A-ending Codons Research Articles

Assembly and comparative analysis of the complete mitochondrial genome of Fritillaria ussuriensis Maxim. (Liliales: Liliaceae), an endangered medicinal plant.

Fritillaria ussuriensis is an endangered medicinal plant known for its notable therapeutic properties. Unfortunately, its population has drastically declined due to the destruction of forest habitats. Thus, effectively protecting F. ussuriensis from extinction poses a significant challenge. A profound understanding of its genetic foundation is crucial. To date, research on the complete mitochondrial genome of F. ussuriensis has not yet been reported. The complete mitochondrial genome of F. ussuriensis was sequenced and assembled by integrating PacBio and Illumina sequencing technologies, revealing 13 circular chromosomes totaling 737,569 bp with an average GC content of 45.41%. A total of 55 genes were annotated in this mitogenome, including 2 rRNA genes, 12 tRNA genes, and 41 PCGs. The mitochondrial genome of F. ussuriensis contained 192 SSRs and 4,027 dispersed repeats. In the PCGs of F. ussuriensis mitogenome, 90.00% of the RSCU values exceeding 1 exhibited a preference for A-ended or U-ended codons. In addition, 505 RNA editing sites were predicted across these PCGs. Selective pressure analysis suggested negative selection on most PCGs to preserve mitochondrial functionality, as the notable exception of the gene nad3 showed positive selection. Comparison between the mitochondrial and chloroplast genomes of F. ussuriensis revealed 20 homologous fragments totaling 8,954 bp. Nucleotide diversity analysis revealed the variation among genes, and gene atp9 was the most notable. Despite the conservation of GC content, mitogenome sizes varied significantly among six closely related species, and colinear analysis confirmed the lack of conservation in their genomic structures. Phylogenetic analysis indicated a close relationship between F. ussuriensis and Lilium tsingtauense. In this study, we sequenced and annotated the mitogenome of F. ussuriensis and compared it with the mitogenomes of other closely related species. In addition to genomic features and evolutionary position, this study also provides valuable genomic resources to further understand and utilize this medicinal plant.

Compositional biases and evolution of the largest plant RNA virus order Patatavirales

Patatavirales is the largest order of plant RNA viruses and exclusively contains the family Potyviridae, accounting for 30 % of all known plant viruses. The composition bias of animal RNA viruses and several plant RNA viruses has been determined. However, the comprehensive nucleic acid composition, codon pair usage patterns, dinucleotide preference and codon pair preference of plant RNA viruses have not been investigated to date. In this study, integrated analysis and discussion of the nucleic acid composition, codon usage patterns, dinucleotide composition and codon pair bias of potyvirids were performed using 3732 complete genome coding sequences. The nucleic acid composition of potyvirids was significantly enriched in A/U. Interestingly, the A/U-rich nucleotide composition of Patatavirales is essential for determining the preferred A-ended and U-ended codons and the overexpression of UpG and CpA dinucleotides. The codon usage patterns and codon pair bias of potyvirids were significantly correlated with their nucleic acid composition. Additionally, the codon usage pattern, dinucleotide composition and codon-pair bias of potyvirids are more dependent on the classification of the virus compared with their hosts. Our analysis provides a better understanding of future research on the origin and evolution patterns of the order Patatavirales.

English

Dengue viruses (DENV) are the most common mosquito-borne RNA virus with high variation and adaptation in tropical and subtropical regions. Exploration of codon usage bias of DENV can be significant to understand their genetic variation and adaptation. In the study, the codon usage pattern of dengue virus type 1 (DENV-1) was analyzed by using codonW, CUSP and SPSS. The extent of codon preference of DENV-1 is weak with a 50.57 mean value of ENC, indicating that the DENV-1 genome has low codon bias. Of the 18 optimal codons of DENV-1, 13 end in A/U, with A ending in the majority. The result shows that DENV-1 prefers A-ended codons, and their codon bias is influenced more by natural selection than by mutations selection, as revealed by ENC-plot and neutrality analysis. Furthermore, comparison of codon usage bias between DENV-1 and host showed that codon usage pattern of DENV-1 is more similar to Home sapiens instead of Aedes aegypti or A. albopictus. Our findings contributed to understanding of the evolution of the DENV-1.   Key words: Dengue virus type 1, Codon bias, RSCU, ENC-plot, neutrality-plot.

Natural Selection Plays an Important Role in Shaping the Codon Usage of Structural Genes of the Viruses Belonging to the Coronaviridae Family.

Viruses belonging to the Coronaviridae family have a single-stranded positive-sense RNA with a poly-A tail. The genome has a length of ~29.9 kbps, which encodes for genes that are essential for cell survival and replication. Different evolutionary constraints constantly influence the codon usage bias (CUB) of different genes. A virus optimizes its codon usage to fit the host environment on which it savors. This study is a comprehensive analysis of the CUB for the different genes encoded by viruses of the Coronaviridae family. Different methods including relative synonymous codon usage (RSCU), an Effective number of codons (ENc), parity plot 2, and Neutrality plot, were adopted to analyze the factors responsible for the genetic evolution of the Coronaviridae family. Base composition and RSCU analyses demonstrated the presence of A-ended and U-ended codons being preferred in the 3rd codon position and are suggestive of mutational selection. The lesser ENc value for the spike ‘S’ gene suggests a higher bias in the codon usage of this gene compared to the other structural genes. Parity plot 2 and neutrality plot analyses demonstrate the role and the extent of mutational and natural selection towards the codon usage pattern. It was observed that the structural genes of the Coronaviridae family analyzed in this study were at the least under 84% influence of natural selection, implying a major role of natural selection in shaping the codon usage.

Bacterial wobble modifications of NNA-decoding tRNAs.

Nucleotides of transfer RNAs (tRNAs) are highly modified, particularly at the anticodon. Bacterial tRNAs that read A-ending codons are especially notable. The U34 nucleotide canonically present in these tRNAs is modified by a wide range of complex chemical constituents. An additional two A-ending codons are not read by U34-containing tRNAs but are accommodated by either inosine or lysidine at the wobble position (I34 or L34). The structural basis for many N34 modifications in both tRNA aminoacylation and ribosome decoding has been elucidated, and evolutionary conservation of modifying enzymes is also becoming clearer. Here we present a brief review of the structure, function, and conservation of wobble modifications in tRNAs that translate A-ending codons. © 2019 IUBMB Life, 2019 © 2019 IUBMB Life, 71(8):1158-1166, 2019.

Analysis of Synonymous Codon Usage Bias in Flaviviridae Virus.

Background Flaviviridae viruses are single-stranded, positive-sense RNA viruses, which threat human constantly mediated by mosquitoes, ticks, and sandflies. Considering the recent increase in the prevalence of the family virus and its risk potential, we investigated the codon usage pattern to understand its evolutionary processes and provide some useful data to develop the medications for most of Flaviviridae viruses. Results The overall extent of codon usage bias in 65 Flaviviridae viruses is low with the average value of GC contents being 50.5% and the highest value being 55.9%; the lowest value is 40.2%. ENC values of Flaviviridae virus genes vary from 48.75 to 57.83 with a mean value of 55.56. U- and A-ended codons are preferred in the Flaviviridae virus. Correlation analysis shows that the positive correlation between ENC value and GC content at the third nucleotide positions was significant in this family virus. The result of analysis of ENC, neutrality plot analysis, and correlation analysis revealed that codon usage bias of all the viruses was affected mainly by natural selection. Meanwhile, according to correspondence analysis (CoA) based on RSCU and phylogenetic analysis, the Flaviviridae viruses mainly are made up of two groups, Group I (Yellow fever virus, Apoi virus, Tembusu virus, Dengue virus 1, and others) and Group II (West Nile virus lineage 2, Japanese encephalitis virus, Usutu virus, Kedougou virus, and others). Conclusions All in, the bias of codon usage pattern is affected not only by compositional constraints but also by natural selection. Phylogenetic analysis also illustrates that codon usage bias of virus can serve as an effective means of evolutionary classification in Flaviviridae virus.

Genetic Analysis and Evolutionary Changes of the Torque teno sus Virus.

The torque teno sus virus (TTSuV) is an emerging virus threating the Suidae species of unclear pathogenicity, although it was previously reported as a worsening factor of other porcine diseases, in particular, porcine circovirus associated disease (PCVAD). Here, a comprehensive codon usage analysis of the open reading frame 1 (ORF1), which encodes the viral capsid protein, was undertaken for the first time to reveal its evolutionary history. We revealed independent phylogenetic processes for the two genera during TTSuV evolution, which was confirmed by principal component analysis (PCA). A low codon usage bias was observed in different genera and different species, with Kappatorquevirus a (TTSuVk2a) displaying the highest, which was mainly driven by mutation pressure and natural selection, especially natural selection. Overall, ATs were more abundant than GCs, along with more A-ended synonymous codons in relative synonymous codon usage (RSCU) analysis. To further confirm the role of natural selection and TTSuV adaptation to the Suidae species, codon adaptation index (CAI), relative codon deoptimization index (RCDI), and similarity index (SiD) analyses were performed, which showed different adaptations for different TTSuVs. Importantly, we identified a more dominant role of Sus scrofa in the evolution of Iotatorquevirus (TTSuV1), with the highest CAI values and lowest RCDI values compared to Sus scrofa domestica. However, in TTSuVk2, the roles of Sus scrofa and Sus scrofa domestica were the same, regarding codon usage, with similar CAI and RCDI values. Our study provides a new perspective of the evolution of TTSuV and valuable information to develop control measures against TTSuV.

Codon usage vis-a-vis start and stop codon context analysis of three dicot species

To understand the variation in genomic composition and its effect on codon usage, we performed the comparative analysis of codon usage and nucleotide usage in the genes of three dicots, Glycine max, Arabidopsis thaliana and Medicago truncatula. The dicot genes were found to be A/T rich and have predominantly A-ending and/or T-ending codons. GC3s directly mimic theusage pattern of global GC content. Relative synonymous codon usage analysis suggests that the high usage frequency of A/T over G/C mononucleotide containing codons in AT-rich dicot genome is due to compositional constraint as a factor of codon usage bias. Odds ratio analysis identified the dinucleotides TpG, TpC, GpA, CpA and CpT as over-represented, where, CpG and TpA as under-represented dinucleotides. The results of (NcExp-NcObs)/NcExp plot suggests that selection pressure other than mutation played a significant role in influencing the pattern of codon usage in these dicots. PR2 analysis revealed the significant role of selection pressure on codon usage. Analysis of varience on codon usage at start and stop site showed variation in codon selection in these sites. This study provides evidence that the dicot genes were subjected to compositional selection pressure.

Translational Fidelity Mediated Regulation of ER-Stress by Dph3

Background: Translational errors can give rise to an accumulation of unfolded proteins leading to the activation of the unfolded protein response. Among the various mechanisms that exist to reduce these errors, uracil modifications, specifically 5-methoxycarbonylmethyluridine (mcm5U), 5-methoxycarbonylmethyl-2- thiouridine (mcm5s2U) and 5-carbamoylmethyluridine (ncm5U), in tRNA increase fidelity by improving binding to A-ending codons in mixed codon boxes. Dph3 is a protein involved in diphthamide synthesis, but also plays a role with the elongator complex. Results: Activation of ER-stress response was observed through the presence of increased splicing of HAC1 mRNA in dph3 mutants Cell viability was decreased in mutants stressed by paromomycin as demonstrated using cytotoxic assays. Dph3 mutants were partially rescued from cell stress by the complementation of DPH3. lacZ reporter assays showed an increased unfolded protein response in mutants lacking Dph3 under both basal and stressed conditions. Conclusion: This experiment revealed the critical role of Dph3 in normal cell?s ability to produce proteins accurately especially when under external stressors. The results showed that the DPH3 gene in Saccharomyces cerevisiae was involved in the prevention of translational errors and the presence of Dph3 lowered ERstress response factors.

Genomic analysis of codon usage shows influence of mutation pressure, natural selection, and host features on Marburg virus evolution.

BackgroundThe Marburg virus (MARV) has a negative-sense single-stranded RNA genome, belongs to the family Filoviridae, and is responsible for several outbreaks of highly fatal hemorrhagic fever. Codon usage patterns of viruses reflect a series of evolutionary changes that enable viruses to shape their survival rates and fitness toward the external environment and, most importantly, their hosts. To understand the evolution of MARV at the codon level, we report a comprehensive analysis of synonymous codon usage patterns in MARV genomes. Multiple codon analysis approaches and statistical methods were performed to determine overall codon usage patterns, biases in codon usage, and influence of various factors, including mutation pressure, natural selection, and its two hosts, Homo sapiens and Rousettus aegyptiacus.ResultsNucleotide composition and relative synonymous codon usage (RSCU) analysis revealed that MARV shows mutation bias and prefers U- and A-ended codons to code amino acids. Effective number of codons analysis indicated that overall codon usage among MARV genomes is slightly biased. The Parity Rule 2 plot analysis showed that GC and AU nucleotides were not used proportionally which accounts for the presence of natural selection. Codon usage patterns of MARV were also found to be influenced by its hosts. This indicates that MARV have evolved codon usage patterns that are specific to both of its hosts. Moreover, selection pressure from R. aegyptiacus on the MARV RSCU patterns was found to be dominant compared with that from H. sapiens. Overall, mutation pressure was found to be the most important and dominant force that shapes codon usage patterns in MARV.ConclusionsTo our knowledge, this is the first detailed codon usage analysis of MARV and extends our understanding of the mechanisms that contribute to codon usage and evolution of MARV.Electronic supplementary materialThe online version of this article (doi:10.1186/s12862-015-0456-4) contains supplementary material, which is available to authorized users.

2-Thiouracil deprived of thiocarbonyl function preferentially base pairs with guanine rather than adenine in RNA and DNA duplexes.

2-Thiouracil-containing nucleosides are essential modified units of natural and synthetic nucleic acids. In particular, the 5-substituted-2-thiouridines (S2Us) present in tRNA play an important role in tuning the translation process through codon–anticodon interactions. The enhanced thermodynamic stability of S2U-containing RNA duplexes and the preferred S2U-A versus S2U-G base pairing are appreciated characteristics of S2U-modified molecular probes. Recently, we have demonstrated that 2-thiouridine (alone or within an RNA chain) is predominantly transformed under oxidative stress conditions to 4-pyrimidinone riboside (H2U) and not to uridine. Due to the important biological functions and various biotechnological applications for sulfur-containing nucleic acids, we compared the thermodynamic stabilities of duplexes containing desulfured products with those of 2-thiouracil-modified RNA and DNA duplexes. Differential scanning calorimetry experiments and theoretical calculations demonstrate that upon 2-thiouracil desulfuration to 4-pyrimidinone, the preferred base pairing of S2U with adenosine is lost, with preferred base pairing with guanosine observed instead. Therefore, biological processes and in vitro assays in which oxidative desulfuration of 2-thiouracil-containing components occurs may be altered. Moreover, we propose that the H2U-G base pair is a suitable model for investigation of the preferred recognition of 3′-G-ending versus A-ending codons by tRNA wobble nucleosides, which may adopt a 4-pyrimidinone-type structural motif.

Codon usage bias analysis for the spermidine synthase gene from Camellia sinensis (L.) O. Kuntze.

The spermidine synthase (SPDS) gene exists widely in all types of plants. In this paper, the codon usage of the SPDS gene from Camellia sinensis (CsSPDS) was analyzed. The results showed that the codon usage of the CsSPDS gene is biased towards the T-ended or A-ended codons, which is similar to that observed in 73 genes selected from the C. sinensis genome. An ENC-plot for 15 SPDS genes from various plant species suggested that mutational bias was the major factor in shaping codon usage in these genes. Codon usage frequency analysis indicated that there was little difference between the CsSPDS gene and dicot genomes, such as Arabidopsis thaliana and Nicotiana tabacum, but significant differences in codon usage were observed between the CsSPDS gene and monocot genomes, such as Triticum aestivum and Zea mays. Therefore, A. thaliana and N. tabacum expression systems may be more suitable for the expression of the CsSPDS gene.

Analysis of synonymous codon usage in FAD7 genes from different plant species.

In this study, the codon bias of the FAD7 genes among 10 different plant species was analyzed to identify general patterns of codon usage in the FAD7 genes. Our results showed that U-ended or A-ended codons were preferentially used in FAD7 for dicots, whereas G-ended or C-ended codons were preferentially used in FAD7 for monocots. An ENC-plot showed that some other factors may influence the codon usage of FAD7, except mutation bias in plant species. A correlation analysis between the codon adaptation index and GC or GC3s contents demonstrated that the codon usage bias of the FAD7 gene in plant species could be influenced by the gene expression level. The cluster analysis of relative synonymous codon usage values and phylogenetic trees of protein sequences for FAD7 genes confirm that the codon preference of FAD7 is influenced by genetic relationships. Moreover, Arabidopsis thaliana and Nicotiana tabacum were predicted to be the most appropriate expression hosts for the FAD7 genes from dicots, and Zea mays may be suitable for the expression of the FAD7 genes from monocots. Our results provide useful insights into the evolutionary relationships of plant species.

Looking into the genome of Thermosynechococcus elongatus (thermophilic cyanobacteria) with codon selection and usage perspective.

Genome analysis of thermophilic cyanobacterium, Thermosynechococcus elongatus BP-1 revealed factors ruling choices of codons in this organism. Multiple parameters like Nc, GC3s, RSCU, Codon Adaptation Index (CAI), optimal and rare codons, codon-pair context and amino acid usage were analysed and compositional constraint was identified as major factor. Wide range of Nc values for the same GC3 content suggested the role of translational selection. Mutational bias is suggested at synonymous position. Among optimal codons for translation, most were GC-ending. Seven codons (AGA, AGG, AUA, UAA, UAG, UCA and UGA) were found to have least occurrence in the entire genome and except stop codons all were A-ending (exception AGG). Most widely used codon-pair in the genome are G-ending or C-ending and A-ending or U-ending codons make pair with G-ending or C-ending codons. Amino acids which are largely distributed in T. elongatus tend to use G-ending or C-ending codons most frequently. Findings showed cumulative role of translational selection, translational accuracy and gene expression levels with mutational bias as key player in codon selection pattern of this organism.

Isolation and expression of CsFAD7 and CsFAD8, two genes encoding ω-3 fatty acid desaturase from Camellia sinensis

The plastidial ω-3 fatty acid desaturase catalyses the production of trienoic fatty acids (TAs) in plant chloroplasts and plays an important role in plant responses to environmental stress. In this study, the full-length cDNAs encoding two plastidial ω-3 desaturases, designated CsFAD7 and CsFAD8 (GenBank Accession No. JX943516 and KC847167, respectively), were isolated from the tea plant (Camellia sinensis L.) using RT-PCR and RACE. Codon usage analysis revealed that U- and A-ended codons were preferentially used in these two genes. Sequence analysis showed that the deduced amino acid sequences of CsFAD7 and CsFAD8 had high homology to plastidial ω-3 desaturases from other plant species. Expression analysis by real-time PCR revealed that both genes are tissue-specific and expressed the highest levels in shoots. Meanwhile, CsFAD7 and CsFAD8 responded to various abiotic stresses and hormones, but in very different manners. Taken together, these results suggest that CsFAD7 and CsFAD8 are both responsive to abiotic stress signals; however, they may play very different roles during stress tolerance in tea plants.

ScnRCA: A Novel Method to Detect Consistent Patterns of Translational Selection in Mutationally-Biased Genomes

Codon usage bias (CUB) results from the complex interplay between translational selection and mutational biases. Current methods for CUB analysis apply heuristics to integrate both components, limiting the depth and scope of CUB analysis as a technique to probe into the evolution and optimization of protein-coding genes. Here we introduce a self-consistent CUB index (scnRCA) that incorporates implicit correction for mutational biases, facilitating exploration of the translational selection component of CUB. We validate this technique using gene expression data and we apply it to a detailed analysis of CUB in the Pseudomonadales. Our results illustrate how the selective enrichment of specific codons among highly expressed genes is preserved in the context of genome-wide shifts in codon frequencies, and how the balance between mutational and translational biases leads to varying definitions of codon optimality. We extend this analysis to other moderate and fast growing bacteria and we provide unified support for the hypothesis that C- and A-ending codons of two-box amino acids, and the U-ending codons of four-box amino acids, are systematically enriched among highly expressed genes across bacteria. The use of an unbiased estimator of CUB allows us to report for the first time that the signature of translational selection is strongly conserved in the Pseudomonadales in spite of drastic changes in genome composition, and extends well beyond the core set of highly optimized genes in each genome. We generalize these results to other moderate and fast growing bacteria, hinting at selection for a universal pattern of gene expression that is conserved and detectable in conserved patterns of codon usage bias.

Characterization of Codon Usage Bias in UL21gene from Duck Enteritis Virus

In this paper, the comprehensive analysis of codon usage bias of Duck enteritis virus (DEV) UL21 gene was performed by using CAI, CHIPS and CUSP program of EMBOSS. Our study showed that codon usage bias of DEV UL21 had strong bias towards the A-ended or T-ended codons, and GC3s contents of the codon usage bias in DEV UL21 gene were significantly varied compared with those of other 27 reference herpesviruses. The CAI, ENC value of DEV CHv strain UL21 gene is 0.615 and 55.167, respectively, indicating that the codon usage bias of this gene is weak and lowly expressed. The plot of ENC versus GC3S content revealed that DEV UL21 gene is subject to GC compositional constraints. The phylogentic analysis about amino acids codon usage bias of DEV UL21 and the27 reference herpesviruses showed that DEV was evolutionarily closer to herpesviruses Mardivirus. In addition, the codon usage bias of DEV UL21 gene was compared with those of E. coli, yeast and humans. There are 42, 45, 39 same codons usage bias between the DEV UL21 to E.coli, Yeast, H.sapiens, respectively, indicaiting that UL21 gene of DEV may be more efficiently expressed in the yeast system.

Synonymous codon usage bias of spike genes of porcine epidemic diarrhea virus

Synonymous codons are not used randomly. Rather, some codons are used more frequently than others. Investigating codon usage bias is essential to the understanding of viral evolution. However the factors shaping synonymous codon usage bias and nucleotide composition in animal viruses have been studied only to a limited extent. In our study, using the 19 complete CDS sequences of Spike (S) genes of porcine epidemic diarrhea virus (PEDV), we analyzed synonymous codon usage bias. The relative synonymous codon usage (RSCU) was used to estimate codon usage variation in each gene, and the results indicated that preferentially used codons were A-ended, G-ended, and U-ended codons. Effective number of codons (ENC) values varies from 48.15 to 49.52, which suggested that the codon usage bias in PEDV S genes was very slightly. Correspondence analysis (COA) was further performed to study the major trend in codon usage variation, and the plot of ENC values against GC3s (at synonymous third codon position) revealed that mutational pressure rather than translational selection was the main factor determining the codon usage bias in PEDV S genes. Moreover, correlation analysis indicated that aromaticity and hydrophobicity of S genes also influenced the codon usage variation in a minor way. Our study provides the most comprehensive analysis to date of PEDV S genes’ codon usage patterns and provides a basic understanding of the mechanisms for codon usage bias. Key words: Porcine epidemic diarrhea virus, Spike gene, relative synonymous codon usage, effective number of codons, correspondence analysis, correlation analysis.

HIV-1 Modulates the tRNA Pool to Improve Translation Efficiency

Despite its poorly adapted codon usage, HIV-1 replicates and is expressed extremely well in human host cells. HIV-1 has recently been shown to package non-lysyl transfer RNAs (tRNAs) in addition to the tRNALys needed for priming reverse transcription and integration of the HIV-1 genome. By comparing the codon usage of HIV-1 genes with that of its human host, we found that tRNAs decoding codons that are highly used by HIV-1 but avoided by its host are overrepresented in HIV-1 virions. In particular, tRNAs decoding A-ending codons, required for the expression of HIV's A-rich genome, are highly enriched. Because the affinity of Gag-Pol for all tRNAs is nonspecific, HIV packaging is most likely passive and reflects the tRNA pool at the time of viral particle formation. Codon usage of HIV-1 early genes is similar to that of highly expressed host genes, but codon usage of HIV-1 late genes was better adapted to the selectively enriched tRNA pool, suggesting that alterations in the tRNA pool are induced late in viral infection. If HIV-1 genes are adapting to an altered tRNA pool, codon adaptation of HIV-1 may be better than previously thought.

Comparative Analysis of Codon Usage and tRNA in Mitochondrial Genomes of Gallus Gallus

Codon usage in mitochondrial genes of 11 Gallus gallus and two Anatidae species was analysed to determine the general patterns in codon choice of Callus gallus species. C3 contents were higher in Gallus gallus than in mammalian mitochondrial genomes that encode protein codon positions. The high C3 contents of Callus gallus might be the result of relatively strong mutational bias that occurred in the lineage of the Callus gallus species. A and C ending codons were detected as the “preferred 77 codons in Callus gallus and Anatidae. The NNR codon families are dominated by the A-ending codons, the NNY codon families are dominated by the C-ending codons and the NNN codon families are dominated by the A-ending or the C-ending codons. A comparison of the relative synonymous codon usage (RSCU) and synonymous codon families (SCF) of tRNA and proteins was made, and two groups can be classified by SCF. The codon usage in Callus gallus species indicates that codons containing A or C at the third position are used preferentially, regardless of whether corresponding tRNAs are encoded in the mtDNA. In both Callus gallus and Anatidae species mtDNA, codon usage biases are highly related to CC-ending binucleotide condons.