酿酒酵母中过表达URA5及URA3基因催化合成UMP的初步研究

为提高乳清酸到尿嘧啶核苷酸(UMP)的转化效率,利用PCR方法扩增酿酒酵母乳清酸磷酸核糖转移酶基因URA5, 并将其连接到携带乳清苷酸脱羧酶基因URA3的表达载体pYX212中,构建了重组质粒pYX212-URA5,然后转化到酿酒酵母BJX12中进行表达,并进行转化乳清酸到UMP的初步研究。试验结果表明: pYX212-URA5/ BJX12发酵培养40h后以32 mM乳清酸为底物催化产生UMP的量约为7 mM。明显高于同等条件下pYX212/ BJX12的UMP产量2.7 mM和对照组野生型BJX12的UMP产量2.4 mM。     

酿酒酵母氧化胁迫应答反应机制

氧化胁迫是生物体面对逆境时的重要反应。在与逆境和活性氧做斗争的过程中,细胞进化出一套完整的应答调控机制,通过调节体内活性氧的代谢平衡,来保护DNA、脂质和蛋白质等免受氧化攻击。本文以酿酒酵母为例,根据近年来国内外研究的进展,围绕其在氧化胁迫应答过程中的三道保护屏障,即抗氧化物质和防御酶系统、转录调节和氧化物降解以及细胞器自噬,综述了其抗氧化代谢机理,为深入认识细胞的抗氧化应答机制奠定基础。     

Engineering membrane asymmetry to increase medium-chain fatty acid tolerance in Saccharomyces cerevisiae

Saccharomyces cerevisiae is an attractive chassis for the production of medium-chain fatty acids, but the toxic effect of these compounds often prevents further improvements in titer, yield, and productivity. To address this issue, Lem3 and Sfk1 were identified from adaptive laboratory evolution mutant strains as membrane asymmetry regulators. Co-overexpression of Lem3 and Sfk1 [Lem3(M)-Sfk1(H) strain] through promoter engineering remodeled the membrane phospholipid distribution, leading to an increased accumulation of phosphatidylethanolamine in the inner leaflet of the plasma membrane. As a result, membrane potential and integrity were increased by 131.5% and 29.2%, respectively; meanwhile, the final OD₆₀₀ in the presence of hexanoic acid, octanoic acid, and decanoic acid was improved by 79.6%, 73.4%, and 57.7%, respectively. In summary, this study shows that membrane asymmetry engineering offers an efficient strategy to enhance medium-chain fatty acids tolerance in S. cerevisiae, thus generating a robust industrial strain for producing high-value biofuels.     

Expression of HPV16 E6 oncoprotein increases resistance to several stress conditions in Saccharomyces cerevisiae

The E6 protein of human papillomavirus type 16 is essential for the oncogenic transformation process induced by these viruses. Here we expressed the E6 protein in Saccharomyces cerevisiae (which lacks p53) in order to determine if E6 interacts with normal cell functioning, independently of the p53 tumour suppressor factor. We observed a higher resistance to caffeine, hydrogen peroxide and to pheromone, but not to high temperature, starvation and osmostress. Measurement of the relative expression levels of target genes of the signalling pathways, involved in the latter stressful stimuli, led us to conclude that such pathways are differently regulated in the presence of E6.     

Hypertonic conditions trigger transient plasmolysis,growth arrest and blockage of transporter endocytosis in Aspergillus nidulans and Saccharomyces cerevisiae

Abstract

By using Aspergillus nidulans strains expressing functional GFP-tagged transporters under hypertonic conditions, we noticed the rapid appearance of cortical, relatively static, fluorescent patches (0.5–2.3 μm). These patches do not correspond to transporter microdomains as they co-localize with other plasma membrane-associated molecules, such as the pleckstrin homology (PH) domain and the SsoA t-Snare, or the lipophilic markers FM4-64 and filipin. In addition, they do not show characteristics of lipid rafts, MCCs or other membrane microdomains. Deconvoluted microscopic images showed that fluorescent patches correspond to plasma membrane invaginations. Transporters remain fully active during this phenomenon of localized plasmolysis. Plasmolysis was however associated with reduced growth rate and a dramatic blockage in transporter and FM4-64 endocytosis. These phenomena are transient and rapidly reversible upon wash-out of hypertonic media. Based on the observation that block in endocytosis by hypertonic treatment altered dramatically the cellular localization of tropomyosin (GFP-TpmA), although it did not affect the cortical appearance of upstream (SlaB-GFP) or downstream (AbpA-mRFP) endocytic components, we conclude that hypertonicity modifies actin dynamics and thus acts indirectly on endocytosis. This was further supported by the effect of latrunculin B, an actin depolymerization agent, on endocytosis. We show that the phenomena observed in A. nidulans also occur in Saccharomyces cerevisiae, suggesting that they constitute basic homeostatic responses of ascomycetes to hypertonic shock. Finally, our work shows that hypertonic treatments can be used as physiological tools to study the endocytic down-regulation of transporters in A. nidulans, as non-conditional genetic blocks affecting endocytic internalization are lethal or severely debilitating.     

Transient increase in Ca2+ influx in Saccharomyces cerevisiae in response to glucose: effects of intracellular acidification and cAMP levels

Influx of 45Ca2+ into Saccharomyces cerevisiae was measured under experimental conditions which enabled measurements of initial rate of transport across the plasma membrane, without interference by the vacuolar Ca2+ transport system. Addition of glucose or glycerol to the cells, after pre-incubation in glucose-free medium for 5 min, caused a rapid, transient increase in 45Ca2+ influx, reaching a peak at 3-5 min after addition of substrate. Ethanol, or glycerol added with antimycin A, had no effect on 45Ca2+ influx. We have shown previously that this increase is not mediated by an effect of the substrates on intracellular ATP levels. Changes in membrane potential accounted for only a part of the glucose-stimulated 45Ca2+ influx. The roles of intracellular acidification and changes in cellular cAMP in mediating the effects of glucose on 45Ca2+ influx were examined. After a short preincubation in glucose-free medium addition of glucose caused a decrease in the intracellular pH, [pH]i, which reached a minimum value after 3 min. A transient increase in the cellular cAMP level was also observed. Addition of glycerol also caused intracellular acidification, but ethanol or glycerol added with antimycin A had no effect on [pH]i. Artificial intracellular acidification induced by exposure to isobutyric acid or to CCCP caused a transient rise in Ca2+ influx but the extent of the increase was smaller than that caused by glucose, and the time-course was different. We conclude that intracellular acidification may be responsible for part of the glucose stimulation of Ca2+ influx.(ABSTRACT TRUNCATED AT 250 WORDS)     

RNA complementary to the 5' UTR of mRNA triggers effective silencing in Saccharomyces cerevisiae

Conditional silencing of target genes in Saccharomyces cerevisiae by antisense RNAs expressed in vivo has been challenged. The MFalpha1::lacZ fusion present in S. cerevisiae SF51-3 was chosen as a model target, and fragments of this gene were cloned in reverse orientation into the expression vector pYES2, bearing the GAL1 promoter. Among the different antisense constructs tested, only the one complementary to the 5' UTR of target mRNA featured effective silencing. Nevertheless, the expression in vivo of this antisense RNA could not be properly tuned by the absence or presence of galactose in the culture medium. Accordingly, conditional silencing could not be attained by this antisense hosted into pYES2. On the contrary, cloning the same antisense construct into the expression vector pSAL4 yielded a fully conditional silencing linked to the control of antisense expression by the absence or presence of Cu(2+) into the culture medium.     

Induction of DNA synthesis in Anopheles albimanus tissue cultures in response to a Saccharomyces cerevisiae challenge

DNA synthesis was detected by the incorporation of 5-bromo-2' deoxy-uridine (BrdU) in adult Anopheles albimanus organs in culture in response to a challenge with Saccharomyces cerevisiae. Abdomens of mosquitoes inoculated with Roswell Park Memorial Institute medium (RPMI, control) or yeast were cultivated in RPMI plus ConA and BrdU for 5 days. DNA was obtained by phenolic extraction and the incorporated BrdU was quantified by ELISA using anti-BrdU peroxidase-labeled antibodies. Abdomen tissues of mosquitoes inoculated with yeast showed higher DNA synthesis than controls. Organs from untreated mosquitoes cultured in the presence of zymosan also synthesized DNA but at a lower level than tissues from yeast-inoculated mosquitoes. In similar experiments, DNA synthesis was inhibited by the addition of colchicine. DNA synthesis, evidenced by epifluorescence using an anti-BrdU fluorescein-labeled antibody, occurred in fat body, epithelial cells in pleural membranes, and the dorsal vessel. Pleural membranes showed the highest number of labeled cells. These tissues were also labeled with anti-PCNA (proliferating cell nuclear antigen) antibodies, two of which were able to produce polytene chromosomes under yeast stimulation. These results demonstrate that different An. albimanus tissues undergo DNA synthesis in response to foreign particles.     

Enhancement of Stress Tolerance in Saccharomyces cerevisiae by Overexpression of Ubiquitin Ligase Rsp5 and Ubiquitin-Conjugating Enzymes

Yeast Saccharomyces cerevisiae cells overexpressing essential ubiquitin ligase Rsp5 or ubiquitin-conjugating enzymes (Ubc1-Ubc13) showed tolerance to various stresses. Co-overexpression of Rsp5 and Ubc1, Ubc2, Ubc3, Ubc5, Ubc6, Ubc9, Ubc10, Ubc11, Ubc12, or Ubc13 further enhanced stress tolerance. These results suggest that overexpression of ubiquitin-related enzymes might be a useful method for breeding novel stress-resistant strains.     

Adenovirus E4orf4 protein induces PP2A-dependent growth arrest in Saccharomyces cerevisiae and interacts with the anaphase-promoting complex/cyclosome

Adenovirus early region 4 open reading frame 4 (E4orf4) protein has been reported to induce p53-independent, protein phosphatase 2A (PP2A)-dependent apoptosis in transformed mammalian cells. In this report, we show that E4orf4 induces an irreversible growth arrest in Saccharomyces cerevisiae at the G2/M phase of the cell cycle. Growth inhibition requires the presence of yeast PP2A-Cdc55, and is accompanied by accumulation of reactive oxygen species. E4orf4 expression is synthetically lethal with mutants defective in mitosis, including Cdc28/Cdk1 and anaphase-promoting complex/cyclosome (APC/C) mutants. Although APC/C activity is inhibited in the presence of E4orf4, Cdc28/Cdk1 is activated and partially counteracts the E4orf4-induced cell cycle arrest. The E4orf4-PP2A complex physically interacts with the APC/C, suggesting that E4orf4 functions by directly targeting PP2A to the APC/C, thereby leading to its inactivation. Finally, we show that E4orf4 can induce G2/M arrest in mammalian cells before apoptosis, indicating that E4orf4-induced events in yeast and mammalian cells are highly conserved.     

Specific early-G1 blocks accompanied with stringent response in Saccharomyces cerevisiae lead to growth arrest in resting state similar to the G0 of higher eucaryotes

Growth arrests of Saccharomyces cerevisiae cells in early G1 phase brought by various means were classified into two types according to the mode of growth recovery after release of the restraints against growth. The first type, including arrests caused by cdc25, cdc33, cdc35, and ils1 mutations at the nonpermissive temperature and also by sulfur starvation, showed a subsequent delay in the onset of budding when shifted back to permissive conditions. The length of the delay was positively correlated with the time that cells had been arrested. The second type, including those caused by cdc28 and cdc24 mutations and by alpha factor, did not affect the mode of growth recovery after the shift to permissive conditions irrespective of the time that cell proliferation had been restricted. Growth arrests of the first type seem to allow yeast cells to enter a resting state equivalent to the G0 state of higher eucaryotes because features of the G0 shown with lymphocytes and other cultured cells including unusually long delay before the growth recovery (L.H. Augenlicht and R. Baserga, 1974, Exp. Cell Res., 89:255-262; and Kumagai, J., H. Akiyama, S. Iwashita, H. lida, and I. Yahara, 1981, J. Immunol., 126:1249-1254) appeared to be associated with this type. We have noted that arrests of the first type were always accompanied with a stringent response of macromolecular synthesis and its partial release by cycloheximide. Mapping of arrest points along the path of the cell cycle by the reciprocal shift experiment suggested that arrest points in G1 that led to the G0-like arrest precede or are near the step sensitive to alpha-factor.     

Engineering responsiveness to cell culture stresses: growth arrest and DNA damage gene 153 (GADD153) and the unfolded protein response (UPR) in NS0 myeloma cells

The successful movement of a newly synthesized protein through the endoplasmic reticulum (ER) and associated membranous compartments is dependent on appropriate recognition by complex processing systems. Failure to perceive appropriately processed or modified intermediates in the pathway can initiate a series of cellular signaling events (ER stress or unfolded protein response, UPR) that can lead to cell apoptosis and loss of biomass in culture processes. We have shown that expression of growth arrest and DNA damage gene 153 (GADD153) is associated with recognition of damaged or mis-processed proteins within the secretory processes of CHO and NS0 myeloma cells. To directly characterize the roles of GADD153 in UPR-directed apoptosis, we have generated stable clones of NS0 myeloma cells with elevated (constitutive and inducible) and deleted GADD153 expression. Although GADD153 is a robust indicator of the onset of ER stress or the UPR, GADD153 expression alone is not sufficient to provoke NS0 myeloma apoptosis and it is not required for apoptosis to occur.     

Roles of the negatively charged N-terminal extension of Saccharomyces cerevisiae ribosomal protein S5 revealed by characterization of a yeast strain containing human ribosomal protein S5

Ribosomal protein (rp) S5 belongs to a family of ribosomal proteins that includes bacterial rpS7. rpS5 forms part of the exit (E) site on the 40S ribosomal subunit and is essential for yeast viability. Human rpS5 is 67% identical and 79% similar to Saccharomyces cerevisiae rpS5 but lacks a negatively charged (pI approximately 3.27) 21 amino acid long N-terminal extension that is present in fungi. Here we report that replacement of yeast rpS5 with its human homolog yielded a viable yeast strain with a 20%-25% decrease in growth rate. This replacement also resulted in a moderate increase in the heavy polyribosomal components in the mutant strain, suggesting either translation elongation or termination defects, and in a reduction in the polyribosomal association of the elongation factors eEF3 and eEF1A. In addition, the mutant strain was characterized by moderate increases in +1 and -1 programmed frameshifting and hyperaccurate recognition of the UAA stop codon. The activities of the cricket paralysis virus (CrPV) IRES and two mammalian cellular IRESs (CAT-1 and SNAT-2) were also increased in the mutant strain. Consistently, the rpS5 replacement led to enhanced direct interaction between the CrPV IRES and the mutant yeast ribosomes. Taken together, these data indicate that rpS5 plays an important role in maintaining the accuracy of translation in eukaryotes and suggest that the negatively charged N-terminal extension of yeast rpS5 might affect the ribosomal recruitment of specific mRNAs.     

Mutations in Saccharomyces cerevisiae sterol C5-desaturase conferring resistance to the CYP51 inhibitor fluconazole

Understanding fluconazole resistance is important as it emerged as a serious clinical problem for this CYP51, sterol 14alpha-demethylase, inhibitor. One mechanism, observed first in Saccharomyces cerevisiae, was through defective sterol C5-desaturase (Erg3p) required to form the fungistatic sterol end-product resulting from CYP51 inhibition, 14alpha-methylergosta-8,24(28)-dien-3beta,6alpha-diol. Here, we report molecular changes resulting in both blocked mutants and also leaky mutants in which reduced ergosterol levels were detected. Blocked mutants exhibited nonsense and frameshift mutations, while leaky mutants contained missense mutations that were generally in conserved positions based on the alignment of sterol C5-desaturases and located mainly between residues 250 and 282.