裂殖酵母作为外源基因表达系统

虽然裂殖酵母与酿酒酵母同属于子囊真菌,但比其它的酵母相比,裂殖酵母与更高等的真核细胞有许多相似的性质,使得裂殖酵母在分子生物学研究中成为一种提供信息的、准确的真核实验模型.它在外源基因表达方面同样具有前景.主要介绍了裂殖酵母的优点,其表达载体的性质,以及外源蛋白表达的例子.     

Differentiated Gene Expression in Cells within Yeast Colonies

Yeast cells growing on solid media organize themselves into multicellular structures, colonies, exhibiting patterns specific for particular yeast strains. With the aim of identifying genes involved in regulations of the colony formation, we applied a new approach enabling the extensive screening of Saccharomyces cerevisiae genes, the expression of which is changed during colony development. We used the library of S. cerevisiae DNA fragments inserted in front of the lacZ gene lacking its own promoter. Colonies of transformants with a blue/white patterned morphotype, implying that the expression of the lacZ gene from the inserted yeast promoter is switched on and off during the colony formation, were isolated. We identified several genes with variable expression during colony morphogenesis, including CCR4, PAM1, MEP3, ADE5,7 and CAT2. S. cerevisiae strain deleted in the CCR4 gene forms colonies with less organized morphology when compared with the isogenic parental strain. The synchronization of the expression patterns of some of the isolated genes in neighboring colonies was observed.     

Modification of the Sunflower Defensin SD2 Gene Sequence and Its Expression in Bacterial and Yeast Cells

To achieve broader range of the defensin antimicrobial activity, based on the sd2 gene sequence, the modified gene, sd2mod, was constructed. Hybrid genes, sd2-licBM2, licBM2-sd2, licBM2-sd2mod, and sd2mod-licBM2, in which the wild-type and modified gene sequences were fused in frame with the reporter gene encoding thermostable lichenase, were constructed. Expression of the wild-type, modified, and hybrid genes was examined in the cells of pro- and eukaryotes. It was demonstrated that these genes were efficiently expressed in the cells of lower eukaryotes, the yeast. Inhibiting effect of the SD2 and SDmod proteins as the components of the hybrid proteins, SD2-LicBM2 and SD2mod-LicBM2, on the growth of the Fusarium culmorum hyphae was similar to that of the wild-type and modified proteins. It was shown that the presence of lichenase in the hybrid proteins facilitated selection and analysis of the hybrid proteins expression in transgenic organisms.     

受水杨酸盐浓度调控的细菌遏制系统

由于存在基因工程微生物(GEMs)不受控制地在环境中释放的风险, 利用GEMs的生物降解能力治理环境污染的方法受到了限制。在大肠杆菌JM109中构建了一个受环境污染物调控的细菌遏制系统, 该系统是由杀伤元件和调控元件组成的双质粒体系, 使细菌的存活受环境中水杨酸盐浓度的调控。当培养基含水杨酸盐时, 阻遏蛋白LacI合成, 阻止自杀基因gef表达, 细菌快速繁殖; 当水杨酸盐不存在时, 自杀基因gef的表达导致细胞杀伤, 菌体大量死亡。该遏制系统可作为模型用于具有生物修复功能的基因工程菌的构建。     

The Biological Role of Glycerol in Yeast Cells. Yeast as Glycerol Producers

Applied Biochemistry and Microbiology - This review contains information on the physiological role of glycerol as an osmoprotective and cryoprotective factor in the vital activity of yeast cells....     

Global Gene Expression Analysis of Yeast Cells during Sake Brewing

During the brewing of Japanese sake, Saccharomyces cerevisiae cells produce a high concentration of ethanol compared with other ethanol fermentation methods. We analyzed the gene expression profiles of yeast cells during sake brewing using DNA microarray analysis. This analysis revealed some characteristics of yeast gene expression during sake brewing and provided a scaffold for a molecular level understanding of the sake brewing process.     

哺乳类细胞基因表达系统

通过适当的设计,可以构建在哺乳类细胞中表达的质粒,将其导入哺乳类细胞后,可以有效地表达外源基因．文章主要就这类质粒的特征、分类及其研究进展等方面予以综述．     

酵母基因中断技术

酵母基因中断技术是研究酵母基因功能的重要手段，自80年代初诞生以来经历了不断的改进和发展.PCR介导的酵母基因中断技术，大大简化了操作，实现了酵母基因的精确缺失；酵母基因的多重中断技术，可在酵母内实现多个基因的中断；可进行大规模基因中断和功能分析的酵母基因中断技术，适应了在酵母全基因组测序完成的情况下进行功能基因组学研究的要求.酵母基因中断技术对人类基因功能研究也有很大启示作用.     

Antimicrobial and Biological Effects of Bomphos and Phomphos on Bacterial and Yeast Cells

In this study, the antimicrobial effects of monophosphazenes such as SM, BOMPHOS, and PHOMPHOS were examined on bacterial and yeast strains. In addition, the biological effects of these compounds were tested on the Saccharomyces cerevisiae and Candida albicans cells. The SM has an antimicrobial effect on the bacterial and yeast strains within the range of 100 and 1500 μg. When the concentration was increased, the inhibition zone expanded on the growth media (P < 0.01; P < 0.001). Like SM, BOMPHOS molecule has antimicrobial activity on the bacterial and yeast cells. The most effective concentrations of BOMPHOS on the microorganisms were observed by 1500 μg (P < 0.001). The PHOMPHOS did not effect on the bacterial and yeast cells between 100 and 1000 range, but it has an antimicrobial effect in 1500 μg. In vitro media, the biological effects of these molecules were compared with vitamin E, melatonin, and fish oil on the yeast cells. In S. cerevisiae growth media, the cell densities were increased SM, BOMPHOS, and PHOMPHOS after 20, 30, and 45 h. The highest increase in the cell density were observed in media of BOMPHOS. In C. albicans growth media, the cell density was increased by melatonin after 20, 30, and 45 h, but were decreased by other supplemental groups. Lipid level of S. cerevisiae was reduced by administered 300 and 1000 μg vitamin E and fish oil (P < 0.01). In addition, the lipid level of the same yeast cell were diminished by the 1000 μg melatonin and 300 μg PHOMPHOS (P < 0.05, P < 0.01). The lipid level of C. albicans were increased by vitamin E and BOMPHOS and fish oil, but was decreased with PHOMPHOS (P < 0.01). In conclusion, while high concentration of PHOMPHOS has antimicrobial effects on the bacterial and yeast cells, the SM and BOMPHOS have antimicrobial effects in all the concentrations. PHOMPHOS decreased the lipid level of C. albicans, but BOMPHOS increased in the the same yeast cell. In addition, the antioxidants such as vitamin E, melatonin, and fish oils have affected on the lipid synthesis of yeast cells. Copyright 2000 Academic Press.     

Development of a Tunable Wide-Range Gene Induction System Useful for the Study of Streptococcal Toxin-Antitoxin Systems

Despite the plethora of genetic tools that have been developed for use in Streptococcus mutans, the S. mutans genetic system still lacks an effective gene induction system exhibiting low basal expression and strong inducibility. Consequently, we created two hybrid gene induction cassettes referred to as Xyl-S1 and Xyl-S2. Both Xyl-S cassettes are xylose inducible and controlled by the Bacillus megaterium xylose repressor. The Xyl-S cassettes each demonstrated >600-fold-increased reporter activity in the presence of 1.2% (wt/vol) xylose. However, the Xyl-S1 cassette yielded a much higher maximum level of gene expression, whereas the Xyl-S2 cassette exhibited much lower uninduced basal expression. The cassettes also performed similarly in Streptococcus sanguinis and Streptococcus gordonii, which suggests that they are likely to be useful in a variety of streptococci. We demonstrate how both Xyl-S cassettes are particularly useful for the study of toxin-antitoxin (TA) modules using both the previously characterized S. mutans mazEF TA module and a previously uncharacterized HicAB TA module in S. mutans. HicAB TA modules are widely distributed among bacteria and archaea, but little is known about their function. We show that HicA serves as the toxin component of the module, while HicB serves as the antitoxin. Our results suggest that, in contrast to that of typical TA modules, HicA toxicity in S. mutans is modest at best. The implications of these results for HicAB function are discussed.     

甲醇酵母表达系统

甲醇酵母基因表达系统是一种最近发展迅速的外源蛋白质生产系统。甲醇酵母是可利用甲醇作为唯一碳源的酵母 ,主要有Ｈ .Ｐｏｌｙｍｏｒｐｈａ、ＣａｎｄｉｄａＢｏｄｉｎｉｉ、ＰｉｃｈｉａＰａｓ ｔｏｒｉｓ三种 ,其中ＰｉｃｈｉａＰａｓｔｏｒｉｓ作为基因表达系统使用得最多、最广泛[1] 。与以往的基因表达系统相比 ,它具有无可匹敌的高表达特性 ,已被认为是最具有发展前景的生产蛋白质的工具之一。1 .外源蛋白质的基因在甲醇酵母中的高效表达目前已有多种蛋白质的基因在该表达系统中克隆成功 ,包括蛋白酶、酶抑制剂、受体、单链抗体等 (…     

Insufficiency of Copper Ion Homeostasis Causes Freeze-Thaw Injury of Yeast Cells as Revealed by Indirect Gene Expression Analysis

Saccharomyces cerevisiae is exposed to freeze-thaw stress in commercial processes, including frozen dough baking. Cell viability and fermentation activity after a freeze-thaw cycle were dramatically decreased due to freeze-thaw injury. Because this type of injury involves complex phenomena, the injury mechanisms are not fully understood. We examined freeze-thaw injury by indirect gene expression analysis during postthaw incubation after freeze-thaw treatment using DNA microarray profiling. The results showed that genes involved in the homeostasis of metal ions were frequently contained in genes that were upregulated, depending on the freezing period. We assessed the phenotype of deletion mutants of the metal ion homeostasis genes that exhibited freezing period-dependent upregulation and found that the strains with deletion of the MAC1 and CTR1 genes involved in copper ion homeostasis exhibited freeze-thaw sensitivity, suggesting that copper ion homeostasis is required for freeze-thaw tolerance. We found that supplementation with copper ions during postthaw incubation increased intracellular superoxide dismutase activity and intracellular levels of reactive oxygen species were decreased. Moreover, cell viability was increased by supplementation with copper ions. These results suggest that insufficiency of copper ion homeostasis may be one of the causes of freeze-thaw injury.Yeast (Saccharomyces cerevisiae) cells are exposed to various environmental stresses such as freeze-thaw, high-temperature, osmotic, and air-drying stresses during commercial processes. Freeze-thaw stress is important in the bread-making process, because frozen dough baking has become a major technology (3). Frozen dough baking improves labor conditions for bakers and enables them to provide fresh baked goods for consumers (3). Because frozen dough baking involves freeze-thaw treatment, it exposes yeast cells to freeze-thaw stress, which leads to a significant decrease in the fermentation ability and viability of yeast cells (called “freeze-thaw injury”) (8). The freeze-thaw injury of yeast cells depends on many factors, including freezing periods, freezing temperature, and the physiology of yeast cells (2, 5, 16, 17). Clarification of the changes in the cell physiology of yeast cells caused by freeze-thaw stress is important, because bakers are eager to extend the shelf life of frozen dough. In this study, we attempted to determine the changes in yeast cell physiology due to freeze-thaw injury by indirect gene expression analysis, which is described below.Freezing subjects yeast cells to low temperature, ice crystal formation in the cells, and dehydration from the cells. This causes both physical damage to cellular components, such as the cell wall, membrane, and proteins, and formation of reactive oxygen species (ROS) (13, 15). Superoxide anions and free radicals are generated in yeast cells during the freeze-thaw process (18), and ROS generation during the thawing process is increased, depending on the freezing period (5, 18). Oxidative stress generated by freeze-thaw treatment enhances the damage to cellular components (9, 25). Because modulation of intracellular levels of ROS after freeze-thaw treatment is required to protect against toxicity, ROS scavenging systems such as glutathione, catalase, and superoxide dismutase (SOD) are believed to be important for freeze-thaw tolerance of yeast cells (1, 2, 17). In particular, copper/zinc SOD (Cu/Zn SOD), which plays a role in oxygen radical detoxification, is necessary to confer full tolerance to freeze-thaw injury (18). Heavy metal ions, such as iron ions and copper ions, are important transition metals for the detoxification of oxygen radicals in yeast cells (6).Although there have been several studies on the mechanisms of freeze-thaw injury (5, 17, 18, 24), the mechanisms are complex and have not yet been fully elucidated. We therefore examined freeze-thaw injury by indirect gene expression analysis, which was conducted during postthaw incubation after freeze-thaw treatment using DNA microarray profiling. Indirect gene expression analysis may be advantageous for such an examination, because changes of gene expression may reflect the physiology of the freezing-state cell. We hypothesized that the genes involved in freeze-thaw injury may upregulate during postthaw incubation. To elucidate the physiological changes during freeze-thaw injury, we carried out indirect gene expression analysis using yeast cells after they had been frozen for different periods. The upregulated genes in the indirect gene expression analysis were extracted by clustering methods. We found that the genes involved in metal ion homeostasis were specifically upregulated. The importance of the genes extracted by the clustering was confirmed by phenotypic analysis using the deletion strains of the extracted genes. We also showed, by physiological analysis, that insufficiency of copper ion homeostasis causes freeze-thaw injury.     

Water Transport in Intact Yeast Cells as Assessed by Fluorescence Self-Quenching

Intact yeast cells loaded with 5- and-6-carboxyfluorescein were used to assess water transport. The results were similar to those previously reported for protoplasts assessed by using either fluorescence or light scattering, and the activation energies were 8.0 and 15.1 kcal mol⁻¹ (33.4 and 63.2 kJ mol⁻¹) for a strain overexpressing AQY1 aquaporin and a parental strain, respectively.     

asr基因酵母双杂交系统的构建及筛选鉴定

为研究asr作用的分子机制,分析该基因表达产物在体内相互作用的靶蛋白,分别构建诱饵质粒载体和转基因拟南芥cDNA文库,通过转入酵母细胞中,初步的鉴定分析证实成功构建了诱饵质拉载体和拟南芥cDNA文库.构建的酵母双杂交系统为发现asr基因表达产物在体内相互作用的靶蛋白奠定了基础.     

Photoreactivation and Gene Dosage in Yeast

The amount of photoreactivating enzyme in tetraploid cells of Saccharomyces cerevisiae and the ability of the cells to be photoreactivated after ultraviolet irradiation are directly proportional to the number of genes per cell involved in the synthesis of photoreactivating enzyme.     

Expression in Yeast of a Fusion Gene Composed of the Promoter of a Heat-Shock Gene from Arabidopsis and a Bacterial Gene for {beta}-Glucuronidase

Production of a functional ß-glucuronidase (GUS) proteinwas induced by exposure of exponentially growing yeast cellsto heat shock after transformation of the GUS gene under thecontrol of the promoter of the heat-shock gene, HSP18.2, fromArabidopsis. Yeast cyr and bcy mutations appeared to have essentiallyno effect. ¹Present Address: Laboratory of Plant Molecular Biology, TheRockefeller University, 1230 York Avenue, New York, NY 10021-6399,U.S.A.     

Frequency of Dividing Cells as an Estimator of Bacterial Productivity

It has recently been proposed that the frequency of dividing bacterial cells (FDC) can be used to predict growth rates of natural aquatic bacterial assemblages. We have examined the relationship between FDC and growth rate in bacteria from southern-temperate, coastal marine waters by using incubation under conditions of manipulated nutrient availability and exclusion of bacterivores. The regression of the natural logarithm of bacterial instantaneous growth rate (μ) on FDC resulted in a better fit than regression of untransformed μ on FDC. The regression equation was ln μ = 0.299FDC − 4.961. The coefficient of variation for predicted ln μ at mean FDC was 7%. The range of FDC-estimated bacterial instantaneous generation times for coastal Georgia waters was 12 to 68 h, and range of calculated bacterial production rates was 0.6 to 17.6 mg of C·m⁻³· h⁻¹. Unresolved problems of and suggested improvements on the FDC method of predicting growth rate are discussed.