Mutations that increase the mitotic stability of minichromosomes in yeast: characterization of RAR1

Summary In an attempt to identify proteins involved in the initiation of DNA replication, we have isolated a series of Saccharomyces cerevisiae mutants in which the function of putative replication origins is affected. The phenotype of these Rar^- (regulation of autonomous replication) mutants is to increase the mitotic stability of plasmids whose replication is dependent on weak ARS elements. These mutations are generally recessive and complementation analysis shows that mutations in several genes may improve the ability of weak ARS elements to function. One mutation (rar1-1) also confers temperature-sensitive growth, and thus an essential gene is affected. We have determined the DNA sequence of the RAR1 gene, which reveals an open reading frame for a 48.5 kDa protein. The RAR1 gene is linked to rna1 on chromosome XIII.     

Glutathione serves an extracellular defence function to decrease arsenite accumulation and toxicity in yeast

Arsenic is an environmental toxin and a worldwide health hazard. Since this metalloid is ubiquitous in nature, virtually all living organisms require systems for detoxification and tolerance acquisition. Here, we show that during chronic exposure to arsenite [As(III)], Saccharomyces cerevisiae (budding yeast) exports and accumulates the low‐molecular‐weight thiol molecule glutathione (GSH) outside of cells. Extracellular accumulation of the arsenite triglutathione complex As(GS)₃ was also detected and direct transport assays demonstrate that As(GS)₃ does not readily enter cells. Yeast cells with increased extracellular GSH levels accumulate less arsenic and display improved growth when challenged with As(III). Conversely, cells defective in export and extracellular accumulation of GSH are As(III) sensitive. Taken together, our data are consistent with a novel detoxification mechanism in which GSH is exported to protect yeast cells from arsenite toxicity by preventing its uptake.     

<Emphasis Type="Italic">Caenorhabditis elegans gcs-1</Emphasis> Confers Resistance to Arsenic-Induced Oxidative Stress

Gamma-glutamylcysteine synthetase (γ-GCS) catalyzes the first, rate-limiting step in the biosynthesis of glutathione (GSH). To evaluate the protective role of cellular GSH against arsenic-induced oxidative stress in Caenorhabditis elegans (C. elegans), we examined the effect of the C. elegans ortholog of GCS(h), gcs-1, in response to inorganic arsenic exposure. We have evaluated the responses of wild-type and gcs-1 mutant nematodes to both inorganic arsenite (As(III)) and arsenate (As(V)) ions and found that gcs-1 mutant nematodes are more sensitive to arsenic toxicity than that of wild-type animals. The amount of metal ion required to kill half of the population of worms falls in the order of wild-type/As(V)>gcs-1/As(V)> wild-type/As(III)>gcs-1/As(III). gcs-1 mutant nematodes also showed an earlier response to the exposure of As(III) and As(V) than that of wild-type animals. Pretreatment with GSH significantly raised the survival rate of gcs-1 mutant worms compared to As(III)- or As(V)-treated worms alone. These results indicate that GCS-1 is essential for the synthesis of intracellular GSH in C. elegans and consequently that the intracellular GSH status plays a critical role in protection of C. elegans from arsenic-induced oxidative stress.     

The function of Alr1p of <Emphasis Type="Italic">Saccharomyces cerevisiae</Emphasis> in cadmium detoxification: Insights from phylogenetic studies and particle-induced X-ray emission

Two genes in Saccharomyces cerevisiae, ALR1 and ALR2, encode transmembrane proteins involved in Mg²⁺ uptake. The present study investigates the phylogenetic relationship of Alr1p/Alr2p with bacterial CorA proteins and some proteins related to Mg²⁺influx/efflux transport in mitochondrial and bacterial zinc transporters; including hydrophobic cluster analysis (HCA). The phylogenetic results indicate that the Alrp sequences of S. cerevisiae share a common carboxy-terminus with proteins related to zinc efflux transport. We also analyse the intracellular metal content by particle-induced X-ray emission (PIXE) after cell exposure to cadmium. The PIXE analysis of cadmium-exposed ALR mutants and wild-type yeast cells suggests that Alrp has a central role in cell survival in a cadmium-rich environment. Published online December 2004 Ana Lúcia Kern, Diego Bonatto: Both authors contributed equally to this work.     

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Abstract

Arsenic-induced tissue damage is a major concern to the human population. An impaired antioxidant defense mechanism followed by oxidative stress is the major cause of arsenic-induced toxicity, which can lead to reproductive failure. The present study was carried out to investigate the preventive role of arjunolic acid, a triterpenoid saponin isolated from the bark of Terminalia arjuna, against arsenic-induced testicular damage in mice. Administration of arsenic (in the form of sodium arsenite, NaAsO₂, at a dose of 10 mg/kg body weight) for 2 days significantly decreased the intracellular antioxidant power, the activities of the antioxidant enzymes, as well as the levels of cellular metabolites. In addition, arsenic intoxication enhanced testicular arsenic content, lipid peroxidation, protein carbonylation and the level of glutathione disulfide (GSSG). Exposure to arsenic also caused significant degeneration of the seminiferous tubules with necrosis and defoliation of spermatocytes. Pretreatment with arjunolic acid at a dose of 20 mg/kg body weight for 4 days could prevent the arsenic-induced testicular oxidative stress and injury to the histological structures of the testes. Arjunolic acid had free radical scavenging activity in a cell-free system and antioxidant power in vivo. In summary, the results suggest that the chemopreventive role of arjunolic acid against arsenic-induced testicular toxicity may be due to its intrinsic antioxidant property.     

酿酒酵母细胞基因组中与转录因子Rim101亚细胞定位相关的磷酸酶和激酶基因的筛选

Rim101是一个具有锌指结构的转录因子,在调控酿酒酵母细胞耐受碱性和高盐环境、钙离子稳态、细胞分裂以及硒毒性方面起作用。前人研究结果显示,细胞周期依赖性激酶基因PHO85的缺失,导致Rim101蛋白在细胞核内积累。为了探索Rim101亚细胞定位的新调节因子,通过荧光显微镜技术对酿酒酵母细胞基因组中编码磷酸酶的73个非必需基因缺失株和编码激酶的139个非必需基因缺失株进行了筛选,发现编码磷脂酰肌醇磷酸(Ptd Ins P)的磷酸酶Sac1调控Rim101的亚细胞定位。     

The RCK1 and RCK2 protein kinase genes from Saccharomyces cerevisiae suppress cell cycle checkpoint mutations in Schizosaccharomyces pombe

The protein kinase-encoding genes RCK1 and RCK2 from Saccharomyces cerevisiae have been identified as suppressors of Schizosaccharomyces pombe cell cycle checkpoint mutations. Upon expression of these genes, radiation resistance is partially restored in S. pombe mutants with checkpoint deficiencies, but not in mutants with DNA repair defects. Some checkpoint mutants are sensitive to the DNA synthesis inhibitor hydroxyurea, and this sensitivity is also suppressed by RCK1 and RCK2. The degree of suppression can be modulated by varying expression levels. Expression of RCK1 or RCK2 in S. pombe causes cell elongation and decelerated growth. Cells expressing these genes have a single nucleus and a 2n DNA content. We conclude that these genes act in S. pombe to prolong the G2 phase of the cell cycle.     

MicroRNA-181b and microRNA-9 mediate arsenic-induced angiogenesis via NRP1

Environmental exposure to inorganic arsenic compounds has been reported to have serious health effects on humans. Recent studies reported that arsenic targets endothelial cells lining blood vessels, and endothelial cell activation or dysfunction, may underlie the pathogenesis of arsenic-induced diseases and developmental toxicity. It has been reported that microRNAs (miRNAs) may act as an angiogenic switch by regulating related genes. The present study was designed to test the hypothesis that arsenite-regulated miRNAs play pivotal roles in arsenic-induced toxicity. Fertilized eggs were injected via the yolk sac with 100 nM sodium arsenite at Hamburger-Hamilton (HH) stages 6, 9, and 12, and harvested at HH stage 18. To identify the individual miRNAs and mRNAs that may regulate the genetic network, the expression profiles of chick embryos were analyzed by microarray analysis. Microarray analyses revealed that the expression of a set of miRNAs changed after arsenite administration, especially miRNA-9, 181b, 124, 10b, and 125b, which exhibited a massive decrease in expression. Integrative analyses of the microarray data revealed that several miRNAs, including miR-9 and miR-181b, might target several key genes involved in arsenic-induced developmental toxicity. A luciferase reporter assay confirmed neuropilin-1 (Nrp1) as a target of mir-9 and mir-181b. Data from the transwell migration assay and the tube-formation assay indicated that miR-9 and mir-181b inhibited the arsenic-induced EA.hy926 cell migration and tube formation by targeting NRP1. Our study demonstrates that the environmental toxin, sodium arsenite, induced angiogenesis by altering the expression of miRNAs and their cognate mRNA targets.     

Induction of reactive oxygen species by diphenyl diselenide is preceded by changes in cell morphology and permeability in Saccharomyces cerevisiae

Organoselenium compounds, such as diphenyl diselenide (PhSe)₂ and phenylselenium zinc chloride (PhSeZnCl), show protective activities related to their thiol peroxidase activity. However, depending on experimental conditions, organoselenium compounds can cause toxicity by oxidising thiol groups of proteins and induce the production of reactive oxygen species (ROS). Here, we analysed the toxicity of (PhSe)₂ and PhSeZnCl in yeast Saccharomyces cerevisiae. Cell growth of S. cerevisiae after 1, 2, 3, 4, 6, and 16?h of treatment with 2, 4, 6, and 10?μM of (PhSe)₂ was evaluated. For comparative purpose, PhSeZnCl was analysed only at 16?h of incubation at equivalent concentrations of selenium (i.e. 4, 8, 12, and 20?μM). ROS production (DCFH-DA), size, granularity, and cell membrane permeability (propidium iodide) were determined by flow cytometry. (PhSe)₂ inhibited cell growth at 2?h (10?μM) of incubation, followed by increase in cell size. The increase of cell membrane permeability and granularity (10?μM) was observed after 3?h of incubation, however, ROS production occurs only at 16?h of incubation (10?μM) with (PhSe)₂, indicating that ROS overproduction is a more likely consequence of (PhSe)₂ toxicity and not its determinant. All tested parameters showed that only concentration of 20?μM induced toxicity in samples incubated with PhSeZnCl. In summary, the results suggest that (PhSe)₂ toxicity in S. cerevisiae is time and concentration dependent, presenting more toxicity when compared with PhSeZnCl.     

Non-enzymatic roles for the URE2 glutathione S-transferase in the response of Saccharomyces cerevisiae to arsenic

The response of Saccharomyces cerevisiae to arsenic involves a large ensemble of genes, many of which are associated with glutathione-related metabolism. The role of the glutathione S-transferase (GST) product of the URE2 gene involved in resistance of S. cerevisiae to a broad range of heavy metals was investigated. Glutathione peroxidase activity, previously reported for the Ure2p protein, was unaffected in cell-free extracts of an ure2Δ mutant of S. cerevisiae. Glutathione levels in the ure2Δ mutant were lowered about threefold compared to the isogenic wild-type strain but, as in the wild-type strain, increased 2–2.5-fold upon addition of either arsenate (As^V) or arsenite (As^III). However, lack of URE2 specifically caused sensitivity to arsenite but not to arsenate. The protective role of URE2 against arsenite depended solely on the GST-encoding 3′-end portion of the gene. The nitrogen source used for growth was suggested to be an important determinant of arsenite toxicity, in keeping with non-enzymatic roles of the URE2 gene product in GATA-type regulation.     

Acetyl-<Emphasis Type="SmallCaps">l</Emphasis>-Carnitine Attenuates Arsenic-Induced Oxidative Stress and Hippocampal Mitochondrial Dysfunction

Augmentation of mitochondrial oxidative stress through activating a series of deadly events has implicated as the main culprit of arsenic toxicity and therapeutic approaches based on improving mitochondrial function hold a great promise for attenuating the arsenic-induced toxicity. Acetyl-l-carnitine (ALC) through balancing the coenzyme A (CoA)/acyl-CoA ratio plays an important role in mitochondrial metabolism and thereby can help protect hippocampal neurons from oxidative damage. In the present study, we aimed to explore the effect of arsenic interactions on the mitochondrial function in the hippocampus of rats. Rats were randomly divided into five groups of control (distilled water), sodium arsenite (NaAsO₂, 20 mg/kg), and co-treatment of NaAsO₂ with various doses of ALC in three groups (100, 200, 300 mg/kg) and were treated orally for 21 consecutive days. Our results point out that arsenic exposure caused oxidative stress in rats’ hippocampus, which led to the reactive oxygen species (ROS) generation, mitochondrial swelling, the collapse of the mitochondrial membrane potential, and release of cytochrome c. It also altered Bcl-2/Bax expression ratio and increased caspase-3 and caspase-9 activities. Furthermore, arsenic exposure via activation of NF-κB and microglia increased inflammation. ALC could concentration-dependently counteract the arsenic-induced oxidative stress, modulate the antioxidant defense capacity, and improve mitochondrial functions. In addition, ALC decreased the expression of both death-associated proteins and of inflammatory markers. These findings indicate that ALC improved the arsenic-induced hippocampal mitochondrial dysfunction which underlines the importance of ALC in providing a possible therapeutic strategy for the prevention of arsenic-induced neurodegeneration.     

Inactivation of SSM4, a new Saccharomyces cerevisiae gene, suppresses mRNA instability due to rna14 mutations

Decay rates of mRNAs depend on many elements and among these, the role of the poly(A) tail is now well established. In the yeast Saccharomyces cerevisiae, thermosensitive mutations in two genes, RNA14 and RNA15, result in mRNAs having shorter poly(A) tails and reduced half-life. To identify other components interacting in the same process, we have used a genetic approach to isolate mutations that suppress the thermosensitivity of an rna14 mutant strain. Mutations in a single locus, named SSM4, not only suppress the cell growth phenotype but also the mRNA instability and extend the short mRNA poly(A) tails. The frequency of appearance and the recessive nature of these mutations suggested that the suppressor effect was probably due to a loss of function. We failed to clone the SSM4 gene directly by complementation, owing to its absence from gene banks; it later emerged that the gene is toxic to Escherichia coli, but we have nevertheless been able to clone the SSM4 sequence by Ty element transposition tagging. Disruption of the SSM4 gene does not affect cell viability and suppresses the rna14 mutant phenotypes. The protein encoded by the SSM4 gene has a calculated molecular mass of 151 kDa and does not contain any known motif or show homology with known proteins. The toxicity of the SSM4 gene in E. coli suggests that a direct biochemical activity is associated with the corresponding protein.     

Plasmid-determined resistance to arsenic and antimony inPseudomonas aeruginosa

Resistance to arsenic salts in aPseudomonas aeruginosa clinical isolate was shown to be determined by a 100 kb transferable plasmid. The resistance pattern included arsenate, arsenite, and antimonate ions. Arsenate and arsenite resistances were inducible by previous exposure of cultures to subinhibitory amounts of either of the two ions. Phosphate ions protectedP. aeruginosa cells from the toxic effects of arsenate but did not alter arsenite toxicity.     

Nup211, the fission yeast homolog of Mlp1/Tpr,is involved in mRNA export

Synthetic lethal mutants have been previously isolated in fission yeast Schizosaccharomyces pombe, which genetically interact with spmex67, in order to identify the genes involved in mRNA export. The nup211 gene was isolated by complementation of the growth defect in one of the synthetic lethal mutants, SLMex2, under synthetic lethal condition. We showed that Nup211, fission yeast homolog of Mlpl/Mlp2/Tpr, is essential for vegetative growth and Nup211-GFP proteins expressed at endogenous level are localized mainly in nuclear periphery. The accumulation of poly(A)⁺ RNA in the nucleus is exhibited when expression of nup211 is repressed or over-expressed. These results suggest that the Nup211 protein plays a pivotal role of mRNA export in fission yeast.     

Immunological evidence for the involvement of cell wall proteins in phosphate uptake in the yeast Saccharomyces cerevisiae

Immunological cross-reactivity between cell wall proteins obtained from two yeast genera (Candida tropicalis and Saccharomyces cerevisiae) is reported. Specific retention of two cell wall proteins from Saccharomyces cerevisiae by an immunoabsorbent column coupled with antibodies against phosphate binding protein 2 (PiBP2) from Candida tropicalis allowed to generate antibodies against the proteins from S. cerevisiae. These antibodies were effective in inhibiting phosphate uptake by S. cerevisiae cells. The proteins from S. cerevisiae displayed a phosphate binding activity which was inhibited in the presence of the forementioned antibodies. These results and the observation that the amount of these proteins in the shock fluid was dependent of the growth conditions (i.e., in the presence or in the absence of phosphate) support the idea that these proteins are involved in the high affinity phosphate transport system.Abbreviations Pi inorganic phosphate - PiBP2 phosphate binding protein 2 obtained from Candida tropicalis - Tris Tris(hydroxymethyl)-aminoethane - MES [2-(N-Morpholino)] ethanesulfonic acid - EDTA ethylene diamine tetraacetic acid, disoldium salt - PMSF phenylmethyl sulfonyl fluoride - SDS sodium dodecyl sulfate - SDS-PAGE sodium dodecyl sulfate polyacrylamide gel electrophoresis     

The effects of vanadate on the yeast Saccharomyces cerevisiae

Growth of Saccharomyces cerevisiae on non-fermentable medium was more sensitive to inhibition by vanadate than growth of fermentable medium. The frequency of petite mutants increased in cultures grown for 18 hours in fermentable medium containing vanadate. However, oxygen uptake markedly increased in yeast cultures grown in the presence of vanadate, a similar effect being produced by phosphate. It was also found that oligomycin toxicity was relieved by vanadate. These results suggest that vanadate may interact with the mitochondria of S. cerevisiae.     

Population growth responses of Tetrahymena shanghaiensis in exposure to rare earth elements

This article presents the population growth responses of Tetrahymena shanghaiensis s₁ in exposure to rare earth elements (REEs). Both the light REEs (La, Sm) and the heavy REEs (Y, Gd) were investigated with 24- and 96-hr population growth assays to evaluate their aquatic toxicity. Four end points, cell count, frequency of neutral red (NR) uptake, total protein, and nucleic acid content were employed in the 24-h assay, and a population growth curve was plotted in the 96-h assay. The results of 24- and 96-h assays suggest a dual effect of REEs on T. shanghaiensis of the stimulated growth at low concentrations and the toxicity at higher ones. In the promoted growth of T. shanghaiensis, however, the cell density increased with an increased growth rate and the protein and nucleic acid content per 10⁴ cells undertook no remarkable changes, which suggests possible cell growth control by REE.     

Multicopy Suppression Screen in the <Emphasis Type="Italic">msb3 msb4 Saccharomyces cerevisiae</Emphasis> Double Mutant,Affected in Ypt/RabGAP Activity

The Msb3p and Msb4p proteins of Saccharomyces cerevisiae are members of the Ypt/Rab-specific GTPase-activating protein (GAP) family. They are essential to vesicular trafficking and involved in the regulation of exocytosis and in the organization of the actin cytoskeleton, but their exact biological roles have yet to be determined. The msb3 msb4 yeast double mutation causes growth inhibition in the presence of DMSO and/or caffeine, affects the organization of the actin cytoskeleton, produces a random budding pattern in diploid cells, and affects segregation of the nucleus. To find cell components that interact genetically with the products of the MSB3 and MSB4 genes, we screened a genomic library for multicopy suppressor genes restoring normal growth of the double mutant in the presence of DMSO and caffeine. Six genes were identified, and the extent to which each gene corrects specific growth defects of the msb3 msb4 mutant is described. The encoded suppressors were classified on the basis of functional features into four groups: vesicular transport proteins (Sec7p, Vps35p, and Uso1p), a protein involved in cell division (Sap155p), a molecular chaperon (Ssz1p), and a protein associated with the 25S proteasome (Cic1p).     

Killer-toxin-resistantkre12 mutants ofSaccharomyces cerevisiae: genetic and biochemical evidence for a secondary K1 membrane receptor

TheSaccharomyces cerevisiae killer toxin K1 is a secreted α/β-heterodimeric protein toxin that kills sensitive yeast cells in a receptor-mediated two-stage process. The first step involves toxin binding to β-1,6-d-glucan-components of the outer yeast cell surface; this step is blocked in yeast mutants bearing nuclear mutations in any of theKRE genes whose products are involved in synthesis and/or assembly of cell wall β-d-glucans. After binding to the yeast cell wall, the killer toxin is transferred to the cytoplasmic membrane, subsequently leading to cell death by forming lethal ion channels. In an attempt to identify a secondary K1 toxin receptor at the plasma membrane level, we mutagenized sensitive yeast strains and isolated killer-resistant (kre) mutants that were resistant as spheroplasts. Classical yeast genetics and successive back-crossings to sensitive wild-type strain indicated that this toxin resistance is due to mutation(s) in a single chromosomal yeast gene (KRE12), renderingkrel2 mutants incapable of binding significant amounts of toxin to the membrane. Sincekrel2 mutants showed normal toxin binding to the cell wall, but markedly reduced membrane binding, we isolated and purified cytoplasmic membranes from akrel2 mutant and from an isogenicKre12+ strain and analyzed the membrane protein patterns by 2D-electrophoresis using a combination of isoelectric focusing and SDS-PAGE. Using this technique, three different proteins (or subunits of a single multimeric protein) were identified that were present in much lower amounts in thekre12 mutant. A model for K1 killer toxin action is presented in which the gene product ofKRE12 functions in vivo as a K1 docking protein, facilitating toxin binding to the membrane and subsequent ion channel formation.     

Enhanced expression of the DNA damage-inducible gene<Emphasis Type="Italic"> DIN7</Emphasis> results in increased mutagenesis of mitochondrial DNA in<Emphasis Type="Italic"> Saccharomyces cerevisiae</Emphasis>

We reported previously that the product of DIN7, a DNA damage-inducible gene of Saccharomyces cerevisiae, belongs to the XPG family of proteins, which are involved in DNA repair and replication. This family includes the S. cerevisiae protein Rad2p and its human homolog XPGC, Rad27p and its mammalian homolog FEN-1, and Exonuclease I (Exo I). Interestingly, Din7p is the only member of the XPG family which specifically functions in mitochondria. We reported previously that overexpression of DIN7 results in a mitochondrial mutator phenotype. In the present study we wished to test the hypothesis that this phenotype is dependent on the nuclease activity of Din7p. For this purpose, we constructed two alleles, din7-D78A and din7-D173A, which encode proteins in which highly conserved aspartates important for the nuclease activity of the XPG proteins have been replaced by alanines. Here, we report that overexpression of the mutant alleles, in contrast to DIN7, fails to increase the frequency of mitochondrial petite mutants or erythromycin-resistant (E^r) mutants. Also, overproduction of din7-D78Ap does not result in destabilization of poly GT tracts in mitochondrial DNA (mtDNA), the phenotype observed in cells that overexpress Din7p. We also show that petite mutants induced by enhanced synthesis of wild-type Din7p exhibit gross rearrangements of mtDNA, and that this correlates with enhanced recombination within the mitochondrial cyt b gene. These results suggest that the stability of the mitochondrial genome of S. cerevisiae is modulated by the level of the nuclease Din7p.Communicated by R. Devoret