Codon replacement in the PGK1 gene of Saccharomyces cerevisiae: experimental approach to study the role of biased codon usage in gene expression.

The coding sequences of genes in the yeast Saccharomyces cerevisiae show a preference for 25 of the 61 possible coding triplets. The degree of this biased codon usage in each gene is positively correlated to its expression level. Highly expressed genes use these 25 major codons almost exclusively. As an experimental approach to studying biased codon usage and its possible role in modulating gene expression, systematic codon replacements were carried out in the highly expressed PGK1 gene. The expression of phosphoglycerate kinase (PGK) was studied both on a high-copy-number plasmid and as a single copy gene integrated into the chromosome. Replacing an increasing number (up to 39% of all codons) of major codons with synonymous minor ones at the 5' end of the coding sequence caused a dramatic decline of the expression level. The PGK protein levels dropped 10-fold. The steady-state mRNA levels also declined, but to a lesser extent (threefold). Our data indicate that this reduction in mRNA levels was due to destabilization caused by impaired translation elongation at the minor codons. By preventing translation of the PGK mRNAs by the introduction of a stop codon 3' and adjacent to the start codon, the steady-state mRNA levels decreased dramatically. We conclude that efficient mRNA translation is required for maintaining mRNA stability in S. cerevisiae. These findings have important implications for the study of the expression of heterologous genes in yeast cells.     

Codon bias as a factor in regulating expression via translation rate in the human genome

We study the interrelations between tRNA gene copy numbers, gene expression levels and measures of codon bias in the human genome. First, we show that isoaccepting tRNA gene copy numbers correlate positively with expression-weighted frequencies of amino acids and codons. Using expression data of more than 14,000 human genes, we show a weak positive correlation between gene expression level and frequency of optimal codons (codons with highest tRNA gene copy number). Interestingly, contrary to non-mammalian eukaryotes, codon bias tends to be high in both highly expressed genes and lowly expressed genes. We suggest that selection may act on codon bias, not only to increase elongation rate by favoring optimal codons in highly expressed genes, but also to reduce elongation rate by favoring non-optimal codons in lowly expressed genes. We also show that the frequency of optimal codons is in positive correlation with estimates of protein biosynthetic cost, and suggest another possible action of selection on codon bias: preference of optimal codons as production cost rises, to reduce the rate of amino acid misincorporation. In the analyses of this work, we introduce a new measure of frequency of optimal codons (FOP'), which is unaffected by amino acid composition and is corrected for background nucleotide content; we also introduce a new method for computing expected codon frequencies, based on the dinucleotide composition of the introns and the non-coding regions surrounding a gene.     

双孢蘑菇基因组密码子的偏好性分析

双孢蘑菇Agaricus bisporus是世界上最广泛栽培的食用菌之一。本研究通过分析双孢蘑菇基因组密码子使用偏性,探讨密码子偏性的影响因素及其对基因表达的影响。以双孢蘑菇基因组和转录组数据为依据,分析了双孢蘑菇基因组基因、高表达基因（high expression gene,HEG）和低表达基因（low expression gene,LEG）的密码子使用性。发现双孢蘑菇基因组编码基因平均GC含量为49.08%,T3s值（35.59%）最高,平均ENC值偏高,多数基因表达潜力较低。共鉴定出14个最优密码子,均以C或T结尾,并且遵循密码子中嘌呤和嘧啶使用的均衡性原则。高表达基因具有更强的密码子偏性,进化过程中受到基因突变和自然选择等多种因素影响。基因表达与G/C碱基含量和CAI值呈极显著正相关。高表达基因编码了多种与真菌生长发育相关的蛋白和酶类。研究结果明确了双孢蘑菇基因密码子的使用偏性,为双孢蘑菇转基因育种和品质改良提供了参考。     

Codon usages in different gene classes of the Escherichia coli genome

A new measure for assessing codon bias of one group of genes with respect to a second group of genes is introduced. In this formulation, codon bias correlations for Escherichia coli genes are evaluated for level of expression, for contrasts along genes, for genes in different 200 kb (or longer) contigs around the genome, for effects of gene size, for variation over different function classes, for codon bias in relation to possible lateral transfer and for dicodon bias for some gene classes. Among the function classes, codon biases of ribosomal proteins are the most deviant from the codon frequencies of the average E. coli gene. Other classes of ‘highly expressed genes’ (e.g. amino acyl tRNA synthetases, chaperonins, modification genes essential to translation activities) show less extreme codon biases. Consistently for genes with experimentally determined expression rates in the exponential growth phase, those of highest molar abundances are more deviant from the average gene codon frequencies and are more similar in codon frequencies to the average ribosomal protein gene. Independent of gene size, the codon biases in the 5′ third of genes deviate by more than a factor of two from those in the middle and 3′ thirds. In this context, there appear to be conflicting selection pressures imposed by the constraints of ribosomal binding, or more generally the early phase of protein synthesis (about the first 50 codons) may be more biased than the complete nascent polypeptide. In partitioning the E. coli genome into 10 equal lengths, pronounced differences in codon site 3 G+C frequencies accumulate. Genes near to oriC have 5% greater codon site 3 G+C frequencies than do genes from the ter region. This difference also is observed between small (100–300 codons) and large (>800 codons) genes. This result contrasts with that for eukaryotic genomes (including human, Caenorhabditis elegans and yeast) where long genes tend to have site 3 more AT rich than short genes. Many of the above results are special for E. coli genes and do not apply to genes of most bacterial genomes. A gene is defined as alien (possibly horizontally transferred) if its codon bias relative to the average gene exceeds a high threshold and the codon bias relative to ribosomal proteins is also appropriately high. These are identified, including four clusters (operons). The bulk of these genes have no known function.     

Drosophila Melanogaster Mitochondrial DNA: Gene Organization and Evolutionary Considerations

The sequence of a 8351-nucleotide mitochondrial DNA (mtDNA) fragment has been obtained extending the knowledge of the Drosophila melanogaster mitochondrial genome to 90% of its coding region. The sequence encodes seven polypeptides, 12 tRNAs and the 3' end of the 16S rRNA and CO III genes. The gene organization is strictly conserved with respect to the Drosophila yakuba mitochondrial genome, and different from that found in mammals and Xenopus. The high A + T content of D. melanogaster mitochondrial DNA is reflected in a reiterative codon usage, with more than 90% of the codons ending in T or A, G + C rich codons being practically absent. The average level of homology between the D. melanogaster and D. yakuba sequences is very high (roughly 94%), although insertion and deletions have been detected in protein, tRNA and large ribosomal genes. The analysis of nucleotide changes reveals a similar frequency for transitions and transversions, and reflects a strong bias against G + C on both strands. The predominant type of transition is strand specific.     

Essential factors determining codon usage in ubiquitin genes

Summary Ubiquitin is ubiquitous in all eukaryotes and its amino acid sequence shows extreme conservation. Ubiquitin genes comprise direct repeats of the ubiquitin coding unit with no spacers. The nucleotide sequences coding for 13 ubiquitin genes from 11 species reported so far have been compiled and analyzed. The G+C content of codon third base reveals a positive linear correlation with the genome G+C content of the corresponding species. The slope strongly suggests that the overall G+C content of codons of polyubiquitin genes clearly reflects the genome G+C content by AT/GC substitutions at the codon third position. The G+C content of ubiquitin codon third base also shows a positive linear correlation with the overall G+C content of coding regions of compiled genes, indicating the codon choices among synonymous codons reflect the average codon usage pattern of corresponding species. On the other hand, the monoubiquitin gene, which is different from the polyubiquitin gene in gene organization, gene expression, and function of the encoding protein, shows a different codon usage pattern compared with that of the polyubiquitin gene. From comparisons of the levels of synonymous substitutions among ubiquitin repeats and the homology of the amino acid sequence of the tail of monomeric ubiquitin genes, we propose that the molecular evolution of ubiquitin genes occurred as follows: Plural primitive ubiquitin sequences were dispersed on genome in ancestral eukaryotes. Some of them situated in a particular environment fused with the tail sequence to produce monomeric ubiquitin genes that were maintained across species. After divergence of species, polyubiquitin genes were formed by duplication of the other primitive ubiquitin sequences on different chromosomes. Differences in the environments in which ubiquitin genes are embedded reflect the differences in codon choice and in gene expression pattern between poly- and monomeric ubiquitin genes.     

Mutation bias is the driving force of codon usage in the Gallus gallus genome

Synonymous codons are used with different frequencies both among species and among genes within the same genome and are controlled by neutral processes (such as mutation and drift) as well as by selection. Up to now, a systematic examination of the codon usage for the chicken genome has not been performed. Here, we carried out a whole genome analysis of the chicken genome by the use of the relative synonymous codon usage (RSCU) method and identified 11 putative optimal codons, all of them ending with uracil (U), which is significantly departing from the pattern observed in other eukaryotes. Optimal codons in the chicken genome are most likely the ones corresponding to highly expressed transfer RNA (tRNAs) or tRNA gene copy numbers in the cell. Codon bias, measured as the frequency of optimal codons (Fop), is negatively correlated with the G + C content, recombination rate, but positively correlated with gene expression, protein length, gene length and intron length. The positive correlation between codon bias and protein, gene and intron length is quite different from other multi-cellular organism, as this trend has been only found in unicellular organisms. Our data displayed that regional G + C content explains a large proportion of the variance of codon bias in chicken. Stepwise selection model analyses indicate that G + C content of coding sequence is the most important factor for codon bias. It appears that variation in the G + C content of CDSs accounts for over 60% of the variation of codon bias. This study suggests that both mutation bias and selection contribute to codon bias. However, mutation bias is the driving force of the codon usage in the Gallus gallus genome. Our data also provide evidence that the negative correlation between codon bias and recombination rates in G. gallus is determined mostly by recombination-dependent mutational patterns.