Patterns of codon usage in two ciliates that reassign the genetic code: Tetrahymena thermophila and Paramecium tetraurelia

We used the recently sequenced genomes of the ciliates Tetrahymena thermophila and Paramecium tetraurelia to analyze the codon usage patterns in both organisms; we have analyzed codon usage bias, Gln codon usage, GC content and the nucleotide contexts of initiation and termination codons in Tetrahymena and Paramecium. We also studied how these trends change along the length of the genes and in a subset of highly expressed genes. Our results corroborate some of the trends previously described in Tetrahymena, but also negate some specific observations. In both genomes we found a strong bias toward codons with low GC content; however, in highly expressed genes this bias is smaller and codons ending in GC tend to be more frequent. We also found that codon bias increases along gene segments and in highly expressed genes and that the context surrounding initiation and termination codons are always AT rich. Our results also suggest differences in the efficiency of translation of the reassigned stop codons between the two species and between the reassigned codons. Finally, we discuss some of the possible causes for such translational efficiency differences.     

Analysis of synonymous codon usage bias in Chlamydia

Chlamydiae are obligate intracellular bacterial pathogens that cause ocular and sexuallytransmitted diseases,and are associated with cardiovascular diseases.The analysis of codon usage mayimprove our understanding of the evolution and pathogenesis of Chlamydia and allow reengineering of targetgenes to improve their expression for gene therapy.Here,we analyzed the codon usage of C.muridarum,C.trachomatis(here indicating biovar trachoma and LGV),C.pneumoniae,and C.psittaci using the codonusage database and the CUSP(Create a codon usage table)program of EMBOSS(The European MolecularBiology Open Software Suite).The results show that the four genomes have similar codon usage patterns,with a strong bias towards the codons with A and T at the third codon position.Compared with Homosapiens,the four chlamydial species show discordant seven or eight preferred codons.The ENC(effectivenumber of codons used in a gene)-plot reveals that the genetic heterogeneity in Chlamydia is constrained bythe G+C content,while translational selection and gene length exert relatively weaker influences.Moreover,mutational pressure appears to be the major determinant of the codon usage variation among the chlamydialgenes.In addition,we compared the codon preferences of C.trachomatis with those of E.coli,yeast,adenovirus and Homo sapiens.There are 23 codons showing distinct usage differences between C.trachomatisand E.coli,24 between C.trachomatis and adenovirus,21 between C.trachomatis and Homo sapiens,butonly six codons between C.trachomatis and yeast.Therefore,the yeast system may be more suitable for theexpression of chlamydial genes.Finally,we compared the codon preferences of C.trachomatis with those ofsix eukaryotes,eight prokaryotes and 23 viruses.There is a strong positive correlation between the differ-ences in coding GC content and the variations in codon bias(r=0.905,P<0,001).We conclude that thevariation of codon bias between C.trachomatis and other organisms is much less influenced by phylogeneticlineage and primarily determined by the extent of disparities in GC content.     

普通野生稻线粒体蛋白质编码基因密码子使用偏好性的分析

以普通野生稻(Oryza rufipogon Griff.)线粒体基因组为对象,分析其蛋白质编码基因的密码子使用特征及与亚洲栽培稻(O. sativa L.)的差异,探讨其密码子偏性形成的影响因素和进化过程。结果显示:普通野生稻线粒体基因组编码序列第1、第2和第3位碱基的GC含量依次为49.18%、42.67%和40.86%;有效密码子数(Nc)分布于45.32～61.00之间,其密码子偏性较弱; Nc值仅与GC_3呈显著相关,密码子第3位的碱基组成对密码子偏性影响较大;第1向量轴上显示9.91%的差异,其与GC_3s、Nc、密码子偏好指数(CBI)和最优密码子使用频率(Fop)的相关性均达到显著水平;而GC_3和GC₁₂的相关性未达到显著水平。因此,普通野生稻线粒体基因组密码子的使用偏性主要受自然选择压力影响而形成。本研究确定了21个普通野生稻线粒体基因组的最优密码子,大多以A或T结尾,与叶绿体密码子具有趋同进化,但是与核基因组具有不同的偏好性。同义密码子相对使用度(RSCU)、PR2偏倚分析和中性绘图分析显示,普通野生稻线粒体基因功能和其密码子使用密切相关,且线粒体密码子使用在普通野生稻、粳稻(O. sativa L. subsp. japonica Kato)和籼稻(O. sativa L. subsp.indica Kato)内具有同质性。     

Constraint on di-nucleotides by codon usage bias in bacterial genomes

It has been reported earlier that the relative di-nucleotide frequency (RDF) in different parts of a genome is similar while the frequency is variable among different genomes. So RDF is termed as genome signature in bacteria. It is not known if the constancy in RDF is governed by genome wide mutational bias or by selection. Here we did comparative analysis of RDF between the inter-genic and the coding sequences in seventeen bacterial genomes, whose gene expression data was available. The constraint on di-nucleotides was found to be higher in the coding sequences than that in the inter-genic regions and the constraint at the 2nd codon position was more than that in the 3rd position within a genome. Further analysis revealed that the constraint on di-nucleotides at the 2nd codon position is greater in the high expression genes (HEG) than that in the whole genomes as well as in the low expression genes (LEG). We analyzed RDF at the 2nd and the 3rd codon positions in simulated coding sequences that were computationally generated by keeping the codon usage bias (CUB) according to genome G+C composition and the sequence of amino acids unaltered. In the simulated coding sequences, the constraint observed was significantly low and no significant difference was observed between the HEG and the LEG in terms of di-nucleotide constraint. This indicated that the greater constraint on di-nucleotides in the HEG was due to the stronger selection on CUB in these genes in comparison to the LEG within a genome. Further, we did comparative analyses of the RDF in the HEG rpoB and rpoC of 199 bacteria, which revealed a common pattern of constraints on di-nucleotides at the 2nd codon position across these bacteria. To validate the role of CUB on di-nucleotide constraint, we analyzed RDF at the 2nd and the 3rd codon positions in simulated rpoB/rpoC sequences. The analysis revealed that selection on CUB is an important attribute for the constraint on di-nucleotides at these positions in bacterial genomes. We believe that this study has come with major findings of the role of CUB on di-nucleotide constraint in bacterial genomes.     

Codon bias signatures, organization of microorganisms in codon space, and lifestyle

New and simple numerical criteria based on a codon adaptation index are applied to the complete genomic sequences of 80 Eubacteria and 16 Archaea, to infer weak and strong genome tendencies toward content bias, translational bias, and strand bias. These criteria can be applied to all microbial genomes, even those for which little biological information is known, and a codon bias signature, that is the collection of strong biases displayed by a genome, can be automatically derived. A codon bias space, where genomes are identified by their preferred codons, is proposed as a novel formal framework to interpret genomic relationships. Principal component analysis confirms that although GC content has a dominant effect on codon bias space, thermophilic and mesophilic species can be identified and separated by codon preferences. Two more examples concerning lifestyle are studied with linear discriminant analysis: suitable separating functions characterized by sets of preferred codons are provided to discriminate: translationally biased (hyper)thermophiles from mesophiles, and organisms with different respiratory characteristics, aerobic, anaerobic, facultative aerobic and facultative anaerobic. These results suggest that codon bias space might reflect the geometry of a prokaryotic "physiology space." Evolutionary perspectives are noted, numerical criteria and distances among organisms are validated on known cases, and various results and predictions are discussed both on methodological and biological grounds.     

敏捷气热菌起始密码子侧翼序列与密码子偏好及基因长度相关性研究

好氧超嗜热古菌敏捷气热菌 (Aeropyrumpernix)mRNA中起始密码子AUG侧翼序列的保守性以及它与密码子使用偏好及基因长度之间具有相关性。AUG侧翼序列的保守性由M1(1)值表示 ,AUG侧翼序列对翻译起始的有效性由AUGCAI值表示。研究表明 :高表达和低表达基因的 - 2 0位到 13位中某些位点的保守性存在差异 ,其中高表达基因的 - 4位和 - 3位可能与其高表达的特性有关 ;在A .pernix中一个普遍的趋势是 :较短的基因有较高的表达效率 ,较长的基因的表达效率较低。与仅使用密码子偏好相比 ,将AUGCAI值引入到研究古菌在翻译水平上的自然选择更准确、更具有广泛适应性     

Synonymous codon usage in forty staphylococcal phages identifies the factors controlling codon usage variation and the phages suitable for phage therapy

The immergence and dissemination of multidrug-resistant strains of Staphylococcus aureus in recent years have expedited the research on the discovery of novel anti-staphylococcal agents promptly. Bacteriophages have long been showing tremendous potentialities in curing the infections caused by various pathogenic bacteria including S. aureus. Thus far, only a few virulent bacteriophages, which do not carry any toxin-encoding gene but are capable of eradicating staphylococcal infections, were reported. Based on the codon usage analysis of sixteen S. aureus phages, previously three phages were suggested to be useful as the anti-staphylococcal agents. To search for additional S. aureus phages suitable for phage therapy, relative synonymous codon usage bias has been investigated in the protein-coding genes of forty new staphylococcal phages. All phages appeared to carry A and T ending codons. Several factors such as mutational pressure, translational selection and gene length seemed to be responsible for the codon usage variation in the phages. Codon usage indeed varied phage to phage. Of the phages, phages G1, Twort, 66 and Sap-2 may be extremely lytic in nature as majority of their genes possess high translational efficiency, indicating that these phages may be employed in curing staphylococcal infections.     

Effect of strong directional selection on weakly selected mutations at linked sites: implication for synonymous codon usage

The fixation of weakly selected mutations can be greatly influenced by strong directional selection at linked loci. Here, I investigate a two-locus model in which weakly selected, reversible mutations occur at one locus and recurrent strong directional selection occurs at the other locus. This model is analogous to selection on codon usage at synonymous sites linked to nonsynonymous sites under strong directional selection. Two approximations obtained here describe the expected frequency of the weakly selected preferred alleles at equilibrium. These approximations, as well as simulation results, show that the level of codon bias declines with an increasing rate of substitution at the strongly selected locus, as expected from the well-understood theory that selection at one locus reduces the efficacy of selection at linked loci. These solutions are used to examine whether the negative correlation between codon bias and nonsynonymous substitution rates recently observed in Drosophila can be explained by this hitchhiking effect. It is shown that this observation can be reasonably well accounted for if a large fraction of the nonsynonymous substitutions on genes in the data set are driven by strong directional selection.     

Evidence for a trade-off between translational efficiency and splicing regulation in determining synonymous codon usage in Drosophila melanogaster

In Drosophila melanogaster, synonymous codons corresponding to the most abundant cognate tRNAs are used more frequently, especially in highly expressed genes. Increased use of such "optimal" codons is considered an adaptation for translational efficiency. Need it always be the case that selection should favor the use of a translationally optimal codon? Here, we investigate one possible confounding factor, namely, the need to specify information in exons necessary to enable correct splicing. As expected from such a model, in Drosophila many codons show different usage near intron-exon boundaries versus exon core regions. However, this finding is in principle also consistent with Hill-Robertson effects modulating usage of translationally optimal codons. However, several results support the splice model over the translational selection model: 1) the trends in codon usage are strikingly similar to those in mammals in which codon usage near boundaries correlates with abundance in exonic splice enhancers (ESEs), 2) codons preferred near boundaries tend to be enriched for A and avoid C (conversely those avoided near boundaries prefer C rather than A), as expected were ESEs involved, and 3) codons preferred near boundaries are typically not translationally optimal. We conclude that usage of translationally optimal codons usage is compromised in the vicinity of splice junctions in intron-containing genes, to the effect that we observe higher levels of usage of translationally optimal codons at the center of exons. On the gene level, however, controlling for known correlates of codon bias, the impact on codon usage patterns is quantitatively small. These results have implications for inferring aspects of the mechanism of splicing given nothing more than a well-annotated genome.     

Synonymous codon usage in Escherichia coli: selection for translational accuracy

In many organisms, selection acts on synonymous codons to improve translation. However, the precise basis of this selection remains unclear in the majority of species. Selection could be acting to maximize the speed of elongation, to minimize the costs of proofreading, or to maximize the accuracy of translation. Using several data sets, we find evidence that codon use in Escherichia coli is biased to reduce the costs of both missense and nonsense translational errors. Highly conserved sites and genes have higher codon bias than less conserved ones, and codon bias is positively correlated to gene length and production costs, both indicating selection against missense errors. Additionally, codon bias increases along the length of genes, indicating selection against nonsense errors. Doublet mutations or replacement substitutions do not explain our observations. The correlations remain when we control for expression level and for conflicting selection pressures at the start and end of genes. Considering each amino acid by itself confirms our results. We conclude that selection on synonymous codon use in E. coli is largely due to selection for translational accuracy, to reduce the costs of both missense and nonsense errors.     

Codon usage in mitochondrial genomes: distinguishing context-dependent mutation from translational selection

We analyze the frequencies of synonymous codons in animal mitochondrial genomes, focusing particularly on mammals and fish. The frequencies of bases at 4-fold degenerate sites are found to be strongly influenced by context-dependent mutation, which causes correlations between pairs of neighboring bases. There is a pattern of excess of certain dinucleotides and deficit of others that is consistent across large numbers of species, despite the wide variation of single-nucleotide frequencies among species. In many bacteria, translational selection is an important influence on codon usage. In order to test whether translational selection also plays a role in mitochondria, we need to control for context-dependent mutation. Selection for translational accuracy can be detected by comparison of codon usage in conserved and variable sites in the same genes. We give a test of this type that works in the presence of context-dependent mutation. There is very little evidence for translational accuracy selection in the mitochondrial genes considered here. Selection for translational efficiency might lead to preference for codons that match the limited repertoire of anticodons on the mitochondrial tRNAs. This is difficult to detect because the effect would usually be in the same direction in comparable to codon families and so would not cause an observable difference in codon usage between families. Several lines of evidence suggest that this type of selection is weak in most cases. However, we found several cases where unusual bases occur at the wobble position of the tRNA, and in these cases, some evidence for selection on codon usage was found. We discuss the way that these unusual cases are associated with codon reassignments in the mitochondrial genetic code.     

Characteristics of codon usage bias in two regions downstream of the initiation codons of foot-and-mouth disease virus

The mechanism of utilization of alternative two AUGs in foot-and-mouth disease virus (FMDV) is still unknown to date. In this study, the characteristics of codon usage bias (CUB) of the region between the two AUGs (the region-La) and of the same-sized region behind the second AUG (the region-Lb) in 94 different FMDV RNA sequences were analyzed using relative synonymous codon usage (RSCU) values. The results indicated that many codons with negative CUB were preferentially used in the region-La. There were two conserved residues (Thr and Cys) on the 4th and 6th residue positions of the region-La. The conserved residues had a general tendency to choose synonymous codons with negative CUB. Although most positions in the region-La did not contain conserved residues, many positions tended to use codons with negative CUB in this region. Among these codons, the majority belonged to the amino acids containing synonymous codons with clearly positive and negative CUB, including Asp, Val, Ile, Leu, Thr, Ala, Ser, Asn and Arg. The presence of many codons with negative CUB in the region-La might impair the efficiency of the first AUG selection. The phylogenetic incongruence of the region-La and the region-Lb implied that intertypic recombination played an important role in the evolution of FMDV. Furthermore, due to the existence of more positions with positive CUB and more widespread phylogenetic incongruence in the region-Lb than the region-La, a probable relationship between the degree of CUB and the evolution of the two target regions was revealed.     

Impact of bias discrepancy and amino acid usage on estimates of the effective number of codons used in a gene, and a test for selection on codon usage

The effective number of codons (Nc) used in a gene is one of the most commonly used measures of synonymous codon usage bias, owing much of its popularity to the fact that it is species independent and that simulation studies have shown that it is less dependent of gene length than other measures. In this paper I provide a clear and practically meaningful definition of bias discrepancy (BD; when the degree of codon bias varies within a degeneracy class). Moreover I evaluate the impact of BD and amino acid usage on estimates of Nc. It is shown that both factors have a significant effect on accuracy and precision. Both amino acid usage and BD influence accuracy considerably, especially in short genes. Finally, I demonstrate how the definition of bias discrepancy can be applied to investigate if codon usage is influenced by selection and I discuss this test in relation to the incongruous literature that exists for Buchnera sp. APS and Borrelia burgdorferi.     

Stress induced MAPK genes show distinct pattern of codon usage in Arabidopsis thaliana,Glycine max and Oryza sativa

Mitogen activated protein kinase (MAPK) genes provide resistance to various biotic and abiotic stresses. Codon usage profiling of the genes reveals the characteristic features of the genes like nucleotide composition, gene expressivity, optimal codons etc. The present study is a comparative analysis of codon usage patterns for different MAPK genes in three organisms, viz. Arabidopsis thaliana, Glycine max (soybean) and Oryza sativa (rice). The study has revealed a high AT content in MAPK genes of Arabidopsis and soybean whereas in rice a balanced AT-GC content at the third synonymous position of codon. The genes show a low bias in codon usage profile as reflected in the higher values (50.83 to 56.55) of effective number of codons (N_c). The prediction of gene expression profile in the MAPK genes revealed that these genes might be under the selective pressure of translational optimization as reflected in the low codon adaptation index (CAI) values ranging from 0.147 to 0.208.     

Genomic characteristics comparisons of 12 food-related filamentous fungi in tRNA gene set, codon usage and amino acid composition

Filamentous fungi are widely exploited in food industry due to their abilities to secrete large amounts of enzymes and metabolites. The recent availability of fungal genome sequences has provided an opportunity to explore the genomic characteristics of these food-related filamentous fungi. In this paper, we selected 12 representative filamentous fungi in the areas of food processing and safety, which were Aspergillus clavatus, A. flavus, A. fumigatus, A. nidulans, A. niger, A. oryzae, A. terreus, Monascus ruber, Neurospora crassa, Penicillium chrysogenum, Rhizopus oryzae and Trichoderma reesei, and did the comparative studies of their genomic characteristics of tRNA gene distribution, codon usage pattern and amino acid composition. The results showed that the copy numbers greatly differed among isoaccepting tRNA genes and the distribution seemed to be related with translation process. The results also revealed that genome compositional variation probably constrained the base choice at the third codon, and affected the overall amino acid composition but seemed to have little effect on the integrated physicochemical characteristics of overall amino acids. The further analysis suggested that the wobble pairing and base modification were the important mechanisms in codon-anticodon interaction. In the scope of authors' knowledge, it is the first report about the genomic characteristics analysis of food-related filamentous fungi, which would be informative for the analysis of filamentous fungal genome evolution and their practical application in food industry.     

Efficient tRNA degradation and quantification in Escherichia coli cell extract using RNase‐coated magnetic beads: A key step toward codon emancipation

Emancipating sense codons toward a minimized genetic code is of significant interest to science and engineering. A key approach toward sense codon emancipation is the targeted in vitro removal of native tRNA. However, challenges remain such as the insufficient depletion of tRNA in lysate‐based in vitro systems and the high cost of the purified components system (PURE). Here we used RNase‐coated superparamagnetic beads to efficiently degrade E. coli endogenous tRNA. The presented method removes >99% of tRNA in cell lysates, while partially preserving cell‐free protein synthesis activity. The resulting tRNA‐depleted lysate is compatible with in vitro‐transcribed synthetic tRNA for the production of peptides and proteins. Additionally, we directly measured residual tRNA using quantitative real‐time PCR. © 2017 American Institute of Chemical Engineers Biotechnol. Prog., 33:1401–1407, 2017     

ANCAC: amino acid, nucleotide, and codon analysis of COGs -- a tool for sequence bias analysis in microbial orthologs

ABSTRACT: BACKGROUND: The COG database is the most popular collection of orthologous proteins from many different completely sequenced microbial genomes. Per definition, a cluster of orthologous groups (COG) within this database exclusively contains proteins that most likely achieve the same cellular function. Recently, the COG database was extended by assigning to every protein both the corresponding amino acid and its encoding nucleotide sequence resulting in the NUCOCOG database. This extended version of the COG database is a valuable resource connecting sequence features with the functionality of the respective proteins. RESULTS: Here we present ANCAC, a web tool and MySQL database for the analysis of amino acid, nucleotide, and codon frequencies in COGs on the basis of freely definable phylogenetic patterns. We demonstrate the usefulness of ANCAC by analyzing amino acid frequencies, codon usage, and GC-content in a species- or function-specific context. With respect to amino acids we, at least in part, confirm the cognate bias hypothesis by using ANCAC's NUCOCOG dataset as the largest one available for that purpose thus far. CONCLUSIONS: Using the NUCOCOG datasets, ANCAC connects taxonomic, amino acid, and nucleotide sequence information with the functional classification via COGs and provides a GUI for flexible mining for sequence-bias. Thereby, to our knowledge, it is the only tool for the analysis of sequence composition in the light of physiological roles and phylogenetic context without requirement of substantial programming-skills.     

Allozyme variation in American ginseng (Panax quinquefolius L.): Variation, breeding system, and implications for current conservation practice

Sustained harvest of wild North Americanginseng (Panax quinquefolius L.) for overtwo centuries has led to heightenedconservation concern and a recent interest inthe population genetics of this species. Thisstudy examined allozyme variation from 32 wildand 12 cultivated populations of Americanginseng to: (1) document the amount anddistribution of genetic variation over a wideportion of the species' natural range, (2)examine genetic differences between wild andcultivated populations, and (3) provideindirect estimates of its breeding system. Strong genetic differences between wild andcultivated populations were found in the amountof variation within populations and thedistribution of variation among populations. Wild populations were significantly lower inall within-population diversity measures, butcontained significantly higher levels ofvariation partitioned among populations. Similarities between wild and cultivatedpopulations were also found. As a whole,cultivated and wild groups shared nearly allalleles, and populations of both groups showedstrong homozygote excess compared toexpectations under random mating. Thehomozygote excess is best explained by highlevels of selfing. In wild populations,significant correlations were found betweengenetic diversity and estimated populationsize, and between interpopulation geneticdistance and geographic distance. Overall, theresults for wild populations suggest that theyare influenced by high levels of genetic driftand low migration among populations. Conservation implications for American ginsengare discussed with particular emphases on: (1)the current debate surrounding the existence ofwild populations, (2) the ongoing practice ofintroducing cultivated seed into wildpopulations, and (3) the collection of geneticmaterial for the establishment of breedingprograms.