ste20基因突变抑制葡萄糖诱导的酿酒酵母细胞凋亡

近年来,酿酒酵母的细胞调亡研究取得了很大进展。多种因素可以诱导其调亡,譬如过氧化氢（H2O2）、醋酸、高渗透压和高盐浓度等。葡萄糖是酿酒酵母生长所必须的重要营养物质之一。同时,在其他营养元素缺乏的条件下,只用葡萄糖培养将迅速的诱导酿酒酵母的细胞凋亡。Ste20是PAK（p21 activated kinase）家族的成员,它参与酿酒酵母的信息素应答、假菌丝生长和侵入生长等途径。有研究表明,ste20突变株能抵抗由信息素和过氧化物诱导的细胞调亡。我们发现STE20基因突变也能抑制葡萄糖诱导的凋亡,用葡萄糖处理时,与野生型相比,ste20突变株细胞能保持完整的细胞膜和细胞核结构。H2O2诱导酿酒酵母细胞凋亡时,需要Ste20激酶磷酸化组蛋白H2B第十号丝氨酸（S10）。因此,葡萄糖诱导的酿酒酵母细胞凋亡作用可能通过类似于过氧化氢诱导的酿酒酵母细胞凋亡的途径进行的。     

Membrane Protein Rim21 Plays a Central Role in Sensing Ambient pH in Saccharomyces cerevisiae

External alkalization activates the Rim101 pathway in Saccharomyces cerevisiae. In this pathway, three integral membrane proteins, Rim21, Dfg16, and Rim9, are considered to be the components of the pH sensor machinery. However, how these proteins are involved in pH sensing is totally unknown. In this work, we investigated the localization, physical interaction, and interrelationship of Rim21, Dfg16, and Rim9. These proteins were found to form a complex and to localize to the plasma membrane in a patchy and mutually dependent manner. Their cellular level was also mutually dependent. In particular, the Rim21 level was significantly decreased in dfg16Δ and rim9Δ cells. Upon external alkalization, the proteins were internalized and degraded. We also demonstrate that the transient degradation of Rim21 completely suppressed the Rim101 pathway but that the degradation of Dfg16 or Rim9 did not. This finding strongly suggests that Rim21 is the pH sensor protein and that Dfg16 and Rim9 play auxiliary functions through maintaining the level of Rim21 and assisting in its plasma membrane localization. Even without external alkalization, the Rim101 pathway was activated in a Rim21-dependent manner by either protonophore treatment or depletion of phosphatidylserine in the inner leaflet of the plasma membrane, both of which caused plasma membrane depolarization like the external alkalization. Therefore, plasma membrane depolarization seems to be one of the key signals for the pH sensor molecule Rim21.     

CaSRB9基因的克隆及其在酿酒酵母形态发生中的作用

白色念珠菌是一种重要的人体致病真菌 ,致病机制与其形态发生紧密相关。酿酒酵母Flo8因子在其形态发生中起重要作用 ,我们把白色念珠菌基因组DNA导入酿酒酵母flo8基因缺失株中 ,筛选能够互补 flo8侵入生长缺陷的基因 ,分离到了一个与酿酒酵母SRB9同源的新基因 ,命名为CaSRB9。该基因全长 4998bp ,编码一种16 6 5个氨基酸的蛋白质。在双倍体酿酒酵母中CaSRB9可以部分互补MAPK途径基因缺失株以及 flo8缺失株的菌丝生长缺陷 ;在单倍体酿酒酵母中表达能够互补 flo8缺失株的侵入生长缺陷 ,但在MAPK途径基因缺失株中不能形成侵入生长     

“Sleeping Beauty”: Quiescence in Saccharomyces cerevisiae

The cells of organisms as diverse as bacteria and humans can enter stable, nonproliferating quiescent states. Quiescent cells of eukaryotic and prokaryotic microorganisms can survive for long periods without nutrients. This alternative state of cells is still poorly understood, yet much benefit is to be gained by understanding it both scientifically and with reference to human health. Here, we review our knowledge of one “model” quiescent cell population, in cultures of yeast grown to stationary phase in rich media. We outline the importance of understanding quiescence, summarize the properties of quiescent yeast cells, and clarify some definitions of the state. We propose that the processes by which a cell enters into, maintains viability in, and exits from quiescence are best viewed as an environmentally triggered cycle: the cell quiescence cycle. We synthesize what is known about the mechanisms by which yeast cells enter into quiescence, including the possible roles of the protein kinase A, TOR, protein kinase C, and Snf1p pathways. We also discuss selected mechanisms by which quiescent cells maintain viability, including metabolism, protein modification, and redox homeostasis. Finally, we outline what is known about the process by which cells exit from quiescence when nutrients again become available.     

L-Carnosine Affects the Growth of Saccharomyces cerevisiae in a Metabolism-Dependent Manner

The dipeptide L-carnosine (β-alanyl-L-histidine) has been described as enigmatic: it inhibits growth of cancer cells but delays senescence in cultured human fibroblasts and extends the lifespan of male fruit flies. In an attempt to understand these observations, the effects of L-carnosine on the model eukaryote, Saccharomyces cerevisiae, were examined on account of its unique metabolic properties; S. cerevisiae can respire aerobically, but like some tumor cells, it can also exhibit a metabolism in which aerobic respiration is down regulated. L-Carnosine exhibited both inhibitory and stimulatory effects on yeast cells, dependent upon the carbon source in the growth medium. When yeast cells were not reliant on oxidative phosphorylation for energy generation (e.g. when grown on a fermentable carbon source such as 2% glucose), 10–30 mM L-carnosine slowed growth rates in a dose-dependent manner and increased cell death by up to 17%. In contrast, in media containing a non-fermentable carbon source in which yeast are dependent on aerobic respiration (e.g. 2% glycerol), L-carnosine did not provoke cell death. This latter observation was confirmed in the respiratory yeast, Pichia pastoris. Moreover, when deletion strains in the yeast nutrient-sensing pathway were treated with L-carnosine, the cells showed resistance to its inhibitory effects. These findings suggest that L-carnosine affects cells in a metabolism-dependent manner and provide a rationale for its effects on different cell types.     

An Order-Disorder Transition Plays a Role in Switching Off the Root Effect in Fish Hemoglobins

The Root effect is a widespread property among fish hemoglobins whose structural basis remains largely obscure. Here we report a crystallographic and spectroscopic characterization of the non-Root-effect hemoglobin isolated from the Antarctic fish Trematomus newnesi in the deoxygenated form. The crystal structure unveils that the T state of this hemoglobin is stabilized by a strong H-bond between the side chains of Asp95α and Asp101β at the α₁β₂ and α₂β₁ interfaces. This unexpected finding undermines the accepted paradigm that correlates the presence of this unusual H-bond with the occurrence of the Root effect. Surprisingly, the T state is characterized by an atypical flexibility of two α chains within the tetramer. Indeed, regions such as the CDα corner and the EFα pocket, which are normally well ordered in the T state of tetrameric hemoglobins, display high B-factors and non-continuous electron densities. This flexibility also leads to unusual distances between the heme iron and the proximal and distal His residues. These observations are in line with Raman micro-spectroscopy studies carried out both in solution and in the crystal state. The findings here presented suggest that in fish hemoglobins the Root effect may be switched off through a significant destabilization of the T state regardless of the presence of the inter-aspartic H-bond. Similar mechanisms may also operate for other non-Root effect hemoglobins. The implications of the flexibility of the CDα corner for the mechanism of the T-R transition in tetrameric hemoglobins are also discussed.     

酿酒酵母ADH3基因的敲除

设计含有与酿酒酵母(Saccharomyces cerevisiae)编码乙醇脱氢酶Ⅲ的ADH3基因ORF两侧序列同源的长引物,以质粒pUG6为模板进行PCR构建带有Cre/loxP系统的敲除组件。转化酿酒酵母YS3(Saccharomyces cerevisiae),并将质粒pSH65转入阳性克隆子。半乳糖诱导表达Cre酶切除Kanr基因,在YPD培养基中连续传代培养丢失pSH65质粒,在原ORF处留下一个loxP位点,获得ADH3单倍体缺陷型菌株。利用同样的方法再次敲除双倍体的另一个等位基因。最终获得ADH3双倍体基因缺陷型突变株YS3-ADH3。     

OsRRMh,a Spen-like Gene,Plays an Important Role during the Vegetative to Reproductive Transition in Rice

OsRRMh, a homologue of OsRRM, encodes a Spen-like protein, and is composed of two N-terminal RNA recognition motifs (RRM) and one C-terminal Spen paralogue and an orthologue C-terminal domain (SPOC). The gene has been found to be constitutively expressed in the root, stem, leaf, spikelet, and immature seed, and alternative splicing patterns were confirmed in different tissues, which may indicate diverse functions for OsRRMh. The OsRRMh dsRNAi lines exhibited late-flowering and a larger panicle phenotype. When full-length OsRRMh and/or its SPOC domain were overexpressed, the fertility rate and number of spikelets per panicle were both markedly reduced. Also, overexpression of OsRRMh in the Arabidopsis fpa mutant did not restore the normal flowering time, and it delayed flowering in Col plants. Therefore, we propose that OsRRMh may confer one of its functions in the vegetative-to-reproductive transition in rice (Oryza sativa L. subsp. japonica cv. Zhonghua No. 11 (ZH11)).     

Ambiguity in a Polypeptide-Synthesizing Extract from Saccharomyces cerevisiae

Environmental factors known to induce ambiguity in bacterial extracts were tested in an in vitro cytoplasmic polypeptide-synthesizing system derived from Saccharomyces cerevisiae. Increasing concentrations of magnesium, spermine, and spermidine resulted in extensive leucine-phenylalanine ambiguity in polyuridylic acid-directed polypeptide synthesis. Kinetic studies showed that spermine-mediated stimulation of leucine incorporation occurred when phenylalanine was being actively incorporated. In addition to leucine, the amino acids isoleucine and serine were incorporated in the presence of added magnesium and spermine. Ambiguity in the presence of a high Mg(2+) concentration was decreased when the pH of the reaction mixture was lowered. Ethanol and neomycin enhanced ambiguity to a small, but significant, extent. Streptomycin and temperature had no effect on ambiguity. Leucine, isoleucine, and serine were not attached to phenylalanine transfer ribonucleic acid (tRNA) when the aminoacylation reaction was performed at increasing Mg(2+) and spermine concentrations. On the other hand, increasing levels of Mg(2+) and spermine stimulated the incorporation of leucine from tRNA into polypeptide during the transfer reaction. The formal similarity between the findings in the yeast and Escherichia coli systems implies the existence of a tRNA-screening site on the yeast ribosome comparable to that suggested for bacteria. A proposal is made as to the manner in which this site may function to produce the ambiguous codon translation observed.     

Growth of Saccharomyces cerevisiae in gaseous fluidized beds

Summary Baker's yeast was aerobically grown in gaseous fluidized beds in the form of solid particles. Air was used as the fluidizing fluid and as a source of oxygen, while the concentrated nutrient solution was sprayed at the top of the bed. Five glucose concentrations 125, 160, 200, 250 und 350 gl^–1 were used. A maximum in the growth rate and in the yield coefficient occurred at 250 and 200 g l^–1, respectively. The calculated growth rates are one order of magnitude less than the growth rates in submerged cultures, but the maintenance energy coefficient is the same in both systems. Alcohol ppm level in the exhaust gases increased with increasing glucose concentration in the nutrient solution. Oscillations in the alcohol production indicated product inhibition of the cell growth under high glucose concentrations in the nutrient feed solution.     

Growth of Saccharomyces cerevisiae in a chemostat under high glucose conditions

Saccharomyces cerevisiae was grown in a chemostat under high glucose conditions (up to 300 g l^–1). The results support the view that higher glucose feed favors higher ethanol production regardless of the existence of osmotic stress. A low glucose utilization and yield coefficient provides an opportunity to improve continuous fermentation performance in the fuel alcohol industry. The possibility exists of reusing yeast cells and subsequently lower operating costs, and by using an optimal glucose feeding concentration between 100 and 200 g l^–1.