酵母SOD1基因缺失突变体应答刚果红胁迫的转录组学分析（英文）

SOD1基因编码的铜锌超氧化物歧化酶是酵母细胞中最重要的抗氧化酶.前期研究发现,SOD1基因缺失(sod1Δ)导致酵母细胞对真菌细胞壁抑制剂刚果红(Congo red,CR)的敏感性增加,提示细胞抗氧化能力与细胞壁稳定性相关.本研究采用酵母全基因组表达谱芯片,比较了CR胁迫条件下,野生型酵母细胞和sod1Δ酵母细胞的转录表达谱.结果表明,与野生型酵母细胞相比,sod1Δ酵母细胞中260个基因发生了显著差异表达(140个基因表达上调、120个基因表达下调).随机选取12个差异表达基因采用定量PCR验证,结果与芯片分析结果一致.差异表达基因功能主要涉及细胞壁(几丁质合成)、细胞代谢、细胞防御(抗氧化和热冲击蛋白)、蛋白质合成以及大量功能未知基因.进一步研究发现,CR处理后,细胞壁几丁质含量和细胞内氧化应激指标丙二醛(MDA)含量在sod1Δ酵母细胞中显著升高,而在野生型酵母细胞中无明显变化,与芯片筛选差异表达基因的生物学功能分析结果一致.本研究提供了在全基因组水平上对SOD1基因与细胞壁应激反应之间关联的新认识.     

酿酒酵母对数生长后期代谢重构的全基因组表达谱芯片分析

为了探讨酵母进入对数生长后期以后酒精生产速度降低的原因,我们利用酵母表达谱芯片技术对酿酒酵母细胞从对数生长中期进入对数生长后期时的全基因组表达谱进行了分析,发现酵母在对数生长中期的表达谱非常稳定,而一旦进入对数生长后期.则出现明显的代谢重构现象.许多氨基酸合成和代谢相关的基因、离子转移以及与能量的生成和储存等功能相关的基因出现了不同程度的上调;而许多涉及酵母转座和DNA重组的基因则表达下调;一些中心代谢途径也发生了代谢重构.包括:琥珀酸和α-酮戊二酸生成途径基因的一致上调,都与氨基酸合成和代谢相关基因表达的结果相吻合.结果表明:由于氨基酸合成的需求量增加,进入对数生长后期酵母的代谢转向TCA循环和乙醛酸循环,导致酒精的生产速率降低.     

RASSF1A对黑色素瘤A375细胞基因网络的影响

RASSF1A(Ras association domain family 1 isoform A)是定位于染色体3p21.3区域的抑瘤基因,编码一个由340个氨基酸残基构成的微管相关蛋白.该基因在包括恶性黑色素瘤在内的多种肿瘤中因启动子高甲基化而表达沉默.本研究建立了RASSF1A稳定表达的恶性黑色素瘤A375细胞系,通过全基因组表达谱基因芯片分析RASSF1A过表达对A375细胞基因表达谱的影响,发现RASSF1A引起184个基因表达上调,26个基因表达下调.通过Realtime RT-PCR对部分差异表达基因进行验证,结果表明与芯片筛选结果一致.RASSF1A影响的差异表达基因功能上归属于细胞生长与增殖、细胞周期、细胞凋亡、细胞间黏附、信号传导等生物过程.采用STRING软件构建了RASSF1A影响的差异表达基因调控网络,结果表明RASSF1A调控的差异表达基因构成一个高连接度的基因网络.其中,炎症细胞因子、转录因子位于网络中央.RASSF1A通过影响炎症细胞因子与转录因子之间的表达,影响A375细胞基因网络,调节黑色素瘤恶性生物学行为.     

应用基因芯片分析甘蓝型油菜柱头特异表达基因

以甘蓝型油菜(Brassica napus)野生型(宁油10号)及其柱头授粉功能缺失突变体FS-M1为材料,使用油菜基因表达谱芯片筛选甘蓝型油菜柱头特异表达基因。在含有16 540个基因的油菜基因表达谱芯片中(43 803探针),获得了4 410条差异表达探针,选择部分差异表达基因进行实时定量PCR,所得结果与芯片检测结果相吻合。其中,野生型较FS-M1显著上调且获得209个功能注释的探针,对应198个基因,这些特异表达的基因主要富集在水解酶、转移酶、氧化还原酶和转录因子中;涉及较大的基因家族包括:细胞色素P450基因、GDSL脂肪酶/水解酶基因、ABC转运蛋白基因、myb转录因子基因、bHLH转录因子基因、过氧化物酶家族和受体激酶基因等。推测这些基因与甘蓝型油菜柱头发育及授粉功能有关。     

高盐低温胁迫下水稻叶细胞ROS清除系统的相关基因表达

采用Affymetrix水稻表达谱芯片,分析高盐和低温胁迫下水稻叶细胞内ROS清除系统的相关基因表达状况,探讨在响应非生物胁迫过程中水稻植株体内抗氧化防卫体系的积极作用。结果表明:(1)水稻叶细胞内ROS清除系统涉及187个基因和/或EST,由抗氧化的非酶类物质如抗坏血酸、谷胱甘肽、生育酚等和抗氧化酶如超氧化物歧化酶(SOD)、抗坏血酸过氧化物酶(APX)、过氧化氢酶(CAT)等组成。(2)在低温逆境下,籼稻(i-93-11)叶细胞表达上调基因(2倍以上,下同)有5个,下调(0.5倍以下,下同)基因有2个;粳稻(j-NJ-1)叶细胞表达上调基因5个,下调基因6个。(3)在高盐胁迫下,籼稻(i-93-11)叶细胞表达上调基因有31个、下调基因13个;粳稻(j-NJ-1)叶细胞表达上调基因27个,下调基因25个。(4)高盐和低温胁迫下水稻叶细胞ROS清除系统相关基因的表达在籼粳稻间存在较大差异,并根据水稻基因表达谱芯片数据构建了水稻响应高盐和低温胁迫ROS清除网络图。     

应用cDNA微阵列技术研究EB病毒LMP1介导的鼻咽癌中转移相关基因的表达

探讨EB病毒基因组编码的癌蛋白LMP1对鼻咽癌细胞中转移相关基因表达的影响.采用蛋白质印迹法检测在强力霉素(Dox)诱导下,鼻咽癌细胞系pTet-on-LMP1 HNE2(L7细胞)中LMP1表达的时效和量效关系.应用cDNA微阵列技术建立诱导性LMP1介导鼻咽癌细胞中转移相关基因差异表达谱;运用RT-PCR验证cDNA微阵列筛选差异基因表达的可靠性.与LMP1不表达的L7细胞比较,LMP1高表达的L7细胞中7个基因的表达显著上调,12个基因的表达显著下调.随机选择其中4个基因进行RT-PCR,结果显示,这些基因表达阳性,且与微阵列中的变化趋势一致.LMP1可能通过激活和/或抑制一些转移相关基因的表达而参与鼻咽癌转移过程.     

氦氧饱和高气压暴露对铜绿假单胞菌PAO1基因表达的诱导调节

研究氦氧饱和高气压暴露对铜绿假单胞菌基因表达谱的系统影响,对急性毒力基因表达的调节。用全基因组DNA芯片分析技术比较菌株暴露前后基因表达谱差异;RT-PCR方法验证部分差异表达基因;用分光光度法在细胞水平验证弹性蛋白酶含量;小鼠染毒法观察暴露组细菌毒力在整体动物水平的变化。基因表达谱分析结果表明,铜绿假单胞菌暴露12 h差异表达基因达243个、72 h差异表达基因为1 168个。72 h差异表达基因中与细菌应激响应、蛋白折叠、转录调节、菌毛和鞭毛合成、毒力因子调节与合成、细菌外膜蛋白和抗原合成的基因大量上调;部分基因的RT-PCR验证结果与芯片结果一致;细胞水平验证结果显示暴露72 h细菌毒力表型增强。因此,氦氧饱和高气压暴露对铜绿假单胞菌基因表达谱有明显影响,对急性侵袭性感染毒力因子基因表达水平有正向诱导调节作用。     

转录因子XBP1S基因表达谱差异分析

目的 应用基因表达谱芯片技术了解XBP1S在肝细胞中可能上调或下调的基因,了解其可能的调节功能线索.方法 构建pcDNA3.1（-）-XBP1S真核表达载体,转染HepG2细胞,同时以空载体pcDNA3.1（-）处理相同细胞系作为对照.48 h后制备细胞裂解液,提取mRNA,应用基因表达谱芯片技术对差异表达mRNA进行检测和分析.结果 构建的表达载体经过限制性内切酶分析和DNA序列测定,证实准确无误,提取高质量的总mRNA并进行逆转录成为cDNA,进行基因表达谱芯片技术分析.经过差异基因表达谱的筛选,发现HepG2细胞转染XBP1S以后,有38个基因表达水平显著上调,30个基因表达水平显著下调.结论 成功构建XBP1S的真核表达载体pcDNA3.1（-）-XBP1S,运用基因表达谱芯片技术成功筛选了XBP1S转染细胞后的差异表达基因,这些差异表达基因包括细胞周期、蛋白质的翻译合成及运输、能量代谢、体内免疫调节、细胞凋亡及细胞内的信号转导等方面起重要作用及肿瘤发生相关的基因,为进一步阐明XBP1S可能存在的调控机制及XBP1S蛋白可能的生物学功能提供理论依据.     

转萝卜过氧化物酶基因Rsprx1提高毕赤酵母抗盐性

为探讨转萝卜过氧化物酶基因（Rsprx1）提高毕赤酵母(Pichia pastoris)抗盐性机理,用不同浓度NaCl处理转基因酵母GSRP25和野生型酵母GS115,检测菌体生长、相对无机盐含量、过氧化物酶活性和同工酶谱及某些抗性基因表达．实验结果表明,在YPD培养条件下,转基因酵母的过氧化物酶活性和菌体生长速率高于野生型酵母,其过氧化氢酶（CTT1）、热休克蛋白（Hsp12）、Rsprx1基因表达和K+/Na+比值均高于野生型．醛脱氢酶（ALD3）的mRNA表达在两者之间没有差异．在BMMY培养条件下,转基因酵母菌体生长速率和过氧化物酶活性显著高于野生型酵母．因此,转基因酵母通过增加过氧化物酶基因表达提高过氧化物酶活性,改变细胞的某些基因表达和无机盐相对含量,从而提高酵母抗盐能力．     

利用表达谱基因芯片筛选溃疡性结肠炎差异表达基因

目的:利用人类全基因组表达谱芯片技术,分析溃疡性结肠炎患者和健康者基因表达谱差异,筛选出溃疡性结肠炎相关基因。方法:采用Trizol法提取8例溃疡性结肠炎患者和8例健康对照者结肠粘膜组织总RNA并纯化,逆转录合成c DNA,利用荧光染料Cy3标记aa UTP,转录合成标记的c RNA,并与Agilent人类全基因组表达谱芯片杂交,扫描荧光信号图像,对芯片原始数据进行归一化处理,利用倍数差异和t检验计算筛选出相关差异表达基因,采用DAVID在线分析系统进行基因的功能注释和关联分析,明确差异基因的生物学功能,并对部分差异表达基因进行实时荧光定量PCR验证。结果:筛查出溃疡性结肠炎结肠粘膜组织差异表达基因4132个,其中上调基因2004个,下调基因2128个。选取6条差异表达基因进行PCR验证,结果有3条基因表达上调,3条基因表达下调,表达趋势与芯片结果一致。结论:溃疡性结肠炎患者与健康对照者基因表达存在明显差异,分析这些差异表达基因有助于我们探索溃疡性结肠炎的发病机制,为疾病的治疗提供理论依据。     

Cu,Zn-superoxide dismutase is required for cell wall structure and for tolerance to cell wall-perturbing agents in Saccharomyces cerevisiae

Here we report that deletion of SOD1, the Cu,Zn-superoxide dismutase in Saccharomyces cerevisiae is sensitive to cell wall-perturbing agents, such as Calcofluor white and Congo red. The sensitivity was restored by retransformation with wild type SOD1 or the addition of N-acetylcysteine or reduced glutathione to the medium. Additionally, the accumulation of reactive oxygen species was observed in sod1Δ mutant in the presence of Calcofluor white or Congo red. Cell wall analysis indicated an increase of cell wall chitin and cell wall thickness in sod1Δ mutant compared to wild type. These results indicate a novel direct connection between antioxidative functions and cell wall homeostasis.     

Genes involved in glucose repression and oxidative stress response in the fission yeast Schizosaccharomyces pombe

We looked for changes in gene expression and novel genes that could be involved in the interaction between glucose repression and oxidative stress response in the fission yeast, Schizosaccharomyces pombe, using a constitutive invertase mutant, ird11, which is resistant to glucose. BLAST analysis was made of the S. pombe genome database of cDNAs whose expression ratios differentially decreased or increased upon exposure to mild oxidative stress in this mutant compared to the wild type. Genes with this type of activity were identified as rpl302, encoding 60S ribosomal protein L3, and mpg1, encoding mannose-1-phosphate guanyltransferase; their expression patterns were measured using quantitative real-time PCR. We found that the expression levels of rpl302 and mpg1 genes in ird11 under unstressed conditions were increased compared to those of the wild type. Under stress conditions, the expression levels of the rpl302 gene were decreased in both strains, while mpg1 expression levels remained unchanged. These results suggest that these genes play a role in the response to oxidative stress in this mutant strain.     

Absence of superoxide dismutase activity causes nuclear DNA fragmentation during the aging process

Superoxide dismutases (SOD) serve as an important antioxidant defense mechanism in aerobic organisms, and deletion of these genes shortens the replicative life span in the budding yeast Saccharomyces cerevisiae. Even though involvement of superoxide dismutase enzymes in ROS scavenging and the aging process has been studied extensively in different organisms, analyses of DNA damages has not been performed for replicatively old superoxide dismutase deficient cells. In this study, we investigated the roles of SOD1, SOD2 and CCS1 genes in preserving genomic integrity in replicatively old yeast cells using the single cell comet assay. We observed that extend of DNA damage was not significantly different among the young cells of wild type, sod1Δ and sod2Δ strains. However, ccs1Δ mutants showed a 60% higher amount of DNA damage in the young stage compared to that of the wild type cells. The aging process increased the DNA damage rates 3-fold in the wild type and more than 5-fold in sod1Δ, sod2Δ, and ccs1Δ mutant cells. Furthermore, ROS levels of these strains showed a similar pattern to their DNA damage contents. Thus, our results confirm that cells accumulate DNA damages during the aging process and reveal that superoxide dismutase enzymes play a substantial role in preserving the genomic integrity in this process.     

The stress response protein Hsp12p increases the flexibility of the yeast Saccharomyces cerevisiae cell wall

The yeast S. cerevisiae cell wall comprising a 10 nm thick layer of polysaccharides, predominantly beta(1,3)-glucan and proteins, is the interface between the cell and the neighbouring environment. As such it is not a static entity but rather one that is dynamically remodelled in response to changes in the environmental conditions. We have recently proposed from studies using yeast cells lacking the gene encoding Hsp12p (Deltahsp12 yeast) and from incorporation of Hsp12p into agarose, used as a model system for the beta-glucan layer of the cell wall, that the hydrophilic stress response cell wall protein Hsp12p acts as a cell wall plasticizer. In this report we have used force spectroscopy to confirm that Deltahsp12 yeast are indeed less flexible than the wild type strain. The spring constant of the cell wall of Deltahsp12 yeast, kcw was determined to be 72+/-3 mN m-1 as compared to 17+/-5 mN m-1 obtained for the wild type strain. A similar result was found on the basis of a quantitative analysis of the electrophoretic mobilities measured for the two yeast strains. Those indicated that the hydrodynamic permeability quantified through the softness parameter of the external layer of Deltahsp12 cells was smaller than the one of wild type cells. We proposed from surface infrared spectroscopy measurements that yeast compensate for the lack of Hsp12p by reducing the carbohydrate/proteins ratio of the cell wall or increasing the cell wall chitin content.     

Mitochondrial protein oxidation in yeast mutants lacking manganese-(MnSOD) or copper- and zinc-containing superoxide dismutase (CuZnSOD): evidence that MnSOD and CuZnSOD have both unique and overlapping functions in protecting mitochondrial proteins from oxidative damage

Saccharomyces cerevisiae expresses two forms of superoxide dismutase (SOD): MnSOD, encoded by SOD2, which is located within the mitochondrial matrix, and CuZnSOD, encoded by SOD1, which is located in both the cytosol and the mitochondrial intermembrane space. Because two different SOD enzymes are located in the mitochondrion, we examined the relative roles of each in protecting mitochondria against oxidative stress. Using protein carbonylation as a measure of oxidative stress, we have found no correlation between overall levels of respiration and the level of oxidative mitochondrial protein damage in either wild type or sod mutant strains. Moreover, mitochondrial protein carbonylation levels in sod1, sod2, and sod1sod2 mutants are not elevated in cells harvested from mid-logarithmic and early stationary phases, suggesting that neither MnSOD nor CuZnSOD is required for protecting the majority of mitochondrial proteins from oxidative damage during these early phases of growth. During late stationary phase, mitochondrial protein carbonylation increases in all strains, particularly in sod1 and sod1sod2 mutants. By using matrix-assisted laser desorption ionization time-of-flight mass spectrometry, we have found that specific proteins become carbonylated in sod1 and sod2 mutants. We identified six mitochondrial protein spots representing five unique proteins that become carbonylated in a sod1 mutant and 19 mitochondrial protein spots representing 11 unique proteins that become carbonylated in a sod2 mutant. Although some of the same proteins are carbonylated in both mutants, other proteins are not. These findings indicate that MnSOD and CuZnSOD have both unique and overlapping functions in the mitochondrion.     

Genetic and biochemical characterization of Cu,Zn superoxide dismutase mutants in Saccharomyces cerevisiae

The allele scd 1 is a recessive chromosomal mutation in Saccharomyces cerevisiae that eliminates Cu,Zn superoxide dismutase (SOD-1) activity. SOD-1- strains are unable to grow in 100% O2 in rich medium and are methionine and lysine auxotrophic when grown in air (Bilinski, T., Krawiec, Z., Liczmanski, A., and Litwinska, J. (1985) Biochem. Biophys. Res. Commun. 130, 533-539). In this report, scd1 was genetically mapped to the right arm of chromosome X, 11 centimorgans proximal to cdc11. The gene for SOD-1 (SOD1) was physically mapped by Southern blot to restriction fragments containing CDC11. scd1 failed to complement a complete deletion of SOD1. Thus, scd1 maps to the SOD1 locus and is designated sod1-1. The molecular basis for the lack of SOD-1 activity in sodl-1 carrying strains has also been established. The size and amount of SOD-1 mRNA in the mutant were essentially the same as in wild type cells. Western blot analysis showed that the SOD-1 dimer and 16-kilodalton subunit that co-migrated electrophoretically with wild type yeast SOD-1 were abundant in mutant cell extracts. However, two additional SOD-1 immunoreactive polypeptides were detected in these extracts in both denaturing and nondenaturing gels. None of the SOD-1 immunoreactive species in the mutant extracts exhibited superoxide dismutase activity. Transformants of the mutant strain carrying episomal, wild type SOD1 expressed wild type, active SOD-1 protein, indicating that the mutant allele had no discernible effect on the correct synthesis and activation of apoSOD-1. Size exclusion chromatography of soluble cell extracts derived from wild type and SOD1 deletion strains identified a copper binding peak that corresponded to SOD-1. This copper-binding fraction was absent in cell extracts from the sod1-1-containing strain although Western blot analysis of the corresponding chromatographic fractions showed that SOD-1 polypeptide was present in these fractions. Sequence data derived from the cloned genes showed that sod1-1 differed from SOD1 only in the adjacent 5'-noncoding region. The biochemical data indicate that this genetic alteration results in the synthesis of a collection of SOD-1 polypeptides that fail to bind copper and may also fail to completely self-associate. Both phenotypes could be due to the inability of these polypeptides to adopt the native SOD-1 conformation.     

Insights into the Role of the Unusual Disulfide Bond in Copper-Zinc Superoxide Dismutase

The functional and structural significance of the intrasubunit disulfide bond in copper-zinc superoxide dismutase (SOD1) was studied by characterizing mutant forms of human SOD1 (hSOD) and yeast SOD1 lacking the disulfide bond. We determined x-ray crystal structures of metal-bound and metal-deficient hC57S SOD1. C57S hSOD1 isolated from yeast contained four zinc ions per protein dimer and was structurally very similar to wild type. The addition of copper to this four-zinc protein gave properly reconstituted 2Cu,2Zn C57S hSOD, and its spectroscopic properties indicated that the coordination geometry of the copper was remarkably similar to that of holo wild type hSOD1. In contrast, the addition of copper and zinc ions to apo C57S human SOD1 failed to give proper reconstitution. Using pulse radiolysis, we determined SOD activities of yeast and human SOD1s lacking disulfide bonds and found that they were enzymatically active at ∼10% of the wild type rate. These results are contrary to earlier reports that the intrasubunit disulfide bonds in SOD1 are essential for SOD activity. Kinetic studies revealed further that the yeast mutant SOD1 had less ionic attraction for superoxide, possibly explaining the lower rates. Saccharomyces cerevisiae cells lacking the sod1 gene do not grow aerobically in the absence of lysine, but expression of C57S SOD1 increased growth to 30–50% of the growth of cells expressing wild type SOD1, supporting that C57S SOD1 retained a significant amount of activity.     

Effect of vitamin A treatment on superoxide dismutase-deficient yeast strains

Vitamin A (Vit A) is widely suggested to be protective against oxidative stress. However, different studies have been demonstrated the pro-oxidant effects of retinoids in several experimental models. In this work, we used the yeast Saccharomyces cerevisiae as a model organism to study the Vit A effects on superoxide dismutase (SOD)-deficient yeast strains. We report here that Vit A (10, 20 and 40 mg/ml) decreases the survival of exponentially growing yeast cells, especially in strains deficient in CuZnSOD (sod1Δ) and CuZnSOD/MnSOD (sod1Δsod2Δ). We also observed the protective effect of vitamin E against the Vit A-induced toxicity. Possible adaptation effects induced by sub-lethal oxidative stress were monitored by pre-, co- and post-treatment with the oxidative agent paraquat. The enzymatic activities of catalase (CAT) and glutathione peroxidase (GPx), and the total glutathione content were determined after Vit A treatment. Our results showed that CuZnSOD represents an important defence against Vit A-generated oxidative damage. In SOD-deficient strains, the main defence against Vit A-produced reactive oxygen species (ROS) is GPx. However, the induction of GPx activity is not sufficient to prevent the Vit A-induced cell death in these mutants in exponential phase growth.