Evolution of Prokaryotic Genes by Shift of Stop Codons

De novo origin of coding sequence remains an obscure issue in molecular evolution. One of the possible paths for addition (subtraction) of DNA segments to (from) a gene is stop codon shift. Single nucleotide substitutions can destroy the existing stop codon, leading to uninterrupted translation up to the next stop codon in the gene’s reading frame, or create a premature stop codon via a nonsense mutation. Furthermore, short indels-caused frameshifts near gene’s end may lead to premature stop codons or to translation past the existing stop codon. Here, we describe the evolution of the length of coding sequence of prokaryotic genes by change of positions of stop codons. We observed cases of addition of regions of 3′UTR to genes due to mutations at the existing stop codon, and cases of subtraction of C-terminal coding segments due to nonsense mutations upstream of the stop codon. Many of the observed stop codon shifts cannot be attributed to sequencing errors or rare deleterious variants segregating within bacterial populations. The additions of regions of 3′UTR tend to occur in those genes in which they are facilitated by nearby downstream in-frame triplets which may serve as new stop codons. Conversely, subtractions of coding sequence often give rise to in-frame stop codons located nearby. The amino acid composition of the added region is significantly biased, compared to the overall amino acid composition of the genes. Our results show that in prokaryotes, shift of stop codon is an underappreciated contributor to functional evolution of gene length.     

载脂蛋白基因家族的密码子空间分析——核苷酸替换的非随机进化选择

ＤＮＡ分子进化中,对核苷酸替换的选择可呈选择中性或选择倾向性。为研究载脂蛋白基因进化过程中对核苷酸变化的选择方式,本文建立了基因的密码子空间分析方法。密码子空间是由密码子３个位置上核苷酸出现机率所组成的矩阵。对该空间中核苷酸分布的非随机性度量可以反映进化过程中核苷酸替换的选择方式。应用该法,我们发现载脂蛋白基因密码子空间第一及第三位的核苷酸分布呈高度非随机性。进一步研究表明：这种核苷酸的非随机分布可能与腺苷酸、胸苷酸对密码子位置的非中性选择有关。此外,还研究了同义密码子的选择使用与分支种系发生的关系。结果显示：载脂蛋白分子演化中存在着同义密码子使用的分子进化钟。这些研究提示密码子空间中核苷酸替换的非随机选择可能是载脂蛋白基因进化的一种特征。     

Sounds of silence: synonymous nucleotides as a key to biological regulation and complexity

Messenger RNA is a key component of an intricate regulatory network of its own. It accommodates numerous nucleotide signals that overlap protein coding sequences and are responsible for multiple levels of regulation and generation of biological complexity. A wealth of structural and regulatory information, which mRNA carries in addition to the encoded amino acid sequence, raises the question of how these signals and overlapping codes are delineated along non-synonymous and synonymous positions in protein coding regions, especially in eukaryotes. Silent or synonymous codon positions, which do not determine amino acid sequences of the encoded proteins, define mRNA secondary structure and stability and affect the rate of translation, folding and post-translational modifications of nascent polypeptides. The RNA level selection is acting on synonymous sites in both prokaryotes and eukaryotes and is more common than previously thought. Selection pressure on the coding gene regions follows three-nucleotide periodic pattern of nucleotide base-pairing in mRNA, which is imposed by the genetic code. Synonymous positions of the coding regions have a higher level of hybridization potential relative to non-synonymous positions, and are multifunctional in their regulatory and structural roles. Recent experimental evidence and analysis of mRNA structure and interspecies conservation suggest that there is an evolutionary tradeoff between selective pressure acting at the RNA and protein levels. Here we provide a comprehensive overview of the studies that define the role of silent positions in regulating RNA structure and processing that exert downstream effects on proteins and their functions.     

Possible multiple origins of replication in primate mitochondria: Alternative role of tRNA sequences

DNA replication in vertebrate mitochondria is usually directional, leaving different portions of the genome single-stranded for different periods of time. During this time, mutations resulting from deaminations of cytosines to thymines and adenines to guanines accumulate on the heavy strand. Therefore, T/C and G/A ratios increase along mitochondrial genomes, proportionally to the time spent single-stranded during replication. Such trends exist at third codon positions for base ratios averaged across genes in individual genomes as well as for gene-specific and site-specific substitution frequencies estimated using phylogenetic methods. We use multiple regressions to test for the potential functioning of all 12 tRNA clusters in 19 primate mitochondrial genomes as alternative origins of light strand replication (OL). We provide a general algorithm for calculating time spent single stranded by a given site for any possible locations of the site and OL. For codon positions 1, 2, and 3, respectively, 23%, 9% and 35% of tRNA gene clusters have significant (p < 0.05) deamination gradients originating from them. The strength of the deamination gradient originating from tRNA gene clusters varies among species, and for five clusters, correlates with the tendency of tRNA genes in each of these clusters to form secondary structures that resemble the OL's structure. This is notably true for all codon positions for tRNA-Lys, which in absence of nuclear regulation, forms secondary structures resembling the hairpin structure of OL. For two tRNA gene clusters, correlations were statistically significant, but opposite to the direction expected by the known unidirectional replication, putatively compatible with bi-directional replication. Few substitutions in tRNA sequences can be neutral at the level of cloverleaf structure and function, yet significantly alter capacities to form OL-like structures, causing sudden evolution of genome-wide nucleotide contents.     

Genetic robustness and selection at the protein level for synonymous codons

Synonymous codons are neutral at the protein level, therefore natural selection at the protein level should have no effect on their frequencies. Synonymous codons, however, differ in their capacity to reduce the effects of errors: after mutation, certain codons keep on coding for the same amino acid or for amino acids with similar properties, while other synonymous codons produce very different amino acids. Therefore, the impact of errors on a coding sequence (genetic robustness) can be measured by analysing its codon usage. I analyse the codon usage of sequenced nuclear and cytoplasmic genomes and I show that there is an extensive variation in genetic robustness at the DNA sequence level, both among genomes and among genes of the same genome. I also show theoretically that robustness can be adaptive, that is natural selection may lead to a preference for codons that reduce the impact of errors. If selection occurs only among the mutants of a codon (e.g. among the progeny before the adult phase), however, the codons that are more sensitive to the effects of mutations may increase in frequency because they manage to get rid more easily of deleterious mutations. I also suggest other possible explanations for the evolution of genetic robustness at the codon level.     

Restoration of a translational stop-start overlap reinstates translational coupling in a mutant trpB''-trpA gene pair of the Escherichia coli tryptophan operon.

The trpB and trpA coding regions of the polycistronic trp mRNA of Escherichia coli are separated by overlapping translation stop and start codons. Efficient translation of the trpA coding region is subject to translational coupling, i.e., maximal trpA expression is dependent on prior translation of the trpB coding region. Previous studies demonstrated that the trpA Shine-Dalgarno sequence (within trpB) and/or the location of the trpB stop codon influenced trpA expression. To examine the effect of stop codon location specifically, we constructed plasmids in which different nucleotide sequences preceding the trpA start codon were retained, and only the reading frame was changed. When trpB translation proceeded in the wild type reading frame and terminated at the normal trpB stop codon, trpA polypeptide levels were elevated over the levels observed when translation stopped before or after the natural trpB stop codon. The proximity of the trpB stop codon to the trpA start codon therefore markedly influences trpA expression.     

Comparative study of overlapping genes in the genomes of Mycoplasma genitalium and Mycoplasma pneumoniae

Overlapping genes are defined, in this paper, as a pair of adjacent genes whose coding regions are partly overlapping. We systematically analyzed all overlapping genes in the genomes of two closely related species: Mycoplasma genitalium and Mycoplasma pneumoniae. Careful comparisons were made for homologous genes that are overlapped in one species but not in the other. This comparative analysis allows us to propose a model of how overlapping genes emerged in the course of evolution. It was found that overlapping genes were generated primarily due to the loss of a stop codon in either gene, in many cases, the absence of which resulted in elongation of the 3' end of the gene's coding region. More specifically, the loss of the stop codon took place as a result of the following events: deletion of the stop codon (64.4%), point mutation at the stop codon (4.4%), and frame shift at the end of the coding region (6.7%). Overlapping genes, in a sense, can be thought of as the results of evolutionary pressure to minimize genome size. However, our analysis indicates that many overlapping genes, at least in the genomes of M.genitalium and M.pneumoniae, are due to incidental elongation of the coding regions.     

The genetic consequences of DNA precursor pool imbalance: sequence analysis of mutations induced by excess thymidine at the hamster aprt locus

To determine the effect of deoxyribonucleoside triphosphate pool imbalances on the accuracy of DNA replication within the cell, we examined the base pair alterations induced by excess intracellular dTTP at the adenine phosphoribosyl transferase (aprt) locus of CHO cells. The mutations were predominantly simple (C----T) transitions (38/44) and transversions (G----T, 5/44) explicable by the misincorporation of the DNA precursor supplied in excess (dTTP). Only one small deletion was observed. The context of the mutations is notable as the nucleotide incorporated after the error was usually the nucleotide in excess for the great majority of the transitions but not the transversions. As next nucleotide effects are characteristic of replication complexes having proofreading exonuclease activity, our data indicate that this mechanism functions within the cell to control the occurrence of some types of replicational errors.     

Potential evolutionary influences on overlapping reading frames in the bovine leukemia virus pXBL region

Bovine leukemia virus contains a pXBL region encoding the 3' parts of four regulatory proteins (Tax, Rex, G4, R3) in overlapping reading frames. Here we report the pXBL polymorphisms of 30 isolates from four countries. Rates of overall and synonymous substitutions were consistently lower, and nucleotide/amino acid composition bias and codon bias higher, in more-overlapped than in less-overlapped regions. Ratios of nonsynonymous/synonymous substitutions were lowest in the tax gene and its subregions. The 5' parts of the four genes showed selection patterns corresponding to their genomic context outside of the pXBL region. Longer G4 variants due to a natural stop codon mutation had additional triple overlap with reduced sequence variability. These data support the concept that a higher level of overlapping in coding regions correlates with greater evolutionary constraint. Tax, the most conserved among the four regulatory proteins, showed purifying selection consistent with its importance in the viral life cycle.     

Nucleotide sequence of the Xdh region in Drosophila pseudoobscura and an analysis of the evolution of synonymous codons

The nucleotide sequence of the Xdh region of Drosophila pseudoobscura is presented. The Xdh gene structure and organization are compared with the homologous region in D. melanogaster. This locus is shown to have similar organization in the two species, although an additional intron and three insertion/deletion events are described for the D. pseudoobscura coding region. The encoded proteins are predicted to have very similar charges and hydrophobic/hydrophilic domains even though 11% of the amino acids are different. A gene 5' to Xdh, putative l(3)s12, is suggested from sequence similarity between the species. Synonymous differences at the Xdh locus between the two species are analyzed using a new method described in the preceding paper by Lewontin. This analysis shows that synonymous positions within the Xdh locus are evolving at very different rates, being dependent on level of codon redundancy. A comparison of synonymous divergence between D. melanogaster and D. pseudoobscura in five additional genes reveals variation in the level of synonymous substitution.      

Reduction of the rate of poliovirus protein synthesis through large-scale codon deoptimization causes attenuation of viral virulence by lowering specific infectivity

Exploring the utility of de novo gene synthesis with the aim of designing stably attenuated polioviruses (PV), we followed two strategies to construct PV variants containing synthetic replacements of the capsid coding sequences either by deoptimizing synonymous codon usage (PV-AB) or by maximizing synonymous codon position changes of the existing wild-type (wt) poliovirus codons (PV-SD). Despite 934 nucleotide changes in the capsid coding region, PV-SD RNA produced virus with wild-type characteristics. In contrast, no viable virus was recovered from PV-AB RNA carrying 680 silent mutations, due to a reduction of genome translation and replication below a critical level. After subcloning of smaller portions of the AB capsid coding sequence into the wt background, several viable viruses were obtained with a wide range of phenotypes corresponding to their efficiency of directing genome translation. Surprisingly, when inoculated with equal infectious doses (PFU), even the most replication-deficient viruses appeared to be as pathogenic in PV-sensitive CD155tg (transgenic) mice as the PV(M) wild type. However, infection with equal amounts of virus particles revealed a neuroattenuated phenotype over 100-fold. Direct analysis indicated a striking reduction of the specific infectivity of PV-AB-type virus particles. Due to the distribution effect of many silent mutations over large genome segments, codon-deoptimized viruses should have genetically stable phenotypes, and they may prove suitable as attenuated substrates for the production of poliovirus vaccines.     

Trends in the codon usage patterns of Chromohalobacter salexigens genes

Chromohalobacter salexigens, a Gammaproteobacterium belonging to the family Halomonadaceae, shows a broad salinity range for growth. In order to reveal the factors influencing architecture of protein coding genes in C. salexigens, pattern of synonymous codon usage bias has been investigated. Overall codon usage analysis of the microorganism revealed that C and G ending codons are predominantly used in all the genes which are indicative of mutational bias. Multivariate statistical analysis showed that the genes are separated along the first major explanatory axis according to their expression levels and their genomic GC content at the synonymous third positions of the codons. Both NC plot and correspondence analysis on Relative Synonymous Codon Usage (RSCU) indicates that the variation in codon usage among the genes may be due to mutational bias at the DNA level and natural selection acting at the level of mRNA translation. Gene length and the hydrophobicity of the encoded protein also influence the codon usage variation of genes to some extent. A comparison of the relative synonymous codon usage between 10% each of highly and lowly expressed genes determines 23 optimal codons, which are statistically over represented in the former group of genes and may provide useful information for salt-stressed gene prediction and gene-transformation. Furthermore, genes for regulatory functions; mobile and extrachromosomal element functions; and cell envelope are observed to be highly expressed. The study could provide insight into the gene expression response of halophilic bacteria and facilitate establishment of effective strategies to develop salt-tolerant crops of agronomic value.     

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.     

Synonymous and Nonsynonymous Substitution Rates in Diatoms: A Comparison Between Chloroplast and Nuclear Genes

Rates of synonymous and nonsynonymous nucleotide substitutions and codon usage bias (ENC) were estimated for a number of nuclear and chloroplast genes in a sample of centric and pennate diatoms. The results suggest that DNA evolution has taken place, on an average, at a slower rate in the chloroplast genes than in the nuclear genes: a rate variation pattern similar to that observed in land plants. Synonymous substitution rates in the chloroplast genes show a negative association with the degree of codon usage bias, suggesting that genes with a higher degree of codon usage bias have evolved at a slower rate. While this relationship has been shown in both prokaryotes and multicellular eukaryotes, it has not been demonstrated before in diatoms. Received: 3 June 1998 / Accepted: 11 August 1998     

Inferring Weak Selection from Patterns of Polymorphism and Divergence at ``silent' Sites in Drosophila DNA

Patterns of codon usage and ``silent'''' DNA divergence suggest that natural selection discriminates among synonymous codons in Drosophila. ``Preferred'''' codons are consistently found in higher frequencies within their synonymous families in Drosophila melanogaster genes. This suggests a simple model of silent DNA evolution where natural selection favors mutations from unpreferred to preferred codons (preferred changes). Changes in the opposite direction, from preferred to unpreferred synonymous codons (unpreferred changes), are selected against. Here, selection on synonymous DNA mutations is investigated by comparing the evolutionary dynamics of these two categories of silent DNA changes. Sequences from outgroups are used to determine the direction of synonymous DNA changes within and between D. melanogaster and Drosophila simulans for five genes. Population genetics theory shows that differences in the fitness effect of mutations can be inferred from the comparison of ratios of polymorphism to divergence. Unpreferred changes show a significantly higher ratio of polymorphism to divergence than preferred changes in the D. simulans lineage, confirming the action of selection at silent sites. An excess of unpreferred fixations in 28 genes suggests a relaxation of selection on synonymous mutations in D. melanogaster. Estimates of selection coefficients for synonymous mutations (3.6 <|N(e)s| < 1.3) in D. simulans are consistent with the reduced efficacy of natural selection (|N(e)s| < 1) in the three- to sixfold smaller effective population size of D. melanogaster. Synonymous DNA changes appear to be a prevalent class of weakly selected mutations in Drosophila.     

Ectopic expression of AID in a non-B cell line triggers A:T and G:C point mutations in non-replicating episomal vectors

Somatic hypermutation (SHM) of immunoglobulin genes is currently viewed as a two step process initiated by the deamination of deoxycytidine (C) to deoxyuridine (U), catalysed by the activation induced deaminase (AID). Phase 1 mutations arise from DNA replication across the uracil residue or the abasic site, generated by the uracil-DNA glycosylase, yielding transitions or transversions at G:C pairs. Phase 2 mutations result from the recognition of the U:G mismatch by the Msh2/Msh6 complex (MutS Homologue), followed by the excision of the mismatched nucleotide and the repair, by the low fidelity DNA polymerase eta, of the gap generated by the exonuclease I. These mutations are mainly focused at A:T pairs. Whereas in activated B cells both G:C and A:T pairs are equally targeted, ectopic expression of AID was shown to trigger only G:C mutations on a stably integrated reporter gene. Here we show that when using non-replicative episomal vectors containing a GFP gene, inactivated by the introduction of stop codons at various positions, a high level of EGFP positive cells was obtained after transient expression in Jurkat cells constitutively expressing AID. We show that mutations at G:C and A:T pairs are produced. EGFP positive cells are obtained in the absence of vector replication demonstrating that the mutations are dependent only on the mismatch repair (MMR) pathway. This implies that the generation of phase 1 mutations is not a prerequisite for the expression of phase 2 mutations.