A new high resolution screening method for study of phenotype stress responses of Saccharomyces cerevisae mutants

A high resolution high throughput screening method has been developed for stress response phenotyping of the global Saccharomyces cerevisiae knock out mutant collection. Stress causing agent is added at three concentrations to individual mutant cultures growing in early exponentially phase in 384-well microplates, and the dynamic effect of stress agent exposure is measured by following subsequent growth profiles of individual mutants with a resolution of three optical density measurements per hour. Software was written for calculation of sensitivity coefficients and efficient visual inspection of the growth and inhibition curves. Three DNA damage response causing agents were chosen to explore the feasibility of the new screening method: methyl methanesulphonate, 5-fluorouracil and cisplatin. They were tested in three biological replicas on a 1400 mutant large sub-library of the homozygote diploid S. cerevisiae gene knock out collection. The sub-library consisted of only mutants with a human ortholog to the inactivated gene. Almost 400 mutants were found more sensitive to one or more of the agents. Forty-nine mutants were sensitive to all three agents. One of the mutants, ERK5, sensitive to all three agents was chosen for follow-up human cell experiments to verify that such yeast screens can be used as hypothesis generator for human cell studies. Similar to yeast, HeLa cells became more sensitive against all three DNA damaging agents when co-treated with the ERK5 inhibitor BIX21088, thus supporting the result from the yeast phenotype screen.     

A new high resolution screening method for study of phenotype stress responses of Saccharomyces cerevisae mutants

A high resolution high throughput screening method has been developed for stress response phenotyping of the global Saccharomyces cerevisiae knock out mutant collection. Stress causing agent is added at three concentrations to individual mutant cultures growing in early exponentially phase in 384-well microplates, and the dynamic effect of stress agent exposure is measured by following subsequent growth profiles of individual mutants with a resolution of three optical density measurements per hour. Software was written for calculation of sensitivity coefficients and efficient visual inspection of the growth and inhibition curves. Three DNA damage response causing agents were chosen to explore the feasibility of the new screening method: methyl methanesulphonate, 5-fluorouracil and cisplatin. They were tested in three biological replicas on a 1400 mutant large sub-library of the homozygote diploid S. cerevisiae gene knock out collection. The sub-library consisted of only mutants with a human ortholog to the inactivated gene. Almost 400 mutants were found more sensitive to one or more of the agents. Forty-nine mutants were sensitive to all three agents. One of the mutants, ERK5, sensitive to all three agents was chosen for follow-up human cell experiments to verify that such yeast screens can be used as hypothesis generator for human cell studies. Similar to yeast, HeLa cells became more sensitive against all three DNA damaging agents when co-treated with the ERK5 inhibitor BIX21088, thus supporting the result from the yeast phenotype screen.     

Microarray blood testing: Pros & cons

Blood donation screening represents rather a unique set of blood grouping-related and pathogen detection assays. We are confronted with continuously growing numbers of testing targets. Ideally, the spectrum of clinically significant blood group antigens and alloantibodies would be wider than allowed by current routine tests. At the same time, we are witnessing an increase in emerging and re-emerging human pathogens due to urbanisation, increased international travel and trade, climate change and other factors. The spectrum of blood-borne infectious agents requiring donation screening is expected to grow correspondingly. Dengue and chikungunya viruses, variant CJD and hepatitis E virus represent just some of the candidate infectious agents for future donation screening. Multiplexing techniques, such as microarrays are well suited to address the growing number of targets, pending the increase in sensitivity of some of the microarrays assays. There are several possible scenarios for future testing algorithms, combining new multiplexing techniques with the existing blood testing assays. New generation testing platforms capable of microbiology screening, blood grouping and potential additional types of targets, are also being developed.     

X-ray structure of yeast Hal2p, a major target of lithium and sodium toxicity, and identification of framework interactions determining cation sensitivity

The product of the yeast HAL2 gene (Hal2p) is an in vivo target of sodium and lithium toxicity and its overexpression improves salt tolerance in yeast and plants. Hal2p is a metabolic phosphatase which catalyses the hydrolysis of 3'-phosphoadenosine-5'-phosphate (PAP) to AMP. It is, the prototype of an evolutionarily conserved family of PAP phosphatases and the engineering of sodium insensitive enzymes of this group may contribute to the generation of salt-tolerant crops. We have solved the crystal structure of Hal2p in complex with magnesium, lithium and the two products of PAP hydrolysis, AMP and Pi, at 1.6 A resolution. A functional screening of random mutations of the HAL2 gene in growing yeast generated forms of the enzyme with reduced cation sensitivity. Analysis of these mutants defined a salt bridge (Glu238 ellipsis Arg152) and a hydrophobic bond (Va170 ellipsis Trp293) as important framework interactions determining cation sensitivity. Hal2p belongs to a larger superfamily of lithium-sensitive phosphatases which includes inositol monophosphatase. The hydrophobic interaction mutated in Hal2p is conserved in this superfamily and its disruption in human inositol monophosphatase also resulted in reduced cation sensitivity.     

Validation of a novel assay for checkpoint responses: characterization of camptothecin derivatives in Saccharomyces cerevisiae

The evolutionary conservation of pathways preserving genetic stability supports the use of a lower eukaryote such as the yeast Saccharomyces cerevisiae in screening for novel anti-neoplastic agents. Yeast is already established as a model system to characterize the cellular effects of the topoisomerase inhibitor and anti-cancer agent camptothecin (CPT). Here, we demonstrate that a recently developed two-hybrid based plate assay that visualizes the DNA damage-induced homomeric complex formation of the yeast checkpoint protein Rad17 correctly predicts the biological activity of the tested camptothecin derivatives. The used criteria for biological activity include lethality, cell cycle arrest and Rad53p phosphorylation, an essential signaling event during checkpoint activation. Surprisingly, although responsive to camptothecin and not without influence on drug sensitivity, Rad17p appears to be dispensable for cell cycle arrest and for Rad53p phosphorylation following treatment with camptothecin. Such a role is only uncovered if double-strand break repair is compromised.     

The effects of “Cell age” upon the lethal effects of physical and chemical mutagens in the yeast, Saccharomyces cerevisiae

Summary SummaryYeast cultures progressing from the exponential to the stationary phase of growth showed changes in cell sensitivity to physical agents such as UV light, heat shock at 52° C and the chemical mutagens ethyl methane sulphonate, nitrous acid and mitomycin C.Exponential phase cells showed maximum resistance to UV light and minimum resistance to heat shock and the three chemicals. The increased resistance of exponential phase cells to UV light was shown to be dependent upon the functional integrity of the RAD ₅₀ gene.Treatment of growing yeast cultures with radioactively labelled ethyl methane sulphonate indicated the preferential uptake of radioactivity during the sensitive exponential stage of growth. The results indicated that the differential uptake of the chemical mutagens was responsible for at least a fraction of the variations in cell sensitivity observed in yeast cultures at different phases of growth.     

Calculation of half-lives of proteins in vivo. Heterogeneity in the rate of degradation of yeast proteins

Summary A method is given for the calculation of half-lives of proteins in vivo from the measurement of the decrease of radioactivity in pulse-labelled proteins with time. This method could be particularly useful for the study of the degradation of proteins in cells that have a low growth rate.The method applied to growing yeast indicates that there are two major classes of protein. The class with low turnover constitutes the bulk of yeast protein and has a half-life of 160 h in a medium with glucose or galactose and of 50 h in a medium with ethanol. The class of proteins with high turnover (half-life between 0.8 and 2.4 hours) represents from 1% of total protein in yeast growing on glucose to 7% in yeast growing on ethanol.It is shown that some proteins which are derepressed during growth on ethanol or induced during growth on galactose are particularly susceptible to degradation in a medium which contains glucose.It is proposed that protein degradation is regulated by a coarse control at the level of protease activity and a fine control on the susceptibility of individual proteins to proteases.     

Acetylcholinesterase assay for rapid expression screening in liquid and solid media

The synaptic enzyme acetylcholinesterase (AChE), which is the target of many insecticides and potential warfare agents, is implied in Alzheimer's disease and is a good potential candidate to be used in biosensors. This promotes a strong demand for production of recombinant AChE to be used in various studies. A promising expression system is the yeast Pichia pastoris, but the expression efficiency needs to be improved. Optimization studies require a rapid and efficient screening test to detect positive yeast colonies after transformation. Using indoxylacetate as a substrate, we designed a chromogenic test that is not interfered with by the culture media background color and, thus, is suitable for microplate screening. Moreover, it was possible to adapt the test for direct on-plate detection of AChE-expressing colonies.     

纤维凝胶固定化增殖酵母连续生产酒精的研究

本文比较了纤维胶固定增殖酵母与海藻酸钙凝胶球、纤维海藻酸铝凝胶与纤维海藻酸钙凝胶、以及不同厚度的纤维凝胶固定化增殖酵母的发酵结果。重点进行了纤维凝胶固定化增殖酵母连续生产酒精试验。采用１．１Ｌ柱式生物反应器,ＣＯ２排出通畅,停留时间为４小时,成熟醪中酒精含量为１０．１－１１．０％（Ｖ／Ｖ）,平均为１０．５５％,酒精生产能力为９．４ｇ／Ｌ．ｈ,总糖利用率为９４．５％。     

The response to chemical mutagens of the individual haploid and homoallelic diploid UV-sensitive mutants of the rad 3 locus of Saccharomyces cerevisiae

Summary Mutant cultures of yeast defective at the generad 3 show increased sensitivity to the lethal effects of UV light. The order of UV sensitivity shown by haploid and homoallelic diploid cultures carrying the variousrad 3 alleles was duplicated by their sensitivity to the action of nitrous acid. In contrast, after treatment with the alkylating agents ethyl-methane sulphonate and methylmethane sulphonate therad 3 cultures showed only small differences in sensitivity compared with the wild-typeRAD culture. These small differences in sensitivity appear to result from variation in the metabolic condition of the cultures when treated with alkylating agents.The results indicate that the product of therad 3 gene in yeast is involved in the repair of UV induced pyrimidine dimers and deaminated bases produced by nitrous acid but does not participate in the repair of single strand DNA breaks produced by alkylating agents.     

Identification and dissection of a complex DNA repair sensitivity phenotype in Baker's yeast

Complex traits typically involve the contribution of multiple gene variants. In this study, we took advantage of a high-density genotyping analysis of the BY (S288c) and RM strains of Saccharomyces cerevisiae and of 123 derived spore progeny to identify the genetic loci that underlie a complex DNA repair sensitivity phenotype. This was accomplished by screening hybrid yeast progeny for sensitivity to a variety of DNA damaging agents. Both the BY and RM strains are resistant to the ultraviolet light–mimetic agent 4-nitroquinoline 1-oxide (4-NQO); however, hybrid progeny from a BY×RM cross displayed varying sensitivities to the drug. We mapped a major quantitative trait locus (QTL), RAD5, and identified the exact polymorphism within this locus responsible for 4-NQO sensitivity. By using a backcrossing strategy along with array-assisted bulk segregant analysis, we identified one other locus, MKT1, and a QTL on Chromosome VII that also link to the hybrid 4-NQO–sensitive phenotype but confer more minor effects. This work suggests an additive model for sensitivity to 4-NQO and provides a strategy for mapping both major and minor QTL that confer background-specific phenotypes. It also provides tools for understanding the effect of genetic background on sensitivity to genotoxic agents.     

Influence of the membrane on T-2 toxin toxicity in Saccharomyces spp

In growing cells of Saccharomyces cerevisiae and Saccharomyces carlsbergensis, T-2 toxin inhibits cell growth. We have examined the role of the yeast membranes in the uptake mechanism(s) of T-2 toxin. The effects of membrane-modulating agents, ethanol, cetyltrimethylammonium bromide, Triton X-100, and heat were studied; these agents were found to increase the sensitivity of the yeasts toward T-2 toxin. In the presence of 5% (vol/vol) ethanol, 2 micrograms of T-2 toxin per ml caused complete inhibition of growth. In the presence of 1 microgram of cetyltrimethylammonium bromide per ml, yeast cells became sensitive to T-2 toxin, starting with a concentration of 0.5 micrograms/ml. Triton X-100 at concentrations below 1% (vol/vol) sensitized the cells toward T-2 toxin, but at higher concentrations it protected the cells from T-2 toxin. Temperatures of incubation between 7 and 30 degrees C influenced the growth reduction caused by T-2 toxin. The greatest observed reduction of growth in T-2 toxin-treated cultures occurred at 30 degrees C. To further prove that the membrane influences the interaction of T-2 toxin with yeasts, we have studied a yeast mutant with a reduced plasma membrane permeability (G. H. Rank et al., Mol. Gen. Genet. 152:13-18, 1977). This yeast mutant proved to be resistant to T-2 toxin concentrations of up to 50 micrograms/ml. These results show that the membrane plays a significant role in the interaction of T-2 toxin with yeast cells.     

Influence of the membrane on T-2 toxin toxicity in Saccharomyces spp.

In growing cells of Saccharomyces cerevisiae and Saccharomyces carlsbergensis, T-2 toxin inhibits cell growth. We have examined the role of the yeast membranes in the uptake mechanism(s) of T-2 toxin. The effects of membrane-modulating agents, ethanol, cetyltrimethylammonium bromide, Triton X-100, and heat were studied; these agents were found to increase the sensitivity of the yeasts toward T-2 toxin. In the presence of 5% (vol/vol) ethanol, 2 micrograms of T-2 toxin per ml caused complete inhibition of growth. In the presence of 1 microgram of cetyltrimethylammonium bromide per ml, yeast cells became sensitive to T-2 toxin, starting with a concentration of 0.5 micrograms/ml. Triton X-100 at concentrations below 1% (vol/vol) sensitized the cells toward T-2 toxin, but at higher concentrations it protected the cells from T-2 toxin. Temperatures of incubation between 7 and 30 degrees C influenced the growth reduction caused by T-2 toxin. The greatest observed reduction of growth in T-2 toxin-treated cultures occurred at 30 degrees C. To further prove that the membrane influences the interaction of T-2 toxin with yeasts, we have studied a yeast mutant with a reduced plasma membrane permeability (G. H. Rank et al., Mol. Gen. Genet. 152:13-18, 1977). This yeast mutant proved to be resistant to T-2 toxin concentrations of up to 50 micrograms/ml. These results show that the membrane plays a significant role in the interaction of T-2 toxin with yeast cells.     

三种转基因酵母法筛选类雌激素活性物质方法的比较

利用转基因酵母菌株,分别采用摇瓶法、96孔板法和单板法筛选类雌激素活性化学品,并对实验参数进行了优化。通过对工作光密度及β半乳糖苷酶反应时间的优化,使改进后的转基因酵母筛选法筛选一批样品的时间从原来需要的几天缩短为半天。并且在时间缩短的同时,仍保留原来检测的高灵敏度和准确性,也节省了大量的试验耗材,达到了能应用于环境样品中类雌激素活性物质的快速测定的目的。       

Study of the specificity of amino acid binding protein fromSaccharomyces cerevisiae membranes

Membranes obtained from exponentially growing yeast were extracted with buffered solution of 1% Tween 80 plus 0.1m MgCl₂. The soluble proteins, saparated from detergent and fine membrane particles on Saphadex G-100 colunn, were fond, to have binding affinity for Val, Lau, Ileu, Asp, Lys, His, Arg, Phe and Met. The occurrence of individual binding activities in different preparations was irregular. This and the fast inactivation by aging showed the extrame sensitivity of the molecules of binding proteins towards deleterious agents. Saturation of the binding proteins with aspartic acid and phenylalanine was of an identical biphasic shape indicating the change ofK _diss and suggesting the existence of several binding sites for each amino acid. In our opinion the above listed observations imply low specificity of amino acid binding proteins in yeast.     

1,8-Substituted anthraquinones,anthrones and bianthrones as potential non-azole leads against fungal infections

The synthesis of a variety of 1,8-substituted anthraquinones, anthrones and bianthrones and their potential as antifungal agents is evaluated. Preliminary screening against Schizosaccharomyces pombe (S. pombe), a fission yeast, and Saccharomyces cerevisiae (S. cerevisiae), a budding yeast, is reported. Both these yeast species demonstrate close homologue to a number of pathogenic fungi.     

Nob1p, a new essential protein, associates with the 26S proteasome of growing saccharomyces cerevisiae cells

Nob1p, which interacts with Nin1p/Rpn12, a subunit of the 19S regulatory particle (RP) of the yeast 26S proteasome, has been identified by two-hybrid screening. NOB1 was found to be an essential gene, encoding a protein of 459 amino acid residues. Nob1p was detected in growing cells but not in cells in the stationary phase. During the transition to the stationary phase, Nob1p was degraded, at least in part, by the 26S proteasome. Nob1p was found only in proteasomal fractions in a glycerol gradient centrifugation profile and immuno-coprecipitated with Rpt1, which is an ATPase component of the yeast proteasomes. These results suggest that association of Nob1p with the proteasomes is essential for the function of the proteasomes in growing cells.     

Induction of the cytoplasmic 'petite' mutation by chemical and physical agents in Saccharomyces cerevisiae.

A range of physical and chemical agents induce the mitochondrial 'petite' mutation in the yeast Saccharomyces cerevisiae. DNA intercalating agents as well as chemicals which can interfere with DNA synthesis induce this mutation, but only in growing cells. Many chemical or physical agents that produce a DNA lesion which is not simply reversed can induce various levels of the petite mutation, and may be more effective in non-growing cells. A limited number of chemicals act like ethidium bromide, inducing a high frequency of petites which is partially reversible with increasing concentration or time. The ability of a specific compound to be transported into mitochondria or its affinity for AT base pairs in DNA may determine whether it acts primarily as a nuclear or mitochondrial mutagen. In mammalian cells, some neoplastic changes occur at the mitochondrial level. Analogies between yeast and mammalian mitochondria suggest that agents which increase petite mutagenesis in yeast may have some carcinogenic potential. Although some types of petite inducer may have potential as antitumour drugs, those which are very effective antimitochondrial agents appear to be too toxic for therapeutic use. A process comparable to early stages in petite mutagensis occurs in human degenerative diseases and it seems possible that a consequence of exposure to petite mutagens could be an increase in the rate of degenerative diseases or of the aging process.     

异源表达脂肪酶的重组酵母筛选培养基的改良

在脂肪酶产生菌的筛选中,最常用的方法是三丁酸甘油酯琼脂平板透明圈法和橄榄油琼脂平板变色圈法。而传统的三丁酸甘油酯平板缺乏有效营养成分,重组酵母无法生长;橄榄油平板灵敏度较低,低脂肪酶活力的重组酵母不能产生变色圈,这给产脂肪酶基因工程菌的鉴定带来了困难。本研究在三丁酸甘油酯平板的基础上,添加适合酵母生长的营养成分,获得了新的改良培养基;重组酵母在该培养基上生长良好、水解圈清晰,检测脂肪酶灵敏度高。