人Elongator复合物Elp3亚基基因可显著补偿酵母elp3缺失菌株的生长缺陷

从HeLa细胞中分离的人的Elongator复合物在组成及与RNAPⅡ的作用方式上与酵母的E- longator复合物十分相似,但对其功能研究极少。为了研究人的Elongator复合物催化亚基Elp3的功能,将人elp3等基因转入酵母elp3基因缺失的突变菌株(elp3Δ菌株),并对转化菌株进行功能互补实验和ssa3和pho5基因表达分析,结果表明人elp3基因可显著恢复突变菌株对高温和Caffeine的敏感性,在低磷条件下显著补偿了突变株pho5基因表达延迟的缺陷,并可在热激条件下提高ssa3基因的表达。含酵母elp3非HAT区和人elp3 HAT区的融合yhelp3对上述缺陷有着更强的补偿能力。而HAT区催化结构域缺失的yhelp3HAT没有任何补偿能力,表明人Elp3亚基可能与酵母的该亚基功能相似,人Elp3的HAT活性也为其行使功能所必需。     

人Elongator复合物Elp3亚基基因可显著补偿酵母elp3缺失菌株的生长缺陷

从HeLa细胞中分离的人的Elongator复合物在组成及与RNAPⅡ的作用方式上与酵母的Elongator复合物十分相似．但对其功能研究极少。为了研究人的Elongator复合物催化亚基Elp3的功能,将人elp3等基因转入酵母elp3基因缺失的突变菌株（elp3△菌株）,并对转化菌株进行功能互补实验和ssa和pho5基因表达分析,结果表明人elp3基因可显著恢复突变菌株对高温和Caffeine的敏感性．在低磷条件下显著补偿了突变株ph05基因表达延迟的缺陷．并可在热激条件下提高ssa3基因的表达。含酵母elp3非HAT区和人elp3 HAT区的融合yhelp3对上述缺陷有着更强的补偿能力。而HAT区催化结构域缺失的yhelp3HAT-没有任何补偿能力．表明人Elp3亚基可能与酵母的该亚基功能相似．人Elp3的HAT活性也为其行使功能所必需。     

酿酒酵母组蛋白H3 K4L和K36L突变对细胞生长和GAL1、SSA3、PHO5表达的影响

组蛋白甲基化和乙酰化修饰对基因表达和细胞生长至关重要,为揭示组蛋白H3第4、36位赖氨酸(K)修饰对酵母生长和诱导基因表达的重要性及两位点功能差异,文章构建了两位点单独或共同突变为亮氨酸(L)的组蛋白突变株S4、S36和D436,对其在正常、半乳糖为单一碳源、高温、高盐等条件下的生长及GAL1、SSA3和PHO5表达进行比较。结果显示:D436对高温最敏感,各突变株对咖啡因显著敏感;3个突变株在高温、高盐、6-AU、咖啡因存在时的生长及GAL1、SSA3和PHO5的激活均明显慢于野生型;S4在高温、高盐条件下生长及GAL1激活慢于S36。H3-K4和H3-K36的翻译后修饰对细胞生长和适应不利环境非常重要,在对高温等逆境快速适应上,K4比K36更重要,组蛋白突变株的表型缺陷是因该条件下细胞生存所必需的诱导基因表达延迟所致,同一位点突变对不同基因表达有不同影响。3个突变株的缺陷表型严格上应是相应位点突变导致组蛋白修饰模式改变所造成的综合影响。     

PHO80、PHO85基因和芽殖酵母金属离子耐受

PHO8 5基因是芽殖酵母中的一个多功能基因。它参与了无机磷酸的代谢、碳源利用、糖原积累、特定蛋白质的降解和细胞周期调控。研究了酵母株YPH499及其衍生的pho85缺失株、pho80缺失株、pap1(pcl7)缺失株在不同浓度的不同金属离子中的存活情况 ,结果表明和芽殖酵母YPH499相比 ,pho85缺失株和pho80缺失株表现出对K 、Mg2 、Zn2 、Ca2 和Mn2 的耐受下降 ,而PAP1基因的缺失则不会导致芽殖酵母对上述金属离子的敏感性的变化 ;而对Cu2 ,3株突变株都表现出和YPH499相同的耐受性。同时测定了各缺失株和YPH499对上述金属离子的半致死浓度以及pho85缺失株、pho80缺失株和YPH499的细胞内总钙量。这些结果显示 ,PHO85蛋白激酶通过和它的PCLPHO80而不是PAP1结合 ,参与了芽殖酵母K 、Mg2 、Zn2 、Ca2 和Mn2 离子平衡的调控。PHO85和PHO80基因的缺失损害了芽殖酵母钙的储存。     

里氏木霉RutC30常温常压等离子体（ARTP）诱变筛选pyr4基因缺陷型菌株及转化系统的建立

以里氏木霉Trichoderma reesei重要工业生产菌RutC30为出发菌株,通过等离子体（ARTP）诱变筛选,以5-氟乳清酸（5-FOA）和尿苷（Uridine）进行筛选,获得一株pyr4基因缺陷株RutC30ΔU3。用含有野生型里氏木霉pyr4基因的互补质粒转化突变株,可回复野生性状。经测序发现其pyr4基因在核酸序列多个位点发生突变,其中包括两个错义突变和一个移码突变,从而导致乳清酸核苷-5′-磷酸脱羧酶失活。经遗传稳定性研究分析,传代5次后仍保持良好的尿苷依赖性、去葡萄糖阻遏以及高产纤维素酶特性。经实验筛选获得了pyr4基因缺陷菌株可作为基因表达系统的受体菌株,建立了以尿苷营养缺陷为筛选标记的木霉转化系统。     

转录因子Pho4参与白念珠菌药物敏感性的调控

目的通过对缺失相应转录因子基因的白念珠菌进行抗真菌药物敏感性的筛选,考察转录因子对白念珠菌耐药性的影响及调控机制。方法通过微量液基稀释法、点板实验(Spot Assay)检测实验菌株对抗真菌药物的敏感性。采用实时定量PCR(RT-PCR)的方法检测白念珠菌耐药性相关MDR1,CDR1以及ERG11的表达,并通过检测菌株对罗丹明6G的外排能力进一步检测菌株对抗真菌药物的外排能力。结果最低抑菌浓度(minimal inhibitory concentration,MIC)测定和Spot Assay实验结果表明,与亲本菌相比,PHO4基因缺失菌对氟康唑、咪康唑的敏感性显著升高。虽然耐药相关基因的表达增加,但对罗丹明6G的外排能力降低,抗氧化应激能力下降。结论转录因子Pho4的缺失可能通过降低白念珠菌的抗氧化应激能力,减弱对药物的外排作用而导致对唑类药物敏感,但其具体的调控机制有待进一步研究。     

拟南芥Rab1家族基因的克隆和功能研究

Yptl蛋白是酵母唯一的Rab1 GTP酶,调控囊泡从内质网到高尔基体的运输.酵母温敏突变株 ASY01是一个Ypt1基因功能部分缺失菌株,在26℃可以正常生长,但在37℃不能生长.拟南芥有4个Rab1基因,分别是AtRab1A1、AtRab1B1、AtRab1B2、AtRab1C1.克隆了所有4个AtRab1基因,构建酵母表达载体,转化温敏突变型酵母ASY01.温度敏感性实验结果表明,所有转基因菌株在37℃都恢复正常生长.说明拟南芥4个Rab1基因都与酵母Ypt1基因功能互补,都具有调节囊泡从内质网到高尔基体运输的功能.     

酵母PAP1与PHO85激酶复合物对YLR190w的磷酸化

酵母基因Pho85编码一个依赖于细胞周期蛋白 (cyclin)的蛋白激酶 (CDK) ,参与多种调控途径。PHO85功能的多效性归于其相关的细胞周期因子 ,现已经鉴定了 10个与PHO85相关的细胞周期因子 (PCL)。为了筛选PAP1 PHO85激酶复合物的特异底物 ,以PAP1为靶分子 ,利用酵母双杂交 (two hybrid)系统从酵母cDNA文库中克隆到一个与PAP1相互作用的蛋白质因子的基因 ,Ylr190w。Ylr190w编码 491个氨基酸的多肽链。体外翻译的YLR190w与纯化的融合蛋白GST PAP1可以被谷胱甘肽亲和柱共同吸附 ,这表明PAP1与YLR190w在体外也可以结合。用免疫沉淀获得的PAP1 PHO85复合物可以磷酸化在大肠杆菌中表达GST YLR190w ;并受到无机磷浓度影响 :高磷条件时磷酸化程度高 ,低磷条件时磷酸化程度低。它能与酵母细胞内YAF9结合 ,YAF9是人具有转录调控活性蛋白质因子AF9的酵母同源物。YLR190w与YAF9的相互作用受到磷条件影响 ,突变YLR190w蛋白S/TP位点的S和T后 ,它们的相互作用明显减弱 ,且不再受到磷条件影响     

延伸因子复合物对真核生物tRNA的修饰作用

延伸因子复合物(Elongator complex, Elp)由6个亚基蛋白Elp1～6组成,在真核细胞生物中呈现高度的进化保守,提示其具有重要的生物学功能。研究表明,Elp涉及多种细胞行为如转录延伸、细胞外分泌、端粒基因沉默和DNA损伤修复、神经系统的发育和功能等。然而越来越多的证据显示,Elp通过介导tRNA修饰影响翻译过程,从而间接调控上述细胞行为。在人类,ELP1/IKBKAP突变可导致家族性植物神经功能障碍症,ELP2、ELP3和ELP4基因的遗传变异也可能与其他神经退行性病变相关。本文对Elp的结构、Elp修饰tRNA和Elp相关疾病等的研究现状及其进展进行综述。     

白假丝酵母CFL1基因通过转录调控参与氧化压力应答

【背景】CFL1基因是白假丝酵母高铁还原酶基因,介导胞外铁离子的还原,在白假丝酵母胞内铁稳态的维持方面发挥着重要作用。【目的】研究CFL1基因调节氧化压力应答的分子机制。【方法】采用液体培养及巨噬细胞模型,测定CFL1缺失对氧化压力耐受性和杀伤巨噬细胞能力的影响;使用羟基自由基清除剂二甲基亚砜(DMSO)分析其对缓解氧化压力敏感性的影响;采用实时荧光定量PCR分析CFL1缺失对氧化压力应答基因表达的影响;采用过氧化氢酶(CAT)活性测定方法研究CFL1缺失对CAT1基因表达的影响;通过构建WT-CAT1-GFP和cfl1Δ/Δ-CAT1-GFP菌株分析过氧化氢酶基因过表达对cfl1Δ/Δ氧化压力敏感性的影响。【结果】白假丝酵母CFL1基因的缺失会造成杀伤巨噬细胞能力的减弱,氧化压力应答基因表达的下降。过氧化氢酶基因的过表达则能恢复与野生型几乎一致的氧化压力水平。【结论】CFL1基因通过转录调控参与白假丝酵母氧化压力应答过程。     

Subunit communications crucial for the functional integrity of the yeast RNA polymerase II elongator (gamma-toxin target (TOT)) complex

In response to the Kluyveromyces lactis zymocin, the gamma-toxin target (TOT) function of the Saccharomyces cerevisiae RNA polymerase II (pol II) Elongator complex prevents sensitive strains from cell cycle progression. Studying Elongator subunit communications, Tot1p (Elp1p), the yeast homologue of human IKK-associated protein, was found to be essentially involved in maintaining the structural integrity of Elongator. Thus, the ability of Tot2p (Elp2p) to interact with the HAT subunit Tot3p (Elp3p) of Elongator and with subunit Tot5p (Elp5p) is dependent on Tot1p (Elp1p). Also, the association of core-Elongator (Tot1-3p/Elp1-3p) with HAP (Elp4-6p/Tot5-7p), the second three-subunit subcomplex of Elongator, was found to be sensitive to loss of TOT1 (ELP1) gene function. Structural integrity of the HAP complex itself requires the ELP4/TOT7, ELP5/TOT5, and ELP6/TOT6 genes, and elp6Delta/tot6Delta as well as elp4Delta/tot7Delta cells can no longer promote interaction between Tot5p (Elp5p) and Tot2p (Elp2p). The association between Elongator and Tot4p (Kti12p), a factor that may modulate the TOT activity of Elongator, requires Tot1-3p (Elp1-3p) and Tot5p (Elp5p), indicating that this contact requires a preassembled holo-Elongator complex. Tot4p also binds pol II hyperphosphorylated at its C-terminal domain Ser(5) raising the possibility that Tot4p bridges the contact between Elongator and pol II.     

组蛋白不同乙酰化位点突变对酵母细胞生长和Ssa3、Gal1基因表达的影响

组蛋白乙酰化对基因表达和细胞生长非常重要.为揭示组蛋白H3K14和H4K8的乙酰化修饰对不同条件下细胞生长和Ssa3、Gal1基因表达的重要性及二者功能差异.构建了H3K14、H4K8分别突变为精氨酸的单突变株S14、S8及二者同时突变的双突变株D814,并对其在正常、高温、咖啡因存在等条件下生长及Ssa3、Gal1表达进行比较.结果表明,所有突变株对咖啡因敏感性增加;D814对温度敏感,且在供试条件下其生长及Ssa3和Gal1激活均明显慢于野生型和单突变株;除半乳糖和葡萄糖为单一碳源,30℃时两单突变株差别不大外,其它条件下S8生长及Ssa3和Gal1激活均慢于S14.表明H3K14、H4K8乙酰化对细胞生长和适应不利环境非常重要,而且在对不利条件的快速适应方面,H4K8的乙酰化修饰可能更为重要.组蛋白突变株的表型缺陷是因该条件下细胞生存所必需的基因激活延迟所致.     

Elongator function in tRNA wobble uridine modification is conserved between yeast and plants

Based on studies in yeast and mammalian cells the Elongator complex has been implicated in functions as diverse as histone acetylation, polarized protein trafficking and tRNA modification. Here we show that Arabidopsis mutants lacking the Elongator subunit AtELP3/ELO3 have a defect in tRNA wobble uridine modification. Moreover, we demonstrate that yeast elp3 and elp1 mutants expressing the respective Arabidopsis Elongator homologues AtELP3/ELO3 and AtELP1/ELO2 assemble integer Elongator complexes indicating a high degree of structural conservation. Surprisingly, in vivo complementation studies based on Elongator‐dependent tRNA nonsense suppression and zymocin tRNase toxin assays indicated that while AtELP1 rescued defects of a yeast elp1 mutant, the most conserved Elongator gene AtELP3, failed to complement an elp3 mutant. This lack of complementation is due to incompatibility with yeast ELP1 as coexpression of both plant genes in an elp1 elp3 yeast mutant restored Elongator's tRNA modification function in vivo. Similarly, AtELP1, not ScELP1 also supported partial complementation by yeast–plant Elp3 hybrids suggesting that AtElp1 has less stringent sequence requirements for Elp3 than ScElp1. We conclude that yeast and plant Elongator share tRNA modification roles and propose that this function might be conserved in Elongator from all eukaryotic kingdoms of life.     

Characterization of a Six-Subunit Holo-Elongator Complex Required for the Regulated Expression of a Group of Genes in Saccharomyces cerevisiae

The Elongator complex associated with elongating RNA polymerase II in Saccharomyces cerevisiae was originally reported to have three subunits, Elp1, Elp2, and Elp3. Using the tandem affinity purification (TAP) procedure, we have purified a six-subunit yeast Holo-Elongator complex containing three additional polypeptides, which we have named Elp4, Elp5, and Elp6. TAP tapping and subsequent purification of any one of the six subunits result in the isolation of all six components. Purification of Elongator in higher salt concentrations served to demonstrate that the complex could be separated into two subcomplexes: one consisted of Elp1, -2, and -3, and the other consisted of Elp4, -5, and -6. Deletions of the individual genes encoding the new Elongator subunits showed that only the ELP5 gene is essential for growth. Disruption of the two nonessential new Elongator-encoding genes, ELP4 and ELP6, caused the same phenotypes observed with knockouts of the original Elongator-encoding genes. Results of microarray analyses demonstrated that the gene expression profiles of strains containing deletions of genes encoding subunits of either Elongator subcomplex, in which we detected significantly altered mRNA expression levels for 96 genes, are very similar, implying that all the Elongator subunits likely function together to regulate a group of S. cerevisiae genes in vivo.     

RNA polymerase II elongator holoenzyme is composed of two discrete subcomplexes.

Elongator is a histone acetyltransferase complex that associates with the elongating form of RNA polymerase II. We purified Elongator to virtual homogeneity via a rapid three-step procedure based largely on affinity chromatography. The purified factor, holo-Elongator, is a labile six-subunit factor composed of two discrete subcomplexes: one comprised of the previously identified Elp1, Elp2, and Elp3 proteins and another comprised of three novel polypeptides, termed Elp4, Elp5, and Elp6. Disruption of the yeast genes encoding the new Elongator proteins confers phenotypes indistinguishable from those previously described for the other elp mutants, and concomitant disruption of genes encoding proteins in either subcomplex does not confer new phenotypes. Taken together, our results indicate that holo-Elongator is a functional entity in vitro as well as in vivo. Metazoan homologues of Elp1 and Elp3 have previously been reported. We cloned the human homologue of yeast ELP4 and show that this gene is ubiquitously expressed in human tissues.