The factors dictating the codon usage variation among the genes in the genome of Burkholderia pseudomallei

Burkholderia pseudomallei is a recognized biothreat agent and the causative agent of melioidosis. Codon usage biases of all protein-coding genes (length greater than or equal to 300 bp) from the complete genome of B. pseudomallei K96243 have been analyzed. As B. pseudomallei is a GC-rich organism (68.5%), overall codon usage data analysis indicates that indeed codons ending in G and/or C are predominant in this organism. But multivariate statistical analysis indicates that there is a single major trend in the codon usage variation among the genes in this organism, which has a strong positively correlation with the expressivities of the genes. The majority of the lowly expressed genes are scattered towards the negative end of the major axis whereas the highly expressed genes are clustered towards the positive end. At the same time, from the results that there were two significant correlations between axis 1 coordinates and the GC, GC3s content at silent sites of each sequence, and clearly significant negatively correlations between the ‘Effective Number of Codons’ values and GC, GC3s content, we inferred that codon usage bias was affected by gene nucleotide composition also. In addition, some other factors such as the lengths of the genes as well as the hydrophobicity of genes also influence the codon usage variation among the genes in this organism in a minor way. At the same time, notably, 21 codons have been defined as ‘optimal codons’ of the B. pseudomallei. In summary, our work have provided a basic understanding of the mechanisms for codon usage bias and some more useful information for improving the expression of target genes in vivo and in vitro. Sheng Zhao and Qin Zhang contributed equally to this work.     

Nucleotide sequence and expression of the two genes encoding D2 protein and the single gene encoding the CP43 protein of Photosystem II in the cyanobacterium synechococcus sp. PCC 7002

The unicellular photoheterotrophic cyanobacterium Synechococcus sp. PCC 7002 was shown to encode two genes for the Photosystem II reaction center core protein D2 and one gene for the reaction center chlorophyhll-binding protein CP43. These three genes were cloned and their DNA sequences determined along with their flanking DNA sequences. Northern hybridization experiments show that both genes which encode D2, psbD1 and psbD2, are expressed at roughly equivalent levels. For each of the two psbD genes, there are 18 nucleotide differences among the 1059 nucleotides which are translated. The DNA sequences surrounding the coding sequences are nearly 70% divergent. Despite the DNA sequence differences in the genes, the proteins encoded by the two genes are predicted to be identical. The proteins encoded by psbD1 and psbD2 are 92% homologous to other sequenced cyanobacterial psbD genes and 86% homologous to sequenced chloroplast-encoded psbD genes.The single gene for CP43, psbC, overlaps the 3 end of psbD1 and is co-transcribed with it. Results from previous sequencing of psbC genes encoded by chloroplasts suggest that the 5 end of the psbC gene overlaps the 3 end of the coding sequence of psbD by 50 nucleotides. In Synechococcus sp. PCC 7002, the methionine codon previously proposed to be the start codon for psbC is replaced by an ACG (threonine) codon. We propose an alternative start for the psbC gene at a GTG codon 36 nucleotides downstream from the threonine codon. This GTG codon is preceded by a consensus E. coli-like ribosome binding sequence. Both the GTG start codon and its preceding ribosome binding sequence are conserved in all psbC genes sequenced from cyanobacteria and chloroplasts. This suggests that all psbC genes start at this alternative GTG codon. Based on this alternative start codon, the gene product is 85% identical to other cyanobacterial psbC gene products and 77% identical to eucaryotic chloroplast-encoded psbC gene products.     

Studies on codon usage in Entamoeba histolytica

Codon usage bias of Entamoeba histolytica, a protozoan parasite, was investigated using the available DNA sequence data. Entamoeba histolytica having AT rich genome, is expected to have A and/or T at the third position of codons. Overall codon usage data analysis indicates that A and/or T ending codons are strongly biased in the coding region of this organism. However, multivariate statistical analysis suggests that there is a single major trend in codon usage variation among the genes. The genes which are supposed to be highly expressed are clustered at one end, while the majority of the putatively lowly expressed genes are clustered at the other end. The codon usage pattern is distinctly different in these two sets of genes. C ending codons are significantly higher in the putatively highly expressed genes suggesting that C ending codons are translationally optimal in this organism. In the putatively lowly expressed genes A and/or T ending codons are predominant, which suggests that compositional constraints are playing the major role in shaping codon usage variation among the lowly expressed genes. These results suggest that both mutational bias and translational selection are operational in the codon usage variation in this organism.     

Codon usage in regulatory genes in Escherichia coli does not reflect selection for ''rare'' codons.

It has often been suggested that differential usage of codons recognized by rare tRNA species, i.e. "rare codons", represents an evolutionary strategy to modulate gene expression. In particular, regulatory genes are reported to have an extraordinarily high frequency of rare codons. From E. coli we have compiled codon usage data for highly expressed genes, moderately/lowly expressed genes, and regulatory genes. We have identified a clear and general trend in codon usage bias, from the very high bias seen in very highly expressed genes and attributed to selection, to a rather low bias in other genes which seems to be more influenced by mutation than by selection. There is no clear tendency for an increased frequency of rare codons in the regulatory genes, compared to a large group of other moderately/lowly expressed genes with low codon bias. From this, as well as a consideration of evolutionary rates of regulatory genes, and of experimental data on translation rates, we conclude that the pattern of synonymous codon usage in regulatory genes reflects primarily the relaxation of natural selection.     

The sequence surrounding the translation initiation codon of the pea plastocyanin gene increases translational efficiency of a reporter gene

The 5-upstream region of the pea plastocyanin gene (petE) directed 5–10-fold higher levels of -glucuronidase (GUS) activity than the cauliflower mosaic virus 35S promoter in transgenic tobacco plants, although the levels of GUS mRNA were similar. The sequence (AAAAAUGG) around the translation initiation codon of petE enhanced translation of the GUS mRNA 10-fold compared to translation from the GUS translation initiation codon in transgenic tobacco plants and transfected protoplasts.     

Mistranslation in cells infected with the bacteriophage MS2: Direct evidence of Lys for Asn substitution

Summary The coat protein of the bacteriophage MS2 was found to show an increased level of charge heterogeneity when synthesized in Escherichia coli starved for Asn or Lys. No such increase was found when the host was starved for Arg, His, Ile or Pro. This is the pattern predicted by two-out-of-three codon misreading in the coat protein gene. In the case of Asn starvation, direct measurements of the relative incorporation of Lys demonstrate that the observed charge heterogeneity is the result of mistranslation. Asn starvation increased the error frequency in coat protein to over 0.3 mistake per asparagine codon. The small amount of charge heterogeneity seen in unstarved cells seems also to be the result of misreading Asn codons.     

Two-dimensional 1H NMR study of two cyclobutane type photodimers of thymidylyl-(3′→5′)-thymidine

2D NMR spectroscopy and J coupling constant analysis are applied to resolve the structure of two photoproducts of thymidylyl-(35)-thymidine. These products are cyclobutane type thymine dimers possessing the cis-syn (the predominant one) and trans-syn geometry. The cis-syn is formed in an ANTI-ANTI conformation about the N-glycosyl linkages and resembles the normal base-stacked configuration. The glycosidic conformation in solution of the 5 terminal fragment differs from the crystal in which the less common SYN conformation is observed. In this isomer only the sugar pucker of the 3 terminal fragment is changed substantially with respect to the dinucleotide. The trans-syn isomer is formed in a SYN-ANTI glycosidic conformation. In this isomer the sugar puckers of both deoxyribose rings are affected and a preference for a pure 2-endo conformation is observed.Abbreviations dTpdT 2-deoxythymidylyl-(35)-2-deoxythymidine - dTp[]dT cyclobutane type photodimers of dTpdT - dTp- and dTp[]- their 5' terminal fragments (fragment A) - -pdT and-[]pdT their 3 terminal fragments (fragment B) - RP-HPLC reversed-phase high-performance liquid chromatography - COSY two-dimensional correlated spectroscopy - 2D NOE two-dimensional nuclear Overhauser spectroscopy     

Heterogeneity in Codon Usage in the Flatworm Schistosoma mansoni

Synonymous codon choices vary considerably among Schistosoma mansoni genes. Principal components analysis detects a single major trend among genes, which highly correlates with GC content in third codon positions and exons, but does not discriminate among putatively highly and lowly expressed genes. The effective number of codons used in each gene, and its distribution when plotted against GC₃, suggests that codon usage is shaped mainly by mutational biases. The GC content of exons, GC₃, 5′, 3′, and flanking (5′+ 3′+ introns) regions are all correlated among them, suggesting that variations in GC content may exist among different regions of the S. mansoni genome. We propose that this genome structure might be among the most important factors shaping codon usage in this species, although the action of selection on certain sequences cannot be excluded. Received: 10 March 1997 / Accepted: 27 June 1997     

Expression inEscherichia coli K12 strains of two synthetic human calcitonin genes differing in codon composition

Two genes coding for a Val⁸-variant of the human calcitonin (hCT) are synthesized on two different codon biases: the native codons for the hCT gene and the codons preferential forEscherichia coli. Both genes are fused to a synthetic human interferon-gamma (IF) gene [6] and expressed in various strains ofE. coli K12. It is found that, in all host strains used, the level of expression of both genes is similar and much lower (1/50–1/100) than that of the IF gene alone.     

Synonymous codon usage variation among Giardia lamblia genes and isolates.

The pattern of codon usage in the amitochondriate diplomonad Giardia lamblia has been investigated. Very extensive heterogeneity was evident among a sample of 65 genes. A discrete group of genes featured unusual codon usage due to the amino acid composition of their products: these variant surface proteins (VSPs) are unusually rich in Cys and, to a lesser extent, Gly and Thr. Among the remaining 50 genes, correspondence analysis revealed a single major source of variation in synonymous codon usage. This trend was related to the extent of use of a particular subset of 21 codons which are inferred to be those which are optimal for translation; at one end of this trend were genes expected to be expressed at low levels with near random codon usage, while at the other extreme were genes expressed at high levels in which these optimal codons are used almost exclusively. These optimal codons all end in C or G so G + C content at silent sites varies enormously among genes, from values around 40%, expected to reflect the background level of the genome, up to nearly 100%. Although VSP genes are occasionally extremely highly expressed, they do not, in general, have high frequencies of optimal codons, presumably because their high expression is only intermittent. These results indicate that natural selection has been very effective in shaping codon usage in G. lamblia. These analyses focused on sequences from strains placed within G. lamblia "assemblage A"; a few sequences from other strains revealed extensive divergence at silent sites, including some divergence in the pattern of codon usage.     

Translational coupling in a penP-lacZ gene fusion in Bacillus subtilis and Escherichia coli: Use of AUA as a restart codon

Summary An out-of-frame fusion between the penicillinase gene (penP) of Bacillus licheniformis and the -galactosidase gene (lacZ) of Escherichia coli was shown to direct the synthesis of an active -galactosidase with the same electrophoretic mobility as the wild-type protein, both in B. subtilis and E. coli. This synthesis was dependent on translation of the truncated penP gene and appeared to result from translational coupling. The fusion point between penP and lacZ contained the sequence AUAG, in which the UAG and AUA codons were in-frame with the penP and lacZ reading units, respectively. N-terminal amino acid sequence analysis of the -galactosidase protein suggested that, both in B. subtilis and E. coli, reinitiation of translation occurred at the AUA codon present at the gene fusion point.     

Role of mitogen-activated protein kinase pathway in reactive oxygen species-mediated endothelin-1-induced β-myosin heavy chain gene expression and cardiomyocyte hypertrophy

Endothelin-1 (ET-1) has been found to increase cardiac -myosin heavy chain (-MyHC) gene expression and induce hypertrophy in cardiomyocytes. ET-1 has been demonstrated to increase intracellular reactive oxygen species (ROS) in cardiomyocytes. The exact molecular mechanism by which ROS regulate ET-1-induced -MyHC gene expression and hypertrophy in cardiomyocytes, however, has not yet been fully described. We aim to elucidate the molecular regulatory mechanism of ROS on ET-1-induced -MyHC gene expression and hypertrophic signaling in neonatal rat cardiomyocytes. Following stimulation with ET-1, cultured neonatal rat cardiomyocytes were examined for ³H-leucine incorporation and -MyHC promoter activities. The effects of antioxidant pretreatment on ET-1-induced cardiac hypertrophy and mitogen-activated protein kinase (MAPKs) phosphorylation were studied to elucidate the redox-sensitive pathway in cardiomyocyte hypertrophy and -MyHC gene expression. ET-1 increased ³H-leucine incorporation and -MyHC promoter activities, which were blocked by the specific ET_A receptor antagonist BQ-485. Antioxidants significantly reduced ET-1-induced ³H-leucine incorporation, -MyHC gene promoter activities and MAPK (extracellular signal-regulated kinase, p38, and c-Jun NH₂ -terminal kinase) phosphorylation. Both PD98059 and SB203580 inhibited ET-1-increased ³H-leucine incorporation and -MyHC promoter activities. Co-transfection of the dominant negative mutant of Ras, Raf, and MEK1 decreased the ET-1-induced -MyHC promoter activities, suggesting that the Ras-Raf-MAPK pathway is required for ET-1 action. Truncation analysis of the -MyHC gene promoter showed that the activator protein-2 (AP-2)/specificity protein-1 (SP-1) binding site(s) were(was) important cis-element(s) in ET-1-induced -MyHC gene expression. Moreover, ET-1-induced AP-2 and SP-1 binding activities were also inhibited by antioxidant. These data demonstrate the involvement of ROS in ET-1-induced hypertrophic responses and -MyHC expression. ROS mediate ET-1-induced activation of MAPK pathways, which culminates in hypertrophic responses and -MyHC expression. Tzu-Hurng Cheng, Neng-Lang Shih: These authors have equally contributed to this work     

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.     

Comparative analysis of genes encoding methyl coenzyme M reductase in methanogenic bacteria

Summary The sequence of the gene cluster encoding the methyl coenzyme M reductase (MCR) in Methanococcus voltae was determined. It contains five open reading frames (ORF), three of which encode the known enzyme subunits. Putative ribosome binding sites were found in front of all ORFs. They differ in their degrees of complementarity to the 3 end of the 16 S rRNA, which is discussed in terms of different translation efficiencies of the respective genes. The codon usage bias is different in the subunit encoding genes compared with the two other ORFs in the cluster and two other known genes of Mc. voltae. This is interpreted in terms of increased translational accuracy of the highly expressed MCR subunit genes. The derived polypeptide sequences encoded by the five ORFs of the MCR cluster were compared to those of the respective genes in Methanobacterium thermoautotrophicum Marburg and Methanosarcina barkeri. Conserved regions were detected in the enzyme subunits, which are candidates for factor binding domains. Conserved hydrophobic sequences found in the and subunits are discussed with respect to the membrane association of the enzyme.     

Canavanine synthesis in thein vitro propagated tissues ofCanavalia lineata

Maximum shoot induction from stem explants ofCanavalia lineata was obtained with an agar-solidified PC medium containing 10 M benzylaminopurine and 1 M naphthaleneacetic acid. Rooting of thesein vitro produced shoots was achieved with hormone-free PC medium. Canavanine was produced almost exclusively in the leaves and was not detected in the roots ofin vitro propagatedC. lineata. To exclude the possibility of imminent translocation of canavanine from the root to leaf, adventitious roots were induced from leaf explants in PC medium supplemented with 1 M kinetin and 20 M indole-3-acetic acid and subcultured in medium lacking growth regulators, and the roots excised from germinated seedlings were cultured in hormone-free PC medium. All the roots were incapable of accumulation of canavanine. These results suggest that leaves ofC. lineata are the possible site of canavanine synthesis.     

Amplified ribosomal DNA in meiotic prophase oocyte nuclei of acipenserid fishes

Summary In situ hybridization has been performed in sections through ovaries ofAcipenser ruthenus andAcipenser güldenstädti in order to detect the rDNA sequences. Hybridization resulted in specific labelling of the caps of extrachromosomal DNA present in pachytene oocyte nuclei and of the chromatin granules distributed beneath the nuclear envelope in early diplotene nuclei. In the same sections, the nuclei of all ovarian cells in both species (oogonia, leptotene, and zygotene stage oocytes, follicular cells, connective tissue cells) showed a very low, but similar labelling.Amplification of genes for rRNA thus occurs at the pachytene stage in early oogenesis ofAcipenseridae. No rDNA amplification could be detected in the previous stages.     

Pollen selection for Alternaria resistance in oilseed brassicas: responses of pollen grains and leaves to a toxin of A. brassicae

Summary The effects of destruxin B, a host-specific toxin of Alternaria brassicae that causes black spot disease in oilseed brassicas, were studied on in vitro pollen germination and pollen-tube growth of Brassica campestris var brown sarson, B. juncea, B. napus cvs Westar and Cresor, B. nigra and Sinapis alba. Pollen grains of B. nigra, B. juncea and B. campestris were the most sensitive and those of S. alba the least sensitive to the toxin. Effects of the toxin were also studied on the leaves of these species, and the degree of sensitivity of leaves of different species was comparable to that of their pollen grains. The results on the responses of pollen grains as well as leaves to the toxin are in agreement with the degree of susceptibility/resistance of these species to A. brassicae reported in the literature, indicating that the genes imparting susceptibility/restistance are expressed in the pollen, a prerequisite for pollen selection. Results are also presented which show that the toxin fed to the cut end of isolated inflorescence axis is readily taken up by the developing pollen and results in the inhibition of germination of susceptible pollen. This technique offers a simple and effective method for application of selection pressure to eliminate pollen grains susceptible to the toxin from effecting fertilization.     

Synonymous codon usage in Lactococcus lactis: mutational bias versus translational selection

In this study codon usage bias of all experimentally known genes of Lactococcus lactis has been analyzed. Since Lactococcus lactis is an AT rich organism, it is expected to occur A and/or T at the third position of codons and detailed analysis of overall codon usage data indicates that A and/or T ending codons are predominant in this organism. However, multivariate statistical analyses based both on codon count and on relative synonymous codon usage (RSCU) detect a large number of genes, which are supposed to be highly expressed are clustered at one end of the first major axis, while majority of the putatively lowly expressed genes are clustered at the other end of the first major axis. It was observed that in the highly expressed genes C and T ending codons are significantly higher than the lowly expressed genes and also it was observed that C ending codons are predominant in the duets of highly expressed genes, whereas the T endings codons are abundant in the quartets. Abundance of C and T ending codons in the highly expressed genes suggest that, besides, compositional biases, translational selection are also operating in shaping the codon usage variation among the genes in this organism as observed in other compositionally skewed organisms. The second major axis generated by correspondence analysis on simple codon counts differentiates the genes into two distinct groups according to their hydrophobicity values, but the same analysis computed with relative synonymous codon usage values could not discriminate the genes according to the hydropathy values. This suggests that amino acid composition exerts constraints on codon usage in this organism. On the other hand the second major axis produced by correspondence analysis on RSCU values differentiates the genes into two groups according to the synonymous codon usage for cysteine residues (rarest amino acids in this organism), which is nothing but a artifactual effect induced by the RSCU values. Other factors such as length of the genes and the positions of the genes in the leading and lagging strand of replication have practically no influence in the codon usage variation among the genes in this organism.     

Codon bias in actin multigene families and effects on the reconstruction of phylogenetic relationships

Codon usage patterns and phylogenetic relationships in the actin multigene family have been analyzed for three dipteran species—Drosophila melanogaster, Bactrocera dorsalis, and Ceratitis capitata. In certain phylogenetic tree reconstructions, using synonymous distances, some gene relationships are altered due to a homogenization phenomenon. We present evidence to show that this homogenization phenomenon is due to codon usage bias. A survey of the pattern of synonymous codon preferences for I I actin genes from these three species reveals that five out of the six Drosophila actin genes show high degrees of codon bias as indicated by scaled ² values. In contrast to this, four out of the five actin genes from the other species have low codon bias values. A Monte Carlo contingency test indicates that for those Drosophila actin genes which exhibit codon bias, the patterns of codon usage are different compared to actin genes from the other species. In addition, the genes exhibiting codon bias also appear to have reduced rates of synonymous substitution. The homogenization phenomenon seen in terms of synonymous substitutions is not observed for nonsynonymous changes. Because of this homogenization phenomenon, trees constructed based on synonymous substitutions will be affected. These effects can be overt in the case of multigene families, but similar distortions may underlie reconstructions based on single-copy genes which exhibit codon usage bias.Correspondence to: M. He