Phosphorylation governs the activity of many proteins. Insight into molecular mechanisms in biology would be immensely improved by robust, sensitive methods for identifying precisely sites phosphate addition. An approach to selective mapping protein phosphorylation on a specific target interest using LC-MS is described here. In this multiple reaction monitoring used as an extremely MS survey scan potential phosphopeptides from known protein. This automatically followed peptide sequencing and...

Mitotic exit in Saccharomyces cerevisiae and septation Schizosaccharomyces pombe are regulated by a conserved signaling network called the mitotic septum initiation networks (SIN), respectively. The is active on one of two anaphase B spindle-pole bodies (SPBs). Whereas inherent asymmetry growth budding accounts for elements S. cerevisiae, it has been unclear how, or why, pathway asymmetric pombe. We show that SPB duplication conservative, SIN new SPB. association with persists after...

Targeted alteration of the genome lies at heart exploitation S. pombe as a model system. The rate analysis is often determined by efficiency with which target locus can be manipulated. For most loci this not problem, however for some loci, such fin1+, rates gene targeting below 5% limit scope and scale manipulations that are feasible within reasonable time frame. We now describe simple modification transformation procedure directing integration genomic sequences leads to 5-fold increase in...

The fission yeast interphase spindle pole body (SPB) is a bipartite structure in which bulky cytoplasmic domain separated from nuclear component by the envelope. During mitosis, SPB incorporated into fenestra that forms within envelope during mitotic commitment. Closure of this anaphase B/mitotic exit returns to face an intact Here we show Brr6 transiently recruited SPBs at both insertion and extrusion. required for integrity exit. Genetic interactions with apq12 defective sterol...

PP1 holoenzymes are composed of a small number catalytic subunits and an array regulatory, targeting, subunits. The Schizosaccharomyces pombe genome encodes two highly related subunits, Dis2 Sds21. gene for either protein can be individually deleted, however, simultaneous deletion both is lethal. We fused enhanced green fluorescent (EGFP) coding sequences to the 5' end endogenous sds21(+) dis2(+) genes. Dis2.NEGFP accumulated in nuclei, associated with centromeres, foci at cell tips...

Strand-specific RNA sequencing of S. pombe revealed a highly structured programme ncRNA expression at over 600 loci. Waves antisense transcription accompanied sexual differentiation. A substantial proportion arose from mechanisms previously considered to be largely artefactual, including improper 3' termination and bidirectional transcription. Constitutive induction the entire spk1+, spo4+, dis1+ spo6+ transcripts an integrated, ectopic, locus disrupted their respective meiotic functions....

The Schizosaccharomyces pombe CLIP170-associated protein (CLASP) Peg1 was identified in a screen for mutants with spindle formation defects and molecules that antagonized EB1 function. conditional peg1.1 mutant enabled us to identify key features of First, required form astral microtubules, yet destabilized interphase microtubules. Second, slow the polymerization rate microtubules establish end-on contact cortex at cell tips. Third, action S. CLIP170 (Tip1) (Mal3). Fourth, although resembled...

Vigorous chromosome movements driven by cytoskeletal assemblies are a widely conserved feature of sexual differentiation to facilitate meiotic recombination. In fission yeast, this process involves the dramatic conversion arrays cytoplasmic microtubules (MTs), generated from multiple MT organizing centers (MTOCs), into single radial (rMT) array associated with spindle pole body (SPB), major MTOC during prophase. The rMT is then dissolved upon onset meiosis I when bipolar emerges conduct...

In carbon-depleted cultures of Penicillium chrysogenum, age-related chitinases were shown to play a crucial role in both autolysis and fragmentation as indicated by vivo enzyme inhibition experiments using allosamidin. This pseudotrisaccharide even hindered significantly the outgrowth new hyphal tips from surviving yeastlike fragments after glucose supplementation. The antifungal effect allosamidin on autolyzing P. chrysogenum mycelia was fungistatic rather than fungicidal. growing hyphae,...

Abstract Genome annotation is a synthesis of computational prediction and experimental evidence. Small genes are notoriously difficult to detect because the patterns used identify them often indistinguishable from chance occurrences, leading an arbitrary cutoff threshold for length protein-coding gene identified solely by in silico analysis. We report systematic reappraisal Schizosaccharomyces pombe genome that ignores thresholds. A complete six-frame translation was compared proteome data...

Schizosaccharomyces pombe is an attractive model organism with which to study core principles of conserved molecular cell biology processes. The ability monitor protein behavior following separation by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) underpins much this activity. Here we describe a robust protocol for the preparation samples analysis SDS-PAGE.

Schizosaccharomyces pombe cells are rod shaped, and they grow by tip elongation. Growth ceases during mitosis cell division; therefore, the length of a septated is direct measure timing mitotic commitment, wild-type an indicator its position in cycle. A large number documented stage-specific changes can be used as landmarks to characterize cycle progression under specific experimental conditions. Conditional mutations permanently or transiently block at almost any stage. Large, synchronously...

Summary: Fission yeast cells grow by extension at the ends (poles) and divide transverse fission. It has previously been reported that Schizosaccharomyces japonicus var. can switch to unipolar, filamentous growth. Here it is shown yeast-to-mycelium transition a gradual process involving changeover unipolar growth associated with asymmetric divisions, development of large polarly located vacuoles, modifications actin microtubular cytoskeleton repression cell separation after division. High...

Genetic analysis of 20 sterile mutants prone to form hyphae revealed 11 novel ste genes (sep6 sep16) Schizosaccharomyces pombe. None the was completely mycelial. Most formed branching and showed normal septation. Aberrant septal structures actin distribution were seen only at 36 degrees C. sep9-307, sep14-576 sep15-598 genetic interactions with sep1-1, a mutation in forkhead transcription factor homologue. Additional detected between sep6-194, cdc16-116, mutant allele an anaphase modulator...

Intracellular movement is a fundamental property of all cell types. Many organelles and molecules are actively transported throughout the cytoplasm by molecular motors, such as dimeric type V myosins. These possess long neck, which contains an IQ motif, that allow it to make 36-nm steps along actin polymer. Live imaging fission yeast myosin Myo52 reveals protein moves rapidly cytoplasm. Here, we describe analysis this have established distances on filaments in ATP-dependent manner at ∼0.5...

The fission yeast scaffold molecule Sid4 anchors the septum initiation network to spindle pole body (SPB, centrosome equivalent) control mitotic exit events. A second SPB-associated scaffold, Cut12, promotes Cdk1–cyclin B drive commitment. Signals emanating from each have been assumed operate independently promote two distinct outcomes. We now find that signals contribute Cut12 commitment switch. Specifically, phosphorylation of by NIMAFin1 reduces affinity for its SPB anchor, Ppc89, while...

ABSTRACT sep1 + encodes a Schizosaccharomyces pombe homolog of the HNF-3/forkhead family tissue-specific and developmental gene regulators identified in higher eukaryotes. Its mutant allele sep1-1 causes defect cytokinesis confers mycelial morphology. Here we report on genetic interactions with M-phase initiation mutations wee1 − , cdc2-1w cdc25-22 . The double mutants form dikaryon cells at high frequency, which is due to nuclear division absence cell division. dikaryosis reversible...

Transient inactivation of the cdc25 + gene product by manipulation culture temperature for cdc25-22 cells is most commonly exploited approach to mitotic synchronization in fission yeast. Because Cdc25 removes inhibitory phosphate placed on Cdk1 Wee1, arrests at G 2 /M boundary. Incubation restrictive 36°C just over one generation time forces all accumulate Restoration function via a return permissive or chemical inhibition Wee1 activity can then promote highly synchronous wave cell division...