Higher Codon Usage Bias Research Articles

Niche differentiation in microbial communities with stable genomic traits over time in engineered systems.

Microbial communities in full-scale engineered systems undergo dynamic compositional changes. However, mechanisms governing assembly of such microbes and succession of their functioning and genomic traits under various environmental conditions are unclear. In this study, we used the activated sludge and anaerobic treatment systems of four full-scale industrial wastewater treatment plants as models to investigate the niches of microbes in communities and the temporal succession patterns of community compositions. High-quality representative metagenome-assembled genomes revealed that taxonomic, functional, and trait-based compositions were strongly shaped by environmental selection, with replacement processes primarily driving variations in taxonomic and functional compositions. Plant-specific indicators were associated with system environmental conditions and exhibited strong determinism and trajectory directionality over time. The partitioning of microbes in a co-abundance network according to groups of plant-specific indicators, together with significant between-group differences in genomic traits, indicated the occurrence of niche differentiation. The indicators of the treatment plant with rich nutrient input and high substrate removal efficiency exhibited a faster predicted growth rate, lower guanine-cytosine content, smaller genome size, and higher codon usage bias than the indicators of the other plants. In individual plants, taxonomic composition displayed a more rapid temporal succession than functional and trait-based compositions. The succession of taxonomic, functional, and trait-based compositions was correlated with the kinetics of treatment processes in the activated sludge systems. This study provides insights into ecological niches of microbes in engineered systems and succession patterns of their functions and traits, which will aid microbial community management to improve treatment performance.

Differential Synonymous Codon Selection in the B56 Gene Family of PP2A Regulatory Subunits.

Protein phosphatase 2A (PP2A) functions as a tumor suppressor and consists of a scaffolding, catalytic, and regulatory subunit. The B56 gene family of regulatory subunits impart distinct functions onto PP2A. Codon usage bias (CUB) involves the selection of synonymous codons, which can affect gene expression by modulating processes such as transcription and translation. CUB can vary along the length of a gene, and differential use of synonymous codons can be important in the divergence of gene families. The N-termini of the gene product encoded by B56α possessed high CUB, high GC content at the third codon position (GC3), and high rare codon content. In addition, differential CUB was found in the sequence encoding two B56γ N-terminal splice forms. The sequence encoding the N-termini of B56γ/γ, relative to B56δ/γ, displayed CUB, utilized more frequent codons, and had higher GC3 content. B56α mRNA had stronger than predicted secondary structure at their 5' end, and the B56δ/γ splice variants had long regions of weaker than predicted secondary structure at their 5' end. The data suggest that B56α is expressed at relatively low levels as compared to the other B56 isoforms and that the B56δ/γ splice variant is expressed more highly than B56γ/γ.

Codon usage bias analysis of the gene encoding NAD+-dependent DNA ligase protein of Invertebrate iridescent virus 6.

The genome of Invertebrate iridescent virus 6 (IIV6) contains a sequence that shows similarity to eubacterial NAD+-dependent DNA ligases. The 615-amino acid open reading frame (ORF 205R) consists of several domains, including an N-terminal domain Ia, followed by an adenylation domain, an OB-fold domain, a helix-hairpin-helix (HhH) domain, and a BRCT domain. Notably, the zinc finger domain, typically present in NAD+-dependent DNA ligases, is absent in ORF 205R. Since the protein encoded by ORF 205R (IIV6 DNA ligase gene) is involved in critical functions such as DNA replication, modification, and repair, it is crucial to comprehend the codon usage associated with this gene. In this paper, the codon usage bias (CUB) in DNA ligase gene of IIV6 and 11 reference iridoviruses was analyzed by comparing the nucleotide contents, relative synonymous codon usage (RSCU), effective number of codons (ENC), codon adaptation index (CAI), relative abundance of dinucleotides and other indices. Both the base content and the RCSU analysis indicated that the A- and T-ending codons were mostly favored in the DNA ligase gene of IIV6. The ENC value of 35.64 implied a high CUB in the IIV6 DNA ligase gene. The ENC plot, neutrality plot, parity rule 2 plot, correspondence analysis revealed that mutation pressure and natural selection had an impact on the CUB of the IIVs DNA ligase genes. Additionally, the analysis of codon adaptation index demonstrated that the IIV6 DNA ligase gene is strongly adapted to its host. These findings will improve our comprehension of the CUB of IIV6 DNA ligase and reference genes, which may provide the required information for a fundamental evolutionary analysis of these genes.

Revisiting SARS-CoV-2: Evolution, polymorphism and compatibility with human tRNA pool in a lineage over a month


 
 
 Introduction: The present study presents a comparative analysis of SARS-CoV-2‟s survival capacity in its human host in an area within a month. Materials and Methods: Codon usage bias study has been carried using Emboss package and evolutionary study has been carried using pn/ps packages in python and R for statistical analysis. Results: The virus has overlapping genes exhibiting a high codon usage bias and optimization with human Lung housekeeping genes. Viral ORFs have near values of minimum folding energies and codon adaptation index with mRNAs of the human Lung housekeeping genes. Then too, viruses showed a greater expression capacity. Polymorphism is in the virus for ORF1ab, surface glycoprotein and nucleocapsid phosphoprotein ORFs. Non-synonymous mutations have shown non-polar substitutions. Out of the twelve mutations nine are for a higher t-RNA copy number. Synonymous mutation simulation mimicking evolution revealed fitter newer strains. Conclusion: Through this study we have explained the inherent codon adaptation of SARS-CoV-2 with human tRNA pool and how the virus shows polymorphism in-order to keep up with its infectious capacity. Hence, giving an insight into viral rapid adaptability.
 
 

Comparative Analysis Reveals Different Evolutionary Fates and Biological Functions in Wheat Duplicated Genes (Triticum aestivum L.).

Wheat (Triticum aestivum L.) is a staple food crop that provides 20% of total human calorie consumption. Gene duplication has been considered to play an important role in evolution by providing new genetic resources. However, the evolutionary fates and biological functions of the duplicated genes in wheat remain to be elucidated. In this study, the resulting data showed that the duplicated genes evolved faster with shorter gene lengths, higher codon usage bias, lower expression levels, and higher tissue specificity when compared to non-duplicated genes. Our analysis further revealed functions of duplicated genes in various biological processes with significant enrichment to environmental stresses. In addition, duplicated genes derived from dispersed, proximal, tandem, transposed, and whole-genome duplication differed in abundance, evolutionary rate, gene compactness, expression pattern, and genetic diversity. Tandem and proximal duplicates experienced stronger selective pressure and showed a more compact gene structure with diverse expression profiles than other duplication modes. Moreover, genes derived from different duplication modes showed an asymmetrical evolutionary pattern for wheat A, B, and D subgenomes. Several candidate duplication hotspots associated with wheat domestication or polyploidization were characterized as potential targets for wheat molecular breeding. Our comprehensive analysis revealed the evolutionary trajectory of duplicated genes and laid the foundation for future functional studies on wheat.

Molecular evolution and population genetics of glutamate decarboxylase acid resistance pathway in lactic acid bacteria.

Glutamate decarboxylase (GAD) pathway (GDP) is a major acid resistance mechanism enabling microorganisms' survival in low pH environments. We aimed to study the molecular evolution and population genetics of GDP in Lactic Acid Bacteria (LAB) to understand evolutionary processes shaping adaptation to acidic environments comparing species where the GDP genes are organized in an operon structure (Levilactobacillus brevis) versus lack of an operon structure (Lactiplantibacillus plantarum). Within species molecular population genetic analyses of GDP genes in L. brevis and L. plantarum sampled from diverse fermented food and other environments showed abundant synonymous and non-synonymous nucleotide diversity, mostly driven by low frequency changes, distributed throughout the coding regions for all genes in both species. GAD genes showed higher level of replacement polymorphism compared to transporter genes (gadC and YjeM) for both species, and GAD genes that are outside of an operon structure showed even higher level of replacement polymorphism. Population genetic tests suggest negative selection against replacement changes in all genes. Molecular structure and amino acid characteristics analyses showed that in none of the GDP genes replacement changes alter 3D structure or charge distribution supporting negative selection against non-conservative amino acid changes. Phylogenetic and between species divergence analyses suggested adaptive protein evolution on GDP genes comparing phylogenetically distant species, but conservative evolution comparing closely related species. GDP genes within an operon structure showed slower molecular evolution and higher conservation. All GAD and transporter genes showed high codon usage bias in examined LAB species suggesting high expression and utilization of acid resistance genes. Substantial discordances between species, GAD, and transporter gene tree topologies were observed suggesting molecular evolution of GDP genes do not follow speciation events. Distribution of operon structure on the species tree suggested multiple independent gain or loss of operon structure in LABs. In conclusion, GDP genes in LABs exhibit a dynamic molecular evolutionary history shaped by gene loss, gene transfer, negative and positive selection to maintain its active role in acid resistance mechanism, and enable organisms to thrive in acidic environments.

Mitogenome-wise codon usage pattern from comparative analysis of the first mitogenome of Blepharipa sp. (Muga uzifly) with other Oestroid flies

Uziflies (Family: Tachinidae) are dipteran endoparasites of sericigenous insects which cause major economic loss in the silk industry globally. Here, we are presenting the first full mitogenome of Blepharipa sp. (Acc: KY644698, 15,080 bp, A + T = 78.41%), a dipteran parasitoid of Muga silkworm (Antheraea assamensis) found in the Indian states of Assam and Meghalaya. This study has confirmed that Blepharipa sp. mitogenome gene content and arrangement is similar to other Tachinidae and Sarcophagidae flies of Oestroidea superfamily, typical of ancestral Diptera. Although, Calliphoridae and Oestridae flies have undergone tRNA translocation and insertion, forming unique intergenic spacers (IGS) and overlapping regions (OL) and a few of them (IGS, OL) have been conserved across Oestroidea flies. The Tachinidae mitogenomes exhibit more AT content and AT biased codons in their protein-coding genes (PCGs) than the Oestroidea counterpart. About 92.07% of all (3722) codons in PCGs of this new species have A/T in their 3rd codon position. The high proportion of AT and repeats in the control region (CR) affects sequence coverage, resulting in a short CR (Blepharipa sp.: 168 bp) and a smaller tachinid mitogenome. Our research unveils those genes with a high AT content had a reduced effective number of codons, leading to high codon usage bias. The neutrality test shows that natural selection has a stronger influence on codon usage bias than directed mutational pressure. This study also reveals that longer PCGs (e.g., nad5, cox1) have a higher codon usage bias than shorter PCGs (e.g., atp8, nad4l). The divergence rates increase nonlinearly as AT content at the 3rd codon position increases and higher rate of synonymous divergence than nonsynonymous divergence causes strong purifying selection. The phylogenetic analysis explains that Blepharipa sp. is well suited in the family of insectivorous tachinid maggots. It's possible that biased codon usage in the Tachinidae family reduces the effective number of codons, and purifying selection retains the core functions in their mitogenome, which could help with efficient metabolism in their endo-parasitic life style and survival strategy.

Understanding the codon usage patterns of mitochondrial CO genes among Amphibians.

A universal phenomenon of using synonymous codons unequally in coding sequences known as codon usage bias (CUB) is observed in all forms of life. Mutation and natural selection drive CUB in many species but the relative role of evolutionary forces varies across species, genes and genomes. We studied the CUB in mitochondrial (mt) CO genes from three orders of Amphibia using bioinformatics approach as no work was reported yet. We observed that CUB of mt CO genes of Amphibians was weak across different orders. Order Caudata had higher CUB followed by Gymnophiona and Anura for all genes and CUB also varied across genes. Nucleotide composition analysis showed that CO genes were AT-rich. The AT content in Caudata was higher than that in Gymnophiona while Anura showed the least content. Multiple investigations namely nucleotide composition, correspondence analysis, parity plot analysis showed that the interplay of mutation pressure and natural selection caused CUB in these genes. Neutrality plot suggested the involvement of natural selection was more than the mutation pressure. The contribution of natural selection was higher in Anura than Gymnophiona and the lowest in Caudata. The codons CGA, TGA, AAA were found to be highly favoured by nature across all genes and orders.

The characteristic of Arachis duranensis-specific genes and their potential function

Arachis species produce flowers aerially, and then grow into the ground, where they develop into fruits; a feature that is unique to Arachis species. We hypothesized that Arachis species evolved genes specifically involved in the control of aerial flowers and the formation of underground fruits. Arachis duranensis is more resistant to biotic and abiotic stressors. Here, we compared different legume species and identified Arachis duranensis-specific genes. We analyzed gene expression patterns, base substitution patterns and sequence features between genes that are conserved across legume plants and A. duranensis-specific genes. Furthermore, we tested the role of A. duranensis-specific genes during seed development, response to nematode Meloidogyne arenaria infection and drought stress. We found that A. duranensis-specific genes had characteristics of young genes. The gene expression level and breadth were lower in the A. duranensis-specific genes compared to conserved genes. The A. duranensis-specific genes had higher codon usage bias than conserved genes, and the polypeptide length and GC content at the three codon sites were lower compared to conserved genes. Of the A. duranensis-specific genes, single-copy and duplicated genes had different features. The RNA-seq result showed A. duranensis-specific genes were involved in seed development, as well as response to nematode infection and drought stress. In addition, we detected asymmetric functions in A. duranensis-specific duplicated genes in response to nematode infection and drought stress.

Factors Affecting Synonymous Codon Usage of Housekeeping Genes in drosophila Melanogaster

Housekeeping genes (HK genes) are required for cell survival and the maintenance of basic cellular functions. The investigation of factors affecting codon usage patterns in HK genes of insects can help in understanding the molecular evolution of insects and aid the development of insect pest management strategies. In this study, we employed bioinformatics approaches to analyze the codon usage bias (CUB) of HK genes in the insect model organism, Drosophila melanogaster. A comparison of CUB between 1107 HK genes and 1084 high tissue specifcity genes suggested that HK genes have higher CUB in D. melanogaster. In addition, we found that CUB inversely correlates with the non-synonymous substitution rate of HK genes. Therefore, we attempted to identify the factors that potentially infuence the codon usage pattern of HK genes. Our results suggest that mutation pressure and natural selection highly correlate with CUB in the HK genes of D. melanogaster and that two topological properties of HK proteins (proportion of protein interacting length and protein connectivity) also correlate with CUB in the HK genes of D. melanogaster. This study provides insight into CUB in the HK genes of D. melanogaster, and the results can support future investigations of potential applications in agricultural and biomedical feld.

Determinants of translation speed are randomly distributed across transcripts resulting in a universal scaling of protein synthesis times.

Ribosome profiling experiments have found greater than 100-fold variation in ribosome density along mRNA transcripts, indicating that individual codon elongation rates can vary to a similar degree. This wide range of elongation times, coupled with differences in codon usage between transcripts, suggests that the average codon translation-rate per gene can vary widely. Yet, ribosome run-off experiments have found that the average codon translation rate for different groups of transcripts in mouse stem cells is constant at 5.6 AA/s. How these seemingly contradictory results can be reconciled is the focus of this study. Here, we combine knowledge of the molecular factors shown to influence translation speed with genomic information from Escherichia coli, Saccharomyces cerevisiae and Homo sapiens to simulate the synthesis of cytosolic proteins in these organisms. The model recapitulates a near constant average translation rate, which we demonstrate arises because the molecular determinants of translation speed are distributed nearly randomly amongst most of the transcripts. Consequently, codon translation rates are also randomly distributed and fast-translating segments of a transcript are likely to be offset by equally probable slow-translating segments, resulting in similar average elongation rates for most transcripts. We also show that the codon usage bias does not significantly affect the near random distribution of codon translation rates because only about 10% of the total transcripts in an organism have high codon usage bias while the rest have little to no bias. Analysis of Ribo-Seq data and an in vivo fluorescent assay supports these conclusions.

Comprehensive analysis of correlations among codon usage bias, gene expression, and substitution rate in Arachis duranensis and Arachis ipa\xebnsis orthologs

The relationship between evolutionary rates and gene expression in model plant orthologs is well documented. However, little is known about the relationships between gene expression and evolutionary trends in Arachis orthologs. We identified 7,435 one-to-one orthologs, including 925 single-copy and 6,510 multiple-copy sequences in Arachis duranensis and Arachis ipaënsis. Codon usage was stronger for shorter polypeptides, which were encoded by codons with higher GC contents. Highly expressed coding sequences had higher codon usage bias, GC content, and expression breadth. Additionally, expression breadth was positively correlated with polypeptide length, but there was no correlation between gene expression and polypeptide length. Inferred selective pressure was also negatively correlated with both gene expression and expression breadth in all one-to-one orthologs, while positively but non-significantly correlated with gene expression in sequences with signatures of positive selection. Gene expression levels and expression breadth were significantly higher for single-copy genes than for multiple-copy genes. Similarly, the gene expression and expression breadth in sequences with signatures of purifying selection were higher than those of sequences with positive selective signatures. These results indicated that gene expression differed between single-copy and multiple-copy genes as well as sequences with signatures of positive and purifying selection.

Differential evolutionary patterns and expression levels between sex-specific and somatic tissue-specific genes in peanut

The patterns of evolution and expression of tissue-specific genes are poorly understood beyond sex-specific genes. Accordingly, we identified 3,191 tissue-specific genes and 38,745 common genes using 22 RNA-seq datasets from cultivated peanut. The expression levels of tissue-specific genes were significantly lower than those of common genes. Further, the expression levels of sex-specific genes were significantly higher than those of somatic tissue-specific genes. Among sex-specific genes, the expression levels of gynoecium-specific genes were significantly higher than those of androecium-specific genes. Function-specific genes were lacking among tissue-specific genes, and tissue-specific gene annotations overlapped among different tissues. Duplicate gene pairs were classified as homogeneous pairs expressed within the same tissue or heterogeneous pairs expressed in different tissues. Heterogeneous gene pairs evolved more rapidly than homogeneous gene pairs. In addition, somatic tissue-specific genes evolved faster than sex-specific genes. Molecular signatures of selection indicated that somatic tissue-specific genes have mainly experienced relaxed selection, while sex-specific genes have been under stronger selective constraint. Somatic tissue-specific genes had higher codon usage bias than sex-specific genes. These contrasting patterns between somatic tissue-specific and sex-specific genes provide new insights into the basic biology and evolution of peanut, an important crop.

Pangenome Evidence for Higher Codon Usage Bias and Stronger Translational Selection in Core Genes of Escherichia coli.

Codon usage bias, as a combined interplay from mutation and selection, has been intensively studied in Escherichia coli. However, codon usage analysis in an E. coli pangenome remains unexplored and the relative importance of mutation and selection acting on core genes and strain-specific genes is unknown. Here we perform comprehensive codon usage analyses based on a collection of multiple complete genome sequences of E. coli. Our results show that core genes that are present in all strains have higher codon usage bias than strain-specific genes that are unique to single strains. We further explore the forces in influencing codon usage and investigate the difference of the major force between core and strain-specific genes. Our results demonstrate that although mutation may exert genome-wide influences on codon usage acting similarly in different gene sets, selection dominates as an important force to shape biased codon usage as genes are present in an increased number of strains. Together, our results provide important insights for better understanding genome plasticity and complexity as well as evolutionary mechanisms behind codon usage bias.

Genome Sequence and Analysis of Peptoclostridium difficile Strain ZJCDC-S82.

Peptoclostridium difficile (Clostridium difficile) is the major pathogen associated with infectious diarrhea in humans. Concomitant with the increased incidence of C. difficile infection worldwide, there is an increasing concern regarding this infection type. This study reports a draft assembly and detailed sequence analysis of C. difficile strain ZJCDC-S82. The de novo assembled genome was 4.19 Mb in size, which includes 4,013 protein-coding genes, 41 rRNA genes, and 84 tRNA genes. Along with the nuclear genome, we also assembled sequencing information for a single plasmid consisting of 11,930 nucleotides. Comparative genomic analysis of C. difficile ZJCDC-S82 and two other previously published strains, such as M120 and CD630, showed extensive similarity. Phylogenetic analysis revealed that genetic diversity among C. difficile strains was not influenced by geographic location. Evolutionary analysis suggested that four genes encoding surface proteins exhibited positive selection in C. difficile ZJCDC-S82. Codon usage analysis indicated that C. difficile ZJCDC-S82 had high codon usage bias toward A/U-ended codons. Furthermore, codon usage patterns in C. difficile ZJCDC-S82 were predominantly affected by mutation pressure. Our results provide detailed information pertaining to the C. difficile genome associated with a strain from mainland China. This analysis will facilitate the understanding of genomic diversity and evolution of C. difficile strains in this region.

Optimization protein productivity of human interleukin-2 through codon usage, gene copy number and intracellular tRNA concentration in CHO cells

Transfer RNA (tRNA) abundance is one of the critical factors for the enhancement of protein productivity in prokaryotic and eukaryotic hosts. Gene copy number of tRNA and tRNA codon usage bias are generally used to match tRNA abundance of protein-expressing hosts and to optimize the codons of recombinant proteins. Because sufficient concentration of intracellular tRNA and optimized codons of recombinant proteins enhanced translation efficiency, we hypothesized that sufficient supplement of host’s tRNA improved protein productivity in mammalian cells. First, the small tRNA sequencing results of CHO-K1 cells showed moderate positive correlation with gene copy number and codon usage bias. Modification of human interleukin-2 (IL-2) through codons with high gene copy number and high codon usage bias (IL-2 HH, modified on Leu, Thr, Glu) significantly increased protein productivity in CHO-K1 cells. In contrast, modification through codons with relatively high gene copy number and low codon usage bias (IL-2 HL, modified on Ala, Thr, Val), or relatively low gene copy number and low codon usage bias (IL-2 LH, modified on Ala, Thr, Val) did not increase IL-2 productivity significantly. Furthermore, supplement of the alanine tRNA or threonine tRNA increased IL-2 productivity of IL-2 HL. In summary, we revealed a potential strategy to enhance productivity of recombinant proteins, which may be applied in production of protein drug or design of DNA vaccine.

The Implication of Codon Usage Design and Expression Level in Determining the Nature of Selection and Functionality Amongst the Amino Acid Biosynthetic Pathway Coding Sequences of Arthrobacter sp. FB24

Amino acid biosynthesis is an immensely important life process requiring the participation of nearly hundred gene products, differing in terms of codon usage pattern, expressivity and selection pressure. In this study an attempt was made to categorize the gene products according to their expression level, nature of selection and functionality analyzing the amino acid biosynthetic pathway genes of the soil bacterium Arthrobacter, known for its varied metabolic activities and a key agent for industrial production of amino acids. The relationship between the different parameters like expression level, codon usage and selection pressure was calculated to find out the correlation of biasness, expression level, nature of selection and functionality amongst the genes involved in amino acid biosynthetic pathways. We found that genes coding for enzymes and proteins with large number of functions have higher codon usage bias and are subjected mostly to purifying selection. Gene sequences of proteins and enzymes with low functionality showed less codon usage bias and are under positive selection. The multifunctional genes mostly show high expression level and it is highly correlated with codon usage bias. The results of this study demonstrate that functionality and nature of selective forces acting on the coding sequences can be determined by studying the correlation between the different parameters like effective number of codon, codon adaptation index and nucleotide substitution ratios and careful study of all these parameters is a pretty good indicator for determining the functionality of the genes and this can be extended towards the genes of unknown function(s). Keywords: Amino acid biosynthetic pathways, Arthrobacter, CAI, Nc, Ka/Ks, synonymous and non-synonymous nucleotide substitution rates, positive selection, purifying selection.

Codon Usage Bias of Human Cytomegalovirus Genes with Different Evolutionary Conservancy

Human cytomegalovirus (HCMV) is a member of beta-herpesvirus and contains a double-stranded genome with longer than 230 Kbp. HCMV infection of human is mostly asymptomatic, but often causes fatal diseases in immunocompromised people. In this study, codon usages of HCMV genes were analyzed and attempted to correlate with evolutionary conservancy. Core genes are the most conserved genes common among herpesvirus family, β-herpes genes are common to β-herpesviruses, and CMV genes are the least conserved found only in CMVs. Core genes had higher codon adaptation index (CAI) and GC content of silent 3rd codon position (GC3s) values and lower effective number of codons (Nc) and Nc/GC3s values than CMV genes. The average length of core genes was statistically longer than CMV genes, and core genes were found to be less varied than CMV genes. β-herpes genes could be placed between core and CMV genes. Higher CAI and GC3s values along with lower Nc and Nc/GC3s values are suggestive of higher codon usage bias and more adaptation to host cells. Thus it is concluded that core genes of HCMV are more biased in codon usage and adapted to host cells compared to CMV genes.

Unusual codon usage bias in low expression genes of Vibrio cholerae.

Positive correlation between gene expression and synonymous codon usage bias is well documented in the literature. However, in the present study of Vibrio cholerae genome, we have identified a group of genes having unusually high codon usage bias despite being low potential expressivity. Our results suggest that codon usage in lowly expressed genes might also be selected on to preferably use non-optimal codons to maintain a low cellular concentration of the proteins that they encode. This would predict that lowly expressed genes are also biased in codon usage, but in a way that is opposite to the bias of highly expressed genes.

Purifying selection drives the evolution of surfactant protein C (SP-C) independently of body temperature regulation in mammals

The pulmonary surfactant system of heterothermic mammals must be capable of dealing with the effect of low body temperatures on the physical state of the lipid components. We have shown previously that there is a modest increase in surfactant cholesterol during periods of torpor, however these changes do not fully explain the capacity of surfactant to function under the wide range of physical conditions imposed by torpor. Here we examine indirectly the role of surfactant protein C (SP-C) in adapting to variable body temperatures by testing for the presence of positive (adaptive) selection during evolutionary transitions between heterothermy and homeothermy. We sequenced SP-C from genomic DNA of 32 mammalian species from groups of closely related heterothermic and homeothermic species (contrasts). We used phylogenetic analysis by maximum likelihood estimates of rates of non-synonymous to synonymous substitutions and fully Bayesian inference of these sequences to determine whether the mode of body temperature regulation exerts a selection pressure driving the molecular adaptation of SP-C. The protein sequence of SP-C is highly conserved with synonymous or highly conservative amino acid substitutions being predominant. The evolution of SP-C among mammals is characterised by high codon usage bias and high rates of transition/transversion. The only contrast to show evidence of positive selection was that of the bears ( Ursus americanus and U. maritimus). The significance of this result is unclear. We show that SP-C is under strong evolutionary constraints, driven by purifying selection, presumably to maintain protein function despite variation in the mode of body temperature regulation.