The Effect of Local Nucleotides on Synonymous Codon Usage in the Honeybee (<Emphasis Type="Italic">Apis mellifera</Emphasis> L.) Genome

Using all currently predicted coding regions in the honeybee genome, a novel form of synonymous codon bias is presented that affects the usage of particular codons dependent on the surrounding nucleotides in the coding region. Nucleotides at the third codon site are correlated, dependent on their weak (adenine [A] or thyamine [T]) versus strong (guanine [G] or cytosine [C]) status, to nucleotides on the first codon site which are dependent on their purine (A/G) versus pyrimidine (C/T) status. In particular, for adjacent third and first site nucleotides, weak–pyrimidine and strong–purine nucleotide combinations occur much more frequently than the underabundant weak–purine and strong–pyrimidine nucleotide combinations. Since a similar effect is also found in the noncoding regions, but is present for all adjacent nucleotides, this coding effect is most likely due to a genome-wide context-dependent mutation error correcting mechanism in combination with selective constraints on adjacent first and second nucleotide pairs within codons. The position-dependent relationship of synonymous codon usage is evidence for a novel form of codon position bias which utilizes the redundancy in the genetic code to minimize the effect of nucleotide mutations within coding regions. [Reviewing Editor: Dr. Brian Morton]     

An Extensive Study of Mutation and Selection on the Wobble Nucleotide in tRNA Anticodons in Fungal Mitochondrial Genomes

Two alternative hypotheses aim to predict the wobble nucleotide of tRNA anticodons in mitochondrion. The codon-anticodon adaptation hypothesis predicts that the wobble nucleotide of tRNA anticodon should evolve toward maximizing the Watson-Crick base pairing with the most frequently used codon within each synonymous codon family. In contrast, the wobble versatility hypothesis argues that the nucleotide at the wobble site should be occupied by a nucleotide most versatile in wobble pairing, i.e., the wobble site of the tRNA anticodon should be G for NNY codon families and U for NNR and NNN codon families (where Y stands for C or U, R for A or G, and N for any nucleotide). We examined codon usage and anticodon wobble sites in 36 fungal genomes to evaluate these two alternative hypotheses and identify exceptional cases that deserve new explanations. While the wobble versatility hypothesis is generally supported, there are interesting exceptions involving tRNA(Arg) translating the CGN codon family, tRNA(Trp) translating the UGR codon family, and tRNA(Met) translating the AUR codon family. Our results suggest that the potential to suppress stop codons, the historical inertia, and the conflict between translation initiation and elongation can all contribute to determining the wobble nucleotide of tRNA anticodons.     

Nucleic acid composition,codon usage,and the rate of synonymous substitution in protein-coding genes

Summary Based on the rates of synonymous substitution in 42 protein-codin gene pairs from rat and human, a correlation is shown to exist between the frequency of the nucleotides in all positions of the codon and the synonymous substitution rate. The correlation coefficients were positive for A and T and negative for C and G. This means that AT-rich genes accumulate more synonymous substitutions than GC-rich genes. Biased patterns of mutation could not account for this phenomenon. Thus, the variation in synonymous substitution rates and the resulting unequal codon usage must be the consequence of selection against A and T in synonymous positions. Most of the varition in rates of synonymous substitution can be explained by the nucleotide composition in synonymous positions. Codon-anticodon interactions, dinucleotide frequencies, and contextual factors influence neither the rates of synonymous substitution nor codon usage. Interestingly, the nucleotide in the second position of codons (always a nonsynonymous position) was found to affect the rate of synonymous substitution. This finding links the rate of nonsynonymous substitution with the synonymous rate. Consequently, highly conservative proteins are expected to be encoded by genes that evolve slowly in terms of synonymous substitutions, and are consequently highly biased in their codon usage.     

Analysis of codon usage pattern in the radioresistant bacterium Deinococcus radiodurans

The main factors shaping codon usage bias in the Deinococcus radiodurans genome were reported. Correspondence analysis (COA) was carried out to analyze synonymous codon usage bias. The results showed that the main trend was strongly correlated with gene expression level assessed by the "Codon Adaptation Index" (CAI) values, a result that was confirmed by the distribution of genes along the first axis. The results of correlation analysis, variance analysis and neutrality plot indicated that gene nucleotide composition was clearly contributed to codon bias. CDS length was also key factor in dictating codon usage variation. A general tendency of more biased codon usage of genes with longer CDS length to higher expression level was found. Further, the hydrophobicity of each protein also played a role in shaping codon usage in this organism, which could be confirmed by the significant correlation between the positions of genes placed on the first axis and the hydrophobicity values (r=-0.100, P<0.01). In summary, gene expression level played a crucial role, nucleotide mutational bias, CDS length and the hydrophobicity of each protein just in a minor way in shaping the codon usage pattern of D. radiodurans. Notably, 19 codons firstly defined as "optimal codons" may provide useful clues for molecular genetic engineering and evolutionary studying.     

The 3' Untranslated Region of Messages in the Rumen Protozoan Entodinium caudatum

The 3' untranslated regions of a number of cDNAs from the rumen protozoal species Entodinium caudatum were studied with a view to characterising their preference for stop codons, general length, nucleotide composition and polyadenylation signals. Unlike a number of ciliates, Entodinium caudatum uses UAA as a stop codon, rather than as a codon for glutamine. In addition, the 3' untranslated region of the message is generally less than 100 nucleotides in length, extremely A+T rich, and does not appear to utilise any of the conventional polyadenylation signals described in other organisms.     

Synonymous substitutions are clustered in enterobacterial genes

The spatial distribution of synonymous substitutions in enterobacterial genes is investigated. It is shown that synonymous substitutions are significantly clustered in such a way that a synonymous substitution in one codon elevates the rate of synonymous substitution in an adjacent codon by about 10%. The level of clustering does not appear to be related to the level of gene expression, and it is restricted to a range of two or three codons. There are at least three possible explanations: (1) sequence-directed mutagenesis, (2) recombination, and (3) selection.     

Selection on the codon bias ofChlamydomonas reinhardtii chloroplast genes and the plantpsbA gene

Plant chloroplast genes have a codon use that reflects the genome compositional bias of a high A+T content with the single exception of the highly translatedpsbA gene which codes for the photosystem II D1 protein. The codon usage of plantpsbA corresponds more closely to the limited tRNA population of the chloroplast and is very similar to the codon use observed in the chloroplast genes of the green algaChlamydomonas reinhardtii. This pattern of codon use may be an adaptation for increased translation efficiency. A correspondence between codon use of plantpsbA andChlamydomonas chloroplast genes and the tRNAs coded by the chloroplast genome, however, is not observed in all synonymous codon groups. It is shown here that the degree of correspondence between codon use and tRNA population in different synonymous groups is correlated with the second codon position composition. Synonymous groups with an A or T at the second codon position have a high representation of codons for which a complementary tRNA is coded by the chloroplast genome. Those with a G or C at the second position have an increased representation of codons that bind a chloroplast tRNA by wobble. It is proposed that the difference between synonymous groups in terms of codon adaptation to the tRNA population in plantpsbA andChlamydomonas chloroplast genes may be the result of differences in second position composition.     

The close proximity ofEscherichia coli genes: Consequences for stop codon and synonymous codon use

It is shown that synonymous codon usage is less biased in favor of those codons preferred by highly expressed genes at the end ofEscherichia coli genes than in the middle. This appears to be due to the close proximity of manyE. coli genes. It is shown that a substantial number of genes overlap either the Shine-Dalgarno sequence or the coding sequence of the next gene on the chromosome and that the codons that overlap have lower synonymous codon bias than those which do not. It is also shown that there is an increase in the frequency of A-ending codons, and a decrease in the frequency of G-ending codons at the end ofE. coli genes that lie close to another gene. It is suggested that these trends in composition could be associated with selection against the formation of mRNA secondary structure near the start of the next gene on the chromosome. Stop codon use is also affected by the close proximity of genes; many genes are forced to use TGA and TAG stop codons because they terminate either within the Shine-Dalgarno or coding sequence of the next gene on the chromosome. The implications these results have for the evolution of synonymous codon use are discussed.     

The nucleotide sequence of a carboxymethylcellulase gene from Pseudomonas fluorescens subsp. cellulosa

Summary The complete nucleotide sequence of the gene coding for one of the carboxymethycellulases (CMCase), expressed by Pseudomonas fluorescens subsp. cellulosa, has been determined. The structural gene consists of an open reading frame, commencing with an ATG start codon, of 2886 base pairs followed by a TAA stop codon. The gene was shown to code for a signal peptide which closely resembles the signal peptides of other secreted proteins. Unlike most pseudomonas genes, the CMCase sequence does not have a high G+C (51%) content and there is no marked preference for codons ending in G or C. Upstream of the structural gene there are no sequences which bear a strong resemblance to consensus Escherichia coli promoters. A sequence is present, however, which exhibits homology to the consensus DNA sequence that binds the catabolic activator protein (CAP). Bal31 deletions of the structural gene revealed the extent by which the gene could be modified and still encode a functional CMCase. Subclones of the cellulase gene have been constructed in pUC18 and pUC19. One of the resultant plasmids, pJHS1 directs a 20-fold increase in CMCase synthesis, when compared to the original construct, pJHH2. Analysis of cells harbouring pJHS1 showed the cellulase polypeptide to have a molecular weight of 106000. This is in close agreement with the predicted size of the enzyme deduced from the nucleotide sequence data.Abbreviations CMCase carboxymethylcellulase - PAGE polyacrylamide gel electrophoresis - IPTG isopropyl--D-thiogalactoside - CAT chloramphenicol acetyl transferase     

Genome-wide comparative analysis of simple sequence coding repeats among 25 insect species

We present a detailed genome-scale comparative analysis of simple sequence repeats within protein coding regions among 25 insect genomes. The repetitive sequences in the coding regions primarily represented single codon repeats and codon pair repeats. The CAG triplet is highly repetitive in the coding regions of insect genomes. It is frequently paired with the synonymous codon CAA to code for polyglutamine repeats. The codon pairs that are least repetitive code for polyalanine repeats. The frequency of hexanucleotide and dinucleotide motifs of codon pair repeats is significantly (p<0.001) different in the Drosophila species compared to the non-Drosophila species. However, the frequency of synonymous and non-synonymous codon pair repeats varies in a correlated manner (r(2)=0.79) among all the species. Results further show that perfect and imperfect repeats have significant association with the trinucleotide and hexanucleotide coding repeats in most of these insects. However, only select species show significant association between the numbers of perfect/imperfect hexamers and repeat coding for single amino acid/amino acid pair runs. Our data further suggests that genes containing simple sequence coding repeats may be under negative selection as they tend to be poorly conserved across species. The sequences of coding repeats of orthologous genes vary according to the known phylogeny among the species. In conclusion, the study shows that simple sequence coding repeats are important features of genome diversity among insects.     

Evolution of prokaryotic DNA: intragenic and extragenic divergences observed with orthologs from three related species

This study compared orthologous gene pairs from Escherichia coli K12, E. coli O157:H7 EDL933, Salmonella typhimurium LT2, and Yersinia pestis CO92 using only homologs of equal length, and differing nucleotides were counted and mapped. The data showed very clearly how the rates of divergence change with intragenic and extragenic position. The rate of synonymous mutation is lowest near start codons and near stop codons, and, a little surprisingly, the opposite seemed to be true for nonsynonymous substitutions. Analysis outside genes reveals that nucleotide divergences occur less frequently upstream of start codons than downstream of stop codons, and a remarkable drop in divergences is seen for two of the data sets around N = 9 nucleotides upstream of start codons; that is, the Shine-Dalgarno region changes at a lower rate. The explanation is likely to be the link between expressivity and sequence complementarity to the 3' end of 16S ribosomal rRNA. The latter is highly conserved across many bacterial and archaebacterial species.     

Intralineage variation in the pattern ofrbcL nucleotide substitution

Variation in chloroplastrbcL sequences was studied in representative species of four different lineages: the tribeRubieae (Rubiaceae), and the generaDrosera (Droseraceae),Nothofagus (Nothofagaceae) andIlex (Aquifoliaceae). Each lineage has its particular non-overlapping set ofrbcL polymorphic sites, indicating that common unconstrainedrbcL sites are not shared. Large differences in the rate and pattern of nucleotide substitution are observed among the four lineages. The genusIlex has the lowest rate of substitution, the lowest transition/transversion ratio, the lowest synonymous/replacement ratio and the lowest number of substitutions at the third codon position. An apparent relationship of these measures to the age of the lineages is observed. The A + T content and codon use among the four lineages are very similar and, apparently, cannot account for the observed differences in patterns of nucleotide substitution. However, the A + T content of the two bases immediately flanking the polymorphic sites is higher inIlex than in the other lineages. This could be correlated with the transversion/transition bias observed inIlex. The particularly low synonymous/replacement ratio found inIlex could also be explained by the small population sizes of species in this genus.     

Molecular evolution of the histone 3 multigene family in the Drosophila melanogaster species subgroup

Molecular evolution of the histone multigene family was studied by cloning and sequencing regions of the histone 3 gene in the Drosophila melanogaster species subgroup. Analysis of the nucleotide substitution pattern showed that in the coding region synonymous changes occurred more frequently to A or T in contrast to the GC-rich base composition, while in the 3' region the nucleotide substitutions were most likely in equilibrium. These results suggested that the base composition at the third codon position of the H3 gene, i.e., codon usage, has been changing to A or T in the Drosophila melanogaster species subgroup.     

On the rate of DNA sequence evolution inDrosophila

Summary Analysis of the rate of nucleotide substitution at silent sites inDrosophila genes reveals three main points. First, the silent rate varies (by a factor of two) among nuclear genes; it is inversely related to the degree of codon usage bias, and so selection among synonymous codons appears to constrain the rate of silent substitution in some genes. Second, mitochondrial genes may have evolved only as fast as nuclear genes with weak codon usage bias (and two times faster than nuclear genes with high codon usage bias); this is quite different from the situation in mammals where mitochondrial genes evolve approximately 5–10 times faster than nuclear genes. Third, the absolute rate of substitution at silent sites in nuclear genes inDrosophila is about three times hihger than the average silent rate in mammals.     

杜仲基因密码子使用模式分析

为了解杜仲基因密码子使用模式,该文以杜仲基因组密码子为研究对象,运用CodonW软件对杜仲的320个蛋白编码基因进行同义密码子相对使用频率(RSCU)分析、ENC-GC3s关联分析编码基因的密码子ENC值、PR2-plot偏倚分析编码基因的密码子碱基使用频率,并运用CUSP软件与Codon Usage Database软件对杜仲基因密码子的GC含量、使用频率与代表性物种烟草、拟南芥、大肠杆菌和酿酒酵母的密码子GC含量和使用频率进行比较。结果表明:杜仲基因密码子的RSCU>1的密码子有30个,其中18个以G/C结尾、12个以A/U结尾,说明杜仲基因密码子偏好以G/C结尾,且偏好性较强;有效密码子数(ENC)范围为30~60,该范围内的密码子距离标准曲线较远,其ENC值小,偏好性较强;PR2-plot偏倚分析碱基使用频率显示,G>C、U>A;杜仲与代表性物种的GC含量分析显示,杜仲的GC12、GC3以及平均GC含量均高于代表性物种;杜仲与代表性物种的密码子使用频率分析显示,杜仲与烟草、酿酒酵母的密码子偏好较为接近,杜仲与拟南芥、大肠杆菌的密码子偏好差距较大。杜仲是我国特有的珍贵中药材,对其进行密码子使用模式分析,并研究其密码子偏好规律,为杜仲植物基因工程中外源基因的改良及表达提供了理论基础。     

Complimentary DNA sequence data analysis of prokaryotic systems

Complimentary DNA sequence data of Φ × 174, fd, f1, G4, Ml3, MS2, λ and T7 phages ofEscherichia coli are analysed at mono-, di-, tri- and tetranucleotide levels. Our analysis shows that, (i) mononucleotides have certain preferences to occur at specific positions X₁, X₂, X₃ of codon, (ii) These nucleotides interact nonlinearly to form dinucleotide and this dinucleotide also interacts nonlinearely with a third nucleotide to form codon, (iii) However, nonlinear interactions are negligible at tetranucleotide level suggesting that, coding regions of complimentary DNA are Markov chains of order two. Trinucleotide potential values in three frames have suggested that, at least thirteen different trinucleotides can be used as a marker to locate coding regions in DNA of prokaryotes. (iv) Parallel paired codons are expressed in such a way that one of the codons in the pair expresses with high frequency while the other with low frequency. On the other hand the complimentary codon pairs express with small frequency difference, (v) In the synonymous codon groups, codon ending with T are found to express with more frequency     

Profiles of purine biosynthesis, salvage and degradation in disks of potato (Solanum tuberosum L.) tubers

To find general metabolic profiles of purine ribo- and deoxyribonucleotides in potato (Solanum tuberosum L.) plants, we looked at the in situ metabolic fate of various ¹⁴C-labelled precursors in disks from growing potato tubers. The activities of key enzymes in potato tuber extracts were also studied. Of the precursors for the intermediates in de novo purine biosynthesis, [¹⁴C]formate, [2-¹⁴C]glycine and [2-¹⁴C]5-aminoimidazole-4-carboxyamide ribonucleoside were metabolised to purine nucleotides and were incorporated into nucleic acids. The rates of uptake of purine ribo- and deoxyribonucleosides by the disks were in the following order: deoxyadenosine > adenosine > adenine > guanine > guanosine > deoxyguanosine > inosine > hypoxanthine > xanthine > xanthosine. The purine ribonucleosides, adenosine and guanosine, were salvaged exclusively to nucleotides, by adenosine kinase (EC 2.7.1.20) and inosine/guanosine kinase (EC 2.7.1.73) and non-specific nucleoside phosphotransferase (EC 2.7.1.77). Inosine was also salvaged by inosine/guanosine kinase, but to a lesser extent. In contrast, no xanthosine was salvaged. Deoxyadenosine and deoxyguanosine, was efficiently salvaged by deoxyadenosine kinase (EC 2.7.1.76) and deoxyguanosine kinase (EC 2.7.1.113) and/or non-specific nucleoside phosphotransferase (EC 2.7.1.77). Of the purine bases, adenine, guanine and hypoxanthine but not xanthine were salvaged for nucleotide synthesis. Since purine nucleoside phosphorylase (EC 2.4.2.1) activity was not detected, adenine phosphoribosyltransferase (EC 2.4.2.7) and hypoxanthine/guanine phosphoribosyltransferase (EC 2.4.2.8) seem to play the major role in salvage of adenine, guanine and hypoxanthine. Xanthine was catabolised by the oxidative purine degradation pathway via allantoin. Activity of the purine-metabolising enzymes observed in other organisms, such as purine nucleoside phosphorylase (EC 2.4.2.1), xanthine phosphoribosyltransferase (EC 2.4.2.22), adenine deaminase (EC 3.5.4.2), adenosine deaminase (EC 3.5.4.4) and guanine deaminase (EC 3.5.4.3), were not detected in potato tuber extracts. These results suggest that the major catabolic pathways of adenine and guanine nucleotides are AMP → IMP → inosine → hypoxanthine → xanthine and GMP → guanosine → xanthosine → xanthine pathways, respectively. Catabolites before xanthosine and xanthine can be utilised in salvage pathways for nucleotide biosynthesis.     

Protein Evolutionary Rates Correlate with Expression Independently of Synonymous Substitutions in Helicobacter pylori

In free-living microorganisms, such as Escherichia coli and Saccharomyces cerevisiae, both synonymous and nonsynonymous substitution frequencies correlate with expression levels. Here, we have tested the hypothesis that the correlation between amino acid substitution rates and expression is a by-product of selection for codon bias and translational efficiency in highly expressed genes. To this end, we have examined the correlation between protein evolutionary rates and expression in the human gastric pathogen Helicobacter pylori, where the absence of selection on synonymous sites enables the two types of substitutions to be uncoupled. The results revealed a statistically significant negative correlation between expression levels and nonsynonymous substitutions in both H. pylori and E. coli. We also found that neighboring genes located on the same, but not on opposite strands, evolve at significantly more similar rates than random gene pairs, as expected by co-expression of genes located in the same operon. However, the two species differ in that synonymous substitutions show a strand-specific pattern in E. coli, whereas the weak similarity in synonymous substitutions for neighbors in H. pylori is independent of gene orientation. These results suggest a direct influence of expression levels on nonsynonymous substitution frequencies independent of codon bias and selective constraints on synonymous sites. Electronic Supplementary Material Electronic Supplementary material is available for this article at and accessible for authorised users. [Reviewing Editor: Dr. Nicolas Galtier]     

Comparative genome sequence analysis of Sulfolobus acidocaldarius and 9 other isolates of its genus for factors influencing codon and amino acid usage

In the present study, major constraints for codon and amino acid usage of Sulfolobus acidocaldarius, Sulfolobus solfataricus, Sulfolobus tokodali, Sulfolobus islandis and 6 other isolates from islandicus species of genus Sulfolobus were investigated. Correspondence analysis revealed high significant correlation between the major trend of synonymous codon usage and gene expression level, as assessed by the “Codon Adaptation Index” (CAI). There is a significant negative correlation between Nc (Effective number of codons) and CAI demonstrating role of codon bias as an important determinant of codon usage. The significant correlation between major trend of synonymous codon usage and GC3s (G + C at third synonymous position) indicated dominant role of mutational bias in codon usage pattern. The result was further supported from SCUO (synonymous codon usage order) analysis. The amino acid usage was found to be significantly influenced by aromaticity and hydrophobicity of proteins. However, translational selection which causes a preference for codons that are most rapidly translated by current tRNA with multiple copy numbers was not found to be highly dominating for all studied isolates. Notably, 26 codons that were found to be optimally used by genes of S. acidocaldarius at higher expression level and its comparative analysis with 9 other isolates may provide some useful clues for further in vivo genetic studies on this genus.     

Expression and secretion of human bone morphogenetic protein-7 in pichia pastoris

The synonymous codons are used in a highly nonrandom manner in hosts of widely divergent species, which is termed ‘codon usage bias’. Several reports suggest that codon usage bias sometimes obstructs attempts to express high levels of exogenous genes. In this study, an attempt was made to express mature peptide of human bone morphogenetic protein-7 with optimized codons in P. pastoris expression system. Three low-usage ARG codons (CGG or CGA) in hBmp7 mature domain have been successfully transformed into P. pastoris-preferred ARG codons (AGA) with overlap extension PCR-based multiple-site-directed mutagenesis for a high level expression of hBMP7 mature peptide. The results of this study showed that the production level (25.45 mg/L) of a codon-optimized strain increased 4.6-fold in comparison with that (5.5 mg/L) of noncodon-optimized strain. A strain harboring multicopy of codon-optimized hbmp7 expression cassette showed an even higher expression level, which was about 2-fold compared with that of the single-copy one. These recombinant hBMP7 mature peptides were produced as 18-kD monomer proteins and were easily purified from culture supernatants using ion-exchange chromatography. Functional assay demonstrated that rhBMP7 could induce ectopic cartilage formation, although its inductive ability was much less active than that of CHO cell-derived hBMP7.