水稻eIF3大亚基(eIF3a)编码基因的克隆及其表达模式分析

真核生物翻译起始因子(eIFs)在蛋白合成中起关键作用,在已经鉴定的13个因子中eIF3(由8个或更多的亚基组成)是分子量最大的一个并在翻译起始过程中起着核心作用。eIF3a是eIF3中最大的亚基并介导了eIF3的大多数的功能的实现。利用荧光-差异显示PCR法对生长素处理后的水稻材料进行分析发现eIF3a的表达可被生长素诱导,进一步通过筛选cDNA文库分离出水稻编码eIF3a的全长cDNA(命名为OseIF3a1),OseIF3a1含3459碱基(含5’和3’非翻译区)并编码一个986氨基酸的蛋白,与基因组序列比较表明OseIF3a1基因存在有12个内含子。OSeIF3a1与玉米和烟草的同源蛋白一致性分别为82.4%和70.1%并与其他真核生物eIF3a蛋白具较高一致性。以组织材料进行的RT-PCR结果表明OseIF3a1在根、幼苗、幼穗、茎和叶中表达,启动子-报告基因的转基因结果进一步表明OseIF3a1在根尖及幼嫩叶片表达较高,进一步的RTPCR分析确证了生长素对OseIF3a1的诱导,表明生长素在调控植物生长时可能涉及了翻译水平上的调节。     

小麦蛋白翻译起始因子5A基因（eIF5A）的克隆与分析

真核生物的翻译起始因子5A （eIF5A）是调控生物生长发育、衰老及环境适应等的重要因子。利用设计的小麦蛋白翻译起始因子5A基因的引物对小麦“中国春”基因组DNA和cDNA进行PCR扩增,并将扩增的特异片段回收、克隆和测序,从基因组DNA中得到长度分别为1 679 bp、1 910 bp两条带,从cDNA扩增得到1条636 bp带,分别命名为eIF5a1（基因登录号：DQ167202）、eIF5a2（基因登录号：DQ167201）和eIF5a3。利用GeneRace方法得到eIF5a3（基因登录号：DQ167203）的全长为768 bp。序列分析表明,eIF5a1、eIF5a2具82.3%相似性,都形成636 bp的转录产物,转录产物仅6个核苷酸差异。将eIF5a1、eIF5a2和 eIF5a3这3个序列的预测氨基酸序列进行比对,发现仅有1～2个氨基酸的差异,证实它们为eIF5A基因家族的成员。进化分析表明它们与报道的玉米、水稻、西红柿、烟草的eIF5A基因序列的遗传关系最近。进一步研究表明eIF5a2位于2B染色体上,并用半定量RT-PCR 研究了小麦eIF5A基因的表达情况。     

月季eIF－5A基因的表达增强宿主大肠杆菌对高温和低温的耐性

真核生物翻译起始因子（eIF－5A）是在调控生物生长发育、衰老与环境响应中起重要作用的蛋白质。设计eIF－5A基因的兼并引物,对月季受高温诱导的叶片cDNA进行PCR扩增,获得特异性片段回收、克隆和测序,确定该cDNA为月季eIF-5A(命名为RceIF5A),含有480bp的核苷酸,编码159个氨基酸。将该cDNA序列克隆到原核表达载体PET32a中,获得重组子pET32a－eIF5A。高温（50℃）和低温（4℃）胁迫下含有该基因的大肠杆菌(Escherichia coli) BL21 (pET32a-eIF5A)比E. coli BL21 (pET32a)有明显的抗性提高,据此认为含有重组子的E. coli BL21 (pET32a-eIF5A)对高低温的抗性可能与eIF-5A基因的表达相关。该基因的GeneBank登录号为 EF177192。     

小麦eIF3c基因片段的克隆及序列分析

根据真核翻译起始因子eIF3c的保守序列,搜索小麦EST,由拼接的序列设计引物,从普通小麦‘川麦107'幼苗总RNA中克隆出1102bp的小麦eIF3c1基因片段命名为WeIF3c1.序列分析表明,该片段推断的氨基酸序列与两个拟南芥、水稻、甜樱桃、人类、线虫、老鼠等物种的真核翻译起始因子eIF3c相比较同源性分别为69、61%、85%、72%、38%、36%和38%.     

eIF3a与肿瘤发生、演进和干预的研究进展

真核翻译起始因子3(Eukaryotic translation factor 3,eIF3)是由多个亚单位组成的复合因子,其中eIF3a是其最大的亚单位。很多研究表明在酵母和哺乳动物细胞中,eIF3都参与了m RNA翻译起始,并对蛋白质的合成有很好的调控作用。值得一提的是eIF3a通过调控一系列与肿瘤的生成、细胞周期的调控DNA修复等过程相关的m RNA的翻译从而在肿瘤的发生、演进和干预中发挥重要作用。此外,研究发现eIF3a对RAF-MEK-ERK信号通路有抑制作用。eIF3a对蛋白质翻译的调节及其对RAF-MEK-ERK信号通路的影响使其有望成为肿瘤治疗的新靶点。本文将着重围绕eIF3a在肿瘤发生、演进和干预中的作用进行概述。     

eIF3g差异表达的乳癌细胞模型的建立

真核翻译起始因子3的亚单4ieIF3g在一些多药耐药肿瘤细胞中表达上调。背景相近而eIF3g表达有明显差异的肿瘤细胞模型的建立对阐明其作用及机制有重要意义。该研究利用四环素调控的Tet-OnAdvanced诱导表达系统,分别构建了可诱导过表达外源性geIF3g和表达eIF3g人工Y-microRNA的载体,并包装为相应的慢病毒,将慢病毒分别感染乳腺癌细胞Bcap37,经400μg／mLG418和0．4gg／mLPuromycin筛选后,分别获得稳定转染的乳癌细胞克隆Bcap37／Tet—On．eIF3g和Bcap37／Tet—On—eIF3gmiR。将这些细胞克隆分别在1gg／mLDOX的作用下诱导培养72h,用West．ernblot检测eIF3g的表达。结果显示,Bcap37／Tet—On-eIF3g中eIF3g表达明显增加,Bcap37／Tet-On—eIF3gmiR中eIF3g表达抑制明显。该工作成功建立了可诱导外源性eIF3g过表达和抑制内源性eIF3g表达的乳腺癌细胞模型,为进一步的研究打下了基础。     

降钙素基因相关肽对肺纤维化大鼠肺组织eIF3a、p27表达的影响

目的：观察降钙素基因相关肽（CGRP）对肺纤维化大鼠肺组织真核翻译起始因子3a （eIF3a）、p27表达的影响,探讨CGRP在肺纤维化中的作用及机制。方法：雄性SD大鼠,体重180~220 g,随机分为3组（n=8）：对照组、博莱霉素组、博莱霉素+辣椒素组。采用气管内注射博莱霉素（5 mg/kg）诱导肺纤维化大鼠模型。造模前4 d大鼠皮下注射辣椒素（Capsaicin）（50 mg/kg·d）,造模后第28天处死动物,颈动脉采血ELISA法测定血浆CGRP含量。细胞实验分6组（n=9）：Control组,转化生长因子-β1（TGF-β1）组,CGRP （1、10、100 nmol/L）组,CGRP8-37 1 μmol/L和CGRP 100 nmol/L组。细胞用CGRP和（或） CGRP8-37预处理1 h,再用TGF-β1（5 ng/ml）处理48 h。5-溴脱氧尿嘧啶核苷（BrdU）法检测细胞增殖。免疫组化、real-time PCR和（或） Western blot检测eIF3a、p27、α-平滑肌肌动蛋白（α-SMA）、collagen Ⅰ mRNA及蛋白表达。结果：博莱霉素诱发肺纤维化动物肺组织eIF3a、α-SMA及Ⅰ胶原表达增高,CGRP及p27的表达明显降低。外源性CGRP可剂量依赖性的抑制TGF-β1诱导的肺成纤维细胞增殖,明显抑制eIF3a、α-SMA、Ⅰ胶原的表达,上调p27的表达,这些作用可以被CGRP阻断剂CGRP8-37所取消。结论：CGRP在博莱霉素诱导的肺纤维化中起着重要作用,可能通过抑制eIF3a、上调p27的表达而抑制肺成纤维细胞的增殖,进而抑制肺纤维化的形成与发展。     

水稻液泡ATPase B亚基基因的克隆及其在低磷胁迫下的表达特征分析

利用抑制性扣除杂交（SSH）技术构建水稻（Oryza sativa L.）根系饥饿诱导cDNA文库,获得编码液泡ATPase（V-ATPase）B亚基的克隆,通过反转录PCR方法获得该基因的完整序列。该基因编码487个氨基酸,含有一个保守的ATP结合位点,其蛋白分子量为54.06kD,等电点为4.99。Southern印迹表明,V-ATPase B亚基基因在水稻基因组中以单拷贝形式存在。氮基酸同源性分析发现,V-ATPase B亚基是一个较为保守的蛋白亚基,其序列变化伴随生物的进化过程同步进行。Northern印迹表明,V-ATPase B亚基在水稻根系中受到磷饥饿诱导表达,磷饥饿6～12h出现表达高峰,而在叶片中表达有所滞后（24～48h）,在缺磷环境条件下,ATPase B亚基可能通过提高其表达量,进而提高质子转运活性,形成跨膜的电化学梯度,为体内储备磷跨液泡膜运输提供能量,从而提高植物体内磷的利用效率及其耐低磷的能力。     

人蛋白激酶CK2α′亚基在大肠杆菌中的克隆、表达及其重组蛋白的纯化与性质鉴定

通过RT PCR从HL 6 0细胞获得人蛋白激酶CK2α′亚基编码区cDNA ,将NdeⅠ HindⅢ双酶切的PCR产物和pT7 7表达载体进行定向克隆、细菌转化、电泳初筛和限制性酶切分析鉴定 .随机挑选阳性克隆进行DNA测序确证 ,筛选含与已知序列完全相符的重组质粒 (命名为pTCKA′) .将其转化BL2 1(DE3)菌 ,IPTG诱导后未见高效特异表达 .然后将人CK2α′cDNA亚克隆至GST融合蛋白表达载体 ,经同样转化和诱导步骤后可见一蛋白特异高效表达 .Western印迹结果证明 :该蛋白能与兔抗人CK2α′3 3 3 3 50 肽段抗血清发生特异性免疫反应 .采用GSH Sepharose 4B柱纯化 ,凝血酶酶切 ,最后从 4g细菌获 4 4mg纯化重组蛋白 .通过性质鉴定和酶动力学分析证明 :克隆、表达和纯化的重组蛋白是有生物学活性的人CK2α′亚基 .     

食蟹猴疟原虫 (Plasmodium cynomolgi)中一个真核翻译起始因子4A(eIF-4A)同源蛋白的cDNA克隆、表达和ATP酶活性测定(英文)

真核翻译起始因子 4A(eukaryoticinitiationfactor 4A ,eIF 4A)是DEAD盒蛋白家族的ATP依赖性的RNA解旋酶类中的一个原型成员 .它在真核细胞的蛋白质合成的起始过程中起着关键性作用 .通过PCR扩增和放射探针杂交相结合的方法筛选食蟹猴疟原虫 (Plasmodiumcynomolgi)的cDNA文库 ,克隆了一个eIF 4A同源蛋白的完整cDNA序列 ,命名为CH1F .CH1F全长 1 75 3bp ,包含一个1 1 97bp的完整阅读框 ,推测编码一个由 398个氨基酸组成的蛋白 .对CH1F的蛋白序列用BlastP进行搜索和分析 ,提示它应该是DEAD盒家族的一个eIF 4A同源蛋白 ;用DNAStar将其与许多典型的DEAD盒蛋白序列进行比对分析 ,结果显示 :比起其它的DEAD盒蛋白 ,它与eIF 4A或eIF 4A的同源蛋白具有更高的同源性和更多序列上的相似结构域 .将包含完整阅读框的片段亚克隆进表达载体pET 2 8a (+) ,在大肠杆菌DH5α中表达 ,产生的融合蛋白大小在 4 5kD左右 .对该融合蛋白进行纯化、重新折叠和初步鉴定 .ATP酶活性检测显示 ,该融合蛋白只有很低的ATP酶活性 ,而且它的ATP酶活性似乎不依赖于核酸底物 .对这一检测结果给出 3种可能的原因 .这一检测结果与根据序列分析得到的推论———CH1F蛋白可能是一个eIF 4A并不矛盾     

Isolation and characterization of two cDNAs encoding translation initiation factor 1A from rice(Oryza sativa L.).

The eukaryotic initiation factor 1A(eIF1A) is essential for transferring of the initiator Met-tRNA to 40S ribosomal subunits to form the 40S pre-initiation complex. In present study, we describe the cloning and characterization of two eIF1A genes from rice, which were designated as Oryza sativa eukaryotic initiation factor 1A genes OseIF1A-1, OseIF1A-2, respectively. Both rice elF1As shared high identities in amino acids with eIF1A proteins from other eukaryotes. The mRNA expression analysis revealed that OseIF1A-2 mRNA was much more accumulated than OseIF1A-1 in all tissues but each gene is expressed in root, stem, leaf and flowering spike in high and nearly equal level, and in immature spike in lower level. These results, together with their different location in unrooted phylogenetic tree inferred from amino acid sequences of all known eIF1As, suggested that there are two types of eIF1A genes with different function or different regulation in rice.     

Generation of multiple isoforms of eukaryotic translation initiation factor 4GI by use of alternate translation initiation codons

Eukaryotic translation initiation factor 4GI (eIF4GI) is an essential protein that is the target for translational regulation in many cellular processes and viral systems. It has been shown to function in both cap-dependent and cap-independent translation initiation by recruiting the 40S ribosomal subunit to the mRNA cap structure or internal ribosome entry site (IRES) element, respectively. Interestingly eIF4GI mRNA itself has been reported to contain an IRES element in its 5' end that facilitates eIF4GI protein synthesis via a cap-independent mechanism. In HeLa cells, eIF4GI exists as several isoforms that differ in their migration in sodium dodecyl sulfate (SDS) gels; however, the nature of these isoforms was unclear. Here, we report a new cDNA clone for eIF4GI that extends the 5' sequence 340 nucleotides beyond the previously published sequence. The new extended sequence of eIF4GI is located on chromosome 3, within two additional exons immediately upstream of the previously published eIF4GI sequence. When mRNA transcribed from this cDNA clone was translated in vitro, five eIF4GI polypeptides were generated that comigrated in SDS-polyacrylamide gels with the five isoforms of native eIF4GI. Furthermore, translation of eIF4GI-enhanced green fluorescent protein fusion constructs in vitro or in vivo generated five isoforms of fusion polypeptides, suggesting that multiple isoforms of eIF4GI are generated by alternative translation initiation in vitro and in vivo. Mutation of two of the five in-frame AUG residues in the eIF4GI cDNA sequence resulted in loss of corresponding polypeptides after translation in vitro, confirming alternate use of AUGs as the source of the multiple polypeptides. The 5' untranslated region of eIF4GI mRNA also contains an out-of-frame open reading frame (ORF) that may down-regulate expression of eIF4GI. Further, data are presented to suggest that a proposed IRES embedded in the eIF4GI ORF is able to catalyze synthesis of multiple eIF4GI isoforms as well. Our data suggest that expression of the eIF4GI isoforms is partly controlled by a complex translation strategy involving both cap-dependent and cap-independent mechanisms.     

Translation initiation factor (iso) 4E interacts with BTF3, the beta subunit of the nascent polypeptide-associated complex

A two-hybrid screen with the translation initiation factor, eIF(iso)4E from Arabidopsis, identified a clone encoding a lipoxygenase type 2 [Freire, M.A., et al., 2000. Plant lipoxygenase 2 is a translation initiation factor-4E-binding protein. Plant Molecular Biology 44, 129-140], and three cDNA clones encoding the homologue of the mammalian BTF3 factor, the beta subunit of the nascent polypeptide-associated complex (NAC). Here we report on the interaction between the translation initiation factor eIF(iso)4E and AtBTF3. AtBTF3 protein is able to interact with the wheat initiation factors eIF4E and eIF(iso)4E. AtBTF3 contains a sequence related to the prototypic motif found on most of the 4E-binding proteins, and competes with the translation initiation factor eIF(iso)4G for eIF4(iso)4E binding, in a two hybrid interference assay. These findings provide a molecular link between the translation initiation mechanism and the emergence of the nascent polypeptide chains.     

Cloning of murine translation initiation factor 6 and functional analysis of the homologous sequence YPR016c in Saccharomyces cerevisiae

The cDNA sequence of a murine gene whose expression was up-regulated after epidermal injury was cloned utilizing differential display. The full-length cDNA was isolated by 3' and 5' rapid amplification of cDNA ends from mouse liver. The predicted protein is >97% identical to the human sequence for eukaryotic translation initiation factor (eIF) 6, thus identifying the gene as murine eIF6. Functional studies of the yeast eIF6 homolog, YPR016c, were initiated in Saccharomyces cerevisiae to determine the cellular role(s) of eIF6. Complete deletion of the YPR016c coding sequence was lethal. Viability was restored in the presence of either YPR016c or murine eIF6, when either was expressed as amino-terminal green fluorescent protein fusion protein. Moreover, both fusion proteins localized to nuclear/perinuclear compartments in their respective yeast strains. When the expression of YPR016c-green fluorescent protein was repressed, there was a dramatic reduction in the 60 S ribosomal subunit and polysome content and decreased 80S monosome content. Additionally, the YPR016c-depleted cells arrested in G1. These studies show that YPR016c, which encodes yeast eIF6, is necessary for maximal polysome formation and plays an important role in determining free 60 S ribosomal subunit content.