蕨类植物问荆DEAD box RNA解旋酶基因的克隆与分析

DEAD box RNA解旋酶参与RNA多方面的代谢,在植物生长发育和逆境反应中起重要作用。本研究从蕨类植物问荆(Equisetum arvense)中克隆到一条DEAD box RNA解旋酶cDNA全长序列,命名为EaRH1,并在GenBank注册登记（KJ734026）。序列分析显示：该cDNA全长3230bp,包含一个从487bp到2799bp编码770个氨基酸的开放读码框,其对应的蛋白序列包含9个保守模块结构。EaRH1与其它物种DEAD box RNA 解旋酶蛋白序列比对结果显示：模块Ⅰa、Ⅱ和Ⅲ序列几乎完全相同,模块Q、Ⅰ和 Ⅳ序列存在一些差异。EaRH1与江南卷柏(Selaginella moellendorffii)基因组一条假定序列相似度高达69%,其中相似度最高的区域集中在包含9个保守模块的结构域。系统进化树分析显示：EaRH1与拟南芥(Arabidopsis thaliana)DEAD box RNA解旋酶At3g22320在氨基酸序列上有相对较高的同源性。序列结构比较和进化分析可推测出EaRH1可能参与植物体生长发育、miRNA生物合成、与RNA结合蛋白的相互作用和非生物胁迫应答。本文的研究为探索问荆DEAD box RNA解旋酶的进一步功能提供参考。     

RNA解旋酶和应激颗粒

应激颗粒（stress granules, SGs）是细胞在环境压力刺激下停止蛋白质翻译后,mRNA与多种细胞蛋白组装而成的胞质颗粒结构.RNA 解旋酶家族作为生物体内普遍存在的一类高度保守的蛋白质酶类,参与了RNA代谢各个环节,近年来其家族成员被陆续发现是一类新的SG重要组分.本文综述了RNA解旋酶参与应激颗粒形成过程,RNA解旋酶家族蛋白的结构和其参与应激颗粒形成的研究进展.     

恶性疟原虫abstrakt同源基因的克隆及DEAD盒家族蛋白的序列分析

DEAD盒蛋白家族的ATP依赖性的RNA解旋酶类参与细胞内几乎所有的RNA代谢过程 ,在几乎所有生物的细胞生长发育过程中扮演着众多不可或缺的角色。在本实验中 ,通过PCR和探针杂交相结合的筛选方法 ,筛选恶性疟原虫 (Plasmodiumfalciparum)的基因组文库 ,克隆了FH1F———abstrakt同源基因的完整序列。通过搜索已经完成测序的恶性疟原虫基因组数据库 ,推测FH1F序列定位在第 5条染色体上。FH1F全长2 80 4bp,包含一个 1 1 6 1bp的完整阅读框 ,编码一个由 386个氨基酸组成的蛋白。对FH1F蛋白序列用BlastP进行搜索和分析以及用DNAStar与许多典型的DEAD盒蛋白序列进行比对分析 ,结果均提示FH1F蛋白应该是DEAD盒家族的一个Abstrakt蛋白。另一方面 ,用DNAStar对已知所有完整的DEAD盒蛋白进行详细的序列分析以及用Mega对这些序列进行系统发育研究的结果都显示 :DEAD盒家族的蛋白聚类成为若干不同的亚群 ;与DEAD盒蛋白的一般保守序列相比 ,Abstrakt,eIF 4A ,Vasa ,P6 8等不同亚群的DEAD盒蛋白在保守区具有各自不同的结构特征。本文对不同的DEAD盒蛋白的结构特征进行了总结并试图给出不同亚群分类上的结构标准 ,对Abstrakt蛋白在本应高度保守的位点上异常于其它DEAD盒蛋白的氨基酸残基的取代也进行了相关的初步分析     

DEAD-box RNA解旋酶家族在天然免疫应答信号通路中的作用

病毒入侵宿主细胞时,宿主细胞启动抑制病毒复制的免疫机制.同样,病毒也会利用多种手段去逃避先天免疫感应机制的监测以及宿主细胞对外来者的降解,同时还会操纵宿主细胞为自身的增殖提供便利.DEAD-box解旋酶家族是一类存在于宿主细胞中的功能蛋白,它们在转录、剪接、mRNA的合成和翻译等多种细胞过程中起着关键作用.该家族成员拥...     

蕨类植物问荆DEAD-box RNA解旋酶基因的克隆与分析

DEAD-box RNA解旋酶参与RNA多方面的代谢,在植物生长发育和逆境反应中起重要作用。本研究从蕨类植物问荆（Equisetum arvense）中克隆到一条DEAD-box RNA解旋酶c DNA全长序列,命名为EaRH1,并在Gen Bank注册登记（KJ734026）。序列分析显示：该c DNA全长3 230 bp,包含一个从487 bp到2 799 bp编码770个氨基酸的开放读码框,其对应的蛋白序列包含9个保守模块结构。EaRH1与其它物种DEAD-box RNA解旋酶蛋白序列比对结果显示：模块Ⅰa、Ⅱ和Ⅲ序列几乎完全相同,模块Q、Ⅰ和Ⅳ序列存在一些差异。EaRH1与江南卷柏（Selaginella moellendorffii）基因组一条假定序列相似度高达69%,其中相似度最高的区域集中在包含9个保守模块的结构域。系统进化树分析显示：EaRH1与拟南芥（Arabidopsis thaliana）DEAD-box RNA解旋酶At3g22320在氨基酸序列上有相对较高的同源性。序列结构比较和进化分析可推测出EaRH1可能参与植物体生长发育、miRNA生物合成、与RNA结合蛋白的相互作用和非生物胁迫应答。本文的研究为探索问荆DEAD-box RNA解旋酶的进一步功能提供参考。     

几种不同细胞系之间P68 RNA解旋酶表型差异的研究

对分别来自4种不同细胞系的细胞,在生长期间胞内P68 RNA解旋酶的动态变化进行了蛋白质和mRNA的比较研究。结果发现随着细胞培养时间的延长,各系细胞中P68 RNA解旋酶均呈现出有规律的改变,并且这种规律具有明显的细胞系特征:肿瘤细胞系之间表现出P68 RNA解旋酶蛋白条带单一的一致性;非肿瘤细胞系P68 RNA解旋酶蛋白出现多条带变化,并表现出明显的适应性;肿瘤细胞系与非肿瘤细胞系之间P68 RNA解旋酶的表型存在着明显的差异,这种差异从分子水平上揭示出P68 RNA解旋酶与细胞生长密切相关,并可能与细胞癌化有关。对产生这种差异的可能原因及其生物学意义进行了讨论。     

猪瘟病毒p80基因NTPase/RNA解旋酶功能区在E.coli中的表达

采用PCR技术扩增出猪瘟病毒GXYL株和C株p80基因的NTPase／RNA解旋酶功能区(sGXNS3和sCNS3),将其克隆到表达载体pET-30a( )中,获得重组质粒pET—sGXNS3和pET～sCNS3。PCR、酶切和序列分析鉴定目的基因插入位置、方向和读码框完全正确。1．0mmol／L IPTG诱导得到分子量为26kD的目的蛋白。Western blot检测表明,表达的目的蛋白能被CSFV阳性血清识别。     

番茄DEAD-box RNA解旋酶基因SlDEAH1的克隆及表达分析

DEAD-box RNA解旋酶是一种特殊的RNA分子伴侣,参与了RNA代谢,包括前体RNA剪接、核糖体合成、RNA降解以及基因表达,并对植物的发育和抗性等也具有重要作用。根据已报道的拟南芥DEAD-box蛋白,通过同源比对,在NCBI据库中筛选得到一个DEAD-box RNA解旋酶同源蛋白,命名为SlDEAH1,并根据其基因序列设计特异引物,应用RT-PCR方法从野生型番茄(Solanum lycopersicum)AC++中克隆得到了该基因的全长编码区序列。利用生物学网站、软件及实时荧光定量PCR方法,对其进行生物信息学、表达模式、胁迫及激素处理分析。结果表明:SlDEAH1包括2 073 bp的开放阅读框,编码690个氨基酸残基,其编码蛋白有9个保守结构基序,其所涉及到的ATP结合、ATP水解及RNA结合等功能对于解旋酶活性是至关重要的;表达模式分析表明SlDEAH1基因可能在野生型番茄萼片、叶片发育及果实成熟方面起到重要作用;高温、低温、脱水、伤害、盐胁迫不同程度的诱导了SlDEAH1的表达,但在根中该基因的表达受盐胁迫抑制;ABA、ACC、IAA、GA3、MeJA和ZT均不同程度诱导了SlDEAH1的表达,其中ABA诱导效应最为明显。这些结果为进一步研究SlDEAH1在番茄发育和胁迫响应中的功能奠定了基础。     

RNA 解旋酶调控rRNA内稳态: 水稻耐热 新机制、分子育种新资源

高温热害是影响水稻(Oryza sativa)产量形成的重要限制因子。DEAD-box RNA解旋酶在核糖体RNA前体加工及植物抗逆中扮演着重要角色。最近, 中国科学家在DEAD-box RNA解旋酶调控水稻耐热性分子机理研究方面取得了突破性进展。     

低温诱导型凡纳滨对虾DEAD-box RNA解旋酶基因的克隆与表达分析

为研究凡纳滨对虾耐寒性状的分子基础, 克隆鉴定了凡纳滨对虾DEAD-box RNA解旋酶的同源基因。根据差减文库中筛选到的一个低温上调表达的DEAD-box RNA解旋酶基因片段, 通过RACE PCR扩增获得两条高度相似的cDNA全长序列, 两条cDNA均为1430 bp, 包含139 bp 5'UTR、79 bp 3'UTR和1212 bp 开放阅读框, 编码403个氨基酸残基。Blast比对结果显示, 两条cDNA与其他物种的DDX5相似性最高, 推测为凡纳滨对虾DDX5基因的两个变异体, 分别命名为LvDDX5variant A (LvDDX5A)和LvDDX5variant B (LvDDX5B), 在系统进化树中, LvDDX5A和LvDDX5B聚为最近的一支, 并与虾类DDX5、文昌鱼DDX、贝类的DDX5距离较近。Real-time PCR分析结果显示, LvDDX5A和LvDDX5B都呈多组织遍在表达, 在精巢、鳃、肠、肌肉和卵巢中,LvDDX5B表达量高于LvDDX5A, 而在肝胰腺中LvDDX5A表达量高于LvDDX5B。在低温胁迫对虾肝胰腺和心中, LvDDX5A呈上调表达而LvDDX5B表达量无明显变化。进一步对LvDDX5A在不同低温条件胁迫对虾的肝胰腺中表达变化进行了分析。结果显示, LvDDX5A在18℃、15℃、13℃和11℃低温胁迫36h均被诱导表达,并随着15℃和13℃低温胁迫时间的增加呈先上升后下降的表达模式, 在48h达到峰值, LvDDX5A的低温诱导表达模式暗示其可能是在凡纳滨对虾低温适应中发挥作用的剪接体。     

MLN51 stimulates the RNA-helicase activity of eIF4AIII

The core of the exon-junction complex consists of Y14, Magoh, MLN51 and eIF4AIII, a DEAD-box RNA helicase. MLN51 stimulates the ATPase activity of eIF4AIII, whilst the Y14-Magoh complex inhibits it. We show that the MLN51-dependent stimulation increases both the affinity of eIF4AIII for ATP and the rate of enzyme turnover; the K(M) is decreased by an order of magnitude and k(cat) increases 30 fold. Y14-Magoh do inhibit the MLN51-stimulated ATPase activity, but not back to background levels. The ATP-bound form of the eIF4AIII-MLN51 complex has a 100-fold higher affinity for RNA than the unbound form and ATP hydrolysis reduces this affinity. MLN51 stimulates the RNA-helicase activity of eIF4AIII, suggesting that this activity may be functionally important.     

Multifunctional nuclear protein NAP57 specifically interacts with dead RNA-helicase p68

NAP57 has been found as a component of nuclear matrix protein complex with ability to specifically bind alphoid DNA. Polyclonal antibodies against NAP57 were raised in order to investigate intranuclear localization and interactions of the protein. Two types of localization were observed: a) nucleoplasmic and b) nucleolar. A bulk of nucleoplasmic fraction is present in splicing factors compartments (SFC). The type of localization pattern does not depend on the cell cycle phase, but we revealed changes in NAP57 localization pattern during S phase. According to immunoprecipitation and immunofluorescence assays, NAP57 specifically interacts with DEAD RNA helicase p68 in vitro and co-localizes with helicase p68 in the nucleus of cultured cells. We suppose participation of both proteins in processing of small nuclear RNA on the SFC periphery, and positioning of the nucleolus according to centromere regions of chromosomes.     

A putative DEAD-box RNA-helicase is required for normal zoospore development in the late blight pathogen Phytophthora infestans

The asexual multinucleated sporangia of Phytophthora infestans can germinate directly through a germ tube or indirectly by releasing zoospores. The molecular mechanisms controlling sporangial cytokinesis or sporangial cleavage, and zoospore release are largely unknown. Sporangial cleavage is initiated by a cold shock that eventually compartmentalizes single nuclei within each zoospore. Comparison of EST representation in different cDNA libraries revealed a putative ATP-dependent DEAD-box RNA-helicase gene in P. infestans, Pi-RNH1, which has a 140-fold increased expression level in young zoospores compared to uncleaved sporangia. RNA interference was employed to determine the role of Pi-RNH1 in zoospore development. Silencing efficiencies of up to 99% were achieved in some transiently-silenced lines. These Pi-RNH1-silenced lines produced large aberrant zoospores that had undergone partial cleavage and often had multiple flagella on their surface. Transmission electron microscopy revealed that cytoplasmic vesicles fused in the silenced lines, resulting in the formation of large vesicles. The Pi-RNH1-silenced zoospores were also sensitive to osmotic pressure and often ruptured upon release from the sporangia. These findings indicate that Pi-RNH1 has a major function in zoospore development and its potential role in cytokinesis is discussed.     

Molecular cloning and characterization of a novel human gene (HERNA) which encodes a putative RNA-helicase

A full-length cDNA encoding a novel protein was isolated and sequenced from a human hepatocellular cDNA library. This cDNA consists of 7037 base pairs and has a predicted open reading frame encoding 1924 amino acids. It possesses an RNA-helicase motif containing a DEXH-box in its amino-terminus and an RNase motif in the carboxy-terminus. From a striking homology to Caenorhabditis elegans K12H4.8, it might be a human homolog of the K12H4.8. PCR-based mapping with both a monochromosomal hybrid panel and radiation hybrid cell panels placed the gene to human chromosome 14q31 near the marker D14S605.     

Saccharomyces cerevisiae

Yeast prions are infectious proteins that spread exclusively by mating. The frequency of prions in the wild therefore largely reflects the rate of spread by mating counterbalanced by prion growth slowing effects in the host. We recently showed that the frequency of outcross mating is about 1% of mitotic doublings with 23–46% of total matings being outcrosses. These findings imply that even the mildest forms of the [PSI+], [URE3] and [PIN+] prions impart > 1% growth/survival detriment on their hosts. Our estimate of outcrossing suggests that Saccharomyces cerevisiae is far more sexual than previously thought and would therefore be more responsive to the adaptive effects of natural selection compared with a strictly asexual yeast. Further, given its large effective population size, a growth/survival detriment of > 1% for yeast prions should strongly select against prion-infected strains in wild populations of Saccharomyces cerevisiae.     

Thermoresistance in Saccharomyces cerevisiae yeasts

Under natural conditions, yeast Saccharomyces cerevisiae reproduce, as a rule, on the surface of solid or liquid medium. Thus, life cycle of yeast populations is substantially influenced by diurnal changes in ambient temperature. The pattern in the response of unrestricted yeast S. cerevisiae culture to changes in the temperature of cultivation is revealed experimentally. Yeast population, in the absence of environmental constraints on the functioning of cell chemosmotic bioenergetic system, demonstrates the ability of thermoresistance when the temperature of cultivation switches from the range of 12-36 degrees C to 37.5-40 degrees C. During the transient period that is associated with the temperature switching and lasts from 1 to 4 turnover cycles, yeast reproduction rate remains 1.5-2 times higher than under stationary conditions. This is due to evolutionary acquired adaptive activity of cell chemosmotic system. After the adaptive resources exhausting, yeast thermoresistance fully recovers at the temperature range of 12-36 degrees C within one generation time under conditions of both restricted and unrestricted nourishment. Adaptive significance of such thermoresistance seems obvious enough--it allows maintaining high reproduction rate in yeast when ambient temperature is reaching a brief maximum shortly after noon.     

Monoterpenoid biosynthesis in Saccharomyces cerevisiae

Plant monoterpenoids belong to a large family of plant secondary metabolites with valuable applications in cosmetics and medicine. Their usual low levels and difficult purification justify the need for alternative fermentative processes for large-scale production. Geranyl diphosphate is the universal precursor of monoterpenoids. In yeast it occurs exclusively as an intermediate of farnesyl diphosphate synthesis. In the present study we investigated the potential use of Saccharomyces cerevisiae as an alternative engineering tool. The expression of geraniol synthase of Ocimum basilicum in yeast allowed a strong and specific excretion of geraniol to the growth medium, in contrast to mutants defective in farnesyl diphosphate synthase which excreted geraniol and linalool in similar amounts. A further increase of geraniol synthesis was obtained using yeast mutants defective in farnesyl diphosphate synthase. We also showed that geraniol synthase expression affects the general ergosterol pathway, but in a manner dependent on the genetic background of the strain.     

Saccharomyces cerevisiae infections in man

Saccharomyces cerevisiae is a yeast commonly used to make food products such as bread, beer and wine, or ingested whole as a 'health' food. We report five cases of infections involving S. cerevisiae, including one in which S. cerevisiae alone was implicated, and review the literature on its pathogenicity.