植物色氨酸一天冬氨酸重复序列蛋白响应逆境胁迫的调控作用

干旱、盐渍、低温等逆境胁迫会严重影响植物的正常生长发育,导致植物的许多响应基因被诱导表达,其蛋白质产物能够保护植物免受胁迫的伤害。色氨酸一天冬氨酸重复序列蛋白（wD40蛋自）在植物中广泛存在,参与植物体内众多代谢反应的调控,如花的发育、开花、花青素的生物合成、激素响应、渗透胁迫等。WD40蛋白含有40-60个氨基酸的保守的wD重复序列,其c末端为色氨酸．天冬氨酸（Trp-Asp,WD）,形成一个p螺旋桨（p—propeller）结构,通过调节多蛋白复合体的组装而影响蛋白质与蛋白质、蛋白质与DNA间的相互作用。本文综述植物WD40蛋白响应逆境胁迫的调控作用。     

植物RACK1蛋白研究进展

RACK1（蛋白激酶C受体）是一种色氨酸-天门冬氨酸域（WD40结构）重复蛋白。它是一种多功能支架蛋白, 结合来自不同转导通路的信号分子并在多种哺乳动物发育过程中起关键作用。在植物中也存在RACK1同源基因, 如拟南芥基因组有3个编码RACK1蛋白质的基因, 这3个蛋白质与哺乳动物RACK1在氨基酸水平的相似性都超过75%。此外, 植物RACK1蛋白质包含的WD40数量、位置和蛋白激酶C结合位点的结构域在很大程度上是保守的。该文对植物RACK1蛋白的发现、结构及其在信号转导方面的功能进行综述。     

人类新基因WDR24的研究初报

WD40家族是一类结构保守、功能复杂的蛋白.目前很多研究显示该家族成员通过参与MAPK信号途径调控细胞内信号转导而影响细胞的基本生命活动.为了鉴定参与细胞生命活动的新基因,运用同源基因克隆法,通过PCR技术扩增获得一个新的人类基因WDR24, 其cDNA全长3 302 bp,2 373 bp长的开放阅读框编码由790个氨基酸残基组成的蛋白质.生物信息学分析表明,WDR24蛋白在进化上高度保守,与其他脊椎动物中的同源蛋白组成了一个功能未知的亚家族.蛋白序列分析显示其中有6个WD40重复序列和1个ERK的停泊位点D-domain.RT-PCR分析表明,该基因在所有被检测的人类胚胎组织中表达.     

海南龙血树WD40转录因子基因DcWD40-1的克隆和表达分析

海南龙血树是国产血竭的主要基源植物,其血竭主要化学成分为类黄酮化合物。为进一步了解DcWD40-1在类黄酮生物合成中的潜在功能和作用机制,该研究根据海南龙血树转录组数据,利用RT-PCR技术在海南龙血树中克隆了一个WD40基因DcWD40-1,该基因全长1 550 bp,包含一个1 353 bp的开放阅读框,编码450个氨基酸,蛋白质分子量50.77 kD,理论等电点5.71。生物信息学分析显示,DcWD40-1属于WD40蛋白家族成员,具有5个保守的WD40结构域,和其他植物WD40蛋白同源性高,保守性强。利用Genome Walking方法分离了1 503 bp的DcWD40-1启动子序列,该区域具有典型真核生物启动子结构特征,并含有多个应答激素和胁迫的响应元件。表达分析显示,血竭诱导剂能够诱导Dc WD40-1的表达,DcWD40-1的变化与血竭形成及类黄酮积累正相关。此外,DcWD40-1也能对茉莉酸甲酯、细胞分裂素、油菜素内酯和UV-B处理做出积极响应。     

千里光脂肪醛脱羰基酶(CER1蛋白)的保守基序(CSM)与功能结构域分析

基于已构建的千里光全长c DNA文库,克隆其脂肪醛脱羰基酶基因(Gen Bank No.:KT895253),并进一步分析该基因所编码蛋白质(CER1蛋白)的氨基酸保守基元序列(conserved sequence motif,CSM)和蛋白质功能结构域的关系。蛋白质一级结构分析发现,千里光CER1蛋白由623个氨基酸残基组成;进化树和序列比对结果提示SH-片段和LH-片段这两个富含His的保守基元序列在不同物种中表现出高度保守性;3-D结构预测结果表明,高等植物CER1蛋白水合状态下具有高效的底物结合能力和脂肪醛脱羰基的催化效率。因此,根据脂肪醛脱羰基酶氨基酸保守基序对蛋白质功能域的决定作用,推测CER1蛋白的保守结构域是形成底物结合部位和酶催化活性中心的结构基础。     

G_β类似蛋白─WDR26对细胞增殖的影响

WD40重复序列蛋白是一类结构保守、功能复杂的蛋白.关于此类蛋白和细胞内信号传导途径的研究显示,该家族成员很可能通过调控胞内信号转导而影响细胞基本生命活动.在此前的研究中,我们报道了一个新基因WD40repeatprotein26的克隆.其初步研究结果显示WDR26蛋白与MAPK信号途径的负调控相关.在最近的研究中,我们构建了稳定转染pCMV-WDR26表达载体的HeLa细胞系,结果显示WDR26蛋白在HeLa细胞系中的过度表达,能够促进细胞分裂,加快细胞的倍增速度.因此,WDR26很可能具有通过MAPK信号途径调节细胞增殖的功能.     

WDR蛋白在肿瘤发生发展中的作用机制

WDR蛋白家族(Trp-asp repeat protein family)是含有多个保守WD基序(WD motif)的蛋白质类,广泛存在于真核生物中。近年,WDR蛋白的研究已成为细胞生物学和病理学研究的新热点。目前已知多种WDR蛋白在肿瘤细胞中异常表达,并通过调节信号转导、细胞周期、泛素化、转录和RNA加工等生物过程促使肿瘤发生。现对WDR蛋白在肿瘤发生发展中的作用机制作一综述。     

BRCA2的BRC4基元与RAD51相互作用位点的研究进展

乳腺癌易感基因2(breast cancer susceptibility gene 2,BRCA2),是人体内一种与乳腺、卵巢、胰腺等部位的肿瘤有关的抑癌基因。人的RAD51(h RAD51)是参与DNA同源重组修复过程的关键蛋白。BRCA2蛋白通过其结构中8个高度保守的BRC重复基元来调控h RAD51通过同源重组对DNA损伤进行的修复,从而阻止细胞癌变。在BRCA2的8个BRC重复基元中,BRC4与同源重组酶h RAD51的相互作用较为明显。综述了BRCA2的BRC4基元与h RAD51相互作用位点的研究进展,为了解BRCA2与RAD51相互作用的分子机理提供基础。     

一个莱茵衣藻WD40蛋白正向调控油脂的积累

WD40蛋白(WD40-repeats)一般存在于真核生物中,具有广泛的生理生化功能。本研究以莱茵衣藻一个WD40(Cre12.g552900)蛋白为研究对象,通过遗传转化的方法实现对该基因的敲除和过量表达。转基因藻株检测结果显示Cr WD40 RNAi转基因藻株油脂含量减少了11%~24%,而Cr WD40过量表达转基因藻株油脂含量增加了14.3%~75%,正反两方面实验说明Cr WD40对油脂积累起到正向调控的作用。     

Excel平台的EST-SSR分析方法

科学工作者在分析EST-SSR特性时需要许多手工操作,这是一个耗时而又容易出错的工作。本文介绍了以Excel平台来计算核苷酸数目、计算特定碱基数量和比例、合并SSR重复基元、筛选基元的重复记录、统计基元的总重复数、计算基元在SSR中最大或最小重复数、截取特定核苷酸序列等方法,这样可以提高分析的效率和准确性,同时对其他DNA或蛋白质序列分析有借鉴作用。     

Isolation, characterization, and mapping of the mouse and human WDR8 genes, members of a novel WD-repeat gene family

The Trp-Asp (WD) motif has been shown to exist in a number of proteins. Genes containing repeats of the WD motif compose a large gene family associated with a variety of cellular functions and can be divided into a number of functional subfamilies. By means of the differential display method using ttw, a mouse model for the early stage of ectopic ossification, we have identified a novel mouse gene, Wdr8 (WD repeat domain 8), which contains two WD repeats, together with its human orthologue. The human and mouse WDR8 genes encode 460 and 462 amino acids, respectively, with 89% identity, and are expressed in almost all tissues, including bone and cartilage, and in bone-forming cells, including osteoblasts and chondrocytes. Wdr8 expression in cartilage was differentially displayed by stimuli for ectopic ossification in ttw and was observed strongly only at a transition period from hypertrophic to mineralizing stages in ATDC5, a chondrogenic cell line that exhibits endochondral ossification, suggesting a potential role for Wdr8 in the process of ossification. The WDR8 protein is highly conserved among a variety of species, but is distinctly different from other WD-repeat proteins, indicating that it represents a novel subfamily of the WD-repeat gene family.     

Wdr12, a mouse gene encoding a novel WD-Repeat Protein with a notchless-like amino-terminal domain

The WD-repeat protein family consists of a large group of structurally related yet functionally diverse proteins found predominantly in eukaryotic cells. These factors contain several (4-16) copies of a recognizable amino-acid sequence motif (the WD unit) thought to be organized into a "propeller-like" structure involved in protein-protein regulatory interactions. Here, we report the cloning of a mouse cDNA, referred to as Wdr12, which encodes a novel WD-repeat protein of 423 amino acids. The WDR12 protein was predicted to contain seven WD units and a nuclear localization signal located within a protruding peptide between the third and fourth WD domains. The amino-terminal region shows similarity to that of the Notchless WD repeat protein. Sequence comparisons revealed WDR12 orthologs in various eukaryotic species. Wdr12 seems to correspond to a single-copy gene in the mouse genome, located within the C1-C2 bands of chromosome 1. These data, together with the results of Wdr12 gene expression studies and evidence of in vitro binding of WDR12 to the cytoplasmic domain of Notch1, led us to postulate a function for the WDR12 protein in the modulation of Notch signaling activity.     

The WD repeat: a common architecture for diverse functions.

Our knowledge of the large family of proteins that contain the WD repeat continues to accumulate. The WD-repeat proteins are found in all eukaryotes and are implicated in a wide variety of crucial functions. The solution of the three-dimensional structure of one WD-repeat protein and the assumption that the structure will be common to all members of this family has allowed subfamilies of WD-repeat proteins to be defined on the basis of probable surface similarity. Proteins that have very similar surfaces are likely to have common binding partners and similar functions.     

The structure of Ski8p, a protein regulating mRNA degradation: Implications for WD protein structure

Ski8p is a 44-kD protein that primarily functions in the regulation of exosome-mediated, 3'--> 5' degradation of damaged mRNA. It does so by forming a complex with two partner proteins, Ski2p and Ski3p, which complete a complex that is capable of recruiting and activating the exosome/Ski7p complex that functions in RNA degradation. Ski8p also functions in meiotic recombination in complex with Spo11 in yeast. It is one of the many hundreds of primarily eukaryotic proteins containing tandem copies of WD repeats (also known as WD40 or beta-transducin repeats), which are short ~40 amino acid motifs, often terminating in a Trp-Asp dipeptide. Genomic analyses have demonstrated that WD repeats are found in 1%-2% of proteins in a typical eukaryote, but are extremely rare in prokaryotes. Almost all structurally characterized WD-repeat proteins are composed of seven such repeats and fold into seven-bladed beta propellers. Ski8p was thought to contain five WD repeats on the basis of primary sequence analysis implying a five-bladed propeller. The 1.9 A crystal structure unexpectedly exhibits a seven-bladed propeller fold with seven structurally authentic WD repeats. Structure-based sequence alignments show additional sequence diversity in the two undetected repeats. This demonstrates that many WD repeats have not yet been identified in sequences and also raises the possibility that the seven-bladed propeller may be the predominant fold for this family of proteins.     

WD40重复蛋白家族基因At1g65030调控拟南芥种子的重量与体积

植物中WD40-repeat蛋白在细胞周期调控等方面具有重要作用。本研究鉴定了一株拟南芥WD40-repeat蛋白基因突变体at1g65030,与野生型植株相比种子重量增重体积变大,营养生长长势较弱,角果种子结实率较低。以突变体作为母本/父本与野生型父本/母本杂交,前者杂交后代未显示有母本的突变表型,后者部分杂交后代显示出父本的突变表型,统计突变体后代分离比符合1:1。用苯胺兰(DAB)、4,6-二氨基-2-苯基吲哚(DAPI)、2,3,5-氯化三苯基四氮唑(TTC)、碘-碘化钾花粉染色,发现花粉部分败育且主要为核败育。爱氏苏木精花粉染色结果显示可观察到正常减数分裂各时期形态。采取热不对称交错PCR(thermal asymmetric interlaced PCR,TAIL-PCR)方法确认突变基因位于第一条染色体65030位置,生物信息学分析表明该基因含有DWD基序。半定量RT-PCR分析发现在拟南芥发育晚期该基因在花器官中大量表达,过表达该基因使种子重量减轻。推测At1g65030影响了拟南芥花粉发育细胞核有丝分裂过程,该研究增加了人们对调控拟南芥花粉发育分子机制的认识。     

Identification of a structural motif that confers specific interaction with the WD40 repeat domain of Arabidopsis COP1

Arabidopsis COP1 is a photomorphogenesis repressor capable of directly interacting with the photomorphogenesis-promoting factor HY5. This interaction between HY5 and COP1 results in targeted deg radation of HY5 by the 26S proteasome. Here we characterized the WD40 repeat domain-mediated interactions of COP1 with HY5 and two new proteins. Mutational analysis of those interactive partners revealed a conserved motif responsible for the interaction with the WD40 domain. This novel motif, with the core sequence V-P-E/D-φ-G (φ = hydrophobic residue) in conjunction with an upstream stretch of 4-5 negatively charged residues, interacts with a defined surface area of the ss-propeller assembly of the COP1 WD40 repeat domain through both hydrophobic and ionic interactions. Several residues in the COP1 WD40 domain that are critical for the interaction with this motif have been revealed. The fact that point mutations either in the COP1 WD40 domain or in the HY5 motif that abolish the interaction between COP1 and HY5 in yeast result in a dramatic reduction of HY5 degradation in transgenic plants validates the biological significance of this defined interaction.