Codon Usage Preference Research Articles

Characterization of Lophiotoma leucotropis Mitochondrial Genome of Family Turridae and Phylogenetic Considerations within the Neogastropoda.

Neogastropoda is a group of marine organisms with an extremely wide distribution that is rich in species and economic and ornamental values, the classification of species in this order has been ongoing for a long time, but there is still a great controversy about whether this order is monophyletic. In this study, we obtained the complete mitogenome of Lophiotoma leucotropis by next-generation sequencing and analyzed the basic structural features of the genome, and we found that the number of genes was consistent with that of most of the Neogastropoda snails, containing 37 genes, including 13 protein-coding genes (PCGs), 2 rRNAs, and 22 tRNAs. Analyzing base content, amino acid content, codon usage preference, and tRNA structure, the mitogenomes of eight species of Turridae were selected for analysis of selection pressures, and it was found that the evolution of species in this family was affected by purifying selection. In addition, by analyzing the rearrangement characteristics, it was found that the sequence of L. leucotropis was consistent with the Conoidea consensus order, and four of the eight species involved in the analysis showed rearrangements. Finally, we constructed a phylogenetic tree by combining PCGs of 60 species within Caenogastropoda and found Neogastropoda to be a monophyletic group, validating the results of morphological classification. The results will provide more references for the classification and species evolution of Neogastropoda, as well as phylogenetic analysis.

Codon usage and evolutionary dynamics of genetic diversity of novel imported porcine reproductive and respiratory syndrome virus in China

Porcine reproductive and respiratory syndrome (PRRS) is a problem that has significant economic impact on the global pig industry. In recent years, there has been an increased importation of pork into China, contributing to the emergence of novely imported porcine reproductive and respiratory syndrome virus (PRRSV) sub-types. Nevertheless, codon usage patterns and their effects on the evolution and adaptation of these new input PRRSV sub-types in hosts remain elusive. To investigate this, we employed a Bayesian approach to analyze two novel imported PRRSV sub-types, namely, NADC30-like and NADC34-like viruses. These sub-types have different codon preferences. Besides, the Effective Number of Codon (ENC) analysis revealed that both NADC30-like and NADC34-like fall within the expected curve distribution, describing a balanced codon usage for both NADC30-like and NADC34-like virus. Based on the Codon Adaptation Index (CAI), NADC30-like showed the highest similarity to the host, aligning with the main prevalence trend of the host. In contrast, NADC34-like exhibited the highest frequency of optimal codon usage; this analysis is based on Frequency of Optimal Codons (FOP). Moreover, the Relative Codon Deoptimization Index (RCDI) indicates that NADC30-like sub-types have a greater degree of inverse optimization sub-type. These findings suggest that mutational pressure affects codon usage preferences of genes in newly imported PRRSV, and that natural selection plays a vital role in determining PRRSV gene codon preferences. Our study provides new insights into the disease, origin, evolutionary patterns, and host adaptation of these newly imported PRRSV sub-types in China. It also contributes to the development of theoretical frameworks for studying genetics and the evolution of PRRSV.

Adaptive evidence of mitochondrial genes in Pteromalidae and Eulophidae (Hymenoptera: Chalcidoidea).

Pteromalidae and Eulophidae are predominant and abundant taxa within Chalcidoidea (Hymenoptera: Apocrita). These taxa are found in diverse ecosystems, ranging from basin deserts (200 m) to alpine grasslands (4500 m). Mitochondria, cellular powerhouses responsible for energy production via oxidative phosphorylation, are sensitive to various environmental factors such as extreme cold, hypoxia, and intense ultraviolet radiation characteristic of alpine regions. Whether the molecular evolution of mitochondrial genes in these parasitoids corresponds to changes in the energy requirements and alpine environmental adaptations remains unknown. In this study, we performed a comparative analysis of mitochondrial protein-coding genes from 11 alpine species of Pteromalidae and Eulophidae, along with 18 lowland relatives, including 16 newly sequenced species. We further examined the codon usage preferences (RSCU, ENC-GC3s, neutrality, and PR2 bias plot) in these mitochondrial protein-coding sequences and conducted positive selection analysis based on their Bayesian phylogenetic relationships, and identified positive selection sites in the ATP6, ATP8, COX1, COX3, and CYTB genes, emphasizing the crucial role of mitochondrial gene adaptive evolution in the adaptation of Pteromalidae and Eulophidae to alpine environments. The phylogenetically independent contrast (PIC) analysis results verified the ω ratio of 13 PCGs from Pteromalidae and Eulophidae increased with elevation, and results from generalized linear model confirm that ATP6, ATP8, COX3, and ND1 are closely correlated with temperature-related environmental factors. This research not only enriched the molecular data of endemic alpine species but also underscores the significance of mitochondrial genes in facilitating the adaptation of these minor parasitoids to plateau habitats.

Subfunctionalisation of paralogous genes and evolution of differential codon usage preferences: The showcase of polypyrimidine tract binding proteins.

Gene paralogs are copies of an ancestral gene that appear after gene or full genome duplication. When two sister gene copies are maintained in the genome, redundancy may release certain evolutionary pressures, allowing one of them to access novel functions. Here, we focused our study on gene paralogs on the evolutionary history of the three polypyrimidine tract binding protein genes (PTBP) and their concurrent evolution of differential codon usage preferences (CUPrefs) in vertebrate species. PTBP1-3 show high identity at the amino acid level (up to 80%) but display strongly different nucleotide composition, divergent CUPrefs and, in humans and in many other vertebrates, distinct tissue-specific expression levels. Our phylogenetic inference results show that the duplication events leading to the three extant PTBP1-3 lineages predate the basal diversification within vertebrates, and genomic context analysis illustrates that local synteny has been well preserved over time for the three paralogs. We identify a distinct evolutionary pattern towards GC3-enriching substitutions in PTBP1, concurrent with enrichment in frequently used codons and with a tissue-wide expression. In contrast, PTBP2s are enriched in AT-ending, rare codons, and display tissue-restricted expression. As a result of this substitution trend, CUPrefs sharply differ between mammalian PTBP1s and the rest of PTBPs. Genomic context analysis suggests that GC3-rich nucleotide composition in PTBP1s is driven by local substitution processes, while the evidence in this direction is thinner for PTBP2-3. An actual lack of co-variation between the observed GC composition of PTBP2-3 and that of the surrounding non-coding genomic environment would raise an interrogation on the origin of CUPrefs, warranting further research on a putative tissue-specific translational selection. Finally, we communicate an intriguing trend for the use of the UUG-Leu codon, which matches the trends of AT-ending codons. Our results are compatible with a scenario in which a combination of directional mutation-selection processes would have differentially shaped CUPrefs of PTBPs in vertebrates: the observed GC-enrichment of PTBP1 in placental mammals may be linked to genomic location and to the strong and broad tissue-expression, while AT-enrichment of PTBP2 and PTBP3 would be associated with rare CUPrefs and thus, possibly to specialized spatio-temporal expression. Our interpretation is coherent with a gene subfunctionalisation process by differential expression regulation associated with the evolution of specific CUPrefs.

Multi-Chromosomal mitochondrial genome of medicinal plant Acorus tatarinowii (Acoraceae): Firstly reported from Acorales Order

Acorus tatarinowii Schott (A. tatarinowii), a well-known traditional Chinese medicinal plant renowned for its high medicinal value, but its mitochondrial genome (mitogenome) is still unexplored. In this study, we meticulously assembled the complete mitochondrial genome of A. tatarinowii using a combination of Illumina short reads and Oxford Nanopore long reads. Our findings revealed that A. tatarinowii possesses a complex chromosomal structural mitogenome, comprising two linear chromosomes and seven circular chromosomes. This mitogenome spans 1.81 Mb in length with a GC content of 38.29 %. Notably, it contained 24 unique mitochondrial core genes, seven unique variable genes, 17 tRNA genes, and three rRNA genes. Analyses of codon usage, most protein-coding genes (PCGs) exhibited a common codon usage preference, with RSCU values greater than 1, and the codon with the highest RSCU value was UAA(End, 1.90). We conducted a thorough analysis of repeat sequences, the distribution of repetitive sequences in nine mitochondrial chromosomes showed distinct patterns. Moreover, we identified 82 and 12 homologous fragments by comparing the sequences of chloroplast and nuclear genomes to the A. tatarinowii mitogenome, respectively. Lastly, We predicted a total of 234 potential RNA editing sites in 28 unique PCGs and discovered that the nad4 gene has been edited the most often, at 26 times. Our results contribute to the enrichment of mitochondrial genome resources for Acoraceae, and the mitogenome also can be used as a reference for other species.

Genome evolution of Buchnera aphidicola (Gammaproteobacteria): Insights into strand compositional asymmetry, codon usage bias, and phylogenetic implications

Taxa of Buchnera aphidicola (hereafter “Buchnera”) are mutualistic intracellular symbionts of aphids, known for their remarkable biological traits such as genome reduction, strand compositional asymmetry, and symbiont-host coevolution. With the growing availability of genomic data, we performed a comprehensive analysis of 103 genomes of Buchnera strains from 12 host subfamilies, focusing on the genomic characterizations, codon usage patterns, and phylogenetic implications. Our findings revealed consistent features among all genomes, including small genome sizes, low GC contents, and gene losses. We also identified strong strand compositional asymmetries in all strains at the genome level. Further investigation suggested that mutation pressure may have played a crucial role in shaping codon usage of Buchnera. Moreover, the genomic asymmetries were reflected in asymmetric codon usage preferences within chromosomal genes. Notably, the levels of these asymmetries were varied among strains and were significantly influenced by the degrees of genome shrinkages. Lastly, our phylogenetic analyses presented an alternative topology of Aphididae, based on the Buchnera symbionts, providing robust confirmation of the paraphylies of Eriosomatinae, and Macrosiphini. Our objectives are to further understand the strand compositional asymmetry and codon usage bias of Buchnera taxa, and provide new perspectives for phylogenetic studies of Aphididae.

Dicodon-based measures for modeling gene expression.

Codon usage preference patterns have been associated with modulation of translation efficiency, protein folding, and mRNA decay. However, new studies support that codon pair usage has also a remarkable effect at the gene expression level. Here, we expand the concept of CAI to answer if codon pair usage patterns can be understood in terms of codon usage bias, or if they offer new information regarding coding translation efficiency. Through the implementation of a weighting strategy to consider the dicodon contributions, we observe that the dicodon-based measure has greater correlations with gene expression level than CAI. Interestingly, we have noted that dicodons associated with a low value of adaptiveness are related to dicodons which mediate strong translational inhibition in yeast. We have also noticed that some codon-pairs have a smaller dicodon contribution than estimated by the product of the respective codon contributions. Scripts, implemented in Python, are freely available for download at https://zenodo.org/record/7738276#.ZBIDBtLMIdU.

Antimicrobial peptides in Dendrobium officinale: Genomic parameters, peptide structures, and gene expression patterns.

A weak codon usage bias was found in Dendrobium catenatum (D. officiale) antimicrobial peptides (AMPs), after the analysis of relative synonymous codon usage, GC contents, and the effective number of codons. The codon usage preference was mainly influenced by natural selection pressure. The self-optimized prediction method and SWISS-MODEL were applied for peptide structural and domain analyses, and some typical antimicrobial domains were found in D. officinale AMP amino sequences, such as knot1 domain, gibberellins-stimulated domain, cupin_1 domain, defensin_like domain, and SLR1-BP (S locus-related glycoprotein 1 binding pollen coat protein) domain. To investigate the AMPs gene expression pattern, abiotic stresses, such as salt stress, drought stress, salicylic acid (SA), and methyl jasmonate (JA), were applied and the gene expression levels were detected by the real-time fluorescent quantitative polymerase chain reaction. Results showed that, even though the basic AMPs gene expressions were low, some AMPs can still be induced by salt dress, while the drought dress did not show the same impact. The SA and JA signaling pathways might be involved in most of the AMPs expressions. The natural selection of the D. officinale AMPs and thus forming diverse types of AMPs enhanced the plant's innate immunity and disease resistance capability, which would lead to a better understanding of the molecular mechanism for D. officinale adapting to the environment. The finding that salt stress, SA, and JA signaling pathways can induce AMP expression lays a foundation for the further development and functional verification of D. officinale AMPs.

Global Spread Dynamics and Intrinsic Evolutionary Dynamics of Apple Chlorotic Leaf Spot Virus Whole-Genome Sequences over the Last 30 Years

Apple chlorotic leaf spot virus (ACLSV; genus Trichovirus) is an economically important virus. Approximately 101 ACLSV whole-genome sequences were obtained from NCBI and used to explore the evolutionary dynamics of ACLSV. The time to the most recent common ancestor of ACLSV based on BEAST analysis appeared on an apple host from Canada in 1918 and then spread around the world in three ways concurrent with host spread. The maximum clade credibility tree of ACLSV shows that the mean evolution rate was 4.92 × 10-4 substitutions per site per year (subs/site/year), and we found that, during host evolution, the rate of evolution was mostly 2.31 × 10-4 to 2.72 × 10-4 subs/site/year. The rate of geographic evolution was 5.51 × 10-4 to 6.17 × 10-4 subs/site/year. To further explore the intrinsic changes in ACLSV during the process of geographic and host spread, we explored the secondary structural changes of the ACLSV coat protein (CP), which were mainly concentrated in four regions-20 to 40, 70 to 90, 120 to 140, and 180 to 193-which are related to the presence or absence and change in length of the β-turn, β strand, coil, and α helix, respectively. We then explored the codon usage preference within the CP across the migration pathways of ACLSV. These comprehensive analyses not only reveal the changes in ACLSV in the last 30 years but also further elucidate the intrinsic evolutionary dynamics of ACLSV. This is also the first report on the intrinsic evolutionary dynamics of ACLSV.

Analysis of Codon Usage Bias in Xyloglucan Endotransglycosylase (XET) Genes

Xyloglucan endotransglycosylase (XET) genes are widely distributed in most plants, but the codon usage bias of XET genes has remained uncharacterized. Thus, we analyzed the codon usage bias using 4500 codons of 20 XET genes to elucidate the genetic and evolutionary patterns. Phylogenetic and hierarchical cluster analyses revealed that the 20 XET genes belonged to two groups. The closer the genetic distance, the more similar the codon usage preference. The codon usage bias of most XET genes was weak, but there was also some codon usage bias. AGA, AGG, AUC, and GUG were the top four codons (RSCU > 1.5) in the 20 XET genes. CitXET had a stronger codon usage bias, and there were eight optimal codons of CitXET (i.e., AGA, AUU, UCU, CUU, CCA, GCU, GUU, and AAA). The RSCU values underwent a correspondence analysis. The two main factors affecting codon usage bias (i.e., Axes 1 and 2) accounted for 54.8% and 17.6% of the total variation, respectively. Multiple correspondence analysis revealed that XET genes were widely distributed, with Group 1 genes being closer to Axis 1 than Group 2 genes, which were closer to Axis 2. Codons with A/U at the third codon position were distributed closer to Axis 1 than codons with G/C at the third codon position. PgXET, ZmXET, VlXET, VrXET, and PcXET were biased toward codons ending with G/C. In contrast, CitXET, DpXET, and BrpXET were strongly biased toward codons ending with A/U, indicating that these XET genes have a strong codon usage bias. Translational selection and base composition (especially A and U at the third codon position), followed by mutation pressure and natural selection, may be the most important factors affecting codon usage of 20 XET genes. These results may be useful in clarifying the codon usage bias of XET genes and the relevant evolutionary characteristics.

The complete mitochondrial genome of Hyotissasinensis (Bivalvia, Ostreoidea) indicates the genetic diversity within Gryphaeidae.

Different from the true oyster (family Ostreidae), the molecular diversity of the gryphaeid oyster (family Gryphaeidae) has never been sufficiently investigated. In the present study, the complete mitochondrial (mt) genome of Hyotissasinensis was sequenced and compared with those of other ostreoids. The total length of H.sinensis mtDNA is 30,385 bp, encoding 12 protein-coding-genes (PCGs), 26 transfer RNA (tRNA) genes and two ribosomal RNA (rRNA) genes. The nucleotide composition and codon usage preference of H.sinensis mtDNA is similar to that of H.hyotis within the same genus. On the other hand, the presence of three trnM and three trnL genes of H.sinensis was not detected neither in H.hyotis nor other ostroid species. Another unique character of H.sinensis mtDNA is that both rrnS and rrnL have a nearly identical duplication. The PCG order of H.sinensis is identical to H.hyotis and the two congener species also share an identical block of 12 tRNA genes. The tRNA rearrangements mostly happen in the region from Cox1 to Nad3, the same area where the duplicated genes are located. The rearrangements within Gryphaeidae could be explained by a "repeat-random loss model". Phylogenetic analyses revealed Gryphaeidae formed by H.sinensis + H.hyotis as sister to Ostreidae, whereas the phylogenetic relationship within the latter group remains unresolved. The present study indicated the mitogenomic diversity within Gryphaeidae and could also provide important data for future better understanding the gene order rearrangements within superfamily Ostreoidea.

Microbacterium Cluster EA Bacteriophages: Phylogenomic Relationships and Host Range Predictions.

Bacteriophages are being widely harnessed as an alternative to antibiotics due to the global emergence of drug-resistant pathogens. To guide the usage of these bactericidal agents, characterization of their host specificity is vital-however, host range information remains limited for many bacteriophages. This is particularly the case for bacteriophages infecting the Microbacterium genus, despite their importance in agriculture, biomedicine, and biotechnology. Here, we elucidate the phylogenomic relationships between 125 Microbacterium cluster EA bacteriophages-including members from 11 sub-clusters (EA1 to EA11)-and infer their putative host ranges using insights from codon usage bias patterns as well as predictions from both exploratory and confirmatory computational methods. Our computational analyses suggest that cluster EA bacteriophages have a shared infection history across the Microbacterium clade. Interestingly, bacteriophages of all sub-clusters exhibit codon usage preference patterns that resemble those of bacterial strains different from ones used for isolation, suggesting that they might be able to infect additional hosts. Furthermore, host range predictions indicate that certain sub-clusters may be better suited in prospective biotechnological and medical applications such as phage therapy.

Mitogenomic Codon Usage Patterns of Superfamily Certhioidea (Aves, Passeriformes): Insights into Asymmetrical Bias and Phylogenetic Implications

The superfamily Certhioidea currently comprises five families. Due to the rapid diversification, the phylogeny of Certhioidea is still controversial. The advent of next generation sequencing provides a unique opportunity for a mitogenome-wide study. Here, we first provided six new complete mitogenomes of Certhioidea (Certhia americana, C. familiaris, Salpornis spilonota, Cantorchilus leucotis, Pheugopedius coraya, and Pheugopedius genibarbis). We further paid attention to the genomic characteristics, codon usages, evolutionary rates, and phylogeny of the Certhioidea mitogenomes. All mitogenomes we analyzed displayed typical ancestral avian gene order with 13 protein-coding genes (PCGs), 22 tRNAs, 2 rRNAs, and one control region (CR). Our study indicated the strand-biased compositional asymmetry might shape codon usage preferences in mitochondrial genes. In addition, natural selection might be the main factor in shaping the codon usages of genes. Additionally, evolutionary rate analyses indicated all mitochondrial genes were under purifying selection. Moreover, MT-ATP8 and MT-CO1 were the most rapidly evolving gene and conserved genes, respectively. According to our mitophylogenetic analyses, the monophylies of Troglodytidae and Sittidae were strongly supported. Importantly, we suggest that Salpornis should be separated from Certhiidae and put into Salpornithidae to maintain the monophyly of Certhiidae. Our findings are useful for further evolutionary studies within Certhioidea.

Comprehensive Analysis of Codon Usage in Quercus Chloroplast Genome and Focus on psbA Gene.

Quercus (oak) is an important economic and ecological tree species in the world, and it is the necessary feed for oak silkworm feeding. Chloroplasts play an important role in green plants but the codon usage of oak chloroplast genomes is not fully studied. We examined the codon usage of the oak chloroplast genomes in detail to facilitate the understanding of their biology and evolution. We downloaded all the protein coding genes of 26 non-redundant chloroplast reference genomes, removed short ones and those containing internal stop codons, and finally retained 50 genes shared by all genomes for comparative analyses. The base composition, codon bias, and codon preference are not significantly different between genomes but are significantly different among genes within these genomes. Oak chloroplast genomes prefer T/A-ending codons and avoid C/G-ending codons, and the psbA gene has the same preference except for the codons encoding amino acid Phe. Complex factors such as context-dependent mutations are the major factors affecting codon usage in these genomes, while selection plays an important role on the psbA gene. Our study provided an important understanding of codon usage in the oak chloroplast genomes and found that the psbA gene has nearly the same codon usage preference as other genes in the oak chloroplasts.

Comparative Plastome Analyses of Ephedra przewalskii and E. monosperma (Ephedraceae)

Ephedra species were erect, branching shrubs found in desert or arid regions worldwide as the source of ephedrine alkaloids. In this study, the complete chloroplast genome of Ephedra przewalskii and E. monosperma on the Qinghai-Tibet Plateau were sequenced, assembled, and annotated. Compared with the other four published Ephedra species, the chloroplast genomes of Ephedra species were highly conservative, with a quadripartite structure. The length of the chloroplast genome was 109,569 bp in E. przewalskii with 36.6% GC and 109,604 bp in E. monosperma with 36.6% GC. We detected 118 genes in both Ephedra species, including 73 PCGs, 37 tRNA genes, and eight rRNA genes. Among them, the ndh family genes were lost, which could be used to study the phylogeny and genetic diversity of the genus Ephedra, combined with multiple highly variable intergenic spacer (IGS) regions. Codon usage preference of Ephedra species was weak. The ratio of non-synonymous substitutions and synonymous substitutions was low, showing that the PCGs of Ephedra may be under the pressure of purifying selection. ML and BI analysis showed similar phylogenetic topologies. Ephedra species clustered together in a well-supported monophyletic clade. E. przewalskii and E. monosperma were not gathered in one clade, consistent with the classification system by Flora of China. This study reveals differences in the chloroplast genomes of Ephedra, providing valuable and abundant data for the phylogenetic analysis and species identification of Ephedra.

Codon Usage Bias for Fatty Acid Genes FAE1 and FAD2 in Oilseed Brassica Species

Codon usage bias (CUB) phenomenon varies with the species and even within the genes of the same species, where few codons are preferred more frequently than their other synonymous codons. It also categorizes the differences between species. Nucleotide compositional analysis reveals the molecular mechanisms of genes and the evolutionary relationship of a gene in dissimilar plant species. In the present study, three orthologous sequences of each FAE1 (FAE1.1, FAE1.2, and FAE1.3) and FAD2 (FAD2.1, FAD2.2, and FAD2.3) genes, from six Brassica species were accessed using the GenBank database. Further, CUB-related parameters such as nucleotide composition (AT and GC content), relative synonymous codon usage (RSCU), the effective number of codons (ENC), frequency of optimal codons (Fop), relative codon usage bias (RCBS), neutrality plot (GC12 vs. GC3), parity rule-2 [(A3/(A3 + T3) vs. (G3/(G3 + C3)], and correspondence analysis (COA) were analyzed to compare codon bias in U’s triangle Brassica species. The FAE1 genes were AT-biased and FAD2 genes were GC-biased across the studied Brassica species. RSCU values indicated that both the genes had moderate codon usage frequency for selected amino acids. The evolutionary study confirmed that codon usage preference is similar within the species grouped into the same cluster for FAE1; however, B. nigra performed differently for FAD2.2 orthologue. The high ENC value, low Fop, and RSCU value highlighted that FAE1 and FAD2 genes had a low level of gene expression and moderate preference for codon usage across the Brassicas. In addition, neutrality plot, parity rule, and correspondence analysis revealed that natural selection pressure had significantly contributed to CUB for FAE1 genes, whereas mutation and selection pressure occurred for FAD2 genes. This study would help to decode codon optimization, improve the level of expression of exogenous genes, and transgenic engineering to increase fatty acid profiling for the betterment of seed oil in Brassica species.

The evolution of N6-methyladenosine regulators in plants.

As a reversible modification, N6-methyladenosine (m6A) plays key roles in series of biological processes. Although it has been found that m6A modification is regulated by writers, erasers and readers, their evolutionary processes are still not clearly and systematically described. In the present work, we identified 1592 m6A modification regulators from 65 representative plant species and performed the phylogenetic relationships, sequence structure, selection pressure, and codon usage analysis across species. The regulators from different species or subfamilies were distinguishable based on the phylogenetic trees. Although the gene structure was structurally and functionally conserved for each kind of regulators, the unique exon/intron structures and motif organizations were observed among different families. The selection pressure analysis demonstrated that the regulators experienced purifying selection. Interestingly, the selection pressure for the regulators in higher plants was more relaxed, indicating that they might have acquired new functions during evolution. In addition, the different codon usage preferences were observed for the different kinds of m6A modification regulators. These results will not only facilitate our understanding of the evolution of m6A regulators, but also shed light on how the evolutionary differences affect their functional divergence.

Strong Selectional Forces Fine-Tune CpG Content in Genes Involved in Neurological Disorders as Revealed by Codon Usage Patterns.

Neurodegenerative disorders cause irreversible damage to the neurons and adversely affect the quality of life. Protein misfolding and their aggregation in specific parts of the brain, mitochondrial dysfunction, calcium load, proteolytic stress, and oxidative stress are among the causes of neurodegenerative disorders. In addition, altered metabolism has been associated with neurodegeneration as evidenced by reductions in glutamine and alanine in transient global amnesia patients, higher homocysteine-cysteine disulfide, and lower methionine decline in serum urea have been observed in Alzheimer’s disease patients. Neurodegeneration thus appears to be a culmination of altered metabolism. The study’s objective is to analyze various attributes like composition, physical properties of the protein, and factors like selectional and mutational forces, influencing codon usage preferences in a panel of genes involved directly or indirectly in metabolism and contributing to neurodegeneration. Various parameters, including gene composition, dinucleotide analysis, Relative synonymous codon usage (RSCU), Codon adaptation index (CAI), neutrality and parity plots, and different protein indices, were computed and analyzed to determine the codon usage pattern and factors affecting it. The correlation of intrinsic protein properties such as the grand average of hydropathicity index (GRAVY), isoelectric point, hydrophobicity, and acidic, basic, and neutral amino acid content has been found to influence codon usage. In genes up to 800 amino acids long, the GC3 content was highly variable, while GC12 content was relatively constant. An optimum CpG content is present in genes to maintain a high expression level as required for genes involved in metabolism. Also observed was a low codon usage bias with a higher protein expression level. Compositional parameters and nucleotides at the second position of codons played essential roles in explaining the extent of bias. Overall analysis indicated that the dominance of selection pressure and compositional constraints and mutational forces shape codon usage.

Establishment of Stable Chinese Hamster Ovary Cells Expressing Codon-Optimized Human Interferon-β Gene

Background: Codon optimization is an efficient approach to achieve a higher level of heterologous gene expression and generate productive recombinant mammalian cell lines. In our previous work, based on the codon usage preference of Chinese hamster ovary (CHO) cells, a codon-optimized human interferon-beta (opt-hIFN-β) gene was redesigned and transiently expressed in a suspension-adapted CHO (CHO-s) cell line. Our results indicated a 2.8-fold increase in the expression level of the codon-optimized gene compared to the unmodified sequence. Objectives: In the current work, based on our previous results, a stable CHO-K1 cell line expressing the opt-hIFN-β gene was engineered, in which the opt-hIFN-β gene expression was confirmed by dot and western blotting analyses. Methods: The designed opt-hIFN-β sequence was digested and cloned into a pcDNA3.0 shuttle vector downstream to the cytomegalovirus (CMV) promoter. The verified recombinant plasmid was then linearized and transfected into a CHO-K1 cell line to integrate the opt-hIFN-β gene into the CHO-K1 genome. The transfected cells were then grown under the selective pressure of 450 µg/mL of G418 to develop a stable CHO-K1 cell line expressing the opt-hIFN-β gene. The enzyme-linked immunosorbent assay (ELISA) and dot and western blotting analyses were carried out to verify hIFN-β protein expression. Results: ELISA and dot and western blotting analyses confirmed the expression of hIFN-β in the stably-transfected CHO-K1 cells. Conclusions: Stable expression of the opt-hIFN-β gene in the CHO-K1 cell line was verified by ELISA and dot and western blotting analyses. This study was a pioneering work for further production of recombinant hIFN-β in the bioreactor.

Isolation and Identification of a Large Green Alga Virus (Chlorella Virus XW01) of Mimiviridae and Its Virophage (Chlorella Virus Virophage SW01) by Using Unicellular Green Algal Cultures.

Virophages are a group of small double-stranded DNA viruses that infect protist hosts and parasitize the viral factory of host giant/large viruses to propagate. Here, we discover a novel cell-virus-virophage (CVv) tripartite interaction system by using unicellular micro-green algae (Chlorella sp.) as eukaryotic hosts for the first time. Viral particles, resembling known virophages and large alga viruses, are detected in culture supernatants and inside algal cells. Complete genomic sequences of the virophage (Chlorella virus virophage SW01 [CVv-SW01]; 24,744 bp) and large virus (Chlorella virus XW01 [CV-XW01]; 407,612 bp) are obtained from the cocultures. Both genomic and phylogenetic analyses show that CVv-SW01 is closely related to virophages previously found in Dishui Lake. CV-XW01 shares the greatest number of homologous genes (n = 82) with Cafeteria roenbergensis virus (CroV) and phylogenetically represents the closest relative to CroV. This is the first report of a large green alga virus being affiliated with a heterotrophic zooplankton-infecting Cafeteriavirus of the family Mimiviridae. Moreover, the codon usage preferences of CV-XW01 and CVv-SW01 are highly similar to those of CroV and its virophage Mavirus, respectively. The discovery of such a novel CVv system with the green alga Chlorella sp. as the single cellular eukaryotic host paves a way to further investigate the potential interaction mechanism of CVv and its significance in the ecology of green algae and the evolution of large/giant viruses and their parasitic viruses. IMPORTANCE Parasitic virophages are small unicellular eukaryotic dsDNA viruses that rely on the viral factories of coinfecting giant/large dsDNA viruses for propagation. Presently, the identified eukaryotic hosts of isolated virophages were restricted to a free-living amoeba, Acanthamoeba polyphaga, and a widespread marine heterotrophic flagellate, Cafeteria roenbergensis. In this study, we successfully discovered and identified a novel tripartite interaction system comprised of a micro-green alga (Chlorella sp.), Mimiviridae large green alga virus, and virophage at the coculture level, with Chlorella sp. as the eukaryotic host, based on combination analysis of infection, morphotype, genome, and phylogeny. The large green alga virus CV-XW01 represents the closest relative to the Mimiviridae giant virus Cafeteria roenbergensis virus, host virus of the virophage Mavirus, as well as a novel large virus of Mimiviridae that infects a non-protozoan protist host. The virophage CVv-SW01 highly resembles Mavirus in its codon usage frequency and preference, although they are phylogenetically distantly related. These findings give novel insights into the diversity of large/giant viruses and their virophages.