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.     

Hyphal Growth in the Fission Yeast Schizosaccharomyces pombe

The fission yeast Schizosaccharomyces pombe grows in a single-celled form or can mate and undergo meiosis and sporulation. Here we show that wild-type S. pombe can also differentiate to form elaborately branched hyphae which invade deep into solid medium. Branches appear in the hyphae adjacent to unseparated septa. Electron microscopy reveals unusual multivesicular structures within the hyphae. Nitrogen deprivation appears to be the main stimulus for hyphal growth. No mitogen-activated protein kinase is necessary for the response. Inhibition of cyclic AMP (cAMP) production or signaling prevents the response, and exogenous cAMP promotes it, suggesting that detection of a good carbon source is required for hyphal growth but not for mating.     

HIV-1 Protease in the Fission Yeast Schizosaccharomyces pombe

Background

HIV-1 protease (PR) is an essential viral enzyme. Its primary function is to proteolyze the viral Gag-Pol polyprotein for production of viral enzymes and structural proteins and for maturation of infectious viral particles. Increasing evidence suggests that PR cleaves host cellular proteins. However, the nature of PR-host cellular protein interactions is elusive. This study aimed to develop a fission yeast (Schizosaccharomyces pombe) model system and to examine the possible interaction of HIV-1 PR with cellular proteins and its potential impact on cell proliferation and viability.

Results

A fission yeast strain RE294 was created that carried a single integrated copy of the PR gene in its chromosome. The PR gene was expressed using an inducible nmt1 promoter so that PR-specific effects could be measured. HIV-1 PR from this system cleaved the same indigenous viral p6/MA protein substrate as it does in natural HIV-1 infections. HIV-1 PR expression in fission yeast cells prevented cell proliferation and induced cellular oxidative stress and changes in mitochondrial morphology that led to cell death. Both these PR activities can be prevented by a PR-specific enzymatic inhibitor, indinavir, suggesting that PR-mediated proteolytic activities and cytotoxic effects resulted from enzymatic activities of HIV-1 PR. Through genome-wide screening, a serine/threonine kinase, Hhp2, was identified that suppresses HIV-1 PR-induced protease cleavage and cell death in fission yeast and in mammalian cells, where it prevented PR-induced apoptosis and cleavage of caspase-3 and caspase-8.

Conclusions

This is the first report to show that HIV-1 protease is functional as an enzyme in fission yeast, and that it behaves in a similar manner as it does in HIV-1 infection. HIV-1 PR-induced cell death in fission yeast could potentially be used as an endpoint for mechanistic studies, and this system could be used for developing a high-throughput system for drug screenings.     

Germ-Tube Formation is Independent of DNA Synthesis during Spore Germination in the Fission Yeast, Schizosaccharomyces pombe

Germ-tube emergence took place at almost the same time as DNAsynthesis during spore germination in the fission yeast, Schizosaccharomycespombe. Neither the emergence nor the elongation of the germ-tubeswas inhibited by hydroxyurea. Spores harboring a temperature-sensitivecdc 10 mutation produced germ-tubes even at a restrictive temperature,which indicates that germ-tube formation is not dependent onDNA synthesis. ¹ Present address: Department of Microbial Genetics, ResearchInstitute for Microbial Diseases, Osaka University, Yamada-Oka,Suita, Osaka 565, Japan. (Received June 3, 1982; Accepted July 5, 1982)     

Phosphatidylethanolamine Is Required for Normal Cell Morphology and Cytokinesis in the Fission Yeast Schizosaccharomyces pombe

To investigate the contributions of phosphatidylethanolamine to the growth and morphogenesis of the fission yeast Schizosaccharomyces pombe, we have characterized three predicted genes in this organism, designated psd1, psd2, and psd3, encoding phosphatidylserine decarboxylases, which catalyze the conversion of phosphatidylserine to phosphatidylethanolamine in both eukaryotic and prokaryotic organisms. S. pombe mutants carrying deletions in any one or two psd genes are viable in complex rich medium and synthetic defined minimal medium. However, mutants carrying deletions in all three psd genes (psd1-3Δ mutants) grow slowly in rich medium and are inviable in minimal medium, indicating that the psd1 to psd3 gene products share overlapping essential cellular functions. Supplementation of growth media with ethanolamine, which can be converted to phosphatidylethanolamine by the Kennedy pathway, restores growth to psd1-3Δ cells in minimal medium, indicating that phosphatidylethanolamine is essential for S. pombe cell growth. psd1-3Δ cells produce lower levels of phosphatidylethanolamine than wild-type cells, even in medium supplemented with ethanolamine, indicating that the Kennedy pathway can only partially compensate for the loss of phosphatidylserine decarboxylase activity in S. pombe. psd1-3Δ cells appear morphologically indistinguishable from wild-type S. pombe cells in medium supplemented with ethanolamine, but when cultured in nonsupplemented medium, they produce high frequencies of abnormally shaped cells as well as cells exhibiting severe septation defects, including multiple, mispositioned, deformed, and misoriented septa. Our results demonstrate that phosphatidylethanolamine is essential for cell growth and for normal cytokinesis and cellular morphogenesis in S. pombe, and they illustrate the usefulness of this model eukaryote for investigating potentially conserved biological and molecular functions of phosphatidylethanolamine.Phosphatidylethanolamine (PE) is a major phospholipid component of cell membranes in both prokaryotic and eukaryotic organisms (34, 35). There are three distinct pathways for PE synthesis in eukaryotic cells: (i) decarboxylation of phosphatidylserine (PS) via reactions catalyzed by PS decarboxylase (PSD) enzymes; (ii) the CDP-ethanolamine branch of the Kennedy pathway, which converts ethanolamine to PE (34); and (iii) acylation of lysophosphatidylethanolamine (21, 29), a reaction that in the budding yeast Saccharomyces cerevisiae is catalyzed by the enzyme Ale1 (22). Genetic studies have demonstrated that PE is essential for cell viability in S. cerevisiae, although the minimal threshold of PE required for cell growth in this organism can apparently be provided by any of the routes of PE synthesis listed above (22). In contrast, the results of mouse knockout experiments indicate that both PSD- and Kennedy pathway-catalyzed pathways for PE synthesis are essential for embryonic development (9, 28, 35).While PE is present in most, if not all, eukaryotic cell membranes, it is particularly enriched in the membranes of mitochondria (32, 35, 37). Indeed, S. cerevisiae mutants carrying a null mutation in the PSD1 gene, which encodes a mitochondrially localized PSD, exhibit phenotypes indicative of mitochondrial dysfunction, as do cells derived from mouse embryos carrying a disruption of the Psid gene, which encodes a protein highly homologous in structure to S. cerevisiae Psd1 (28, 32). A second PSD enzyme in S. cerevisiae, encoded by the PSD2 gene, is localized to Golgi and vacuolar membranes (33, 37). Consistent with a role in vacuolar function, PE has been implicated in the process of autophagy by genetic studies utilizing S. cerevisiae vacuolar targeting mutants and by studies showing that Atg8, a ubiquitin-like protein required for yeast autophagy, is conjugated to PE, as are several related mammalian proteins (19, 20, 27).Interestingly, studies utilizing a streptavidin-conjugated form of the PE-binding peptide cinnamycin demonstrated that PE is enriched at cell division sites in S. cerevisiae, the fission yeast Schizosaccharomyces pombe, and mammalian cells (6, 11). Moreover, streptavidin-conjugated cinnamycin was shown to inhibit the disassembly of the contractile ring and the completion of cytokinesis in cultures of Chinese hamster ovary cells, and a PE-deficient cell line from the same species was found to arrest growth in cytokinesis with an intact contractile ring (7). PE has also been shown to be enriched at the growing ends of interphase S. pombe cells and at the emerging bud cortex in dividing cells of S. cerevisiae, findings that implicate PE in processes controlling polarized cell growth (11).Although S. pombe mutants defective in enzymes that directly catalyze PE synthesis have not been described previously, we recently showed that mutants carrying a null mutation in the PS synthase gene pps1 are ethanolamine auxotrophs that exhibit severe morphology- and cytokinesis-defective phenotypes under ethanolamine-limited growth conditions (17). These findings implicated PE in the regulation of cellular morphogenesis and cytokinesis in S. pombe. To investigate the biological functions of PE in S. pombe, in particular its contributions to the control of cell morphology and cytokinesis, we have in the present study generated and characterized mutants carrying null mutations in three open reading frames predicted to encode PSD enzymes in this organism. In this paper, we describe the phenotypes of S. pombe PSD-null mutants, which demonstrate central roles for PE in the regulation of cell morphology and cytokinesis in this model eukaryote.     

Ascospore Formation in the Yeast Saccharomyces cerevisiae

Sporulation of the baker's yeast Saccharomyces cerevisiae is a response to nutrient depletion that allows a single diploid cell to give rise to four stress-resistant haploid spores. The formation of these spores requires a coordinated reorganization of cellular architecture. The construction of the spores can be broadly divided into two phases. The first is the generation of new membrane compartments within the cell cytoplasm that ultimately give rise to the spore plasma membranes. Proper assembly and growth of these membranes require modification of aspects of the constitutive secretory pathway and cytoskeleton by sporulation-specific functions. In the second phase, each immature spore becomes surrounded by a multilaminar spore wall that provides resistance to environmental stresses. This review focuses on our current understanding of the cellular rearrangements and the genes required in each of these phases to give rise to a wild-type spore.     

De Novo Biosynthesis of Vanillin in Fission Yeast (Schizosaccharomyces pombe) and Baker's Yeast (Saccharomyces cerevisiae)

Vanillin is one of the world''s most important flavor compounds, with a global market of 180 million dollars. Natural vanillin is derived from the cured seed pods of the vanilla orchid (Vanilla planifolia), but most of the world''s vanillin is synthesized from petrochemicals or wood pulp lignins. We have established a true de novo biosynthetic pathway for vanillin production from glucose in Schizosaccharomyces pombe, also known as fission yeast or African beer yeast, as well as in baker''s yeast, Saccharomyces cerevisiae. Productivities were 65 and 45 mg/liter, after introduction of three and four heterologous genes, respectively. The engineered pathways involve incorporation of 3-dehydroshikimate dehydratase from the dung mold Podospora pauciseta, an aromatic carboxylic acid reductase (ACAR) from a bacterium of the Nocardia genus, and an O-methyltransferase from Homo sapiens. In S. cerevisiae, the ACAR enzyme required activation by phosphopantetheinylation, and this was achieved by coexpression of a Corynebacterium glutamicum phosphopantetheinyl transferase. Prevention of reduction of vanillin to vanillyl alcohol was achieved by knockout of the host alcohol dehydrogenase ADH6. In S. pombe, the biosynthesis was further improved by introduction of an Arabidopsis thaliana family 1 UDP-glycosyltransferase, converting vanillin into vanillin β-d-glucoside, which is not toxic to the yeast cells and thus may be accumulated in larger amounts. These de novo pathways represent the first examples of one-cell microbial generation of these valuable compounds from glucose. S. pombe yeast has not previously been metabolically engineered to produce any valuable, industrially scalable, white biotech commodity.In 2007, the global market for flavor and fragrance compounds was an impressive $20 billion, with an annual growth of 11 to 12%. The isolation and naming of vanillin (3-methoxy-4-hydroxybenzaldehyde) as the main component of vanilla flavor in 1859 (8), and the ensuing chemical synthesis in 1874 (41), in many ways marked the true birth of this industry, and this compound remains the global leader in aroma compounds. The original source of vanillin is the seed pod of the vanilla orchid (Vanilla planifolia), which was grown by the Aztecs in Mexico and brought to Europe by the Spaniards in 1520. Production of natural vanillin from the vanilla pod is a laborious and slow process, which requires hand pollination of the flowers and a 1- to 6-month curing process of the harvested green vanilla pods (37). Production of 1 kg of vanillin requires approximately 500 kg of vanilla pods, corresponding to the pollination of approximately 40,000 flowers. Today, only about 0.25% (40 tons out of 16,000) of vanillin sold annually originates from vanilla pods, while most of the remainder is synthesized chemically from lignin or fossil hydrocarbons, in particular guaiacol. Synthetically produced vanillin is sold for approximately $15 per kg, compared to prices of $1,200 to $4,000 per kg for natural vanillin (46).An attractive alternative is bioconversion or de novo biosynthesis of vanillin; for example, vanillin produced by microbial conversion of the plant constituent ferulic acid is marketed at $700 per kilogram under the trade name Rhovanil Natural (produced by Rhodia Organics). Ferulic acid and eugenol are the most attractive plant secondary metabolites amenable for bioconversion into vanillin, since they can be produced at relatively low costs: around $5 per kilogram (37). For the bioconversion of eugenol or ferulic acid into vanillin, several microbial species have been tested, including gram-negative bacteria of the Pseudomonas genus, actinomycetes of the genera Amycolatopsis and Streptomyces, and the basidiomycete fungus Pycnoporus cinnabarinus (19, 23, 25, 27, 31, 34, 35, 36, 45, 48). In experiments where the vanillin produced was absorbed on resins, Streptomyces cultures afforded very high vanillin yields (up to 19.2 g/liter) and conversion rates as high as 55% were obtained (15). Genes for the responsible enzymes from some of these organisms were isolated and expressed in Escherichia coli, and up to 2.9 g/liter of vanillin were obtained by conversion of eugenol or ferulic acid (1, 3, 32, 49).Compared to bioconversion, de novo biosynthesis of vanillin from a primary metabolite like glucose is much more attractive, since glucose costs less than $0.30/kilogram (42). One route for microbial production of vanillin from glucose was devised by Frost and coworker Li (6, 20), combining de novo biosynthesis of vanillic acid in E. coli with enzymatic in vitro conversion of vanillic acid to vanillin. 3-Dehydroshikimic acid is an intermediate in the shikimate pathway for biosynthesis of aromatic amino acids, and the recombinant E. coli was engineered to dehydrate this compound to form protocatechuic acid (3,4-dihydroxybenzoic acid) and methylate this to form vanillic acid. The vanillic acid was subsequently converted into vanillin in vitro using carboxylic acid reductase isolated from Neurospora crassa. The main products of the in vivo step were protocatechuic acid, vanillic acid, and isovanillic acid in an approximate ratio of 9:4:1, indicating a bottleneck at the methylation reaction and nonspecificity of the OMT (O-methyltransferase) enzyme for the meta-hydroxyl group of protocatechuic acid. Serious drawbacks of this scheme are the lack of an in vivo step for the enzymatic reduction of vanillic acid, demanding the addition of isolated carboxylic acid reductase and costly cofactors such as ATP, NADPH, and Mg²⁺, and the generation of isovanillin as a contaminating side product.In this study, we have genetically engineered single-recombination microorganisms to synthesize vanillin from glucose, according to the metabolic route depicted in Fig. ​Fig.1.1. To avoid the synthesis of isovanillin as an undesired side product, a large array of OMTs was screened for the desired high substrate specificity, and an appropriate enzyme was identified. A synthetic version of an aromatic carboxylic acid reductase (ACAR) gene, optimized for yeast codon usage, was introduced to achieve the reduction step. The vanillin pathway was introduced into both Saccharomyces cerevisiae and Schizosaccharomyces pombe yeast, and significant levels of vanillin production were obtained in both organisms. Vanillin β-d-glucoside is the form in which vanillin accumulates and is stored in the fresh pod of the vanilla orchid (Vanilla planifolia). During the “curing” process of the pod, β-glucosidases are liberated and facilitate a partial conversion of the vanillin β-d-glucoside into vanillin. Upon consumption or application, the conversion of vanillin β-d-glucoside into free vanillin by enzymes in the saliva or in the skin microflora can provide for a slow-release effect that prolongs and augments the sensory event, as is the case for other flavor glycosides investigated, such as menthol glucoside (14, 16). In addition to the increased value of vanillin β-d-glucoside as an aroma or flavor compound, production of the glucoside in yeast may offer several advantages. Vanillin β-d-glucoside is more water soluble than vanillin, but most importantly, compounds such as vanillin in high concentrations are toxic to many living cells (4). It has been shown that glucosides of toxic compounds are less toxic to yeasts (24). We found this to be the case with vanillin and S. cerevisiae yeast as well. Thus, to facilitate storage and accumulation of higher vanillin yields, we introduced a step for vanillin glucosylation in S. pombe.Open in a separate windowFIG. 1.Biosynthetic scheme for de novo biosynthesis of vanillin in Schizosaccharomyces pombe and outline of the different vanillin catabolites and metabolic side products observed in different yeast strains and constructs. Gray arrows, primary metabolic reactions in yeast; black arrows, enzyme reactions introduced by metabolic engineering; diagonally striped arrows, undesired inherent yeast metabolic reactions.     

Methionine Induces Sexual Development in the Fission Yeast Schizosaccharomyces pombe via an ste11-Dependent Signalling Pathway

Methionine added to minimal medium overcomes the repressing effects of ammonium and cyclic AMP (cAMP) on sexual development and efficiently induces mating and sporulation in homothallic strains of Schizosaccharomyces pombe. In heterothallic strains it induces G₁ arrest when cells enter stationary phase. We show that methionine reduces the intracellular cAMP pool and induces the expression of at least two cAMP-repressible genes, including fbp1 and ste11. The easiest interpretation of the results is that methionine induces sexual development via a cAMP-dependent ste11 signalling pathway.     

An Analysis of Interference in the Fission Yeast Schizosaccharomyces Pombe

The evaluation of three-point crosses at the tetrad and random spore level leads to the conclusion that both chiasma and chromatid interference are absent in the fission yeast Schizosaccharomyces pombe.     

Study of Adenine Aminohydrolase in the Yeast, Schizosaccharomyces pombe

Observation of the growth of some adenineless mutants of Schizosaccharomyces pombe on six substituted purine analogs leads to the hypothesis that an enzyme is present which catalyzes the conversion of these analogs into hypoxanthine. The enzyme adenase (adenine aminohydrolase, EC 3.5.4.2) has been found to be active in cell-free extracts of S. pombe. Results are reported which are in agreement with the hypothesis that this enzyme is responsible for the in vivo utilization of 6-chloropurine. This evidence comes mainly from a study of adenine aminohydrolase in two mutants selected for partial inability to grow on 6-chloropurine.     

Absolute Proteome and Phosphoproteome Dynamics during the Cell Cycle of Schizosaccharomyces pombe (Fission Yeast)

To quantify cell cycle-dependent fluctuations on a proteome-wide scale, we performed integrative analysis of the proteome and phosphoproteome during the four major phases of the cell cycle in Schizosaccharomyces pombe. In highly synchronized cells, we identified 3753 proteins and 3682 phosphorylation events and relatively quantified 65% of the data across all phases. Quantitative changes during the cell cycle were infrequent and weak in the proteome but prominent in the phosphoproteome. Protein phosphorylation peaked in mitosis, where the median phosphorylation site occupancy was 44%, about 2-fold higher than in other phases. We measured copy numbers of 3178 proteins, which together with phosphorylation site stoichiometry enabled us to estimate the absolute amount of protein-bound phosphate, as well as its change across the cell cycle. Our results indicate that 23% of the average intracellular ATP is utilized by protein kinases to phosphorylate their substrates to drive regulatory processes during cell division. Accordingly, we observe that phosphate transporters and phosphate-metabolizing enzymes are phosphorylated and therefore likely to be regulated in mitosis.Cell replication involves a complex series of highly regulated and evolutionary conserved events, called the “cell cycle.” Aberrations in the cell cycle have severe implications and can cause cancerous growth. A detailed understanding of the cell cycle and its regulation may identify additional targets for cancer therapy (1–3). The cell cycle has been the subject of previous proteomics studies. Olsen et al. (4) measured the dynamics of thousands of proteins and phosphorylation events across cell cycle phases of HeLa cells, providing insights into the underlying regulatory mechanisms and pointing to a general increase in phosphorylation site occupancy during M phase. In a targeted study, Pagliuca et al. (5) investigated interactors of cyclins E1, A2, and B1 in HeLa cells, revealing key mechanistic links between DNA replication and mitosis.Schizosaccharomyces pombe (fission yeast) is a unicellular organism, which can easily be genetically manipulated and carries many cell cycle features similar to metazoan cells. It is an important model organism to study the cell cycle and its checkpoint controls (6). Recent global proteomics studies of yeasts and their cell cycle (7–13) have mainly focused on Saccharomyces cerevisiae (budding yeast), with only a few studies of fission yeast (14, 15), although the fission yeast cell cycle may be more representative of eukaryotic cell cycles (16). However, attention of the proteomics community toward S. pombe is increasing. Recent proteomics studies covered up to 4087 S. pombe proteins (71% of the predicted proteome) and 1544 phosphoproteins in both asynchronous and synchronized cell cultures (17–22); however, a comprehensive analysis of the S. pombe cell cycle is so far missing.Here, we use high resolution mass spectrometry in combination with stable isotope labeling by amino acids in the cell culture (SILAC)¹ method, termed super-SILAC (23), and intensity-based absolute quantification (iBAQ) (24) to measure relative and absolute dynamics of the proteome and phosphoproteome during the cell cycle of fission yeast. We estimate copy numbers for 3178 S. pombe proteins, and we combine these data with calculated phosphorylation site stoichiometry to estimate the total amount of protein-bound phosphate and its dynamics across the cell cycle. Providing the global absolute dynamics and stoichiometry of proteins and their modifications will be a valuable resource for classical and systems biologists alike.     

Double-Strand Break-Induced Mitotic Intrachromosomal Recombination in the Fission Yeast Schizosaccharomyces Pombe

The Saccharomyces cerevisiae HO gene and MATa cutting site were used to introduce site-specific double-strand breaks (DSBs) within intrachromosomal recombination substrates in Schizosaccharomyces pombe. The recombination substrates consisted of nontandem direct repeats of ade6 heteroalleles. DSB induction stimulated the frequency of recombinants 2000-fold. The spectrum of DSB-induced recombinants depended on whether the DSB was introduced within one of the ade6 repeats or in intervening unique DNA. When the DSB was introduced within unique DNA, over 99.8% of the recombinants lacked the intervening DNA but retained one copy of ade6 that was wild type or either one of the heteroalleles. When the DSB was located in duplicated DNA, 77% of the recombinants were similar to the deletion types described above, but the single ade6 copy was either wild type or exclusively that of the uncut repeat. The remaining 23% of the induced recombinants were gene convertants with two copies of ade6 and the intervening sequences; the ade6 heteroallele in which the DSB was induced was the recipient of genetic information. Half-sectored colonies were isolated, analyzed and interpreted as evidence of heteroduplex DNA formation. The results are discussed in terms of current models for recombination.     

Population Genomics of the Fission Yeast Schizosaccharomyces pombe

The fission yeast Schizosaccharomyces pombe has been widely used as a model eukaryote to study a diverse range of biological processes. However, population genetic studies of this species have been limited to date, and we know very little about the evolutionary processes and selective pressures that are shaping its genome. Here, we sequenced the genomes of 32 worldwide S. pombe strains and examined the pattern of polymorphisms across their genomes. In addition to introns and untranslated regions (UTRs), intergenic regions also exhibited lower levels of nucleotide diversity than synonymous sites, suggesting that a considerable amount of noncoding DNA is under selective constraint and thus likely to be functional. A number of genomic regions showed a reduction of nucleotide diversity probably caused by selective sweeps. We also identified a region close to the end of chromosome 3 where an extremely high level of divergence was observed between 5 of the 32 strains and the remain 27, possibly due to introgression, strong positive selection, or that region being responsible for reproductive isolation. Our study should serve as an important starting point in using a population genomics approach to further elucidate the biology of this important model organism.     

Multiple Orientation-Dependent, Synergistically Interacting, Similar Domains in the Ribosomal DNA Replication Origin of the Fission Yeast, Schizosaccharomyces pombe

Previous investigations have shown that the fission yeast, Schizosaccharomyces pombe, has DNA replication origins (500 to 1500 bp) that are larger than those in the budding yeast, Saccharomyces cerevisiae (100 to 150 bp). Deletion and linker substitution analyses of two fission yeast origins revealed that they contain multiple important regions with AT-rich asymmetric (abundant A residues in one strand and T residues in the complementary strand) sequence motifs. In this work we present the characterization of a third fission yeast replication origin, ars3001, which is relatively small (~570 bp) and responsible for replication of ribosomal DNA. Like previously studied fission yeast origins, ars3001 contains multiple important regions. The three most important of these regions resemble each other in several ways: each region is essential for origin function and is at least partially orientation dependent, each region contains similar clusters of A+T-rich asymmetric sequences, and the regions can partially substitute for each other. These observations suggest that ars3001 function requires synergistic interactions between domains binding similar proteins. It is likely that this requirement extends to other fission yeast origins, explaining why such origins are larger than those of budding yeast.     

芽殖酵母和裂殖酵母中异染色质形成机制

芽殖酵母(Saccharomyces cerevisiae)和裂殖酵母(Schizosaccharomyces pombe)是用来研究异染色质形成、细胞周期、DNA复制等重要细胞功能的理想单细胞真核生物.本文主要介绍这2种酵母中异染色质形成的机制.异染色质是一种抑制基因转录和DNA重组的特殊染色质结构.尽管在芽殖酵母和裂殖酵母中异染色质形成都需要组蛋白修饰,但异染色质建立的机制不同.在芽殖酵母中参与异染色质形成的主要蛋白是Sir1-4蛋白（其中Sir2为组蛋白H3去乙酰化酶）,而组蛋白H3赖氨酸9甲基化酶Clr4和异染色质蛋白Swi6在裂殖酵母异染色质形成中起关键的作用.在这两个酵母中,参与异染色质形成的组蛋白修饰蛋白由DNA结合蛋白招募到异染色质.此外,裂殖酵母也利用RNA干扰系统招募组蛋白修饰蛋白.     

d-Amino Acid-Induced Expression of d-Amino Acid Oxidase in the Yeast Schizosaccharomyces pombe

We investigated d-amino acid oxidase (DAO) induction in the popular model yeast Schizosaccharomyces pombe. The product of the putative DAO gene of the yeast expressed in E.?coli displayed oxidase activity to neutral and basic d-amino acids, but not to an l-amino acid or acidic d-amino acids, showing that the putative DAO gene encodes catalytically active DAO. DAO activity was weakly detected in yeast cells grown on a culture medium without d-amino acid, and was approximately doubled by adding d-alanine. The elimination of ammonium chloride from culture medium induced activity by up to eight-fold. l-Alanine also induced the activity, but only by about half of that induced by d-alanine. The induction by d-alanine reached a maximum level at 2?h cultivation; it remained roughly constant until cell growth reached a stationary phase. The best inducer was d-alanine, followed by d-proline and then d-serine. Not effective were N-carbamoyl-d,l-alanine (a better inducer of DAO than d-alanine in the yeast Trigonopsis variabilis), and both basic and acidic d-amino acids. These results showed that S. pombe DAO could be a suitable model for analyzing the regulation of DAO expression in eukaryotic organisms.