance of these solutions to other DNA-protein regulatory systems are discussed.

  9. Genetic aspects of targeted insertion mutagenesis in yeasts.

    Science.gov (United States)

    Klinner, U; Schäfer, B

    2004-05-01

    Targeted insertion mutagenesis is a main molecular tool of yeast science initially applied in Saccharomyces cerevisiae. The method was extended to fission yeast Schizosaccharomyces pombe and to "non-conventional" yeast species, which show specific properties of special interest to both basic and applied research. Consequently, the behaviour of such non-Saccharomyces yeasts is reviewed against the background of the knowledge of targeted insertion mutagenesis in S. cerevisiae. Data of homologous integration efficiencies obtained with circular, ends-in or ends-out vectors in several yeasts are compared. We follow details of targeted insertion mutagenesis in order to recognize possible rate-limiting steps. The route of the vector to the target and possible mechanisms of its integration into chromosomal genes are considered. Specific features of some yeast species are discussed. In addition, similar approaches based on homologous recombination that have been established for the mitochondrial genome of S. cerevisiae are described.

  10. Fork rotation and DNA precatenation are restricted during DNA replication to prevent chromosomal instability.

    Science.gov (United States)

    Schalbetter, Stephanie A; Mansoubi, Sahar; Chambers, Anna L; Downs, Jessica A; Baxter, Jonathan

    2015-08-18

    Faithful genome duplication and inheritance require the complete resolution of all intertwines within the parental DNA duplex. This is achieved by topoisomerase action ahead of the replication fork or by fork rotation and subsequent resolution of the DNA precatenation formed. Although fork rotation predominates at replication termination, in vitro studies have suggested that it also occurs frequently during elongation. However, the factors that influence fork rotation and how rotation and precatenation may influence other replication-associated processes are unknown. Here we analyze the causes and consequences of fork rotation in budding yeast. We find that fork rotation and precatenation preferentially occur in contexts that inhibit topoisomerase action ahead of the fork, including stable protein-DNA fragile sites and termination. However, generally, fork rotation and precatenation are actively inhibited by Timeless/Tof1 and Tipin/Csm3. In the absence of Tof1/Timeless, excessive fork rotation and precatenation cause extensive DNA damage following DNA replication. With Tof1, damage related to precatenation is focused on the fragile protein-DNA sites where fork rotation is induced. We conclude that although fork rotation and precatenation facilitate unwinding in hard-to-replicate contexts, they intrinsically disrupt normal chromosome duplication and are therefore restricted by Timeless/Tipin.

  11. Genetic diversity of the yeast Candida utilis.

    Science.gov (United States)

    Stoltenburg, R; Klinner, U; Ritzerfeld, P; Zimmermann, M; Emeis, C C

    1992-12-01

    The electrophoretic karyotypes and some mtDNA restriction fragment patterns of 13 strains of Candida utilis and one strain of Hansenula jadinii were compared. PFGE separations revealed remarkable chromosome length polymorphisms between two groups of strains suggesting that perhaps they do not belong to the same species. However, all strains had the same or similar EcoRI, HindIII and BamHI mtDNA restriction patterns. The mtDNA genomes had an average size range of 55 kb. These results support the supposition that C. utilis is a yeast with a highly variable electrophoretic karyotype as already known for another imperfect yeast species, Candida albicans.

  12. CHROMOSOME SEGREGATION: NOVEL INSIGHTS INTO THE MECHANISM AND REGULATION

    Directory of Open Access Journals (Sweden)

    Miroslava Pozgajova

    2013-02-01

    Full Text Available A crucial feature of every healthy living organism is accurate segregation of chromosomes. Errors in this process may lead to aneuploidy, which is responsible for diverse genetic defects and diseases such as Down syndrome, miscarriages, cancer and others. Although, chromosome segregation has been studied intensively in the past, the exact mechanism of accurate chromosome segregation still remains unclear. Identification and characterization of proteins and protein complexes involved in this process is essential for understanding of processes that lead to chromosome missegregation. Basic molecular mechanism share common principles in animals, humans, plants and unicellular organisms; it is therefore possible to study these mechanisms in simple model organisms such as yeasts. The fission yeast Schizosaccharomyces pombe is an excellent model organism to study the function and regulation of chromosome segregation in both mitosis and meiosis.

  13. Genetic constitution of industrial yeast.

    Science.gov (United States)

    Benítez, T; Martínez, P; Codón, A C

    1996-09-01

    Saccharomyces cerevisiae industrial yeast strains are highly heterogeneous. These industrial strains, including bakers', wine, brewing and distillers', have been compared with respect to their DNA content, number and size of chromosomes, homologies between their genes and those of laboratory strains, and restriction fragment lengths of their mitDNA. A high variability, and the presence of multigenic families, were observed in some industrial yeast groups. The occurrence or the lack of chromosomal polymorphism, as well as the presence of multiple copies of some genes, could be related to a selective process occurring under specific industrial conditions. This polymorphism is generated by reorganization events, that take place mainly during meiosis and are mediated by repetitive Y' and Ty elements. These elements give rise to ectopic and asymmetric recombination and to gene conversion. The polymorphism displayed by the mitDNA could also result from specific industrial conditions. However, in enological strains the selective process is masked by the mutagenic effect that ethanol exerts on this DNA.

  14. The fission yeast inhibitor of growth (ING) protein Png1p functions in response to DNA damage.

    Science.gov (United States)

    Chen, Jian-Qiang; Li, Yang; Pan, Xian; Lei, Bing-Kun; Chang, Cheng; Liu, Zheng-Xun; Lu, Hong

    2010-05-21

    In budding yeast and human cells, ING (inhibitor of growth) tumor suppressor proteins play important roles in response to DNA damage by modulating chromatin structure through collaborating with histone acetyltransferase or histone deacetylase complexes. However, the biological functions of ING family proteins in fission yeast are poorly defined. Here, we report that Png1p, a fission yeast ING homolog protein, is required for cell growth under normal and DNA-damaged conditions. Png1p was further confirmed to regulate histone H4 acetylation through collaboration with the MYST family histone acetyltransferase 1 (Mst1). Additionally, both fission yeast PNG1 and MST1 can functionally complement their budding yeast correspondence homologs YNG2 and ESA1, respectively. These results suggest that ING proteins in fission yeast might also conserve function, similar to ING proteins in budding yeast and human cells. We also showed that decreased acetylation in Deltapng1 cells resulted in genome-wide down-regulation of 756 open reading frames, including the central DNA repair gene RAD22. Overexpression of RAD22 partially rescued the png1 mutant phenotype under both normal and DNA-damaged conditions. Furthermore, decreased expression of RAD22 in Deltapng1 cells was confirmed to be caused by decreased H4 acetylation at its promoter. Altogether, these results indicate that Png1p is required for histone H4 acetylation and functions upstream of RAD22 in the DNA damage response pathway.

  15. Sequence analysis of a 9873 bp fragment of the left arm of yeast chromosome XV that contains the ARG8 and CDC33 genes, a putative riboflavin synthase beta chain gene, and four new open reading frames.

    Science.gov (United States)

    Casas, C; Aldea, M; Casamayor, A; Lafuente, M J; Gamo, F J; Gancedo, C; Ariño, J; Herrero, E

    1995-09-15

    The DNA sequence of a 9873 bp fragment located near the left telomere of chromosome XV has been determined. Sequence analysis reveals seven open reading frames. One is the ARG8 gene coding for N-acetylornithine aminotransferase. Another corresponds to CDC33, which codes for the initiation factor 4E or cap binding protein. The open reading frame AOE169 can be considered as the putative gene for the Saccharomyces cerevisiae riboflavin synthase beta chain, since its translation product shows strong homology with four prokaryotic riboflavin synthase beta chains.

  16. The sequence of a 8 kb segment on the right arm of yeast chromosome VII identifies four new open reading frames and the genes for yTAFII145.

    Science.gov (United States)

    Ruzzi, M; Marconi, A; Saliola, M; Fabiani, L; Montebove, F; Frontali, L

    1997-03-30

    We report the sequence of a 8,061 bp fragment of Saccharomyces cerevisiae chromosome VII. Five open reading frames (ORFs) of at least 100 amino acids were identified. Three show similarities to the amino-acid sequence of known gene products. ORF G9374 corresponds to the gene coding for the yTAFII145 protein: a TBP-associated factor whose amino-acid sequence was previously reported (Reese et al., 1994). The remaining ORF does not display similarities to known sequences.

  17. Chromosome Analysis

    Science.gov (United States)

    1998-01-01

    Perceptive Scientific Instruments, Inc., provides the foundation for the Powergene line of chromosome analysis and molecular genetic instrumentation. This product employs image processing technology from NASA's Jet Propulsion Laboratory and image enhancement techniques from Johnson Space Center. Originally developed to send pictures back to earth from space probes, digital imaging techniques have been developed and refined for use in a variety of medical applications, including diagnosis of disease.

  18. Use of the BioGRID Database for Analysis of Yeast Protein and Genetic Interactions.

    Science.gov (United States)

    Oughtred, Rose; Chatr-aryamontri, Andrew; Breitkreutz, Bobby-Joe; Chang, Christie S; Rust, Jennifer M; Theesfeld, Chandra L; Heinicke, Sven; Breitkreutz, Ashton; Chen, Daici; Hirschman, Jodi; Kolas, Nadine; Livstone, Michael S; Nixon, Julie; O'Donnell, Lara; Ramage, Lindsay; Winter, Andrew; Reguly, Teresa; Sellam, Adnane; Stark, Chris; Boucher, Lorrie; Dolinski, Kara; Tyers, Mike

    2016-01-04

    The BioGRID database is an extensive repository of curated genetic and protein interactions for the budding yeast Saccharomyces cerevisiae, the fission yeast Schizosaccharomyces pombe, and the yeast Candida albicans SC5314, as well as for several other model organisms and humans. This protocol describes how to use the BioGRID website to query genetic or protein interactions for any gene of interest, how to visualize the associated interactions using an embedded interactive network viewer, and how to download data files for either selected interactions or the entire BioGRID interaction data set. © 2016 Cold Spring Harbor Laboratory Press.

  19. Generation of micronuclei during interphase by coupling between cytoplasmic membrane blebbing and nuclear budding.

    Directory of Open Access Journals (Sweden)

    Koh-ichi Utani

    Full Text Available Micronucleation, mediated by interphase nuclear budding, has been repeatedly suggested, but the process is still enigmatic. In the present study, we confirmed the previous observation that there are lamin B1-negative micronuclei in addition to the positive ones. A large cytoplasmic bleb was found to frequently entrap lamin B1-negative micronuclei, which were connected to the nucleus by a thin chromatin stalk. At the bottom of the stalk, the nuclear lamin B1 structure appeared broken. Chromatin extrusion through lamina breaks has been referred to as herniation or a blister of the nucleus, and has been observed after the expression of viral proteins. A cell line in which extrachromosomal double minutes and lamin B1 protein were simultaneously visualized in different colors in live cells was established. By using these cells, time-lapse microscopy revealed that cytoplasmic membrane blebbing occurred simultaneously with the extrusion of nuclear content, which generated lamin B1-negative micronuclei during interphase. Furthermore, activation of cytoplasmic membrane blebbing by the addition of fresh serum or camptothecin induced nuclear budding within 1 to 10 minutes, which suggested that blebbing might be the cause of the budding. After the induction of blebbing, the frequency of lamin-negative micronuclei increased. The budding was most frequent during S phase and more efficiently entrapped small extrachromosomal chromatin than the large chromosome arm. Based on these results, we suggest a novel mechanism in which cytoplasmic membrane dynamics pulls the chromatin out of the nucleus through the lamina break. Evidence for such a mechanism was obtained in certain cancer cell lines including human COLO 320 and HeLa. The mechanism could significantly perturb the genome and influence cancer cell phenotypes.

  20. Genetics and breeding of brewers yeast

    Energy Technology Data Exchange (ETDEWEB)

    Nilsson-Tillgren, T.; Gjermansen, C.; Petersen, J.G.L.; Holmberg, S.; Kielland-Brandt, M.C.

    1984-01-01

    Yeasts, used for beer production, can be divided into two groups, top fermenters and bottom fermenters and Saccharomyces carlsbergensis has been chosen as the name for the bottom fermenting yeasts which are used in lager beer production. The key for the analysis of the chromosomes of Saccharomyces carlsbergensis was provided by the discovery that single chromosomes of meiotic segregants of these strains can be transferred to genetically marked Saccharomyces cerevisiae strains and subsequently analyzed by tetrad analysis and molecular hybridization. It is proposed that Saccharomyces carlsbergensis is composed of two rather divergent genotypes. Breeding can be accomplished by cross breeding and mutagenesis and possibly by introducing in vitro modified cloned genes into meiotic segregants of Saccharomyces carlsbergensis.

  1. Yeast Interacting Proteins Database: YMR124W, YLR031W [Yeast Interacting Proteins Database

    Lifescience Database Archive (English)

    Full Text Available in localizes to the cell periphery, cytoplasm, bud, and bud neck; interacts with Crm1p in two-hybrid assay; ... periphery, cytoplasm, bud, and bud neck; interacts with Crm1p in two-hybrid assay

  2. Mechanism of supercooling in flower bud of Camellia oleifea

    Institute of Scientific and Technical Information of China (English)

    苏维埃; 潘良文

    1995-01-01

    It is the first time for MRI to be used in the research of flower buds supercooling. Directobservation on freezing course of living flower buds of Camellia yuhsienensis by MRI and tissue browning test showed that freezing order of the flower organs is bud axis, scale, petal, pistil and stamen. It is coincident with the direction of ice development from bud axes to flower organs upwards. The corresponding results from MRI and freezing-fixation showed that the water translocation from flower organs to axes and scales is carried on in the course of bud freezing. ’H spectral measurement of NMR was used to follow the decrease of unfrozen water in the buds during the cooling.

  3. The size of the nucleus increases as yeast cells grow.

    Science.gov (United States)

    Jorgensen, Paul; Edgington, Nicholas P; Schneider, Brandt L; Rupes, Ivan; Tyers, Mike; Futcher, Bruce

    2007-09-01

    It is not known how the volume of the cell nucleus is set, nor how the ratio of nuclear volume to cell volume (N/C) is determined. Here, we have measured the size of the nucleus in growing cells of the budding yeast Saccharomyces cerevisiae. Analysis of mutant yeast strains spanning a range of cell sizes revealed that the ratio of average nuclear volume to average cell volume was quite consistent, with nuclear volume being approximately 7% that of cell volume. At the single cell level, nuclear and cell size were strongly correlated in growing wild-type cells, as determined by three different microscopic approaches. Even in G1-phase, nuclear volume grew, although it did not grow quite as fast as overall cell volume. DNA content did not appear to have any immediate, direct influence on nuclear size, in that nuclear size did not increase sharply during S-phase. The maintenance of nuclear size did not require continuous growth or ribosome biogenesis, as starvation and rapamycin treatment had little immediate impact on nuclear size. Blocking the nuclear export of new ribosomal subunits, among other proteins and RNAs, with leptomycin B also had no obvious effect on nuclear size. Nuclear expansion must now be factored into conceptual and mathematical models of budding yeast growth and division. These results raise questions as to the unknown force(s) that expand the nucleus as yeast cells grow.

  4. Lipid raft involvement in yeast cell growth and death.

    Science.gov (United States)

    Mollinedo, Faustino

    2012-01-01

    The notion that cellular membranes contain distinct microdomains, acting as scaffolds for signal transduction processes, has gained considerable momentum. In particular, a class of such domains that is rich in sphingolipids and cholesterol, termed as lipid rafts, is thought to compartmentalize the plasma membrane, and to have important roles in survival and cell death signaling in mammalian cells. Likewise, yeast lipid rafts are membrane domains enriched in sphingolipids and ergosterol, the yeast counterpart of mammalian cholesterol. Sterol-rich membrane domains have been identified in several fungal species, including the budding yeast Saccharomyces cerevisiae, the fission yeast Schizosaccharomyces pombe as well as the pathogens Candida albicans and Cryptococcus neoformans. Yeast rafts have been mainly involved in membrane trafficking, but increasing evidence implicates rafts in a wide range of additional cellular processes. Yeast lipid rafts house biologically important proteins involved in the proper function of yeast, such as proteins that control Na(+), K(+), and pH homeostasis, which influence many cellular processes, including cell growth and death. Membrane raft constituents affect drug susceptibility, and drugs interacting with sterols alter raft composition and membrane integrity, leading to yeast cell death. Because of the genetic tractability of yeast, analysis of yeast rafts could be an excellent model to approach unanswered questions of mammalian raft biology, and to understand the role of lipid rafts in the regulation of cell death and survival in human cells. A better insight in raft biology might lead to envisage new raft-mediated approaches to the treatment of human diseases where regulation of cell death and survival is critical, such as cancer and neurodegenerative diseases.

  5. Lipid raft involvement in yeast cell growth and death

    Directory of Open Access Journals (Sweden)

    Faustino eMollinedo

    2012-10-01

    Full Text Available The notion that cellular membranes contain distinct microdomains, acting as scaffolds for signal transduction processes, has gained considerable momentum. In particular, a class of such domains that is rich in sphingolipids and cholesterol, termed as lipid rafts, is thought to compartmentalize the plasma membrane, and to have important roles in survival and cell death signaling in mammalian cells. Likewise, yeast lipid rafts are membrane domains enriched in sphingolipids and ergosterol, the yeast counterpart of mammalian cholesterol. Sterol-rich membrane domains have been identified in several fungal species, including the budding yeast Saccharomyces cerevisiae, the fission yeast Schizosaccharomyces pombe as well as the pathogens Candida albicans and Crytococcus neoformans. Yeast rafts have been mainly involved in membrane trafficking, but increasing evidence implicates rafts in a wide range of additional cellular processes. Yeast lipid rafts house biologically important proteins involved in the proper function of yeast, such as proteins that control Na+, K+ and pH homeostasis, which influence many cellular processes, including cell growth and death. Membrane raft constituents affect drug susceptibility, and drugs interacting with sterols alter raft composition and membrane integrity, leading to yeast cell death. Because of the genetic tractability of yeast, analysis of yeast rafts could be an excellent model to approach unanswered questions of mammalian raft biology, and to understand the role of lipid rafts in the regulation of cell death and survival in human cells. A better insight in raft biology might lead to envisage new raft-mediated approaches to the treatment of human diseases where regulation of cell death and survival is critical, such as cancer and neurodegenerative diseases.

  6. 5'-end sequences of budding yeast full-length cDNA clones - Budding yeast cDNA sequencing project | LSDB Archive [Life Science Database Archive metadata

    Lifescience Database Archive (English)

    Full Text Available [ Credits ] BLAST Search Image Search Home About Archive Update History Contact us ...a entries 83,706 entries - Joomla SEF URLs by Artio About This Database Database Description Download License Update

  7. Genome sequence of the lager brewing yeast, an interspecies hybrid.

    Science.gov (United States)

    Nakao, Yoshihiro; Kanamori, Takeshi; Itoh, Takehiko; Kodama, Yukiko; Rainieri, Sandra; Nakamura, Norihisa; Shimonaga, Tomoko; Hattori, Masahira; Ashikari, Toshihiko

    2009-04-01

    This work presents the genome sequencing of the lager brewing yeast (Saccharomyces pastorianus) Weihenstephan 34/70, a strain widely used in lager beer brewing. The 25 Mb genome comprises two nuclear sub-genomes originating from Saccharomyces cerevisiae and Saccharomyces bayanus and one circular mitochondrial genome originating from S. bayanus. Thirty-six different types of chromosomes were found including eight chromosomes with translocations between the two sub-genomes, whose breakpoints are within the orthologous open reading frames. Several gene loci responsible for typical lager brewing yeast characteristics such as maltotriose uptake and sulfite production have been increased in number by chromosomal rearrangements. Despite an overall high degree of conservation of the synteny with S. cerevisiae and S. bayanus, the syntenies were not well conserved in the sub-telomeric regions that contain lager brewing yeast characteristic and specific genes. Deletion of larger chromosomal regions, a massive unilateral decrease of the ribosomal DNA cluster and bilateral truncations of over 60 genes reflect a post-hybridization evolution process. Truncations and deletions of less efficient maltose and maltotriose uptake genes may indicate the result of adaptation to brewing. The genome sequence of this interspecies hybrid yeast provides a new tool for better understanding of lager brewing yeast behavior in industrial beer production.

  8. Image processing and classification algorithm for yeast cell morphology in a microfluidic chip.

    Science.gov (United States)

    Yang Yu, Bo; Elbuken, Caglar; Ren, Carolyn L; Huissoon, Jan P

    2011-06-01

    The study of yeast cell morphology requires consistent identification of cell cycle phases based on cell bud size. A computer-based image processing algorithm is designed to automatically classify microscopic images of yeast cells in a microfluidic channel environment. The images were enhanced to reduce background noise, and a robust segmentation algorithm is developed to extract geometrical features including compactness, axis ratio, and bud size. The features are then used for classification, and the accuracy of various machine-learning classifiers is compared. The linear support vector machine, distance-based classification, and k-nearest-neighbor algorithm were the classifiers used in this experiment. The performance of the system under various illumination and focusing conditions were also tested. The results suggest it is possible to automatically classify yeast cells based on their morphological characteristics with noisy and low-contrast images.

  9. Regeneration of Blue Honeysuckle via Dormant Axillary Buds

    Institute of Scientific and Technical Information of China (English)

    QU Guiqin; HUANG Longshuang; HUO Junwei

    2008-01-01

    The optimum medium for dormant axillary buds culture of blue honeysuckle was screened according to the growth rate and elongation rate by inoculating the buds on culture medium with various 6-BA and iron-salt concentration. About 35 days, the stretched stem buds were divided into strong root system after inoculated on 1/2 MS+1.0 mg·L-1 IBA rooting medium. Amount of qualified tissue-cultured young plants could be obtained by the stretched stem buds reproduction.

  10. Development Correlations of the Buds of Grapevine (Vitis vinifera L.

    Directory of Open Access Journals (Sweden)

    Liliana ROTARU

    2010-06-01

    Full Text Available The development characteristics of different buds of the grapevine are mainly related by stimulation and/or inhibition effects, the action of which is still inexplicable. The present study examines the development dynamics of the buds of a one-year old branch after excision of different buds and the application of ?-naphtyl acetic acid (ANA, as well as the growth capacity of each bud individually. We verified the effects of acrotony cited previously by various researchers. These effects are due to different developmental characteristics of which could to lay the groundwork for the improvement of different productions methods.

  11. BioGRID: A Resource for Studying Biological Interactions in Yeast.

    Science.gov (United States)

    Oughtred, Rose; Chatr-aryamontri, Andrew; Breitkreutz, Bobby-Joe; Chang, Christie S; Rust, Jennifer M; Theesfeld, Chandra L; Heinicke, Sven; Breitkreutz, Ashton; Chen, Daici; Hirschman, Jodi; Kolas, Nadine; Livstone, Michael S; Nixon, Julie; O'Donnell, Lara; Ramage, Lindsay; Winter, Andrew; Reguly, Teresa; Sellam, Adnane; Stark, Chris; Boucher, Lorrie; Dolinski, Kara; Tyers, Mike

    2016-01-04

    The Biological General Repository for Interaction Datasets (BioGRID) is a freely available public database that provides the biological and biomedical research communities with curated protein and genetic interaction data. Structured experimental evidence codes, an intuitive search interface, and visualization tools enable the discovery of individual gene, protein, or biological network function. BioGRID houses interaction data for the major model organism species--including yeast, nematode, fly, zebrafish, mouse, and human--with particular emphasis on the budding yeast Saccharomyces cerevisiae and the fission yeast Schizosaccharomyces pombe as pioneer eukaryotic models for network biology. BioGRID has achieved comprehensive curation coverage of the entire literature for these two major yeast models, which is actively maintained through monthly curation updates. As of September 2015, BioGRID houses approximately 335,400 biological interactions for budding yeast and approximately 67,800 interactions for fission yeast. BioGRID also supports an integrated posttranslational modification (PTM) viewer that incorporates more than 20,100 yeast phosphorylation sites curated through its sister database, the PhosphoGRID. © 2016 Cold Spring Harbor Laboratory Press.

  12. Circular permutation of a synthetic eukaryotic chromosome with the telomerator

    Science.gov (United States)

    Mitchell, Leslie A.; Boeke, Jef D.

    2014-01-01

    Chromosome engineering is a major focus in the fields of systems biology, genetics, synthetic biology, and the functional analysis of genomes. Here, we describe the “telomerator,” a new synthetic biology device for use in Saccharomyces cerevisiae. The telomerator is designed to inducibly convert circular DNA molecules into mitotically stable, linear chromosomes replete with functional telomeres in vivo. The telomerator cassette encodes convergent yeast telomere seed sequences flanking the I-SceI homing endonuclease recognition site in the center of an intron artificially transplanted into the URA3 selectable/counterselectable auxotrophic marker. We show that inducible expression of the homing endonuclease efficiently generates linear molecules, identified by using a simple plate-based screening method. To showcase its functionality and utility, we use the telomerator to circularly permute a synthetic yeast chromosome originally constructed as a circular molecule, synIXR, to generate 51 linear variants. Many of the derived linear chromosomes confer unexpected phenotypic properties. This finding indicates that the telomerator offers a new way to study the effects of gene placement on chromosomes (i.e., telomere proximity). However, that the majority of synIXR linear derivatives support viability highlights inherent tolerance of S. cerevisiae to changes in gene order and overall chromosome structure. The telomerator serves as an important tool to construct artificial linear chromosomes in yeast; the concept can be extended to other eukaryotes. PMID:25378705

  13. Model selection emphasises the importance of non-chromosomal information in genetic studies.

    Directory of Open Access Journals (Sweden)

    Reda Rawi

    Full Text Available Ever since the case of the missing heritability was highlighted some years ago, scientists have been investigating various possible explanations for the issue. However, none of these explanations include non-chromosomal genetic information. Here we describe explicitly how chromosomal and non-chromosomal modifiers collectively influence the heritability of a trait, in this case, the growth rate of yeast. Our results show that the non-chromosomal contribution can be large, adding another dimension to the estimation of heritability. We also discovered, combining the strength of LASSO with model selection, that the interaction of chromosomal and non-chromosomal information is essential in describing phenotypes.

  14. Molecular mechanism of arenavirus assembly and budding.

    Science.gov (United States)

    Urata, Shuzo; Yasuda, Jiro

    2012-10-10

    Arenaviruses have a bisegmented negative-strand RNA genome, which encodes four viral proteins: GP and NP by the S segment and L and Z by the L segment. These four viral proteins possess multiple functions in infection, replication and release of progeny viruses from infected cells. The small RING finger protein, Z protein is a matrix protein that plays a central role in viral assembly and budding. Although all arenaviruses encode Z protein, amino acid sequence alignment showed a huge variety among the species, especially at the C-terminus where the L-domain is located. Recent publications have demonstrated the interactions between viral protein and viral protein, and viral protein and host cellular protein, which facilitate transportation and assembly of viral components to sites of virus egress. This review presents a summary of current knowledge regarding arenavirus assembly and budding, in comparison with other enveloped viruses. We also refer to the restriction of arenavirus production by the antiviral cellular factor, Tetherin/BST-2.

  15. Molecular Mechanism of Arenavirus Assembly and Budding

    Directory of Open Access Journals (Sweden)

    Shuzo Urata

    2012-10-01

    Full Text Available Arenaviruses have a bisegmented negative-strand RNA genome, which encodes four viral proteins: GP and NP by the S segment and L and Z by the L segment. These four viral proteins possess multiple functions in infection, replication and release of progeny viruses from infected cells. The small RING finger protein, Z protein is a matrix protein that plays a central role in viral assembly and budding. Although all arenaviruses encode Z protein, amino acid sequence alignment showed a huge variety among the species, especially at the C-terminus where the L-domain is located. Recent publications have demonstrated the interactions between viral protein and viral protein, and viral protein and host cellular protein, which facilitate transportation and assembly of viral components to sites of virus egress. This review presents a summary of current knowledge regarding arenavirus assembly and budding, in comparison with other enveloped viruses. We also refer to the restriction of arenavirus production by the antiviral cellular factor, Tetherin/BST-2.

  16. Tumor budding in upper gastrointestinal carcinomas

    Directory of Open Access Journals (Sweden)

    Viktor Hendrik Koelzer

    2014-08-01

    Full Text Available The basis of personalized medicine in oncology is the prediction of an individual’s risk of relapse and death from disease. The presence of tumor budding (TB at the tumor-host interface of gastrointestinal cancers has been recognized as a hallmark of unfavorable disease biology. TB is defined as the presence of dedifferentiated cells or small clusters of up to five cells at the tumor invasive front and can be observed in aggressive carcinomas of the esophagus, stomach, pancreas, ampulla, colon and rectum. Presence of TB reproducibly correlates with advanced tumor stage, frequent lymphovascular invasion, nodal and distant metastasis. The UICC has officially recognized TB as additional independent prognostic factor in cancers of the colon and rectum. Recent studies have also characterized TB as a promising prognostic indicator for clinical management of esophageal squamous cell carcinoma, adenocarcinoma of the gastro-esophageal junction and gastric adenocarcinoma. However, several important issues have to be addressed for application in daily diagnostic practice: 1 Validation of prognostic scoring systems for tumor budding in large, multi-center studies 2 Consensus on the optimal assessment method 3 Inter-observer reproducibility. This review provides a comprehensive analysis of TB in cancers of the upper gastrointestinal tract including critical appraisal of perspectives for further study.

  17. Hypermutability of damaged single-strand DNA formed at double-strand breaks and uncapped telomeres in yeast Saccharomyces cerevisiae.

    Directory of Open Access Journals (Sweden)

    Yong Yang

    2008-11-01

    Full Text Available The major DNA repair pathways operate on damage in double-strand DNA because they use the intact strand as a template after damage removal. Therefore, lesions in transient single-strand stretches of chromosomal DNA are expected to be especially threatening to genome stability. To test this hypothesis, we designed systems in budding yeast that could generate many kilobases of persistent single-strand DNA next to double-strand breaks or uncapped telomeres. The systems allowed controlled restoration to the double-strand state after applying DNA damage. We found that lesions induced by UV-light and methyl methanesulfonate can be tolerated in long single-strand regions and are hypermutagenic. The hypermutability required PCNA monoubiquitination and was largely attributable to translesion synthesis by the error-prone DNA polymerase zeta. In support of multiple lesions in single-strand DNA being a source of hypermutability, analysis of the UV-induced mutants revealed strong strand-specific bias and unexpectedly high frequency of alleles with widely separated multiple mutations scattered over several kilobases. Hypermutability and multiple mutations associated with lesions in transient stretches of long single-strand DNA may be a source of carcinogenesis and provide selective advantage in adaptive evolution.

  18. Hypermutability of Damaged Single-Strand DNA Formed at Double-Strand Breaks and Uncapped Telomeres in Yeast Saccharomyces cerevisiae

    Science.gov (United States)

    Yang, Yong; Sterling, Joan; Storici, Francesca; Resnick, Michael A.; Gordenin, Dmitry A.

    2008-01-01

    The major DNA repair pathways operate on damage in double-strand DNA because they use the intact strand as a template after damage removal. Therefore, lesions in transient single-strand stretches of chromosomal DNA are expected to be especially threatening to genome stability. To test this hypothesis, we designed systems in budding yeast that could generate many kilobases of persistent single-strand DNA next to double-strand breaks or uncapped telomeres. The systems allowed controlled restoration to the double-strand state after applying DNA damage. We found that lesions induced by UV-light and methyl methanesulfonate can be tolerated in long single-strand regions and are hypermutagenic. The hypermutability required PCNA monoubiquitination and was largely attributable to translesion synthesis by the error-prone DNA polymerase ζ. In support of multiple lesions in single-strand DNA being a source of hypermutability, analysis of the UV-induced mutants revealed strong strand-specific bias and unexpectedly high frequency of alleles with widely separated multiple mutations scattered over several kilobases. Hypermutability and multiple mutations associated with lesions in transient stretches of long single-strand DNA may be a source of carcinogenesis and provide selective advantage in adaptive evolution. PMID:19023402

  19. A sphingolipid-dependent diffusion barrier confines ER stress to the yeast mother cell

    Science.gov (United States)

    Clay, Lori; Caudron, Fabrice; Denoth-Lippuner, Annina; Boettcher, Barbara; Buvelot Frei, Stéphanie; Snapp, Erik Lee; Barral, Yves

    2014-01-01

    In many cell types, lateral diffusion barriers compartmentalize the plasma membrane and, at least in budding yeast, the endoplasmic reticulum (ER). However, the molecular nature of these barriers, their mode of action and their cellular functions are unclear. Here, we show that misfolded proteins of the ER remain confined into the mother compartment of budding yeast cells. Confinement required the formation of a lateral diffusion barrier in the form of a distinct domain of the ER-membrane at the bud neck, in a septin-, Bud1 GTPase- and sphingolipid-dependent manner. The sphingolipids, but not Bud1, also contributed to barrier formation in the outer membrane of the dividing nucleus. Barrier-dependent confinement of ER stress into the mother cell promoted aging. Together, our data clarify the physical nature of lateral diffusion barriers in the ER and establish the role of such barriers in the asymmetric segregation of proteotoxic misfolded proteins during cell division and aging. DOI: http://dx.doi.org/10.7554/eLife.01883.001 PMID:24843009

  20. Field and laboratory investigations of budding in the tetillid sponge Cinachyrella cavernosa

    Digital Repository Service at National Institute of Oceanography (India)

    Singh, A.; Thakur, N.L.

    regression analysis of physico-chemical factors revealed temperature as the most prominent factor regulating the intensity of budding. Based on size and morphology, three stages of sponge buds were defined. The production of buds was found to be asynchronous...

  1. Cell to cell signalling during vertebrate limb bud development

    NARCIS (Netherlands)

    Panman, Lia

    2004-01-01

    Communication between cells is essential during embryonic development. The vertebrate limb bud provides us a model to study signalling interactions between cells during patterning of embryonic tissues and organogenesis. In chapter 1 I give an introduction about limb bud development that is focussed

  2. Photosynthetic leaf area modulates tiller bud outgrowth in sorghum.

    Science.gov (United States)

    Kebrom, Tesfamichael H; Mullet, John E

    2015-08-01

    Shoot branches or tillers develop from axillary buds. The dormancy versus outgrowth fates of buds depends on genetic, environmental and hormonal signals. Defoliation inhibits bud outgrowth indicating the role of leaf-derived metabolic factors such as sucrose in bud outgrowth. In this study, the sensitivity of bud outgrowth to selective defoliation was investigated. At 6 d after planting (6 DAP), the first two leaves of sorghum were fully expanded and the third was partially emerged. Therefore, the leaves were selectively defoliated at 6 DAP and the length of the bud in the first leaf axil was measured at 8 DAP. Bud outgrowth was inhibited by defoliation of only 2 cm from the tip of the second leaf blade. The expression of dormancy and sucrose-starvation marker genes was up-regulated and cell cycle and sucrose-inducible genes was down-regulated during the first 24 h post-defoliation of the second leaf. At 48 h, the expression of these genes was similar to controls as the defoliated plant recovers. Our results demonstrate that small changes in photosynthetic leaf area affect the propensity of tiller buds for outgrowth. Therefore, variation in leaf area and photosynthetic activity should be included when integrating sucrose into models of shoot branching.

  3. Desiccation tolerance of dormant buds from selected Prunus species

    Science.gov (United States)

    Dormant buds of woody plant species present a convenient material for backing-up of germplasm in liquid nitrogen. Routinely, this type of material is used in long-term preservation of only a few species (e.g. apple and sour cherry). Cryopreservation procedures of dormant buds are species dependent, ...

  4. Development of Crystalline Peroxisomes in Methanol-Grown Cells of the Yeast Hansenula polymorpha and Its Relation to Environmental Conditions

    NARCIS (Netherlands)

    Veenhuis, M.; Dijken, J.P. van; Pilon, S.A.F.; Harder, W.

    1978-01-01

    The development of peroxisomes has been studied in cells of the yeast Hansenula polymorpha during growth on methanol in batch and chemostat cultures. During bud formation, new peroxisomes were generated by the separation of small peroxisomes from mature organelles in the mother cells. The number of

  5. Development of Crystalline Peroxisomes in Methanol-Grown Cells of the Yeast Hansenula polymorpha and Its Relation to Environmental Conditions

    NARCIS (Netherlands)

    Veenhuis, M.; Dijken, J.P. van; Pilon, S.A.F.; Harder, W.

    1978-01-01

    The development of peroxisomes has been studied in cells of the yeast Hansenula polymorpha during growth on methanol in batch and chemostat cultures. During bud formation, new peroxisomes were generated by the separation of small peroxisomes from mature organelles in the mother cells. The number of

  6. Decellularized Tooth Bud Scaffolds for Tooth Regeneration.

    Science.gov (United States)

    Zhang, W; Vazquez, B; Oreadi, D; Yelick, P C

    2017-01-01

    Whole tooth regeneration approaches currently are limited by our inability to bioengineer full-sized, living replacement teeth. Recently, decellularized organ scaffolds have shown promise for applications in regenerative medicine by providing a natural extracellular matrix environment that promotes cell attachment and tissue-specific differentiation leading to full-sized organ regeneration. We hypothesize that decellularized tooth buds (dTBs) created from unerupted porcine tooth buds (TBs) can be used to guide reseeded dental cell differentiation to form whole bioengineered teeth, thereby providing a potential off-the-shelf scaffold for whole tooth regeneration. Porcine TBs were harvested from discarded 6-mo-old pig jaws, and decellularized by successive sodium dodecyl sulfate/Triton-X cycles. Four types of replicate implants were used in this study: 1) acellular dTBs; 2) recellularized dTBs seeded with porcine dental epithelial cells, human dental pulp cells, and human umbilical vein endothelial cells (recell-dTBs); 3) dTBs seeded with bone morphogenetic protein (BMP)-2 (dTB-BMPs); and 4) freshly isolated nondecellularized natural TBs (nTBs). Replicate samples were implanted into the mandibles of host Yucatan mini-pigs and grown for 3 or 6 mo. Harvested mandibles with implanted TB constructs were fixed in formalin, decalcified, embedded in paraffin, sectioned, and analyzed via histological methods. Micro-computed tomography (CT) analysis was performed on harvested 6-mo samples prior to decalcification. All harvested constructs exhibited a high degree of cellularity. Significant production of organized dentin and enamel-like tissues was observed in dTB-recell and nTB implants, but not in dTB or dTB-BMP implants. Micro-CT analyses of 6-mo implants showed the formation of organized, bioengineered teeth of comparable size to natural teeth. To our knowledge, these results are the first to describe the potential use of dTBs for functional whole tooth regeneration.

  7. Yeast That Smell

    Directory of Open Access Journals (Sweden)

    Eugenia Y Xu

    2008-08-01

    Full Text Available The fundamental mechanism of olfactory receptor activation has been conserved from yeast to humans. Engineered yeast cells can smell some of the same odorants as humans can, which makes yeast an ideal model system for studying human olfaction. Furthermore, if engineered yeast cells are incorporated into sensory arrays, they can be used as biosensors or artificial noses.Keywords: Yeast, olfactory receptor, G protein-coupled receptor, biosensor, smellReceived: 31 July 2008 / Received in revised form: 6 August 2008, Accepted: 13 August 2008, Published online: 17 August 2008

  8. Recent advances in yeast molecular biology: recombinant DNA. [Lead abstract

    Energy Technology Data Exchange (ETDEWEB)

    1982-09-01

    Separate abstracts were prepared for the 25 papers presented at a workshop focusing on chromosomal structure, gene regulation, recombination, DNA repair, and cell type control, that have been obtained by experimental approaches incorporating the new technologies of yeast DNA transformation, molecular cloning, and DNA sequence analysis. (KRM)

  9. A Predictive Model for Yeast Cell Polarization in Pheromone Gradients.

    Science.gov (United States)

    Muller, Nicolas; Piel, Matthieu; Calvez, Vincent; Voituriez, Raphaël; Gonçalves-Sá, Joana; Guo, Chin-Lin; Jiang, Xingyu; Murray, Andrew; Meunier, Nicolas

    2016-04-01

    Budding yeast cells exist in two mating types, a and α, which use peptide pheromones to communicate with each other during mating. Mating depends on the ability of cells to polarize up pheromone gradients, but cells also respond to spatially uniform fields of pheromone by polarizing along a single axis. We used quantitative measurements of the response of a cells to α-factor to produce a predictive model of yeast polarization towards a pheromone gradient. We found that cells make a sharp transition between budding cycles and mating induced polarization and that they detect pheromone gradients accurately only over a narrow range of pheromone concentrations corresponding to this transition. We fit all the parameters of the mathematical model by using quantitative data on spontaneous polarization in uniform pheromone concentration. Once these parameters have been computed, and without any further fit, our model quantitatively predicts the yeast cell response to pheromone gradient providing an important step toward understanding how cells communicate with each other.

  10. A permeability barrier surrounds taste buds in lingual epithelia.

    Science.gov (United States)

    Dando, Robin; Pereira, Elizabeth; Kurian, Mani; Barro-Soria, Rene; Chaudhari, Nirupa; Roper, Stephen D

    2015-01-01

    Epithelial tissues are characterized by specialized cell-cell junctions, typically localized to the apical regions of cells. These junctions are formed by interacting membrane proteins and by cytoskeletal and extracellular matrix components. Within the lingual epithelium, tight junctions join the apical tips of the gustatory sensory cells in taste buds. These junctions constitute a selective barrier that limits penetration of chemosensory stimuli into taste buds (Michlig et al. J Comp Neurol 502: 1003-1011, 2007). We tested the ability of chemical compounds to permeate into sensory end organs in the lingual epithelium. Our findings reveal a robust barrier that surrounds the entire body of taste buds, not limited to the apical tight junctions. This barrier prevents penetration of many, but not all, compounds, whether they are applied topically, injected into the parenchyma of the tongue, or circulating in the blood supply, into taste buds. Enzymatic treatments indicate that this barrier likely includes glycosaminoglycans, as it was disrupted by chondroitinase but, less effectively, by proteases. The barrier surrounding taste buds could also be disrupted by brief treatment of lingual tissue samples with DMSO. Brief exposure of lingual slices to DMSO did not affect the ability of taste buds within the slice to respond to chemical stimulation. The existence of a highly impermeable barrier surrounding taste buds and methods to break through this barrier may be relevant to basic research and to clinical treatments of taste.

  11. Mitochondrial localization of fission yeast manganese superoxide dismutase is required for its lysine acetylation and for cellular stress resistance and respiratory growth

    Energy Technology Data Exchange (ETDEWEB)

    Takahashi, Hidekazu, E-mail: hidetakahashi@riken.jp [Chemical Genetics Laboratory/Chemical Genomics Research Group, RIKEN Advanced Science Institute, Wako, Saitama 351-0198 (Japan); Suzuki, Takehiro [Biomolecular Characterization Team, RIKEN Advanced Science Institute, Wako, Saitama 351-0198 (Japan); CREST Research Project, Japan Science and Technology Corporation, Kawaguchi, Saitama 332-0012 (Japan); Shirai, Atsuko; Matsuyama, Akihisa [Chemical Genetics Laboratory/Chemical Genomics Research Group, RIKEN Advanced Science Institute, Wako, Saitama 351-0198 (Japan); Dohmae, Naoshi [Biomolecular Characterization Team, RIKEN Advanced Science Institute, Wako, Saitama 351-0198 (Japan); CREST Research Project, Japan Science and Technology Corporation, Kawaguchi, Saitama 332-0012 (Japan); Yoshida, Minoru, E-mail: yoshidam@riken.jp [Chemical Genetics Laboratory/Chemical Genomics Research Group, RIKEN Advanced Science Institute, Wako, Saitama 351-0198 (Japan); CREST Research Project, Japan Science and Technology Corporation, Kawaguchi, Saitama 332-0012 (Japan)

    2011-03-04

    Research highlights: {yields} Fission yeast manganese superoxide dismutase (MnSOD) is acetylated. {yields} The mitochondrial targeting sequence (MTS) is required for the acetylation of MnSOD. {yields} The MTS is not crucial for MnSOD activity, but is important for respiratory growth. {yields} Posttranslational regulation of MnSOD differs between budding and fission yeast. -- Abstract: Manganese-dependent superoxide dismutase (MnSOD) is localized in the mitochondria and is important for oxidative stress resistance. Although transcriptional regulation of MnSOD has been relatively well studied, much less is known about the protein's posttranslational regulation. In budding yeast, MnSOD is activated after mitochondrial import by manganese ion incorporation. Here we characterize posttranslational modification of MnSOD in the fission yeast Schizosaccharomyces pombe. Fission yeast MnSOD is acetylated at the 25th lysine residue. This acetylation was diminished by deletion of N-terminal mitochondrial targeting sequence, suggesting that MnSOD is acetylated after import into mitochondria. Mitochondrial localization of MnSOD is not essential for the enzyme activity, but is crucial for oxidative stress resistance and growth under respiratory conditions of fission yeast. These results suggest that, unlike the situation in budding yeast, S. pombe MnSOD is already active even before mitochondrial localization; nonetheless, mitochondrial localization is critical to allow the cell to cope with reactive oxygen species generated inside or outside of mitochondria.

  12. Physical mapping of the split hand/split foot (SHSF) locus on chromosome 7 reveals a relationship between SHSF and the syndromic ectrodactylies

    Energy Technology Data Exchange (ETDEWEB)

    Poorkaj, P.; Nunes, M.E.; Geshuri, D. [Univ. of Washington, Seattle, WA (United States)] [and others

    1994-09-01

    Split hand/split foot (also knows as ectrodactyly) is a human developmental malformation characterized by missing digits and claw-like extremities. An autosomal dominant form of this disorder has been mapped to 7q21.3-q22.1 on the basis of SHSF-associated chromosomal rearrangements: this locus has been designated SHFD1. We have constructed a physical map of the SHFD1 region that consists of contiguous yeast artificial chromosome clones and spans approximately 8 Mb. Somatic cell hybrid and fluorescent in situ hybridization analyses were used to define SHSF-associated chromosomal breakpoints in fourteen patients. A critical interval of about 1 Mb was established for SHFD1 by analysis of six patients with deletions. Translocation and inversion breakpoints in seven other patients were found to localize within a 500-700 kb interval within the critical region. Several candidate genes including DLX5 and DLX6 (members of the Drosophilia Distal-less homeobox-containing gene family) localize to this region. At least four of these genes are expressed in the developing mouse limb bud. Of particular interest is the observation that 8 of the 14 patients studied have syndromic ectrodactyly, which is characterized by the association of SHSF with a variety of other anomalies including cleft lip/palate, ectodermal dysplasia, and renal anomalies. Thus, these data implicate a single gene or cluster of genes at the SHFD1 locus in a wide range of developmental processes and serve to establish a molecular genetic relationship between simple SHSF and a broad group of human birth defects.

  13. Assignment of ten DNA repair genes from Schizosaccharomyces pombe to chromosomal NotI restriction fragments

    NARCIS (Netherlands)

    B.C. Broughton; N.C. Barbet; J. Murray (Johanne); F.Z. Watts (Felicity); M.H.M. Koken (Marcel); A.R. Lehmann (Alan); A.M. Carr (Anthony)

    1991-01-01

    textabstractTen DNA repair (rad) genes from the fission yeast, Schizosaccharomyces pombe were mapped to the 17 NotI fragments of the three chromosomes. Nine of the genes map to chromosome I, but there is no evidence for significant clustering.

  14. The electrophoretic banding pattern of the chromosomes of Pichia stipitis and Candida shehatae.

    Science.gov (United States)

    Passoth, V; Hansen, M; Klinner, U; Emeis, C C

    1992-11-01

    The electrophoretic karyotype of fu1ur strains of P. stipitis and five strains of C. shehatae were compared by means of OFAGE and TAFE techniques. Although the number of chromosomal bands was six in all cases except one, P. stipitis revealed a clearly distinct pattern in comparison to C. shehatae. Both yeasts showed remarkable chromosome length polymorphism.

  15. Genotyping 1000 yeast strains by next-generation sequencing

    Directory of Open Access Journals (Sweden)

    Wilkening Stefan

    2013-02-01

    Full Text Available Abstract Background The throughput of next-generation sequencing machines has increased dramatically over the last few years; yet the cost and time for library preparation have not changed proportionally, thus representing the main bottleneck for sequencing large numbers of samples. Here we present an economical, high-throughput library preparation method for the Illumina platform, comprising a 96-well based method for DNA isolation for yeast cells, a low-cost DNA shearing alternative, and adapter ligation using heat inactivation of enzymes instead of bead cleanups. Results Up to 384 whole-genome libraries can be prepared from yeast cells in one week using this method, for less than 15 euros per sample. We demonstrate the robustness of this protocol by sequencing over 1000 yeast genomes at ~30x coverage. The sequence information from 768 yeast segregants derived from two divergent S. cerevisiae strains was used to generate a meiotic recombination map at unprecedented resolution. Comparisons to other datasets indicate a high conservation of recombination at a chromosome-wide scale, but differences at the local scale. Additionally, we detected a high degree of aneuploidy (3.6% by examining the sequencing coverage in these segregants. Differences in allele frequency allowed us to attribute instances of aneuploidy to gains of chromosomes during meiosis or mitosis, both of which showed a strong tendency to missegregate specific chromosomes. Conclusions Here we present a high throughput workflow to sequence genomes of large number of yeast strains at a low price. We have used this workflow to obtain recombination and aneuploidy data from hundreds of segregants, which can serve as a foundation for future studies of linkage, recombination, and chromosomal aberrations in yeast and higher eukaryotes.

  16. Checkpoint genes and Exo1 regulate nearby inverted repeat fusions that form dicentric chromosomes in Saccharomyces cerevisiae.

    Science.gov (United States)

    Kaochar, Salma; Shanks, Lisa; Weinert, Ted

    2010-12-14

    Genomic rearrangements are common, occur by largely unknown mechanisms, and can lead to human diseases. We previously demonstrated that some genome rearrangements occur in budding yeast through the fusion of two DNA sequences that contain limited sequence homology, lie in inverted orientation, and are within 5 kb of one another. This inverted repeat fusion reaction forms dicentric chromosomes, which are well-known intermediates to additional rearrangements. We have previously provided evidence indicating that an error of stalled or disrupted DNA replication forks can cause inverted repeat fusion. Here we analyze how checkpoint protein regulatory pathways known to stabilize stalled forks affect this form of instability. We find that two checkpoint pathways suppress inverted repeat fusion, and that their activities are distinguishable by their interactions with exonuclease 1 (Exo1). The checkpoint kinase Rad53 (Chk2) and recombination protein complex MRX(MRN) inhibit Exo1 in one pathway, whereas in a second pathway the ATR-like kinases Mec1 and Tel1, adaptor protein Rad9, and effector kinases Chk1 and Dun1 act independently of Exo1 to prevent inverted repeat fusion. We provide a model that indicates how in Rad53 or MRX mutants, an inappropriately active Exo1 may facilitate faulty template switching between nearby inverted repeats to form dicentric chromosomes. We further investigate the role of Rad53, using hypomorphic alleles of Rad53 and null mutations in Rad9 and Mrc1, and provide evidence that only local, as opposed to global, activity of Rad53 is sufficient to prevent inverted repeat fusion.

  17. CHROMOSOME ABNORMALITIES IN INFERTILITY

    Directory of Open Access Journals (Sweden)

    Mateja Smogavec

    2009-08-01

    Conclusions Chromosomal analysis is an important method in diagnostic procedures of infertility, because chromosomal abnormalities could play the important role in etiology of infertility and are more frequently detected in this group of patients compared to general population. In the infertile couples balanced chromosomal abnormalities are the main cause of spontaneous abortions. Sex chromosome aneuploidies are highly correlated to infertility of females and males.

  18. Recent advances in yeast organelle and membrane proteomics.

    Science.gov (United States)

    Premsler, Thomas; Zahedi, René Peiman; Lewandrowski, Urs; Sickmann, Albert

    2009-10-01

    Yeast proteome research comprises two different aspects: with respect to systemic fungal infections (fungemias), invasive candidiasis, for instance by Candida albicans, is among the most common causes of morbidity and mortality particularly in the expanding population of immunocompromised patients, which rises a high medical and pharmaceutical interest in this facultative pathogenic organism. Apart from its clinical relevance, yeast research moreover provides an indispensable source of knowledge regarding fundamental biochemical processes of eukaryotic cells. In this context, the budding yeast Saccharomyces cerevisiae is, in addition to its multiple industrial applications, one of the most extensively used microorganisms and serves as the best understood eukaryotic model system so far. Consequently, numerous studies have focused on gaining insight into the yeast proteome, with protein MS providing a very efficient technology to cope with this task since it enables both protein identification and differential quantification of cellular material. In this review we present an overview of recent advances in yeast organelle and membrane proteomics focusing on the cell wall, plasma membrane, mitochondria and vacuole.

  19. CRISPR-PCS: a powerful new approach to inducing multiple chromosome splitting in Saccharomyces cerevisiae

    Science.gov (United States)

    Sasano, Yu; Nagasawa, Koki; Kaboli, Saeed; Sugiyama, Minetaka; Harashima, Satoshi

    2016-01-01

    PCR-mediated chromosome splitting (PCS) was developed in the yeast Saccharomyces cerevisiae. It is based on homologous recombination and enables division of a chromosome at any point to form two derived and functional chromosomes. However, because of low homologous recombination activity, PCS is limited to a single site at a time, which makes the splitting of multiple loci laborious and time-consuming. Here we have developed a highly efficient and versatile chromosome engineering technology named CRISPR-PCS that integrates PCS with the novel genome editing CRISPR/Cas9 system. This integration allows PCS to utilize induced double strand breaks to activate homologous recombination. CRISPR-PCS enhances the efficiency of chromosome splitting approximately 200-fold and enables generation of simultaneous multiple chromosome splits. We propose that CRISPR-PCS will be a powerful tool for breeding novel yeast strains with desirable traits for specific industrial applications and for investigating genome function. PMID:27530680

  20. Isolation and characterization of DNA probes for human chromosome 21.

    Science.gov (United States)

    Watkins, P C

    1990-01-01

    A coordinated effort to map and sequence the human genome has recently become a national priority. Chromosome 21, the smallest human chromosome accounting for less than 2% of the human genome, is an attractive model system for developing and evaluating genome mapping technology. Several strategies are currently being explored including the development of chromosome 21 libraries from somatic cell hybrids as reported here, the cloning of chromosome 21 in yeast artificial chromosomes (McCormick et al., 1989b), and the construction of chromosome 21 libraries using chromosome flow-sorting techniques (Fuscoe et al., 1989). This report describes the approaches used to identify DNA probes that are useful for mapping chromosome 21. Probes were successfully isolated from both phage and cosmid libraries made from two somatic cell hybrids that contain human chromosome 21 as the only human chromosome. The 15 cosmid clones from the WA17 library, reduced to cloned DNA sequences of an average size of 3 kb, total 525 kb of DNA which is approximately 1% of chromosome 21. From these clones, a set of polymorphic DNA markers that span the length of the long arm of chromosome 21 has been generated. All of the probes thus far analyzed from the WA17 libraries have been mapped to chromosome 21 both by physical and genetic mapping methods. It is therefore likely that the WA17 hybrid cell line contains human chromosome 21 as the only human component, in agreement with cytogenetic observation. The 153E7b cosmid libraries will provide an alternative source of cloned chromosome 21 DNA. Library screening techniques can be employed to obtain cloned DNA sequences from the same genetic loci of the two different chromosome 21s. Comparative analysis will allow direct estimation of DNA sequence variation for different regions of chromosome 21. Mapped DNA probes make possible the molecular analysis of chromosome 21 at a level of resolution not achievable by classical cytogenetic techniques (Graw et al

  1. Nutrient sensing and TOR signaling in yeast and mammals.

    Science.gov (United States)

    González, Asier; Hall, Michael N

    2017-02-15

    Coordinating cell growth with nutrient availability is critical for cell survival. The evolutionarily conserved TOR (target of rapamycin) controls cell growth in response to nutrients, in particular amino acids. As a central controller of cell growth, mTOR (mammalian TOR) is implicated in several disorders, including cancer, obesity, and diabetes. Here, we review how nutrient availability is sensed and transduced to TOR in budding yeast and mammals. A better understanding of how nutrient availability is transduced to TOR may allow novel strategies in the treatment for mTOR-related diseases. © 2017 The Authors.

  2. Cryptococcus friedmannii, a new species of yeast from the Antarctic

    Science.gov (United States)

    Vishniac, H. S.

    1985-01-01

    Cryptococcus friedmannii Vishniac sp. nov. from an Antarctic cryptoendolithic community is a psychrophilic basidioblastomycete characterized by cream-colored colonies of cells with smooth, layered walls, budding monopolarly, producing amylose and extracellular proteinase, utilizing nitrate and D-alanine (inter alia) as nitrogen sources and L-arabinose, arbutin, cellobiose, D-glucuronate, maltose, melezitose, salicin, soluble starch, trehalose, and D-xylose as carbon sources. This species differs from all other basidiomycetous yeasts in possessing the following combination of characters: amylose production (positive), assimilation of cellobiose (positive), D-galactose (negative), myo-inositol (negative), D-mannitol (negative), and sucrose (negative).

  3. ORDERING OF MARKERS IN THE PERICENTROMERIC REGION OF CHROMOSOME-10

    NARCIS (Netherlands)

    HOFSTRA, RMW; STELWAGEN, T; PASINI, B; VANDERVEEN, AY; PONDER, BAJ; NAKAMURA, Y; ROMEO, G; BUYS, CHCM

    Seven polymorphic cosmids previously assigned to 10cen-q11.2 were mapped between D10S34 and RBP3, and ordered by interphase in situ hybridization and yeast artificial chromosome analysis. Some of the presumed unique sequences from the centromeric region have homologies either within the same region

  4. Micropropagation of Helleborus through axillary budding.

    Science.gov (United States)

    Beruto, Margherita; Viglione, Serena; Bisignano, Alessandro

    2013-01-01

    Helleborus genus, belonging to the Ranunculaceae family, has 20 species of herbaceous perennial flowering plants. The commercial exploitation of this plant is dependent on the selection and propagation of appropriate lines. High propagation rate could be accomplished by using a suitable tissue culture method enabling the rapid introduction of valuable selections in the market. However, in vitro cultivation of Helleborus is still very difficult. Thereby the development of reliable in vitro propagation procedures is crucial for future production systems. Axillary buds cultured on agar-solidified Murashige and Skoog medium supplemented with 1 mg/L benzyladenine, 0.1 mg/L β-naphthoxyacetic acid, and 2 mg/L isopentenyl adenine develop shoots after 16 weeks of culture under 16 h light regime, 50-60 μmol/s/m(2), and 19 ± 1°C. The multiplication rate ranges from 1.4 to 2.1. However, the genotype and the number of subcultures affect the efficiency of the micropropagation process. The rooting of shoots is about 80% in solidified MS medium containing 1 mg/L 1-naphthaleneacetic acid and 3 mg/L indole-3-butyric acid. The described protocol provides information which can contribute to the commercial production of Helleborus plants.

  5. Flower Bud Differentiation in Quercus suber L.

    Directory of Open Access Journals (Sweden)

    Maria Carolina Varela

    2016-06-01

    Full Text Available Background and Purpose: Cork oak (Quercus suber L. is one of the most important forest species growing in the Western Mediterranean region. This investigation intends to assess the timing of flowering differentiation of cork oak and contribute to the deepening of the knowledge about the process of the sexual reproduction of the species. Materials and Methods: In 2010 four trees were selected (9, 14, 24, 25 from a plot of 25 trees located at Quinta da Serra, Portugal. A total of 240 buds were collected from these four trees, on three days (8, 14 and 23 March, from 4 branches per tree and 5 positions per branch for the assessment of meristem differentiation. Results: Meristem differentiation analysed on the sampling days revealed there were only vegetative structures by 8 March; a few male and female primordia on 14 March; and fully differentiated reproductive structures on 23 March. Conclusions: Flowering sex determination of cork oak occurs about one month before the flowering onset.

  6. Phase Transition and dissipation driven budding in lipid vesicles

    CERN Document Server

    Franke, Thomas; Wixforth, Achim; Dan, Nily; Schneider, Matthias F

    2013-01-01

    Membrane budding has been extensively studied as an equilibrium process attributed to the formation of coexisting domains or changes in the vesicle area to volume ratio (reduced volume). In contrast, non-equilibrium budding remains experimentally widely unexplored especially when time scales fall well below the characteristic diffusion time of lipids{\\tau} . We show that localized mechanical perturbations, initiated by driving giant unilamellar vesicles (GUVs) through their lipid phase transition, leads to the immediate formation of rapidly growing, multiply localized, non-equilibrium buds, when the transition takes place at short timescales (<{\\tau}). We show that these buds arise from small fluid-like perturbations and grow as spherical caps in the third dimension, since in plane spreading is obstructed by the continuous rigid gel-like matrix. Accounting for both three and two dimensional viscosity, we demonstrate that dissipation decreases the size scale of the system and therefore favours the formation...

  7. Grapevine bud break prediction for cool winter climates

    Science.gov (United States)

    Nendel, Claas

    2010-05-01

    Statistical analysis of bud break data for grapevine ( Vitis vinifera L. cvs. Riesling and Müller-Thurgau) at 13 sites along the northern boundary of commercial grapevine production in Europe revealed that, for all investigated sites, the heat summation method for bud break prediction can be improved if the starting date for the accumulation of heat units is specifically determined. Using the coefficient of variance as a criterion, a global minimum for each site can be identified, marking the optimum starting date. Furthermore, it was shown that the application of a threshold temperature for the heat summation method does not lead to an improved prediction of bud break. Using site-specific parameters, bud break of grapevine can be predicted with an accuracy of ± 2.5 days. Using average parameters, the prediction accuracy is reduced to ± 4.5 days, highlighting the sensitivity of the heat summation method to the quality and the representativeness of the driving temperature data.

  8. Factors influencing axillary shoot proliferation and adventitious budding in cedar.

    Science.gov (United States)

    Renau-Morata, Begoña; Ollero, Javier; Arrillaga, Isabel; Segura, Juan

    2005-04-01

    We developed procedures for in vitro cloning of Cedrus atlantica Manetti and C. libani A. Rich explants from juvenile and mature plants. Explant size was one determinant of the frequency of axillary bud break in both species. Shoot tips and nodal explants mainly developed calli, whereas bud sprouting occurred in defoliated microcuttings cultured on a modified Murashige and Skoog medium without growth regulators. Isolation and continuous subculture of sprouted buds on the same medium allowed cloning of microcuttings from C. atlantica and C. libani seedlings and bicentennial C. libani trees, thus providing a desirable alternative for multiplying mature trees that have demonstrated superior characteristics. We also report adventitious bud differentiation from isolated embryos of C. atlantica. Neither auxin treatments nor other methods tested, including infection with Agrobacterium rhizogenes, were effective in inducing root initiation.

  9. Functional cell types in taste buds have distinct longevities.

    Directory of Open Access Journals (Sweden)

    Isabel Perea-Martinez

    Full Text Available Taste buds are clusters of polarized sensory cells embedded in stratified oral epithelium. In adult mammals, taste buds turn over continuously and are replenished through the birth of new cells in the basal layer of the surrounding non-sensory epithelium. The half-life of cells in mammalian taste buds has been estimated as 8-12 days on average. Yet, earlier studies did not address whether the now well-defined functional taste bud cell types all exhibit the same lifetime. We employed a recently developed thymidine analog, 5-ethynil-2'-deoxyuridine (EdU to re-evaluate the incorporation of newly born cells into circumvallate taste buds of adult mice. By combining EdU-labeling with immunostaining for selected markers, we tracked the differentiation and lifespan of the constituent cell types of taste buds. EdU was primarily incorporated into basal extragemmal cells, the principal source for replenishing taste bud cells. Undifferentiated EdU-labeled cells began migrating into circumvallate taste buds within 1 day of their birth. Type II (Receptor taste cells began to differentiate from EdU-labeled precursors beginning 2 days after birth and then were eliminated with a half-life of 8 days. Type III (Presynaptic taste cells began differentiating after a delay of 3 days after EdU-labeling, and they survived much longer, with a half-life of 22 days. We also scored taste bud cells that belong to neither Type II nor Type III, a heterogeneous group that includes mostly Type I cells, and also undifferentiated or immature cells. A non-linear decay fit described these cells as two sub-populations with half-lives of 8 and 24 days respectively. Our data suggest that many post-mitotic cells may remain quiescent within taste buds before differentiating into mature taste cells. A small number of slow-cycling cells may also exist within the perimeter of the taste bud. Based on their incidence, we hypothesize that these may be progenitors for Type III cells.

  10. Bioprofiling of Salicaceae bud extracts through high-performance thin-layer chromatography hyphenated to biochemical, microbiological and chemical detections.

    Science.gov (United States)

    Hage, Salim; Morlock, Gertrud E

    2017-03-24

    The buds of poplars (Populus L.) and willows (Salix L.), both from the same family (Salicaceae Mirbel), are increasingly used in gemmotherapy and importantly contribute to the production of the physiologically active propolis by European bee Apis mellifera L. In order to study their phenolic profiles, polar extracts of buds from P. nigra L. were compared to those of P. alba L. and S. alba L. through high-performance thin-layer chromatography (HPTLC). Five chemotypical patterns were distinguished after derivatisation with the Natural Product reagent and confirmed by principal component analysis. The HPTLC analysis was directly hyphenated to various microbiological and biochemical assays as well as spectrometric techniques, directly linking to active molecules in the chromatograms. At a glance, polyvalent compounds were evident when all derivatisation and activity assays, to which HPTLC was hyphenated at ease, were combined together. In Populus buds, at least three antimicrobial compound zones were detected using Aliivibrio fischeri and Bacillus subtilis bioassays, and one phyto-œstrogen with the planar yeast œstrogen screen. In all samples, several inhibitors of acetyl- and butyrylcholinesterase and rabbit liver esterase were detected. Hyphenation to high resolution mass spectrometry supported the assignment of bioactive compounds, as shown for chrysin as selective cholinesterase inhibitor as well as caffeic acid and galangin as antimicrobials in P. nigra and P. alba. This fast and cost-efficient method can be appropriately extended and applied to the botanical origin determination and quality control of bud extracts and propolis samples. Copyright © 2017 Elsevier B.V. All rights reserved.

  11. Longleaf pine bud development: influence of seedling nutrition

    Science.gov (United States)

    J. P. Barnett; D. P. Jackson; R. K. Dumroese

    2010-01-01

    A subset of seedlings from a larger study (Jackson and others 2006, 2007) were selected and evaluated for two growing seasons to relate bud development, and root-collar diameter (RCD), and height growth with three nursery fertilization rates. We chose seedlings in the 0.5 (lowest), 2.0 (mid-range), and 4.0 (highest) mg of nitrogen per seedling treatments. Buds moved...

  12. A buckling mechanism for ESCRT-III budding

    CERN Document Server

    Lenz, Martin; Joanny, Jean-François

    2009-01-01

    The ESCRT-III protein complex binds to the membrane of eukaryotic cells, causing it to bud into long tubes. Here we propose that this budding is akin to a buckling instability. We analyze the linear stability of flat ESCRT-III-dressed membranes and account for the formation of long tubes. We study strongly deformed dressed membranes and their bifurcation diagram numerically. Our mechanism is compatible with reasonable in vivo parameter values and we propose an experiment allowing its validation.

  13. Centromeric DNA Facilitates Nonconventional Yeast Genetic Engineering.

    Science.gov (United States)

    Cao, Mingfeng; Gao, Meirong; Lopez-Garcia, Carmen Lorena; Wu, Yutong; Seetharam, Arun Somwarpet; Severin, Andrew Josef; Shao, Zengyi

    2017-08-18

    Many nonconventional yeast species have highly desirable features that are not possessed by model yeasts, despite that significant technology hurdles to effectively manipulate them lay in front. Scheffersomyces stipitis is one of the most important exemplary nonconventional yeasts in biorenewables industry, which has a high native xylose utilization capacity. Recent study suggested its much better potential than Saccharomyces cerevisiae as a well-suited microbial biomanufacturing platform for producing high-value compounds derived from shikimate pathway, many of which are associated with potent nutraceutical or pharmaceutical properties. However, the broad application of S. stipitis is hampered by the lack of stable episomal expression platforms and precise genome-editing tools. Here we report the success in pinpointing the centromeric DNA as the partitioning element to guarantee stable extra-chromosomal DNA segregation. The identified centromeric sequence not only stabilized episomal plasmid, enabled homogeneous gene expression, increased the titer of a commercially relevant compound by 3-fold, and also dramatically increased gene knockout efficiency from <1% to more than 80% with the expression of CRISPR components on the new stable plasmid. This study elucidated that establishment of a stable minichromosome-like expression platform is key to achieving functional modifications of nonconventional yeast species in order to expand the current collection of microbial factories.

  14. Valsts policijas budžeta analīze

    OpenAIRE

    Spūle, Lāsma

    2014-01-01

    Valsts policijas pamatbudžeta strukturālais sadalījums ir neatņemama Iekšlietu ministrijas kopējā budžeta sastāvdaļa. Ikviena plānošanas darbība ietekmē kopējā budžeta izstrādi, lai atspoguļotu valsts budžeta iestāžu darbību, tika analizēta Valsts policijas budžeta struktūra, tā sastādīšanas un izpildes nepilnības. Maģistra darba mērķis ir analizēt Valsts policijas budžetu, ar tā veidošanu un izlietošanu saistītās problēmas, kā arī izstrādāt priekšlikumus Valsts policijas budžeta procesa u...

  15. HSC90 is required for nascent hepatitis C virus core protein stability in yeast cells.

    Science.gov (United States)

    Kubota, Naoko; Inayoshi, Yasutaka; Satoh, Naoko; Fukuda, Takashi; Iwai, Kenta; Tomoda, Hiroshi; Kohara, Michinori; Kataoka, Kazuhiro; Shimamoto, Akira; Furuichi, Yasuhiro; Nomoto, Akio; Naganuma, Akira; Kuge, Shusuke

    2012-07-30

    Hepatitis C virus core protein (Core) contributes to HCV pathogenicity. Here, we demonstrate that Core impairs growth in budding yeast. We identify HSP90 inhibitors as compounds that reduce intracellular Core protein level and restore yeast growth. Our results suggest that HSC90 (Hsc82) may function in the protection of the nascent Core polypeptide against degradation in yeast and the C-terminal region of Core corresponding to the organelle-interaction domain was responsible for Hsc82-dependent stability. The yeast system may be utilized to select compounds that can direct the C-terminal region to reduce the stability of Core protein. Copyright © 2012 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.

  16. Undetected sex chromosome aneuploidy by chromosomal microarray.

    Science.gov (United States)

    Markus-Bustani, Keren; Yaron, Yuval; Goldstein, Myriam; Orr-Urtreger, Avi; Ben-Shachar, Shay

    2012-11-01

    We report on a case of a female fetus found to be mosaic for Turner syndrome (45,X) and trisomy X (47,XXX). Chromosomal microarray analysis (CMA) failed to detect the aneuploidy because of a normal average dosage of the X chromosome. This case represents an unusual instance in which CMA may not detect chromosomal aberrations. Such a possibility should be taken into consideration in similar cases where CMA is used in a clinical setting.

  17. Rfc5p regulates alternate RFC complex functions in sister chromatid pairing reactions in budding yeast

    OpenAIRE

    Maradeo, Marie E.; Garg, Anisha; Skibbens, Robert V.

    2010-01-01

    Sister chromatid pairing reactions, termed cohesion establishment, occur during S phase and appear to be regulated by replication factor C (RFC) complexes. For instance, RFCs that contain Ctf18p exhibit pro-establishment activities while those that contain Elg1p exhibit anti-establishment activities. It remains unknown whether Ctf18p-RFC and Elg1p-RFC functions are simply opposing or instead reveal complicated and non-parallel regulatory mechanisms. To better understand the nature of these no...

  18. Rfc5p regulates alternate RFC complex functions in sister chromatid pairing reactions in budding yeast.

    Science.gov (United States)

    Maradeo, Marie E; Garg, Anisha; Skibbens, Robert V

    2010-11-01

    Sister chromatid pairing reactions, termed cohesion establishment, occur during S-phase and appear to be regulated by Replication Factor C (RFC) complexes. For instance, RFCs that contain Ctf18p exhibit pro-establishment activities while those that contain Elg1p exhibit anti-establishment activities. It remains unknown whether Ctf18p-RFC and Elg1p-RFC functions are simply opposing or instead reveal complicated and non-parallel regulatory mechanisms. To better understand the nature of these novel pathways, we analyzed the small RFC subunit Rfc5p that is common to both Ctf18p-RFC and Elg1p-RFC. Despite this commonality, the data show that diminished Rfc5p function rescues ctf7/eco1 mutant cell phenotypes, revealing that Rfc5p promotes anti-establishment activities. This rescue is specific to establishment pathways in that rfc5-1 greatly accentuates growth defects when expressed in scc2 (deposition), mcd1/scc1 or smc3 (cohesion maintenance) mutated cells. Our results reveal for the first time a role for small RFC subunits in directing RFC complex functions-in this case towards anti-establishment pathways. We further report that Pds5p exhibits both establishment and anti-establishment functions in cohesion. This duality suggests that categorizations of establishment and anti-establishment activities require further examination.

  19. Cell mass and cell cycle dynamics of an asynchronous budding yeast population

    DEFF Research Database (Denmark)

    Lencastre Fernandes, Rita; Carlquist, Magnus; Lundin, Luisa

    2013-01-01

    consumption observed during batch cultivation. The good agreement between the proposed multi-scale model (a population balance model [PBM] coupled to an unstructured model) and experimental data (both the overall physiology and cell size and cell cycle distributions) indicates that a mechanistic model...... of model predictions for cell property distributions against experimental data is scarce. This study focuses on the experimental and mathematical description of the dynamics of cell size and cell cycle position distributions, of a population of Saccharomyces cerevisiae, in response to the substrate......Despite traditionally regarded as identical, cells in a microbial cultivation present a distribution of phenotypic traits, forming a heterogeneous cell population. Moreover, the degree of heterogeneity is notably enhanced by changes in micro-environmental conditions. A major development...

  20. High-resolution transcription atlas of the mitotic cell cycle in budding yeast

    DEFF Research Database (Denmark)

    Granovskaia, Marina V; Jensen, Lars J; Ritchie, Matthew E

    2010-01-01

    Extensive transcription of non-coding RNAs has been detected in eukaryotic genomes and is thought to constitute an additional layer in the regulation of gene expression. Despite this role, their transcription through the cell cycle has not been studied; genome-wide approaches have only focused on...

  1. Coevolution trumps pleiotropy: carbon assimilation traits are independent of metabolic network structure in budding yeast.

    Science.gov (United States)

    Opulente, Dana A; Morales, Christopher M; Carey, Lucas B; Rest, Joshua S

    2013-01-01

    Phenotypic traits may be gained and lost together because of pleiotropy, the involvement of common genes and networks, or because of simultaneous selection for multiple traits across environments (multiple-trait coevolution). However, the extent to which network pleiotropy versus environmental coevolution shapes shared responses has not been addressed. To test these alternatives, we took advantage of the fact that the genus Saccharomyces has variation in habitat usage and diversity in the carbon sources that a given strain can metabolize. We examined patterns of gain and loss in carbon utilization traits across 488 strains of Saccharomyces to investigate whether the structure of metabolic pathways or selection pressure from common environments may have caused carbon utilization traits to be gained and lost together. While most carbon sources were gained and lost independently of each other, we found four clusters that exhibit non-random patterns of gain and loss across strains. Contrary to the network pleiotropy hypothesis, we did not find that these patterns are explained by the structure of metabolic pathways or shared enzymes. Consistent with the hypothesis that common environments shape suites of phenotypes, we found that the environment a strain was isolated from partially predicts the carbon sources it can assimilate.

  2. Coevolution trumps pleiotropy: carbon assimilation traits are independent of metabolic network structure in budding yeast.

    Directory of Open Access Journals (Sweden)

    Dana A Opulente

    Full Text Available Phenotypic traits may be gained and lost together because of pleiotropy, the involvement of common genes and networks, or because of simultaneous selection for multiple traits across environments (multiple-trait coevolution. However, the extent to which network pleiotropy versus environmental coevolution shapes shared responses has not been addressed. To test these alternatives, we took advantage of the fact that the genus Saccharomyces has variation in habitat usage and diversity in the carbon sources that a given strain can metabolize. We examined patterns of gain and loss in carbon utilization traits across 488 strains of Saccharomyces to investigate whether the structure of metabolic pathways or selection pressure from common environments may have caused carbon utilization traits to be gained and lost together. While most carbon sources were gained and lost independently of each other, we found four clusters that exhibit non-random patterns of gain and loss across strains. Contrary to the network pleiotropy hypothesis, we did not find that these patterns are explained by the structure of metabolic pathways or shared enzymes. Consistent with the hypothesis that common environments shape suites of phenotypes, we found that the environment a strain was isolated from partially predicts the carbon sources it can assimilate.

  3. Expression of budding yeast FKBP12 confers rapamycin susceptibility to the unicellular red alga Cyanidioschyzon merolae.

    Science.gov (United States)

    Imamura, Sousuke; Ishiwata, Aiko; Watanabe, Satoru; Yoshikawa, Hirofumi; Tanaka, Kan

    2013-09-20

    The target of rapamycin (TOR) is serine/threonine protein kinase that is highly conserved among eukaryotes and can be inactivated by the antibiotic rapamycin through the formation of a ternary complex composed of rapamycin and two proteins, TOR and FKBP12. Differing from fungi and animals, plant FKBP12 proteins are unable to form the ternary complex, and thus plant TORs are insensitive to rapamycin. This has led to a poor understanding of TOR functions in plants. As a first step toward the understanding of TOR function in a rapamycin-insensitive unicellular red alga, Cyanidioschyzon merolae, we constructed a rapamycin-susceptible strain in which the Saccharomyces cerevisiae FKBP12 protein (ScFKBP12) was expressed. Treatment with rapamycin resulted in growth inhibition and decreased polysome formation in this strain. Binding of ScFKBP12 with C. merolae TOR in the presence of rapamycin was demonstrated in vivo and in vitro by pull-down experiments. Moreover, in vitro kinase assay showed that inhibition of C. merolae TOR kinase activity was dependent on ScFKBP12 and rapamycin. Copyright © 2013 Elsevier Inc. All rights reserved.

  4. Database Description - Budding yeast cDNA sequencing project | LSDB Archive [Life Science Database Archive metadata

    Lifescience Database Archive (English)

    Full Text Available or Creator Name: Fumihito Miura Creator Affiliation: Department of Computational Biology, Graduate School of...Ito* Creator Affiliation: Department of Computational Biology, Graduate School of Frontier Sciences, The Uni

  5. License - Budding yeast cDNA sequencing project | LSDB Archive [Life Science Database Archive metadata

    Lifescience Database Archive (English)

    Full Text Available n the Standard License. Standard License The Standard License for this database is the license specified in the Creative Commons...ght© 2009 Takashi Ito(the University of Tokyo) licensed under CC Attribution-Share Alike 2.1 Japan . The summary of the Creative Comm...ons Attribution-Share Alike 2.1 Japan is found here . Wi

  6. Computational Methods for Estimation of Cell Cycle Phase Distributions of Yeast Cells

    Directory of Open Access Journals (Sweden)

    Jalovaara Henna

    2007-01-01

    Full Text Available Two computational methods for estimating the cell cycle phase distribution of a budding yeast (Saccharomyces cerevisiae cell population are presented. The first one is a nonparametric method that is based on the analysis of DNA content in the individual cells of the population. The DNA content is measured with a fluorescence-activated cell sorter (FACS. The second method is based on budding index analysis. An automated image analysis method is presented for the task of detecting the cells and buds. The proposed methods can be used to obtain quantitative information on the cell cycle phase distribution of a budding yeast S. cerevisiae population. They therefore provide a solid basis for obtaining the complementary information needed in deconvolution of gene expression data. As a case study, both methods are tested with data that were obtained in a time series experiment with S. cerevisiae. The details of the time series experiment as well as the image and FACS data obtained in the experiment can be found in the online additional material at http://www.cs.tut.fi/sgn/csb/yeastdistrib/.

  7. Live longer on MARS: a yeast paradigm of mitochondrial adaptive ROS signaling in aging

    Directory of Open Access Journals (Sweden)

    Gerald S. Shadel

    2014-04-01

    Full Text Available Adaptive responses to stress, including hormesis, have been implicated in longevity, but their mechanisms and out comes are not fully understood. Here, I briefly summarize a longevity mechanism elucidated in the budding yeast chronological lifespan model by which Mitochondrial Adaptive ROS Signaling (MARS promotes beneficial epigenetic and metabolic remodeling. The potential relevance of MARS to the human disease Ataxia-Telangiectasia and as a potential anti-aging target is discussed.

  8. Developing a biomimetic tooth bud model.

    Science.gov (United States)

    Smith, Elizabeth E; Zhang, Weibo; Schiele, Nathan R; Khademhosseini, Ali; Kuo, Catherine K; Yelick, Pamela C

    2017-01-08

    A long-term goal is to bioengineer, fully functional, living teeth for regenerative medicine and dentistry applications. Biologically based replacement teeth would avoid insufficiencies of the currently used dental implants. Using natural tooth development as a guide, a model was fabricated using post-natal porcine dental epithelial (pDE), porcine dental mesenchymal (pDM) progenitor cells, and human umbilical vein endothelial cells (HUVEC) encapsulated within gelatin methacrylate (GelMA) hydrogels. Previous publications have shown that post-natal DE and DM cells seeded onto synthetic scaffolds exhibited mineralized tooth crowns composed of dentin and enamel. However, these tooth structures were small and formed within the pores of the scaffolds. The present study shows that dental cell-encapsulated GelMA constructs can support mineralized dental tissue formation of predictable size and shape. Individually encapsulated pDE or pDM cell GelMA constructs were analysed to identify formulas that supported pDE and pDM cell attachment, spreading, metabolic activity, and neo-vasculature formation with co-seeded endothelial cells (HUVECs). GelMa constructs consisting of pDE-HUVECS in 3% GelMA and pDM-HUVECs within 5% GelMA supported dental cell differentiation and vascular mineralized dental tissue formation in vivo. These studies are the first to demonstrate the use of GelMA hydrogels to support the formation of post-natal dental progenitor cell-derived mineralized and functionally vascularized tissues of specified size and shape. These results introduce a novel three-dimensional biomimetic tooth bud model for eventual bioengineered tooth replacement teeth in humans. Copyright © 2017 John Wiley & Sons, Ltd.

  9. Rapid isolation of yeast genomic DNA: Bust n' Grab

    Directory of Open Access Journals (Sweden)

    Peterson Kenneth R

    2004-04-01

    Full Text Available Abstract Background Mutagenesis of yeast artificial chromosomes (YACs often requires analysis of large numbers of yeast clones to obtain correctly targeted mutants. Conventional ways to isolate yeast genomic DNA utilize either glass beads or enzymatic digestion to disrupt yeast cell wall. Using small glass beads is messy, whereas enzymatic digestion of the cells is expensive when many samples need to be analyzed. We sought to develop an easier and faster protocol than the existing methods for obtaining yeast genomic DNA from liquid cultures or colonies on plates. Results Repeated freeze-thawing of cells in a lysis buffer was used to disrupt the cells and release genomic DNA. Cell lysis was followed by extraction with chloroform and ethanol precipitation of DNA. Two hundred ng – 3 μg of genomic DNA could be isolated from a 1.5 ml overnight liquid culture or from a large colony. Samples were either resuspended directly in a restriction enzyme/RNase coctail mixture for Southern blot hybridization or used for several PCR reactions. We demonstrated the utility of this method by showing an analysis of yeast clones containing a mutagenized human β-globin locus YAC. Conclusion An efficient, inexpensive method for obtaining yeast genomic DNA from liquid cultures or directly from colonies was developed. This protocol circumvents the use of enzymes or glass beads, and therefore is cheaper and easier to perform when processing large numbers of samples.

  10. Biofuels. Altered sterol composition renders yeast thermotolerant

    DEFF Research Database (Denmark)

    Caspeta, Luis; Chen, Yun; Ghiaci, Payam

    2014-01-01

    adaptive laboratory evolution to select yeast strains with improved growth and ethanol production at ≥40°C. Sequencing of the whole genome, genome-wide gene expression, and metabolic-flux analyses revealed a change in sterol composition, from ergosterol to fecosterol, caused by mutations in the C-5 sterol...... desaturase gene, and increased expression of genes involved in sterol biosynthesis. Additionally, large chromosome III rearrangements and mutations in genes associated with DNA damage and respiration were found, but contributed less to the thermotolerant phenotype....

  11. Sphingolipids Facilitate Age Asymmetry of Membrane Proteins in Dividing Yeast Cells.

    Science.gov (United States)

    Singh, Pushpendra; Ramachandran, Sree Kumar; Zhu, Jin; Kim, Byoung Choul; Biswas, Debojyoti; Ha, Taekjip; Iglesias, Pablo A; Li, Rong

    2017-08-02

    A mechanism of cellular aging was proposed to be the gradual loss of certain cellular components that are insufficiently renewed. In an earlier study, multidrug resistance transporters (MDRs) were postulated to be such aging determinants during yeast replicative life span (RLS). Aged MDR proteins are asymmetrically retained by the aging mother cell and do not diffuse freely into the bud, whereas newly synthesized MDR proteins were thought to be deposited mostly in the bud prior to cytokinesis. In this study, we further demonstrate the proposed age asymmetry of MDR proteins in dividing yeast cells and investigated the mechanism that control diffusive properties of MDR proteins to maintain this asymmetry. We found that long-chain sphingolipids, but not the septin/ER-based membrane diffusion barrier, are important for restricting MDR diffusion. Depletion of sphingolipids or shortening of their long acyl chains resulted in an increase in the lateral mobility of MDR proteins, causing aged MDR protein in the mother cell to enter the bud. We used a mathematical model to understand the effect of diminished MDR age asymmetry on yeast cell aging, the result of which was qualitatively consistent with the observed RLS shortening in sphingolipid mutants. © 2017 by The American Society for Cell Biology.

  12. [Bud population dynamics of Phragmites australis in heterogeneous habitats of Northeast grassland, China].

    Science.gov (United States)

    2015-02-01

    To adapt ecological environment, typical clonal plants can occur continuously by means of buds. The changes in the bud bank and bud flow in the heterogeneous habitats become the foundation for deep understanding the characteristics of vegetative propagation. By sampling soil from the unit area, a comparative analysis was performed for rhizome bud population dynamics of Phragmites australis community in both meadow soil and saline-alkali soil habitats in meadow grassland of Northeast China. The one-age class rhizome buds formed in the current year were used as input, with the other age classes rhizome buds as output, counting the dormancy buds and death buds. The results showed that the storage, input, output, dormancy, death and the input rates of P. australis rhizome bud populations in meadow soil habitat were significantly higher than that in saline-alkali habitat. There was no significant difference in output rate between the two habitats. The dormant rate in saline-alkali habitat was significantly greater than that in meadow soil habitat. The death rates remained at relatively low levels in both, less than 2%. With the going of growing season, the input buds and input rate of bud bank increased in the two habitats, while the output buds remained relatively stable. The output rate increased first and decreased later, the dormancy buds and dormant rate decreased. Bud bank and bud flow were positively related to soil moisture, soil organic matter and soil available nitrogen content. However, they were negatively related to soil pH value and soil available phosphorus content. Bud bank and bud flow had a similar seasonal variation. Constantly for both habitats, P. australis populations generated new rhizome buds supplied to the bud bank and kept a stable output to maintain their vegetative propagation.

  13. Acentric chromosome ends are prone to fusion with functional chromosome ends through a homology-directed rearrangement.

    Science.gov (United States)

    Ohno, Yuko; Ogiyama, Yuki; Kubota, Yoshino; Kubo, Takuya; Ishii, Kojiro

    2016-01-08

    The centromeres of many eukaryotic chromosomes are established epigenetically on potentially variable tandem repeats; hence, these chromosomes are at risk of being acentric. We reported previously that artificially created acentric chromosomes in the fission yeast Schizosaccharomyces pombe can be rescued by end-to-end fusion with functional chromosomes. Here, we show that most acentric/functional chromosome fusion events in S. pombe cells harbouring an acentric chromosome I differed from the non-homologous end-joining-mediated rearrangements that result in deleterious dicentric fusions in normal cells, and were elicited by a previously unidentified homologous recombination (HR) event between chromosome end-associated sequences. The subtelomere repeats associated with the non-fusogenic ends were also destabilized in the surviving cells, suggesting a causal link between general subtelomere destabilization and acentric/functional chromosome fusion. A mutational analysis indicated that a non-canonical HR pathway was involved in the rearrangement. These findings are indicative of a latent mechanism that conditionally induces general subtelomere instability, presumably in the face of accidental centromere loss events, resulting in rescue of the fatal acentric chromosomes by interchromosomal HR.

  14. Unique geometry of sister kinetochores in human oocytes during meiosis I may explain maternal age-associated increases in chromosomal abnormalities.

    Science.gov (United States)

    Patel, Jessica; Tan, Seang Lin; Hartshorne, Geraldine M; McAinsh, Andrew D

    2015-12-30

    The first meiotic division in human oocytes is highly error-prone and contributes to the uniquely high incidence of aneuploidy observed in human pregnancies. A successful meiosis I (MI) division entails separation of homologous chromosome pairs and co-segregation of sister chromatids. For this to happen, sister kinetochores must form attachments to spindle kinetochore-fibres emanating from the same pole. In mouse and budding yeast, sister kinetochores remain closely associated with each other during MI, enabling them to act as a single unified structure. However, whether this arrangement also applies in human meiosis I oocytes was unclear. In this study, we perform high-resolution imaging of over 1900 kinetochores in human oocytes, to examine the geometry and architecture of the human meiotic kinetochore. We reveal that sister kinetochores in MI are not physically fused, and instead individual kinetochores within a pair are capable of forming independent attachments to spindle k-fibres. Notably, with increasing female age, the separation between kinetochores increases, suggesting a degradation of centromeric cohesion and/or changes in kinetochore architecture. Our data suggest that the differential arrangement of sister kinetochores and dual k-fibre attachments may explain the high proportion of unstable attachments that form in MI and thus indicate why human oocytes are prone to aneuploidy, particularly with increasing maternal age.

  15. Unique geometry of sister kinetochores in human oocytes during meiosis I may explain maternal age-associated increases in chromosomal abnormalities

    Directory of Open Access Journals (Sweden)

    Jessica Patel

    2016-02-01

    Full Text Available The first meiotic division in human oocytes is highly error-prone and contributes to the uniquely high incidence of aneuploidy observed in human pregnancies. A successful meiosis I (MI division entails separation of homologous chromosome pairs and co-segregation of sister chromatids. For this to happen, sister kinetochores must form attachments to spindle kinetochore-fibres emanating from the same pole. In mouse and budding yeast, sister kinetochores remain closely associated with each other during MI, enabling them to act as a single unified structure. However, whether this arrangement also applies in human meiosis I oocytes was unclear. In this study, we perform high-resolution imaging of over 1900 kinetochores in human oocytes, to examine the geometry and architecture of the human meiotic kinetochore. We reveal that sister kinetochores in MI are not physically fused, and instead individual kinetochores within a pair are capable of forming independent attachments to spindle k-fibres. Notably, with increasing female age, the separation between kinetochores increases, suggesting a degradation of centromeric cohesion and/or changes in kinetochore architecture. Our data suggest that the differential arrangement of sister kinetochores and dual k-fibre attachments may explain the high proportion of unstable attachments that form in MI and thus indicate why human oocytes are prone to aneuploidy, particularly with increasing maternal age.

  16. The Double-Strand Break Landscape of Meiotic Chromosomes Is Shaped by the Paf1 Transcription Elongation Complex in Saccharomyces cerevisiae.

    Science.gov (United States)

    Gothwal, Santosh K; Patel, Neem J; Colletti, Meaghan M; Sasanuma, Hiroyuki; Shinohara, Miki; Hochwagen, Andreas; Shinohara, Akira

    2016-02-01

    Histone modification is a critical determinant of the frequency and location of meiotic double-strand breaks (DSBs), and thus recombination. Set1-dependent histone H3K4 methylation and Dot1-dependent H3K79 methylation play important roles in this process in budding yeast. Given that the RNA polymerase II associated factor 1 complex, Paf1C, promotes both types of methylation, we addressed the role of the Paf1C component, Rtf1, in the regulation of meiotic DSB formation. Similar to a set1 mutation, disruption of RTF1 decreased the occurrence of DSBs in the genome. However, the rtf1 set1 double mutant exhibited a larger reduction in the levels of DSBs than either of the single mutants, indicating independent contributions of Rtf1 and Set1 to DSB formation. Importantly, the distribution of DSBs along chromosomes in the rtf1 mutant changed in a manner that was different from the distributions observed in both set1 and set1 dot1 mutants, including enhanced DSB formation at some DSB-cold regions that are occupied by nucleosomes in wild-type cells. These observations suggest that Rtf1, and by extension the Paf1C, modulate the genomic DSB landscape independently of H3K4 methylation.

  17. Analysis of stress-induced duplex destabilization (SIDD properties of replication origins, genes and intergenes in the fission yeast, Schizosaccharomyces pombe

    Directory of Open Access Journals (Sweden)

    Yadav Mukesh P

    2012-11-01

    Full Text Available Abstract Background Replication and transcription, the two key functions of DNA, require unwinding of the DNA double helix. It has been shown that replication origins in the budding yeast, Saccharomyces cerevisiae contain an easily unwound stretch of DNA. We have used a recently developed method for determining the locations and degrees of stress-induced duplex destabilization (SIDD for all the reported replication origins in the genome of the fission yeast, Schizosaccharomyces pombe. Results We have found that the origins are more susceptible to SIDD as compared to the non-origin intergenic regions (NOIRs and genes. SIDD analysis of many known origins in other eukaryotes suggests that SIDD is a common property of replication origins. Interestingly, the previously shown deletion-dependent changes in the activities of the origins of the ura4 origin region on chromosome 3 are paralleled by changes in SIDD properties, suggesting SIDD’s role in origin activity. SIDD profiling following in silico deletions of some origins suggests that many of the closely spaced S. pombe origins could be clusters of two or three weak origins, similar to the ura4 origin region. Conclusion SIDD appears to be a highly conserved, functionally important property of replication origins in S. pombe and other organisms. The distinctly low SIDD scores of origins and the long range effects of genetic alterations on SIDD properties provide a unique predictive potential to the SIDD analysis. This could be used in exploring different aspects of structural and functional organization of origins including interactions between closely spaced origins.

  18. Chromosome Disorder Outreach

    Science.gov (United States)

    ... BLOG Join Us Donate You are not alone. Chromosome Disorder Outreach, Inc. is a non-profit organization, ... Support For all those diagnosed with any rare chromosome disorder. Since 1992, CDO has supported the parents ...

  19. Chromosome painting in plants.

    NARCIS (Netherlands)

    Schubert, I.; Fransz, P.F.; Fuchs, J.; Jong, de J.H.

    2001-01-01

    The current 'state-of-art' as to chromosome painting in plants is reviewed. We define different situations described as painting so far: i) Genomic in situ hybridisation (GISH) with total genomic DNA to distinguish alien chromosomes on the basis of divergent dispersed repeats, ii) 'Chromosomal in si

  20. ZEBRAFISH CHROMOSOME-BANDING

    NARCIS (Netherlands)

    PIJNACKER, LP; FERWERDA, MA

    1995-01-01

    Banding techniques were carried out on metaphase chromosomes of zebrafish (Danio rerio) embryos. The karyotypes with the longest chromosomes consist of 12 metacentrics, 26 submetacentrics, and 12 subtelocentrics (2n = 50). All centromeres are C-band positive. Eight chromosomes have a pericentric C-b

  1. Chromosome painting in plants.

    NARCIS (Netherlands)

    Schubert, I.; Fransz, P.F.; Fuchs, J.; Jong, de J.H.

    2001-01-01

    The current 'state-of-art' as to chromosome painting in plants is reviewed. We define different situations described as painting so far: i) Genomic in situ hybridisation (GISH) with total genomic DNA to distinguish alien chromosomes on the basis of divergent dispersed repeats, ii) 'Chromosomal in

  2. Unidirectional P-body transport during the yeast cell cycle.

    Directory of Open Access Journals (Sweden)

    Cecilia Garmendia-Torres

    Full Text Available P-bodies belong to a large family of RNA granules that are associated with post-transcriptional gene regulation, conserved from yeast to mammals, and influence biological processes ranging from germ cell development to neuronal plasticity. RNA granules can also transport RNAs to specific locations. Germ granules transport maternal RNAs to the embryo, and neuronal granules transport RNAs long distances to the synaptic dendrites. Here we combine microfluidic-based fluorescent microscopy of single cells and automated image analysis to follow p-body dynamics during cell division in yeast. Our results demonstrate that these highly dynamic granules undergo a unidirectional transport from the mother to the daughter cell during mitosis as well as a constrained "hovering" near the bud site half an hour before the bud is observable. Both behaviors are dependent on the Myo4p/She2p RNA transport machinery. Furthermore, single cell analysis of cell size suggests that PBs play an important role in daughter cell growth under nutrient limiting conditions.

  3. Analysis of aging in lager brewing yeast during serial repitching.

    Science.gov (United States)

    Bühligen, Franziska; Lindner, Patrick; Fetzer, Ingo; Stahl, Frank; Scheper, Thomas; Harms, Hauke; Müller, Susann

    2014-10-10

    Serial repitching of brewing yeast inoculates is an important economic factor in the brewing industry, as their propagation is time and resource intensive. Here, we investigated whether replicative aging and/or the population distribution status changed during serial repitching in three different breweries with the same brewing yeast strain but different abiotic backgrounds and repitching regimes with varying numbers of reuses. Next to bud scar numbers the DNA content of the Saccharomyces pastorianus HEBRU cells was analyzed. Gene expression patterns were investigated using low-density microarrays with genes for aging, stress, storage compound metabolism and cell cycle. Two breweries showed a stable rejuvenation rate during serial repitching. In a third brewery the fraction of virgin cells varied, which could be explained with differing wort aeration rates. Furthermore, the number of bud scars per cell and cell size correlated in all 3 breweries throughout all runs. Transcriptome analyses revealed that from the 6th run on, mainly for the cells positive gene expression could be seen, for example up-regulation of trehalose and glycogen metabolism genes. Additionally, the cells' settling in the cone was dependent on cell size, with the lowest and the uppermost cone layers showing the highest amount of dead cells. In general, cells do not progressively age during extended serial repitching.

  4. Trichoplusia ni Kinesin-1 Associates with Autographa californica Multiple Nucleopolyhedrovirus Nucleocapsid Proteins and Is Required for Production of Budded Virus

    Science.gov (United States)

    Biswas, Siddhartha; Blissard, Gary W.

    2016-01-01

    ABSTRACT The mechanism by which nucleocapsids of Autographa californica multiple nucleopolyhedrovirus (AcMNPV) egress from the nucleus to the plasma membrane, leading to the formation of budded virus (BV), is not known. AC141 is a nucleocapsid-associated protein required for BV egress and has previously been shown to be associated with β-tubulin. In addition, AC141 and VP39 were previously shown by fluorescence resonance energy transfer by fluorescence lifetime imaging to interact directly with the Drosophila melanogaster kinesin-1 light chain (KLC) tetratricopeptide repeat (TPR) domain. These results suggested that microtubule transport systems may be involved in baculovirus nucleocapsid egress and BV formation. In this study, we investigated the role of lepidopteran microtubule transport using coimmunoprecipitation, colocalization, yeast two-hybrid, and small interfering RNA (siRNA) analyses. We show that nucleocapsid AC141 associates with the lepidopteran Trichoplusia ni KLC and kinesin-1 heavy chain (KHC) by coimmunoprecipitation and colocalization. Kinesin-1, AC141, and microtubules colocalized predominantly at the plasma membrane. In addition, the nucleocapsid proteins VP39, FP25, and BV/ODV-C42 were also coimmunoprecipitated with T. ni KLC. Direct analysis of the role of T. ni kinesin-1 by downregulation of KLC by siRNA resulted in a significant decrease in BV production. Nucleocapsids labeled with VP39 fused with three copies of the mCherry fluorescent protein also colocalized with microtubules. Yeast two-hybrid analysis showed no evidence of a direct interaction between kinesin-1 and AC141 or VP39, suggesting that either other nucleocapsid proteins or adaptor proteins may be required. These results further support the conclusion that microtubule transport is required for AcMNPV BV formation. IMPORTANCE In two key processes of the replication cycle of the baculovirus Autographa californica multiple nucleopolyhedrovirus (AcMNPV), nucleocapsids are

  5. Electron tomography reveals the steps in filovirus budding.

    Science.gov (United States)

    Welsch, Sonja; Kolesnikova, Larissa; Krähling, Verena; Riches, James D; Becker, Stephan; Briggs, John A G

    2010-04-29

    The filoviruses, Marburg and Ebola, are non-segmented negative-strand RNA viruses causing severe hemorrhagic fever with high mortality rates in humans and nonhuman primates. The sequence of events that leads to release of filovirus particles from cells is poorly understood. Two contrasting mechanisms have been proposed, one proceeding via a "submarine-like" budding with the helical nucleocapsid emerging parallel to the plasma membrane, and the other via perpendicular "rocket-like" protrusion. Here we have infected cells with Marburg virus under BSL-4 containment conditions, and reconstructed the sequence of steps in the budding process in three dimensions using electron tomography of plastic-embedded cells. We find that highly infectious filamentous particles are released at early stages in infection. Budding proceeds via lateral association of intracellular nucleocapsid along its whole length with the plasma membrane, followed by rapid envelopment initiated at one end of the nucleocapsid, leading to a protruding intermediate. Scission results in local membrane instability at the rear of the virus. After prolonged infection, increased vesiculation of the plasma membrane correlates with changes in shape and infectivity of released viruses. Our observations demonstrate a cellular determinant of virus shape. They reconcile the contrasting models of filovirus budding and allow us to describe the sequence of events taking place during budding and release of Marburg virus. We propose that this represents a general sequence of events also followed by other filamentous and rod-shaped viruses.

  6. Electron tomography reveals the steps in filovirus budding.

    Directory of Open Access Journals (Sweden)

    Sonja Welsch

    2010-04-01

    Full Text Available The filoviruses, Marburg and Ebola, are non-segmented negative-strand RNA viruses causing severe hemorrhagic fever with high mortality rates in humans and nonhuman primates. The sequence of events that leads to release of filovirus particles from cells is poorly understood. Two contrasting mechanisms have been proposed, one proceeding via a "submarine-like" budding with the helical nucleocapsid emerging parallel to the plasma membrane, and the other via perpendicular "rocket-like" protrusion. Here we have infected cells with Marburg virus under BSL-4 containment conditions, and reconstructed the sequence of steps in the budding process in three dimensions using electron tomography of plastic-embedded cells. We find that highly infectious filamentous particles are released at early stages in infection. Budding proceeds via lateral association of intracellular nucleocapsid along its whole length with the plasma membrane, followed by rapid envelopment initiated at one end of the nucleocapsid, leading to a protruding intermediate. Scission results in local membrane instability at the rear of the virus. After prolonged infection, increased vesiculation of the plasma membrane correlates with changes in shape and infectivity of released viruses. Our observations demonstrate a cellular determinant of virus shape. They reconcile the contrasting models of filovirus budding and allow us to describe the sequence of events taking place during budding and release of Marburg virus. We propose that this represents a general sequence of events also followed by other filamentous and rod-shaped viruses.

  7. Chromosomal targeting of replicating plasmids in the yeast Hansenula polymorpha

    NARCIS (Netherlands)

    Faber, Klaas Nico; Swaving, Gert Jan; Faber, Folkert; Ab, Geert; Harder, Willem; Veenhuis, Marten; Haima, Pieter

    1992-01-01

    Using an optimized transformation protocol we have studied the possible interactions between transforming plasmid DNA and the Hansenula polymorpha genome. Plasmids consisting only of a pBR322 replicon, an antibiotic resistance marker for Escherichia coli and the Saccharomyces cerevisiae LEU2 gene we

  8. Yeast identification in floral nectar of Mimulus aurantiacus (Invited)

    Science.gov (United States)

    Kyauk, C.; Belisle, M.; Fukami, T.

    2009-12-01

    Nectar is such a sugar-rich resource that serves as a natural habitat in which microbes thrive. As a result, yeasts arrive to nectar on the bodies of pollinators such as hummingbirds and bees. Yeasts use the sugar in nectar for their own needs when introduced. This research focuses on the identification of different types of yeast that are found in the nectar of Mimulus aurantiacus (commonly known as sticky monkey-flower). Unopened Mimulus aurantiacus flower buds were tagged at Jasper Ridge and bagged three days later. Floral nectar was then extracted and plated on potato dextrose agar. Colonies on the plates were isolated and DNA was extracted from each sample using QIAGEN DNeasy Plant Mini Kit. The DNA was amplified through PCR and ran through gel electrophoresis. The PCR product was used to clone the nectar samples into an E.coli vector. Finally, a phylogenetic tree was created by BLAST searching sequences in GenBank using the Internal Transcribed Space (ITS) locus. It was found that 18 of the 50 identified species were Candida magnifica, 14 was Candida rancensis, 6 were Crytococcus albidus and there were 3 or less of the following: Starmella bombicola, Candida floricola, Aureobasidium pullulans, Pichia kluyvera, Metschnikowa cibodaserisis, Rhodotorua colostri, and Malassezia globosa. The low diversity of the yeast could have been due to several factors: time of collection, demographics of Jasper Ridge, low variety of pollinators, and sugar concentration of the nectar. The results of this study serve as a necessary first step for a recently started research project on ecological interactions between plants, pollinators, and nectar-living yeast. More generally, this research studies the use of the nectar-living yeast community as a natural microcosm for addressing basic questions about the role of dispersal and competitive and facilitative interactions in ecological succession.

  9. Mapping strategies: Chromosome 16 workshop

    Energy Technology Data Exchange (ETDEWEB)

    1989-01-01

    The following topics from a workshop on chromosome 16 are briefly discussed: genetic map of chromosome 16; chromosome breakpoint map of chromosome 16; integrated physical/genetic map of chromosome 16; pulsed field map of the 16p13.2--p13.3 region (3 sheets); and a report of the HGM10 chromosome 16 committee.

  10. Repellence of the red bud borer Resseliella oculiperda from grafted apple trees by impregnation of rubber budding strips with essential oils.

    Science.gov (United States)

    van Tol, Rob W H M; Swarts, Henk J; van der Linden, Anton; Visser, J H

    2007-05-01

    The red bud borer Resseliella oculiperda (Rübs.) is a pest insect of apple trees when rootstocks are grafted with scion buds by 'shield budding'. The female midges are attracted to the wounds of the grafted buds where they lay their eggs. The larvae feed on the cambium and destroy the buds completely or partially, leading to bad union of the buds with the rootstocks. Budding strips are used very often by growers to bind scion buds to rootstocks. These strips cannot prevent midges from reaching the damaged tissue. Chemical treatments applied to the grafts and other types of strip do not provide better protection against the pest and may cause other risks for growers. In orchard experiments in 2000 and 2001, the authors evaluated the repellent action provided by three essential oils and five compounds of plant origin against the midges by impregnating budding strips with them. The essential oils of lavender, Lavandula angustifolia (P. Mill.), and alpha-terpineol decreased the infestation of buds by more than 95 and 80% respectively. The other potential repellents tested [the essential oil of Juniperus virginiana (L.), citronellal, the essential oil of Cinnamomum camphora (L.) J. Presl, R-carvone, linalool and R-fenchone] decreased infestation by 67, 66, 51, 45, 37 and 25% respectively. The formulation and commercial development of budding strips impregnated with lavender oil is discussed.

  11. Adventitious bud regeneration from the stigma of Sinapis alba L.

    Directory of Open Access Journals (Sweden)

    Elżbieta Zenkteler

    2012-12-01

    Full Text Available Stigmas isolated from flower buds of 'Nakielska' variety of Sinapis alba were used to develop a micropropagation method suitable for breeding of new cultivars. The origin of adventitious bud regeneration was studied on MS medium, under stimulation by bezylaminopurine (BAP in combination with 2,4-D - dichlorophenoxyacetic acid (2,4-D. Histological analysis showed the structure of Sinapis stigma (composed from four types of tissue: papillae, transmitting tissue, parenchyma and vascular bundles and revealed that numerous meristematic centers developed from parenchyma cells in close vicinity of vascular bundles. Buds very quickly appeared on the surface of initial explants and later formed multiplantlets that were easily rooted in the soil.

  12. The Mechanism of Budding of Retroviruses from Cell Membranes

    Directory of Open Access Journals (Sweden)

    Andrew Pincetic

    2009-01-01

    Full Text Available Retroviruses have evolved a mechanism for the release of particles from the cell membrane that appropriates cellular protein complexes, referred to as ESCRT-I, -II, -III, normally involved in the biogenesis of multivesicular bodies. Three different classes of late assembly (L domains encoded in Gag, with core sequences of PPXY, PTAP, and YPXL, recruit different components of the ESCRT machinery to form a budding complex for virus release. Here, we highlight recent progress in identifying the role of different ESCRT complexes in facilitating budding, ubiquitination, and membrane targeting of avian sarcoma and leukosis virus (ASLV and human immunodeficiency virus, type 1 (HIV-1. These findings show that retroviruses may adopt parallel budding pathways by recruiting different host factors from common cellular machinery for particle release.

  13. Respiratory Response of Dormant Nectarine Floral Buds on Chilling Deficiency

    Institute of Scientific and Technical Information of China (English)

    TAN Yue; GAO Dong-sheng; LI Ling; CHEN Xiu-de; XU Ai-hong

    2010-01-01

    Changes in main biochemical respiratory pathways in dormant nectarine floral buds were studied with nectarine trees (Prunus persica.var,nectariana cv.Shuguang) in order to determine the function of respiration in dormancy release.Oxygen-electrode system and respiratory inhibitors were used to measure total respiratory rates and rates of respiratory pathways.Results showed that chilling deficiency blocked the transition of respiratory mode,and made buds stay in a state of high level pentose phosphate pathway (PPP) and low level tricarboxylic acid cycle (TCA).The decline of PPP and activation of TCA occurred synchronously with the release of dormancy.In addition,the inhibition of PPP stimulated a respiration increase related with TCA.It could be concluded that the function of PPP activation in dormancy release might be limited and PPP declination inducing TCA activation might be part of respiration mode transition mechanism during bud sprouting.

  14. alpha-Synuclein fission yeast model: concentration-dependent aggregation without plasma membrane localization or toxicity.

    Science.gov (United States)

    Brandis, Katrina A; Holmes, Isaac F; England, Samantha J; Sharma, Nijee; Kukreja, Lokesh; DebBurman, Shubhik K

    2006-01-01

    Despite fission yeast's history of modeling salient cellular processes, it has not yet been used to model human neurodegeneration-linked protein misfolding. Because alpha-synuclein misfolding and aggregation are linked to Parkinson's disease (PD), here, we report a fission yeast (Schizosaccharomyces pombe) model that evaluates alpha-synuclein misfolding, aggregation, and toxicity and compare these properties with those recently characterized in budding yeast (Saccharomyces cerevisiae). Wild-type alpha-synuclein and three mutants (A30P, A53T, and A30P/A53T) were expressed with thiamine-repressible promoters (using vectors of increasing promoter strength: pNMT81, pNMT41, and pNMT1) to test directly in living cells the nucleation polymerization hypothesis for alpha-synuclein misfolding and aggregation. In support of the hypothesis, wild-type and A53T alpha-synuclein formed prominent intracellular cytoplasmic inclusions within fission yeast cells in a concentration- and time-dependent manner, whereas A30P and A30P/A53T remained diffuse throughout the cytoplasm. A53T alpha-synuclein formed aggregates faster than wild-type alpha-synuclein and at a lower alpha-synuclein concentration. Unexpectedly, unlike in budding yeast, wild-type and A53T alpha-synuclein did not target to the plasma membrane in fission yeast, not even at low alpha-synuclein concentrations or as a precursor step to forming aggregates. Despite alpha-synuclein's extensive aggregation, it was surprisingly nontoxic to fission yeast. Future genetic dissection might yield molecular insight into this protection against toxicity. We speculate that alpha-synuclein toxicity might be linked to its membrane binding capacity. To conclude, S. pombe and S. cerevisiae model similar yet distinct aspects of alpha-synuclein biology, and both organisms shed insight into alpha-synuclein's role in PD pathogenesis.

  15. Engineering of plant chromosomes.

    Science.gov (United States)

    Mette, Michael Florian; Houben, Andreas

    2015-02-01

    Engineered minimal chromosomes with sufficient mitotic and meiotic stability have an enormous potential as vectors for stacking multiple genes required for complex traits in plant biotechnology. Proof of principle for essential steps in chromosome engineering such as truncation of chromosomes by T-DNA-mediated telomere seeding and de novo formation of centromeres by cenH3 fusion protein tethering has been recently obtained. In order to generate robust protocols for application in plant biotechnology, these steps need to be combined and supplemented with additional methods such as site-specific recombination for the directed transfer of multiple genes of interest on the minichromosomes. At the same time, the development of these methods allows new insight into basic aspects of plant chromosome functions such as how centromeres assure proper distribution of chromosomes to daughter cells or how telomeres serve to cap the chromosome ends to prevent shortening of ends over DNA replication cycles and chromosome end fusion.

  16. Yeast genome sequencing:

    DEFF Research Database (Denmark)

    Piskur, Jure; Langkjær, Rikke Breinhold

    2004-01-01

    For decades, unicellular yeasts have been general models to help understand the eukaryotic cell and also our own biology. Recently, over a dozen yeast genomes have been sequenced, providing the basis to resolve several complex biological questions. Analysis of the novel sequence data has shown...... of closely related species helps in gene annotation and to answer how many genes there really are within the genomes. Analysis of non-coding regions among closely related species has provided an example of how to determine novel gene regulatory sequences, which were previously difficult to analyse because...... they are short and degenerate and occupy different positions. Comparative genomics helps to understand the origin of yeasts and points out crucial molecular events in yeast evolutionary history, such as whole-genome duplication and horizontal gene transfer(s). In addition, the accumulating sequence data provide...

  17. Vaginal Yeast Infections

    Science.gov (United States)

    ... tight or made of materials like nylon that trap heat and moisture might make yeast infections more ... Nemours Foundation, iStock, Getty Images, Corbis, Veer, Science Photo Library, Science Source Images, Shutterstock, and Clipart.com

  18. Modeling brewers' yeast flocculation

    Science.gov (United States)

    van Hamersveld EH; van der Lans RG; Caulet; Luyben

    1998-02-01

    Flocculation of yeast cells occurs during the fermentation of beer. Partway through the fermentation the cells become flocculent and start to form flocs. If the environmental conditions, such as medium composition and fluid velocities in the tank, are optimal, the flocs will grow in size large enough to settle. After settling of the main part of the yeast the green beer is left, containing only a small amount of yeast necessary for rest conversions during the next process step, the lagering. The physical process of flocculation is a dynamic equilibrium of floc formation and floc breakup resulting in a bimodal size distribution containing single cells and flocs. The floc size distribution and the single cell amount were measured under the different conditions that occur during full scale fermentation. Influences on flocculation such as floc strength, specific power input, and total number of yeast cells in suspension were studied. A flocculation model was developed, and the measured data used for validation. Yeast floc formation can be described with the collision theory assuming a constant collision efficiency. The breakup of flocs appears to occur mainly via two mechanisms, the splitting of flocs and the erosion of yeast cells from the floc surface. The splitting rate determines the average floc size and the erosion rate determines the number of single cells. Regarding the size of the flocs with respect to the scale of turbulence, only the viscous subrange needs to be considered. With the model, the floc size distribution and the number of single cells can be predicted at a certain point during the fermentation. For this, the bond strength between the cells, the fractal dimension of the yeast, the specific power input in the tank and the number of yeast cells that are in suspension in the tank have to be known. Copyright 1998 John Wiley & Sons, Inc.

  19. Nitrile Metabolizing Yeasts

    Science.gov (United States)

    Bhalla, Tek Chand; Sharma, Monica; Sharma, Nitya Nand

    Nitriles and amides are widely distributed in the biotic and abiotic components of our ecosystem. Nitrile form an important group of organic compounds which find their applications in the synthesis of a large number of compounds used as/in pharmaceutical, cosmetics, plastics, dyes, etc>. Nitriles are mainly hydro-lyzed to corresponding amide/acid in organic chemistry. Industrial and agricultural activities have also lead to release of nitriles and amides into the environment and some of them pose threat to human health. Biocatalysis and biotransformations are increasingly replacing chemical routes of synthesis in organic chemistry as a part of ‘green chemistry’. Nitrile metabolizing organisms or enzymes thus has assumed greater significance in all these years to convert nitriles to amides/ acids. The nitrile metabolizing enzymes are widely present in bacteria, fungi and yeasts. Yeasts metabolize nitriles through nitrilase and/or nitrile hydratase and amidase enzymes. Only few yeasts have been reported to possess aldoxime dehydratase. More than sixty nitrile metabolizing yeast strains have been hither to isolated from cyanide treatment bioreactor, fermented foods and soil. Most of the yeasts contain nitrile hydratase-amidase system for metabolizing nitriles. Transformations of nitriles to amides/acids have been carried out with free and immobilized yeast cells. The nitrilases of Torulopsis candida>and Exophiala oligosperma>R1 are enantioselec-tive and regiospecific respectively. Geotrichum>sp. JR1 grows in the presence of 2M acetonitrile and may have potential for application in bioremediation of nitrile contaminated soil/water. The nitrilase of E. oligosperma>R1 being active at low pH (3-6) has shown promise for the hydroxy acids. Immobilized yeast cells hydrolyze some additional nitriles in comparison to free cells. It is expected that more focus in future will be on purification, characterization, cloning, expression and immobilization of nitrile metabolizing

  20. Forces in yeast flocculation.

    Science.gov (United States)

    El-Kirat-Chatel, Sofiane; Beaussart, Audrey; Vincent, Stéphane P; Abellán Flos, Marta; Hols, Pascal; Lipke, Peter N; Dufrêne, Yves F

    2015-02-07

    In the baker's yeast Saccharomyces cerevisiae, cell-cell adhesion ("flocculation") is conferred by a family of lectin-like proteins known as the flocculin (Flo) proteins. Knowledge of the adhesive and mechanical properties of flocculins is important for understanding the mechanisms of yeast adhesion, and may help controlling yeast behaviour in biotechnology. We use single-molecule and single-cell atomic force microscopy (AFM) to explore the nanoscale forces engaged in yeast flocculation, focusing on the role of Flo1 as a prototype of flocculins. Using AFM tips labelled with mannose, we detect single flocculins on Flo1-expressing cells, showing they are widely exposed on the cell surface. When subjected to force, individual Flo1 proteins display two distinct force responses, i.e. weak lectin binding forces and strong unfolding forces reflecting the force-induced extension of hydrophobic tandem repeats. We demonstrate that cell-cell adhesion bonds also involve multiple weak lectin interactions together with strong unfolding forces, both associated with Flo1 molecules. Single-molecule and single-cell data correlate with microscale cell adhesion behaviour, suggesting strongly that Flo1 mechanics is critical for yeast flocculation. These results favour a model in which not only weak lectin-sugar interactions are involved in yeast flocculation but also strong hydrophobic interactions resulting from protein unfolding.

  1. Yeast Interacting Proteins Database: YGR218W, YMR124W [Yeast Interacting Proteins Database

    Lifescience Database Archive (English)

    Full Text Available ry, cytoplasm, bud, and bud neck; interacts with Crm1p in two-hybrid assay; YMR12...bud, and bud neck; interacts with Crm1p in two-hybrid assay; YMR124W is not an essential gene Rows with this

  2. Chromosomal instability in meningiomas.

    Science.gov (United States)

    van Tilborg, Angela A G; Al Allak, Bushra; Velthuizen, Sandra C J M; de Vries, Annie; Kros, Johan M; Avezaat, Cees J J; de Klein, Annelies; Beverloo, H Berna; Zwarthoff, Ellen C

    2005-04-01

    Approximately 60% of sporadic meningiomas are caused by inactivation of the NF2 tumor suppressor gene on chromosome 22. No causative gene is known for the remaining 40%. Cytogenetic analysis shows that meningiomas caused by inactivation of the NF2 gene can be divided into tumors that show monosomy 22 as the sole abnormality and tumors with a more complex karyotype. Meningiomas not caused by the NF2 gene usually have a diploid karyotype. Here we report that, besides the clonal chromosomal aberrations, the chromosome numbers in many meningiomas varied from one metaphase spread to the other, a feature that is indicative of chromosomal instability. Unexpectedly and regardless of genotype, a subgroup of tumors was observed with an average number of 44.9 chromosomes and little variation in the number of chromosomes per metaphase spread. In addition, a second subgroup was recognized with a hyperdiploid number of chromosomes (average 48.5) and considerable variation in numbers per metaphase. However, this numerical instability resulted in a clonal karyotype with chromosomal gains and losses in addition to loss of chromosome 22 only in meningiomas caused by inactivation of the NF2 gene. In cultured cells of all tumor groups, bi- and multinucleated cells were seen, as well as anaphase bridges, residual chromatid strings, multiple spindle poles, and unseparated chromatids, suggesting defects in the mitotic apparatus or kinetochore. Thus, we conclude that even a benign and slow-growing tumor like a meningioma displays chromosomal instability.

  3. Taste bud development and patterning in sighted and blind morphs of Astyanax mexicanus.

    Science.gov (United States)

    Varatharasan, Nirupa; Croll, Roger P; Franz-Odendaal, Tamara

    2009-12-01

    In the blind cave-dwelling morph of A. mexicanus, the eye degenerates while other sensory systems, such as gustation, are expanded compared to their sighted (surface-dwelling) ancestor. This study compares the development of taste buds along the jaws of each morph. To determine whether cavefish have an altered onset or rate of taste bud development, we fluorescently labeled basal and receptor cells within taste buds over a developmental series. Our results show that taste bud number increases during development in both morphs. The rate of development is, however, accelerated in cavefish; a small difference in taste bud number exists at 5 dpf reaching threefold by 22 dpf. The expansion of taste buds in cavefish is, therefore, detectable after the onset of eye degeneration. This study provides important insights into the timing of taste bud expansion in cavefish as well as enhances our understanding of taste bud development in teleosts in general. (c) 2009 Wiley-Liss, Inc.

  4. RESEARCH OF SOPHORA JAPONICA L. FLOWER BUDS VOLATILE COMPOUNDS WITH GAS-CHROMATOGRAPHY/MASS- SPECTROMETRY METHOD

    Directory of Open Access Journals (Sweden)

    Cholak I.S.

    2013-10-01

    Full Text Available This work represents the results of the research ofessential oil contained in Sophora japonica L. flowerbuds volatile compounds collected during the nextstages of their development: green flower buds, formedflower buds and the beginning of flower buds opening.Essential oil assay content in Sophora japonica L.flower buds was determined with hydrodistillationmethod. Content of essential oil in the raw material isless than 0,1%. Qualitative composition and assaycontent of Sophora japonica L. flower buds essential oilconstituents were determined with chromato-massspectrometry method. In consequence of the research 80constituents were identified in Sophora japonica L.flower buds out of which 61 substances are during thegreen flower buds and beginning of flower budsopening stages, 66 substances are during formed flowerbuds stage. Substances are represented by aliphatic andcyclic terpenoids, their alcohols and ketones. Mostvolatile substances were extracted on the stage offormed buds.

  5. Analysis of plant meiotic chromosomes by chromosome painting.

    Science.gov (United States)

    Lysak, Martin A; Mandáková, Terezie

    2013-01-01

    Chromosome painting (CP) refers to visualization of large chromosome regions, entire chromosome arms, or entire chromosomes via fluorescence in situ hybridization (FISH). For CP in plants, contigs of chromosome-specific bacterial artificial chromosomes (BAC) from the target species or from a closely related species (comparative chromosome painting, CCP) are typically applied as painting probes. Extended pachytene chromosomes provide the highest resolution of CP in plants. CP enables identification and tracing of particular chromosome regions and/or entire chromosomes throughout all meiotic stages as well as corresponding chromosome territories in premeiotic interphase nuclei. Meiotic pairing and structural chromosome rearrangements (typically inversions and translocations) can be identified by CP. Here, we describe step-by-step protocols of CP and CCP in plant species including chromosome preparation, BAC DNA labeling, and multicolor FISH.

  6. Lager yeasts possess dynamic genomes that undergo rearrangements and gene amplification in response to stress.

    Science.gov (United States)

    James, Tharappel C; Usher, Jane; Campbell, Susan; Bond, Ursula

    2008-03-01

    A long-term goal of the brewing industry is to identify yeast strains with increased tolerance to the stresses experienced during the brewing process. We have characterised the genomes of a number of stress-tolerant mutants, derived from the lager yeast strain CMBS-33, that were selected for tolerance to high temperatures and to growth in high specific gravity wort. Our results indicate that the heat-tolerant strains have undergone a number of gross chromosomal rearrangements when compared to the parental strain. To determine if such rearrangements can spontaneously arise in response to exposure to stress conditions experienced during the brewing process, we examined the chromosome integrity of both the stress-tolerant strains and their parent during a single round of fermentation under a variety of environmental stresses. Our results show that the lager yeast genome shows tremendous plasticity during fermentation, especially when fermentations are carried out in high specific gravity wort and at higher than normal temperatures. Many localised regions of gene amplification were observed especially at the telomeres and at the rRNA gene locus on chromosome XII, and general chromosomal instability was evident. However, gross chromosomal rearrangements were not detected, indicating that continued selection in the stress conditions are required to obtain clonal isolates with stable rearrangements. Taken together, the data suggest that lager yeasts display a high degree of genomic plasticity and undergo genomic changes in response to environmental stress.

  7. Quantitative resistance to peanut bud necrosis tospovirus in groudnut.

    NARCIS (Netherlands)

    Buiel, A.A.M.

    1996-01-01

    Quantitative resistance to peanut bud necrosis virus (PBNV) is expressed as a reduced disease incidence (percentage of infected plants) in the groundnut crop. An increased plant density reduced this incidence, but the number of infected plants per unit area increased, maintaining high levels of PBNV

  8. In vitro PROLIFERATION ABILITY OF AXILLARY BUDS IN Musa spp

    African Journals Online (AJOL)

    AISA

    2University of Yaounde I, Faculty of Sciences, Laboratory of Biotechnology and ... As axillary buds have shown mass propagation abilities in Big Ebanga, this explant is tested .... The data obtained were subjected to a two-ways ANOVA to determine significant differences between .... «Kluaileb Mue Nang and the analysis of.

  9. Axillary Bud Proliferation Approach for Plant Biodiversity Conservation and Restoration

    Directory of Open Access Journals (Sweden)

    F. Ngezahayo

    2014-01-01

    Full Text Available Due to mainly human population pressure and activities, global biodiversity is getting reduced and particularly plant biodiversity is becoming at high risk of extinction. Consequently, many efforts have been deployed to develop conservation methods. Because it does not involve cell dedifferentiation of differentiated cells but rather the development and growth of new shoots from preexisting meristems, the axillary bud proliferation approach is the method offering least risk of genetic instability. Indeed, meristems are more resistant to genetic changes than disorganized tissues. The present review explored through the scientific literature the axillary bud proliferation approach and the possible somaclonal variation that could arise from it. Almost genetic stability or low level of genetic variation is often reported. On the contrary, in a few cases studied to date, DNA methylation alterations often appeared in the progenies, showing epigenetic variations in the regenerated plants from axillary bud culture. Fortunately, epigenetic changes are often temporary and plants may revert to the normal phenotype. Thus, in the absence of genetic variations and the existence of reverting epigenetic changes over time, axillary bud culture can be adopted as an alternative nonconventional way of conserving and restoring of plant biodiversity.

  10. A New Compound from the Bud of Chrysanthemum indicum L.

    Institute of Scientific and Technical Information of China (English)

    2005-01-01

    A new bicyclic spiroketone was isolated from the bud of Chrysanthemum indicum L.The chemical structure was elucidated as (1R, 9S, 10S)-10-hydroxyl-8 (2', 4'-diynehexylidene)-9-isovaleryloxy-2, 7-dioxaspiro [5, 4] decane based on the X-ray crystallography.

  11. Small Molecule Inhibitors of the Candida albicans Budded-to-Hyphal Transition Act through Multiple Signaling Pathways

    Science.gov (United States)

    Midkiff, John; Borochoff-Porte, Nathan; White, Dylan; Johnson, Douglas I.

    2011-01-01

    The ability of the pathogenic yeast Candida albicans to interconvert between budded and hyphal growth states, herein termed the budded-to-hyphal transition (BHT), is important for C. albicans development and virulence. The BHT is under the control of multiple cell signaling pathways that respond to external stimuli, including nutrient availability, high temperature, and pH. Previous studies identified 21 small molecules that could inhibit the C. albicans BHT in response to carbon limitation in Spider media. However, the studies herein show that the BHT inhibitors had varying efficacies in other hyphal-inducing media, reflecting their varying abilities to block signaling pathways associated with the different media. Chemical epistasis analyses suggest that most, but not all, of the BHT inhibitors were acting through either the Efg1 or Cph1 signaling pathways. Notably, the BHT inhibitor clozapine, a FDA-approved drug used to treat atypical schizophrenia by inhibiting G-protein-coupled dopamine receptors in the brain, and several of its functional analogs were shown to act at the level of the Gpr1 G-protein-coupled receptor. These studies are the first step in determining the target and mechanism of action of these BHT inhibitors, which may have therapeutic anti-fungal utility in the future. PMID:21966518

  12. Small molecule inhibitors of the Candida albicans budded-to-hyphal transition act through multiple signaling pathways.

    Directory of Open Access Journals (Sweden)

    John Midkiff

    Full Text Available The ability of the pathogenic yeast Candida albicans to interconvert between budded and hyphal growth states, herein termed the budded-to-hyphal transition (BHT, is important for C. albicans development and virulence. The BHT is under the control of multiple cell signaling pathways that respond to external stimuli, including nutrient availability, high temperature, and pH. Previous studies identified 21 small molecules that could inhibit the C. albicans BHT in response to carbon limitation in Spider media. However, the studies herein show that the BHT inhibitors had varying efficacies in other hyphal-inducing media, reflecting their varying abilities to block signaling pathways associated with the different media. Chemical epistasis analyses suggest that most, but not all, of the BHT inhibitors were acting through either the Efg1 or Cph1 signaling pathways. Notably, the BHT inhibitor clozapine, a FDA-approved drug used to treat atypical schizophrenia by inhibiting G-protein-coupled dopamine receptors in the brain, and several of its functional analogs were shown to act at the level of the Gpr1 G-protein-coupled receptor. These studies are the first step in determining the target and mechanism of action of these BHT inhibitors, which may have therapeutic anti-fungal utility in the future.

  13. Mechanisms for chromosome segregation.

    Science.gov (United States)

    Bouet, Jean-Yves; Stouf, Mathieu; Lebailly, Elise; Cornet, François

    2014-12-01

    Bacteria face the problem of segregating their gigantic chromosomes without a segregation period restricted in time and space, as Eukaryotes do. Segregation thus involves multiple activities, general or specific of a chromosome region and differentially controlled. Recent advances show that these various mechanisms conform to a “pair and release” rule, which appears as a general rule in DNA segregation. We describe the latest advances in segregation of bacterial chromosomes with emphasis on the different pair and release mechanisms.

  14. The Precarious Prokaryotic Chromosome

    OpenAIRE

    Kuzminov, Andrei

    2014-01-01

    Evolutionary selection for optimal genome preservation, replication, and expression should yield similar chromosome organizations in any type of cells. And yet, the chromosome organization is surprisingly different between eukaryotes and prokaryotes. The nuclear versus cytoplasmic accommodation of genetic material accounts for the distinct eukaryotic and prokaryotic modes of genome evolution, but it falls short of explaining the differences in the chromosome organization. I propose that the t...

  15. Genome scale models of yeast: towards standardized evaluation and consistent omic integration

    DEFF Research Database (Denmark)

    Sanchez, Benjamin J.; Nielsen, Jens

    2015-01-01

    Genome scale models (GEMs) have enabled remarkable advances in systems biology, acting as functional databases of metabolism, and as scaffolds for the contextualization of high-throughput data. In the case of Saccharomyces cerevisiae (budding yeast), several GEMs have been published...... and are currently used for metabolic engineering and elucidating biological interactions. Here we review the history of yeast's GEMs, focusing on recent developments. We study how these models are typically evaluated, using both descriptive and predictive metrics. Additionally, we analyze the different ways...... in which all levels of omics data (from gene expression to flux) have been integrated in yeast GEMs. Relevant conclusions and current challenges for both GEM evaluation and omic integration are highlighted....

  16. Condensin-mediated remodeling of the mitotic chromatin landscape in fission yeast.

    Science.gov (United States)

    Kakui, Yasutaka; Rabinowitz, Adam; Barry, David J; Uhlmann, Frank

    2017-08-21

    The eukaryotic genome consists of DNA molecules far longer than the cells that contain them. They reach their greatest compaction during chromosome condensation in mitosis. This process is aided by condensin, a structural maintenance of chromosomes (SMC) family member. The spatial organization of mitotic chromosomes and how condensin shapes chromatin architecture are not yet fully understood. Here we use chromosome conformation capture (Hi-C) to study mitotic chromosome condensation in the fission yeast Schizosaccharomyces pombe. This showed that the interphase landscape characterized by small chromatin domains is replaced by fewer but larger domains in mitosis. Condensin achieves this by setting up longer-range, intrachromosomal DNA interactions, which compact and individualize chromosomes. At the same time, local chromatin contacts are constrained by condensin, with profound implications for local chromatin function during mitosis. Our results highlight condensin as a major determinant that changes the chromatin landscape as cells prepare their genomes for cell division.

  17. File list: NoD.Pan.05.AllAg.Pancreatic_bud [Chip-atlas[Archive

    Lifescience Database Archive (English)

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  1. Induction of ectopic taste buds by SHH reveals the competency and plasticity of adult lingual epithelium.

    Science.gov (United States)

    Castillo, David; Seidel, Kerstin; Salcedo, Ernesto; Ahn, Christina; de Sauvage, Frederic J; Klein, Ophir D; Barlow, Linda A

    2014-08-01

    Taste buds are assemblies of elongated epithelial cells, which are innervated by gustatory nerves that transmit taste information to the brain stem. Taste cells are continuously renewed throughout life via proliferation of epithelial progenitors, but the molecular regulation of this process remains unknown. During embryogenesis, sonic hedgehog (SHH) negatively regulates taste bud patterning, such that inhibition of SHH causes the formation of more and larger taste bud primordia, including in regions of the tongue normally devoid of taste buds. Here, using a Cre-lox system to drive constitutive expression of SHH, we identify the effects of SHH on the lingual epithelium of adult mice. We show that misexpression of SHH transforms lingual epithelial cell fate, such that daughter cells of lingual epithelial progenitors form cell type-replete, onion-shaped taste buds, rather than non-taste, pseudostratified epithelium. These SHH-induced ectopic taste buds are found in regions of the adult tongue previously thought incapable of generating taste organs. The ectopic buds are composed of all taste cell types, including support cells and detectors of sweet, bitter, umami, salt and sour, and recapitulate the molecular differentiation process of endogenous taste buds. In contrast to the well-established nerve dependence of endogenous taste buds, however, ectopic taste buds form independently of both gustatory and somatosensory innervation. As innervation is required for SHH expression by endogenous taste buds, our data suggest that SHH can replace the need for innervation to drive the entire program of taste bud differentiation.

  2. Possible role of catalase in post-dormancy bud break in grapevines.

    Science.gov (United States)

    Pérez, Francisco J; Lira, Waldo

    2005-03-01

    Changes in the activity of catalase (Cat) and in the levels of H2O2 were followed throughout dormancy in buds of grapevines (Vitis vinifera L.). In grapevines grown in the Elqui valley in Chile, a region with warm-winters, the activity of Cat increased during the recess period of buds, reaching a maximum and thereafter decreased to less than one third of its maximal activity. Three isoforms of Cat were detected in extracts of buds by native PAGE analysis, and the extracted activity was inhibited competitively by hydrogen cyanamide (HC), a potent bud-break agent. Furthermore, HC applications to field-grown grapevines in addition to the expected effect on advancing bud break, reduced the Cat activity during bud dormancy. Similar reductions were observed during dormancy in buds of grapevines grown in the Central valley in Chile, a region with temperate winters, suggesting that HC and winter chilling inhibits the activity of the main H2O2 degrading enzyme in grape buds. A transient rise in H2O2 levels preceded the release of buds from endodormancy, moreover, the peak of H2O2 and the onset of bud break occurred earlier in HC treated than in control grapevines, suggesting the participation of H2O2 as a signal molecule in the release of endodormancy in grape buds. The relationship between Cat inhibition, rise in H2O2 levels and initiation of bud break are discussed.

  3. Induction of ectopic taste buds by SHH reveals the competency and plasticity of adult lingual epithelium

    Science.gov (United States)

    Castillo, David; Seidel, Kerstin; Salcedo, Ernesto; Ahn, Christina; de Sauvage, Frederic J.; Klein, Ophir D.; Barlow, Linda A.

    2014-01-01

    Taste buds are assemblies of elongated epithelial cells, which are innervated by gustatory nerves that transmit taste information to the brain stem. Taste cells are continuously renewed throughout life via proliferation of epithelial progenitors, but the molecular regulation of this process remains unknown. During embryogenesis, sonic hedgehog (SHH) negatively regulates taste bud patterning, such that inhibition of SHH causes the formation of more and larger taste bud primordia, including in regions of the tongue normally devoid of taste buds. Here, using a Cre-lox system to drive constitutive expression of SHH, we identify the effects of SHH on the lingual epithelium of adult mice. We show that misexpression of SHH transforms lingual epithelial cell fate, such that daughter cells of lingual epithelial progenitors form cell type-replete, onion-shaped taste buds, rather than non-taste, pseudostratified epithelium. These SHH-induced ectopic taste buds are found in regions of the adult tongue previously thought incapable of generating taste organs. The ectopic buds are composed of all taste cell types, including support cells and detectors of sweet, bitter, umami, salt and sour, and recapitulate the molecular differentiation process of endogenous taste buds. In contrast to the well-established nerve dependence of endogenous taste buds, however, ectopic taste buds form independently of both gustatory and somatosensory innervation. As innervation is required for SHH expression by endogenous taste buds, our data suggest that SHH can replace the need for innervation to drive the entire program of taste bud differentiation. PMID:24993944

  4. File list: ALL.Pan.05.AllAg.Pancreatic_bud [Chip-atlas[Archive

    Lifescience Database Archive (English)

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  5. File list: NoD.Pan.20.AllAg.Pancreatic_bud [Chip-atlas[Archive

    Lifescience Database Archive (English)

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  6. File list: ALL.Pan.10.AllAg.Pancreatic_bud [Chip-atlas[Archive

    Lifescience Database Archive (English)

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  7. File list: NoD.Pan.50.AllAg.Pancreatic_bud [Chip-atlas[Archive

    Lifescience Database Archive (English)

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  8. Chromosome oscillations in mitosis

    Science.gov (United States)

    Campas, Otger

    2008-03-01

    Successful cell division necessitates a tight regulation of chromosome movement via the activity of molecular motors. Many of the key players at the origin of the forces generating the motion have been identified, but their spatial and temporal organization remains elusive. In animal cells, chromosomes periodically switch between phases of movement towards and away from the pole. This characteristic oscillatory behaviour cannot be explained by the current models of chromosome positioning and congression. We perform a self-contained theoretical analysis in which the motion of mono-oriented chromosomes results from the competition between the activity of the kinetochore and chromokinesin motors on the chromosome arms. Our analysis, consistent with the available experimental data, proposes that the interplay between the aster-like morphology of the spindle and the collective kinetics of molecular motors is at the origin of chromosome oscillations, positioning and congression. It provides a natural explanation for the so-called chromosome directional instability and for the mechanism by which chromosomes sense their position in space. In addition, we estimate the in vivo velocity of chromokinesins at vanishing load and propose new experiments to assess the mechanism at the origin of chromosome movement in cell division.

  9. Bacterial chromosome segregation.

    Science.gov (United States)

    Possoz, Christophe; Junier, Ivan; Espeli, Olivier

    2012-01-01

    Dividing cells have mechanisms to ensure that their genomes are faithfully segregated into daughter cells. In bacteria, the description of these mechanisms has been considerably improved in the recent years. This review focuses on the different aspects of bacterial chromosome segregation that can be understood thanks to the studies performed with model organisms: Escherichia coli, Bacillus subtilis, Caulobacter crescentus and Vibrio cholerae. We describe the global positionning of the nucleoid in the cell and the specific localization and dynamics of different chromosomal loci, kinetic and biophysic aspects of chromosome segregation are presented. Finally, a presentation of the key proteins involved in the chromosome segregation is made.

  10. Molecular tools and protocols for engineering the acid-tolerant yeast Zygosaccharomyces bailii as a potential cell factory.

    Science.gov (United States)

    Branduardi, Paola; Dato, Laura; Porro, Danilo

    2014-01-01

    Microorganisms offer a tremendous potential as cell factories, and they are indeed used by humans for centuries for biotransformations. Among them, yeasts combine the advantage of unicellular state with a eukaryotic organization, and, in the era of biorefineries, their biodiversity can offer solutions to specific process constraints. Zygosaccharomyces bailii, an ascomycetales budding yeast, is widely known for its peculiar tolerance to various stresses, among which are organic acids. Despite the possibility to apply with this yeast some of the molecular tools and protocols routinely used to manipulate Saccharomyces cerevisiae, adjustments and optimizations are necessary. Here, we describe in detail protocols for transformation, for target gene disruption or gene integration, and for designing episomal expression plasmids helpful for developing and further studying the yeast Z. bailii.

  11. Mapping yeast transcriptional networks.

    Science.gov (United States)

    Hughes, Timothy R; de Boer, Carl G

    2013-09-01

    The term "transcriptional network" refers to the mechanism(s) that underlies coordinated expression of genes, typically involving transcription factors (TFs) binding to the promoters of multiple genes, and individual genes controlled by multiple TFs. A multitude of studies in the last two decades have aimed to map and characterize transcriptional networks in the yeast Saccharomyces cerevisiae. We review the methodologies and accomplishments of these studies, as well as challenges we now face. For most yeast TFs, data have been collected on their sequence preferences, in vivo promoter occupancy, and gene expression profiles in deletion mutants. These systematic studies have led to the identification of new regulators of numerous cellular functions and shed light on the overall organization of yeast gene regulation. However, many yeast TFs appear to be inactive under standard laboratory growth conditions, and many of the available data were collected using techniques that have since been improved. Perhaps as a consequence, comprehensive and accurate mapping among TF sequence preferences, promoter binding, and gene expression remains an open challenge. We propose that the time is ripe for renewed systematic efforts toward a complete mapping of yeast transcriptional regulatory mechanisms.

  12. Investigating the interactions of yeast prions: [SWI+], [PSI+], and [PIN+].

    Science.gov (United States)

    Du, Zhiqiang; Li, Liming

    2014-06-01

    Multiple prion elements, which are transmitted as heritable protein conformations and often linked to distinct phenotypes, have been identified in the budding yeast, Saccharomyces cerevisiae. It has been shown that overproduction of a prion protein Swi1 can promote the de novo conversion of another yeast prion [PSI(+)] when Sup35 is co-overproduced. However, the mechanism underlying this Pin(+) ([PSI(+)] inducible) activity is not clear. Moreover, how the Swi1 prion ([SWI(+)]) interacts with other yeast prions is unknown. Here, we demonstrate that the Pin(+) activity associated with Swi1 overproduction is independent of Rnq1 expression or [PIN(+)] conversion. We also show that [SWI(+)] enhances the appearance of [PSI(+)] and [PIN(+)]. However, [SWI(+)] significantly compromises the Pin(+) activity of [PIN(+)] when they coexist. We further demonstrate that a single yeast cell can harbor three prions, [PSI(+)], [PIN(+)], and [SWI(+)], simultaneously. However, under this condition, [SWI(+)] is significantly destabilized. While the propensity to aggregate underlies prionogenesis, Swi1 and Rnq1 aggregates resulting from overproduction are usually nonheritable. Conversely, prion protein aggregates formed in nonoverexpressing conditions or induced by preexisting prion(s) are more prionogenic. For [PSI(+)] and [PIN(+)] de novo formation, heterologous "facilitators," such as preexisting [SWI(+)] aggregates, colocalize only with the newly formed ring-/rod-shaped Sup35 or Rnq1 aggregates, but not with the dot-shaped mature prion aggregates. Their colocalization frequency is coordinated with their prion inducibility, indicating that prion-prion interactions mainly occur at the early initiation stage. Our results provide supportive evidence for the cross-seeding model of prionogenesis and highlight a complex interaction network among prions in yeast.

  13. Fetal chromosome analysis: screening for chromosome disease?

    DEFF Research Database (Denmark)

    Philip, J; Tabor, Ann; Bang, J

    1983-01-01

    The aim of the study was to investigate the rationale of the current indications for fetal chromosome analysis. 5372 women had 5423 amniocentesis performed, this group constituting a consecutive sample at the chromosome laboratory, Rigshospitalet, Copenhagen from March 1973 to September 1980 (Group...... to women having amniocentesis, although considered not to have any increased risk of fetal chromosome abnormality (1390 pregnancies, group B). They were also compared with 750 consecutive pregnancies in women 25-34 years of age, in whom all heritable diseases were excluded (group C). The risk of unbalanced...... with women without elevated risk. Spontaneous abortion rate and prematurity rate did not differ from rates expected without amniocentesis. It is concluded that current indications may be characterized as a mixture of evident high risk factors and factors with only a minor influence on risk. Indications...

  14. The yeast telomerase RNA, TLC1, participates in two distinct modes of TLC1-TLC1 association processes in vivo

    OpenAIRE

    Tet Matsuguchi; Elizabeth Blackburn

    2016-01-01

    Telomerase core enzyme minimally consists of the telomerase reverse transcriptase domain-containing protein (Est2 in budding yeast S. cerevisiae) and telomerase RNA, which contains the template specifying the telomeric repeat sequence synthesized. Here we report that in vivo, a fraction of S. cerevisiae telomerase RNA (TLC1) molecules form complexes containing at least two molecules of TLC1, via two separable modes: one requiring a sequence in the 3′ region of the immature TLC1 precursor and ...

  15. [Fructose transporter in yeasts].

    Science.gov (United States)

    Lazar, Zbigniew; Dobrowolski, Adam; Robak, Małgorzata

    2014-01-01

    Study of hexoses transporter started with discovery of galactose permease in Saccharomyces cerevisiae. Glucose, fructose and mannose assimilation is assumed by numerous proteins encoded by different genes. To date over 20 hexoses transporters, belonging to Sugar Porter family and to Major Facilitator Superfamily, were known. Genome sequence analysis of Candida glabrata, Kluyveromyces lactis, Yarrowia lipolytica, S. cerevisaie and Debaryomyces hansenii reveled potential presence of 17-48 sugar porter proteins. Glucose transporters in S. cerevisiae have been already characterized. In this paper, hexoses transporters, responsible for assimilation of fructose by cells, are presented and compared. Fructose specific transporter are described for yeasts: Zygosaccharomyces rouxii, Zygosaccharomyces bailli, K. lactis, Saccharomyces pastorianus, S. cerevisiae winemaking strain and for fungus Botritys cinerea and human (Glut5p). Among six yeasts transporters, five are fructose specific, acting by facilitated diffusion or proton symport. Yeasts monosaccharides transporter studies allow understanding of sugars uptake and metabolism important aspects, even in higher eukaryotes cells.

  16. Reserve carbohydrates metabolism in the yeast Saccharomyces cerevisiae.

    Science.gov (United States)

    François, J; Parrou, J L

    2001-01-01

    Glycogen and trehalose are the two glucose stores of yeast cells. The large variations in the cell content of these two compounds in response to different environmental changes indicate that their metabolism is controlled by complex regulatory systems. In this review we present information on the regulation of the activity of the enzymes implicated in the pathways of synthesis and degradation of glycogen and trehalose as well as on the transcriptional control of the genes encoding them. cAMP and the protein kinases Snf1 and Pho85 appear as major actors in this regulation. From a metabolic point of view, glucose-6-phosphate seems the major effector in the net synthesis of glycogen and trehalose. We discuss also the implication of the recently elucidated TOR-dependent nutrient signalling pathway in the control of the yeast glucose stores and its integration in growth and cell division. The unexpected roles of glycogen and trehalose found in the control of glycolytic flux, stress responses and energy stores for the budding process, demonstrate that their presence confers survival and reproductive advantages to the cell. The findings discussed provide for the first time a teleonomic value for the presence of two different glucose stores in the yeast cell.

  17. L-arabinose fermenting yeast

    Science.gov (United States)

    Zhang, Min; Singh, Arjun; Knoshaug, Eric; Franden, Mary Ann; Jarvis, Eric; Suominen, Pirkko

    2010-12-07

    An L-arabinose utilizing yeast strain is provided for the production of ethanol by introducing and expressing bacterial araA, araB and araD genes. L-arabinose transporters are also introduced into the yeast to enhance the uptake of arabinose. The yeast carries additional genomic mutations enabling it to consume L-arabinose, even as the only carbon source, and to produce ethanol. Methods of producing ethanol include utilizing these modified yeast strains. ##STR00001##

  18. XYY chromosome anomaly and schizophrenia.

    Science.gov (United States)

    Rajagopalan, M; MacBeth, R; Varma, S L

    1998-02-07

    Sex chromosome anomalies have been associated with psychoses, and most of the evidence is linked to the presence of an additional X chromosome. We report a patient with XYY chromosome anomaly who developed schizophrenia.

  19. Electochemical detection of chromosome translocation

    DEFF Research Database (Denmark)

    Kwasny, Dorota; Dimaki, Maria; Silahtaroglu, Asli

    2014-01-01

    Cytogenetics is a study of the cell structure with a main focus on chromosomes content and their structure. Chromosome abnormalities, such as translocations may cause various genetic disorders and heametological malignancies. Chromosome translocations are structural rearrangements of two chromoso...

  20. Tumor budding is an independent adverse prognostic factor in pancreatic ductal adenocarcinoma.

    Science.gov (United States)

    O'Connor, Kate; Li-Chang, Hector H; Kalloger, Steven E; Peixoto, Renata D; Webber, Douglas L; Owen, David A; Driman, David K; Kirsch, Richard; Serra, Stefano; Scudamore, Charles H; Renouf, Daniel J; Schaeffer, David F

    2015-04-01

    Tumor budding is a well-established adverse prognostic factor in colorectal cancer. However, the significance and diagnostic reproducibility of budding in pancreatic carcinoma requires further study. We aimed to assess the prognostic significance of tumor budding in pancreatic ductal adenocarcinoma, determine its relationship with other clinicopathologic features, and assess interobserver variability in its diagnosis. Tumor budding was assessed in 192 archival cases of pancreatic ductal adenocarcinoma using hematoxylin and eosin (H&E) sections; tumor buds were defined as single cells or nonglandular clusters composed of <5 cells. The presence of budding was determined through assessment of all tumor-containing slides, and associations with clinicopathologic features and outcomes were analyzed. Six gastrointestinal pathologists participated in an interobserver variability study of 120 images of consecutive tumor slides stained with H&E and cytokeratin. Budding was present in 168 of 192 cases and was associated with decreased overall survival (P=0.001). On multivariable analysis, tumor budding was prognostically significantly independent of stage, grade, tumor size, nodal status, lymphovascular invasion, and perineural invasion. There was substantial agreement among pathologists in assessing the presence of tumor budding using both H&E (K=0.63) and cytokeratin (K=0.63) stains. The presence of tumor budding is an independent adverse prognostic factor in pancreatic ductal carcinoma. The assessment of budding with H&E is reliable and could be used to better risk stratify patients with pancreatic ductal adenocarcinoma.

  1. Membrane-elasticity model of Coatless vesicle budding induced by ESCRT complexes.

    Directory of Open Access Journals (Sweden)

    Bartosz Różycki

    Full Text Available The formation of vesicles is essential for many biological processes, in particular for the trafficking of membrane proteins within cells. The Endosomal Sorting Complex Required for Transport (ESCRT directs membrane budding away from the cytosol. Unlike other vesicle formation pathways, the ESCRT-mediated budding occurs without a protein coat. Here, we propose a minimal model of ESCRT-induced vesicle budding. Our model is based on recent experimental observations from direct fluorescence microscopy imaging that show ESCRT proteins colocalized only in the neck region of membrane buds. The model, cast in the framework of membrane elasticity theory, reproduces the experimentally observed vesicle morphologies with physically meaningful parameters. In this parameter range, the minimum energy configurations of the membrane are coatless buds with ESCRTs localized in the bud neck, consistent with experiment. The minimum energy configurations agree with those seen in the fluorescence images, with respect to both bud shapes and ESCRT protein localization. On the basis of our model, we identify distinct mechanistic pathways for the ESCRT-mediated budding process. The bud size is determined by membrane material parameters, explaining the narrow yet different bud size distributions in vitro and in vivo. Our membrane elasticity model thus sheds light on the energetics and possible mechanisms of ESCRT-induced membrane budding.

  2. Florigen is involved in axillary bud development at multiple stages in Arabidopsis.

    Science.gov (United States)

    Niwa, Masaki; Endo, Motomu; Araki, Takashi

    2013-11-01

    The wide variety of plant architectures is largely based on diverse and flexible modes of axillary shoot development. In Arabidopsis, floral transition (flowering) stimulates axillary bud development. The mechanism that links flowering and axillary bud development is, however, largely unknown. We recently showed that FLOWERING LOCUS T (FT) protein, which acts as florigen, promotes the phase transition of axillary meristems, whereas BRANCHED1 (BRC1) antagonizes the florigen action in axillary buds. Here, we present evidences for another possible role of florigen in axillary bud development. Ectopic overexpression of FT or another florigen gene TWIN SISTER OF FT (TSF) with LEAFY (LFY) induces ectopic buds at cotyledonary axils, confirming the previous proposal that these genes are involved in formation of axillary buds. Taken together with our previous report that florigen promotes axillary shoot elongation, we propose that florigen regulates axillary bud development at multiple stages to coordinate it with flowering in Arabidopsis.

  3. Tumor budding is a strong and reproducible prognostic marker in T3N0 colorectal cancer.

    LENUS (Irish Health Repository)

    Wang, Lai Mun

    2012-02-01

    BACKGROUND: Tumor budding along the advancing front of colorectal adenocarcinoma is an early event in the metastatic process. A reproducible, prognostic budding scoring system based on outcomes in early stage colorectal cancer has not been established. DESIGN: One hundred twenty-eight T3N0M0 colorectal carcinoma patients with known outcome were identified. Tumor budding was defined as isolated tumor cells or clusters of <5 cells at the invasive tumor front. Tumor bud counts were generated in 5 regions at 200x by 2 pathologists (conventional bud count method). The median bud count per case was used to divide cases into low (median=0) and high budding (median > or =1) groups. Forty cases were reevaluated to assess reproducibility using the conventional and a novel rapid bud count method. RESULTS: Fifty-seven (45%) carcinomas had high and 71 (55%) had low budding scores. High budding was associated with an infiltrative growth pattern (P<0.0001) and lymphovascular invasion (P=0.005). Five-year cancer-specific survival was significantly poorer in high compared with low budding groups: 63% versus 91%, respectively, P<0.0001. Multivariate analysis demonstrated tumor budding to be independently prognostic (hazard ratio=4.76, P<0.001). Interobserver agreement was at least equivalent comparing the conventional to the rapid bud count methods: 87.5% agreement (kappa=0.75) versus 92.5% agreement (kappa=0.85), respectively. CONCLUSIONS: Tumor budding is a strong, reproducible, and independent prognostic marker of outcome that is easily assessed on hematoxylin and eosin slides. This may be useful for identifying the subset of T3N0M0 patients at high risk of recurrence who may benefit from adjuvant therapy.

  4. Budding Transition of Asymmetric Two-component Lipid Domains

    CERN Document Server

    Wolff, Jean; Andelman, David

    2016-01-01

    We propose a model that accounts for the budding transition of asymmetric two-component lipid domains, where the two monolayers (leaflets) have different average compositions controlled by independent chemical potentials. Assuming a coupling between the local curvature and local lipid composition in each of the leaflets, we discuss the morphology and thermodynamic behavior of asymmetric lipid domains. The membrane free-energy contains three contributions: the bending energy, the line tension, and a Landau free-energy for a lateral phase separation. Within a mean-field treatment, we obtain various phase diagrams containing fully budded, dimpled, and flat states as a function of the two leaflet compositions. The global phase behavior is analyzed, and depending on system parameters, the phase diagrams include one-phase, two-phase and three-phase regions. In particular, we predict various phase coexistence regions between different morphologies of domains, which may be observed in multi-component membranes or ves...

  5. Proteolytic activities in yeast.

    Science.gov (United States)

    Saheki, T; Holzer, H

    1975-03-28

    Studies on the mechanism and time course of the activation of proteinases A (EC 3.4.23.8), B (EC 3.4.22.9) and C (EC 3.4.12.--) in crude yeast extracts at pH 5.1 and 25 degrees C showed that the increase in proteinase B activity is paralleled with the disappearance of proteinase B inhibitor. Addition of purified proteinase A to fresh crude extracts accelerates the inactivation of the proteinase B inhibitor and the appearance of maximal activities of proteinases B and C. The decrease of proteinase B inhibitor activity and the increase of proteinase B activity are markedly retarded by the addition of pepstatin. Because 10-minus 7 M pepstatin completely inhibits proteinase A without affecting proteinase B activity, this is another indication for the role of proteinase A during the activation of proteinase B. Whereas extracts of yeast grown on minimal medium reached maximal activation of proteinases B and C after 20 h of incubation at pH 5.1 and 25 degrees C, extracts of yeast grown on complete medium had to be incubated for about 100 h. In the latter case, the addition of proteinas A results in maximal activation of proteinases B and C and disappearance of proteinase B inhibitor activity only after 10--20 h of incubation. With the optimal conditions, the maximal activities of proteinases A, B and C, as well as of the proteinase B inhibitor, were determined in crude extracts of yeast that had been grown batchwise for different lengths of time either on minimal or on complete medium. Upon incubation, all three proteinases were activated by several times their initial activity. This reflects the existence of proteolytically degradable inhibitors of the three proteinases and together with the above mentioned observations it demonstrates that the "activation" of yeast proteinases A, B and C upon incubation results from the proteolytic digestion of inhibitors rather than from activation of inactive zymogens by limited proteolysis.

  6. L-arabinose fermenting yeast

    Energy Technology Data Exchange (ETDEWEB)

    Zhang, Min; Singh, Arjun; Suominen, Pirkko; Knoshaug, Eric; Franden, Mary Ann; Jarvis, Eric

    2014-09-23

    An L-arabinose utilizing yeast strain is provided for the production of ethanol by introducing and expressing bacterial araA, araB and araD genes. L-arabinose transporters are also introduced into the yeast to enhance the uptake of arabinose. The yeast carries additional genomic mutations enabling it to consume L-arabinose, even as the only carbon source, and to produce ethanol. A yeast strain engineered to metabolize arabinose through a novel pathway is also disclosed. Methods of producing ethanol include utilizing these modified yeast strains.

  7. L-arabinose fermenting yeast

    Energy Technology Data Exchange (ETDEWEB)

    Zhang, Min; Singh, Arjun; Suominen, Pirkko; Knoshaug, Eric; Franden, Mary Ann; Jarvis, Eric

    2013-02-12

    An L-arabinose utilizing yeast strain is provided for the production of ethanol by introducing and expressing bacterial araA, araB and araD genes. L-arabinose transporters are also introduced into the yeast to enhance the uptake of arabinose. The yeast carries additional genomic mutations enabling it to consume L-arabinose, even as the only carbon source, and to produce ethanol. A yeast strain engineered to metabolize arabinose through a novel pathway is also disclosed. Methods of producing ethanol include utilizing these modified yeast strains.

  8. Endogenous peripheral neuromodulators of the mammalian taste bud.

    Science.gov (United States)

    Dando, Robin

    2010-10-01

    The sensitivity of the mammalian taste system displays a degree of plasticity based on short-term nutritional requirements. Deficiency in a particular substance may lead to a perceived increase in palatability of this substance, providing an additional drive to redress this nutritional imbalance through modification of intake. This alteration occurs not only in the brain but also, before any higher level processing has occurred, in the taste buds themselves. A brief review of recent advances is offered.

  9. Spiking Neural P Systems with Neuron Division and Budding

    OpenAIRE

    Pan, Linqiang; Paun, Gheorghe; Pérez Jiménez, Mario de Jesús

    2009-01-01

    In order to enhance the e±ciency of spiking neural P systems, we introduce the features of neuron division and neuron budding, which are processes inspired by neural stem cell division. As expected (as it is the case for P systems with active membranes), in this way we get the possibility to solve computationally hard problems in polynomial time. We illustrate this possibility with SAT problem.

  10. The analysis of mutant alleles of different strength reveals multiple functions of topoisomerase 2 in regulation of Drosophila chromosome structure.

    Science.gov (United States)

    Mengoli, Valentina; Bucciarelli, Elisabetta; Lattao, Ramona; Piergentili, Roberto; Gatti, Maurizio; Bonaccorsi, Silvia

    2014-10-01

    Topoisomerase II is a major component of mitotic chromosomes but its role in the assembly and structural maintenance of chromosomes is rather controversial, as different chromosomal phenotypes have been observed in various organisms and in different studies on the same organism. In contrast to vertebrates that harbor two partially redundant Topo II isoforms, Drosophila and yeasts have a single Topo II enzyme. In addition, fly chromosomes, unlike those of yeast, are morphologically comparable to vertebrate chromosomes. Thus, Drosophila is a highly suitable system to address the role of Topo II in the assembly and structural maintenance of chromosomes. Here we show that modulation of Top2 function in living flies by means of mutant alleles of different strength and in vivo RNAi results in multiple cytological phenotypes. In weak Top2 mutants, meiotic chromosomes of males exhibit strong morphological abnormalities and dramatic segregation defects, while mitotic chromosomes of larval brain cells are not affected. In mutants of moderate strength, mitotic chromosome organization is normal, but anaphases display frequent chromatin bridges that result in chromosome breaks and rearrangements involving specific regions of the Y chromosome and 3L heterochromatin. Severe Top2 depletion resulted in many aneuploid and polyploid mitotic metaphases with poorly condensed heterochromatin and broken chromosomes. Finally, in the almost complete absence of Top2, mitosis in larval brains was virtually suppressed and in the rare mitotic figures observed chromosome morphology was disrupted. These results indicate that different residual levels of Top2 in mutant cells can result in different chromosomal phenotypes, and that the effect of a strong Top2 depletion can mask the effects of milder Top2 reductions. Thus, our results suggest that the previously observed discrepancies in the chromosomal phenotypes elicited by Topo II downregulation in vertebrates might depend on slight differences

  11. The analysis of mutant alleles of different strength reveals multiple functions of topoisomerase 2 in regulation of Drosophila chromosome structure.

    Directory of Open Access Journals (Sweden)

    Valentina Mengoli

    2014-10-01

    Full Text Available Topoisomerase II is a major component of mitotic chromosomes but its role in the assembly and structural maintenance of chromosomes is rather controversial, as different chromosomal phenotypes have been observed in various organisms and in different studies on the same organism. In contrast to vertebrates that harbor two partially redundant Topo II isoforms, Drosophila and yeasts have a single Topo II enzyme. In addition, fly chromosomes, unlike those of yeast, are morphologically comparable to vertebrate chromosomes. Thus, Drosophila is a highly suitable system to address the role of Topo II in the assembly and structural maintenance of chromosomes. Here we show that modulation of Top2 function in living flies by means of mutant alleles of different strength and in vivo RNAi results in multiple cytological phenotypes. In weak Top2 mutants, meiotic chromosomes of males exhibit strong morphological abnormalities and dramatic segregation defects, while mitotic chromosomes of larval brain cells are not affected. In mutants of moderate strength, mitotic chromosome organization is normal, but anaphases display frequent chromatin bridges that result in chromosome breaks and rearrangements involving specific regions of the Y chromosome and 3L heterochromatin. Severe Top2 depletion resulted in many aneuploid and polyploid mitotic metaphases with poorly condensed heterochromatin and broken chromosomes. Finally, in the almost complete absence of Top2, mitosis in larval brains was virtually suppressed and in the rare mitotic figures observed chromosome morphology was disrupted. These results indicate that different residual levels of Top2 in mutant cells can result in different chromosomal phenotypes, and that the effect of a strong Top2 depletion can mask the effects of milder Top2 reductions. Thus, our results suggest that the previously observed discrepancies in the chromosomal phenotypes elicited by Topo II downregulation in vertebrates might depend on

  12. Chromosomal mosaicism goes global

    Directory of Open Access Journals (Sweden)

    Yurov Yuri B

    2008-11-01

    Full Text Available Intercellular differences of chromosomal content in the same individual are defined as chromosomal mosaicism (alias intercellular or somatic genomic variations or, in a number of publications, mosaic aneuploidy. It has long been suggested that this phenomenon poorly contributes both to intercellular (interindividual diversity and to human disease. However, our views have recently become to change due to a series of communications demonstrated a higher incidence of chromosomal mosaicism in diseased individuals (major psychiatric disorders and autoimmune diseases as well as depicted chromosomal mosaicism contribution to genetic diversity, the central nervous system development, and aging. The later has been produced by significant achievements in the field of molecular cytogenetics. Recently, Molecular Cytogenetics has published an article by Maj Hulten and colleagues that has provided evidences for chromosomal mosaicism to underlie formation of germline aneuploidy in human female gametes using trisomy 21 (Down syndrome as a model. Since meiotic aneuploidy is suggested to be the leading genetic cause of human prenatal mortality and postnatal morbidity, these data together with previous findings define chromosomal mosaicism not as a casual finding during cytogenetic analyses but as a more significant biological phenomenon than previously recognized. Finally, the significance of chromosomal mosaicism can be drawn from the fact, that this phenomenon is involved in genetic diversity, normal and abnormal prenatal development, human diseases, aging, and meiotic aneuploidy, the intrinsic cause of which remains, as yet, unknown.

  13. Sequential cloning of chromosomes

    Science.gov (United States)

    Lacks, S.A.

    1995-07-18

    A method for sequential cloning of chromosomal DNA of a target organism is disclosed. A first DNA segment homologous to the chromosomal DNA to be sequentially cloned is isolated. The first segment has a first restriction enzyme site on either side. A first vector product is formed by ligating the homologous segment into a suitably designed vector. The first vector product is circularly integrated into the target organism`s chromosomal DNA. The resulting integrated chromosomal DNA segment includes the homologous DNA segment at either end of the integrated vector segment. The integrated chromosomal DNA is cleaved with a second restriction enzyme and ligated to form a vector-containing plasmid, which is replicated in a host organism. The replicated plasmid is then cleaved with the first restriction enzyme. Next, a DNA segment containing the vector and a segment of DNA homologous to a distal portion of the previously isolated DNA segment is isolated. This segment is then ligated to form a plasmid which is replicated within a suitable host. This plasmid is then circularly integrated into the target chromosomal DNA. The chromosomal DNA containing the circularly integrated vector is treated with a third, retrorestriction (class IIS) enzyme. The cleaved DNA is ligated to give a plasmid that is used to transform a host permissive for replication of its vector. The sequential cloning process continues by repeated cycles of circular integration and excision. The excision is carried out alternately with the second and third enzymes. 9 figs.

  14. Arenavirus budding: a common pathway with mechanistic differences.

    Science.gov (United States)

    Wolff, Svenja; Ebihara, Hideki; Groseth, Allison

    2013-01-31

    The Arenaviridae is a diverse and growing family of viruses that includes several agents responsible for important human diseases. Despite the importance of this family for public health, particularly in Africa and South America, much of its biology remains poorly understood. However, in recent years significant progress has been made in this regard, particularly relating to the formation and release of new enveloped virions, which is an essential step in the viral lifecycle. While this process is mediated chiefly by the viral matrix protein Z, recent evidence suggests that for some viruses the nucleoprotein (NP) is also required to enhance the budding process. Here we highlight and compare the distinct budding mechanisms of different arenaviruses, concentrating on the role of the matrix protein Z, its known late domain sequences, and the involvement of cellular endosomal sorting complex required for transport (ESCRT) pathway components. Finally we address the recently described roles for the nucleoprotein NP in budding and ribonucleoprotein complex (RNP) incorporation, as well as discussing possible mechanisms related to its involvement.

  15. Arenavirus Budding: A Common Pathway with Mechanistic Differences

    Directory of Open Access Journals (Sweden)

    Svenja Wolff

    2013-01-01

    Full Text Available The Arenaviridae is a diverse and growing family of viruses that includes several agents responsible for important human diseases. Despite the importance of this family for public health, particularly in Africa and South America, much of its biology remains poorly understood. However, in recent years significant progress has been made in this regard, particularly relating to the formation and release of new enveloped virions, which is an essential step in the viral lifecycle. While this process is mediated chiefly by the viral matrix protein Z, recent evidence suggests that for some viruses the nucleoprotein (NP is also required to enhance the budding process. Here we highlight and compare the distinct budding mechanisms of different arenaviruses, concentrating on the role of the matrix protein Z, its known late domain sequences, and the involvement of cellular endosomal sorting complex required for transport (ESCRT pathway components. Finally we address the recently described roles for the nucleoprotein NP in budding and ribonucleoprotein complex (RNP incorporation, as well as discussing possible mechanisms related to its involvement.

  16. Bud dormancy in apple trees after thermal fluctuations

    Directory of Open Access Journals (Sweden)

    Rafael Anzanello

    2014-06-01

    Full Text Available The objective of this work was to evaluate the effect of heat waves on the evolution of bud dormancy, in apple trees with contrasting chilling requirements. Twigs of 'Castel Gala' and 'Royal Gala' were collected in orchards in Papanduva, state of Santa Catarina, Brazil, and were exposed to constant (3°C or alternating (3 and 15°C for 12/12 hours temperature, combined with zero, one or two days a week at 25°C. Two additional treatments were evaluated: constant temperature (3°C, with a heat wave of seven days at 25°C, in the beginning or in the middle of the experimental period. Periodically, part of the twigs was transferred to 25°C for daily budburst evaluation of apical and lateral buds. Endodormancy (dormancy induced by cold was overcome with less than 330 chilling hours (CH of constant cold in 'Castel Gala' and less than 618 CH in 'Royal Gala'. A daily 15°C-temperature cycle did not affect the endodormancy process. Heat waves during endodormancy resulted in an increased CH to achieve bud requirements. The negative effect of high temperature depended on the lasting of this condition. Chilling was partly cancelled during dormancy when the heat wave lasted 36 continuous hours or more. Therefore, budburst prediction models need adjustments, mainly for regions with mild and irregular winters, such as those of Southern Brazil.

  17. Ecological conditions favoring budding in colonial organisms under environmental disturbance.

    Directory of Open Access Journals (Sweden)

    Mayuko Nakamaru

    Full Text Available Dispersal is a topic of great interest in ecology. Many organisms adopt one of two distinct dispersal tactics at reproduction: the production of small offspring that can disperse over long distances (such as seeds and spawned eggs, or budding. The latter is observed in some colonial organisms, such as clonal plants, corals and ants, in which (superorganisms split their body into components of relatively large size that disperse to a short distance. Contrary to the common dispersal viewpoint, short-dispersal colonial organisms often flourish even in environments with frequent disturbances. In this paper, we investigate the conditions that favor budding over long-distance dispersal of small offspring, focusing on the life history of the colony growth and the colony division ratio. These conditions are the relatively high mortality of very small colonies, logistic growth, the ability of dispersers to peacefully seek and settle unoccupied spaces, and small spatial scale of environmental disturbance. If these conditions hold, budding is advantageous even when environmental disturbance is frequent. These results suggest that the demography or life history of the colony underlies the behaviors of the colonial organisms.

  18. Sequential cloning of chromosomes

    Energy Technology Data Exchange (ETDEWEB)

    Lacks, S.A.

    1991-12-31

    A method for sequential cloning of chromosomal DNA and chromosomal DNA cloned by this method are disclosed. The method includes the selection of a target organism having a segment of chromosomal DNA to be sequentially cloned. A first DNA segment, having a first restriction enzyme site on either side. homologous to the chromosomal DNA to be sequentially cloned is isolated. A first vector product is formed by ligating the homologous segment into a suitably designed vector. The first vector product is circularly integrated into the target organism`s chromosomal DNA. The resulting integrated chromosomal DNA segment includes the homologous DNA segment at either end of the integrated vector segment. The integrated chromosomal DNA is cleaved with a second restriction enzyme and ligated to form a vector-containing plasmid, which is replicated in a host organism. The replicated plasmid is then cleaved with the first restriction enzyme. Next, a DNA segment containing the vector and a segment of DNA homologous to a distal portion of the previously isolated DNA segment is isolated. This segment is then ligated to form a plasmid which is replicated within a suitable host. This plasmid is then circularly integrated into the target chromosomal DNA. The chromosomal DNA containing the circularly integrated vector is treated with a third, retrorestriction enzyme. The cleaved DNA is ligated to give a plasmid that is used to transform a host permissive for replication of its vector. The sequential cloning process continues by repeated cycles of circular integration and excision. The excision is carried out alternately with the second and third enzymes.

  19. Structure of the Bro1 domain protein BROX and functional analyses of the ALIX Bro1 domain in HIV-1 budding.

    Directory of Open Access Journals (Sweden)

    Qianting Zhai

    Full Text Available BACKGROUND: Bro1 domains are elongated, banana-shaped domains that were first identified in the yeast ESCRT pathway protein, Bro1p. Humans express three Bro1 domain-containing proteins: ALIX, BROX, and HD-PTP, which function in association with the ESCRT pathway to help mediate intraluminal vesicle formation at multivesicular bodies, the abscission stage of cytokinesis, and/or enveloped virus budding. Human Bro1 domains share the ability to bind the CHMP4 subset of ESCRT-III proteins, associate with the HIV-1 NC(Gag protein, and stimulate the budding of viral Gag proteins. The curved Bro1 domain structure has also been proposed to mediate membrane bending. To date, crystal structures have only been available for the related Bro1 domains from the Bro1p and ALIX proteins, and structures of additional family members should therefore aid in the identification of key structural and functional elements. METHODOLOGY/PRINCIPAL FINDINGS: We report the crystal structure of the human BROX protein, which comprises a single Bro1 domain. The Bro1 domains from BROX, Bro1p and ALIX adopt similar overall structures and share two common exposed hydrophobic surfaces. Surface 1 is located on the concave face and forms the CHMP4 binding site, whereas Surface 2 is located at the narrow end of the domain. The structures differ in that only ALIX has an extended loop that projects away from the convex face to expose the hydrophobic Phe105 side chain at its tip. Functional studies demonstrated that mutations in Surface 1, Surface 2, or Phe105 all impair the ability of ALIX to stimulate HIV-1 budding. CONCLUSIONS/SIGNIFICANCE: Our studies reveal similarities in the overall folds and hydrophobic protein interaction sites of different Bro1 domains, and show that a unique extended loop contributes to the ability of ALIX to function in HIV-1 budding.

  20. Flavour-active wine yeasts.

    Science.gov (United States)

    Cordente, Antonio G; Curtin, Christopher D; Varela, Cristian; Pretorius, Isak S

    2012-11-01

    The flavour of fermented beverages such as beer, cider, saké and wine owe much to the primary fermentation yeast used in their production, Saccharomyces cerevisiae. Where once the role of yeast in fermented beverage flavour was thought to be limited to a small number of volatile esters and higher alcohols, the discovery that wine yeast release highly potent sulfur compounds from non-volatile precursors found in grapes has driven researchers to look more closely at how choice of yeast can influence wine style. This review explores recent progress towards understanding the range of 'flavour phenotypes' that wine yeast exhibit, and how this knowledge has been used to develop novel flavour-active yeasts. In addition, emerging opportunities to augment these phenotypes by engineering yeast to produce so-called grape varietal compounds, such as monoterpenoids, will be discussed.