Ageing in yeast does not enhance prion generation

The yeast prions [URE3] and [PSI(+)] are self-propagating amyloids of Ure2p and Sup35p, respectively. The analogous transmissible spongiform encephalopathies of mammals and other amyloidoses are largely diseases of later life. From normal strains lacking the prions, we isolated old cells and measured the frequency of de novo [URE3] and [PSI(+)] prion generation. We find no evidence that ageing of yeast increases the frequency of prion occurrence.     

Most,but not all,yeast strains in the deletion library contain the [PIN+] prion

The yeast deletion library is a collection of over 5100 single gene deletions that has been widely used by the yeast community. The presence of a non‐Mendelian element, such as a prion, within this library could affect the outcome of many large‐scale genomic studies. We previously showed that the deletion library parent strain contained the [PIN⁺] prion. [PIN⁺] is the misfolded infectious prion form of the Rnq1 protein that displays distinct fluorescent foci in the presence of RNQ1–GFP and exists in different physical conformations, called variants. Here, we show that over 97% of the library deletion strains are [PIN⁺]. Of the 141 remaining strains that have completely (58) or partially (83) lost [PIN⁺], 139 deletions were able to efficiently maintain three different [PIN⁺] variants despite extensive growth and storage at 4 °C. One strain, cue2Δ, displayed an alteration in the RNQ1–GFP fluorescent shape, but the Rnq1p prion aggregate shows no biochemical differences from the wild‐type. Only strains containing a deletion of either HSP104 or RNQ1 are unable to maintain [PIN⁺], indicating that 5153 non‐essential genes are not required for [PIN⁺] propagation. Copyright © 2009 John Wiley & Sons, Ltd.     

Psi(+)] prion generation in yeast: characterization of the 'strain' difference

The yeast cytoplasmically-inherited nonsense suppressor [PSI(+)] determinant is presumed to be a manifestation of the aggregated prion-like state of the Sup35 protein. Overexpression of the Sup35 protein induces generation of [PSI(+)] determinants with various suppressor efficiency and mitotic stabilities. Here, we demonstrate that the relative frequency of appearance of [PSI(+)] with different properties depends on the SUP35 allele used to induce their generation. The difference in properties of [PSI(+)] determinants was preserved after their transmission from one yeast strain to another. This difference correlated with variation in properties of the Sup35 protein. A novel type of prion instability was observed: some [PSI(+)] with weak suppressor efficiency could convert spontaneously into strong suppressor determinants.     

An engineered nonsense URA3 allele provides a versatile system to detect the presence, absence and appearance of the [PSI+] prion in Saccharomyces cerevisiae

Common methods to identify yeast cells containing the prion form of the Sup35 translation termination factor, [PSI+], involve a nonsense suppressor phenotype. Decreased function of Sup35p in [PSI+] cells leads to read-through of certain nonsense mutations in a few auxotrophic markers, e.g. ade1-14. This read-through results in growth on adenine-deficient media. While this powerful tool has dramatically facilitated the study of [PSI+], it is limited to a narrow range of laboratory strains and cannot easily be used to screen for cells that have lost the [PSI+] prion. Therefore we have engineered a nonsense mutation in the widely used URA3 gene, termed the ura3-14 allele. Introduction of the ura3-14 allele into an array of genetic backgrounds, carrying a loss-of-function URA3 mutation and [PSI+], allows for growth on media lacking uracil, indicative of decreased translational termination efficiency. This ura3-14 allele is able to distinguish various forms of the [PSI+] prion, called variants, and is able to detect the de novo appearance of [PSI+] in strains carrying the prion form of Rnq1p, [PIN+]. Furthermore, 5-fluoroorotic acid, which kills cells making functional Ura3p, provides a means to select for [psi-] derivatives in a population of [PSI+] cells marked with the ura3-14 allele, making this system much more versatile than previous methods.     

Mutations in Fks1p affect the cell wall content of beta-1,3- and beta-1,6-glucan in Saccharomyces cerevisiae

Fks1p and Fks2p are related proteins thought to be catalytic subunits of the beta-1,3-glucan synthase. Analysis of fks1 delta mutants showed a partial K1 killer toxin-resistant phenotype and a 30% reduction in alkali-soluble beta-1,3-glucan that was accompanied by a modest reduction in beta-1,6-glucan. The gas1 delta mutant lacking a 1,3-beta-glucanosyltransferase displayed a similar reduction in alkali-soluble beta-1,3-glucan but did not share the beta-1,6-glucan defect, indicating that beta-1,6-glucan reduction is not a general phenotype among beta-1,3-glucan biosynthetic mutants. Overexpression of FKS2 suppressed the killer toxin phenotype of fks1 delta mutants, implicating Fks2p in the biosynthesis of the residual beta-1,6-glucan present in fks1 delta cells. In addition, eight out of 12 fks1ts fks2 delta mutants had altered beta-glucan levels at the permissive temperature: the partial killer resistant FKS1F1258Y N1520D allele was severely affected in both polymers and displayed a 55% reduction in beta-1,6-glucan, while the in vitro hyperactive allele FKS1T605I M761T increased both beta-glucan levels. These beta-1,6-glucan phenotypes may be due to altered availability of, and structural changes in, the beta-1,3-glucan polymer, which might serve as a beta-1,6-glucan acceptor at the cell surface. Alternatively, Fks1p and Fks2p could actively participate in the biosynthesis of both polymers as beta-glucan transporters. We analysed Fks1p and Fks2p in beta-1,6-glucan deficient mutants and found that they were mislocalized and that the mutants had reduced in vitro glucan synthase activity, possibly contributing to the observed beta-1,6-glucan defects.     

Role of Tea1p, Tea3p and Pom1p in the determination of cell ends in Schizosaccharomyces pombe

Schizosaccharomyces pombe cells are rod-shaped and grow along a single axis from their two ends. Microtubules extend from the cell centre terminating at the cell ends. The ERM(ezrin/radixin/moesin)-like proteins Tea1p and Tea3p, and the Dyrk-like kinase Pom1p are cell end markers involved in the regulation of growth and microtubular dynamics at the cell ends. We have analysed the relative contribution of these three proteins to the determination of cell ends as sites both for cell growth and for microtubular termination. Pom1Delta, in combination with Tea1Delta or Tea3Delta, has the greatest difficulty in relocalizing actin to the cell ends following actin depolymerization and generates the most defective growth pattern. Tea1Delta, in combination with Pom1Delta or Tea3Delta, displays the highest number of microtubules bending round the cell ends. Tea1DeltaPom1Delta, which has the most defective growth pattern and microtubules, also displays the highest number of branched cells. We show that Tea1p, Tea3p and Pom1p all contribute, to different extents, to the determination of cell ends, as sites for both cell growth and microtubular termination. We also show that the fission yeast cell relies on both the positioning of landmarks and a properly organized microtubule cytoskeleton to direct cell growth.     

Cloning, characterization and functionality of the orotidine-5'-phosphate decarboxylase gene (URA3) of the glycolipid-producing yeast Candida bombicola

Candida bombicola is a yeast species known to synthesize glycolipids. Although these glycolipids find several industrial, cosmetic and pharmaceutical applications, very little is known about the genetics of C. bombicola. A basic tool for genetic study and modification is the availability of an efficient transformation and selection system. In order to develop such a system, the URA3 gene of Candida bombicola was isolated using degenerate PCR and genomic walking. The gene encodes for an enzyme of 262 amino acids and shows high homology with the known orotidine-5'-phosphate decarboxylases of several other yeast species. The functionality of the gene was proved by complementation of a URA3-negative Saccharomyces cerevisiae strain.     

Cloning and disruption of the Pichia pastoris ARG1, ARG2, ARG3, HIS1, HIS2, HIS5, HIS6 genes and their use as auxotrophic markers

Screening of a partial genomic database of Pichia pastoris allowed us to identify the ARG1, ARG2, ARG3, HIS1, HIS2, HIS5 and HIS6 genes, based on homology to their Saccharomyces cerevisiae counterparts. Based on the cloned sequences, a set of disruption vectors was constructed, using the previously described PpURA5-blaster as a selectable marker, and the cloned genes were individually disrupted. All disruptants exhibited the expected auxotrophic phenotypes, with only the his2 knockouts displaying a bradytroph phenotype. To allow their use as auxotrophic markers, we amplified the open reading frames and respective promoters and terminator regions of PpARG1, PpARG2, PpARG3, PpHIS1, PpHIS2 and PpHIS5. We then designed a set of integration vectors harbouring cassettes of the ARG pathway as selectable markers, to disrupt the genes of the HIS pathway and vice versa. Employing this strategy, we devised a scheme allowing for the rapid and stable introduction of several heterologous genes into the genome of P. pastoris without the need for recyclable markers or strains with multiple auxotrophies. Furthermore, simple replica-plating, instead of cost-consuming and labour-intensive colony PCR or Southern analysis, can be used to identify positive transformants, making this approach amendable for initial high-throughput applications, which can then be followed up by a more careful analysis of the selected transformants.     

3,3,6,6-四苄基-1,2,4,5-四硫环己烷的合成及香气研究

以中间体二苄基二硫醇和三氯化铁为原料,乙醇和水为溶剂,合成了新型香料3,3,6,6-四苄基-1,2,4,5-四硫环己烷,产率为50.3%。由IR,HNMR和MS证明了目标化合结构,并对面,3,6,6-四苄基-1,2,4,5-四硫环己烷的香气进行了鉴定,结果表明3,3,6,6-四苄基-1,2,4,5-四硫环己烷具有浓郁的葱蒜气味,其阈值在水中为0.22×10-6,在植物油中为12×10-6。     

Amino acid-dependent formation pathways of 2-acetylfuran and 2,5-dimethyl-4-hydroxy-3[2H]-furanone in the Maillard reaction

The formation pathways of two furanoids, 2-acetylfuran and 2,5-dimethyl-4-hydroxy-3[2H]-furanone (DMHF) were studied by GC–MS in the Maillard-type model system based on glucose and selected amino acids. The reaction was performed in 0.01 M phosphate buffer by heating a 1:1 mixture of [¹³C6] glucose and [¹²C6] glucose with amino acid. There is only one major formation pathway for DMHF in which the glucose carbon skeleton stayed intact. Formation pathways for 2-acetylfuran were more complicated. They formed either from glucose or from glucose and glycine. In the presence of glycine, the [C-5] unit of glucose combined with formaldehyde from glycine leads to 2-acetylfuran. For other amino acids, either cyclisation of intact glucose or recombination of glucose fragments can lead to 2-acetylfuran formation. These results indicate a competitive trend in controlling Maillard reaction. Therefore, besides changing Miallard reaction impact factors (temperature, time, pH etc.), inhibiting or preventing the competitive reaction cascade may direct desired pathways of Maillard reaction.     

The sequence of a 17 933 bp segment of Saccharomyces cerevisiae chromosome XIV contains the RHO2, TOP2, MKT1 and END3 genes and five new open reading frames

We report the DNA sequence of a 17 933 bp fragment from the left arm of chromosome XIV of Saccharomyces cerevisiae. Analysis of the sequence reveals the presence of ten open reading frames (ORFs) larger than 100 codons. Four of these were previously identified as genes RHO2, TOP2, MKT1 and END3. Additionally, the NH₂ end coding region of PMS1 is found in the 3′ end of the sequence. No significant homology to any known protein has been found for the other five ORFs. The nucleotide sequence has been deposited at EMBL, with Accession Number X89016.     

“水解植物蛋白液 葡萄糖”模型热反应中 3-氯-1,2-丙二醇的研究

通过设计“水解植物蛋白液 葡萄糖”模型热反应,考察了不同3-氯-1,2-丙二醇 (3-MCPD)含量的HVP液、葡萄糖用量、反应温度、反应时间等因素对热反应产物中 3-MCPD 含量的影响。结果表明：模型热反应产物中 3-MCPD 含量随着 HVP 液中 3-MCPD 的含量增加而增加;随着葡萄糖用量增加而减少;随着反应温度的升高,3-MCPD 的量先增加后减少;热反应时间越长,3-MCPD 的含量越高。     

甘油与氯化钠模型热反应中生成3-氯-1,2-丙二醇的研究

以甘油、氯化钠、水为原料建立热反应模型,结合气相色谱-质谱法（gas chromatography-mass spectrometry,GC-MS）分析,对其生成的3-氯-1,2-丙二醇（3-MCPD）进行了研究。通过正交实验,考察了反应时间、温度和各原料用量对3-MCPD生成量的影响,同时研究了在热反应模型中分别加入不同种类单糖和氨基酸后3-MCPD生成量的变化规律,并对其反应机理进行了探讨。结果表明,反应时间2h、反应温度140℃、甘油用量0.05g（占原料总量0.31%）、NaCl用量1.0g（6.23%）、水用量15g（93.46%）,3-MCPD生成量最少;葡萄糖、果糖、木糖、甘露糖、半乳糖、缬氨酸、精氨酸、赖氨酸、丝氨酸、亮氨酸、苯丙氨酸、苏氨酸、组氨酸使3-MCPD的生成量减少;谷氨酸、蛋氨酸、丙氨酸、天冬氨酸、甘氨酸使3-MCPD的生成量增加;半胱氨酸、酪氨酸、脯氨酸、色氨酸对3-MCPD的生成无明显作用。      

A 37·5 kb region of yeast chromosome X includes the SME1, MEF2, GSH1 and CSD3 genes,a TCP-1-related gene,an open reading frame similar to the DAL80 gene,and a tRNAArg

The complete DNA sequence of cosmid clone p59 comprising 37,549 bp derived from chromsome X was determined from an ordered set of subclones. The sequence contains 14 open reading frames (ORFs) containing at least 100 consecutive sense codons. Four of the ORFs represent already known and sequenced yeast genes: B645 is identical to the SME1 gene encoding a protein kinase, required for induction of meiosis in yeast, D819 represents the MEF2 gene probably encoding a second mitochondrial elongation factor-like protein, D678 is identical to the yeast GSH1 gene encoding γ-glutamylcysteine synthetase and B746 is identical to the CSD3 gene, which plays an as yet unidentified role in chitin biosynthesis and/or its regulation. The deduced amino acid sequence of A550 is 63% identical to the Ccη subunit of a murine TCP-1-containing chaperonin and more than 35% identical to thermophilic factor 55 from Sulfolobus shibatae, as well as to a number of proteins belonging to the chaperonin TCP-1 family. Open reading frame F551 exhibits homology to two regions of the DAL80 gene located on yeast chromosome XI encoding a pleiotropic negative regulatory protein. In addition, extensive homology was detected in three regions including parts of ORFs A560, B746/CSD3 and the incomplete ORF C852 to three consecutive ORFs of unknown function in the middle of the right arm of chromosome XI. Finally, the sequence contained a tRNA^Arg3 (AGC) gene. The nucleotide sequence data reported in this paper have been deposited in the EMBL and GenBank databases under the accession number X85021.