Enrichment of rare codons at 5' ends of genes is a spandrel caused by evolutionary sequence turnover and does not improve translation.

Previously, Tuller et al. found that the first 30-50 codons of the genes of yeast and other eukaryotes are slightly enriched for rare codons. They argued that this slowed translation, and was adaptive because it queued ribosomes to prevent collisions. Today, the translational speeds of different codons are known, and indeed rare codons are translated slowly. We re-examined this 5' slow translation 'ramp.' We confirm that 5' regions are slightly enriched for rare codons; in addition, they are depleted for downstream Start codons (which are fast), with both effects contributing to slow 5' translation. However, we also find that the 5' (and 3') ends of yeast genes are poorly conserved in evolution, suggesting that they are unstable and turnover relatively rapidly. When a new 5' end forms de novo, it is likely to include codons that would otherwise be rare. Because evolution has had a relatively short time to select against these codons, 5' ends are typically slightly enriched for rare, slow codons. Opposite to the expectation of Tuller et al., we show by direct experiment that genes with slowly translated codons at the 5' end are expressed relatively poorly, and that substituting faster synonymous codons improves expression. Direct experiment shows that slow codons do not prevent downstream ribosome collisions. Further informatic studies suggest that for natural genes, slow 5' ends are correlated with poor gene expression, opposite to the expectation of Tuller et al. Thus, we conclude that slow 5' translation is a 'spandrel'--a non-adaptive consequence of something else, in this case, the turnover of 5' ends in evolution, and it does not improve translation.

Unveiling the translational dynamics of lychee (Litchi chinesis Sonn.) in response to cold stress.

Cold stress poses a significant threat to the quality and productivity of lychee (Litchi chinensis Sonn.). While previous research has extensively explored the genomic and transcriptomic responses to cold stress in lychee, the translatome has not been thoroughly investigated. This study delves into the translatomic landscape of the 'Xiangjinfeng' cultivar under both control and low-temperature conditions using RNA sequencing and ribosome profiling. We uncovered a significant divergence between the transcriptomic and translatomic responses to cold exposure. Additionally, bioinformatics analyses underscored the crucial role of codon occupancy in lychee's cold tolerance mechanisms. Our findings reveal that the modulation of translation via codon occupancy is a vital strategy to abiotic stress. Specifically, the study identifies ribosome stalling, particularly at the E site AAU codon, as a key element of the translation machinery in lychee's response to cold stress. This work enhances our understanding of the molecular dynamics of lychee's reaction to cold stress and emphasizes the essential role of translational regulation in the plant's environmental adaptability.

m3C32 tRNA modification controls serine codon-biased mRNA translation, cell cycle, and DNA-damage response.

The epitranscriptome includes a diversity of RNA modifications that influence gene expression. N3-methylcytidine (m3C) mainly occurs in the anticodon loop (position C32) of certain tRNAs yet its role is poorly understood. Here, using HAC-Seq, we report comprehensive METTL2A/2B-, METTL6-, and METTL2A/2B/6-dependent m3C profiles in human cells. METTL2A/2B modifies tRNA-arginine and tRNA-threonine members, whereas METTL6 modifies the tRNA-serine family. However, decreased m3C32 on tRNA-Ser-GCT isodecoders is only observed with combined METTL2A/2B/6 deletion. Ribo-Seq reveals altered translation of genes related to cell cycle and DNA repair pathways in METTL2A/2B/6-deficient cells, and these mRNAs are enriched in AGU codons that require tRNA-Ser-GCT for translation. These results, supported by reporter assays, help explain the observed altered cell cycle, slowed proliferation, and increased cisplatin sensitivity phenotypes of METTL2A/2B/6-deficient cells. Thus, we define METTL2A/2B/6-dependent methylomes and uncover a particular requirement of m3C32 tRNA modification for serine codon-biased mRNA translation of cell cycle, and DNA repair genes.

Human parainfluenza virus 3 vaccine candidates attenuated by codon-pair deoptimization are immunogenic and protective in hamsters.

Human parainfluenza virus type 3 (HPIV3) is a major pediatric respiratory pathogen lacking available vaccines or antiviral drugs. We generated live-attenuated HPIV3 vaccine candidates by codon-pair deoptimization (CPD). HPIV3 open reading frames (ORFs) encoding the nucleoprotein (N), phosphoprotein (P), matrix (M), fusion (F), hemagglutinin-neuraminidase (HN), and polymerase (L) were modified singly or in combination to generate 12 viruses designated Min-N, Min-P, Min-M, Min-FHN, Min-L, Min-NP, Min-NPM, Min-NPL, Min-PM, Min-PFHN, Min-MFHN, and Min-PMFHN. CPD of N or L severely reduced growth in vitro and was not further evaluated. CPD of P or M was associated with increased and decreased interferon (IFN) response in vitro, respectively, but had little effect on virus replication. In Vero cells, CPD of F and HN delayed virus replication, but final titers were comparable to wild-type (wt) HPIV3. In human lung epithelial A549 cells, CPD F and HN induced a stronger IFN response, viral titers were reduced 100-fold, and the expression of F and HN proteins was significantly reduced without affecting N or P or the relative packaging of proteins into virions. Following intranasal infection in hamsters, replication in the nasal turbinates and lungs tended to be the most reduced for viruses bearing CPD F and HN, with maximum reductions of approximately 10-fold. Despite decreased in vivo replication (and lower expression of CPD F and HN in vitro), all viruses induced titers of serum HPIV3-neutralizing antibodies similar to wt and provided complete protection against HPIV3 challenge. In summary, CPD of HPIV3 yielded promising vaccine candidates suitable for further development.

Molecular Evolution of Protein Sequences and Codon Usage in Monkeypox Viruses.

The monkeypox virus (mpox virus, MPXV) epidemic in 2022 has posed a significant public health risk. Yet, the evolutionary principles of MPXV remain largely unknown. Here, we examined the evolutionary patterns of protein sequences and codon usage in MPXV. We first demonstrated the signal of positive selection in OPG027, specifically in the Clade I lineage of MPXV. Subsequently, we discovered accelerated protein sequence evolution over time in the variants responsible for the 2022 outbreak. Furthermore, we showed strong epistasis between amino acid substitutions located in different genes. The codon adaptation index (CAI) analysis revealed that MPXV genes tended to use more non-preferred codons compared to human genes, and the CAI decreased over time and diverged between clades, with Clade I > IIa and IIb-A > IIb-B. While the decrease in fatality rate among the three groups aligned with the CAI pattern, it remains unclear whether this correlation was coincidental or if the deoptimization of codon usage in MPXV led to a reduction in fatality rates. This study sheds new light on the mechanisms that govern the evolution of MPXV in human populations.

GNN Codon Adjacency Tunes Protein Translation.

The central dogma treats the ribosome as a molecular machine that reads one mRNA codon at a time as it adds each amino acid to its growing peptide chain. However, this and previous studies suggest that ribosomes actually perceive pairs of adjacent codons as they take three-nucleotide steps along the mRNA. We examined GNN codons, which we find are surprisingly overrepresented in eukaryote protein-coding open reading frames (ORFs), especially immediately after NNU codons. Ribosome profiling experiments in yeast revealed that ribosomes with NNU at their aminoacyl (A) site have particularly elevated densities when NNU is immediately followed (3') by a GNN codon, indicating slower mRNA threading of the NNU codon from the ribosome's A to peptidyl (P) sites. Moreover, if the assessment was limited to ribosomes that have only recently arrived at the next codon, by examining 21-nucleotide ribosome footprints (21-nt RFPs), elevated densities were observed for multiple codon classes when followed by GNN. This striking translation slowdown at adjacent 5'-NNN GNN codon pairs is likely mediated, in part, by the ribosome's CAR surface, which acts as an extension of the A-site tRNA anticodon during ribosome translocation and interacts through hydrogen bonding and pi stacking with the GNN codon. The functional consequences of 5'-NNN GNN codon adjacency are expected to influence the evolution of protein coding sequences.

Assembly and comparative analysis of the first complete mitochondrial genome of Setaria italica.

MAIN CONCLUSION: In this study, we assembled the first complete mitochondrial genome of Setaria italica and confirmed the multi-branched architecture. The foxtail millet (Setaria italica) holds significant agricultural importance, particularly in arid and semi-arid regions. It plays a pivotal role in diversifying dietary patterns and shaping planting strategies. Although the chloroplast genome of S. italica has been elucidated in recent studies, the complete mitochondrial genome remains largely unexplored. In this study, we employed PacBio HiFi sequencing platforms to sequence and assemble the complete mitochondrial genome. The mitochondrial genome spans a total length of 446,614 base pairs and harbors a comprehensive set of genetic elements, including 33 unique protein-coding genes (PCGs), encompassing 24 unique mitochondrial core genes and 9 variable genes, along with 20 transfer RNA (tRNA) genes and 3 ribosomal RNA (rRNA) genes. Our analysis of mitochondrial PCGs revealed a pronounced codon usage preference. For instance, the termination codon exhibits a marked preference for UAA, while alanine (Ala) exhibits a preference for GCU, and glutamine (Gln) favors CAA. Notably, the maximum Relative Synonymous Codon Usage (RSCU) values for cysteine (Cys) and phenylalanine (Phe) are both below 1.2, indicating a lack of strong codon usage preference for these amino acids. Phylogenetic analyses consistently place S. italica in close evolutionary proximity to Chrysopogon zizanioides, relative to other Panicoideae plants. Collinearity analysis showed that a total of 39 fragments were identified to display homology with both the mitochondrial and chloroplast genomes. A total of 417 potential RNA-editing sites were discovered across the 33 mitochondrial PCGs. Notably, all these editing events involved the conversion of cytosine (C) to uracil (U). Through the employment of PCR validation coupled with Sanger sequencing for the anticipated editing sites of these codons, RNA-editing events were conclusively identified at two specific loci: nad4L-2 and atp6-1030. The results of this study provide a pivotal foundation for advanced genomic breeding research in foxtail millet. Furthermore, they impart essential insights that will be instrumental for forthcoming investigations into the evolutionary and molecular dynamics of Panicoideae species.

Orb2 enables rare-codon-enriched mRNA expression during Drosophila neuron differentiation.

Regulation of codon optimality is an increasingly appreciated layer of cell- and tissue-specific protein expression control. Here, we use codon-modified reporters to show that differentiation of Drosophila neural stem cells into neurons enables protein expression from rare-codon-enriched genes. From a candidate screen, we identify the cytoplasmic polyadenylation element binding (CPEB) protein Orb2 as a positive regulator of rare-codon-dependent mRNA stability in neurons. Using RNA sequencing, we reveal that Orb2-upregulated mRNAs in the brain with abundant Orb2 binding sites have a rare-codon bias. From these Orb2-regulated mRNAs, we demonstrate that rare-codon enrichment is important for mRNA stability and social behavior function of the metabotropic glutamate receptor (mGluR). Our findings reveal a molecular mechanism by which neural stem cell differentiation shifts genetic code regulation to enable critical mRNA stability and protein expression.

Comparative mitochondrial genomics in Nematoda reveal astonishing variation in compositional biases and substitution rates indicative of multi-level selection.

BACKGROUND: Nematodes are the most abundant and diverse metazoans on Earth, and are known to significantly affect ecosystem functioning. A better understanding of their biology and ecology, including potential adaptations to diverse habitats and lifestyles, is key to understanding their response to global change scenarios. Mitochondrial genomes offer high species level characterization, low cost of sequencing, and an ease of data handling that can provide insights into nematode evolutionary pressures. RESULTS: Generally, nematode mitochondrial genomes exhibited similar structural characteristics (e.g., gene size and GC content), but displayed remarkable variability around these general patterns. Compositional strand biases showed strong codon position specific G skews and relationships with nematode life traits (especially parasitic feeding habits) equal to or greater than with predicted phylogeny. On average, nematode mitochondrial genomes showed low non-synonymous substitution rates, but also high clade specific deviations from these means. Despite the presence of significant mutational saturation, non-synonymous (dN) and synonymous (dS) substitution rates could still be significantly explained by feeding habit and/or habitat. Low ratios of dN:dS rates, particularly associated with the parasitic lifestyles, suggested the presence of strong purifying selection. CONCLUSIONS: Nematode mitochondrial genomes demonstrated a capacity to accumulate diversity in composition, structure, and content while still maintaining functional genes. Moreover, they demonstrated a capacity for rapid evolutionary change pointing to a potential interaction between multi-level selection pressures and rapid evolution. In conclusion, this study helps establish a background for our understanding of the potential evolutionary pressures shaping nematode mitochondrial genomes, while outlining likely routes of future inquiry.

The systematic codon usage bias has an important effect on genetic adaption in native species.

Random mutations increase genetic variety and natural selection enhances adaption over generations. Codon usage biases (CUB) provide clues about the genome adaptation mechanisms of native species and extremophile species. Significant numbers of gene (CDS) of nine classes of endangered, native species, including extremophiles and mesophiles were utilised to compute CUB. Codon usage patterns differ among the lineages of endangered and extremophiles with native species. Polymorphic usage of nucleotides with codon burial suggests parallelism of native species within relatively confined taxonomic groups. Utilizing the deviation pattern of CUB of endangered and native species, I present a calculation parameter to estimate the extinction risk of endangered species. Species diversity and extinction risk are both positively associated with the propensity of random mutation in CDS (Coding DNA sequence). Codon bias tenet profoundly selected and it governs to adaptive evolution of native species.

Profiling expression strategies for a type III polyketide synthase in a lysate-based, cell-free system.

Some of the most metabolically diverse species of bacteria (e.g., Actinobacteria) have higher GC content in their DNA, differ substantially in codon usage, and have distinct protein folding environments compared to tractable expression hosts like Escherichia coli. Consequentially, expressing biosynthetic gene clusters (BGCs) from these bacteria in E. coli often results in a myriad of unpredictable issues with regard to protein expression and folding, delaying the biochemical characterization of new natural products. Current strategies to achieve soluble, active expression of these enzymes in tractable hosts can be a lengthy trial-and-error process. Cell-free expression (CFE) has emerged as a valuable expression platform as a testbed for rapid prototyping expression parameters. Here, we use a type III polyketide synthase from Streptomyces griseus, RppA, which catalyzes the formation of the red pigment flaviolin, as a reporter to investigate BGC refactoring techniques. We applied a library of constructs with different combinations of promoters and rppA coding sequences to investigate the synergies between promoter and codon usage. Subsequently, we assess the utility of cell-free systems for prototyping these refactoring tactics prior to their implementation in cells. Overall, codon harmonization improves natural product synthesis more than traditional codon optimization across cell-free and cellular environments. More importantly, the choice of coding sequences and promoters impact protein expression synergistically, which should be considered for future efforts to use CFE for high-yield protein expression. The promoter strategy when applied to RppA was not completely correlated with that observed with GFP, indicating that different promoter strategies should be applied for different proteins. In vivo experiments suggest that there is correlation, but not complete alignment between expressing in cell free and in vivo. Refactoring promoters and/or coding sequences via CFE can be a valuable strategy to rapidly screen for catalytically functional production of enzymes from BCGs, which advances CFE as a tool for natural product research.

Comparative analysis of codon usage bias in chloroplast genomes of ten medicinal species of Rutaceae.

Rutaceae family comprises economically important plants due to their extensive applications in spices, food, oil, medicine, etc. The Rutaceae plants is able to better utilization through biotechnology. Modern biotechnological approaches primarily rely on the heterologous expression of functional proteins in different vectors. However, several proteins are difficult to express outside their native environment. The expression potential of functional genes in heterologous systems can be maximized by replacing the rare synonymous codons in the vector with preferred optimal codons of functional genes. Codon usage bias plays a critical role in biogenetic engineering-based research and development. In the current study, 727 coding sequences (CDSs) obtained from the chloroplast genomes of ten Rutaceae plant family members were analyzed for codon usage bias. The nucleotide composition analysis of codons showed that these codons were rich in A/T(U) bases and preferred A/T(U) endings. Analyses of neutrality plots, effective number of codons (ENC) plots, and correlations between ENC and codon adaptation index (CAI) were conducted, which revealed that natural selection is a major driving force for the Rutaceae plant family's codon usage bias, followed by base mutation. In the ENC vs. CAI plot, codon usage bias in the Rutaceae family had a negligible relationship with gene expression level. For each sample, we screened 12 codons as preferred and high-frequency codons simultaneously, of which GCU encoding Ala, UUA encoding Leu, and AGA encoding Arg were the most preferred codons. Taken together, our study unraveled the synonymous codon usage pattern in the Rutaceae family, providing valuable information for the genetic engineering of Rutaceae plant species in the future.

In-silico Codon Context and Synonymous Usage Analysis of Genes for Molecular Mechanisms Inducing Autophagy and Apoptosis with Reference to Neurodegenerative Disorders.

Background: Autophagy and apoptosis are cellular processes that maintain cellular homeostasis and remove damaged or aged organelles or aggregated and misfolded proteins. Stress factors initiate the signaling pathways common to autophagy and apoptosis. An imbalance in the autophagy and apoptosis, led by cascade of molecular mechanism prior to both processes culminate into neurodegeneration. Objective: In present study, we urge to investigate the codon usage pattern of genes which are common before initiating autophagy and apoptosis. Methods: In the present study, we took up eleven genes (DAPK1, BECN1, PIK3C3 (VPS34), BCL2, MAPK8, BNIP3 L (NIX), PMAIP1, BAD, BID, BBC3, MCL1) that are part of molecular signaling mechanism prior to autophagy and apoptosis. We analyzed dinucleotide odds ratio, codon bias, usage, context, and rare codon analysis. Results: CpC and GpG dinucleotides were abundant, with the dominance of G/C ending codons as preferred codons. Clustering analysis revealed that MAPK8 had a distinct codon usage pattern compared to other envisaged genes. Both positive and negative contexts were observed, and GAG-GAG followed by CTG-GCC was the most abundant codon pair. Of the six synonymous arginine codons, two codons CGT and CGA were the rarest. Conclusions: The information presented in the study may be used to manipulate the process of autophagy and apoptosis and to check the pathophysiology associated with their dysregulation.

High-level expression of codon-optimized Taq DNA polymerase under the control of rhaBAD promoter.

A DNA polymerase from Thermus aquaticus remains the most popular among DNA polymerases. It was widely applied in various fields involving the application of polymerase chain reaction (PCR), implying the high commercial value of this enzyme. For this reason, an attempt to obtain a high yield of Taq DNA polymerase is continuously conducted. In this study, the l-rhamnose-inducible promoter rhaBAD was utilized due to its ability to produce recombinant protein under tight control in E. coli expression system. Instead of full-length Taq polymerase, an N-terminal deletion of Taq polymerase was selected. To obtain a high-level expression, we attempted to optimize the codon by reducing the rare codon and GC content, and in a second attempt, we optimized the culture conditions for protein expression. The production of Taq polymerase using the optimum culture condition improved the level of expression by up to 3-fold. This approach further proved that a high level of recombinant protein expression could be achieved by yielding a purified Taq polymerase of about 8.5 mg/L of culture. This is the first research publication on the production of Taq polymerase with N-terminal deletion in E. coli with the control of the rhaBAD promoter system.

Analysis of chloroplast genome characteristics and codon usage bias in 14 species of Annonaceae.

For the study of species evolution, chloroplast gene expression, and transformation, the chloroplast genome is an invaluable resource. Codon usage bias (CUB) analysis is a tool that is utilized to improve gene expression and investigate evolutionary connections in genetic transformation. In this study, we analysed chloroplast genome differences, codon usage patterns and the sources of variation on CUB in 14 Annonaceae species using bioinformatics tools. The study showed that there was a significant variation in both gene sizes and numbers between the 14 species, but conservation was still maintained. It's worth noting that there were noticeable differences in the IR/SC sector boundary and the types of SSRs among the 14 species. The mono-nucleotide repeat type was the most common, with A/T repeats being more prevalent than G/C repeats. Among the different types of repeats, forward and palindromic repeats were the most abundant, followed by reverse repeats, and complement repeats were relatively rare. Codon composition analysis revealed that all 14 species had a frequency of GC lower than 50%. Additionally, it was observed that the proteins in-coding sequences of chloroplast genes tend to end with A/T at the third codon position. Among these species, 21 codons exhibited bias (RSCU > 1), and there were 8 high-frequency (HF) codons and 5 optimal codons that were identical across the species. According to the ENC-plot and Neutrality plot analysis, natural selection had less impact on the CUB of A. muricate and A. reticulata. Based on the PR2-plot, it was evident that base G had a higher frequency than C, and T had a higher frequency A. The correspondence analysis (COA) revealed that codon usage patterns different in Annonaceae.

mRNA context and translation factors determine decoding in alternative nuclear genetic codes.

The genetic code is a set of instructions that determine how the information in our genetic material is translated into amino acids. In general, it is universal for all organisms, from viruses and bacteria to humans. However, in the last few decades, exceptions to this rule have been identified both in pro- and eukaryotes. In this review, we discuss the 16 described alternative eukaryotic nuclear genetic codes and observe theories of their appearance in evolution. We consider possible molecular mechanisms that allow codon reassignment. Most reassignments in nuclear genetic codes are observed for stop codons. Moreover, in several organisms, stop codons can simultaneously encode amino acids and serve as termination signals. In this case, the meaning of the codon is determined by the additional factors besides the triplets. A comprehensive review of various non-standard coding events in the nuclear genomes provides a new insight into the translation mechanism in eukaryotes.

DNA damage induces p53-independent apoptosis through ribosome stalling.

In response to excessive DNA damage, human cells can activate p53 to induce apoptosis. Cells lacking p53 can still undergo apoptosis upon DNA damage, yet the responsible pathways are unknown. We observed that p53-independent apoptosis in response to DNA damage coincided with translation inhibition, which was characterized by ribosome stalling on rare leucine-encoding UUA codons and globally curtailed translation initiation. A genetic screen identified the transfer RNAse SLFN11 and the kinase GCN2 as factors required for UUA stalling and global translation inhibition, respectively. Stalled ribosomes activated a ribotoxic stress signal conveyed by the ribosome sensor ZAKα to the apoptosis machinery. These results provide an explanation for the frequent inactivation of SLFN11 in chemotherapy-unresponsive tumors and highlight ribosome stalling as a signaling event affecting cell fate in response to DNA damage.

Genomic analysis and phylogenetic characterization of Himalayan snow trout, Schizothorax esocinus based on mitochondrial protein-coding genes.

BACKGROUND: Mitochondrial DNA (mtDNA) has become a significant tool for exploring genetic diversity and delineating evolutionary links across diverse taxa. Within the group of cold-water fish species that are native to the Indian Himalayan region, Schizothorax esocinus holds particular importance due to its ecological significance and is potentially vulnerable to environmental changes. This research aims to clarify the phylogenetic relationships within the Schizothorax genus by utilizing mitochondrial protein-coding genes. METHODS: Standard protocols were followed for the isolation of DNA from S. esocinus. For the amplification of mtDNA, overlapping primers were used, and then subsequent sequencing was performed. The genetic features were investigated by the application of bioinformatic approaches. These approaches covered the evaluation of nucleotide composition, codon usage, selective pressure using nonsynonymous substitution /synonymous substitution (Ka/Ks) ratios, and phylogenetic analysis. RESULTS: The study specifically examined the 13 protein-coding genes of Schizothorax species which belongs to the Schizothoracinae subfamily. Nucleotide composition analysis showed a bias towards A + T content, consistent with other cyprinid fish species, suggesting evolutionary conservation. Relative Synonymous Codon Usage highlighted leucine as the most frequent (5.18%) and cysteine as the least frequent (0.78%) codon. The positive AT-skew and the predominantly negative GC-skew indicated the abundance of A and C. Comparative analysis revealed significant conservation of amino acids in multiple genes. The majority of amino acids were hydrophobic rather than polar. The purifying selection was revealed by the genetic distance and Ka/Ks ratios. Phylogenetic study revealed a significant genetic divergence between S. esocinus and other Schizothorax species with interspecific K2P distances ranging from 0.00 to 8.87%, with an average of 5.76%. CONCLUSION: The present study provides significant contributions to the understanding of mitochondrial genome diversity and genetic evolution mechanisms in Schizothoracinae, hence offering vital insights for the development of conservation initiatives aimed at protecting freshwater fish species.

Codon Optimization-based Whole-gene Scanning Identifies Hidden Nucleotides Essential for Bombyx mori Nucleopolyhedrovirus polyhedrin Hyperexpression.

During the late stage of infection, alphabaculoviruses produce many occlusion bodies (OBs) in the nuclei of the insect host's cells through the hyperexpression of polyhedrin (POLH), a major OB component encoded by polh. The strong polh promoter has been used to develop a baculovirus expression vector system for recombinant protein expression in cultured insect cells and larvae. However, the relationship between POLH accumulation and the polh coding sequence remains largely unelucidated. This study aimed to assess the importance of polh codon usage and/or nucleotide sequences in POLH accumulation by generating a baculovirus Bombyx mori nucleopolyhedrovirus (BmNPV) expressing mutant polh (co-polh) optimized according to the codon preference of its host insect. Although the deduced amino acid sequence of CO-POLH was the same as that of wild-type POLH, POLH accumulation was significantly lower in cells infected with the co-polh mutant. This reduction was due to decreased polh mRNA levels rather than translational repression. Analysis of mutant viruses with chimeric polh revealed that a 30 base-pair (bp) 5' proximal polh coding region was necessary for maintaining high polh mRNA levels. Sequence comparison of wild-type polh and co-polh identified five nucleotide differences in this region, indicating that these nucleotides were critical for polh hyperexpression. Furthermore, luciferase reporter assays showed that the 30 bp 5' coding region was sufficient for maintaining the polh promoter-driven high level of polh mRNA. Thus, our whole-gene scanning by codon optimization identified important hidden nucleotides for polh hyperexpression in alphabaculoviruses.

Decoding Evolution of Rubioideae: Plastomes Reveal Sweet Secrets of Codon Usage, Diagnostides, and Superbarcoding.

Galium genus belongs to the Rubiaceae family, which consists of approximately 14,000 species. In comparison to its well-known relatives, the plastomes of the Galium genus have not been explored so far. The plastomes of this genus have a typical, quadripartite structure, but differ in gene content, since the infA gene is missing in Galium palustre and Galium trfidum. An evaluation of the effectiveness of using entire chloroplast genome sequences as superbarcodes for accurate plant species identification revealed the high potential of this method for molecular delimitation within the genus and tribe. The trnE-UUC-psbD region showed the biggest number of diagnostides (diagnostic nucleotides) which might be new potential barcodes, not only in Galium, but also in other closely related genera. Relative synonymous codon usage (RSCU) appeared to be connected with the phylogeny of the Rubiaceae family, showing that during evolution, plants started preferring specific codons over others.