A knockout screen for protein kinases required for the proper meiotic segregation of chromosomes in the fission yeast Schizosaccharomyces pombe.

The reduction of chromosome number during meiosis is achieved by two successive rounds of chromosome segregation after just single round of DNA replication. To identify novel proteins required for the proper segregation of chromosomes during meiosis, we analyzed the consequences of deleting Schizosaccharomyces pombe genes predicted to encode protein kinases that are not essential for cell viability. We show that Mph1, a member of the Mps1 family of spindle assembly checkpoint kinases, is required to prevent meiosis I homolog non-disjunction.

Generation of a set of conditional analog-sensitive alleles of essential protein kinases in the fission yeast Schizosaccharomyces pombe.

The genome of the fission yeast Schizosaccharomyces pombe encodes for 17 protein kinases that are essential for viability. Studies of the essential kinases often require the use of mutant strains carrying conditional alleles. To inactivate these kinases conditionally, we applied a recently developed chemical genetic strategy.

Laser microsurgery provides evidence for merotelic kinetochore attachments in fission yeast cells lacking Pcs1 or Clr4.

In order to segregate chromosomes properly, the cell must prevent merotelic kinetochore attachment, an error that occurs when a single kinetochore is attached to microtubules emanating from both spindle poles. Merotelic kinetochore orientation represents a major mechanism of aneuploidy in mitotic mammalian cells and it is the primary mechanism of chromosome instability in cancer cells. Fission yeast mutants defective in putative microtubule-site clamp Pcs1/Mde4 or Clr4/Swi6-dependent centromeric heterochromatin display high frequencies of lagging chromosomes during anaphase.

Casein kinase 1 is required for efficient removal of Rec8 during meiosis I.

Segregation of chromosomes during meiosis depends on separase cleavage of Rec8, the meiosis-specific alpha-kleisin subunit of cohesin. We mapped Rec8 phosphorylation sites by mass spectrometry and show that Rec8 phosphorylation is required for proper chromosome disjunction during meiosis. We further show that the fission yeast casein kinase 1 (CK1) delta/epsilon isoforms Hhp1 and Hhp2 are required for full levels of Rec8 phosphorylation and for efficient removal of Rec8 at the onset of anaphase I.

S. pombe genome deletion project: an update.

The fission yeast Schizosaccharomyces pombe is a model organism used widely to study various aspects of eukaryotic biology. A collection of heterozygous diploid strains containing individual deletions in nearly all S. pombe genes has been created using a PCR based strategy.

High-throughput knockout screen in Schizosaccharomyces pombe identifies a novel gene required for efficient homolog disjunction during meiosis I.

Meiosis is the process which produces haploid gametes from diploid precursor cells. This reduction of chromosome number is achieved by two successive divisions. Whereas homologs segregate during meiosis I, sister chromatids segregate during meiosis II.

An improved strategy for tandem affinity purification-tagging of Schizosaccharomyces pombe genes.

Tandem affinity purification (TAP) is a method that allows rapid purification of native protein complexes. We developed an improved technique to fuse the fission yeast genes with a TAP tag. Our technique is based on tagging constructs that contain regions homologous to the target gene cloned into vectors carrying a TAP tag.

Solving the shugoshin puzzle.

Shugoshin proteins form a complex with protein phosphatase 2A (PP2A) that protects centromeric cohesin from separase-mediated cleavage during yeast meiosis I. Recent work shows that this mechanism is conserved from yeast to mammals. Importantly, a model emerges that explains a long-standing puzzle, namely why the shugoshin-PP2A complex mediates protection of centromeric cohesin from separase cleavage specifically during meiosis I, but not during meiosis II or mitosis.

What makes centromeric cohesion resistant to separase cleavage during meiosis I but not during meiosis II?

Segregation of chromosomes during meiosis I is triggered by separase cleavage of the cohesin's Rec8 subunit along chromosome arms. Centromeric cohesin is protected from separase cleavage during meiosis I by Sgo1/MEI-S332 proteins in complex with protein phosphatase 2A (PP2A). This retention of centromeric sister chromatid cohesion is essential for faithful segregation of chromatids during the second meiotic division.

Construction of conditional analog-sensitive kinase alleles in the fission yeast Schizosaccharomyces pombe.

Reversible protein phosphorylation is a major regulatory mechanism in a cell. A chemical-genetic strategy to conditionally inactivate protein kinases has been developed recently. Mutating a single residue in the ATP-binding pocket confers sensitivity to small-molecule inhibitors.

The kinetochore proteins Pcs1 and Mde4 and heterochromatin are required to prevent merotelic orientation.

Background: Accurate chromosome segregation depends on the establishment of correct-amphitelic-kinetochore orientation. Merotelic kinetochore orientation is an error that occurs when a single kinetochore attaches to microtubules emanating from opposite spindle poles, a condition that hinders segregation of the kinetochore to a spindle pole in anaphase. To avoid chromosome missegregation resulting from merotelic kinetochore orientation, cells have developed mechanisms to prevent or correct merotelic attachment.

Tandem affinity purification of functional TAP-tagged proteins from human cells.

Tandem affinity purification (TAP) is a generic two-step affinity purification protocol for isolation of TAP-tagged proteins together with associated proteins. We used bacterial artificial chromosome to heterologously express TAP-tagged murine Sgo1 protein in human HeLa cells. This allowed us to test the functionality of the Sgo1-TAP protein by RNA interference-mediated depletion of the endogenous human Sgo1.

High-throughput knockout screen in fission yeast.

We have designed the most efficient strategy to knock out genes in fission yeast Schizosaccharomyces pombe on a large scale. Our technique is based on knockout constructs that contain regions homologous to the target gene cloned into vectors carrying dominant drug-resistance markers. Most of the steps are carried out in a 96-well format, allowing simultaneous deletion of 96 genes in one batch.

Pichia pastoris "just in time" alternative respiration.

Alternative oxidases (Aox or Aod) are present in the mitochondria of plants, fungi and many types of yeast. These enzymes transfer electrons from the ubiquinol pool directly to oxygen without contributing to the proton transfer across the mitochondrial membrane. Alternative oxidases are involved in stress responses, programmed cell death and maintenance of the cellular redox balance.