红色荧光蛋白在酿酒酵母中的表达特性研究

目的研究红色荧光蛋白编码基因Dsred在酿酒酵母菌中的快速克隆与表达。方法根据已发表基因序列设计引物,采用连续重叠PCR方法快速克隆获得全长Dsred基因,将其与pMD-18T载体连接后进行测序鉴定。通过In-fusion方法将鉴定正确的pMD-Dsred重组载体与酿酒酵母表达载体pYeDP60进行连接,测序后利用LiAc方法将鉴定正确的pYeDP60-Dsred重组表达载体转化至酿酒酵母菌W303-1B中,PCR扩增筛选阳性克隆,获得的W303B[pYeDP60-Dsred]工程菌经诱导培养后进行SDS-PAGE电泳分析和绿光激发荧光成像检测。将工程菌分别接种至YPD、YPG、SCG和SCD培养基,培养48、72、96、120和144h后分别测定吸光度(A600)值。取诱导表达后的工程菌(菌液组)进行离心(菌体组)、离心后加甘油(高渗组)处理,分别置于–70、–20、4、28和37℃条件培养,荧光显微镜观察其表达特性。结果连续重叠PCR扩增和测序鉴定结果表明,扩增获得的Dsred基因长为678bp,其序列与已发表的基因序列完全一致;Dsred基因已成功插入pYeDP60-Dsred重组表达载体,且受酵母诱导型启动子GAL10-CYC1调控表达。PCR扩增筛选和SDS-PAGE分析显示,pYeDP60-Dsred重组表达载体已成功导入酿酒酵母菌中,诱导表达产物相对分子质量与预期相符,且诱导后工程菌可在绿光激发下发出红色荧光。4种培养基内工程菌的菌体生长情况无明显差别,重组Dsred红色荧光蛋白表达不会抑制酿酒酵母菌体生长。荧光显微镜观察显示,工程菌经不同处理后,高渗组红色荧光蛋白成熟时间最短,菌液组时间最长;红色荧光蛋白在37℃条件下培养时成熟最早,但降解速度较快。结论成功构建了pYeDP60-Dsred重组酿酒酵母表达载体,实现了Dsred基因在酿酒酵母菌体内的异源表达。红色荧光蛋白表达对酿酒酵母菌体生长无明显影响,且离心保留菌体和加入甘油等缺水高渗处理都有利于红色荧光蛋白的成熟。     

狂犬病病毒糖蛋白在酿酒酵母中的分泌表达

目的使狂犬病病毒糖蛋白在酿酒酵母中获得分泌表达,为制备口服狂犬病疫苗奠定基础。方法克隆狂犬病病毒糖蛋白基因,与菊粉酶信号肽序列连接为融合基因InG,该融合基因克隆至pYes2载体Gal1启动子下游,构建成诱导分泌表达载体pYes2-InU-G,该重组载体转化酿酒酵母筛选阳性克隆,阳性重组子经半乳糖诱导12h后,收集上清进行SDS-PAGE电泳和Western-blot鉴定。结果 RT-PCR和Western-blot鉴定显示狂犬病病毒糖蛋白已获得分泌表达,并且具有良好的抗原性。结论 CVS24病毒株的糖蛋白在酿酒酵母中获得分泌表达且具有良好的抗原性,为进一步研究重组酵母能否在消化道中存活并继续进行分泌表达提供了研究条件。     

Protective role of l-threonine against cadmium toxicity in Saccharomyces cerevisiae

Environment and food contamination with cadmium (Cd) can cause serious toxicity, posing a severe threat to agricultural production and human health. However, how amino acids contribute to defenses against oxidative stress caused by Cd in cells is not fully understood. As a model eukaryote with a relatively clear genetic background, Saccharomyces cerevisiae has been commonly used in Cd toxicity research. To gain insight into Cd toxicity and cell defenses against it, 20 amino acids were screened for protective roles against Cd stress in S. cerevisiae. The results showed that threonine (Thr, T) had the strongest protective effect against Cd-induced mortality and membrane damage in the cells. Compared to the antioxidant vitamin C (VC), Thr exhibited a higher efficacy in restoring the superoxide dismutase (SOD) activity that was inhibited by Cd but not by H₂O₂ in vivo. Thr exhibited evident DPPH (2,2-diphenyl-1-picrylhydrazyl) activity but weak ABTS (2,2′-azino-bis(3-ethylbenzothiazoline-6-9 sulfonic acid)) scavenging activity, giving it a weaker effect against Cd-induced lipid peroxidation and superoxide radical O²⁻, compared to VC. More importantly, compared to the chelating agent EDTA, Thr showed stronger chelation of Cd, giving it a stronger protective effect on SOD against Cd than VC in vitro. The results of the in vivo and in vitro experiments revealed that the role Thr plays in cell defenses against Cd may be attributed to its protection of the SOD enzyme, predominantly through the preferential chelation of Cd. Our results provide insights into the protective mechanisms of amino acid Thr that ameliorate Cd toxicity and suggest that a supplement of Thr might help to reduce Cd-induced oxidative damage.     

鼠疫耶尔森氏菌F1抗原在酿酒酵母中的表达及鉴定

目的：构建含鼠疫杆菌F1抗原结构基因caf1的重组载体，并在酿酒酵母中表达。方法：将鼠疫杆菌的F1抗原结构基因caf1插入到pHSS6-mTn-3xHA／locz质粒中，构建重组载体pHSS6-mTn-3xHA／locz—caf1。将重组载体经NotⅠ酶切后，以醋酸锂法转化酿酒酵母，将转化的酿酒酵母接种于SCUra平板上筛选阳性克隆，表达产物用SDS—PAGE和Western blot进行鉴定。结果：经SDS—PAGE鉴定和Western blot分析表明，转化的酿酒酵母可表达鼠疫杆菌F1表面抗原蛋白。结论：成功地构建了重组载体pHSS6-mTn-3xHA／lacZ—caf1，并在酿酒酵母中稳定表达鼠疫杆菌F1表面抗原，为制备可经消化道途径免疫的鼠疫杆菌基因疫苗奠定了基础。     

Comparative study of Saccharomyces cerevisiae LPS

LPS of yeast strains producing human epidermal growth factor were studied. Experiments demonstrated the absence of essential differences in the characteristics of these LPS and LPS of nonrecombinant Saccharomyces cerevisiae strains, which allowed us to develop a universal complex technology of simultaneous preparation of heterologous proteins and highly active immunomodulating LPS.     

Sensitivity of sup35 and sup45 suppressor mutants in Saccharomyces cerevisiae to the anti-microtubule drug benomyl

 SUP35 and SUP45 genes determine the accuracy of translation at the stage of termination. We present indirect evidence indicating that these genes may also control some cellular process mediated by microtubules. A majority of sup35 and sup45 suppressor mutations confer supersensitivity to benomyl, the drug which de-polymerizes microtubules. In addition, data correlating phenotypic manifestations of sup45 suppressor mutations, involving sensitivity to benomyl, respiratory deficiency and a suppressor effect, are also presented. Received: 16 October 1995/7 December 1995     

Molecular and biochemical analysis of Saccharomyces cerevisiae cox1 mutants

We report on the molecular and biochemical analysis of a set of 13 respiratory deficient mutants of Saccharomyces cerevisiae which are specifically altered in COX1, the gene encoding the subunit Cox1p of cytochrome c oxidase. DNA sequence analysis shows that three are due to frameshift mutations, two to nonsense mutations, and eight to missense mutations. All, except the missense mutant S157L, have impaired electron transfer and respiratory activity. Analysis of the mitochondrial translation products shows that when Cox1p is absent, Cox2p and Cox3p are still synthesized. In the missense mutants, the steady state levels in the mitochondrial membranes of the three mitochondrially encoded subunits Cox1p, Cox2p and Cox3p and the nuclear-encoded subunit Cox4p are reduced. In the frameshift and nonsense mutants, Cox1p is absent and Cox2p, Cox3p and Cox4p are considerably decreased or undetectable. A comparison of the steady state levels of Cox1p through Cox4p in the COX1, COX2, COX3 and COX4 mutants shows the interdependance of the accumulation of these four subunits in the mitochondrial membranes. Received: 20 January / 23 June 1998     

产虾青素酿酒酵母工程菌的构建

目的 对酿酒酵母固有的β-胡萝卜素代谢途径进行扩展,构建能够合成虾青素的酿酒酵母工程菌。 方法 对不同来源的虾青素合成基因 crtZ（β-胡萝卜素羟化酶）与 crtW （β-胡萝卜素酮化酶）进行随机组合,采用overlap PCR 技术构建串联表达质粒,并将其导入产β-胡萝卜素酿酒酵母工程菌内,发酵产物经 HPLC 和 UPLC-MS鉴定,确定其成分。 结果 构建出8种 TEF1p-crtZ-ADH1t-PGK1P-crtW-CYC1t组合的串联表达模式,其中基因组合分别为 crtZ1-crtW1, crtZ1-crtW4工程菌 HCCB08571、HCCB08572产生了虾青素;工程菌 HCCB08566、HCCB08568、HCCB08573未检测到产物虾青素,而出现了中间产物玉米黄质和斑蝥黄质。 结论 成功构建了产生虾青素及其中间体的酿酒酵母工程菌。     

Analysis of exon and intron mutants in the cytochrome b mitochondrial gene of Saccharomyces cerevisiae

The nucleotide changes present in a group of five cytochrome b mit^– mutants were analyzed at the sequence level. Two single-base changes were found: one (M10-152) generated a nonsense codon in the first exon while the other (M8-181) created a missense substitution in the second exon. The other mutants all have multiple (three) substitutions that either resulted in a missense mutation in a coding region (M17-162) or else changed nucleotides in the last intron of the gene, so blocking its excision (M6-200 and M8-53). The synthesis of mitochondrial polypeptides and the steady state concentration of the complex-III subunits were examined. The Rieske protein and the core-4 and core-5 subunits were much reduced in all mutants. Consequently the overall stability of complex III is very sensitive even to amino-acid substitutions in the cytochrome b protein. Mutant M8-53 provides direct evidence for the proposed role of the P9.1 stem in the core structure of the group-I type last intron of this gene. Received: 12 February 1996 / 28 March 1996     

Endopolygalacturonase genes and enzymes from Saccharomyces cerevisiae and Kluyveromyces marxianus

The gene encoding endopolygalacturonase (EC 3.2.1.15) has been cloned, sequenced and expressed from three strains of Saccharomyces cerevisiae (including non-secretors) and three strains of Kluyveromyces marxianus. Both control and coding regions showed small differences within each species, one including loss of a potential glycosylation site. Two non-secreting S. cerevisiae strains (FY1679 and var. uvarum) had non-transcribed copies of functional genes. Maximum enzyme activity was achieved with the S. cerevisiae FY1679 gene in an expressing vector, with an enzyme activity of 51 μmol of reducing sugar released from polygalacturonic acid μg protein⁻¹ min⁻¹, the highest so far reported for a yeast. Received: 19 May 2000 / Accepted: 6 August 2000     

Reciprocal translocation at duplicated RPL2 loci might cause speciation of Saccharomyces bayanus and Saccharomyces cerevisiae

By a genomic comparison of two sibling yeasts, Saccharomyces bayanus and S. cerevisiae, we previously demonstrated that chromosomes II and IV of S. cerevisiae were rearranged into chromosomes 12 and 14 of S. bayanus or vice versa. In the present study we have delimited the translocation break sites in chromosomes II and IV by Southern hybridization using DNA fragments of S. cerevisiae cosmid clones as probes. The results suggest that the reciprocal translocation of chromosomes II and IV had occurred at duplicated RPL2 loci. Furthermore, the translocation sites in S. bayanus were confirmed by the cloning and sequence analysis of the regions flanking RPL2 loci. Several genes in the regions flanking the RPL2 loci were present in the order expected for a translocation at these loci between the two species. These results indicated that the reciprocal translocation between chromosomes II and IV was generated by homologous recombination at duplicated RPL2 loci on the two chromosomes. Therefore, we propose that duplicated genes or duplicated regions play an important role in altering genomic organization during the speciation of S. bayanus and S. cerevisiae. Received: 20 October 1997 / 2 March 1998     

Structure and regulation of the isocitrate lyase gene ICL1 from the yeast Saccharomyces cerevisiae

The ICL1 gene encoding the isocitrate lyase from Saccharomyces cerevisiae was cloned and sequenced. A reading frame of 557 amino acids showing significant similarity to isocitrate lyases from seven other species could be identified. Construction of icl1 null mutants led to growth defects on C₂ carbon sources while utilization of sugars or C₃ substrates remained unaffected. Using an ICL1-lacZ fusion integrated at the ICL1 locus, a more than 200-fold induction of -galactosidase activity was observed after growth on ethanol when compared with glucose-repressed conditions. A preliminary analysis of the ICL1 upstream region identified a 364-bp fragment necessary and sufficient for this regulatory phenotype. Sequence motifs also present in the upstream regions of co-regulated genes were found within this region.     

6-Azauracil inhibition of GTP biosynthesis in Saccharomyces cerevisiae

Summary The addition of 6-azauracil to the growth medium causes a strong reduction of the GTP level in the nucleotide pool of Saccharomyces cerevisiae. In-vitro experiments show a strong inhibition of IMP dehydrogenase activity by 6-azaUMP explaining the preceeding effect. PPR2 mutants, previously characterized by an increased sensitivity to 6-azauracil compared to the wildtype, are specifically susceptible to the lowering of the GTP pool, and are able to grow in presence of 6-azauracil when guanine is added to the medium.     

Spontaneous and protein‐induced secretion of proteinases from Saccharomyces cerevisiae

Many fungi are capable of secreting the wide spectrum of hydrolytic enzymes. We characterized an inducible proteinase secretion in yeasts, Saccharomyces cerevisiae. The proteinase secretion by S. cerevisiae was induced in the presence of yeast extract, or of purified proteins, such as bovine serum albumin, casein, or ovalbumin, and some proteolytic activity was present also without protein inducer. We found that properties of proteinases induced under cultivation conditions were different in various aspects (temperature‐ and pH‐dependencies, substrate specificities, sensitivities to proteinase inhibitors). Proteinase activities were also characterized by gelatin zymography. Multiple proteinase bands with wide‐molecular weights (ranging from 45 to 240 kDa) were detected and patterns of proteinase bands were different. S. cerevisiae cells were able to retain the information about previous contacts with protein inducer resulting in faster and more intensive proteinase secretion response after repeated induction. (© 2009 WILEY‐VCH Verlag GmbH & Co. KGaA, Weinheim)     

Characterization of the Saccharomyces cerevisiae RTA1 gene involved in 7-aminocholesterol resistance

 7-aminocholesterol has been described as being a strong inhibitor of yeast and of Gram⁺-bacteria proliferation. In order to determine the precise molecular target of the toxicity of this compound, we searched for yeast resistance linked to gene over-expression. We named the new yeast gene that was isolated RTA1 (EMBL X84736). This gene led to strong resistance to the inhibitor. Gene sequencing revealed that RTA1 is adjacent to the NAB1 gene which is orientated in an opposite direction and localized on chromosome VII. The RTA1 gene, which encodes a putative protein with seven potential membrane-spanning segments, was shown to be a non-essential gene in yeast. Received: 25 December 1995/22 March 1996