核糖核酸酶基因超家族分子进化

核糖核酸酶基因(Ribonuclease A, RNASE A)超家族是进化生物学中研究新基因起源及新功能演变的重要模式系统之一。RNASE A超家族中的很多成员表现出基因复制的进化模式, 而且在适应性(正)选择的驱动下, 发生了功能分化。文章综述了RNASE A超家族成员在不同动物类群中进化模式的研究进展, 包括近年来越来越多在基因组水平上开展的相关研究, 显示该基因超家族可能具有比人们以往认识的更为复杂的基因进化模式。随着越来越多动物基因组数据的产生, 对更多动物代表类群进行RNASE A超家族研究, 将有望揭示新的进化机制和功能分化, 为系统认识动物适应进化的遗传机制奠定基础。     

GAGE基因家族的分子进化特征的研究

GAGE基因通常表达于睾丸组织和部分恶性肿瘤组织中, 被认为可能是理想的癌症诊断的标记和治疗的靶位。我们对GAGE基因家族作了生物信息学分析, 发现它们在X染色体上串联成簇排列, 为灵长类所独有, 各拷贝序列趋异度很低。在人类有15个以上的拷贝, 在黑猩猩和猕猴分别有3个和4个。对GAGE基因家族构建进化树, 并估算了复制事件发生的时间, 结果显示在近400万年内陆续发生。用两种方法计算了GAGE各拷贝间的Ka/Ks值, 结果为显著大于1, 表明该基因家族受到正选择作用。这些结果提示该基因可能与灵长类的特征有关, 其在进化上的地位和在配子发育和肿瘤发生中承担的功能值得深入研究。     

胰脏核糖核酸酶的生理功能和分子结构与进化

各种生物中广泛存在核糖核酸酶(RNase).自1939年Kunitz.首次得到结晶的牛胰脏RNase以来,生物化学家开始对动物胰脏RNase进行多方面的研究.首先是因为胰脏RNase是研究RNA结构的一种重要工具酶.其次,比较胰脏RNase分子中氨基酸的异同和性质,也是深入了解蛋白质结构与功能关系的重要手段,另外,研究进化地位不同的动物     

啮齿目核糖酸酶5基因(RNase5)的分子进化

RNase5是RNASE A基因超家族中的一个重要成员,是分子进化研究的理想模型之一。基于基因组水平,我们对啮齿目的3个进化枝10科17个物种开展RNase5的分子进化研究。利用TBlastN及BlastN方法鉴定每个基因组的RNase5基因,发现该基因在啮齿目的Ctenohystrica所有物种发生丢失,时间是在Ctenohystrica形成之后;邻接法和最大似然法构建的系统发育树均支持RNase5在“与小家鼠相关的进化枝”的小家鼠、褐家鼠和拉布拉多白足鼠发生三次独立基因复制事件;利用PAML软件的枝模型、位点模型及枝-位点模型计算选择压力,均检测到RNase5基因受到强烈的正选择作用。总之,我们的研究深入系统开展了RNase5在啮齿目中的分子进化,增加了该基因研究的多样性,为进一步系统认识该基因在动物的适应性进化遗传机制奠定了基础。     

栝楼核糖核酸酶的性质研究

关于栝楼核糖核酸酶的性质已经研究.它对核糖核酸和多聚尿嘧喧核苷酸所显示的酶活性,有很大的相似性:(1)栝楼核糖核酸酶的最适度应pH在5左右;(2)稳定的pH范围在5-12之间;(3)最佳反应温度为55℃左右;(4) 热稳定性:在15分钟内,60℃以下酶活性基本稳定,在65℃左右时大约有50%的活性丧失;(5)金属离子中,除CU~(2+)和Ag~+有明显的抑制作用外、一般作用不明显.另外,酸对其它合成的多聚核苷酸的水解作用,显示有碱基特异性;对双链RNA(CPV RNA)也显有活性.实验中未发现该酶有磷酸单酯酶的活性及降解DNA的活性.因此,该酶可能是一种具有碱基特异性的核糖核酸酶.     

宫颈癌患者核糖核酸酶抑制因子基因突变的分析

目的通过对宫颈癌患者血液核糖核酸酶抑制因子(ribonuclease inhibitor,RI)基因RNH的突变分析,探讨RNH基因与肿瘤生长的关系。方法针对RNH全基因序列设计21对引物,PCR扩增后采用SSCP对突变进行检测。结果正常人和宫颈癌患者均扩增出相应的21个条带,未发现异常;经过SSCP分析未发现所收集的18例宫颈癌患者血液中的RNH基因有突变发生。结论尚不能肯定RNH基因的突变是否与肿瘤无关,也不能肯定其他肿瘤没有发生突变。需缩小检测片段进一步实验。     

植物颗粒结合淀粉合酶GBSS基因家族的进化

为全面理解植物颗粒结合淀粉合酶(GBSS)基因在植物中的进化模式并重建其进化历史, 利用20种陆生植物和2种藻类植物的基因组数据, 通过生物信息学手段, 深入挖掘和分析植物类群基因组中GBSS基因家族的构成和基因特点, 推测其可能的扩增和丢失规律。结果共识别42条同源序列。系统发育和进化分析表明, GBSS基因起源古老, 可能在所有绿色植物的祖先中就已经出现, 之后在进化过程中不断发生谱系的特异扩张和拷贝丢失, 并最终通过功能分化的形式在植物类群中被固定。     

原核生物核糖核酸酶HII基因的克隆及在大肠杆菌中的高效表达

核糖核酸酶HII (RNaseHII)能有效降解RNA和DNA杂交链中的RNA链。为进一步研究其功能 ,利用大肠杆菌XL1blue为模板 ,相应的寡聚脱氧核苷酸为引物 ,PCR扩增大肠杆菌RNaseHII(rnh 2 )基因 ,并将目的基因连接到克隆载体 pUC18上 ,经测序确认无误 ,分别亚克隆到能够进行IPTG诱导的表达载体pTrcHisC和进行温度诱导的表达载体pBV2 2 0上。重组质粒转化到大肠杆菌DH5α细胞中获得高效表达。在载体pTrcHisC和 pBV2 2 0中目的蛋白RNaseHII的表达量均超过菌体总蛋白的 2 0 % ,且表达产物以稳定的包涵体形式存在。此项工作为以后目的蛋白的纯化提供了有利条件 ,并为研究其结构和功能奠定了基础。     

物分子系统发育与进化研究的基因选择、引物设计与应用策略

引物选择、设计与应用策略是植物分子系统发育与进化研究的关键环节。本文综述了基因选择的原则、引物设计的技巧以及如何有效地利用所涉及的片段获取相应的PCR片段的方法。     

Episodic Molecular Evolution of Pituitary Growth Hormone in Cetartiodactyla

The sequence of growth hormone (GH) is generally strongly conserved in mammals, but episodes of rapid change occurred during the evolution of primates and artiodactyls, when the rate of GH evolution apparently increased substantially. As a result the sequences of higher primate and ruminant GHs differ markedly from sequences of other mammalian GHs. In order to increase knowledge of GH evolution in Cetartiodactyla (Artiodactyla plus Cetacea) we have cloned and characterized GH genes from camel (Camelus dromedarius), hippopotamus (Hippopotamus amphibius), and giraffe (Giraffa camelopardalis), using genomic DNA and a polymerase chain reaction technique. As in other mammals, these GH genes comprise five exons and four introns. Two very similar GH gene sequences (encoding identical proteins) were found in each of hippopotamus and giraffe. The deduced sequence for the mature hippopotamus GH is identical to that of dolphin, in accord with current ideas of a close relationship between Cetacea and Hippopotamidae. The sequence of camel GH is identical to that reported previously for alpaca GH. The sequence of giraffe GH is very similar to that of other ruminants but differs from that of nonruminant cetartiodactyls at about 18 residues. The results demonstrate that the apparent burst of rapid evolution of GH occurred largely after the separation of the line leading to ruminants from other cetartiodactyls.     

Molecular evolution of growth hormone gene family in old world monkeys and hominoids

Growth hormone is a classic molecule in the study of the molecular clock hypothesis as it exhibits a relatively constant rate of evolution in most mammalian orders except primates and artiodactyls, where dramatically enhanced rate of evolution (25–50-fold) has been reported. The rapid evolution of primate growth hormone occurred after the divergence of tarsiers and simians, but before the separation of old world monkeys (OWM) from new world monkeys (NWM). Interestingly, this event of rapid sequence evolution coincided with multiple duplications of the growth hormone gene, suggesting gene duplication as a possible cause of the accelerated sequence evolution. Here we determined 21 different GH-like sequences from four species of OWM and hominoids. Combining with published sequences from OWM and hominoids, our analysis demonstrates that multiple gene duplications and several gene conversion events both occurred in the evolutionary history of this gene family in OWM/hominoids. The episode of recent duplications of CSH-like genes in gibbon is accompanied with rapid sequence evolution likely resulting from relaxation of purifying selection. GHN genes in both hominoids and OWM are under strong purifying selection. In contrast, CSH genes in both lineages are probably not. GHV genes in OWM and hominoids evolved at different evolutionary rates and underwent different selective constraints. Our results disclosed the complex history of the primate growth hormone gene family and raised intriguing questions on the consequences of these evolutionary events.     

The evolution of the RNase P- and RNase MRP-associated RNAs: Phylogenetic analysis and nucleotide substitution rate

We report a detailed evolutionary study of the RNase P- and RNase MRP- associated RNAs. The analyses were performed on all the available complete sequences of RNase MRP (vertebrates, yeast, plant), nuclear RNase P (vertebrates, yeast), and mitochondrial RNase P (yeast) RNAs. For the first time the phylogenetic distance between these sequences and the nucleotide substitution rates have been quantitatively measured. The analyses were performed by considering the optimal multiple alignments obtained mostly by maximizing similarity between primary sequences. RNase P RNA and MRP RNA display evolutionary dynamics following the molecular clock. Both have similar rates and evolve about one order of magnitude faster than the corresponding small rRNA sequences which have been, so far, the most common gene markers used for phylogeny. However, small rRNAs evolve too slowly to solve close phylogenetic relationships such as those between mammals. The quicker rate of RNase P and MRP RNA allowed us to assess phylogenetic relationships between mammals and other vertebrate species and yeast strains. The phylogenetic data obtained with yeasts perfectly agree with those obtained by functional assays, thus demonstrating the potential offered by this approach for laboratory experiments. Correspondence to: E. Sbisà     

Minimal protein-only RNase P structure reveals insights into tRNA precursor recognition and catalysis

Ribonuclease P (RNase P) is an endoribonuclease that catalyzes the processing of the 5′ leader sequence of precursor tRNA (pre-tRNA). Ribonucleoprotein RNase P and protein-only RNase P (PRORP) in eukaryotes have been extensively studied, but the mechanism by which a prokaryotic nuclease recognizes and cleaves pre-tRNA is unclear. To gain insights into this mechanism, we studied homologs of Aquifex RNase P (HARPs), thought to be enzymes of approximately 23 kDa comprising only this nuclease domain. We determined the cryo-EM structure of Aq880, the first identified HARP enzyme. The structure unexpectedly revealed that Aq880 consists of both the nuclease and protruding helical (PrH) domains. Aq880 monomers assemble into a dimer via the PrH domain. Six dimers form a dodecamer with a left-handed one-turn superhelical structure. The structure also revealed that the active site of Aq880 is analogous to that of eukaryotic PRORPs. The pre-tRNA docking model demonstrated that 5′ processing of pre-tRNAs is achieved by two adjacent dimers within the dodecamer. One dimer is responsible for catalysis, and the PrH domains of the other dimer are responsible for pre-tRNA elbow recognition. Our study suggests that HARPs measure an invariant distance from the pre-tRNA elbow to cleave the 5′ leader sequence, which is analogous to the mechanism of eukaryotic PRORPs and the ribonucleoprotein RNase P. Collectively, these findings shed light on how different types of RNase P enzymes utilize the same pre-tRNA processing.     

miR156靶基因SPL在植物中的系统分布和进化模式分析

Squamosa promoterbinding proteinlike genes （SPLs）在植物发育过程中具有重要作用。很多SPLs被miR156调节,然而,对于它们在植物中的系统分布和进化模式还知之甚少。本文对9个测序物种（藻类,苔藓,石松,单子叶和双子叶植物）的183个SPLs进行了生物信息学分析。结果表明miR156应答元件（MREs）仅在陆生植物SPLs中发现,藻类中不存在。系统进化分析显示陆生植物SPLs分为两大分支：group I和group II。 MiR156靶基因仅分布于group II,表明它们有着共同的祖先。Group II进一步分为7个亚支（IIaIIg）,miR156靶基因分布在除IId外的其余6个亚支的特定SPLs。系统分类与基因结构的相关性反映了SPL靶基因结构上的变化。在进化过程中,它们可能发生外显子的丢失且伴随MRE的丢失。另外,基因重复对SPL靶基因的丰度变化影响很大,尤其是被子植物与低等植物分歧后它们数量明显增加。以拟南芥为模式植物分析发现串联重复和片段重复是SPL靶基因扩张的主要机制。     

Analysis of the tertiary structure of bacterial RNase P RNA

The ubiquitous occurrence of ribonuclease P (RNase P) as a ribonucleoprotein and the catalytic properties of bacterial RNase P RNAs indicate that RNA fulfills an ancient and important role in the function of this enzyme. This review focuses on efforts to determine the structure of the bacterial RNase P RNA ribozyme. Phylogenetic comparative analysis of a library of bacterial RNase P RNA sequences has resulted in a well-developed secondary structure model and allowed identification of some elements of tertiary structure. The native structure has been redesigned by circular permutation to facilitate intra- and inter-molecular crosslinking experiments in order to gain further structural information. The crosslinking constraints, together with the constraints provided by comparative analyses, have been incorporated into a first-order model of the structure of the ribozyme-substrate complex. The developing structural perspective allows the design of self-cleaving pre-tRNA-RNase P RNA conjugates which are useful tools for additional structure-probing experiments.Abbreviations cpRNA circularly permuted RNA     

Identification of Nucleotide Residues Essential for RNase P Activity from the Hyperthermophilic Archaeon Pyrococcus horikoshii OT3

Ribonuclease P (RNase P) is involved in the processing of the 5′ leader sequence of precursor tRNA (pre-tRNA). We have found that RNase P RNA (PhopRNA) and five proteins (PhoPop5, PhoRpp21, PhoRpp29, PhoRpp30, and PhoRpp38) reconstitute RNase P activity with enzymatic properties similar to those of the authentic ribozyme from the hyperthermophilic archaeon Pyrococcus horikoshii OT3. We report here that nucleotides A40, A41, and U44 at helix P4, and G269 and G270 located at L15/16 in PhopRNA, are, like the corresponding residues in Esherichia coli RNase P RNA (M1RNA), involved in hydrolysis by coordinating catalytic Mg²⁺ ions, and in the recognition of the acceptor end (CCA) of pre-tRNA by base-pairing, respectively. The information reported here strongly suggests that PhopRNA catalyzes the hydrolysis of pre-tRNA in approximately the same manner as eubacterial RNase P RNAs, even though it has no enzymatic activity in the absence of the proteins.     

Molecular evolution and functional specialization of chalcone synthase superfamily from Phalaenopsis Orchid

Plant genomes appear to exploit the process of gene duplication as a primary means of acquiring biochemical and developmental flexibility. The best example is the gene encoding chalcone synthase (CHS, EC2.3.1.74), the first committed step in flavonoid biosynthesis. In this study, we examined the molecular evolution of three CHS family members of Phalaenopsis including a novel chs gene (phchs5), which is slowly evolved. The inferred phylogeny of the chs genes of Phalaenopsis with other two orchid plants, Bromoheadia finlaysoniana and Dendrobium hybrid, suggested that gene duplication and divergence have occurred before divergence of these three genera. Relatively quantitative RT-PCR analysis identified expression patterns of these three chs genes in different floral tissues at different developmental stages. Phchs5 was the most abundantly expressed chs gene in floral organs and it was specifically transcribed in petal and lip at the stages when anthocyanin accumulated (stage1–4). Phchs3 and phchs4 were expressed at much lower levels than phchs5. Phchs3 was expressed in pigmented tissue (including lip, petal and sepal) at middle stages (stages 2–4) and in colorless reproductive tissue at late stage (stage 5). Phchs4 was only expressed in petal at earlier stages (stage 1–3) and in lip at middle stage (stage 4). These results present new data on differentiation of gene expression among duplicate copies of chs genes in Phalaenopsis.