共查询到20条相似文献，搜索用时 47 毫秒
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Skowyra D Koepp DM Kamura T Conrad MN Conaway RC Conaway JW Elledge SJ Harper JW 《Science (New York, N.Y.)》1999,284(5414):662-665
Control of cyclin levels is critical for proper cell cycle regulation. In yeast, the stability of the G1 cyclin Cln1 is controlled by phosphorylation-dependent ubiquitination. Here it is shown that this reaction can be reconstituted in vitro with an SCF E3 ubiquitin ligase complex. Phosphorylated Cln1 was ubiquitinated by SCF (Skp1-Cdc53-F-box protein) complexes containing the F-box protein Grr1, Rbx1, and the E2 Cdc34. Rbx1 promotes association of Cdc34 with Cdc53 and stimulates Cdc34 auto-ubiquitination in the context of Cdc53 or SCF complexes. Rbx1, which is also a component of the von Hippel-Lindau tumor suppressor complex, may define a previously unrecognized class of E3-associated proteins. 相似文献
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Excess cyclin E-Cdk2 accelerates entry into S phase of the cell cycle and promotes polyploidy, which may contribute to genomic instability in cancer cells. We identified 20 amino acids in cyclin E as a centrosomal localization signal (CLS) essential for both centrosomal targeting and promoting DNA synthesis. Expressed wild-type, but not mutant, CLS peptides localized on the centrosome, prevented endogenous cyclin E and cyclin A from localizing to the centrosome, and inhibited DNA synthesis. Ectopic cyclin E localized to the centrosome and accelerated S phase entry even with mutations that abolish Cdk2 binding, but not with a mutation in the CLS. These results suggest that cyclin E has a modular centrosomal-targeting domain essential for promoting S phase entry in a Cdk2-independent manner. 相似文献
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用基因工程大肠杆菌E.coliP84A/MC1061生产卤醇脱卤酶,此后,进一步进行了酵母浸出物FM888与酵母蛋白胨FP101的联合调控作用研究。结果表明,用高溶解度酵母浸出物FM888替代原配方中的牛骨蛋白胨,可使E.coliP84A/MC1061生长速率增大,发酵稳定期的菌体浓度保持在较高水平,酶活提高了约2.4倍。当补料培养基中的FM888与酵母蛋白胨FP101的比例为7∶3时,菌体浓度保持在较高水平(OD600≥120),酶活进一步提高约50%,大大提高了单罐产率。此研究可为其他酶制剂的发酵生产提供借鉴。 相似文献
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木质素是绿色植物生长发育所需的重要化合物之一。肉桂酰辅酶A还原酶(CCR)是在木质素合成过程中,催化苯丙烷类合成途径第1步的关键酶。本文以其底物之一——对香豆酰辅酶A酯为例,以现有的辅酶A酯合成纯化的文献报道为参考,介绍经过整合改进并不断试验而得到的CCR底物的合成与纯化方法。从大肠杆菌中提取出来的重组酶4-香豆酸辅酶A连接酶(4CL)用于合成CCR底物,这比从植物组织中提取更易获得。提纯后的4CL可以在-80 ℃下保存3、4个月。4CL反应后的产物在2个不同梯度下通过C18反相柱进一步纯化,并用高效液相色谱仪监测。在整个纯化过程中,所有用于装载对香豆酸和对香豆酰辅酶A酯的容器都用锡箔纸完全包裹起来。最后提纯出来的产物用质谱和核磁共振检测。检测的结果表明,提纯出来的辅酶A酯单一性非常好。 相似文献
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【目的】确定FSH是否能调节支持细胞cyclin D1 mRNA和cyclin E1 mRNA的表达及可能的机制。【方法】以培养的仔猪睾丸支持细胞为试验材料,通过添加各种信号通路的抑制剂,应用实时荧光定量PCR 检测cyclin D1 mRNA和cyclin E1 mRNA的相对表达量。【结果】不同浓度的FSH（0—100 ng•mL-1）均可促进cyclin D1 mRNA和cyclin E1 mRNA的表达（P＜0.05）,在FSH浓度为50 ng•mL-1时两种基因的表达量最大（P＜0.05）;FSH（50 ng•mL-1）也以时间依赖的方式促进了cyclin D1 mRNA和cyclin E1 mRNA的表达（P＜0.05）,在作用30 min时其表达量达到高峰（P＜0.05）。不同浓度的Foskolin （0—20 μmol•L-1）均可以促进cyclin D1 mRNA和cyclin E1 mRNA的表达（P＜0.05）,但均低于FSH单独作用时两种基因的表达量;Rp-cAMP（0—40 μmol•L-1）、H-89（0—30 μmol•L-1）和Verapamil（0—100 μg•mL-1）以剂量依赖的方式抑制了FSH诱导的cyclin D1 mRNA和cyclin E1 mRNA的表达（P＜0.05）,而且Rp-cAMP和Verapamil联合作用对FSH的抑制效果高于单独的抑制效果（P＜0.05）,但低于两者之和。此外,不同浓度的U0126（0—10 μmol•L-1）降低了FSH诱导的cyclin D1 mRNA和cyclin E1 mRNA的表达（P＜0.05）,而Rp-cAMP、H-89、Verapamil和U0126单独作用时,cyclin D1 mRNA和cyclin E1 mRNA的表达与对照相比没有显著差异（P＞0.05）。【结论】FSH以剂量依赖和时间依赖的方式诱导了cyclin D1 mRNA和cyclin E1 mRNA的表达;cAMP-PKA、Ca2+和ERK1/2参与了FSH对cyclin D1 mRNA和cyclin E1 mRNA表达的调节。 相似文献
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Nagai S Dubrana K Tsai-Pflugfelder M Davidson MB Roberts TM Brown GW Varela E Hediger F Gasser SM Krogan NJ 《Science (New York, N.Y.)》2008,322(5901):597-602
Recent findings suggest important roles for nuclear organization in gene expression. In contrast, little is known about how nuclear organization contributes to genome stability. Epistasis analysis (E-MAP) using DNA repair factors in yeast indicated a functional relationship between a nuclear pore subcomplex and Slx5/Slx8, a small ubiquitin-like modifier (SUMO)-dependent ubiquitin ligase, which we show physically interact. Real-time imaging and chromatin immunoprecipitation confirmed stable recruitment of damaged DNA to nuclear pores. Relocation required the Nup84 complex and Mec1/Tel1 kinases. Spontaneous gene conversion can be enhanced in a Slx8- and Nup84-dependent manner by tethering donor sites at the nuclear periphery. This suggests that strand breaks are shunted to nuclear pores for a repair pathway controlled by a conserved SUMO-dependent E3 ligase. 相似文献
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COP1 E3连接酶是一个光形态建成的抑制子和光调控植物发育的分子开关。对山核桃Carya cathayensis花芽454测序获得CcCOP1 E3连接酶的片段, 通过cDNA末端快速扩增技术(RACE), 分别获得该基因的全长, 大小为2 331 bp, 它由2个特殊的结构域组成即环形锌指结合域和WD-40重复序列, 其编码的蛋白质有较强的亲水性, 在氨基端主要是亲水性氨基酸, 而羧基端主要是疏水性氨基酸。CcCOP1 E3连接酶与毛果杨Populus trichocarpa等的COP1 E3连接酶同源基因相似度较高, 总体高达77.80%。实时荧光定量聚合酶链式反应(real-timePCR)结果显示:CcCOP1 E3连接酶的表达贯穿于在山核桃雌雄花的发育过程, CcCOP1 E3连接酶在山核桃的茎、叶、果实、花芽中均有表达, 但在花芽中表达量最高, 3月中旬表达量最高, 在5月中旬雄花表达量相对较高。CcCOP1 E3连接酶与山核桃雌雄花分化有关。 相似文献
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Dorrello NV Peschiaroli A Guardavaccaro D Colburn NH Sherman NE Pagano M 《Science (New York, N.Y.)》2006,314(5798):467-471
The tumor suppressor programmed cell death protein 4 (PDCD4) inhibits the translation initiation factor eIF4A, an RNA helicase that catalyzes the unwinding of secondary structure at the 5' untranslated region (5'UTR) of messenger RNAs (mRNAs). In response to mitogens, PDCD4 was rapidly phosphorylated on Ser67 by the protein kinase S6K1 and subsequently degraded via the ubiquitin ligase SCF(betaTRCP). Expression in cultured cells of a stable PDCD4 mutant that is unable to bind betaTRCP inhibited translation of an mRNA with a structured 5'UTR, resulted in smaller cell size, and slowed down cell cycle progression. We propose that regulated degradation of PDCD4 in response to mitogens allows efficient protein synthesis and consequently cell growth. 相似文献
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COP1 E3连接酶是一个光形态建成的抑制子和光调控植物发育的分子开关.对山核桃Carya cathayensis花芽454测序获得CcCOP1 E3连接酶的片段,通过cDNA末端快速扩增技术（RACE）,分别获得该基因的全长,大小为2 331 bp,它由2个特殊的结构域组成即环形锌指结合域和WD-40重复序列,其编码的蛋白质有较强的亲水性,在氨基端主要是亲水性氨基酸,而羧基端主要是疏水性氨基酸.CeCOP1 E3连接酶与毛果杨Populus trichocarpa等的COP1 E3连接酶同源基因相似度较高,总体高达77.80％.实时荧光定量聚合酶链式反应（real-timePCR）结果显示：CcCOP1 E3连接酶的表达贯穿于在山核桃雌雄花的发育过程,CcCOP1 E3连接酶在山核桃的茎、叶、果实、花芽中均有表达,但在花芽中表达量最高,3月中旬表达量最高,在5月中旬雄花表达量相对较高.CcCOP1 E3连接酶与山核桃雌雄花分化有关. 相似文献
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通过现代分子生物学技术,从三角帆蚌(Hyriopsis cumingii)中克隆了细胞周期蛋白B(cyclin B)基因的cDNA,得到长度为1 024 bp的序列信息,包括768 bp的ORF,197 bp的3′-UTR,编码255个Aa。进一步分析表明,三角帆蚌cyclin B基因与牡蛎、扇贝具有较高的相似性,其氨基酸序列中具有1个典型的周期蛋白框(cyclin-box),并存在2个周期蛋白依赖性蛋白激酶(CDK)的作用位点。通过RT-qPCR研究显示:三角帆蚌cyclin B基因在性腺中的表达最高,在雌性中的表达量显著高于雄性(P0.05);而cyclin B基因在其他组织中的表达量均显著低于性腺(P0.05),且无显著雌雄差异(P0.05);沉默cyclin B基因后,除外套膜外,其在性腺和鳃的表达显著降低(P0.05),表明RNA干扰(RNAi)对三角帆蚌不同组织具有不同的沉默效果。流式细胞仪测定RNAi后细胞的分裂时相变化,发现性腺和鳃细胞中G2/M期占比下降,表明cyclin B基因在三角帆蚌中能调控细胞分裂。初步研究了三角帆蚌cyclin B基因及其功能,为三角帆蚌细胞体外建系奠定分子基础。 相似文献
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Phosphorylation by cyclin B-Cdk underlies release of mitotic exit activator Cdc14 from the nucleolus
Azzam R Chen SL Shou W Mah AS Alexandru G Nasmyth K Annan RS Carr SA Deshaies RJ 《Science (New York, N.Y.)》2004,305(5683):516-519
Budding yeast protein phosphatase Cdc14 is sequestered in the nucleolus in an inactive state during interphase by the anchor protein Net1. Upon entry into anaphase, the Cdc14 early anaphase release (FEAR) network initiates dispersal of active Cdc14 throughout the cell. We report that the FEARnetwork promotes phosphorylation of Net1 by cyclin-dependent kinase (Cdk) complexed with cyclin B1 or cyclin B2. These phosphorylations appear to be required for FEAR and sustain the proper timing of late mitotic events. Thus, a regulatory circuit exists to ensure that the arbiter of the mitotic state, Cdk, sets in motion events that culminate in exit from mitosis. 相似文献
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In eukaryotes, it is unknown whether mismatch repair (MMR) is temporally coupled to DNA replication and how strand-specific MMR is directed. We fused Saccharomyces cerevisiae MSH6 with cyclins to restrict the availability of the Msh2-Msh6 mismatch recognition complex to either S phase or G2/M phase of the cell cycle. The Msh6-S cyclin fusion was proficient for suppressing mutations at three loci that replicate at mid-S phase, whereas the Msh6-G2/M cyclin fusion was defective. However, the Msh6-G2/M cyclin fusion was functional for MMR at a very late-replicating region of the genome. In contrast, the heteroduplex rejection function of MMR during recombination was partially functional during both S phase and G2/M phase. These results indicate a temporal coupling of MMR, but not heteroduplex rejection, to DNA replication. 相似文献
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按照肝脏受 TAA损伤的程度把试验动物 Wistar大白鼠分为两组 ( TAA- 1和 TAA- 2 )。Northern杂交分析表明 ,在 TAA- 1的窦内皮细胞中 ,bcl- x和 bax基因的表达水平轻微降低 ;随着肝损伤的增加 ,表达水平再度下降。核 Run- off转录分析表明 ,两组的窦内皮细胞、总的 RNA合成率下降约 30 %( P<0 .0 0 1 ) ;TAA- 1、bad、bax和 cyclin E的 m RNA合成有所降低 ,而 cyclin A的 m RNA合成则明显增加 ;TAA- 2、bad、bax和 bcl- 2的 m RNA合成增加了 2～ 3倍 ,bcl- x的 m RNA合成增加了 7倍多 相似文献
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Dueber EC Schoeffler AJ Lingel A Elliott JM Fedorova AV Giannetti AM Zobel K Maurer B Varfolomeev E Wu P Wallweber HJ Hymowitz SG Deshayes K Vucic D Fairbrother WJ 《Science (New York, N.Y.)》2011,334(6054):376-380
Inhibitor of apoptosis (IAP) proteins are negative regulators of cell death. IAP family members contain RING domains that impart E3 ubiquitin ligase activity. Binding of endogenous or small-molecule antagonists to select baculovirus IAP repeat (BIR) domains within cellular IAP (cIAP) proteins promotes autoubiquitination and proteasomal degradation and so releases inhibition of apoptosis mediated by cIAP. Although the molecular details of antagonist-BIR domain interactions are well understood, it is not clear how this binding event influences the activity of the RING domain. Here biochemical and structural studies reveal that the unliganded, multidomain cIAP1 sequesters the RING domain within a compact, monomeric structure that prevents RING dimerization. Antagonist binding induces conformational rearrangements that enable RING dimerization and formation of the active E3 ligase. 相似文献
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Chung KK Thomas B Li X Pletnikova O Troncoso JC Marsh L Dawson VL Dawson TM 《Science (New York, N.Y.)》2004,304(5675):1328-1331
Parkin is an E3 ubiquitin ligase involved in the ubiquitination of proteins that are important in the survival of dopamine neurons in Parkinson's disease (PD). We show that parkin is S-nitrosylated in vitro, as well as in vivo in a mouse model of PD and in brains of patients with PD and diffuse Lewy body disease. Moreover, S-nitrosylation inhibits parkin's ubiquitin E3 ligase activity and its protective function. The inhibition of parkin's ubiquitin E3 ligase activity by S-nitrosylation could contribute to the degenerative process in these disorders by impairing the ubiquitination of parkin substrates. 相似文献
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The Pseudomonas syringae protein AvrPtoB is translocated into plant cells, where it inhibits immunity-associated programmed cell death (PCD). The structure of a C-terminal domain of AvrPtoB that is essential for anti-PCD activity reveals an unexpected homology to the U-box and RING-finger components of eukaryotic E3 ubiquitin ligases, and we show that AvrPtoB has ubiquitin ligase activity. Mutation of conserved residues involved in the binding of E2 ubiquitin-conjugating enzymes abolishes this activity in vitro, as well as anti-PCD activity in tomato leaves, which dramatically decreases virulence. These results show that Pseudomonas syringae uses a mimic of host E3 ubiquitin ligases to inactivate plant defenses. 相似文献
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Volarevic S Stewart MJ Ledermann B Zilberman F Terracciano L Montini E Grompe M Kozma SC Thomas G 《Science (New York, N.Y.)》2000,288(5473):2045-2047
Because ribosome biogenesis plays an essential role in cell proliferation, control mechanisms may have evolved to recognize lesions in this critical anabolic process. To test this possibility, we conditionally deleted the gene encoding 40S ribosomal protein S6 in the liver of adult mice. Unexpectedly, livers from fasted animals deficient in S6 grew in response to nutrients even though biogenesis of 40S ribosomes was abolished. However, liver cells failed to proliferate or induce cyclin E expression after partial hepatectomy, despite formation of active cyclin D-CDK4 complexes. These results imply that abrogation of 40S ribosome biogenesis may induce a checkpoint control that prevents cell cycle progression. 相似文献