伪狂犬病病毒Ea株基因组UL区的克隆与序列分析

从伪狂犬病病毒Ea株基因组DNA中扩增到3．76kb的基因组片段,该片段包含UL31、UL32、UL33和UL34基因完整编码区,以及UL30和UL35基因部分序列。UL31、UL32、UL33和UL34基因G C含量为69．5％～73．4％,偏向于使用富含GC特别是第三密码子位置上核苷酸是C或G的密码子,Ala、Leu、Arg的利用率最高,占氨基酸残基总数的36．4％。PRV Ea株UL31和UL32基因与PRV Ka株核苷酸与氨基酸序列同源性都很高,在98％以上;而UL33和UL34基因与Ka株的氨基酸序列同源性较低,分别为95．7％和94．8％。UL31基因在疱疹病毒α—亚科所有成员之间都很保守,并且UL31基因与马疱疹病毒IV型同源程度最高。UL32、UL33和UL34基因均与牛疱疹病毒I型同源程度最高。UL31、UL32、UL33与UL34基因产物均有酪蛋白激酶2磷酸化位点和蛋白激酶C磷酸化位点,表明UL31、UL32、UL33、UL34蛋白质可能都是磷酸化蛋白质。     

杜仲基因密码子使用模式分析

为了解杜仲基因密码子使用模式,该文以杜仲基因组密码子为研究对象,运用CodonW软件对杜仲的320个蛋白编码基因进行同义密码子相对使用频率(RSCU)分析、ENC-GC3s关联分析编码基因的密码子ENC值、PR2-plot偏倚分析编码基因的密码子碱基使用频率,并运用CUSP软件与Codon Usage Database软件对杜仲基因密码子的GC含量、使用频率与代表性物种烟草、拟南芥、大肠杆菌和酿酒酵母的密码子GC含量和使用频率进行比较。结果表明:杜仲基因密码子的RSCU>1的密码子有30个,其中18个以G/C结尾、12个以A/U结尾,说明杜仲基因密码子偏好以G/C结尾,且偏好性较强;有效密码子数(ENC)范围为30~60,该范围内的密码子距离标准曲线较远,其ENC值小,偏好性较强;PR2-plot偏倚分析碱基使用频率显示,G>C、U>A;杜仲与代表性物种的GC含量分析显示,杜仲的GC12、GC3以及平均GC含量均高于代表性物种;杜仲与代表性物种的密码子使用频率分析显示,杜仲与烟草、酿酒酵母的密码子偏好较为接近,杜仲与拟南芥、大肠杆菌的密码子偏好差距较大。杜仲是我国特有的珍贵中药材,对其进行密码子使用模式分析,并研究其密码子偏好规律,为杜仲植物基因工程中外源基因的改良及表达提供了理论基础。     

伪狂犬病病毒Ea株基因组UL区的克隆与序列分析

从伪狂犬病病毒Ea株基因组DNA中扩增到3.76 kb的基因组片段,该片段包含UL31、UL32、UL33和UL34基因完整编码区,以及UL30和UL35基因部分序列.UL31、UL32、UL33和UL34基因G+C含量为69.5%~73.4%,偏向于使用富含GC特别是第三密码子位置上核苷酸是C或G的密码子,Ala、Leu、Arg的利用率最高,占氨基酸残基总数的36.4%.PRV Ea株UL31和UL32基因与PRV Ka株核苷酸与氨基酸序列同源性都很高,在98%以上;而UL33和UL34基因与Ka株的氨基酸序列同源性较低,分别为95.7%和94.8%.UL31基因在疱疹病毒α-亚科所有成员之间都很保守,并且UL31基因与马疱疹病毒IV型同源程度最高.UL32、UL33和UL34基因均与牛疱疹病毒I型同源程度最高.UL31、UL32、UL33与UL34基因产物均有酪蛋白激酶2磷酸化位点和蛋白激酶C磷酸化位点,表明UL31、UL32、UL33、UL34蛋白质可能都是磷酸化蛋白质.     

人类基因同义密码子偏好的特征以及与基因GC含量的关系

对人类的728个基因,按其编码区中GC的含量分成四组(从GC＜0.43到GC＞0.58),分别考察了这四组样本对同义密码子偏好的特征,发现在全部样本中都呈现NTG(N代表四种碱基中的任一种)特受偏爱和NCG尽量避免的特征.基因环境中GC含量与C3/G3含量(密码子第三位C和G的含量)的相关分析,以及四组样本对密码子的偏好都支持以C结尾的密码子在编码中有特殊的优势,这种优势有利于保证翻译的准确性.还考察了各种氨基酸含量随编码区GC含量不同而变化的趋势.     

亚心型扁藻叶绿体psbA基因的克隆及序列分析

psbA基因是叶绿体基因组中一个重要的光调控基因,编码光和系统Ⅱ反应中心的D1蛋白。根据叶绿体基因组序列高度保守的特性,利用菜茵衣藻（Chlamydomonasreinhardtii）psbA基因的保守序列（基因登录号：HQ667991．1）设计引物,采用PCR步移的方法从亚心型扁藻（Platymonassubcordiformis）基因组DNA中克隆到psbA基因全长（基因登录号：KF528742）。序列分析表明,亚心型扁藻psbA基因全长1939bp,编码区长度为1062bp,推导编码353个氨基酸,包括4个赖氨酸残基。有效密码子数显示脚删基因具有明显的密码子偏好性,并且偏好使用以A／T结尾的密码子。相对同义密码子使用度表明25个密码子在编码使用时具有偏好性,其中20个密码子以A／T碱基结尾,占到80％。其终止密码子使用了TAG。     

短短芽孢杆菌GZDF3全基因组密码子偏好性分析

为了解短短芽孢杆菌GZDF3全基因组密码子的使用特性,采用Codon W 1.4.2软件和Mobyle portal在线软件分析菌株GZDF3全基因组密码子偏好性,分析该菌的相对同义密码子使用度(relative synonymous codon usage,RSCU)、密码子适应指数(codon adaption index,CAI)、有效密码子数(effective number of codon,Nc)、同义密码子第3位中G/C含量(GC3s)和高表达优越密码子。结果表明,短短芽孢杆菌GZDF3密码子第2位密码子的GC含量明显低于第1位和第3位,为37.56%,第1位和第3位的GC含量差异较小,分别为55.50%和51.13%;基因组大部分Nc值介于30~60,说明密码子偏好性普遍偏弱;RSCU值大于1的密码子共有27个,其中以A或T结尾的14个,占51.9%,并确定了27个最优密码子,为外源基因表达提供理论基础。     

红松查尔酮合成酶基因CHS密码子偏好性分析

查尔酮合成酶(Chalcone synthase,CHS)广泛存在于植物体内,是花色素形成过程中一种重要的酶,可以进一步催化生成黄酮类化合物。本研究采用Codon W和EMBOSS在线软件对红松查尔酮合成酶基因CHS的密码子使用偏好性进行分析,并与北美乔松等其他24种植物的CHS基因以及模式植物基因组进行比较,对认识红松CHS基因的密码子使用偏好性,为选择适宜的表达系统奠定了一定的基础。研究结果表明:红松CHS基因编码区的有效密码子数(ENC)和GC含量分别为48.92和0.548,C+G含量高于A+T含量,密码子偏好以A/T结尾;多数植物CHS基因的G+C含量高于A+T含量,且密码子更偏好C/G结尾;聚类分析表明,红松与马尾松和赤松的密码子使用偏好性的相似性较高;密码子使用频率研究发现,红松CHS遗传转化与异源表达较优的受体可能是大肠杆菌和拟南芥。     

茄腐镰孢(Fusarium solani)线粒体基因组密码子偏好性分析

密码子使用偏好性作为功能基因组重要的进化特征而被广泛报道,但是有关真菌线粒体基因组密码子使用偏好性的研究相对较少。本试验以茄腐镰孢线粒体基因组为研究对象,运用Codon W软件及RSCU在线软件对所筛选到的22条编码基因密码子3个位置上的GC含量、有效密码子数、同义密码子使用频率和最优密码子进行了分析。同时,确定了茄腐镰孢线粒体基因组的最优密码子,并探讨了影响密码子偏性形成的相关因素。分析结果表明,茄腐镰孢线粒体密码子3位的GC含量为GC1(32.7%)GC2(28.0%)GC3(19.7%),第3位GC含量明显小于第1、2位,表现出对以A或者T结尾的密码子发生较强烈的偏向使用;且确定的21个最优密码子中有20个以A或者T结尾。通过中性绘图及ENC分析得出茄腐镰孢线粒体基因组密码子的使用偏好受到选择作用和突变压力的共同影响,选择作用为主要影响因素。     

籼稻品种93-11同义密码子的使用偏性

利用籼稻品种93－11的全基因组序列及相应的EST数据,对影响同义密码子用法的若干因子进行了详细分析。指出93－11基因的表达水平（mRNA丰度）与3个同义密码子偏性指标CAI、CPP和ENC相关极显著（r=0.227^**,0.145^**和-0.147^**),表明高表达的基因其同义密码子非随机使用的程度越大;基因长度与CAI和CPP极显著负相关（r=-0.413^**和-0.480^**),与ENC极显著正相关（r=0.210^**),暗示较短的基因具有更高的转录活性;编码区G＋C含量对其同义密码子偏性的贡献率远高于mRNA丰度和基因长度,G＋C含量与CAI、CPP和ENC相关系数分别高达0.877^**,0.832^**和-0.740^**;起始编码区内A、T、C、G4种碱基呈明显的3周期振荡,尤以ATG下游第一个密码子所在的3个位点（＋4、＋5和＋6）偏置最强烈,由此认为在这3个特殊位点有较高的自然选择压存在;93－11中25个最优密码子的首次确定将对水稻转基因具有指导意义。     

基于同义密码子使用的原核生物基因组大、小染色体及质粒进化特征分析北大核心CSCD

基于同义密码子偏好分析,对54个原核基因组大、小染色体及质粒中蛋白质编码基因的序列特征进行了对比分析。结果表明,大、小染色体中蛋白质编码基因的GC含量分布相近,质粒中蛋白质编码基因的GC含量分布与所在物种全基因组的GC含量差别较大。进一步的分析表明,大、小染色体共同偏好的密码子最多,且具有相近的起始密码子和终止密码子使用特征。基于对应分析的同义密码子使用模式分析表明,大、小染色体具有相近的序列特征,且大、小染色体及质粒之间具有不尽相同的影响因素。这些结果可为今后原核生物基因组进化研究提供可靠的方法和理论依据。     

Translation in Bacillus subtilis: roles and trends of initiation and termination, insights from a genome analysis.

We analysed the Bacillus subtilis protein coding sequences termini, and compared it to other genomes. The analysis focused on signals, com-positional biases of nucleotides, oligonucleotides, codons and amino acids and mRNA secondary structure. AUG is the preferred start codon in all genomes, independent of their G+C content, and seems to induce less stable mRNA structures. However, it is not conserved between homologous genes neither is it preferred in highly expressed genes. In B.subtilis the ribosome binding site is very strong. We found that downstream boxes do not seem to exist either in Escherichia coli or in B.subtilis. UAA stop codon usage is correlated with the G+C content and is strongly selected in highly expressed genes. We found less stable mRNA structures at both termini, which we related to mRNA-ribosome and mRNA-release-factor interactions. This pattern seems to impose a peculiar A-rich nucleotide and codon usage bias in these regions. Finally the analysis of all proteins from B.subtilis revealed a similar amino acid bias near both termini of proteins consisting of over-representation of hydrophilic residues. This bias near the stop codon is partially release-factor specific.     

Characterization of synonymous codon usage bias in the pseudorabies virus US1 gene

In the present study, we examined the codon usage bias between pseudorabies virus (PRV) US1 gene and the US1-like genes of 20 reference alphaherpesviruses. Comparative analysis showed noticeable disparities of the synonymous codon usage bias in the 21 alphaherpesviruses, indicated by codon adaptation index, effective number of codons (ENc) and GC3s value. The codon usage pattern of PRV US1 gene was phylogenetically conserved and similar to that of the US1-like genes of the genus Varicellovirus of alphaherpesvirus, with a strong bias towards the codons with C and G at the third codon position. Cluster analysis of codon usage pattern of PRV US1 gene with its reference alphaherpesviruses demonstrated that the codon usage bias of US1-like genes of 21 alphaherpesviruses had a very close relation with their gene functions. ENc-plot revealed that the genetic heterogeneity in PRV US1 gene and the 20 reference alphaherpesviruses was constrained by G+C content, as well as the gene length. In addition, comparison of codon preferences in the US1 gene of PRV with those of E. coli, yeast and human revealed that there were 50 codons showing distinct usage differences between PRV and yeast, 49 between PRV and human, but 48 between PRV and E. coli. Although there were slightly fewer differences in codon usages between E.coli and PRV, the difference is unlikely to be statistically significant, and experimental studies are necessary to establish the most suitable expression system for PRV US1. In conclusion, these results may improve our understanding of the evolution, pathogenesis and functional studies of PRV, as well as contributing to the area of herpesvirus research or even studies with other viruses.     

Codon Usage in Plastid Genes Is Correlated with Context, Position Within the Gene, and Amino Acid Content

Highly expressed plastid genes display codon adaptation, which is defined as a bias toward a set of codons which are complementary to abundant tRNAs. This type of adaptation is similar to what is observed in highly expressed Escherichia coli genes and is probably the result of selection to increase translation efficiency. In the current work, the codon adaptation of plastid genes is studied with regard to three specific features that have been observed in E. coli and which may influence translation efficiency. These features are (1) a relatively low codon adaptation at the 5′ end of highly expressed genes, (2) an influence of neighboring codons on codon usage at a particular site (codon context), and (3) a correlation between the level of codon adaptation of a gene and its amino acid content. All three features are found in plastid genes. First, highly expressed plastid genes have a noticeable decrease in codon adaptation over the first 10–20 codons. Second, for the twofold degenerate NNY codon groups, highly expressed genes have an overall bias toward the NNC codon, but this is not observed when the 3′ neighboring base is a G. At these sites highly expressed genes are biased toward NNT instead of NNC. Third, plastid genes that have higher codon adaptations also tend to have an increased usage of amino acids with a high G + C content at the first two codon positions and GNN codons in particular. The correlation between codon adaptation and amino acid content exists separately for both cytosolic and membrane proteins and is not related to any obvious functional property. It is suggested that at certain sites selection discriminates between nonsynonymous codons based on translational, not functional, differences, with the result that the amino acid sequence of highly expressed proteins is partially influenced by selection for increased translation efficiency. Received: 21 July 1999 / Accepted: 5 November 1999     

Two Aspects of DNA Base Composition: G+C Content and Translation-Coupled Deviation from Intra-Strand Rule of A=T and G=C

The relative contribution of mutation and selection to the G+C content of DNA was analyzed in bacterial species having widely different G+C contents. The analysis used two methods that were developed previously. The first method was to plot the average G+C content of a set of nucleotides against the G+C content of the third codon position for each gene. This method was used to present the G+C distribution of the third codon position and to assess the relative neutrality of a set of nucleotides to that of the G+C content of the third codon position. The second method was to plot the intrastrand bias of the third codon position from Parity Rule 2 (PR2), where A=T and G=C. It was found that whereas intragenomic distributions of the DNA G+C content of these bacteria are narrow in the majority of species, in some species the G+C content of the minor class of genes distributes over wider ranges than the major class of genes. On the other hand, ubiquitous PR2 biases are amino acid specific and independent of the G+C content of DNA, so that when averaged over the amino acids, the biases are small and not correlated with the DNA G+C content. Therefore, translation coupled PR2-biases are unlikely to explain the wide range of G+C contents among different species. Considering all data available, it was concluded that the amino acid-specific PR2 bias has only a minor effect, if any, on the average G+C content. In addition, PR2 bias patterns of different species show phylogenetic relationships, and the pattern can be as a taxal fingerprint. Received: 5 November 1998 / Accepted: 1 March 1999     

Detecting anomalous gene clusters and pathogenicity islands in diverse bacterial genomes.

A gene in a genome is defined as putative alien (pA) if its codon usage difference from the average gene exceeds a high threshold and codon usage differences from ribosomal protein genes, chaperone genes and protein-synthesis-processing factors are also high. pA gene clusters in bacterial genomes are relevant for detecting genomic islands (GIs), including pathogenicity islands (PAIs). Four other analyses appropriate to this task are G+C genome variation (the standard method); genomic signature divergences (dinucleotide bias); extremes of codon bias; and anomalies of amino acid usage. For example, the cagA domain of Helicobacter pylori is highly deviant in its genome signature and codon bias from the rest of the genome. Using these methods we can detect two potential PAIs in the Neisseria meningitidis genome, which contain hemagglutinin and/or hemolysin-related genes. Additionally, G+C variation and genome signature differences of the Mycobacterium tuberculosis genome indicate two pA gene clusters.     

Codon usage and gene expression level in Dictyostelium discoideum: highly expressed genes do ''prefer'' optimal codons.

Codon usage patterns in the slime mould Dictyostelium discoideum have been re-examined (a total of 58 genes have been analysed). Considering the extreme A + T-richness of this genome (G + C = 22%), there is a surprising degree of codon usage variation among genes. For example, G + C content at silent sites varies from less than 10% to greater than 30%. It was previously suggested [Warrick, H.M. and Spudich, J.A. (1988) Nucleic Acids Res. 16: 6617-6635] that highly expressed genes contain fewer 'optimal' codons than genes expressed at lower levels. However, it appears that the optimal codons were misidentified. Multivariate statistical analysis shows that the greatest variation among genes is in relative usage of a particular subset of codons (about one per amino acid), many of which are C-ending. We have identified these as optimal codons, since (i) their frequency is positively correlated with gene expression level, and (ii) there is a strong mutation bias in this genome towards A and T nucleotides. Thus, codon usage in D. discoideum can be explained by a balance between the forces of mutational bias and translational selection.     

Evolutionary patterns of codon usage in the chloroplast gene rbcL

In this study we reconstruct the evolution of codon usage bias in the chloroplast gene rbcL using a phylogeny of 92 green-plant taxa. We employ a measure of codon usage bias that accounts for chloroplast genomic nucleotide content, as an attempt to limit plausible explanations for patterns of codon bias evolution to selection- or drift-based processes. This measure uses maximum likelihood-ratio tests to compare the performance of two models, one in which a single codon is overrepresented and one in which two codons are overrepresented. The measure allowed us to analyze both the extent of bias in each lineage and the evolution of codon choice across the phylogeny. Despite predictions based primarily on the low G + C content of the chloroplast and the high functional importance of rbcL, we found large differences in the extent of bias, suggesting differential molecular selection that is clade specific. The seed plants and simple leafy liverworts each independently derived a low level of bias in rbcL, perhaps indicating relaxed selectional constraint on molecular changes in the gene. Overrepresentation of a single codon was typically plesiomorphic, and transitions to overrepresentation of two codons occurred commonly across the phylogeny, possibly indicating biochemical selection. The total codon bias in each taxon, when regressed against the total bias of each amino acid, suggested that twofold amino acids play a strong role in inflating the level of codon usage bias in rbcL, despite the fact that twofolds compose a minority of residues in this gene. Those amino acids that contributed most to the total codon usage bias of each taxon are known through amino acid knockout and replacement to be of high functional importance. This suggests that codon usage bias may be constrained by particular amino acids and, thus, may serve as a good predictor of what residues are most important for protein fitness.     

Mutation bias is the driving force of codon usage in the Gallus gallus genome

Synonymous codons are used with different frequencies both among species and among genes within the same genome and are controlled by neutral processes (such as mutation and drift) as well as by selection. Up to now, a systematic examination of the codon usage for the chicken genome has not been performed. Here, we carried out a whole genome analysis of the chicken genome by the use of the relative synonymous codon usage (RSCU) method and identified 11 putative optimal codons, all of them ending with uracil (U), which is significantly departing from the pattern observed in other eukaryotes. Optimal codons in the chicken genome are most likely the ones corresponding to highly expressed transfer RNA (tRNAs) or tRNA gene copy numbers in the cell. Codon bias, measured as the frequency of optimal codons (Fop), is negatively correlated with the G + C content, recombination rate, but positively correlated with gene expression, protein length, gene length and intron length. The positive correlation between codon bias and protein, gene and intron length is quite different from other multi-cellular organism, as this trend has been only found in unicellular organisms. Our data displayed that regional G + C content explains a large proportion of the variance of codon bias in chicken. Stepwise selection model analyses indicate that G + C content of coding sequence is the most important factor for codon bias. It appears that variation in the G + C content of CDSs accounts for over 60% of the variation of codon bias. This study suggests that both mutation bias and selection contribute to codon bias. However, mutation bias is the driving force of the codon usage in the Gallus gallus genome. Our data also provide evidence that the negative correlation between codon bias and recombination rates in G. gallus is determined mostly by recombination-dependent mutational patterns.     

Reduction of wobble-position GC bases in Corynebacteria genes and enhancement of PCR and heterologous expression

Corynebacteria codon usage exhibits an overall GC content of 67%, and a wobble-position GC content of 88%. Escherichia coli, on the other hand has an overall GC content of 51%, and a wobble-position GC content of 55%. The high GC content of Corynebacteria genes results in an unfavorable codon preference for heterologous expression, and can present difficulties for polymerase-based manipulations due to secondary-structure effects. Since these characteristics are due primarily to base composition at the wobble-position, synthetic genes can, in principle, be designed to eliminate these problems and retain the wild-type amino acid sequence. Such genes would obviate the need for special additives or bases during in vitro polymerase-based manipulation and mutant host strains containing uncommon tRNA's for heterologous expression. We have evaluated synthetic genes with reduced wobble-position G/C content using two variants of the enzyme 2,5-diketo-D-gluconic acid reductase (2,5-DKGR A and B) from Corynebacterium. The wild-type genes are refractory to polymerase-based manipulations and exhibit poor heterologous expression in enteric bacteria. The results indicate that a subset of codons for five amino acids (alanine, arginine, glutamate, glycine and valine) contribute the greatest contribution to reduction in G/C content at the wobble-position. Furthermore, changes in codons for two amino acids (leucine and proline) enhance bias for expression in enteric bacteria without affecting the overall G/C content. The synthetic genes are readily amplified using polymerase-based methodologies, and exhibit high levels of heterologous expression in E. coli.