Identification and refinement of two strong constitutive promoters for gene expression system of Schizosaccharomyces pombe

Fission yeast Schizosaccharomyces pombe shares various important properties with higher eukaryotes and is now considered a useful host for elevated production of mammalian proteins for medicinal applications. The full-length nmt1 promoter has been widely used as a strong promoter in S. pombe expression system. In the present study, the promoters of the eno101 and gpd3 genes in S. pombe were identified as strong constitutive promoters. For convenient applications in the plasmids of S. pombe, these promoters were refined to 276-bp eno and 273-bp gpd promoters by deleting undesired sequences and examining the expression of reporter genes including lacZ and xynA. Both the refined eno and gpd promoters provided approximately 1.5-fold higher expression of LacZ than nmt1 promoter. Furthermore, gene expression under the control of the eno or gpd promoter was not repressed by the components of YES medium while nmt1 promoter was inhibited by thiamine in yeast extract. Therefore, both eno and gpd promoters offer opportunities for efficient production of recombinant proteins by S. pombe in high cell-density fermentation.     

Assembly of normal actomyosin rings in the absence of Mid1p and cortical nodes in fission yeast

Cytokinesis in many eukaryotes depends on the function of an actomyosin contractile ring. The mechanisms regulating assembly and positioning of this ring are not fully understood. The fission yeast Schizosaccharomyces pombe divides using an actomyosin ring and is an attractive organism for the study of cytokinesis. Recent studies in S. pombe (Wu, J.Q., V. Sirotkin, D.R. Kovar, M. Lord, C.C. Beltzner, J.R. Kuhn, and T.D. Pollard. 2006. J. Cell Biol. 174:391–402; Vavylonis, D., J.Q. Wu, S. Hao, B. O'Shaughnessy, and T.D. Pollard. 2008. Science. 319:97–100) have suggested that the assembly of the actomyosin ring is initiated from a series of cortical nodes containing several components of this ring. These studies have proposed that actomyosin interactions bring together the cortical nodes to form a compacted ring structure. In this study, we test this model in cells that are unable to assemble cortical nodes. Although the cortical nodes play a role in the timing of ring assembly, we find that they are dispensable for the assembly of orthogonal actomyosin rings. Thus, a mechanism that is independent of cortical nodes is sufficient for the assembly of normal actomyosin rings.     

Defining the Epigenetic Mechanism of Asymmetric Cell Division of Schizosaccharomyces japonicus Yeast

A key question in developmental biology addresses the mechanism of asymmetric cell division. Asymmetry is crucial for generating cellular diversity required for development in multicellular organisms. As one of the potential mechanisms, chromosomally borne epigenetic difference between sister cells that changes mating/cell type has been demonstrated only in the Schizosaccharomyces pombe fission yeast. For technical reasons, it is nearly impossible to determine the existence of such a mechanism operating during embryonic development of multicellular organisms. Our work addresses whether such an epigenetic mechanism causes asymmetric cell division in the recently sequenced fission yeast, S. japonicus (with 36% GC content), which is highly diverged from the well-studied S. pombe species (with 44% GC content). We find that the genomic location and DNA sequences of the mating-type loci of S. japonicus differ vastly from those of the S. pombe species. Remarkably however, similar to S. pombe, the S. japonicus cells switch cell/mating type after undergoing two consecutive cycles of asymmetric cell divisions: only one among four “granddaughter” cells switches. The DNA-strand–specific epigenetic imprint at the mating-type locus1 initiates the recombination event, which is required for cellular differentiation. Therefore the S. pombe and S. japonicus mating systems provide the first two examples in which the intrinsic chirality of double helical structure of DNA forms the primary determinant of asymmetric cell division. Our results show that this unique strand-specific imprinting/segregation epigenetic mechanism for asymmetric cell division is evolutionary conserved. Motivated by these findings, we speculate that DNA-strand–specific epigenetic mechanisms might have evolved to dictate asymmetric cell division in diploid, higher eukaryotes as well.     

Pheromone communication in the fission yeast Schizosaccharomyces pombe

Conjugation between two haploid yeast cells is generally controlled by the reciprocal action of diffusible mating pheromones, cells of each mating type releasing pheromones that induce mating-specific changes in cells of the opposite type. Recent studies into pheromone signalling in the fission yeast Schizosaccharomyces pombe have revealed significant parallels with processes in higher eukaryotes and could provide the opportunity for investigating communication in an organism that is amenable to both biochemical and genetic manipulation.     

A Link between Aurora Kinase and Clp1/Cdc14 Regulation Uncovered by the Identification of a Fission Yeast Borealin-Like Protein

The chromosomal passenger complex (CPC) regulates various events in cell division. This complex is composed of a catalytic subunit, Aurora B kinase, and three nonenzymatic subunits, INCENP, Survivin, and Borealin. Together, these four subunits interdependently regulate CPC function, and they are highly conserved among eukaryotes. However, a Borealin homologue has never been characterized in the fission yeast, Schizosaccharomyces pombe. Here, we isolate a previously uncharacterized S. pombe protein through association with the Cdc14 phosphatase homologue, Clp1/Flp1, and identify it as a Borealin-like member of the CPC. Nbl1 (novel Borealin-like 1) physically associates with known CPC components, affects the kinase activity and stability of the S. pombe Aurora B homologue, Ark1, colocalizes with known CPC subunits during mitosis, and shows sequence similarity to human Borealin. Further analysis of the Clp1–Nbl1 interaction indicates that Clp1 requires CPC activity for proper accumulation at the contractile ring (CR). Consistent with this, we describe negative genetic interactions between mutant alleles of CPC and CR components. Thus, this study characterizes a fission yeast Borealin homologue and reveals a previously unrecognized connection between the CPC and the process of cytokinesis in S. pombe.     

Identification and characterization of a complex chromosomal replication origin in<Emphasis Type="Italic">Schizosaccharomyces pombe</Emphasis>

In the budding yeast,S. cerevisiae, two-dimensional (2D) gel electrophoresis techniques permit mapping of DNA replication origins to short stretches of DNA (±300 bp). In contrast, in mammalian cells andDrosophila, 2D gel techniques do not permit precise origin localization; the results have been interpreted to suggest that replication initiates in broad zones (several kbp or more). However, alternative techniques (replication timing, nascent strand polarity analysis, nascent strand size analysis) suggest that mammalian origins can be mapped to short DNA stretches, just likeS. cerevisiae origins. Because the fission yeast,Schizosaccharomyces pombe, resembles higher organisms in several ways to a greater extent than doesS. cerevisiae, we thought thatS. pombe replication origins might prove to resemble — and thus be helpful models for — animal cell origins. An attempt to test this possibility using 2D gel techiques resulted in identification of a replication origin near theura4 gene on chromosome III ofS. pombe. The 2D gel patterns produced by thisS. pombe origin indeed resemble the patterns produced by animal cell origins and show that theS. pombe origin cannot be precisely located. The data suggest an initiation zone of 3–5 kbp. Some aspects of the 2D gel patterns detected at theS. pombe origin cannot be explained by the rationale of initiation in broad zones, suggesting that future biochemical and genetic studies of this complex origin are likely to provide information useful in helping to understand the apparent conflict between the 2D gel mapping techniques and other mapping techniques at animal cell origins.     

The Spontaneous Mutation Rate in the Fission Yeast Schizosaccharomyces pombe

The rate at which new mutations arise in the genome is a key factor in the evolution and adaptation of species. Here we describe the rate and spectrum of spontaneous mutations for the fission yeast Schizosaccharomyces pombe, a key model organism with many similarities to higher eukaryotes. We undertook an ∼1700-generation mutation accumulation (MA) experiment with a haploid S. pombe, generating 422 single-base substitutions and 119 insertion-deletion mutations (indels) across the 96 replicates. This equates to a base-substitution mutation rate of 2.00 × 10⁻¹⁰ mutations per site per generation, similar to that reported for the distantly related budding yeast Saccharomyces cerevisiae. However, these two yeast species differ dramatically in their spectrum of base substitutions, the types of indels (S. pombe is more prone to insertions), and the pattern of selection required to counteract a strong AT-biased mutation rate. Overall, our results indicate that GC-biased gene conversion does not play a major role in shaping the nucleotide composition of the S. pombe genome and suggest that the mechanisms of DNA maintenance may have diverged significantly between fission and budding yeasts. Unexpectedly, CpG sites appear to be excessively liable to mutation in both species despite the likely absence of DNA methylation.     

Hydrogen peroxide-induced oxidative damages in <Emphasis Type="Italic">Schizosaccharomyces pombe</Emphasis>

Oxidative stress causes damage to proteins, lipids and nucleic acids, and thereby compromises cell viability. Some of the oxidative stress markers in an eukaryotic model organism, fission yeast Schizosaccharomyces pombe, were evaluated in this study. Intracellular oxidation, protein carbonyls, lipid peroxidation and reduced glutathione (GSH) levels were investigated in H₂O₂-treated and non-treated control cells. It was observed that increased H₂O₂ concentration proportionally lowered the cell number and increased the intracellular oxidation, lipid peroxidation and protein carbonyl levels in S. pombe. A dose-dependent decrease in GSH level was also detected. The fission yeast S. pombe is best known for its contribution to understanding of eukaryotic cell cycle control. S. pombe displays a different physiology from Saccharomyces cerevisiae in several ways and is thus probably more closely related to higher eukaryotes. The purpose of this study was to provide some data about the effects of hydrogen peroxide on the proteins and lipids in the fission yeast. The data obtained here is expected to constitute a basis for the further studies on redox balance and related processes in yeast and mammalian cells.     

Nonmedially assembled F-actin cables incorporate into the actomyosin ring in fission yeast

In many eukaryotes, cytokinesis requires the assembly and constriction of an actomyosin-based contractile ring. Despite the central role of this ring in cytokinesis, the mechanism of F-actin assembly and accumulation in the ring is not fully understood. In this paper, we investigate the mechanism of F-actin assembly during cytokinesis in Schizosaccharomyces pombe using lifeact as a probe to monitor actin dynamics. Previous work has shown that F-actin in the actomyosin ring is assembled de novo at the division site. Surprisingly, we find that a significant fraction of F-actin in the ring was recruited from formin-Cdc12p nucleated long actin cables that were generated at multiple nonmedial locations and incorporated into the ring by a combination of myosin II and myosin V activities. Our results, together with findings in animal cells, suggest that de novo F-actin assembly at the division site and directed transport of F-actin cables assembled elsewhere can contribute to ring assembly.     

Isolation and characterization of the fission yeast gene rpa42 +, which encodes a subunit shared by RNA polymerases I and III

Eukaryotic RNA polymerases I and III share two distinct α-related subunits that show limited homology to the α subunit of Escherichia coli RNA polymerase, which forms a homodimer to nucleate the assembly of prokaryotic RNA polymerase. To gain insight into the functions of α-related subunits in eukaryotes, we have previously identified the α-related small subunit RPA17 of RNA polymerase I (and III) in Schizosaccharomyces pombe, and have shown that it is a functional homolog of Saccharomyces cerevisiae AC19. In an extension of that study, we have now isolated and characterized rpa42 ⁺, which encodes the α-related large subunit RPA42 of S. pombe RNA polymerase I, by virtue of the fact that its product interacts with RPA17 in the yeast two-hybrid system. We have found that rpa42 ⁺ encodes a polypeptide with an apparent molecular mass of 42?kDa, which shows 58% identity to the AC40 subunit shared by RNA polymerases I and III in S. cerevisiae. Furthermore, we have shown that rpa42 ⁺ complements a temperature-sensitive mutation in RPC40 the gene that encodes AC40 in S. cerevisiae and which is essential for cell growth. Finally, we have shown that neither RPA42 nor RPA17 can self-associate. These results provide evidence that the two distinct α-related subunits, RPA42 and RPA17, of RNA polymerases I and III are functionally conserved between S. pombe and S. cerevisiae, and suggest that heterodimer formation between them is essential for the assembly of RNA polymerases I and III in eukaryotes.     

Role of Polo Kinase and Mid1p in Determining the Site of Cell Division in Fission Yeast

The fission yeast Schizosaccharomyces pombe divides symmetrically using a medial F-actin– based contractile ring to produce equal-sized daughter cells. Mutants defective in two previously described genes, mid1 and pom1, frequently divide asymmetrically. Here we present the identification of three new temperature-sensitive mutants defective in localization of the division plane. All three mutants have mutations in the polo kinase gene, plo1, and show defects very similar to those of mid1 mutants in both the placement and organization of the medial ring. In both cases, ring formation is frequently initiated near the cell poles, indicating that Mid1p and Plo1p function in recruiting medial ring components to the cell center. It has been reported previously that during mitosis Mid1p becomes hyperphosphorylated and relocates from the nucleus to a medial ring. Here we show that Mid1p first forms a diffuse cortical band during spindle formation and then coalesces into a ring before anaphase. Plo1p is required for Mid1p to exit the nucleus and form a ring, and Pom1p is required for proper placement of the Mid1p ring. Upon overexpression of Plo1p, Mid1p exits the nucleus prematurely and displays a reduced mobility on gels similar to that of the hyperphosphorylated form observed previously in mitotic cells. Genetic and two-hybrid analyses suggest that Plo1p and Mid1p act in a common pathway distinct from that involving Pom1p. Plo1p localizes to the spindle pole bodies and spindles of mitotic cells and also to the medial ring at the time of its formation. Taken together, the data indicate that Plo1p plays a role in the positioning of division sites by regulating Mid1p. Given its previously known functions in mitosis and the timing of cytokinesis, Plo1p is thus implicated as a key molecule in the spatial and temporal coordination of cytokinesis with mitosis.     

Schizosaccharomyces pombe cardiolipin synthase is part of a mitochondrial fusion protein regulated by intron retention

Cardiolipin (CL) is a unique lipid component of mitochondria in all eukaryotes. It is important for the architecture of mitochondrial membranes and for mitochondrial dynamics. CL also creates a highly specific microenvironment of mitochondrial protein machineries. CL biosynthetic pathway is, however, only partially characterized in the fission yeast Schizosaccharomyces pombe. Here we show that CL synthase is an essential protein in S. pombe. It is encoded by the ORF SPAC22A12.08c as a C terminal part of a tandem fusion protein together with a mitochondrial hydrolase of unknown function. Expression of S. pombe CL synthase is able to complement deletion of the CRD1 gene of Saccharomyces cerevisiae and, vice versa, S. cerevisiae CRD1 gene complements deletion of S. pombe SPAC22A12.08c. The proper expression of CL synthase and its partner in the tandem protein, the mitochondrial hydrolase, is regulated at the level of alternate intron splicing. The first part of the SPAC22A12.08c fusion protein could be translated from both major SPAC22A12.08c derived mRNAs, with and without intron IV. Functional CL synthase, however, is produced only from the minor SPAC22A12.08c derived mRNA that has intron IV retained. Thus, intron retention is a novel mechanism for the differential expression of two proteins that evolved as a fusion protein and are under the control of the same promoter.     

Proteomic analysis of the U1 snRNP of Schizosaccharomyces pombe reveals three essential organism-specific proteins

Characterization of spliceosomal complexes in the fission yeast Schizosaccharomyces pombe revealed particles sedimenting in the range of 30–60S, exclusively containing U1 snRNA. Here, we report the tandem affinity purification (TAP) of U1-specific protein complexes. The components of the complexes were identified using (LC-MS/MS) mass spectrometry. The fission yeast U1 snRNP contains 16 proteins, including the 7 Sm snRNP core proteins. In both fission and budding yeast, the U1 snRNP contains 9 and 10 U1 specific proteins, respectively, whereas the U1 particle found in mammalian cells contains only 3. Among the U1-specific proteins in S. pombe, three are homolog to the mammalian and six to the budding yeast Saccharomyces cerevisiae U1-specific proteins, whereas three, called U1H, U1J and U1L, are proteins specific to S. pombe. Furthermore, we demonstrate that the homolog of U1-70K and the three proteins specific to S. pombe are essential for growth. We will discuss the differences between the U1 snRNPs with respect to the organism-specific proteins found in the two yeasts and the resulting effect it has on pre-mRNA splicing.     

The role of AtMUS81 in DNA repair and its genetic interaction with the helicase AtRecQ4A

The endonuclease MUS81 has been shown in a variety of organisms to be involved in DNA repair in mitotic and meiotic cells. Homologues of the MUS81 gene exist in the genomes of all eukaryotes, pointing to a conserved role of the protein. However, the biological role of MUS81 varies between different eukaryotes. For example, while loss of the gene results in strongly impaired fertility in Saccharomyces cerevisiae and nearly complete sterility in Schizosaccharomyces pombe, it is not essential for meiosis in mammals. We identified a functional homologue (AtMUS81/At4g30870) in the genome of Arabidopsis thaliana and isolated a full-length cDNA of this gene. Analysing two independent T-DNA insertion lines of AtMUS81, we found that they are sensitive to the mutagens MMS and MMC. Both mutants have a deficiency in homologous recombination in somatic cells but only after induction by genotoxic stress. In contrast to yeast, no meiotic defect of AtMUS81 mutants was detectable and the mutants are viable. Crosses with a hyperrecombinogenic mutant of the AtRecQ4A helicase resulted in synthetic lethality in the double mutant. Thus, the nuclease AtMUS81 and the helicase AtRecQ4A seem to be involved in two alternative pathways of resolution of replicative DNA structures in somatic cells.