Characterization of the cyrl-2 UGA mutation in Saccharomyces cerevisiae

Summary cyrl-2 is a temperature-sensitive mutation of the yeast adenylate cyclase structural gene, CYR1. The cyrl-2 mutation has been suggested to be a UGA mutation since a UGA suppressor SUP201 has been isolated as a suppressor of the cyrl-2 mutation. Construction of chimeric genes restricted the region containing the cyrl-2 mutation, and the cyrl-2 UGA mutation was identified at codon 1282, which lies upstream of the region coding for the catalytic domain of adenylate cyclase. Alterations in the region upstream of the cyrl-2 mutation site result in null mutations. The complete open reading frame of the cyrl-2 gene expressed under the control of the GAL1 promoter complemented cyrl-dl in a galactose-dependent manner. These results suggest that at the permissive temperature weak readthrough occurs at the cyrl-2 mutation site to produce low levels of active adenylate cyclase. An endogenous suppressor in yeast cells is assumed to be responsible for this readthrough.     

酵母PMT1和TED1基因在细胞壁应激反应中的作用

实验室前期研究表明,蛋白质O-甘露糖转移酶1 (Protein O-mannosyltransferase 1,Pmt1p) 和Ted1p（Traffcking of Emp24p/Erv25p-dependent cargo disrupted）在细胞寿命和内质网应激反应方面存在相互调控关系。进一步研究酵母Pmt1p和Ted1p在细胞壁应激反应中（诱导剂为荧光增白剂或刚果红）的作用。观察PMT1基因缺失（pmt1Δ）酵母菌株和TED1基因缺失（ted1Δ）酵母菌株,以及PMT1和TED1双基因缺失（pmt1Δ ted1Δ）酵母菌株在细胞壁应激反应条件下的克隆形成能力和细胞分裂增殖活性;qRT-PCR检测细胞壁应激反应通路中Slt2p、Ssd1p和Mpt5p等效应蛋白的转录水平。结果表明,在细胞壁应激反应条件下,与对照菌株的生长状态比较,pmt1Δ菌株生长缓慢,ted1Δ菌株生长较快;进一步缺失PMT1基因使得ted1Δ菌株生长缓慢。与对照菌株中效应蛋白的转录水平比较,pmt1Δ菌株和pmt1Δted1Δ菌株中SLT2、SSD1和MPT5基因的转录水平明显上调,ted1Δ菌株中的无明显变化;与pmt1Δ菌株比较,pmt1Δted1Δ菌株中SLT2和MPT5表达明显下调,SSD1表达无明显变化。缺失TED1基因增强酵母细胞对细胞壁应激反应的抵抗性;进一步缺失PMT1基因增强ted1Δ菌株对应激反应的敏感性,上调细胞壁应激反应通路中效应蛋白的转录表达水平。     

Man8GlcNAc2糖基化的酿酒酵母菌株的构建

酿酒酵母糖蛋白的N-糖基化经过高尔基体的修饰后形成聚合度约150-200的甘露寡糖,高尔基体N-糖基化的糖基转移酶Mnn1p和Och1p在甘露寡糖的形成过程中起关键作用。通过同源重组置换敲除了酵母中的MNN1和OCH1基因阻断高尔基体N-糖基化修饰,分离纯化了mnn1 och1突变株中的N-糖蛋白,糖酰胺酶PNGaseF酶解释放的N-糖链经过2-氨基吡啶衍生后,利用HPLC和MALDITOF/MS结合的方法分析了突变株糖蛋白上的N-糖链。结果显示mnn1 och1突变株中的糖蛋白的N-糖链为结构单一的糖链,分子量为1794.66,推测为Man₈GlcNAc₂。     

木糖醇脱氢酶基因高拷贝表达载体构建及在酿酒酵母中的表达

木糖醇脱氢酶（xylitol dehydrogenase, XDH）可以氧化木糖醇生成木酮糖,处于木糖代谢的节点位置。利用PCR方法克隆得到了休哈塔假丝酵母（Candida shehatae） 20335的木糖醇脱氢酶基因、质粒pKT0150的ADH1终止子序列和G418抗性基因（KanR）,以及酿酒酵母(Saccharomyces cerevisiae) W5特定的2.2 kb的rDNA片段。以酿酒酵母整合载体p406ADH1为骨架,利用基因工程手段构建一个多拷贝整合表达载体pLX-AGRX。将重组载体pLX-AGRX线性化转入到酿酒酵母W5后,通过高浓度G418筛选和PCR双重鉴定,证实重组载体pLX-AGRX已整合到酿酒酵母W5基因组上,测定木糖醇脱氢酶酶活可达65.957 4 U/mg。     

A DNA sequence from Dictyostelium discoideum complements ura3 and ura5 mutations of Saccharomyces cerevisiae

Summary A 3.7 kilobase fragment of Dictyostelium discoideum genomic DNA has been cloned by its ability to complement a yeast ura5 mutation affecting the activity of orotidine-5-phosphate carboxy-lyase (EC 4.1.1.23). This fragment also complements a yeast ura5 mutation that leads to a defect in orotate phosphoribosyl transferase (EC 2.4.2.10). The orotidine-5-phosphate carboxy-lyase and the orotate phosphoribosyl transferase activities that result from Dictyostelium gene expression in yeast have been detected. The size of the DNA required for both complementations has been localised to a segment of less than 2 kb. A unique Dictyostelium RNA species of 1,600 base pairs hybridises to this fragment. In vitro deletions in this fragment lead to the simultaneous loss of the two activities. The two enzymatic activities coelute as a protein of 120.000 daltons during gel filtration of a Dictyostelium extract. These results favour the existence, on the cloned Dictyostelium DNA fragment, of a unique structural gene which codes for a bifunctional enzyme carrying the two activities, orotidine-5-phosphate carboxy-lyase and orotate phosphoribosyl transferase.Abbreviations bp basepair - kb kilobasepair - MOPS Morpholino propane sulfonic acid     

Differential effects of the mismatch repair genes MSH2 and MSH3 on homeologous recombination in Saccharomyces cerevisiae

The products of the yeast mismatch repair genes MSH2 and MSH3 participate in the inhibition of genetic recombination between homeologous (divergent) DNA sequences. In strains deficient for these genes, homeologous recombination rates between repeated elements are elevated due to the loss of this inhibition. In this study, the effects of these mutations were further analyzed by quantitation of mitotic homeologous recombinants as crossovers, gene conversions or exceptional events in wild-type, msh2, msh3 and msh2 msh3 mutant strains. When homeologous sequences were present as a direct repeat in one orientation, crossovers and gene conversions were elevated in msh2, msh3 and msh2 msh3 strains. The increases were greater in the msh2 msh3 double mutant than in either single mutant. When the order of the homeologous sequences was reversed, the msh2 mutation again yielded increased rates of crossovers and gene conversions. However, in an msh3 strain, gene conversions occurred at higher levels but interchromosomal crossovers were not increased and intrachromosomal crossovers were reduced relative to wild type. The msh2 msh3 double mutant behaved like the msh2 single mutant in this orientation. Control strains harboring homologous duplications were largely but not entirely unaffected in mutant strains, suggesting specificity for the mismatched intermediates of homeologous recombination. In all strains, very few (<10%) recombinants could be attributed to exceptional events. These results suggest that MSH2 and MSH3 can function differentially to control homeologous exchanges. Received: 24 December 1996 / Accepted: 24 July 1997     

酿酒酵母中双链RNA介导基因沉默技术研究GRE3基因表达抑制

RNA沉默技术作为探索基因功能的实验手段应用于多种生物. 以编码酿酒酵母NADPH依赖型醛糖还原酶的GRE3基因为对象，检测酿酒酵母双链RNA介导的基因沉默效应. 以pESC-LEU为骨架，构建重组质粒psiLENT-GRE3并用于转化酿酒酵母YPH499. 用RT-PCR检测到诱导1 kb RNA双螺旋和136 bp loop结构引起的GRE3基因表达下调. 结果表明，双链RNA介导的基因沉默技术，能够用作降低酿酒酵母某一特定基因表达水平的工具. 并有助于理解芽殖酵母的RNA干扰现象.     

Response of ad-2 mutants of Saccharomyces cerevisiae to carbon dioxide

Summary Confirmation that the ad-2 locus of yeast controls the carboxylation of aminoimidazole ribonucleotide (AIR) to 5-amino-4-imidazole carboxylate ribonucleotide (CAIR) is provided by the observation that 21 out of a sample of 113 ad-2 mutants were affected by CO₂. 19 of the mutants were stimulated by CO₂ and 2 were inhibited. The majority of the CO₂-stimulated mutants were confined to one section of the complementation map of the ad-2 locus.     

Synthesis and structure of a lead(II)-citrate: {Na(H2O)3}[Pb5(C6H5O7)3(C6H6O7)(H2O)3]·9.5H2O

The reaction of lead(II) nitrate with trisodium citrate Na₃(C₆H₅O₇) in a 1:22.5 ratio at pH 4.8 provides crystals of {Na(H₂O)₃}[Pb₅(H₂O)₃(C₆H₅O₇)₃(C₆H₆O₇)]·9.5H₂O (1). The structure of 1 is two-dimensional and exhibits five distinct Pb(II) sites and four different modes of citrate bonding. The five lead sites all display hemidirected coordination geometries, that is, irregular distribution of neighboring oxygen atoms resulting in obvious gaps in the coordination spheres. Consequently, the lead coordination geometries exhibit proximal bonding to a number of oxygen donors, as well as distal interactions with nearest neighbors. The coordination numbers vary from 8 to 10, with ‘5+3’, ‘5+4’, ‘6+4’ and ‘7+3’ coordination modes where the first number refers to the proximal ligands and the second to the distal set. The four crystallographically distinct citrate groups include three with deprotonated carboxylate groups (C₆H₅O₇)³⁻ and one with a single protonated carboxyl group (C₆H₆O₇)². The citrate ligands bridge 3, 5, 7 and 7 lead sites. Three of the citrate groups exhibit tridentate chelation coordination to a lead site through two carboxylate oxygen donors and the hydroxyl groups. One citrate group projects an uncoordinated -OH group and a pendant protonated carboxyl group into the interlamellar domain. This latter carboxyl group coordinates to a sodium cation, which exhibits five coordinate geometry defined by three aqua ligands and the carbonyl oxygen of the -CO₂H groups in the basal plane and a citrate -OH donor in the apical position.     

Synthesis and characterisation of a trinuclear uranyl complex: Crystal structure of (CN3H6)5[(UO2)3O(OH)2(CH3COO)(C2O4)3]

The reaction of uranyl oxalate trihydrate with guanidinium acetate at room temperature in water yields known uranyl complex with composition (CN₃H₆)₂[UO₂(C₂O₄)₂(H₂O)]·H₂O as a first phase and a novel complex (CN₃H₆)₅[(UO₂)₃O(OH)₂(CH₃COO)(C₂O₄)₃] as a second. The second phase was investigated by means of IR spectroscopy and X-ray diffraction. The trinuclear discrete complex contains two symmetrically independent uranyl ions with a pentagonal bipyramid structure and has a nonplanar geometry. The distortion of its equatorial plane is caused by substitution of a monodentate bridge hydroxide anion by a bidentate bridge acetate-anion. The acidic ligands found in the complex are usually in competition for a place in coordination sphere of an uranyl ion, thus peculiarities of the complex formation are discussed in terms of ‘crystallochemical analysis’.     

Methionine biosynthesis in Saccharomyces cerevisiae: Mutations at the regulatory locus ETH2

Summary Homoallelic and heteroallelic diploids involving the eth2-1, eth2-2 and eth2-7 alleles have been studied on the basis of several criteria used for the study of haploid strains: resistance towards ethionine, overproduction of either methionine or/and S-adenosylmethionine, repressibility of methionine biosynthetic enzymes. Complete recessivity of the three alleles over the wild type allele has been observed, when resistance and methionine synthesis are considered. However, with the eth2-2 allele, repressibility corresponds more to a dose effect of the ETH2 allele than to recessivity. The implications of these findings have been discussed. Results obtained for heteroallelic combinations show significant deviations from the expected values. These results have been interpreted as indicating possible interactions between two differently impaired products of gene ETH2. They render likely that the product of this gene is at least an homopolymer.     

Comparative reactivity of triorganosilanes, HSi(OEt)3 and HSiEt3, with IrCl(CO)(PPh3)2. Formation of IrCl(H)2(CO)(PPh3)2 or Ir(H)2(SiEt3)(CO)(PPh3)2 depending on the substituents at Si

Reaction of HSi(OEt)₃ with IrCl(CO)(PPh₃)₂ (5:1 molar ratio) at room temperature for 1 h gives IrCl(H){Si(OEt)₃}(CO)(PPh₃)₂ (1), which is observed by the ¹H and ³¹P{¹H} NMR spectra of the reaction mixture. The same reaction, but in 20:1 molar ratio at 50 °C for 24 h produces IrCl(H)₂(CO)(PPh₃)₂ (2) rather than the expected product Ir(H)₂{Si(OEt)₃}(CO)(PPh₃)₂ (3) that was previously reported to be formed by this reaction. Accompanying formation of Si(OEt)₄, (EtO)₃SiOSi(OEt)₃, and (EtO)₂HSiOSi(OEt)₃ is observed. On the other hand, trialkylhydrosilane HSiEt₃ reacts with IrCl(CO)(PPh₃)₂ (10:1 molar ratio) at 80 °C for 84 h to give Ir(H)₂(SiEt₃)(CO)(PPh₃)₂ (4) in a high yield, accompanying with a release of ClSiEt₃.     

Cyano-bridged cluster-metal coordination compound: synthesis and crystal structure of [Cu(NH3)3][Cu(NH3)4][Cu(NH3)5][W4Te4(CN)12]·5H2O

Dark-brown single crystals of the title compound 1 were obtained in high yield by layering a CuCl₂ solution in 25% aqueous ammonia on a glycerol solution of K₆[W₄Te₄(CN)₁₂]·5H₂O. The complex 1 was characterized by single crystal X-ray diffraction analysis and IR spectroscopy. The X-ray structure of 1 reveals a polymeric chain cyano-bridged cluster-metal coordination compound. The [W₄Te₄(CN)₁₂]⁶⁻ cluster anions are linked one to another by Cu²⁺ cations through coordination by nitrogen atoms of the CN groups.     

A novel ferromagnetic coupling one-dimensional complex {Cu(II)(NIT3Py)2[N(CN)2]2(H2O)2}

The novel ferromagnetic coupling one-dimensional complex {Cu(NIT3Py)₂[N(CN)₂]₂(H₂O)₂} (NIT3Py=2-(3^′-pyridyl)-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide) was synthesized and characterized structurally and magnetically. It crystallizes in the monoclinic space group C2/c. The Cu(II) ion is in a distorted octahedral environment. The units of {Cu(NIT3Py)₂[N(CN)₂]₂(H₂O)₂} were connected as one-dimensional structure by the intermolecular hydrogen bonds. Magnetic measurements show that there are intramolecular ferromagnetic interactions and intermolecular antiferromagnetic interactions within the chain.     

分别利用产甘油假丝酵母抗逆转录因子CgSTB5和CgSEF1对酿酒酵母菌株糠醛耐受改造

【背景】纤维素是生物转化解决能源问题的主要原料之一,其水解物中存在严重影响抑制菌株生长的糠醛,需脱毒才可应用于发酵,提高菌株耐受性是解决纤维素水解液实际生产应用的关键。【目的】酿酒酵母(Saccharomyces cerevisiae)是主要的纤维素水解液发酵工业菌株,但糠醛耐受性较低,通过分子改造获得具有高糠醛耐受性的菌株。【方法】利用新获得的产甘油假丝酵母(Candidaglycerinogenes)的相关抗逆转录因子CgSTB5、CgSEF1和CgCAS5,通过分子技术进行S.cerevisiae改造,考察其对酿酒酵母糠醛耐受性的影响,并尝试应用于未脱毒纤维素乙醇发酵。【结果】单个表达CgSTB5和CgSEF1的酿酒酵母,通过菌株点板实验表明菌株的糠醛耐受性提高25%以上,并且摇瓶发酵结果显示糠醛降解性能明显提高,生长延滞期明显缩短,S.cerevisiae W303/p414-CgSTB5的未脱毒纤维素乙醇发酵生产效率提高12.5%左右。【结论】转录因子CgSTB5和CgSEF1均能对提高酿酒酵母糠醛耐受性起到重要作用,并且有助于提高酿酒酵母菌株未脱毒纤维素乙醇发酵性能。     

Supramolecular assemblies of new 2-D complexes: Syntheses, crystal structures, spectroscopic and magnetic properties of {[MnAu2(CN)4(NITpPy)2(H2O)2]}n and {[Co(N(CN)2)2(NITpPy)2(H2O)2]}n

Two new complexes, {[MnAu₂(CN)₄(NITpPy)₂(H₂O)₂]}_n (1) and {[Co(N(CN)₂)₂(NITpPy)₂(H₂O)₂]}_n (2), have been synthesized and characterized. The single-crystal X-ray analysis for the complexes 1 and 2 demonstrates that each M(II) (M = Mn or Co) ion assumes a distorted octahedral MN₄O₂ coordination polyhedron. Four nitrogen atoms come from the cyanide groups and the pyridyl rings in a common plane, and two oxygen atoms come from the H₂O molecules in trans-positions. The structures of complexes 1 and 2 illustrate that aurophilicity and/or hydrogen bonding interactions play important roles in increasing dimensionality. Magnetic investigations on complexes 1 and 2 show the presence of weak antiferromagnetic interactions.