鳜传染性脾肾坏死病毒主衣壳蛋白基因结构及序列分析

分析了鳜传染性脾肾坏死病毒(infectious spleen and kidney necrosis virus,ISKNV)的主衣壳蛋白(MCP)基因结构及其序列。对ISKNV DNA Kpn I L酶切片段的序列分析结果发现,该序列中含有完整的MCP基因。ISKNV MCP基因完整读码框为1362bp,比含量为56．24％,编码一个长为453aa、分子量为49．61kD、等电点为6．25的推定蛋白。结构分析发现,该基因具有启动子元件TATA框和CAAT基序。根据对虹彩病毒MCP系统进化树和脊椎动物虹彩病毒的生物学特性的分析比较发现,ISKNV、RSIV、SBIV、GIV和ALIV等在养殖海、淡水鱼类中引起其脾、肾、固有层和表皮细胞肿大的虹彩病毒,是独立于蛙病毒属和淋巴囊肿病毒属的又一新脊椎动物虹彩病毒类群。     

水生动物虹彩病毒的分子生物学

近年来在世界范围出现的由虹彩病毒感染引起的水生动物高发病率和高死亡率现象给水产业造成了重大经济损失,引起了病毒学家们的热切关注,根据ICTV第七次报告,虹彩病毒科(Iridoviridae)分为4个属,包括感染脊椎动物的蛙病毒属(Ranavirus)和淋巴囊肿病毒属(Lymphocystivirus),以及感染无脊椎动物的虹彩病毒属(Iridovirus)和绿虹彩病毒属(Chloriridovirus).       

黄鳝cGnRH-Ⅱ的cDNA克隆与序列分析

促性腺激素释放激素（Conadotropin-releasing hormone,GnRH）是一个保守的十肽神经家族激素,在脊椎动物的性腺发育和繁殖功能的维持方面起着重要的调控作用。本文通过运用RACE和RT-PCR方法,从黄鳝脑组织中克隆得到cGnRH-ⅡcDNA全序列,其核苷酸序列长度为617bp。该cDNA编码的cGnRH-Ⅱ的前体氨基酸序列结构组成与其他物种的cGnRH-Ⅱ前体结构一致,其推导的蛋白前体长度为83个氨基酸,包括一个信号肽、cGnRH-Ⅱ十肽和一个由蛋白水解位点（Gly—Lys—Arg）连接的GnRH联接肽,其中信号肽和联接肽的长度分别为21和49个氨基酸。cGnRH-Ⅱ的氨基酸序列和其他相关物种cGnRH-Ⅱ氨基酸序列比较结果显示,cGnRH-Ⅱ cDNA的蛋白编码区高度保守,而非编码区的保守性程度很低。     

大黄鱼虹彩病毒PCR快速检测试剂盒的研制

针对近年来由虹彩病毒所引起的海水养殖鱼类疾病呈日趋严重的态势,该文在证实了引发我国大黄鱼大规模流行病的病原为一种虹彩病毒及测定病毒全基因组序列(111,760bp;GenBank accession numberl．AY779031)的基础上,通过与已报道虹彩病毒核酸序列进行分析比较,结合生物信息学手段,确定了以虹彩病毒ATPase基因保守区序列(295bp)作为扩增靶序列,设计合成了一对特异性引物,通过改进PCR模板的制备方法和优化扩增条件,建立了大黄鱼虹彩病毒PCR快速检测技术,并开发成简便、快速、实用的检测试剂盒,该试剂盒的检测灵敏度相当于30个病毒粒子,模板制备时间约30min、回收率为52％、半个工作日即可得到准确的结果,无非特异性扩增带,适用于大黄鱼虹彩病毒病的早期快速诊断、苗种的检疫及水质环境的监测,目前正在推广应用。     

云斑尖塘鳢肿大细胞病毒属虹彩病毒的分离与鉴定

2009年10月, 广东顺德地区一云斑尖塘鳢养殖场暴发不明病因疾病, 发病尖塘鳢体长15-18 cm不等,死亡率约85%, 濒死尖塘鳢从池塘底层游至水面, 呈现游动失衡状态直至死亡。死亡尖塘鳢腹部膨大,剖检可见肝脏、脾脏、肾脏肿大, 有出血斑点, 从内脏器官肝脏、脾脏和肾脏未分离到致病菌。病鱼内脏组织研磨过滤除菌后,腹腔注射20尾尖塘鳢, 7d后开始出现死亡, 10d后全部死亡, 对照组无死亡。自然发病鱼和人工感染鱼的病理切片显示肝脏、脾脏和肾脏出现大量肿大细胞,超薄切片经电子显微镜观察, 肝脏、脾脏和肾脏观察到大量病毒颗粒。电镜下病毒呈六边形,直径约135 nm,形态与虹彩病毒相似。针对虹彩病毒主衣壳蛋白(Major capsid protein,MCP)序列设计引物,提取自然发病鱼和人工感染鱼的DNA作为模板, 均能扩增出预期大小的特异性产物。利用NCBI的Blast搜索, 结果显示扩增序列与肿大细胞病毒属的传染性脾肾坏死病毒(ISKNV)、闪电丽鱼虹彩病毒(DGIV)和条石鲷虹彩病毒(RBIV)MCP核苷酸序列同源性分别为98.8%、98.1%和94.7%。利用MCP序列构建的系统发育树显示, 导致云斑尖塘鳢发病死亡的病毒为肿大细胞病毒属虹彩病毒, 暂命名云斑尖塘鳢虹彩病毒(MSGIV)。       

棉铃虫核型多角体病毒几丁质酶基因及杆状病毒 几丁质酶基因的分子进化

用PCR方法扩增了棉铃虫Helicoverpa armigera单粒包埋型核型多角体病毒（HaSNPV）几丁质酶基因,测定了基因编码区的核苷酸全序列。基因编码区全长1.713 bp,可编码570个氨基酸残基组成的多肽,预计分子量为63.6 kD。将所推导的HaSNPV几丁质酶氨基酸序列与其它已知杆状病毒几丁质酶氨基酸序列进行联配比较,结果表明HaSNPV 与谷实夜蛾H.zea单粒包埋型核型多角体病毒（HzSNPV）的氨基酸序列非常相似,同源性高达90.7%,与苜蓿丫纹夜蛾Autographa californica多粒包埋型核型多角体病毒（AcMNPV）、家蚕Bombyx mori核型多角体病毒（BmNPV）、美国白蛾Hyphantria cunea核型多角体病毒（HcNPV）、舞毒蛾Lymantria dispar多粒包埋型核型多角体病毒（LdMNPV）、黄杉毒蛾Orgyia pseudotsugata多粒包埋型核型多角体病毒（OpMNPV）和云杉卷叶蛾Choristoneura fumiferana核型多角体病毒（CfMNPV）氨基酸序列同源性分别为64.4%、64.9%、64.2%、62.9%、66.2%和61.5%。根据氨基酸序列用PC＼GENE程序绘制已知杆状病毒几丁质酶的分子系统树,并与杆状病毒中最为保守的多角体蛋白基因系统树作了比较,结果表明几丁质酶基因和多角体蛋白基因的进化速率是不尽相同的。     

鳜肌酸激酶M-CK cDNA的克隆与组织表达分析

利用RT–PCR和cDNA末端快速扩增法（RACE）克隆了鳜（Siniperca chuatsi）肌酸激酶（creatine kinase,CK）cDNA序列,并分析了该基因的结构特征和系统关系。鳜CK cDNA序列全长1586 bp,包括5′端非翻译区92 bp,3′端非翻译区348 bp和开放阅读框（ORF）1 146 bp,共编码381个氨基酸。鳜CK具有脊椎动物CK共有的保守结构域和肌型肌酸激酶（M-CK）同工酶的特异识别位点;氨基酸序列与M-CK型的相似度最高,而与脑型肌酸激酶（B-CK）和线粒体型肌酸激酶（Mi-CKs）的相似度较低;在CK系统关系树中鳜CK与M-CK群聚类。这些均表明,鳜CK属脊椎动物M-CK型。RT-PCR分析表明,鳜M-CK在成体不同组织中的表达量不同,其中,在皮肤、卵巢、肾脏、胃、肌肉和心脏中表达较强;而在眼和脑、肝胰脏中表达较弱。     

鲤鱼线粒体tRNAphe基因结构的特异性

鲤鱼线粒体ｔＲＮＡｐｈｅ基因的核酸序列已被测定。在鲸、人、抓蟾、牛、小鼠、鸡和锂鱼中对此基因序列比较发现在Ｄ茎存在一个奇怪的保守结构,然而Ｄ茎在其余种类的已经测定的脊椎动物线粒体ｔＲＮＡ基因和细胞质ｔＲＮＡ基因中是极不保守的。这一保结构饮食有１３ｂｐ碱基,我们将此保守区前７个碱基与真核生物ＲＮＡＰｏｌⅢ识别的Ａ区要比较, 此不同物种的两种序列存在部分的同源性。考虑到ｔＲＮＡｐｈｅ基因在编码区之间这     

小西葫芦黄化花叶病毒分离物的3′末端序列多态性研究

研究了来自中国大陆9个小西葫芦黄化花叶病毒（ZYMV）分离物的基因组3′末端核苷酸序列及所推导的外壳蛋白（CP）氨基酸序列以及3′末端非编码区（UTR）序列,并与其它地区所报道的16个ZMYV分离物进行了同源性比较。ZYMV CP基因核苷酸序列具有一定的寄主相关性和地域相关性,但总体上其关联程度不明显;同时,CP氨基酸序列的寄主适应性程度明显高于地域相关性。25个ZYMV分离物的CP氨基酸序列根据其变异程度分为2个区： N端约41个氨基酸为高度变异区,CP核心区和C端氨基酸序列为保守区。研究结果初步揭示了ZYMV作为单链RNA病毒通过与寄主相互作用而表现寄主适应性变异的趋势。     

基于第二代测序技术兰州鲇线粒体基因组全序列测定与分析

研究采用高通量第二代测序技术,构建获得兰州鲇（Silurus lanzhouensis）线粒体基因组全序列,并对全序列特征和结构进行了分析。研究结果表明,兰州鲇线粒体基因组全序列长度为16523 bp,碱基组成具有高A+T低G+C含量的偏向性,具有脊椎动物典型的结构组成。13个PCG基因中存在2种启动子（ATG、GTG）、3种终止子（TAG、TAA和T或TA）。除tRNA-Ser（AGN）基因二级结构中DHU臂缺失,其余21个tRNA基因可折叠成典型三叶草结构。12S rRNA二级结构由45个茎环结构组成4个结构域,16S rRNA由54个茎环结构组成6个结构域。含有关键序列标签的控制区（CR）可分为3个不同的结构域:终止序列区（TAS1、TAS2）、中央保守区（CSB-F、CSB-E和CSB-D）和保守序列区（CSB1、CSB2和CSB3）。非编码区含有一段保守的控制轻链复制起始的序列区（O_L）。基于线粒体基因组全序列和通用标签COX1基因标记可区分兰州鲇同其他鲇形目鱼类种质进化关系。     

DsaV methyltransferase and its isoschizomers contain a conserved segment that is similar to the segment in Hhai methyltransferase that is in contact with DNA bases.

The methyltransferase (MTase) in the DsaV restriction--modification system methylates within 5'-CCNGG sequences. We have cloned the gene for this MTase and determined its sequence. The predicted sequence of the MTase protein contains sequence motifs conserved among all cytosine-5 MTases and is most similar to other MTases that methylate CCNGG sequences, namely M.ScrFI and M.SsoII. All three MTases methylate the internal cytosine within their recognition sequence. The 'variable' region within the three enzymes that methylate CCNGG can be aligned with the sequences of two enzymes that methylate CCWGG sequences. Remarkably, two segments within this region contain significant similarity with the region of M.HhaI that is known to contact DNA bases. These alignments suggest that many cytosine-5 MTases are likely to interact with DNA using a similar structural framework.     

AdoMet-dependent methylation, DNA methyltransferases and base flipping

Twenty AdoMet-dependent methyltransferases (MTases) have been characterized structurally by X-ray crystallography and NMR. These include seven DNA MTases, five RNA MTases, four protein MTases and four small molecule MTases acting on the carbon, oxygen or nitrogen atoms of their substrates. The MTases share a common core structure of a mixed seven-stranded beta-sheet (6 downward arrow 7 upward arrow 5 downward arrow 4 downward arrow 1 downward arrow 2 downward arrow 3 downward arrow) referred to as an 'AdoMet-dependent MTase fold', with the exception of a protein arginine MTase which contains a compact consensus fold lacking the antiparallel hairpin strands (6 downward arrow 7 upward arrow). The consensus fold is useful to identify hypothetical MTases during structural proteomics efforts on unannotated proteins. The same core structure works for very different classes of MTase including those that act on substrates differing in size from small molecules (catechol or glycine) to macromolecules (DNA, RNA and protein). DNA MTases use a 'base flipping' mechanism to deliver a specific base within a DNA molecule into a typically concave catalytic pocket. Base flipping involves rotation of backbone bonds in double-stranded DNA to expose an out-of-stack nucleotide, which can then be a substrate for an enzyme-catalyzed chemical reaction. The phenomenon is fully established for DNA MTases and for DNA base excision repair enzymes, and is likely to prove general for enzymes that require access to unpaired, mismatched or damaged nucleotides within base-paired regions in DNA and RNA. Several newly discovered MTase families in eukaryotes (DNA 5mC MTases and protein arginine and lysine MTases) offer new challenges in the MTase field.     

Are there two DNA methyltransferase gene families in plant cells? A new potential methyltransferase gene isolated from an Arabidopsis thaliana genomic library.

Using the 1kb 3' terminal DNA fragment of the mouse methyltransferase cDNA as a probe and low stringent hybridisation conditions, a new potential methyltransferase (MTase) gene family was isolated from an Arabidopsis thaliana genomic DNA library. One clone (MTase-11), which gave the strongest signal at the Northern blot, was entirely sequenced (11483 bp) and further characterised. Under consideration of the likely open reading frames and our preliminary cDNA experiments we propose that the clone 11 gene encodes for an approximately 90 kD protein. As deduced form the DNA sequence this protein contains all conserved sequence motifs specific for the 5m cytosine MTases. MTase-11 gene expression was demonstrable in callus and during germination but not in one month old plants or in leaves.     

Identification and preliminary characterization of an O6-methylguanine DNA repair methyltransferase in the yeast Saccharomyces cerevisiae

Saccharomyces cerevisiae contains a DNA repair methyltransferase (MTase) that repairs O6-methylguanine. Methyl groups are irreversibly transferred from O6-methylguanine in DNA to a 25-kilodalton protein in S. cerevisiae cell extracts, and methyl transfer is accompanied by the formation of S-methylcysteine. The yeast MTase is expressed at approximately 150 molecules/cell in exponentially growing yeast cultures but is not detectable in stationary phase cells. Unlike mammalian and bacterial MTases, the yeast MTase is very temperature-sensitive, having a half-life of about 4 min at 37 degrees C, which may explain why others have failed to detect it. Like other DNA repair MTases, the S. cerevisiae MTase repairs O6-methylguanine more efficiently in double-stranded DNA than in single-stranded DNA. Synthesis of the yeast DNA MTase is apparently not inducible by sublethal exposures to alkylating agent, but rather MTase activity is depleted in cells exposed to low doses of alkylating agent. Judging from its molecular weight and substrate specificity, the yeast DNA MTase is more closely related to mammalian MTases than to Escherichia coli MTases.     

HpaII methyltransferase is mutagenic in Escherichia coli.

A genetic reversion assay to study C-to-T mutations within CG sites in DNA is described. It was used to demonstrate that the presence of HpaII methyltransferase (MTase) in Escherichia coli causes a substantial increase in C-to-T mutations at CG sites. This is similar to the known mutagenic effects of E. coli MTase Dcm within its own recognition sequence. With this genetic system, a homolog of an E. coli DNA repair gene in Haemophilus parainfluenzae was tested for antimutagenic activity. Unexpectedly, the homolog was found to have little effect on the reversion frequency. The system was also used to show that HpaII and SssI MTases can convert cytosine to uracil in vitro. These studies define 5-methylcytosine as an intrinsic mutagen and further elaborate the mutagenic potential of cytosine MTases.     

Structure, function and mechanism of exocyclic DNA methyltransferases

DNA MTases (methyltransferases) catalyse the transfer of methyl groups to DNA from AdoMet (S-adenosyl-L-methionine) producing AdoHcy (S-adenosyl-L-homocysteine) and methylated DNA. The C5 and N4 positions of cytosine and N6 position of adenine are the target sites for methylation. All three methylation patterns are found in prokaryotes, whereas cytosine at the C5 position is the only methylation reaction that is known to occur in eukaryotes. In general, MTases are two-domain proteins comprising one large and one small domain with the DNA-binding cleft located at the domain interface. The striking feature of all the structurally characterized DNA MTases is that they share a common core structure referred to as an 'AdoMet-dependent MTase fold'. DNA methylation has been reported to be essential for bacterial virulence, and it has been suggested that DNA adenine MTases (Dams) could be potential targets for both vaccines and antimicrobials. Drugs that block Dam could slow down bacterial growth and therefore drug-design initiatives could result in a whole new generation of antibiotics. The transfer of larger chemical entities in a MTase-catalysed reaction has been reported and this represents an interesting challenge for bio-organic chemists. In general, amino MTases could therefore be used as delivery systems for fluorescent or other reporter groups on to DNA. This is one of the potential applications of DNA MTases towards developing non-radioactive DNA probes and these could have interesting applications in molecular biology. Being nucleotide-sequence-specific, DNA MTases provide excellent model systems for studies on protein-DNA interactions. The focus of this review is on the chemistry, enzymology and structural aspects of exocyclic amino MTases.     

Sequence-structure-function studies of tRNA:m5C methyltransferase Trm4p and its relationship to DNA:m5C and RNA:m5U methyltransferases

Three types of methyltransferases (MTases) generate 5-methylpyrimidine in nucleic acids, forming m5U in RNA, m5C in RNA and m5C in DNA. The DNA:m5C MTases have been extensively studied by crystallographic, biophysical, biochemical and computational methods. On the other hand, the sequence-structure-function relationships of RNA:m5C MTases remain obscure, as do the potential evolutionary relationships between the three types of 5-methylpyrimidine-generating enzymes. Sequence analyses and homology modeling of the yeast tRNA:m5C MTase Trm4p (also called Ncl1p) provided a structural and evolutionary platform for identification of catalytic residues and modeling of the architecture of the RNA:m5C MTase active site. The analysis led to the identification of two invariant residues that are important for Trm4p activity in addition to the conserved Cys residues in motif IV and motif VI that were previously found to be critical. The newly identified residues include a Lys residue in motif I and an Asp in motif IV. A conserved Gln found in motif X was found to be dispensable for MTase activity. Locations of essential residues in the model of Trm4p are in very good agreement with the X-ray structure of an RNA:m5C MTase homolog PH1374. Theoretical and experimental analyses revealed that RNA:m5C MTases share a number of features with either RNA:m5U MTases or DNA:m5C MTases, which suggested a tentative phylogenetic model of relationships between these three classes of 5-methylpyrimidine MTases. We infer that RNA:m5C MTases evolved from RNA:m5U MTases by acquiring an additional Cys residue in motif IV, which was adapted to function as the nucleophilic catalyst only later in DNA:m5C MTases, accompanied by loss of the original Cys from motif VI, transfer of a conserved carboxylate from motif IV to motif VI and sequence permutation.     

High plasticity of multispecific DNA methyltransferases in the region carrying DNA target recognizing enzyme modules.

Multispecific cytosine C5 DNA methyltransferases (MTases) methylate more than one specific DNA target. This is due to the presence of several target recognizing domains (TRDs) in these enzymes. Such TRDs form part of a variable centre in the MTase primary sequence, which separates conserved enzyme core sequences responsible for general steps in the methylation reaction. By deleting, rearranging and exchanging several TRDs of multispecific MTases, we demonstrate their modular character; they mediate target recognition independent of a particular TRD or core sequence context. We show also that multispecific MTases can accommodate inert material of non-MTase origin within their variable region without losing their activity. The remarkable plasticity with respect to the material that can be integrated into this region suggests that the enzyme core sequences preceding or following it form separable functional domains. In spite of the documented flexibility multispecific MTases could not be endowed with novel specificities by integration of putative TRDs of monospecific MTases, pointing to differences between multi- and monospecific MTases in the way their core and TRD sequences interact.     

Isolation of mutants in a DNA methyltransferase through mcrB-mediated restriction

A procedure has been developed that permits the positive selection of mutants in a DNA methyltransferase (MTase) gene. The stringency of this selection can be varied so as to yield null mutants only, or a mixture of null and partially defective mutants. The procedure was developed with the PvuII MTase gene (pvuIIM), which was subcloned into a bacteriophage lambda vector. Growth of this lambda pvuIIM construct on an mcrB+ host selected for non-methylating mutants, and the stringency of selection was proportional to the number of consecutive lytic cycles. Many cytosine MTases have been found to generate substrates for mcrB-mediated restriction, and this procedure should be applicable to a number of cytosine MTase genes.     

Diversity of DNA methyltransferases that recognize asymmetric target sequences

DNA methyltransferases (MTases) are a group of enzymes that catalyze the methyl group transfer from S-adenosyl-L-methionine in a sequence-specific manner. Orthodox Type II DNA MTases usually recognize palindromic DNA sequences and add a methyl group to the target base (either adenine or cytosine) on both strands. However, there are a number of MTases that recognize asymmetric target sequences and differ in their subunit organization. In a bacterial cell, after each round of replication, the substrate for any MTase is hemimethylated DNA, and it therefore needs only a single methylation event to restore the fully methylated state. This is in consistent with the fact that most of the DNA MTases studied exist as monomers in solution. Multiple lines of evidence suggest that some DNA MTases function as dimers. Further, functional analysis of many restriction-modification systems showed the presence of more than one or fused MTase genes. It was proposed that presence of two MTases responsible for the recognition and methylation of asymmetric sequences would protect the nascent strands generated during DNA replication from cognate restriction endonuclease. In this review, MTases recognizing asymmetric sequences have been grouped into different subgroups based on their unique properties. Detailed characterization of these unusual MTases would help in better understanding of their specific biological roles and mechanisms of action. The rapid progress made by the genome sequencing of bacteria and archaea may accelerate the identification and study of species- and strain-specific MTases of host-adapted bacteria and their roles in pathogenic mechanisms.