The effects of differential gene expression on coding sequence features: analysis by one-way ANOVA

It is well-established that non-random patterns in coding DNA sequence (CDS) features can be partially explained by translational selection. Recent extensions of microarray and proteomic expression data have stimulated many genome-wide investigations of the relationships between gene expression and various CDS features. However, only modest correlations have been found. Here we introduced the one-way ANOVA, a more powerful extension of previous grouping methods, to re-examine these relationships at the whole genome scale for Saccharomyces cerevisiae, where genome-wide protein abundance has been recently quantified. Our results clarify that coding sequence features are inappropriate for use as genome-wide estimators for protein expression levels. This analysis also demonstrates that one-way ANOVA is a powerful and simple method to explore the influence of gene expression on CDS features.     

花生MYB转录因子的鉴定与生物信息学分析

MYB转录因子是植物中重要的基因家族之一,参与多种生物学功能的调控.目前对花生(Arachis hypogaea)MYB转录因子家族的功能仍知之甚少,对花生中MYB转录因子的鉴定及生物信息学分析具有重要的意义.本研究在栽培花生中共鉴定出MYB转录因子443个,包括219个1R-MYB、209个2R-MYB、12个3R-...     

Comprehensive sequence analysis of translation termination sites in various eukaryotes

Recent investigations into the translation termination sites of various organisms have revealed that not only stop codons but also sequences around stop codons have an effect on translation termination. To investigate the relationship between these sequence patterns and translation as well as its termination efficiency, we analysed the correlation between strength of consensus and translation efficiency, as predicted according to Codon Adaptation Index (CAI) value. We used RIKEN full-length mouse cDNA sequences and ten other eukaryotic UniGene datasets from NCBI for the analyses. First, we conducted sequence profile analyses following translation termination sites. We found base G and A at position +1 as a strong consensus for mouse cDNA. A similar consensus was found for other mammals, such as Homo sapiens, Rattus norvegicus and Bos taurus. However, some plants had different consensus sequences. We then analysed the correlation between the strength of consensus at each position and the codon biases of whole coding regions, using information content and CAI value. The results showed that in mouse cDNA, CAI value had a positive correlation with information content at positions +1. We also found that, for positions with strong consensus, the strength of the consensus is likely to have a positive correlation with CAI value in some other eukaryotes. Along with these observations, biological insights into the relationship between gene expression level, codon biases and consensus sequence around stop codons will be discussed.     

Identification and characterization of phospholipase D and its association with drought susceptibilities in peanut (Arachis hypogaea)

Preharvest aflatoxin contamination has been identified by the peanut industry as a serious issue in food safety and human health because of the carcinogenic toxicity. Drought stress is the most important environmental factor exacerbating Aspergillus infection and aflatoxin contamination in peanut. The development of drought-tolerant peanut cultivars could reduce aflatoxin contamination and would represent a major advance in the peanut industry. In this study, we identified a novel PLD gene in peanut (Arachis hypogaea), encoding a putative phospholipase D (PLD, EC 3.1.4.4). The completed cDNA sequence was obtained by using the consensus-degenerated hybrid oligonucleotide primer strategy. The deduced amino acid sequence shows high identity with known PLDs, and has similar conserved domains. The PLD gene expression under drought stress has been studied using four peanut lines: Tifton 8 and A13 (both drought tolerant) and Georgia Green (moderate) and PI 196754 (drought sensitive). Northern analysis showed that PLD gene expression was induced faster by drought stress in the drought-sensitive lines than the drought tolerance lines. Southern analysis showed that cultivated peanut has multiple copies (3 to 5 copies) of the PLD gene. These results suggest that peanut PLD may be involved in drought sensitivity and tolerance responses. Peanut PLD gene expression may be useful as a tool in germplasm screening for drought tolerance. The nucleotide sequence, reported in this paper, have been submitted to GenBank under accession number AY274834.     

Optimization protein productivity of human interleukin-2 through codon usage,gene copy number and intracellular tRNA concentration in CHO cells

Transfer RNA (tRNA) abundance is one of the critical factors for the enhancement of protein productivity in prokaryotic and eukaryotic hosts. Gene copy number of tRNA and tRNA codon usage bias are generally used to match tRNA abundance of protein-expressing hosts and to optimize the codons of recombinant proteins. Because sufficient concentration of intracellular tRNA and optimized codons of recombinant proteins enhanced translation efficiency, we hypothesized that sufficient supplement of host’s tRNA improved protein productivity in mammalian cells. First, the small tRNA sequencing results of CHO-K1 cells showed moderate positive correlation with gene copy number and codon usage bias. Modification of human interleukin-2 (IL-2) through codons with high gene copy number and high codon usage bias (IL-2 HH, modified on Leu, Thr, Glu) significantly increased protein productivity in CHO-K1 cells. In contrast, modification through codons with relatively high gene copy number and low codon usage bias (IL-2 HL, modified on Ala, Thr, Val), or relatively low gene copy number and low codon usage bias (IL-2 LH, modified on Ala, Thr, Val) did not increase IL-2 productivity significantly. Furthermore, supplement of the alanine tRNA or threonine tRNA increased IL-2 productivity of IL-2 HL. In summary, we revealed a potential strategy to enhance productivity of recombinant proteins, which may be applied in production of protein drug or design of DNA vaccine.     

Codon bias and gene expression of mitochondrial ND2 gene in chordates

Background: Mitochondrial ND gene, which encodes NADH dehydrogenase, is the first enzyme of the mitochondrial electron transport chain. Leigh syndrome, a neurodegenerative disease caused by mutation in the ND2 gene (T4681C), is associated with bilateral symmetric lesions in basal ganglia and subcortical brain regions. Therefore, it is of interest to analyze mitochondrial DNA to glean information for evolutionary relationship. This study highlights on the analysis of compositional dynamics and selection pressure in shaping the codon usage patterns in the coding sequence of MT-ND2 gene across pisces, aves and mammals by using bioinformatics tools like effective number of codons (ENC), codon adaptation index (CAI), relative synonymous codon usage (RSCU) etc. Results: We observed a low codon usage bias as reflected by high ENC values in MT-ND2 gene among pisces, aves and mammals. The most frequently used codons were ending with A/C at the 3^rd position of codon and the gene was AT rich in all the three classes. The codons TCA, CTA, CGA and TGA were over represented in all three classes. The F1 correspondence showed significant positive correlation with G, T3 and CAI while the F2 axis showed significant negative correlation with A and T but significant positive correlation with G, C, G3, C3, ENC, GC, GC1, GC2 and GC3. Conclusions: The codon usage bias in MTND2 gene is not associated with expression level. Mutation pressure and natural selection affect the codon usage pattern in MT-ND 2 gene.     

Bioinformatic analysis of the link between gene composition and expressivity in Saccharomyces cerevisiae and Schizosaccharomyces pombe

The compositional non-randomness was studied in genes of Saccharomyces cerevisiae and Schizosaccharomyces pombe. In both species, codon usage is well correlated with expressivity (measured as the codon adaptation index). Both species generally display higher nucleotide non-randomness in the group of highly expressed genes than in the lowly expressed genes. The highly expressed genes in both species are furthermore characterized by marked peaks in non-randomness at N=3 upstream of start codons, N=2 downstream of start codons and at N=1 and N=7 downstream of stop codons, indicating that these nucleotides may be key elements in translational regulation. Intragenic variation in codon usage was also observed to be linked to expressivity. It is suggested that the firm link between expressivity and codon usage calls for codon optimization. Based on bioinformatic calculations, examples of proteins are given for which codon optimizations might be relevant.     

棉铃虫HaTO-like基因的克隆、序列分析和表达特征

【目的】克隆和分析了棉铃虫Helicoverpa armigera HaTO-like基因的编码框序列,检测了该基因的时空表达谱以及在棉铃虫感染核型多角体病毒HaSNPV后的转录变化,为深入研究该基因的功能提供理论依据。【方法】本研究利用RT-PCR的方法首次克隆获得HaTO-like基因的全长cDNA序列,通过几种生物信息学软件对该基因的核苷酸序列和氨基酸序列进行了分析,并利用荧光定量PCR技术检测了该基因在棉铃虫不同发育阶段、幼虫组织和成虫组织的表达情况,以及HaSNPV感染对HaTO-like基因表达的影响。【结果】棉铃虫HaTO-like基因cDNA全长为994 bp,开放阅读框为756 bp,编码251个氨基酸,其蛋白序列的N端含有23个氨基酸的信号肽。进一步的序列分析表明棉铃虫HaTO-like与其他昆虫同源蛋白的氨基酸序列一致性不是太高,大概在39%~61%之间,其中与家蚕和脐橙螟在系统进化上关系最近。荧光定量PCR结果表明该基因在棉铃虫的5龄0 h和成虫第1天的的表达量相对较高,在幼虫的头部和表皮内的表达量较其他幼虫组织较高,在成虫的头部和足的表达量也相对较高。而病毒感染则显著地诱导了该基因在棉铃虫幼虫头部和表皮内的表达。【讨论】本研究克隆了棉铃虫HaTO-like基因的全长cDNA序列,分析了该基因的序列特征和表达谱,为进一步阐释该基因的功能奠定理论基础。     

The role of super-relaxed myosin in skeletal and cardiac muscle

The super-relaxed (SRX) state of myosin was only recently reported in striated muscle. It is characterised by a sub-population of myosin heads with a highly inhibited rate of ATP turnover. Myosin heads in the SRX state are bound to each other along the thick filament core producing a highly ordered arrangement. Upon activation, these heads project into the interfilament space where they can bind to the actin filaments. Thus far, the population and lifetimes of myosin heads in the SRX state have been characterised in rabbit cardiac, and fast and slow skeletal muscle, as well as in the skeletal muscle of the tarantula. These studies suggest that the role of SRX in cardiac and skeletal muscle regulation is tailored to their specific functions. In skeletal muscle, the SRX modulates the resting metabolic rate. Cardiac SRX represents a “reserve” of inactive myosin heads that may protect the heart during times of stress, e.g. hypoxia and ischaemia. These heads may also be called up when there is a sustained demand for increased power. The SRX in cardiac muscle provides a potential target for novel therapies.     

Abberent expression analysis of LMNA gene in hutchinson-gilford progeria syndrome

Hutchinson-Gilford progeria syndrome (HGPS) is caused by de novo dominant point mutations of the genes encoding nuclear lamina proteins, leading towards premature aging. A protein sequence is subjected to mutations in nature which can affect the function and folding pattern of the protein by different ways. Mutations involved in HGPS were identified and were substituted in the seed sequence retrieved from the UniProt database to get the mutated versions. Tertiary structure of the Lamin A protein was previously unpredicted so was performed for all the mutated as well as for the seed protein to analyze the effects of mutations on the protein structure, folding and interactions. All the predicted models were refined and validated through multiple servers for multiple parameters. The validated 3D structure of seed protein was then successfully submitted to the Protein Model Database and was assigned with the PMDB ID PM0077829. All the predicted structures were superimposed with a root mean square deviation value of 7.0 Å and a high Dali Z-score of 1.9. It was observed that mutations affected physiochemical properties as well as instability index and thus is affecting the domains in specific and the whole structure in general. It was further analyzed that HGPS is the result of affected Lamin a protein interactions with other integral and binding proteins in the inner nuclear membrane affecting the link in between the nuclear membrane and the network of the lamina.     

Highly potent inhibitors of cathepsin K with a differently positioned cyanohydrazide warhead: structural analysis of binding mode to mature and zymogen-like enzymes

Cathepsin K (CatK) is a target for the treatment of osteoporosis, arthritis, and bone metastasis. Peptidomimetics with a cyanohydrazide warhead represent a new class of highly potent CatK inhibitors; however, their binding mechanism is unknown. We investigated two model cyanohydrazide inhibitors with differently positioned warheads: an azadipeptide nitrile Gü1303 and a 3-cyano-3-aza-β-amino acid Gü2602. Crystal structures of their covalent complexes were determined with mature CatK as well as a zymogen-like activation intermediate of CatK. Binding mode analysis, together with quantum chemical calculations, revealed that the extraordinary picomolar potency of Gü2602 is entropically favoured by its conformational flexibility at the nonprimed-primed subsites boundary. Furthermore, we demonstrated by live cell imaging that cyanohydrazides effectively target mature CatK in osteosarcoma cells. Cyanohydrazides also suppressed the maturation of CatK by inhibiting the autoactivation of the CatK zymogen. Our results provide structural insights for the rational design of cyanohydrazide inhibitors of CatK as potential drugs.     

芹菜韧皮部蛋白基因的分离与表达分析

韧皮部蛋白在维持植物形态,物质转运以及植物伤口保护等方面起着重要作用。本研究以地域来源和性状特性差异均较大的两个芹菜品种‘六合黄心芹’和‘美国西芹’为试验材料,利用RT-PCR技术获得这两种芹菜韧皮部蛋白基因的cDNA序列。结果显示：这两种芹菜来源的韧皮部蛋白基因全长均为546 bp,编码181个氨基酸。两者核苷酸序列有3个位点的不同,分别为：88G/A、399T/C和489T/C;在氨基酸序列上有一个位点的不同,为30T/A。预测其蛋白质分子量为19 kD,pI值为9.18。‘六合黄心芹’和‘美国西芹’的韧皮部蛋白与忽地笑等植物的韧皮部蛋白相似度较高,在保守位置分别具有5个亮氨酸残基和4个色氨酸残基。实时定量PCR表达分析表明,该基因主要在芹菜的茎和根等部位表达,具有明显的组织特异性。     

Comprehensive expression analysis of rice phospholipase D gene family during abiotic stresses and development

Phospholipase D is one of the crucial enzymes involved in lipid mediated signaling, triggered during various developmental and physiological processes. Different members of PLD gene family have been known to be induced under different abiotic stresses and during developmental processes in various plant species. In this report, we are presenting a detailed microarray based expression analysis and expression profiles of entire set of PLD genes in rice genome, under three abiotic stresses (salt, cold and drought) and different developmental stages (3-vegetative stages and 11-reproductive stages). Seven and nine PLD genes were identified, which were expressed differentially under abiotic stresses and during reproductive developmental stages, respectively. PLD genes, which were expressed significantly under abiotic stresses exhibited an overlapping expression pattern and were also differentially expressed during developmental stages. Moreover, expression pattern for a set of stress induced genes was validated by real time PCR and it supported the microarray expression data. These findings emphasize the role of PLDs in abiotic stress signaling and development in rice. In addition, expression profiling for duplicated PLD genes revealed a functional divergence between the duplicated genes and signify the role of gene duplication in the evolution of this gene family in rice. This expressional study will provide an important platform in future for the functional characterization of PLDs in crop plants.