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Author McPheeters, D.S.; Wise, J.A. file  url
openurl 
  Title Measurement of in vivo RNA synthesis rates Type Journal Article
  Year 2013 Publication Methods in Enzymology Abbreviated Journal Methods Enzymol  
  Volume 530 Issue Pages 117-135  
  Keywords Gene Expression Regulation, Fungal; RNA, Fungal/*genetics; Saccharomyces cerevisiae/*genetics; Schizosaccharomyces/*genetics; Transcription, Genetic; Immobilized DNA/RNA; Immobilized probes; In vivo RNA synthesis rates; Labeled RNA; Nascent transcripts  
  Abstract A technique is described to directly measure ongoing transcription from individual genes in permeabilized cells of either the budding yeast Saccharomyces cerevisiae or the fission yeast Schizosaccharomyces pombe. Transcription run-on (TRO) analysis is used to compare the relative rates of synthesis for specific transcripts in cells grown under different environmental conditions or harvested at different stages of development. As the amount of an individual RNA species present at any given time is determined by its net rate of synthesis and degradation, an accurate picture of transcription per se can be obtained only by directly measuring de novo synthesis of RNA (if you are interested in RNA degradation, see Method for measuring mRNA decay rate in Saccharomyces cerevisiae). Most techniques employed to measure changes in the relative levels of individual transcripts present under different conditions, including Northern analysis (see Northern blotting), RT-PCR (see Reverse-transcription PCR (RT-PCR)), nuclease protection assays (see Explanatory Chapter: Nuclease Protection Assays), and genome-wide assays, such as microarray analysis and high throughput RNA sequencing, measure changes in the steady-state level of a transcript, which may or may not reflect the actual changes in transcription of the gene. Recent studies carried out in fission yeast have demonstrated that increases in the steady-state level (accumulation) of many individual mRNAs occur without any significant changes in transcription rates (McPheeters et al., 2009), highlighting the important role of regulated RNA stability in determining gene expression programs (Harigaya et al., 2006).  
  Call Number Serial 1345  
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Author Durieu-Trautmann, O.; Delavier-Klutchko, C. file  url
openurl 
  Title Effect of ammonia and glutamine on macromolecule synthesis and breakdown during sporulation of Saccharomyces cerevisiae Type Journal Article
  Year 1977 Publication Biochemical and Biophysical Research Communications Abbreviated Journal Biochem Biophys Res Commun  
  Volume 79 Issue 2 Pages 438-442  
  Keywords  
  Abstract The effect of two known inhibitors of sporulation in yeast, ammonia and glutamine, on certain biochemical events during sporogenesis have been studied using sporulating and non sporulating cells. Both strains gave similar results on the increase in dry cell weight, protein and RNA breakdown and the suppression of the intensive RNA and protein syntheses occurring after 4 hours. The inhibitory effect of ammonia and glutamine on RNA and protein syntheses is reversible under the same conditions which do so for sporulation.

Subject Headings: Ammonia/*pharmacology; DNA/biosynthesis; Fungal Proteins/biosynthesis; Glutamine/*pharmacology; Kinetics; RNA/biosynthesis; Saccharomyces cerevisiae/drug effects/*metabolism; Spores, Fungal/drug effects/metabolism
 
  Call Number Serial 2319  
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Author Wiltzius, J.J.W.; Hohl, M.; Fleming, J.C.; Petrini, J.H.J. file  url
openurl 
  Title The Rad50 hook domain is a critical determinant of Mre11 complex functions Type Journal Article
  Year 2005 Publication Nature Structural & Molecular Biology Abbreviated Journal Nat Struct Mol Biol  
  Volume 12 Issue 5 Pages 403-407  
  Keywords Cell Division; DNA, Fungal/genetics/metabolism; DNA-Binding Proteins/*chemistry/genetics/*metabolism; Endodeoxyribonucleases/*metabolism; Exodeoxyribonucleases/*metabolism; Ligands; Meiosis/genetics; Mutation/genetics; Phenotype; Protein Binding; Protein Structure, Tertiary; Recombination, Genetic/genetics; Saccharomyces cerevisiae/genetics/*metabolism; Saccharomyces cerevisiae Proteins/*chemistry/genetics/*metabolism; Telomere/metabolism  
  Abstract The Mre11 complex (in Saccharomyces cerevisiae: Mre11, Rad50 and Xrs2) influences multiple facets of chromosome break metabolism. A conserved feature of the Mre11 complex is a zinc-coordinating motif in Rad50 called the Rad50 hook. We established a diploid yeast strain, rad50(hook), in which Rad50 is encoded in halves, one from each of the two RAD50 alleles, with the residues constituting the hook deleted. In all respects, rad50(hook) phenocopies complete Rad50 deficiency. Replacing the hook domain with a ligand-inducible FKBP dimerization cassette partially mitigated all phenotypes in a ligand-dependent manner. The data indicate that the Rad50 hook is critical for Mre11 complex-dependent DNA repair, telomere maintenance and meiotic double-strand break formation. Sister chromatid cohesion was unaffected by Rad50 deficiency, suggesting that molecular bridging required for recombinational DNA repair is qualitatively distinct from cohesin-mediated sister chromatid cohesion.  
  Call Number Serial 1716  
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Author Schmidt, B.H.; Osheroff, N.; Berger, J.M. file  url
openurl 
  Title Structure of a topoisomerase II-DNA-nucleotide complex reveals a new control mechanism for ATPase activity Type Journal Article
  Year 2012 Publication Nature Structural & Molecular Biology Abbreviated Journal Nat Struct Mol Biol  
  Volume 19 Issue 11 Pages 1147-1154  
  Keywords Adenylyl Imidodiphosphate/*chemistry/metabolism; Amino Acid Sequence; Antigens, Neoplasm/*chemistry/metabolism; Chromatography, Gel; Crystallization; DNA/*chemistry/metabolism; DNA Topoisomerases, Type II/*chemistry/metabolism; DNA-Binding Proteins/*chemistry/metabolism; Dimerization; *Models, Molecular; Molecular Sequence Data; Multiprotein Complexes/*chemistry/metabolism; *Protein Conformation; Saccharomyces cerevisiae/*enzymology  
  Abstract Type IIA topoisomerases control DNA supercoiling and disentangle chromosomes through a complex ATP-dependent strand-passage mechanism. Although a general framework exists for type IIA topoisomerase function, the architecture of the full-length enzyme has remained undefined. Here we present the structure of a fully catalytic Saccharomyces cerevisiae topoisomerase II homodimer complexed with DNA and a nonhydrolyzable ATP analog. The enzyme adopts a domain-swapped configuration wherein the ATPase domain of one protomer sits atop the nucleolytic region of its partner subunit. This organization produces an unexpected interaction between bound DNA and a conformational transducing element in the ATPase domain, which we show is critical for both DNA-stimulated ATP hydrolysis and global topoisomerase activity. Our data indicate that the ATPase domains pivot about each other to ensure unidirectional strand passage and that this state senses bound DNA to promote ATP turnover and enzyme reset.  
  Call Number Serial 2189  
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Author Williams, R.P.; Gott, C.L.; Qadri, S.M.; Scott, R.H. file  url
openurl 
  Title Influence of temperature of incubation and type of growth medium on pigmentation in Serratia marcescens Type Journal Article
  Year 1971 Publication Journal of Bacteriology Abbreviated Journal J Bacteriol  
  Volume 106 Issue 2 Pages 438-443  
  Keywords Anti-Bacterial Agents/*biosynthesis; Bacterial Proteins/biosynthesis; Bacteriological Techniques; Caseins; Cell Division; Chloramphenicol/pharmacology; *Culture Media; Hot Temperature; Hydrogen-Ion Concentration; Oxygen; Pigments, Biological/*biosynthesis; Prodigiosin/biosynthesis; Protein Hydrolysates; Pyrroles/*biosynthesis; Saccharomyces; Serratia marcescens/cytology/drug effects/growth & development/*metabolism; Spectrophotometry; *Temperature; Time Factors  
  Abstract Maximal amounts of prodigiosin were synthesized in either minimal or complete medium after incubation of cultures at 27 C for 7 days. Biosynthesis of prodigiosin began earlier and the range of temperature for formation was greater in complete medium. No prodigiosin was formed in either medium when cultures were incubated at 38 C; however, after a shift to 27 C, pigmentation ensued, provided the period of incubation at 38 C was not longer than 36 hr for minimal medium or 48 hr for complete medium. Washed, nonpigmented cells grown in either medium at 38 C for 72 hr could synthesize prodigiosin when suspended in saline at 27 C when casein hydrolysate was added. These suspensions produced less prodigiosin at a slower rate than did cultures growing in casein hydrolysate at 27 C without prior incubation at 38 C. Optimal concentration of casein hydrolysate for pigment formation by suspensions was 0.4%; optimal temperature was 27 C. Anaerobic incubation, shift back to 38 C, killing cells by heating, or chloramphenicol (25 mug/ml) inhibited pigmentation. Suspensions of washed cells forming pigment reached pH 8.0 to 8.3 rapidly and maintained this pH throughout incubation for 7 days. Measurements of viable count and of protein, plus other data, indicated that cellular multiplication did not occur in suspensions of washed cells during pigment formation. By this procedure utilizing a shift down in temperature, biosynthesis of prodigiosin by washed cells could be separated from multiplication of bacteria.  
  Call Number Serial 1615  
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Author Westermann, S.; Wang, H.-W.; Avila-Sakar, A.; Drubin, D.G.; Nogales, E.; Barnes, G. file  url
openurl 
  Title The Dam1 kinetochore ring complex moves processively on depolymerizing microtubule ends Type Journal Article
  Year 2006 Publication Nature Abbreviated Journal Nature  
  Volume 440 Issue 7083 Pages 565-569  
  Keywords  
  Abstract Chromosomes interact through their kinetochores with microtubule plus ends and they are segregated to the spindle poles as the kinetochore microtubules shorten during anaphase A of mitosis. The molecular natures and identities of coupling proteins that allow microtubule depolymerization to pull chromosomes to poles during anaphase have long remained elusive. In budding yeast, the ten-protein Dam1 complex is a critical microtubule-binding component of the kinetochore that oligomerizes into a 50-nm ring around a microtubule in vitro. Here we show, with the use of a real-time, two-colour fluorescence microscopy assay, that the ring complex moves processively for several micrometres at the ends of depolymerizing microtubules without detaching from the lattice. Electron microscopic analysis of 'end-on views' revealed a 16-fold symmetry of the kinetochore rings. This out-of-register arrangement with respect to the 13-fold microtubule symmetry is consistent with a sliding mechanism based on an electrostatically coupled ring-microtubule interface. The Dam1 ring complex is a molecular device that can translate the force generated by microtubule depolymerization into movement along the lattice to facilitate chromosome segregation.

Subject Headings: Cell Cycle Proteins/*physiology; Chromosome Segregation/physiology; Kinetochores/*physiology/ultrastructure; Microscopy, Fluorescence; Microtubule-Associated Proteins/*physiology; Microtubules/*physiology/ultrastructure; Movement; Saccharomyces cerevisiae; Saccharomyces cerevisiae Proteins/*physiology; Spindle Apparatus/*physiology/ultrastructure
 
  Call Number Serial 2219  
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Author Bieling, P.; Laan, L.; Schek, H.; Munteanu, E.L.; Sandblad, L.; Dogterom, M.; Brunner, D.; Surrey, T. file  url
openurl 
  Title Reconstitution of a microtubule plus-end tracking system in vitro Type Journal Article
  Year 2007 Publication Nature Abbreviated Journal Nature  
  Volume 450 Issue 7172 Pages 1100-1105  
  Keywords  
  Abstract The microtubule cytoskeleton is essential to cell morphogenesis. Growing microtubule plus ends have emerged as dynamic regulatory sites in which specialized proteins, called plus-end-binding proteins (+TIPs), bind and regulate the proper functioning of microtubules. However, the molecular mechanism of plus-end association by +TIPs and their ability to track the growing end are not well understood. Here we report the in vitro reconstitution of a minimal plus-end tracking system consisting of the three fission yeast proteins Mal3, Tip1 and the kinesin Tea2. Using time-lapse total internal reflection fluorescence microscopy, we show that the EB1 homologue Mal3 has an enhanced affinity for growing microtubule end structures as opposed to the microtubule lattice. This allows it to track growing microtubule ends autonomously by an end recognition mechanism. In addition, Mal3 acts as a factor that mediates loading of the processive motor Tea2 and its cargo, the Clip170 homologue Tip1, onto the microtubule lattice. The interaction of all three proteins is required for the selective tracking of growing microtubule plus ends by both Tea2 and Tip1. Our results dissect the collective interactions of the constituents of this plus-end tracking system and show how these interactions lead to the emergence of its dynamic behaviour. We expect that such in vitro reconstitutions will also be essential for the mechanistic dissection of other plus-end tracking systems.

Subject Heading: Cell-Free System; Heat-Shock Proteins/metabolism; Intermediate Filament Proteins/metabolism; Microscopy, Fluorescence; Microtubule-Associated Proteins/*metabolism; Microtubules/*chemistry/*metabolism; *Schizosaccharomyces/chemistry/cytology; Schizosaccharomyces pombe Proteins/metabolism
 
  Call Number Serial 2223  
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Author Heinisch, J.J. file  url
doi  openurl
  Title Baker's yeast as a tool for the development of antifungal kinase inhibitors--targeting protein kinase C and the cell integrity pathway Type Journal Article
  Year 2005 Publication Biochimica et Biophysica Acta Abbreviated Journal Biochim Biophys Acta  
  Volume 1754 Issue 1-2 Pages 171-182  
  Keywords Antifungal Agents/*chemistry/metabolism/pharmacology; Cell Cycle/*drug effects; Cell Wall/drug effects/metabolism; Enzyme Inhibitors/*chemistry/metabolism/pharmacology; Humans; MAP Kinase Signaling System/drug effects; Models, Biological; Protein Kinase C/*antagonists & inhibitors/chemistry/drug effects/metabolism; Protein Kinases/genetics/metabolism; Recombinant Fusion Proteins/chemistry/*metabolism; Saccharomyces cerevisiae/chemistry/enzymology/*metabolism; Saccharomyces cerevisiae Proteins/*antagonists & inhibitors/chemistry/drug effects/metabolism  
  Abstract Today, the yeast Saccharomyces cerevisiae is probably the best-studied eukaryotic organism. This review first focuses on the signaling process which is mediated by the unique yeast protein kinase C (Pkc1p) and a downstream mitogen-activated protein kinase (MAPK) cascade. This pathway ensures cellular integrity by sensing cell surface stress and controlling cell wall biosynthesis and progression through the cell cycle. The domain structure of Pkc1p is conserved from yeast to humans. A yeast system for heterologous expression of specific domains in a chimeric yeast/mammalian PKC enzyme (“domain shuffling”) is depicted. It is also proposed how this system could be employed for the study of protein kinase inhibitors in high-throughput screens. Moreover, a reporter assay that allows a quantitative readout of the activity of the cell integrity signaling pathway is introduced. Since a variety of protein kinases take part in the signal transduction, this broadens the range of targets for potential inhibitors.  
  Call Number Serial 554  
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Author Weaver, T.; Lees, M.; Zaitsev, V.; Zaitseva, I.; Duke, E.; Lindley, P.; McSweeny, S.; Svensson, A.; Keruchenko, J.; Keruchenko, I.; Gladilin, K.; Banaszak, L. file  url
openurl 
  Title Crystal structures of native and recombinant yeast fumarase Type Journal Article
  Year 1998 Publication Journal of Molecular Biology Abbreviated Journal J Mol Biol  
  Volume 280 Issue 3 Pages 431-442  
  Keywords Binding Sites; Crystallography, X-Ray; Fumarate Hydratase/*chemistry; Fungal Proteins/*chemistry; Models, Molecular; Polymers/chemistry; *Protein Conformation; Saccharomyces cerevisiae/*enzymology; Water/chemistry  
  Abstract Crystal structures for both native and recombinant forms of yeast fumarase from Saccharomyces cerevisiae have been completed to moderate resolution by two separate laboratories. The recombinant form was obtained by the construction of an expression plasmid for Escherichia coli. Despite a high level of amino acid sequence similarity, purification of the eukaryotic enzyme from the wild-type prokaryotic enzyme was feasible. The crystal structure of the native form, NY-fumarase, encompasses residues R22 through M484, while the recombinant form, RY-fumarase, consists of residues S27 through L485. Both crystal structures lack the N-terminal translocation segment. Each subunit of the homo-tetrameric protein has three domains. The active site is formed by segments from each of three polypeptide chains. The results of these studies on the eukaryotic proteins are unique, since the recombinant form was done in the absence of dicarboxylic acid and has an unoccupied active site. As a comparison, native fumarase was crystallized in the presence of the competitive inhibitor, meso-tartrate. Meso-tartrate occupies a position close to that of the bound citrate molecule found in the active site of the E. coli enzyme. This inhibitor participates in hydrogen bonding to an active-site water molecule. The independent determination of the two structures provides further evidence that an active-site water molecule may play an active role in the fumarase-catalyzed reaction.  
  Call Number Serial 1178  
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Author Downing, K.J.; Thomson, J.A. file  url
openurl 
  Title Introduction of the Serratia marcescens chiA gene into an endophytic Pseudomonas fluorescens for the biocontrol of phytopathogenic fungi Type Journal Article
  Year 2000 Publication Canadian Journal of Microbiology Abbreviated Journal Can J Microbiol  
  Volume 46 Issue 4 Pages 363-369  
  Keywords Chitinases/*genetics/metabolism; DNA-Binding Proteins/genetics/metabolism; Escherichia coli/genetics; Fabaceae/microbiology; *Pest Control, Biological; Plant Diseases/microbiology; Plants, Medicinal; Plasmids/genetics; Polymerase Chain Reaction/methods; Promoter Regions, Genetic; Pseudomonas fluorescens/*enzymology/*genetics/growth & development/isolation & purification; Repressor Proteins/genetics/metabolism; Rhizoctonia/*growth & development; *Saccharomyces cerevisiae Proteins; Serratia marcescens/enzymology/*genetics; *Telomere-Binding Proteins  
  Abstract An endophytic strain of Pseudomonas fluorescens was isolated from micropropagated apple plantlets and introduced into beans (Phaseolus vulgaris) via their root tips. It was shown to be present as an endophyte in the roots at a level of 1.2 x 10(5) CFU/g fresh weight. The gene coding for the major chitinase of Serratia marcescens, chiA, was cloned under the control of the tac promoter into the broad-host-range plasmid pKT240 and the integration vector pJFF350. Pseudomonas fluorescens carrying tacchiA either on the plasmid or integrated into the chromosome is an effective biocontrol agent of the phytopathogenic fungus Rhizoctonia solani on bean seedlings under plant growth chamber conditions.  
  Call Number Serial 1662  
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