The complete chloroplast genome sequence of Quercus ningangensis and its phylogenetic implication

Quercus ningangensis is an economically and ecologically important tree species belonging to the family Fagaceae. In this study, the complete chloroplast (cp) genome of Q. ningangensis was sequenced and assembled, and 18 published cp genomes of Quercus were retrieved for genomic analyses (including sequence divergence, repeat elements, and structure) and phylogenetic inference. With this study, we found that complete cp genomes in Quercus are conserved, and we discovered a codon composition bias, which may be related to genomic content and genetic characteristics. In addition, we detected considerable structural variations in the expansion and contraction of inverted repeat regions. Six regions with relatively high variable (matK-rps16, psbC, ycf3 intron, rbcL, petA-psbJ, and ycf1) were detected by conducting a sliding window analysis, which has a high potential for developing effective genetic markers. Phylogenetic analysis based on Bayesian inference and maximum likelihood methods resulted in a robust phylogenetic tree of Quercus with high resolution for nearly all identified nodes. The phylogenetic relationships showed that the phylogenetic position of Q. ningangensis was located between Q. sichourensis and Q. acuta. The results of this study contribute to future research into the phylogenetic evolution of Quercus section Cyclobalanopsis (Fagaceae).

iCodon: ideal codon design for customized gene expression

Messenger RNA (mRNA) stability substantially impacts steady-state gene expression levels in a cell. mRNA stability, in turn, is strongly affected by codon composition in a translation dependent manner across species, through a mechanism termed codon optimality. We have developed iCodon (www.iCodon.org), an algorithm for customizing mRNA expression through the introduction of synonymous codon substitutions into the coding sequence. iCodon is optimized for four vertebrate transcriptomes: mouse, human, frog, and fish. Users can predict the mRNA stability of any coding sequence based on its codon composition and subsequently generate more stable (optimized) or unstable (deoptimized) variants encoding for the same protein. Further, we show that codon optimality predictions correlate with expression levels using fluorescent reporters and endogenous genes in human cells and zebrafish embryos. Therefore, iCodon will benefit basic biological research, as well as a wide range of applications for biotechnology and biomedicine.

Mutational Asymmetries in the SARS-CoV-2 Genome May Lead to Increased Hydrophobicity of Virus Proteins

The genomic diversity of SARS-CoV-2 has been a focus during the ongoing COVID-19 pandemic. Here, we analyzed the distribution and character of emerging mutations in a data set comprising more than 95,000 virus genomes covering eight major SARS-CoV-2 lineages in the GISAID database, including genotypes arising during COVID-19 therapy. Globally, the C>U transitions and G>U transversions were the most represented mutations, accounting for the majority of single-nucleotide variations. Mutational spectra were not influenced by the time the virus had been circulating in its host or medical treatment. At the amino acid level, we observed about a 2-fold excess of substitutions in favor of hydrophobic amino acids over the reverse. However, most mutations constituting variants of interests of the S-protein (spike) lead to hydrophilic amino acids, counteracting the global trend. The C>U and G>U substitutions altered codons towards increased amino acid hydrophobicity values in more than 80% of cases. The bias is explained by the existing differences in the codon composition for amino acids bearing contrasting biochemical properties. Mutation asymmetries apparently influence the biochemical features of SARS CoV-2 proteins, which may impact protein–protein interactions, fusion of viral and cellular membranes, and virion assembly.

Transient ribosome slowdown at the decoding step triggers mRNA degradation independent of Znf598 in zebrafish

The control of mRNA stability plays a central role in regulating gene expression patterns. Recent studies have revealed that codon composition in the open reading frame (ORF) determines mRNA stability in multiple organisms. Based on genome-wide correlation approaches, this previously unrecognized role of the genetic code is attributable to the kinetics of the codon-decoding process by the ribosome. However, complementary experimental analysis is required to define the codon effects on mRNA stability apart from the related cotranslational mRNA decay pathways such as those triggered by aberrant ribosome stalls. In the current study, we performed a set of reporter-based analyses to define codon-mediated mRNA decay and ribosome stall-dependent mRNA decay in zebrafish embryos. Our analysis showed that the effect of codons on mRNA stability stems from the decoding process, independent of Znf598 and stall-dependent mRNA decay. We propose that codon-mediated mRNA decay is triggered by transiently slowed ribosomes engaging in a productive translation cycle in zebrafish embryos.

iCodon: ideal codon design for customized gene expression

Messenger RNA (mRNA) stability substantially impacts steady-state gene expression levels in a cell. mRNA stability, in turn, is strongly affected by codon composition in a translation-dependent manner across species, through a mechanism termed codon optimality. We have developed iCodon (www.iCodon.org), an algorithm for customizing mRNA expression through the introduction of synonymous codon substitutions into the coding sequence. iCodon is optimized for four vertebrate transcriptomes: mouse, human, frog, and fish. Users can predict the mRNA stability of any coding sequence based on its codon composition and subsequently generate more stable (optimized) or unstable (deoptimized) variants encoding for the same protein. Further, we show that codon optimality predictions correlate with expression levels using fluorescent reporters and endogenous genes in human cells and zebrafish embryos. Therefore, iCodon will benefit basic biological research, as well as a wide range of applications for biotechnology and biomedicine.

Advances for the Hepatitis A Virus Antigen Production Using a Virus Strain With Codon Frequency Optimization Adjustments in Specific Locations

The available cell-adapted hepatitis A virus (HAV) strains show a very slow replication phenotype hampering the affordable production of antigen. A fast-growing strain characterized by the occurrence of mutations in the internal ribosome entry site (IRES), combined with changes in the codon composition has been selected in our laboratory. A characterization of the IRES activity of this fast-growing strain (HM175-HP; HP) vs. its parental strain (HM175; L0) was assessed in two cell substrates used in vaccine production (MRC-5 and Vero cells) compared with the FRhK-4 cell line in which its selection was performed. The HP-derived IRES was significantly more active than the L0-derived IRES in all cells tested and both IRES were more active in the FRhK-4 cells. The translation efficiency of the HP-derived IRES was also much higher than the L0-derived IRES, particularly, in genes with a HP codon usage background. These results correlated with a higher virus production in a shorter time for the HP strain compared to the L0 strain in any of the three cell lines tested, and of both strains in the FRhK-4 cells compared to Vero and MRC-5 cells. The addition of wortmannin resulted in the increase of infectious viruses and antigen in the supernatant of FRhK-4 infected cells, independently of the strain. Finally, the replication of both strains in a clone of FRhK-4 cells adapted to grow with synthetic sera was optimal and again the HP strain showed higher yields.

A Modelling Framework Linking Resource-Based Stochastic Translation to the Optimal Design of Synthetic Constructs

The effect of gene expression burden on engineered cells has motivated the use of “whole-cell models” (WCMs) that use shared cellular resources to predict how unnatural gene expression affects cell growth. A common problem with many WCMs is their inability to capture translation in sufficient detail to consider the impact of ribosomal queue formation on mRNA transcripts. To address this, we have built a “stochastic cell calculator” (StoCellAtor) that combines a modified TASEP with a stochastic implementation of an existing WCM. We show how our framework can be used to link a synthetic construct’s modular design (promoter, ribosome binding site (RBS) and codon composition) to protein yield during continuous culture, with a particular focus on the effects of low-efficiency codons and their impact on ribosomal queues. Through our analysis, we recover design principles previously established in our work on burden-sensing strategies, namely that changing promoter strength is often a more efficient way to increase protein yield than RBS strength. Importantly, however, we show how these design implications can change depending on both the duration of protein expression, and on the presence of ribosomal queues.

Codon composition in human oocytes reveals age-associated defects in mRNA decay

ABSTRACTOocytes from women of advanced reproductive age have lower developmental potential, yet the underlying mechanisms of this phenomena are incompletely understood. Oocyte maturation is dependent upon translational control of stored maternal mRNA that were synthesized during oocyte growth. We observed that GC content of mRNA was negatively associated with half-life in oocytes from reproductively young women (< 30 years), contrastingly directly with oocytes from reproductively aged women (≥ 40 years) where mRNA half-lives were positively associated with GC nucleotide content. Additionally, we observed that mRNA half-lives were negatively associated with protein abundance in young oocytes, while GC content was positively associated with protein abundance in aged oocytes. Examination of codon composition during the GV-to-MII transition revealed that codons that facilitate rapid translation promoted mRNA stability and are considered optimal, while codons that slow translation destabilized mRNA, and are considered non-optimal. GC-containing codons were more optimal in reproductive aging, and also correlated positively with protein abundance. This study indicates that reproductive aging coincides with the stabilization of a subset of mRNA that have the potential to be over-translated during oocyte maturation, this is likely to lead to observed decreases in oocyte quality in older women. Because oocyte mRNA decay is translationally linked, this suggests that maternal aging causes defects in translation, which results in reduced translational efficiency and the retention of maternal mRNA that are normally degraded in oocytes from young women. In the case of oocytes, defects in translation can alter the RNA decay pathways and result in incorrect maternal mRNA dosage, which may negatively impact embryonic development.

EXPRESSION OF NUCLEOCAPSID VIRAL PROTEINS IN THE BACTERIAL SYSTEM OF Escherichia coli: THE INFLUENCE OF THE CODON COMPOSITION AND THE UNIFORMITY OF ITS DISTRIBUTION WITHIN GENE

A heterologous host has got a unique expression ability of each gene. Differences between the synonymous sequences play an important role in regulation of protein expression in organisms from Escherichia coli to human, and many details of this process remain unclear. The work was aimed to study the composition of codons, its distribution over the sequence and the effect of rare codons on the expression of viral nucleocapsid proteins and their fragments in the heterologous system of E.coli. The plasmid vector pJC 40 and the BL 21 (DE 3) E. coli strain were used for protein expression. The codon composition analysis was performed using the online resource (www.biologicscorp.com). 10 recombinant polypeptides were obtained encoding the complete nucleotide sequence of nucleocapsid proteins (West Nile and hepatitis C viruses) and the fragments including antigenic determinants (Lassa virus, Marburg, Ebola, Crimean-Congo hemorrhagic fever (CCHF), Puumaravala, Hantaan, and lymphocytic choriomeningitis (LHM)). Hybrid plasmid DNAs provide efficient production of these proteins in the prokaryotic system with the recombinant protein yield varying by a factor of 8: from 5 to 40 mg per 1 liter of bacterial culture. No correlation was found between the level of protein expression and the frequency of occurrence of rare codons in the cloned sequence: the maximum frequency of occurrence of rare codons per cloned sequence was observed for the West Nile virus (14.6%), the minimum was for the CCHF virus (6.6%), whereas the expression level for these proteins was 30 and 5 mg/L culture, respectively. The codon adaptation index (CAI) values, calculated on the basis of the codon composition in E. coli, for the cloned viral sequences were in the range from 0.50 to 0.58, which corresponded to the average expressed proteins. The analysis of the distribution profiles of CAI in the cloned sequences indicated the absence of clusters of rare codons that could create difficulties in translation. A statistically significant difference between the frequencies of the distribution of amino acids in the cloned sequences and their content in E. coli was observed for the nucleocapsid proteins of the Marburg, Ebola, West Nile, and hepatitis C viruses.