SILAC-Based Proteomic Analysis of Meiosis in the Fission Yeast Schizosaccharomyces pombe.

Stable isotope labeling by amino acids in cell culture (SILAC) provides a powerful tool to quantify proteins and posttranslational modifications. Here we describe how to apply SILAC for protein identification and quantification in synchronous meiotic cultures induced by inactivation of the Pat1 kinase in the fission yeast Schizosaccharomyces pombe.

Identification of Nrl1 Domains Responsible for Interactions with RNA-Processing Factors and Regulation of Nrl1 Function by Phosphorylation.

Pre-mRNA splicing is a key process in the regulation of gene expression. In the fission yeast , Nrl1 regulates splicing and expression of several genes and non-coding RNAs, and also suppresses the accumulation of R-loops. Here, we report analysis of interactions between Nrl1 and selected RNA-processing proteins and regulation of Nrl1 function by phosphorylation.

Proteomic analysis of meiosis and characterization of novel short open reading frames in the fission yeast .

Meiosis is the process by which haploid gametes are produced from diploid precursor cells. We used stable isotope labeling by amino acids in cell culture (SILAC) to characterize the meiotic proteome in the fission yeast . We compared relative levels of proteins extracted from cells harvested around meiosis I with those of meiosis II, and proteins from premeiotic S phase with the interval between meiotic divisions, when S phase is absent.

Probing Mechanical Properties in Biology Using Brillouin Microscopy.

Brillouin microscopy can be used to map the mechanical properties of samples in a noncontact and label-free manner, with potential applications in cell biology. Here, we provide an overview of the underlying principles and technology as well as the current challenges and outlook.

Mutations that prevent methylation of cohesin render sensitivity to DNA damage in .

The canonical role of cohesin is to mediate sister chromatid cohesion. In addition, cohesin plays important roles in processes such as DNA repair and regulation of gene expression. Mounting evidence suggests that various post-translational modifications, including phosphorylation, acetylation and sumoylation regulate cohesin functions.

The evolutionary history of Saccharomyces species inferred from completed mitochondrial genomes and revision in the 'yeast mitochondrial genetic code'.

The yeast Saccharomyces are widely used to test ecological and evolutionary hypotheses. A large number of nuclear genomic DNA sequences are available, but mitochondrial genomic data are insufficient. We completed mitochondrial DNA (mtDNA) sequencing from Illumina MiSeq reads for all Saccharomyces species.

Dbl2 Regulates Rad51 and DNA Joint Molecule Metabolism to Ensure Proper Meiotic Chromosome Segregation.

To identify new proteins required for faithful meiotic chromosome segregation, we screened a Schizosaccharomyces pombe deletion mutant library and found that deletion of the dbl2 gene led to missegregation of chromosomes during meiosis. Analyses of both live and fixed cells showed that dbl2Δ mutant cells frequently failed to segregate homologous chromosomes to opposite poles during meiosis I. Removing Rec12 (Spo11 homolog) to eliminate meiotic DNA double-strand breaks (DSBs) suppressed the segregation defect in dbl2Δ cells, indicating that Dbl2 acts after the initiation of meiotic recombination.

Casein Kinase 1 and Phosphorylation of Cohesin Subunit Rec11 (SA3) Promote Meiotic Recombination through Linear Element Formation.

Proper meiotic chromosome segregation, essential for sexual reproduction, requires timely formation and removal of sister chromatid cohesion and crossing-over between homologs. Early in meiosis cohesins hold sisters together and also promote formation of DNA double-strand breaks, obligate precursors to crossovers. Later, cohesin cleavage allows chromosome segregation.

Post-zygotic sterility and cytonuclear compatibility limits in S. cerevisiae xenomitochondrial cybrids.

Nucleo-mitochondrial interactions, particularly those determining the primary divergence of biological species, can be studied by means of xenomitochondrial cybrids, which are cells where the original mitochondria are substituted by their counterparts from related species. Saccharomyces cerevisiae cybrids are prepared simply by the mating of the ρ(0) strain with impaired karyogamy and germinating spores from other Saccharomyces species and fall into three categories. Cybrids with compatible mitochondrial DNA (mtDNA) from Saccharomyces paradoxus CBS 432 and Saccharomyces cariocanus CBS 7994 are metabolically and genetically similar to cybrids containing mtDNA from various S.

Synchronized fission yeast meiosis using an ATP analog-sensitive Pat1 protein kinase.

Synchronous cultures are often indispensable for studying meiosis. Here we present an optimized protocol for induction of synchronous meiosis in the fission yeast Schizosaccharomyces pombe. Chemical inactivation of an ATP analog-sensitive form of the Pat1 kinase (pat1-as2) by adding the ATP analog 1-NM-PP1 in G1-arrested cells allows the induction of synchronous meiosis at optimal temperature (25°C).

Mal3, the Schizosaccharomyces pombe homolog of EB1, is required for karyogamy and for promoting oscillatory nuclear movement during meiosis.

Two successive rounds of chromosome segregation following a single round of DNA replication enable the production of haploid gametes during meiosis. In the fission yeast Schizosaccharomyces pombe, karyogamy is the process where the nuclei from 2 haploid cells fuse to create a diploid nucleus, which then undergoes meiosis to produce 4 haploid spores. By screening a collection of S.

Small chromosomes among Danish Candida glabrata isolates originated through different mechanisms.

We analyzed 192 strains of the pathogenic yeast Candida glabrata from patients, mainly suffering from systemic infection, at Danish hospitals during 1985-1999. Our analysis showed that these strains were closely related but exhibited large karyotype polymorphism. Nine strains contained small chromosomes, which were smaller than 0.

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.

New evidence that SAC can tolerate misaligned chromosomes in mouse oocytes.

Comment on: Sebestova J, et al. Cell Cycle 2012; 11:3011-8.

A complete sequence of Saccharomyces paradoxus mitochondrial genome that restores the respiration in S. cerevisiae.

We determined the complete sequence of 71 355-bp-long mitochondrial genome from Saccharomyces paradoxus entirely by direct sequencing of purified mitochondrial DNA (mtDNA). This mtDNA possesses the same features as its close relative Saccharomyces cerevisiae - A + T content 85.9%, set of genes coding for the three components of cytochrome oxidase, cytochrome b, three subunits of ATPase, both ribosomal subunits, gene for ribosomal protein, rnpB gene, tRNA package (24) and yeast genetic code.

Chromosome segregation: monopolin attracts condensin.

To segregate chromosomes properly, the cell must prevent merotely, an error that occurs when a single kinetochore is attached to microtubules emanating from both spindle poles. Recent evidence suggests that cooperation between Pcs1/Mde4 and condensin complexes plays an important role in preventing merotely.

Merotelic kinetochore attachment: causes and effects.

Accurate chromosome segregation depends on the proper attachment of sister kinetochores to microtubules emanating from opposite spindle poles. Merotelic kinetochore orientation is an error in which a single kinetochore is attached to microtubules emanating from both spindle poles. Despite correction mechanisms, merotelically attached kinetochores can persist until anaphase, causing chromatids to lag on the mitotic spindle and hindering their timely segregation.

Mitochondrial genome from the facultative anaerobe and petite-positive yeast Dekkera bruxellensis contains the NADH dehydrogenase subunit genes.

The progenitor of the Dekkera/Brettanomyces clade separated from the Saccharomyces/Kluyveromyces clade over 200 million years ago. However, within both clades, several lineages developed similar physiological traits. Both Saccharomyces cerevisiae and Dekkera bruxellensis are facultative anaerobes; in the presence of excess oxygen and sugars, they accumulate ethanol (Crabtree effect) and they both spontaneously generate respiratory-deficient mutants (petites).

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.

Geotrichum bryndzae sp. nov., a novel asexual arthroconidial yeast species related to the genus Galactomyces.

Ten strains of an asexual arthroconidial yeast species were isolated from Bryndza, a traditional Slovak artisanal sheep cheese, which was manufactured from raw milk during a 4-month summer production period at two Slovakian sites (the northern RuZomberok and the central-southern Tisovec areas). Sequence comparison of the D1/D2 domains of the large-subunit rRNA gene revealed that this yeast represents a novel species of the genus Geotrichum, which contains anamorphs of the ascogenous genus Galactomyces, for which the name Geotrichum bryndzae sp. nov.

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

In eukaryotes, the number and rough organization of chromosomes is well preserved within isolates of the same species. Novel chromosomes and loss of chromosomes are infrequent and usually associated with pathological events. Here, we analyzed 40 pathogenic isolates of a haploid and asexual yeast, Candida glabrata, for their genome structure and stability.

A second pathway to degrade pyrimidine nucleic acid precursors in eukaryotes.

Pyrimidine bases are the central precursors for RNA and DNA, and their intracellular pools are determined by de novo, salvage and catabolic pathways. In eukaryotes, degradation of uracil has been believed to proceed only via the reduction to dihydrouracil. Using a yeast model, Saccharomyces kluyveri, we show that during degradation, uracil is not reduced to dihydrouracil.

Transition of the ability to generate petites in the Saccharomyces/Kluyveromyces complex.

Petite-positivity - the ability to tolerate the loss of mtDNA - was examined after the treatment with ethidium bromide (EB) in over hundred isolates from the Saccharomyces/Kluyveromyces complex. The identity of petite mutants was confirmed by the loss of specific mtDNA DAPI staining patterns. Besides unequivocal petite-positive and petite-negative phenotypes, a few species exhibited temperature sensitive petite positive phenotype and petiteness of a few other species could be observed only at the elevated EB concentrations.