Synonymous Codon Usage Bias Research Articles

Transgene-host cell interactions mediate significant influences on the production, stability, and function of recombinant canine FVIII

Recombinant FVIII manufacturing is characterized by poor product stability and low yields. Codon-optimization of transgenes accelerates translation by exploiting the synonymous codon usage bias of a species. However, this can alter the performance of the final product. Additionally, the effects of transgene design across diverse cell types are not well understood and are of interest for next-generation protein and gene therapies. To investigate the effects of transgene design across different host cells, B-domain-deleted (BDD) and modified codon-optimized (CO-N6) transgenes were inserted via lentiviral delivery into cBOECs, HEK293T, and MDCK cells. The CO-N6 cFVIII transgene produced threefold more protein per transgene in HEK293T cells, and sixfold more protein in the two canine cell lines. However, pharmacokinetic analysis in hemophilia A dogs demonstrated that cFVIII produced from cBOECs transduced with the CO-N6 transgene had significantly reduced in vivo recovery. Furthermore, this product showed reduced in vitro stability and activity on thrombin activation versus the BDD product. This trend was reversed in HEK293T lines. Overall, our results demonstrate the need for an integrated approach that not only assesses protein expression levels but also considers the influence that host-cells have on preserving the molecular and biochemical properties of the naturally occurring FVIII.

Mutational Pressure Drives Evolution of Synonymous Codon Usage in Genetically Distinct Oenothera plastomes

Background: Most of the amino acids are encoded by more than one codon, termed as synonymous codons. Synonymous codon usage is not random as it is unique to species. In each amino acid family, some synonymous codons are preferred and this is referred to as synonymous codon usage bias (SCUB). Trends associated with evolution of SCUB and factors influencing its diversification in plastomes of genetically distinct Oenothera plastomes have not been investigated so far. Objectives: In the present study, major forces that shape SCUB in Oenothera plastomes and putative preferred codons in the protein coding genes (PCG) of plastomes were identified. Materials and Methods: To unravel various features of SCUB across selected Oenothera plastomes, commonly used codon usage indices such as relative synonymous codon usage (RSCU), synonymous codon usage order (SCUO), effective number of codons (ENC) and codon adaptation index (CAI) were calculated. Correspondence analysis (COA) on RSCU was performed to identify various characteristics of SCUB across different PCG in Oenothera plastomes. Spearman’s rank correlation analysis was adopted to correlate nucleotide contents, codon usage indices and major axes of COA to find out critical parameters in shaping SCUB. Results: Mutational bias due to compositional constraints played crucial role in shaping SCUB as T3 and GC3 contents were in strong negative correlation with all axes of COA. Nevertheless, significant negative correlations between axis 1 and 3 with ENC and CAI respectively, in all species, and narrow distribution of GC contents in neutrality plot, indicate the role of natural selection. Hydropathy score of proteins was found to be influencing SCUB in O. glazioviana as it showed strong negative correlation with axis 2. Conclusion: We concluded that mutational pressure coupled with weak selection influenced SCUB in the examined plastomes of Oenothera. In addition, all examined species of Oenothera exist as disjunct populations in different parts of North America and these populations might have experienced genetic drift as random mutations in small populations that have been fixed over a period of time.

Species based synonymous codon usage in fusion protein gene of Newcastle disease virus.

Newcastle disease is highly pathogenic to poultry and many other avian species. However, the Newcastle disease virus (NDV) has also been reported from many non-avian species. The NDV fusion protein (F) is a major determinant of its pathogenicity and virulence. The functionalities of F gene have been explored for the development of vaccine and diagnostics against NDV. Although the F protein is well studied but the codon usage and its nucleotide composition from NDV isolated from different species have not yet been explored. In present study, we have analyzed the factors responsible for the determination of codon usage in NDV isolated from four major avian host species. The F gene of NDV is analyzed for its base composition and its correlation with the bias in codon usage. Our result showed that random mutational pressure is responsible for codon usage bias in F protein of NDV isolates. Aromaticity, GC3s, and aliphatic index were not found responsible for species based synonymous codon usage bias in F gene of NDV. Moreover, the low amount of codon usage bias and expression level was further confirmed by a low CAI value. The phylogenetic analysis of isolates was found in corroboration with the relatedness of species based on codon usage bias. The relationship between the host species and the NDV isolates from the host does not represent a significant correlation in our study. The present study provides a basic understanding of the mechanism involved in codon usage among species.

Seforta, an integrated tool for detecting the signature of selection in coding sequences.

BackgroundThe majority of amino acid residues are encoded by more than one codon, and a bias in the usage of such synonymous codons has been repeatedly demonstrated. One assumption is that this phenomenon has evolved to improve the efficiency of translation by reducing the time required for the recruitment of isoacceptors. The most abundant tRNA species are preferred at sites on the protein which are key for its functionality, a behavior which has been termed “translational accuracy”. Although observed in many species, as yet no public domain software has been made available for its quantification.FindingsWe present here Seforta (Selection for Translational Accuracy), a program designed to quantify translational accuracy. It searches for synonymous codon usage bias in both conserved and non-conserved regions of coding sequences and computes a cumulative odds ratio and a Z-score. The specification of a set of preferred codons is desirable, but the program can also generate these. Finally, a randomization protocol calculates the probability that preferred codon combinations could have arisen by chance.ConclusionsSeforta is the first public domain program able to quantify translational accuracy. It comes with a simple graphical user interface and can be readily installed and adjusted to the user's requirements.

System analysis of synonymous codon usage biases in archaeal virus genomes

Recent studies of geothermally heated aquatic ecosystems have found widely divergent viruses with unusual morphotypes. Archaeal viruses isolated from these hot habitats usually have double-stranded DNA genomes, linear or circular, and can infect members of the Archaea domain. In this study, the synonymous codon usage bias (SCUB) and dinucleotide composition in the available complete archaeal virus genome sequences have been investigated. It was found that there is a significant variation in SCUB among different Archaeal virus species, which is mainly determined by the base composition. The outcome of correspondence analysis (COA) and Spearman׳s rank correlation analysis shows that codon usage of selected archaeal virus genes depends mainly on GC richness of genome, and the gene׳s function, albeit with smaller effects, also contributes to codon usage in this virus. Furthermore, this investigation reveals that aromaticity of each protein is also critical in affecting SCUB of these viral genes although it was less important than that of the mutational bias. Especially, mutational pressure may influence SCUB in SIRV1, SIRV2, ARV1, AFV1, and PhiCh1 viruses, whereas translational selection could play a leading role in HRPV1׳s SCUB. These conclusions not only can offer an insight into the codon usage biases of archaeal virus and subsequently the possible relationship between archaeal viruses and their host, but also may help in understanding the evolution of archaeal viruses and their gene classification, and more helpful to explore the origin of life and the evolution of biology.

Bioinformatics Study Involving Characterization of Synonymous Codon Usage Bias in the Duck Enteritis Virus Glycoprotein D (gD) Gene

Bioinformatics Study Involving Characterization of Synonymous Codon Usage Bias in the Duck Enteritis Virus Glycoprotein D (gD) Gene

Characteristics of synonymous codon usage bias in the beginning region of West Nile virus.

Adaptation in the overall codon usage pattern of West Nile virus (WNV) to that of two hosts was estimated based on the synonymous codon usage value (RSCU). Synonymous codon usage biases for the beginning coding sequence of this virus were also analyzed by calculating the usage fluctuation for each synonymous codon along the target region (the first 270 codon sites of the whole coding sequence of WNV). Adaptation of WNV to Anopheles gambiae regarding the overall codon usage revealed a mixture of synonymous codon usage patterns between this virus and its vector. Regarding the adaptation of WNV to its dead-end host and codon usage, although a mixture of overall codon usage patterns exists, the number of codons with reversed tendency codon usage is lower than that between the virus and its vector. In addition, some codons with low RSCU values for this virus are highly selected in the beginning translation region of WNV, while codons with low RSCU values in this region tend to pair with tRNAs present in low abundance in the host, suggesting that highly selected codons in a specific region in the beginning region of WNV are, to some degree, influenced by the corresponding low tRNA abundance of hosts to regulate the translation speed of the WNV polyprotein.

A Comparative Analysis of Synonymous Codon Usage Bias Pattern in Human Albumin Superfamily

Synonymous codon usage bias is an inevitable phenomenon in organismic taxa across the three domains of life. Though the frequency of codon usage is not equal across species and within genome in the same species, the phenomenon is non random and is tissue-specific. Several factors such as GC content, nucleotide distribution, protein hydropathy, protein secondary structure, and translational selection are reported to contribute to codon usage preference. The synonymous codon usage patterns can be helpful in revealing the expression pattern of genes as well as the evolutionary relationship between the sequences. In this study, synonymous codon usage bias patterns were determined for the evolutionarily close proteins of albumin superfamily, namely, albumin, α-fetoprotein, afamin, and vitamin D-binding protein. Our study demonstrated that the genes of the four albumin superfamily members have low GC content and high values of effective number of codons (ENC) suggesting high expressivity of these genes and less bias in codon usage preferences. This study also provided evidence that the albumin superfamily members are not subjected to mutational selection pressure.

Synonymous Codon Usage in TTSuV2: Analysis and Comparison with TTSuV1

Two species of the DNA virus Torque teno sus virus (TTSuV), TTSuV1 and TTSuV2, have become widely distributed in pig-farming countries in recent years. In this study, we performed a comprehensive analysis of synonymous codon usage bias in 41 available TTSuV2 coding sequences (CDS), and compared the codon usage patterns of TTSuV2 and TTSuV1. TTSuV codon usage patterns were found to be phylogenetically conserved. Values for the effective number of codons (ENC) indicated that the overall extent of codon usage bias in both TTSuV2 and TTSuV1 was not significant, the most frequently occurring codons had an A or C at the third codon position. Correspondence analysis (COA) was performed and TTSuV2 and TTSuV1 sequences were located in different quadrants of the first two major axes. A plot of the ENC revealed that compositional constraint was the major factor determining the codon usage bias for TTSuV2. In addition, hierarchical cluster analysis of 41 TTSuV2 isolates based on relative synonymous codon usage (RSCU) values suggested that there was no association between geographic distribution and codon bias of TTSuV2 sequences. Finally, the comparison of RSCU for TTSuV2, TTSuV1 and the corresponding host sequence indicated that the codon usage pattern of TTSuV2 was similar to that of TTSuV1. However the similarity was low for each virus and its host. These conclusions provide important insight into the synonymous codon usage pattern of TTSuV2, as well as better understangding of the molecular evolution of TTSuV2 genomes.

Identification of Synonymous Codon Usage Bias in the Pseudorabies Virus UL31 Gene

Background: Little knowledge of synonymous codon usage pattern of pseudorabies virus (PRV) genome, especially the UL31 gene in the process for its evolution is available. Objectives: In the present study, the codon usage bias between PRV UL31 sequence and the UL31-like sequences was identified. Materials and Methods: We used a comprehensive analysis on codon usage pattern in the PRV UL31 gene and the UL31-like genes of 48 reference herpesviruses by calculating codon adaptation index, ENc, RSCU and EMBOSS assays. Results: Cluster analysis demonstrated that the codon usage bias of UL31-like genes of 49 herpesviruses had a very close relation to their gene functions. In addition, comparison of codon preferences in the UL31 gene of PRV with those of E. coli, yeast and human showed that there were 33 codons showing discrete usage differences between PRV and yeast, 24 between PRV and E. coli, but 22 between PRV and human. Although there were slightly fewer differences in codon usages between PRV and human, the difference is unlikely to be statistically significant, and experimental studies are necessary to establish the most suitable expression system for PRV UL31. Conclusion: These results may further our comprehending of the evolution, pathogenesis and functional studies of PRV.

Analysis of synonymous codon usage in Newcastle disease virus hemagglutinin–neuraminidase (HN) gene and fusion protein (F) gene

Newcastle disease virus (NDV) hemagglutinin-neuraminidase (HN) is a multifunctional protein, which possesses both the receptor recognition and neuraminidase activities. The fusion (F) protein is a type I membrane glycoprotein that mediates the merger of the viral envelope to the host cell membrane. Although the functions of the HN and F proteins have been well studied, however, the factors shaping synonymous codon usage bias and nucleotide composition in HN and F genes have been few reported. In our study, we analyzed synonymous codon usage using the 69 NDV HN and F genes, respectively. The general correlation between base composition and codon usage bias suggests that mutational pressure rather than natural selection is the main factor that determines the codon usage bias in HN and F genes. In addition, other factors, such as the aromaticity and hydrophobicity, also influence the codon usage variation among HN and F genes. This study represents the most comprehensive analysis to date of NDV HN and F genes codon usage patterns and provides a basic understanding of the mechanisms for codon usage bias.

Genetic Code-guided Protein Synthesis and Folding in Escherichia coli

Universal genetic codes are degenerated with 61 codons specifying 20 amino acids, thus creating synonymous codons for a single amino acid. Synonymous codons have been shown to affect protein properties in a given organism. To address this issue and explore how Escherichia coli selects its "codon-preferred" DNA template(s) for synthesis of proteins with required properties, we have designed synonymous codon libraries based on an antibody (scFv) sequence and carried out bacterial expression and screening for variants with altered properties. As a result, 342 codon variants have been identified, differing significantly in protein solubility and functionality while retaining the identical original amino acid sequence. The soluble expression level varied from completely insoluble aggregates to a soluble yield of ~2.5 mg/liter, whereas the antigen-binding activity changed from no binding at all to a binding affinity of > 10(-8) m. Not only does our work demonstrate the involvement of genetic codes in regulating protein synthesis and folding but it also provides a novel screening strategy for producing improved proteins without the need to substitute amino acids.

Clustering of low usage codons in the translation initiation region of hepatitis C virus

The adaptation of the overall codon usage pattern of hepatitis C virus (HCV) to that of human is estimated by the synonymous codon usage value (RSCU). The synonymous codon usage biases for the translation initiation region (TIR) of this virus are also analyzed by calculation of usage fluctuation of each synonymous codon along the TIR (the first 30 codon sites of the whole coding sequence of HCV). As for the overall codon usage pattern of HCV, this virus has a significant tendency to delete the codons with CpG or TpA dinucleotides. Turning to the adaptation of the overall codon usage of HCV to that of human, over half part of codons has a similar usage pattern between this virus and human, suggesting that the host cellular environment of the overall codon usage pattern influences the formation of codon usage for HCV. In addition, there is no obvious phenomenon that the codons with relatively low energy tend to be highly selected in the TIR of HCV, suggesting that the synonymous codon usage patterns for the TIR of HCV might be not affected by the secondary structure of nucleotide sequence, however, the formation of synonymous codons usage in the TIR of HCV is influenced by the overall codon usage patterns of human to some degree.

Synonymous codon usage bias is correlative to intron number and shows disequilibrium among exons in plants

BackgroundEvidence has been assembled to suggest synonymous codon usage bias (SCUB) has close relationship with intron. However, the relationship (if any) between SCUB and intron number as well as exon position is at present rather unclear.ResultsTo explore this relationship, the sequences of a set of genes containing between zero and nine introns was extracted from the published genome sequences of three algal species, one moss, one fern and six angiosperms (three monocotyledonous species and three dicotyledonous species). In the algal genomes, the frequency of synonymous codons of the form NNG/NNC (codons with G and C at the third position) was positively related to intron number, but that of NNA/NNT was inversely correlated; the opposite was the case in the land plant genomes. The frequency of NNC/NNG was higher and that of NNA/NNT lower in two terminal exons than in the interstitial exons in the land plant genes, but the rule showed to be opposite in the algal genes. SCUB patterns in the interstitial and two terminal exons mirror the different evolutionary relationships between these plant species, while the first exon shows the highest level of conservation is therefore concluded to be the one which experiences the heaviest selection pressure. The phenomenon of SCUB may also be related to DNA methylation induced conversion of CG to AT.ConclusionsThese data provide some evidence of linkage between SCUB, the evolution of introns and DNA methylation, which brings about a new perspective for understanding how genomic variation is created during plant evolution.

Characterization of Synonymous Codon Usage Bias in the Riemerella Anatipestifer OmpA/MotB Gene

The analysis on codon usage bias of OmpA/MotB gene of Riemerella anatipestifer (RA) may provide a basis for understanding the evolution and pathogenesis of RA and for selecting appropriate host expression systems to improve the expression of target genes in vivo and in vitro. In our study, a comparative analysis of the codon usage bias in the newly discovered RA OmpA/MotB gene and the OmpA/MotB gene of 20 reference flavobacteriaceae was performed. The results of the codon adaptation indes (CAI), effective number of codon (ENC), and GC3s values indicated that synonymous codon usage bias in the OmpA/MotB gene of flavobacteriaceae. The results showed that codon usage bias in the RA OmpA/MotB gene was strong bias towards the synonymous codons with A and T at the third codon position. A high level of diversity in codon usage bias existed, and the effective number of codons used in a gene plot revealed that the G+C compositional constraint is the main factor that determines the codon usage bias in OmpA/MotB gene of flavobacteriaceae. Comparison of the codon usage in the OmpA/MotB gene of different organisms revealed that there were 31 codons showing distinct codon usage differences between the RA and E. coli, 41 between the RA and humans, but 29 between the RA and yeast. Therefore the yeast expression system may be more suitable for the expression of RA OmpA/MotB gene. These results may improve our understanding of the evolution, pathogenesis and functional studies of RA, as well as contribute significantly to the area of flavobacteriaceae research.

The Influence of Synonymous Codon Bias on Protein Folding Rates

Based on the present small data of protein folding rates and related works, a bigger protein folding rates data were constructed, and the information of mRNA of each protein was added in the new data. For each protein, the codon usage bias of its corresponding mRNA sequence was calculated, then, for each kind of proteins, the correlation between protein coding rates and the codon usage bias of their corresponding mRNA sequence were analyzed. The results showed that, for some codons, protein coding rates significantly correlate with the codon usage bias of their corresponding mRNA sequence. It means that synonymous codon usage bias may play an important role in protein folding.

Characterization of Codon Usage Bias in the RA Ragb/SusD Gene

The analysis on codon usage bias of Riemerella anatipestifer (RA) RagB/SusD gene (GenBank accession No. NC_017045.1) may improve our understanding of the evolution and pathogenesis of RA and provide a basis for understanding the relevant mechanism for biased usage of synonymous codons and for selecting appropriate expression systems to improve the expression of target genes. In this study, the synonymous codon usage in the RagB/SusD gene of RA and 19 reference bacteroidetes have been investigated. The results showed that codon usage bias in the RagB/SusD gene was strong bias towards the synonymous codons with A and T at the third codon position. A high level of diversity in codon usage bias existed, and the effective number of codons used in a gene plot revealed that the genetic heterogeneity in RagB/SusD gene of bacteroidetes was constrained by the G + C content. The codon adaptation index (CAI), effective number of codons (ENC), and GC3S values indicated synonymous codon usage bias in the RagB/SusD gene of bacteroidetes, and this synonymous bias was correlated with host evolution. The phylogentic analysis suggested that RagB/SusD was evolutionarily closer to Ornithobacterium rhinotracheale and that there was no significant deviation in codon usage in different bacteroidetes. There are 25 codons showing distinct usage differences between RA RagB/SusD and E. coli, 30 between RA RagB/SusD and Homo sapiens, 26 codons between RA RagB/SusD and yeast. Therefore the yeast and E. coli expression system may be suitable for the expression of RA RagB/SusD gene if some codons could be optimized.

A Comparison of Synonymous Codon Usage Bias Patterns in DNA and RNA Virus Genomes: Quantifying the Relative Importance of Mutational Pressure and Natural Selection

Codon usage bias patterns have been broadly explored for many viruses. However, the relative importance of mutation pressure and natural selection is still under debate. In the present study, I tried to resolve controversial issues on determining the principal factors of codon usage patterns for DNA and RNA viruses, respectively, by examining over 38000 ORFs. By utilizing variation partitioning technique, the results showed that 27% and 21% of total variation could be attributed to mutational pressure, while 5% and 6% of total variation could be explained by natural selection for DNA and RNA viruses, respectively, in codon usage patterns. Furthermore, the combined effect of mutational pressure and natural selection on influencing codon usage patterns of viruses is substantial (explaining 10% and 8% of total variation of codon usage patterns). With respect to GC variation, GC content is always negatively and significantly correlated with aromaticity. Interestingly, the signs for the significant correlations between GC, gene lengths, and hydrophobicity are completely opposite between DNA and RNA viruses, being positive for DNA viruses while being negative for RNA viruses. At last, GC12 versus G3s plot suggests that natural selection is more important than mutational pressure on influencing the GC content in the first and second codon positions.

Characterization of Synonymous Codon Usage Bias in the Riemerella Anatipestifer Major Facilitator Superfamily Mfs_1 Gene Encoded 492 Amino Acids Protein

Objective: The aim was to identify the codon usage bias between the newly identified Riemerella anatipestifer (RA) major facilitator superfamily mfs_1 gene (GenBank accession No.CP003388.1) and other reference bacteriums. Methods: A comparative analysis of the codon usage bias of the 18 bacteriums was performed by using the CodonW 1.4 program and CUSP (create a codon usage table) program of EMBOSS (The European Molecular Biology Open Software Suite). Results: The results showed obvious differences of the synonymous codon usage bias in the 18 bacteriums indicated by the Codon Adaptation Index (CAI), effective number of codons (ENc), and the value of G+C content at the 3rd codon position. The codon usage pattern of the major facilitator superfamily mfs_1 gene was phylogenetically conserved and similar to that of the major facilitator superfamily mfs_1 gene of the Elizabethkingia anophelis Ag1 Contig17, with a strong bias towards the codons with A and T at the 3rd codon position. A cluster analysis of codon usage pattern of the RA major facilitator superfamily mfs_1 gene with other reference bacteriums demonstrated that the codon usage bias of the major facilitator superfamily mfs_1 genes of the 18 bacteriums had a very close relation with their gene function. The ENc-plot revealed that the genetic heterogeneity in the RA major facilitator superfamily mfs_1 gene and the 18 reference bacteriums were constrained by G+C content, while gene length exerted relatively weaker influences. In addition, comparisons of the codon preferences in the major facilitator superfamily mfs_1 gene of RA with those of Escherichia coli, yeast and humans revealed that there were 36 codons showing distinct usage differences between the RA and E. coli, and 37 between the RA and humans, but only 28 between the RA and yeast. Therefore, the yeast system may be more suitable for the expression of the RA major facilitator superfamily mfs_1 gene. Conclusion: Together, these results may improve our understanding of the evolution, pathogenesis and functional studies of RA and possibly contribute significantly to the area of other bacteriums.

Characterization of Synonymous Codon Usage Bias in the UL1 Gene of Duck Plague Virus

In this study, we calculated the codon usage bias in DPV CHv UL1 gene and performed a comparative analysis of synonymous codon patterns in UL1 of DPV CHv strain and other 19 reference herpesviruses. The results revealed that the synonymous codons with A and T at the third codon positon have widely usage in the codon of UL1 gene of DPV CHv. G + C compositional constraint was the main factor that determined the codon usage bias in UL1 gene. In addition, the codon usage bias of DPV CHv UL1 gene was compared with those of E. coli, yeast and human. There are 25 codons showing distinct usage differences between DPV and E. coli, 26 codons between DPV and yeast, and 21 codons between DPV and human. Therefore, the Human expression system is more suitable for heterologous expression of the DPV UL1 gene.