Identification and Characterization of MAE1, the Saccharomyces cerevisiae Structural Gene Encoding Mitochondrial Malic Enzyme

Pyruvate, a precursor for several amino acids, can be synthesized from phosphoenolpyruvate by pyruvate kinase. Nevertheless, pyk1 pyk2 mutants of Saccharomyces cerevisiae devoid of pyruvate kinase activity grew normally on ethanol in defined media, indicating the presence of an alternative route for pyruvate synthesis. A candidate for this role is malic enzyme, which catalyzes the oxidative decarboxylation of malate to pyruvate. Disruption of open reading frame YKL029c, which is homologous to malic enzyme genes from other organisms, abolished malic enzyme activity in extracts of glucose-grown cells. Conversely, overexpression of YKL029c/MAE1 from the MET25 promoter resulted in an up to 33-fold increase of malic enzyme activity. Growth studies with mutants demonstrated that presence of either Pyk1p or Mae1p is required for growth on ethanol. Mutants lacking both enzymes could be rescued by addition of alanine or pyruvate to ethanol cultures. Disruption of MAE1 alone did not result in a clear phenotype. Regulation of MAE1 was studied by determining enzyme activities and MAE1 mRNA levels in wild-type cultures and by measuring β-galactosidase activities in a strain carrying a MAE1::lacZ fusion. Both in shake flask cultures and in carbon-limited chemostat cultures, MAE1 was constitutively expressed. A three- to fourfold induction was observed during anaerobic growth on glucose. Subcellular fractionation experiments indicated that malic enzyme in S. cerevisiae is a mitochondrial enzyme. Its regulation and localization suggest a role in the provision of intramitochondrial NADPH or pyruvate under anaerobic growth conditions. However, since null mutants could still grow anaerobically, this function is apparently not essential.     

Regulation and Physiological Role of the DAS1 Gene, Encoding Dihydroxyacetone Synthase, in the Methylotrophic Yeast Candida boidinii

The physiological role of dihydroxyacetone synthase (DHAS) in Candida boidinii was evaluated at the molecular level. The DAS1 gene, encoding DHAS, was cloned from the host genome, and regulation of its expression by various carbon and nitrogen sources was analyzed. Western and Northern analyses revealed that DAS1 expression was regulated mainly at the mRNA level. The regulatory pattern of DHAS was similar to that of alcohol oxidase but distinct from that of two other enzymes in the formaldehyde dissimilation pathway, glutathione-dependent formaldehyde dehydrogenase and formate dehydrogenase. The DAS1 gene was disrupted in one step in the host genome (das1Δ strain), and the growth of the das1Δ strain in various carbon and nitrogen sources was compared with that of the wild-type strain. The das1Δ strain had completely lost the ability to grow on methanol, while the strain with a disruption of the formate dehydrogenase gene could survive (Y. Sakai et al., J. Bacteriol. 179:4480–4485, 1997). These and other experiments (e.g., those to determine the expression of the gene and the growth ability of the das1Δ strain on media containing methylamine or choline as a nitrogen source) suggested that DAS1 is involved in assimilation rather than dissimilation or detoxification of formaldehyde in the cells.     

Glutamate Synthase: Properties of the Reduced Nicotinamide Adenine Dinucleotide-Dependent Enzyme from Saccharomyces cerevisiae

A reduced nicotinamide adenine dinucleotide (NADH)-dependent glutamate synthase has been detected and partially purified from crude extracts of Saccharomyces cerevisiae. The enzyme is specific for NADH, glutamine, and alpha-ketoglutarate (K(m) values of 2.6 muM, 1.0 mM, and 140 muM, respectively) and has a pH optimum between 7.1 and 7.7. The stoichiometry of the reaction has been determined as 2 mol of glutamate synthesized per mol of glutamine consumed. Glutamate synthase can be distinguished from either of the glutamate dehydrogenases of yeast on the basis of its substrate requirements and behavior during agarose gel and ion exchange chromatography. Variations in the specific activity of glutamate synthase, which occur in response to changes in the growth medium, are similar in character to those observed with the nicotinamide adenine dinucleotide phosphate-dependent (anabolic) glutamate dehydrogenase.     

酿酒葡萄分支酸合成酶基因(VvCS)的克隆与表达分析

本研究以酿酒葡萄(Vitis vinifera)品种赤霞珠(Cabernet Sauvignon)及霞多丽(Chardonnay)为试材,采用in silico克隆和分子克隆相结合的策略,从果实中克隆到分支酸合成酶基因,命名为VvCS。该基因的cDNA编码区全长1312bp,编码436个氨基酸残基,预测其编码蛋白质分子量为46.9kD,等电点为7.8;生物信息学分析显示VvCS的DNA全长7117bp,包含13个外显子和12个内含子,定位于葡萄的第13号染色体上。VvCS编码的蛋白与其它植物来源的分支酸合成酶在氨基酸水平上的同源性为75%左右;实时荧光定量PCR分析表明VvCS在葡萄果实、茎、叶和叶柄组织中均有表达,且在果皮、果肉和种子中的表达变化趋势相似,与盛花后5周的果实相比,盛花后11周果实各部位中VvCS表达丰度均有不同程度增加。     

与人体ε-珠蛋白基因5''''旁侧正调控元件(ε-PREI)专一结合的调控因子的初步研究

人体ε－珠蛋白基因５′旁侧ＤＮＡ序列对该基因时空表达与调控起着十分重要的作用。本文运用凝胶电泳阻抑法,ＤＮａｓｅＩ足印法和Ｓｏｕｔｈｗｅｓｔｅｒｎｂｌｏｔ分析法发现在胚胎型红白血病细胞株Ｋ５６２细胞中有一个特异的核蛋白因子（简称ε－ＳＳＰ,其分子量大约为８０ｋＤ）,它能专一地与人体ε－珠蛋白基因５′旁侧一个红细胞专一和发育时期特异的正调控元件（ε－ＰＲＥＩＩ,－４４６ｂｐ到－４１９ｂｐ）相结合。竞争实验表明该因子与ε－ＰＲＥＩＩ的结合能被人体ε－珠蛋白基因启动子区ＤＮＡ片段（－１７７ｂｐ到＋１ｂｐ）所竞争;同时也能被人体β－类珠蛋白基因远侧端调控元件ＬＣＲ中的ＤＮａｓｅＩ超敏感点Ｉ核心区ＤＮＡ片段（－１０９６５ｂｐ到－１０６８１ｂｐ）与超敏感点ＩＩ核心区ＤＮＡ片段（－１４９９３ｂｐ到－１４７１８ｂｐ）所竞争。我们的结果提示了ε－ＳＳＰ不仅是一个与红细胞专一性和发育时期特异性相关的反式调控因子,而且它可能介导远侧端调控元件（ＬＣＲ）与近侧端调控元件启动子之间的相互作用,共同调节ε－珠蛋白基因在胚胎期的表达。     

Improved production of ethanol by deleting FPS1 and over-expressing GLT1 in Saccharomyces cerevisiae

To improve ethanol production in Saccharomyces cerevisiae, two yeast strains were constructed. In the mutant KAM-3, the FPS1 gene, which encodes a channel protein responsible for glycerol export, was deleted. The mutant KAM-11 had the GLT1 gene (encoding glutamate synthase) placed under the PGK1 promoter while having the FPS1 deletion. Growth rate and biomass concentration remained virtually unchanged with the mutant KAM-11, compared to that of the parent. Over-expression of GLT1 by the PGK1 promoter along with FPS1 deletion resulted in a 14% higher ethanol production and a 30% lower glycerol formation compared to the parental strain under anaerobic fermentation conditions. Furthermore, acetate and pyruvic acid formation was also reduced in order for cells to maintain redox balance.     

Regulation of Ceramide Synthase by Casein Kinase 2-dependent Phosphorylation in Saccharomyces cerevisiae

Complex sphingolipids are important components of eukaryotic cell membranes and, together with their biosynthetic precursors, including sphingoid long chain bases and ceramides, have important signaling functions crucial for cell growth and survival. Ceramides are produced at the endoplasmic reticulum (ER) membrane by a multicomponent enzyme complex termed ceramide synthase (CerS). In budding yeast, this complex is composed of two catalytic subunits, Lac1 and Lag1, as well as an essential regulatory subunit, Lip1. Proper formation of ceramides by CerS has been shown previously to require the Cka2 subunit of casein kinase 2 (CK2), a ubiquitous enzyme with multiple cellular functions, but the precise mechanism involved has remained unidentified. Here we present evidence that Lac1 and Lag1 are direct targets for CK2 and that phosphorylation at conserved positions within the C-terminal cytoplasmic domain of each protein is required for optimal CerS activity. Our data suggest that phosphorylation of Lac1 and Lag1 is important for proper localization and distribution of CerS within the ER membrane and that phosphorylation of these sites is functionally linked to the COP I-dependent C-terminal dilysine ER retrieval pathway. Together, our data identify CK2 as an important regulator of sphingolipid metabolism, and additionally, because both ceramides and CK2 have been implicated in the regulation of cancer, our findings may lead to an enhanced understanding of their relationship in health and disease.     

Cloning and Sequencing of the Gene Encoding the Soluble Fumarate Reductase from Saccharomyces cerevisiae

A gene of the soluble fumarate reductase (FRDS) that binds FADnon-covalently was cloned by polymerase chain reaction (PCR)using degenerate oligonucleotides designed from partial aminoacid sequences of highly purified enzyme. The nucleotide sequenceof a 0.99-kb amplified product was found to be nearly identicalto a partial sequence of an open reading frame (ORF) previouslyreported (EMBL database accession number S-30830). Accordingto the sequence in the EMBL database, we cloned 1.7-kb fragmentcontaining entire sequence of this ORF by PCR and found thatthis fragment contained a perfect match to the 0.99-kb sequenceamplified with the degenerate primers. From these results, weconcluded that this ORF is the FRDS gene. The amino acid sequencesof the regions involved in the non-covalent binding of FAD andthe active site, which are conserved among the flavoproteinsubunits of membrane-bound fumarate reductase and succinatedehydrogenase, were found in FRDS. However, unlike the membrane-boundenzymes, FRDS did not contain the histidine residue that covalentlybinds the isoalloxazine ring of FAD at or near the correspondingposition. FRDS showed high homology to the product of S. cerevisiaeOSM1 gene which was reported to be required for growth in hypertonicmedia.     

Neuronal Soluble Factors Differentially Regulate the Expression of the GLT1 and GLAST Glutamate Transporters in Cultured Astroglia

Abstract: The glutamate transporters in the plasma membranes of neural cells secure termination of the glutamatergic synaptic transmission and keep the glutamate levels below toxic concentrations. Astrocytes express two types of glutamate transporters, GLAST (EAAT1) and GLT1 (EAAT2). GLT1 predominates quantitatively and is responsible for most of the glutamate uptake activity in the juvenile and adult brain. However, GLT1 is severely down-regulated in amyotrophic lateral sclerosis, a progressive neurodegenerative disease. Furthermore, selective loss of this transporter occurs in cultured astroglia. Expression of GLAST, but not of GLT1, seems to be regulated via the glutamate receptor signalling. The present study was undertaken to examine whether neuronal factors, other than glutamate, influence the expression of astroglial glutamate transporters. The expression of GLT1 and GLAST was examined in primary cultures of cerebellar granule neurons, cortical neurons, and astrocytes under different experimental conditions, including those that mimic neuron-astrocyte interactions. Pure astroglial cultures expressed only GLAST, whereas astrocytes grown in the presence of neurons expressed both GLAST (at increased levels) and GLT1. The induction of GLT1 protein and its mRNA was reproduced in pure cortical astroglial cultures supplemented with conditioned media from cortical neuronal cultures or from mixed neuron-glia cultures. This treatment did not change the levels of GLAST. These results suggest that soluble neuronal factors differentially regulate the expression of GLT1 and GLAST in cultured astroglia. Further elucidation of the molecular nature of the secreted neuronal factors and corresponding signalling pathways regulating the expression of the astroglial glutamate transporters in vitro may reveal mechanisms important for the understanding and treatment of neurological diseases.     

酵母豆蔻酰辅酶A：蛋白质N端转酰基酶基因的克隆与表达

利用ＰＣＲ技术,从酵母染色体中扩增得到酵母豆蔻酰－ＣｏＡ：蛋白质Ｎ端转酰基酶（ＹＳＣＮＭＴ）基因,并克隆到ｐＢｌｕｅｓｃｒｉｐｔＫＳ＋载体中。由ＤＮＡ全序测定表明,获得了ＹＳＣＮＭＴ编码基因。进一步构建了Ｔ７Ｐｒｏｍｏｔｅｒ控制下的含上述完整ＹＳＣＮＭＴ编码基因的表达质粒ｐＭＦＴ７－５－ＮＭＴ,转化大肠杆菌ＢＬ２１（ＤＥ３）,进行ＩＰＴＧ诱导表达研究。通过ＳＤＳ－ＰＡＧＥ分析,观察到一与理论分子量一致的诱导条带（约５３ｋＤ）,占全菌蛋白的３９％左右,且可溶性部分约占上清液中全部蛋白的３４％。经一步Ｐ１１磷酸纤维素阳离子交换柱层析,将其纯化到纯度达９７％以上．纯化的表达产物经Ｎ端氨基酸序列分析,所测定的Ｎ端５个氨基酸的序列,与从克隆的ＹＳＣＮＭＴ基因推出的氨基酸序列完全一致（不含Ｎ端Ｍｅｔ）。对所得的ＹＳＣＮＭＴ进行酶活力鉴定,观察到了明显的活力。