乙肝相关性肝癌中新基因CHCHD2的生物信息学分析

应用基因芯片技术获取以稳定转染HBx基因的肝癌细胞HepG2(HepG2-X)及非转染的肝癌细胞HepG2的差异表达的基因,利用生物信息学方法对新基因CHCHD2进行初步分析表明,该蛋白开放性读码框长456 bp,编码151个氨基酸残基,相对分子量为15.55 kD,等电点9.43,是主要定位于线粒体中的亲水性蛋白,二级结构均以α-螺旋和无规则卷曲为主要构件。同源性比较分析结果表明,其碱基序列与已经报道的其他17个物种相似性为64%-99%,且符合种属之间的进化关系。     

草鱼AMBP基因cDNA的克隆与序列分析

α1-微球蛋白和Bikunin是由同一基因翻译表达出的两种功能不相关联的血浆蛋白。本文通过快速扩增cDNA末端的方法,首次从草鱼肝脏组织克隆了α1-微球蛋白和Bikunin前体蛋白(α1-microglobulin/Bulinin precursor, AMBP）基因全长cDNA。其cDNA全长1230bp,包含5′非翻译区23 bp,3′非翻译区160 bp和开放读码框1047 bp。开放读码框编码348个氨基酸,包含182个氨基酸的α1-微球蛋白和145个氨基酸的Bikunin。草鱼AMBP与其他物种的氨基酸序列分析结果表明,它们具有较高的同源性(44.7%－84.4%),其中草鱼与斑马鱼同源性最高(84.4%)。结果表明AMBP序列结构和α1-微球蛋白与Bikunin共翻译表达特点在动物机体中具有着重要的生理意义。     

通过新基因计算机识别与实验确认对NCBI人类基因数据库一些模式参考序列错误的分析与纠正

采用生物信息学分析与实验确认相结合的技术路线,通过所识别的基因在非冗余数据库比对发现了网上公布的计算机注释人类基因组编码序列存在各种类型的多处错误,包括cDNA水平的一个或一段碱基插入、缺失或突变,或是这些错误的不同排列组合,其中以错误插入为多,往往导致编码氨基酸的移码突变。最先举证了NCBIGENOME Annotation Project预测人类新基因的下列错误类型：(1)开放读码框架(0RF)中错误插入一个碱基造成编码氨基酸移码;(2)错误拼接;(3)开放读框中错误插入一个或一段碱基造成该读框提前终止。只编码N-端氨基酸的cDNA序列而不完整;(4)只有编码c一端氨基酸序列的cDNA而不完整;(5)只是正确基因0RF中间的一段编码蛋白cDNA序列而不完整,缺N-端与C-端氨基酸序列,并且将不完整蛋白氨基酸序列的第一个非起始码氨基酸错误地预测为起始码氨基酸,如将L错误地预测为M;(6)开放读框中错误插入一个或一段碱基造成前面出现不该有的终止码,因而编码蛋白缺开头部分氨基酸;(7)可能将污染基因组序列当作完整基因cDNA序列对待而预测出所谓单一外显子基因。即便真是基因,也只是较长单一外显子mRNA中有一小0RF,而0RF起始码上游同一相位确实存在终止码,无其他特点符合基因条件;(8)所预测基因只有0RF,而0RF两端没有任何EST证据,可据此0RF拼接出受EST和人类基因组双重支持的完整基因cDNA(开放读框上游同一相位有终止码),预示所预测0RF参考序列可能不正确;(9)有EST实验证据支持存在基因的人类基因组序列范围内又被预测出一条相似但更小的蛋白编码基因,因而新预测基因有可能是错误的。     

SARS-CoV 基因组编码蛋白研究进展

严重急性呼吸综合征(severe acute respiratory syndrome,SARS)2002年底暴发于中国广东,后蔓延至全球的传染性疾病．其病原体为一种新型的未知冠状病毒,基因组长度约30 kb,预测具有14个开放读码框．至今为止,对 SARS 冠状病毒(SARS-COV)基因组编码蛋白质的研究已取得显著进展,其研究主要集中在复制酶 1a/1b、结构蛋白及“附属”蛋白(SARS-CoV 特异性蛋白)结构与功能的研究．以 SARS-CoV 的蛋白组成及功能研究为主要内容,系统介绍了 SARS-CoV 蛋白质研究进展．     

丹参非特异性脂质转移蛋白基因（SmLTP1）的克隆及其表达分析

对丹参EST序列进行Blast分析,获得一个新的非特异性脂质转移蛋白基因,命名为SmLTP1（GenBank注册号为EF187461）。该基因cDNA全长593bp,包含一个长为357bp的开放读码框,编码118个氨基酸。生物信息学结构分析表明,该蛋白具有植物nsLTP的典型结构,即4对二硫键,4个a-螺旋,1个可结合和容纳脂肪酸分子的类似口袋状的疏水结构。实时荧光定量PCR分析结果表明,SmLTP1基因在丹参不同组织器官中差异表达,其表达受病原菌和茉莉酸甲酯的诱导,显示SmLTP1基因在植物防御反应中发挥作用。     

小桐子非特异性脂质转移蛋白A的基因克隆及其表达和功能分析

基于我们最近获得的小桐子低温驯化转录组和数字基因表达谱数据,本工作研究了低温驯化条件下差异表达变化较大的非特异性脂质转移蛋白A基因JcnsLTPA。克隆到该基因的cDNA序列全长833 bp,开放阅读框长度513 bp,编码170个氨基酸,存在ATT_LTSS典型保守功能基序。其启动子区域中鉴定到了TATA框、CAAT框、CATA框、W框等顺式作用元件以及CRT/DRE低温响应元件。半定量RT-PCR分析表明,该基因在茎、根、叶中都有表达,以茎中表达量最高、且受低温诱导最显著。同时,酵母表达JcnsLTPA也提高了重组酵母菌的抗低温能力。这些结果充分说明了小桐子JcnsLTPA是与抗冷性密切相关的基因,可以用于小桐子的抗冷性遗传改良。     

烟草FIE和MSI1基因的克隆及序列分析

采用电子克隆与实验克隆结合的方法获得了烟草胚乳发育相关基因NTFIE和NTMSI1的cDNA序列,序列号分别为EU375458和EU375459.序列分析结果表明,这两个cDNA序列均含有完整的开放读码框,分别编码370和424个氨基酸,含有保守的WD基序.氨基酸序列比对和系统发育分析结果显示,不同物种之间FIE和MSI1基因编码氨基酸序列同源性都较高.组织表达分析结果表明,这两个基因均具有一定程度的组织表达特异性,NTFIE cDNA基因在花中的表达量最多,但在根和茎中未检测到表达,而NTMSI1 cDNA基因只在离体培养的细胞和根中特异性表达.     

斜纹夜蛾核型多角体病毒BamHI—J片段序列分析

报道了斜纹夜蛾核型多角体病毒（SpltMNPV)BamHI-J片段的序列结构。该片段定位于SpltMNPV基因组25.8-29.9图单位（msp unit）,包括4个完整的开放读码框,几丁质酶基因（chiA）的3′端部分序列和一个同源区（hr）的部分序列。4个完整的读码框包括lef-8基因,杆状病毒J结构域蛋白基因（baculovirus J domain protein gene,bjdp),ORF570和ORF165。序列分离表明：ORF570与毒蛾核型多角体病毒（Lymantria dispar MNPV）的解旋酶－2基因有31％的氨基酸同源性。ORF165为SpltMNPV特有。J结构域蛋白在其他杆状病毒基因组中尚未见报道,其氨基酸序列N端存在J结构域,推断该蛋白质具有与DnaJ蛋白类似特征。lef-8基因编码的氨基酸与已报道的杆状病毒基因组中的lef-8基因编码的氨基酸具有高的同源性,且其C端具有与其他杆状病毒LEF－8类似的保守序列CIKICGIHGQKG。     

基于结构的单绕蛋白聚类图构建与分析

蛋白质分子进化规律研究是分子进化研究的重点,对揭示生命起源与进化机制有重要意义。本文对已知空间结构及物种信息的单绕蛋白,利用结构比对信息,构建了不同层次单绕样本系统聚类图。分析发现:功能相似蛋白存在明显聚集现象,同一超家族样本基本聚在一个大支中,同一家族样本集中在所属超家族下的小支中,功能约束下单绕样本聚类图与物种进化图有较好对应关系。结果表明:单绕蛋白的结构演化反映了蛋白质功能的约束,特定功能单绕样本的结构差异具有种属特异性,结构演化包含了物种进化信息。     

小黑杨环锌指蛋白基因的克隆与表达分析

用cDNA-AFLP技术从小黑杨中克隆与盐胁迫反应相关的cDNA片段,进一步应用RACE技术克隆出具有完整开放读码框的小黑杨环锌指蛋白基因（PsnRZF）,该基因全长1061bp,其中5非翻译区为184bp,3非翻译区为82bp,开放读码框为795bp,编码264个氨基酸,预测蛋白的分子量为30.25kDa,理论等电点为8.04。实时定量PCR检测的结果显示,正常生长条件下该基因在根、茎、叶中都表达;NaCl胁迫下,该基因在根、茎、叶中的表达量升高。在叶中的表达量随着处理时间的延长而逐渐升高,胁迫处理后第6天表达量达到最高。     

小蛋白质富集法鉴定酿酒酵母“漏检蛋白”

小蛋白质 (Small proteins,SPs) 是由小开放阅读框 (Short open reading frames,sORFs) 编码长度小于100个氨基酸的多肽。研究发现小蛋白质参与了基因表达调控、细胞信号转导和代谢等重要生物学过程。然而,生命体中大多数的已注释小蛋白质尚缺少蛋白水平存在的实验证据,被称为漏检蛋白 (Missing proteins,MPs)。小蛋白质的高效鉴定是其功能研究的前提,也有助于挖掘“漏检蛋白”。文中采用小蛋白质富集策略鉴定到72个酵母小蛋白质,验证9个“漏检蛋白”,发现低分子量、高疏水性、膜结合、弱密码子使用偏性及不稳定性是蛋白漏检的主要原因,对进一步的技术优化具有指导意义。     

植物小开放阅读框编码肽的研究进展

小开放阅读框（small open reading frame,sORF）一般指基因组中能够编码长度在100个氨基酸左右或以内短肽的开放阅读框。它们广泛存在于植物基因组,却因编码短肽而常被基因组注释忽视。随着翻译组学和蛋白质组学测序技术的发展,具有翻译活性的sORF被证实广泛存在于植物基因组,且参与植物生长发育等重要过程的调控。该文归纳了近些年来植物领域sORF的一些研究进展,主要包括sORF的来源与分类、信息学预测方法和生物学功能等,并基于此对植物sORF未来的研究方向进行了展望。     

SmProt: A Reliable Repository with Comprehensive Annotation of Small Proteins Identified from Ribosome Profiling

Small proteins specifically refer to proteins consisting of less than 100 amino acids translated from small open reading frames (sORFs), which were usually missed in previous genome annotation. The significance of small proteins has been revealed in current years, along with the discovery of their diverse functions. However, systematic annotation of small proteins is still insufficient. SmProt was specially developed to provide valuable information on small proteins for scientific community. Here we present the update of SmProt, which emphasizes reliability of translated sORFs, genetic variants in translated sORFs, disease-specific sORF translation events or sequences, and remarkably increased data volume. More components such as non-ATG translation initiation, function, and new sources are also included. SmProt incorporated 638,958 unique small proteins curated from 3,165,229 primary records, which were computationally predicted from 419 ribosome profiling (Ribo-seq) datasets or collected from literature and other sources from 370 cell lines or tissues in 8 species (Homo sapiens, Mus musculus, Rattus norvegicus, Drosophila melanogaster, Danio rerio, Saccharomyces cerevisiae, Caenorhabditis elegans, and Escherichia coli). In addition, small protein families identified from human microbiomes were also collected. All datasets in SmProt are free to access, and available for browse, search, and bulk downloads at http://bigdata.ibp.ac.cn/SmProt/.     

Uncovering small membrane proteins in pathogenic bacteria: Regulatory functions and therapeutic potential

Bacterial small proteins (below 50 amino acids) encoded by small open reading frames (sORFs) are recognized as an emerging class of functional molecules that have been largely overlooked in the past. While some were uncovered serendipitously, global approaches have recently been developed to detect these sORFs. A large portion of small proteins appears to be hydrophobic and located in the bacterial membrane. In the present review, we describe functional small hydrophobic proteins discovered in pathogenic bacteria and report recent advances in the discovery of additional ones. Small membrane proteins contribute to bacterial adaptation to changing environments and often appear to be implicated in negative feedback regulation loops by modulating the function or stability of larger membrane proteins. A subset of these proteins belongs to toxin-antitoxin modules. We highlight the features of characterized hydrophobic small proteins that may pave the way for identification of the functional small proteins among novel sORFs discovered. Besides providing new insights into bacterial pathogenesis, identification of naturally occurring small hydrophobic proteins of pathogenic bacteria can lead to new therapeutic interventions, as recently shown with the development of synthetic peptides derived from natural small proteins that display antibacterial or antivirulence properties.     

Long signal peptides of RGMa and DCBLD2 are dissectible into subdomains according to the NtraC model

Targeting of proteins to the endoplasmic reticulum (ER) usually requires N-terminal signal peptides (SP) of approximately 22 amino acids in length. However, a substantial number of proteins contain exceptionally long SPs of 40 amino acids and more, an example being protein shrew-1/AJAP1. Using shrew-1's SP as example, the NtraC model has been developed by dissecting long SPs into two functionally distinct subdomains ("N" and "C") separated by a β-turn rich transition area ("tra"). Further proteins have been identified by computational analysis complying with the NtraC model. Here we used the SPs of two of these proteins, DCBLD2 and RGMa (including three isoforms), to show that the NtraC model applies to a growing group of SPs. We demonstrate that the full-length SPs of RGMa and DCBLD2 are functional and furthermore that the C-domains are sufficient and essential for ER targeting, whereas the N-domains are dispensable. Thus, the N-domains are available for additional functions.     

微生物小开放阅读框:小蛋白及翻译调控研究进展

小开放阅读框(small open reading frame, sORF)广泛存在于不同生物基因组中,由于其序列短,以及编码的产物小蛋白(smallprotein,或称微蛋白;microprotein或迷你蛋白miniprotein)检测困难等原因,小开放阅读框长期未得到充分注释和研究。近年来,随着高通量测序、翻译组和质谱分析等技术的不断发展,在不同生物中发现大量新的小开放阅读框,其编码的小蛋白及介导的翻译调控已应用于药物开发及植物抗病机理等研究。但是,目前对微生物的小开放阅读框相关研究和应用还相对有限。本文综述了小开放阅读框编码产物小蛋白的发现和鉴定,以及上游开放阅读框(upstream open reading frame, uORF)对mRNA翻译调控等最新研究进展,重点介绍了微生物基因组中小开放阅读框的鉴定和功能研究进展,为深入认识微生物中小开放阅读框的功能和作用机制,以及植物和动物等高等其他生物的小蛋白和翻译调控相关研究提供参考。     

Geometric differences in the ribosome exit tunnel impact the escape of small nascent proteins

The exit tunnel is the subcompartment of the ribosome that contains the nascent polypeptide chain and, as such, is involved in various vital functions, including regulation of translation and protein folding. As the geometry of the tunnel shows important differences across species, we focus on key geometrical features of eukaryote and prokaryote tunnels. We used a simple coarse-grained molecular dynamics model to study the role of the tunnel geometry in the post-translational escape of short proteins (short open reading frames [sORFs]) with lengths ranging from 6 to 56 amino acids. We found that the probability of escape for prokaryotes is one for all but the 12-mer chains. Moreover, proteins of this length have an extremely low escape probability in eukaryotes. A detailed examination of the associated single trajectories and energy profiles showed that these variations can be explained by the interplay between the protein configurational space and the confinement effects introduced by the constriction sites of the ribosome exit tunnel. For certain lengths, either one or both of the constriction sites can lead to the trapping of the protein in the “pocket” regions preceding these sites. As the distribution of existing sORFs indicates some bias in length that is consistent with our findings, we finally suggest that the constraints imposed by the tunnel geometry have impacted the evolution of sORFs.     

Complete nucleotide sequence of the mitochondrial genome of a salamander, Mertensiella luschani

The complete nucleotide sequence (16,650 bp) of the mitochondrial genome of the salamander Mertensiella luschani (Caudata, Amphibia) was determined. This molecule conforms to the consensus vertebrate mitochondrial gene order. However, it is characterized by a long non-coding intervening sequence with two 124-bp repeats between the tRNA(Thr) and tRNA(Pro) genes. The new sequence data were used to reconstruct a phylogeny of jawed vertebrates. Phylogenetic analyses of all mitochondrial protein-coding genes at the amino acid level recovered a robust vertebrate tree in which lungfishes are the closest living relatives of tetrapods, salamanders and frogs are grouped together to the exclusion of caecilians (the Batrachia hypothesis) in a monophyletic amphibian clade, turtles show diapsid affinities and are placed as sister group of crocodiles+birds, and the marsupials are grouped together with monotremes and basal to placental mammals. The deduced phylogeny was used to characterize the molecular evolution of vertebrate mitochondrial proteins. Amino acid frequencies were analyzed across the main lineages of jawed vertebrates, and leucine and cysteine were found to be the most and least abundant amino acids in mitochondrial proteins, respectively. Patterns of amino acid replacements were conserved among vertebrates. Overall, cartilaginous fishes showed the least variation in amino acid frequencies and replacements. Constancy of rates of evolution among the main lineages of jawed vertebrates was rejected.     

Expression and molecular characterization of spherical particles derived from the genome of the hyperthermophilic euryarchaeote Pyrococcus furiosus

Spherical particles (SPs) of approximately 30 nm in diameter were found in the hyperthermophilic archaeon Pyrococcus furiosus. The SPs contained no nucleic acid and were composed of a single 39-kDa protein. The amino acid sequences of the amino-terminal and internal fragments were identical to portions of the deduced amino acid sequence of the putative 38.7-kDa protein encoded by the genome of P. furiosus, suggesting that the protein was expressed from the genome of P. furiosus. This possibility was confirmed by the observation that the 38.7-kDa protein expressed in Escherichia coli reacted specifically with the antibody against purified SPs, and it also formed SPs similar to those found in P. furiosus. Of the 345 amino acid residues in the 38.7-kDa protein, the amino-terminal 100 amino acids exhibited strong homology to putative proteins from other species of Pyrococcus, while the remaining 245 carboxy-terminal residues were not significantly homologous to putative proteins from other members of archaea. Thus, the carboxy-terminal region might be the product of a foreign gene that was incorporated relatively recently into the genome of P. furiosus.