基因组中重复序列的意义

从原核生物到真核生物,其基因组中的重复序列呈递增趋势.重复序列的作用也被各种实验所揭示.各种重复序列的类型与它在染色体上的分布密切相关.重复序列不是垃圾,而是影响着生命的进化、遗传、变异;同时它对基因表达、转录调控、染色体的构建以及生理代谢都起着不可或缺的作用.它们的功能及演化也正在被逐步阐明.     

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。     

神经富亮氨酸重复蛋白家族研究进展

神经富亮氨酸重复(neural leucine-rich repeat,NLRR)蛋白家族是一类进化上保守、功能多样的Ⅰ型跨膜蛋白.NLRR主要表达于神经组织,肺部、心脏、肝、肾也有微量表达.自1996年Taguchi在发育的小鼠神经系统中发现了新的LRR蛋白质mNLRR-1和mNLRR-2后,一系列含有类似结构的NLRR蛋白质在小鼠、大鼠、爪蟾、斑马鱼和人类等种属中被发现.NLRR蛋白家族成员同源性高,主要含有一些共同的蛋白质结构域:LRR(leucine-rich repeat)、AFR(amino-flanking region)、CFR(carboxy-flanking region)、IgC2(immunoglobulin-like C2 type domain)和FNⅢ(fibronectin type Ⅲ-like domain)等.迄今为止,已有实验证明NLRR作为细胞黏附分子或配体受体分子在神经发育与再生过程中发挥着重要作用.主要对NLRR的时空表达模式、结构及主要生物学功能等进行了综述.     

基于表达序列标签的简单重复序列标记开发策略

作为一种新型分子标记,表达序列标签-简单重复序列(EST-SSR)来自表达基因,因此除具备来源于传统基因组的SSR标记的所有优势外,还与基因功能表达具有直接或间接的关系,从而强化了SSR标记在遗传研究中的应用。我们简要介绍了EST-SSR标记的开发策略、方法及其应用进展,总结了其中存在的一些问题,目的是为今后该领域的研究提供一定的参考。     

重复序列ERIC(IRU)研究进展

重复序列几乎存在于所有生物的基因组中。"肠道细菌基因间重复序列"(Enterobacterial Repetitive Intergenic Consensus,ERIC)是主要存在于肠道细菌的一类基因间重复序列,也称为"基因间重复单位"(Intergenic Repetitive Unit,IRU)。ERIC(IRU)首先在大肠杆菌(Escherichia coli)中发现,后来又在多种其他细菌中发现。ERIC(IRU)长127bp,有的还有插入序列。绝大多数ERIC(IRU)都可以转录,mRNA形成茎环结构。ERIC(IRU)局限于基因组可转录区,即多顺反子操纵子基因间区域,或开放阅读框架上、下游非翻译区。ERIC(IRU)很可能调节侧翼基因(flanking gene)的表达。ERIC(IRU)高度保守,可能其变异受到自然选择压力的限制或它本身就可能是"自私的DNA"(selfish DNA)。Versalovic等建立起ERIC-PCR,它可以有效地同时平行分析不同生态系统的结构差异以及动态监测同一生态系统微生物群落结构的变化。近年来这一技术逐渐运用到对动物肠道菌群的研究上。     

HIV—1长末端重复序列对重组痘苗病毒表达Gag蛋白的影响

将系列缺失的ＨＩＶ１长末端重复序列（ＬＴＲ）和全长的ｇａｇＯＲＦ置于痘苗病毒载体中,经同源重组和血球吸附试验,成功地构建了６株重组痘苗病毒。免疫印迹和免疫酶试验检测均表明,６株重组病毒的Ｇａｇ蛋白表达量因ＬＴＲ不同而有明显差异,表明ＨＩＶ１的ＬＴＲ及其下游基因置于痘病毒启动子控制下,在痘苗病毒中表达时有下述特点：（１）不同的痘苗病毒启动子与全长ＬＴＲ相互作用,对ｇａｇ基因表达有显著不同的调控效果;（２）ＮＲ序列对Ｇａｇ蛋白表达没有明显影响;（３）ＥＮ序列不能被重组痘苗病毒表达系统识别;（４）ＴＡＲ序列可提高Ｇａｇ蛋白的表达量;（５）Ｕ５区及下游非翻译序列不影响Ｇａｇ蛋白的表达。     

衔接重复序列对酵母菌絮凝蛋白功能的调控作用

【目的】了解絮凝基因FLO1中重复DNA序列B和D对絮凝蛋白Flo1p功能的影响,为构建遗传稳定的最小絮凝功能基因奠定理论基础。【方法】通过PCR和融合PCR方法分别克隆到完整的絮凝基因FLO1、重复DNA序列B和D分别缺失的衍生基因FLO1b和FLO1d,分析这些基因在非絮凝酵母中表达对细胞絮凝特性的影响。【结果】与完整絮凝基因相比,重复DNA序列B和D分别缺失后对酵母细胞絮凝强度没有明显影响,但不同基因在酵母菌中表达产生的絮凝特性受环境因素,如甘露糖浓度和pH等的影响有明显差异。FLO1中重复DNA序列B和D缺失后,细胞絮凝特性受甘露糖抑制的敏感性降低;同时对环境pH的改变具有更广泛的适应性。【结论】重复DNA序列B和D对絮凝蛋白Flo1p结构和功能具有调控作用,二者缺失后,特别是D缺失后会使絮凝蛋白在极端酸碱环境下更稳定。     

水稻锚蛋白基因家族分析和锚定膜蛋白的表达模式研究

锚蛋白重复序列模体是生物体内最普遍的蛋白质序列模体之一,在多种细胞活动中主要介导蛋白质与蛋白质的相互作用。采用生物信息学的方法,在基因组水平上搜索和鉴定了水稻锚定重复序列蛋白,通过分析蛋白质二级结构,进行水稻锚蛋白基因家族的聚类分析,并在此基础上,用RT-PCR的方法分析水稻锚定膜蛋白的表达模式,为水稻锚蛋白基因的研究提供了理论依据。     

发育调控信号蛋白Hh的研究进展

发育调控信号蛋白Ｈｈ的研究进展邱嵘（解放军兰州医学高等专科学校,７３００２０）洪水根（厦门大学细胞生物学研究室,３６１００５）Ｈｈ蛋白是发育调控基因ｈｅｄｇｅｈｏｇ（ｈｈ）的产物。ｈｈ基因是由Ｎｉｓｓｌｅｉｎ—ｖｏｌｈａｒｄ和Ｗｉｅｓｃｈａｕｓ（１９...     

Biophysical characterisation of the small ankyrin repeat protein myotrophin

The 118 residue protein myotrophin is composed of four ankyrin repeats that stack linearly to form an elongated, predominantly α-helical structure. The protein folds via a two-state mechanism at equilibrium. The free energy change of unfolding in water (ΔG_U-N^H₂O) is 5.8 kcal.mol⁻¹. The chevron plot reveals that the folding reaction has a broad energy barrier and that it conforms to a two-state mechanism. The rate of folding in water (k_f^H₂O) of 95 s⁻¹ is several orders of magnitude slower than the value predicted by topological calculations. Proline mutants were used to show that the minor kinetic phases observed for myotrophin arise from heterogeneity of the ground states due to cis-trans isomerisation of prolyl as well as non-prolyl peptide bonds. Myotrophin is the first example of a naturally occurring ankyrin repeat protein that conforms to an apparent two-state mechanism at equilibrium and under kinetic conditions, making it highly suitable for high resolution protein folding studies.     

Folding of a designed simple ankyrin repeat protein

Ankyrin repeats (AR) are 33-residue motifs containing a beta-turn, followed by two alpha-helices connected by a loop. AR occur in tandem arrangements and stack side-by-side to form elongated domains involved in very different cellular tasks. Recently, consensus libraries of AR repeats were constructed. Protein E1_5 represents a member of the shortest library, and consists of only a single consensus repeat flanked by designed N- and C-terminal capping repeats. Here we present a biophysical characterization of this AR domain. The protein is compactly folded, as judged from the heat capacity of the native state and from the specific unfolding enthalpy and entropy. From spectroscopic data, thermal and urea-induced unfolding can be modeled by a two-state transition. However, scanning calorimetry experiments reveal a deviation from the two-state behavior at elevated temperatures. Folding and unfolding at 5 degrees C both follow monoexponential kinetics with k(folding) = 28 sec(-1) and k(unfolding) = 0.9 sec(-1). Kinetic and equilibrium unfolding parameters at 5 degrees C agree very well. We conclude that E1_5 folds in a simple two-state manner at low temperatures while equilibrium intermediates become populated at higher temperatures. A chevron-plot analysis indicates that the protein traverses a very compact transition state along the folding/unfolding pathway. This work demonstrates that a designed minimal ankyrin repeat protein has the thermodynamic and kinetic properties of a compactly folded protein, and explains the favorable properties of the consensus framework.     

The ankyrin repeat as molecular architecture for protein recognition

The ankyrin repeat is one of the most frequently observed amino acid motifs in protein databases. This protein-protein interaction module is involved in a diverse set of cellular functions, and consequently, defects in ankyrin repeat proteins have been found in a number of human diseases. Recent biophysical, crystallographic, and NMR studies have been used to measure the stability and define the various topological features of this motif in an effort to understand the structural basis of ankyrin repeat-mediated protein-protein interactions. Characterization of the folding and assembly pathways suggests that ankyrin repeat domains generally undergo a two-state folding transition despite their modular structure. Also, the large number of available sequences has allowed the ankyrin repeat to be used as a template for consensus-based protein design. Such projects have been successful in revealing positions responsible for structure and function in the ankyrin repeat as well as creating a potential universal scaffold for molecular recognition.     

Crystal structure of a consensus-designed ankyrin repeat protein: implications for stability

Consensus-designed ankyrin repeat (AR) proteins are thermodynamically very stable. The structural analysis of the designed AR protein E3_5 revealed that this stability is due to a regular fold with highly conserved structural motifs and H-bonding networks. However, the designed AR protein E3_19 exhibits a significantly lower stability than E3_5 (9.6 vs. 14.8 kcal/mol), despite 88% sequence identity. To investigate the structural correlations of this stability difference between E3_5 and E3_19, we determined the crystal structure of E3_19 at 1.9 A resolution. E3_19 as well has a regular AR domain fold with the characteristic H-bonding patterns. All structural features of the E3_5 and E3_19 molecules appear to be virtually identical (RMSD(Calpha) approximately 0.7 A). However, clear differences are observed in the surface charge distribution of the two AR proteins. E3_19 features clusters of charged residues and more exposed hydrophobic residues than E3_5. The atomic coordinates of E3_19 have been deposited in the Protein Data Bank. PDB ID: 2BKG.     

Stabilizing ionic interactions in a full-consensus ankyrin repeat protein

Full-consensus designed ankyrin repeat proteins (DARPins), in which randomized positions of the previously described DARPin library have been fixed, are characterized. They show exceptionally high thermodynamic stabilities, even when compared to members of consensus DARPin libraries and even more so when compared to naturally occurring ankyrin repeat proteins. We determined the crystal structure of a full-consensus DARPin, containing an N-capping repeat, three identical internal repeats and a C-capping repeat at 2.05 Å resolution, and compared its structure with that of the related DARPin library members E3_5 and E3_19. This structural comparison suggests that primarily salt bridges on the surface, which arrange in a network with almost crystal-like regularity, increase thermostability in the full-consensus NI₃C DARPin to make it resistant to boiling. In the crystal structure, three sulfate ions complement this network. Thermal denaturation experiments in guanidine hydrochloride directly indicate a contribution of sulfate binding to the stability, providing further evidence for the stabilizing effect of surface-exposed electrostatic interactions and regular charge networks. The charged residues at the place of randomized residues in the DARPin libraries were selected based on sequence statistics and suggested that the charge interaction network is a hidden design feature of this protein family. Ankyrins can therefore use design principles from proteins of thermophilic organisms and reach at least similar stabilities.     

Equilibrium folding and stability of myotrophin: a model ankyrin repeat protein

Proteins containing stretches of repeating amino acid sequences are prevalent throughout nature, yet little is known about the general folding and assembly mechanisms of these systems. Here we propose myotrophin as a model system to study the folding of ankyrin repeat proteins. Myotrophin is folded over a large pH range and is soluble at high concentrations. Thermal and urea denaturation studies show that the protein displays cooperative two-state folding properties despite its modular nature. Taken together with previous studies on other ankyrin repeat proteins, our data suggest that the two-state folding pathway may be characteristic of ankyrin repeat proteins and other integrated alpha-helical repeat proteins in general.     

A binding free energy hot spot in the ankyrin repeat protein GABPbeta mediated protein-protein interaction

The frequently observed ankyrin repeat motif represents a structural scaffold evolved for mediating protein-protein interactions. As such, these repeats modulate a diverse range of cellular functions. We thermodynamically characterized the heterodimeric GA-binding protein (GABP) alphabeta complex and focused specifically on the interaction mediated by the ankyrin repeat domain of the GABPbeta. Our isothermal titration calorimetric analysis of the interaction between the GABP subunits determined an association constant (K(A)) of 6.0 x 10(8) M(-1) and that the association is favorably driven by a significant change in enthalpy (DeltaH) and a minor change in entropy (-TDeltaS). A total of 16 GABPbeta interface residues were chosen for alanine scanning mutagenesis. The calorimetrically measured differences in the free energy of binding were compared to computationally calculated values resulting in a correlation coefficient r = 0.71. We identified three spatially contiguous hydrophobic and aromatic residues that form a binding free energy hot spot (DeltaDeltaG > 2.0 kcal/mol). One residue provides structural support to the hot spot residues. Three non-hot spot residues are intermediate contributors (DeltaDeltaG approximately 1.0 kcal/mol) and create a canopy-like structure over the hot spot residues to possibly occlude solvent and orientate the subunits. The remaining interface residues are located peripherally and have weak contributions. Finally, our mutational analysis revealed a significant entropy-enthalpy compensation for this interaction.     

Folding and unfolding mechanism of highly stable full-consensus ankyrin repeat proteins

Full-consensus designed ankyrin repeat proteins were designed with one to six identical repeats flanked by capping repeats. These proteins express well in Escherichia coli as soluble monomers. Compared to our previously described designed ankyrin repeat protein library, randomized positions have now been fixed according to sequence statistics and structural considerations. Their stability increases with length and is even higher than that of library members, and those with more than three internal repeats are resistant to denaturation by boiling or guanidine hydrochloride. Full denaturation requires their heating in 5 M guanidine hydrochloride. The folding and unfolding kinetics of the proteins with up to three internal repeats were analyzed, as the other proteins could not be denatured. Folding is monophasic, with a rate that is nearly identical for all proteins (∼ 400-800 s^− 1), indicating that essentially the same transition state must be crossed, possibly the folding of a single repeat. In contrast, the unfolding rate decreases by a factor of about 10⁴ with increasing repeat number, directly reflecting thermodynamic stability in these extraordinarily slow denaturation rates. The number of unfolding phases also increases with repeat number. We analyzed the folding thermodynamics and kinetics both by classical two-state and three-state cooperative models and by an Ising-like model, where repeats are considered as two-state folding units that can be stabilized by interacting with their folded nearest neighbors. This Ising model globally describes both equilibrium and kinetic data very well and allows for a detailed explanation of the ankyrin repeat protein folding mechanism.     

Crystal structure of the human TRPV2 channel ankyrin repeat domain

TRPV channels are important polymodal integrators of noxious stimuli mediating thermosensation and nociception. An ankyrin repeat domain (ARD), which is a common protein-protein recognition domain, is conserved in the N-terminal intracellular domain of all TRPV channels and predicted to contain three to four ankyrin repeats. Here we report the first structure from the TRPV channel subfamily, a 1.7 A resolution crystal structure of the human TRPV2 ARD. Our crystal structure reveals a six ankyrin repeat stack with multiple insertions in each repeat generating several unique features compared with a canonical ARD. The surface typically used for ligand recognition, the ankyrin groove, contains extended loops with an exposed hydrophobic patch and a prominent kink resulting from a large rotational shift of the last two repeats. The TRPV2 ARD provides the first structural insight into a domain that coordinates nociceptive sensory transduction and is likely to be a prototype for other TRPV channel ARDs.