Developmental stage and level of codon usage bias in Drosophila

Codon usage bias (CUB) is a ubiquitous observation in molecular evolution. As a model, Drosophila has been particularly well-studied and indications show that selection at least partially controls codon usage, probably through selection for translational efficiency. Although many aspects of Drosophila CUB have been studied, this is the first study relating codon usage to development in this holometabolous insect with very different life stages. Here we ask the question: What developmental stage of Drosophila melanogaster has the greatest CUB? Genes with maximum expression in the larval stage have the greatest overall CUB when compared with embryos, pupae, and adults. (The same pattern was observed in Drosophila pseudoobscura, see Supplementary Material online.) We hypothesize this is related to the very rapid growth of larvae, placing increased selective pressure to produce large amounts of protein: a 300-fold increase requiring an approximate doubling of protein content every 10 h. Genes with highest expression in adult males and early embryos, stages with the least de novo protein synthesis, display the least CUB. These results are consistent with the hypothesis that CUB is caused (at least in part) by selection for efficient protein production. This seems to hold on the individual gene level (highly expressed genes are more biased than lowly expressed genes) as well as on a more global scale where genes with maximum expression during times of very rapid growth and protein synthesis are more biased than genes with maximum expression during times of low growth.     

Across bacterial phyla, distantly-related genomes with similar genomic GC content have similar patterns of amino acid usage

The GC content of bacterial genomes ranges from 16% to 75% and wide ranges of genomic GC content are observed within many bacterial phyla, including both gram negative and gram positive phyla. Thus, divergent genomic GC content has evolved repeatedly in widely separated bacterial taxa. Since genomic GC content influences codon usage, we examined codon usage patterns and predicted protein amino acid content as a function of genomic GC content within eight different phyla or classes of bacteria. We found that similar patterns of codon usage and protein amino acid content have evolved independently in all eight groups of bacteria. For example, in each group, use of amino acids encoded by GC-rich codons increased by approximately 1% for each 10% increase in genomic GC content, while the use of amino acids encoded by AT-rich codons decreased by a similar amount. This consistency within every phylum and class studied led us to conclude that GC content appears to be the primary determinant of the codon and amino acid usage patterns observed in bacterial genomes. These results also indicate that selection for translational efficiency of highly expressed genes is constrained by the genomic parameters associated with the GC content of the host genome.     

Evidence of selectively driven codon usage in rice: implications for GC content evolution of Gramineae genes

Two gene classes characterized by high and low GC content have been found in rice and other cereals, but not dicot genomes. We used paralogs with high and low GC contents in rice and found: (a) a greater increase in GC content at exonic fourfold-redundant sites than at flanking introns; (b) with reference to their orthologs in Arabidopsis, most substitution sites between the two kinds of paralogs are found at 2- and 4-degenerate sites with a T-->C mode, while A-->C and A-->G play major roles at 0-degenerate sites; and (c) high-GC genes have greater bias and codon usage is skewed toward codons that are preferred in highly expressed genes. We believe this is strong evidence for selectively driven codon usage in rice. Another cereal, maize, also showed the same trend as in rice. This represents a potential evolutionary process for the origin of genes with a high GC content in rice and other cereals.     

XFINGER: A tool for searching and visualising protein fingerprints and patterns

A tool for searching pattern and fingerprint databases is described.Fingerprints are groups of motifs excised from conserved regionsof sequence alignments and used for iterative database scanning.The constituent motifs are thus encoded as small alignmentsin which sequence information is maximised with each databasepass; they therefore differ from regular-expression patterns,in which alignments are reduced to single consensus sequences.Different database formats have evolved to store these disparatetypes of information, namely the PROSITE dictionary of patternsand the PRINTS fingerprint database, but programs have not beenavailable with the flexibility to search them both. We havedeveloped a facility to do this: the system allows query sequencesto be scanned against either PROSITE, the full PRINTS database,or against individual fingerprints. The results of fingerprintsearches are displayed simultaneously in both text and graphicalwindows to render them more tangible to the user. Where structuralcoordinates are available, identified motifs may be visualisedin a 3D context. The program runs on Silicon Graphics machinesusing GL graphics libraries and on machines with X servers supportingthe PEX extension: its use is illustrated here by depictingthe location of low-density lipoprotein-binding (LDL) motifsand leucine-rich repeats in a mosaic G-protein-coupled receptor(GPCR).     

Thermophilic prokaryotes have characteristic patterns of codon usage, amino acid composition and nucleotide content

A number of recent studies have shown that thermophilic prokaryotes have distinguishable patterns of both synonymous codon usage and amino acid composition, indicating the action of natural selection related to thermophily. On the other hand, several other studies of whole genomes have illustrated that nucleotide bias can have dramatic effects on synonymous codon usage and also on the amino acid composition of the encoded proteins. This raises the possibility that the thermophile-specific patterns observed at both the codon and protein levels are merely reflections of a single underlying effect at the level of nucleotide composition. Moreover, such an effect at the nucleotide level might be due entirely to mutational bias. In this study, we have compared the genomes of thermophiles and mesophiles at three levels: nucleotide content, codon usage and amino acid composition. Our results indicate that the genomes of thermophiles are distinguishable from mesophiles at all three levels and that the codon and amino acid frequency differences cannot be explained simply by the patterns of nucleotide composition. At the nucleotide level, we see a consistent tendency for the frequency of adenine to increase at all codon positions within the thermophiles. Thermophiles are also distinguished by their pattern of synonymous codon usage for several amino acids, particularly arginine and isoleucine. At the protein level, the most dramatic effect is a two-fold decrease in the frequency of glutamine residues among thermophiles. These results indicate that adaptation to growth at high temperature requires a coordinated set of evolutionary changes affecting (i) mRNA thermostability, (ii) stability of codon-anticodon interactions and (iii) increased thermostability of the protein products. We conclude that elevated growth temperature imposes selective constraints at all three molecular levels: nucleotide content, codon usage and amino acid composition. In addition to these multiple selective effects, however, the genomes of both thermophiles and mesophiles are often subject to superimposed large changes in composition due to mutational bias.     

Dissecting the contributions of GC content and codon usage to gene expression in the model alga Chlamydomonas reinhardtii

The efficiency of gene expression in all organisms depends on the nucleotide composition of the coding region. GC content and codon usage are the two key sequence features known to influence gene expression, but the underlying molecular mechanisms are not entirely clear. Here we have determined the relative contributions of GC content and codon usage to the efficiency of nuclear gene expression in the unicellular green alga Chlamydomonas reinhardtii. By comparing gene variants that encode an identical amino acid sequence but differ in their GC content and/or codon usage, we show that codon usage is the key factor determining translational efficiency and, surprisingly, also mRNA stability. By contrast, unfavorable GC content affects gene expression at the level of the chromatin structure by triggering heterochromatinization. We further show that mutant algal strains that permit high‐level transgene expression are less susceptible to epigenetic transgene suppression and do not establish a repressive chromatin structure at the transgenic locus. Our data disentangle the relationship between GC content and codon usage, and suggest simple strategies to overcome the transgene expression problem in Chlamydomonas.     

A large-scale analysis of codon usage bias in 4868 bacterial genomes shows association of codon adaptation index with GC content,protein functional domains and bacterial phenotypes

Multiple synonymous codons code for the same amino acid, resulting in the degeneracy of the genetic code and in the preferred used of some codons called codon bias usage (CBU). We performed a large-scale analysis of codon usage bias analysing the distribution of the codon adaptation index (CAI) and the codon relative adaptiveness index (RA) in 4868 bacterial genomes. We found that CAI values differ significantly between protein functional domains and part of the protein outside domains and show how CAI, GC content and preferred usage of polymerase III alpha subunits are related. Additionally, we give evidence of the association between CAI and bacterial phenotypes.     

Analysis of phylogeny and codon usage bias and relationship of GC content,amino acid composition with expression of the structural nif genes

Bacteria and archaea have evolved with the ability to fix atmospheric dinitrogen in the form of ammonia, catalyzed by the nitrogenase enzyme complex which comprises three structural genes nifK, nifD and nifH. The nifK and nifD encodes for the beta and alpha subunits, respectively, of component 1, while nifH encodes for component 2 of nitrogenase. Phylogeny based on nifDHK have indicated that Cyanobacteria is closer to Proteobacteria alpha and gamma but not supported by the tree based on 16SrRNA. The evolutionary ancestor for the different trees was also different. The GC1 and GC2% analysis showed more consistency than GC3% which appeared to below for Firmicutes, Cyanobacteria and Euarchaeota while highest in Proteobacteria beta and clearly showed the proportional effect on the codon usage with a few exceptions. Few genes from Firmicutes, Euryarchaeota, Proteobacteria alpha and delta were found under mutational pressure. These nif genes with low and high GC3% from different classes of organisms showed similar expected number of codons. Distribution of the genes and codons, based on codon usage demonstrated opposite pattern for different orientation of mirror plane when compared with each other. Overall our results provide a comprehensive analysis on the evolutionary relationship of the three structural nif genes, nifK, nifD and nifH, respectively, in the context of codon usage bias, GC content relationship and amino acid composition of the encoded proteins and exploration of crucial statistical method for the analysis of positive data with non-constant variance to identify the shape factors of codon adaptation index.     

Combining models of protein translation and population genetics to predict protein production rates from codon usage patterns

Genes are often biased in their codon usage. The degree of bias displayed often changes with expression level and intragenic position. Numerous indices, such as the codon adaptation index, have been developed to measure this bias. Although the expression level of a gene and index values are correlated, the heuristic nature of these metrics limits their ability to explain this relationship. As an alternative approach, this study integrates mechanistic models of cellular and population processes in a nested manner to develop a stochastic evolutionary model of a protein's production rate (SEMPPR). SEMPPR assumes that the evolution of codon bias is driven by selection to reduce the cost of nonsense errors and that this selection is counteracted by mutation and drift. Through the application of Bayes' theorem, SEMPPR generates a posterior probability distribution for the protein production rate of a given gene. Conceptually, SEMPPR's predictions are based on the degree of adaptation to reduce the cost of nonsense errors observed in the codon usage pattern of the gene. As an illustration, SEMPPR was parameterized using the Saccharomyces cerevisiae genome and its predictions tested using available empirical data. The results indicate that SEMPPR's predictions are as reliable index based ones. In addition, SEMPPR's output is more easily interpreted and its predictions could be improved through refinements of the models upon which it is built.     

Organization and codon usage of the streptomycin operon in Micrococcus luteus, a bacterium with a high genomic G + C content.

The DNA sequence of the Micrococcus luteus str operon, which includes genes for ribosomal proteins S12 (str or rpsL) and S7 (rpsG) and elongation factors (EF) G (fus) and Tu (tuf), has been determined and compared with the corresponding sequence of Escherichia coli to estimate the effect of high genomic G + C content (74%) of M. luteus on the codon usage pattern. The gene organization in this operon and the deduced amino acid sequence of each corresponding protein are well conserved between the two species. The mean G + C content of the M. luteus str operon is 67%, which is much higher than that of E. coli (51%). The codon usage pattern of M. luteus is very different from that of E. coli and extremely biased to the use of G and C in silent positions. About 95% (1,309 of 1,382) of codons have G or C at the third position. Codon GUG is used for initiation of S12, EF-G, and EF-Tu, and AUG is used only in S7, whereas GUG initiates only one of the EF-Tu's in E. coli. UGA is the predominant termination codon in M. luteus, in contrast to UAA in E. coli.     

Measurements of genomic GC content in plant genomes with flow cytometry: a test for reliability

? Knowledge of the phylogenetic pattern and biological relevance of the base composition of large eukaryotic genomes (including those of plants) is poor. With the use of flow cytometry (FCM), the amount of available data on the guanine + cytosine (GC) content of plants has nearly doubled in the last decade. However, skepticism exists concerning the reliability of the method because of uncertainty in some input parameters. ? Here, we tested the reliability of FCM for estimating GC content by comparison with the biochemical method of DNA temperature melting analysis (TMA). We conducted measurements in 14 plant species with a maximum currently known GC content range (33.6-47.5% as measured by FCM). We also compared the estimations of the GC content by FCM with genomic sequences in 11 Oryza species. ? FCM and TMA data exhibited a high degree of correspondence which remained stable over the relatively wide range of binding lengths (3.39-4.09) assumed for the base-specific dye used. A high correlation was also observed between FCM results and the sequence data in Oryza, although the latter GC contents were consistently lower. ? Reliable estimates of the genomic base composition in plants by FCM are comparable with estimates obtained using other methods, and so wider application of FCM in future plant genomic research, although it would pose a challenge, would be supported by these findings.     

Phenotypic evolutionary models in stem cell biology: replacement, quiescence, and variability

Phenotypic evolutionary models have been used with great success in many areas of biology, but thus far have not been applied to the study of stem cells except for investigations of cancer. We develop a framework that allows such modeling techniques to be applied to stem cells more generally. The fundamental modeling structure is the stochastic kinetics of stem cells in their niche and of transit amplifying and fully differentiated cells elsewhere in the organism, with positive and negative feedback. This formulation allows graded signals to be turned into all or nothing responses, and shows the importance of looking beyond the niche for understanding how stem cells behave. Using the deterministic version of this framework, we show how competition between different stem cell lines can be analyzed, and under what circumstances stem cells in a niche will be replaced by other stem cells with different phenotypic characteristics. Using the stochastic version of our framework and state dependent life history theory, we show that the optimal behavior of a focal stem cell will involve long periods of quiescence and that a population of identical stem cells will show great variability in the times at which activity occurs; we compare our results with classic ones on quiescence and variability in the hematopoietic system.     

Detecting genomic features under weak selective pressure: the example of codon usage in animals and plants

Large scale experiments of gene inactivation in yeast have shown that 50% of genes have no detectable impact on the phenotype, and similar observations have been made in other model organisms. This apparent paradox is probably due to the fact that many genes only have a marginal contribution to the fitness of organisms. Because of the size of populations and the number of generations that can be studied in laboratories, experimental approaches only permit to detect functional elements that have a strong phenotypic impact. Comparative sequence analysis can help to solve this problem: the analysis of sequences evolution permits to detect the action of selection, and hence to reveal functional features of genomes. This approach will be illustrated by the study of synonymous codon usage in animals and plants.     

Molecular evolution of bacteriophages: Discrete patterns of codon usage in T4 genes are related to the time of gene expression

Summary Patterns of codon usage in certain coliphages are adapted to expression inEscherichia coli. Bacteriophage T4 may be an exception to test the rule, as it produces eight tRNAs with specificities that are otherwise rare inE. coli. A database of all known T4 DNA sequences has been compiled, comprising 174 genes and a total of 115 kb (approximately 70% of the T4 genome). Codon usage has been examined in all T4 genes; some of these are known to be expressed before, and some after, the production of phage tRNAs. The results show two different patterns of codon usage: by comparison with the early genes, the late genes exhibit a shift in preference toward those codons recognized by the phage-encoded tRNAs. The T4 tRNAs translate A-ending codons, and it is possible that the phage acquired the tRNA genes because the mutation bias of the T4 DNA polymerase forces the T4 genome toward A+T-richness.Presented at the NATO Advanced Workshop on Genome Organization and Evolution, held in Spetses, Greece, September 1990     

Novel anticodon composition of transfer RNAs in Micrococcus luteus, a bacterium with a high genomic G + C content. Correlation with codon usage.

The number and relative amount of isoacceptor tRNAs for each amino acid in Micrococcus luteus, a Gram-positive bacterium with high genomic G + C content, have been determined by sequencing their anticodon loop and its adjacent regions and by selective labelling of tRNAs. Thirty-one tRNA species with 29 different anticodon sequences have been detected. All the tRNAs have G or C at the anticodon first position except for tRNA(ICGArg) and tRNA(NGASer), in response to the abundant usage of NNC and NNG codons. No tRNA with the anticodon UNN capable of translating codon NNA has been detected, in accordance with a very low or zero usage of NNA codons. The relative amount of isoacceptor tRNAs for an amino acid determined by selective labelling strongly correlates with usage of the corresponding codons. On the basis of these and other observations in this and other eubacterial species, we conclude that the relative amount and anticodon composition of isoacceptor tRNA species are flexible, and their changes are mainly adaptive phenomena that have been primarily affected by codon usage, which in turn is affected by directional mutation pressure.     

Convergence of normal stem cell and cancer stem cell developmental stage: Implication for differential therapies

Increased evidence shows that normal stem cells may contribute to cancer development and progression by acting as cancer-initiating cells through their interactions with abnormal environmental elements.We postulate that normal stem cells and cancer stem cells (CSC) possess similar mechanisms of self-renewal and differentiation.CSC can be the key to the elaboration of anti-cancer-based therapy.In this article,we focus on a controversial new theme relating to CSC.Tumorigenesis may have a critical stage characterized as a "therapeutic window",which can be identified by asso-ciation of molecular,biochemical and biological events.Identifying such a stage can allow the production of more effective therapies (e.g.manipulated stem cells) to treat several cancers.More importantly,confirming the existence of a similar therapeutic window during the conversion of normal stem cells to malignant CSC may lead to targeted therapy specifically against CSC.This conversion information may be derived from investigating the biological behaviour of both normal stem cells and cancerous stem cells.Currently,there is little knowledge about the cellular and molecular mechanisms that govern the initiation and maintenance of CSC.Studies on co-evolution and interdependence of cancer with normal tissues may lead to a useful treatment paradigm of cancer.The crosstalk between normal stem cells and cancer formation may converge developmental stages of different types of stem cells (e.g.normal stem cells,CSC and embryonic stem cells).The differential studies of the convergence may result in novel therapies for treating cancers.     

Embryonic stem cell differentiation models: cardiogenesis, myogenesis, neurogenesis, epithelial and vascular smooth muscle cell differentiation in vitro

Embryonic stem cells, totipotent cells of the early mouse embryo, were established as permanent cell lines of undifferentiated cells. ES cells provide an important cellular system in developmental biology for the manipulation of preselected genes in mice by using the gene targeting technology. Embryonic stem cells, when cultivated as embryo-like aggregates, so-called ‘embryoid bodies’, are able to differentiate in vitro into derivatives of all three primary germ layers, the endoderm, ectoderm and mesoderm. We established differentiation protocols for the in vitro development of undifferentiated embryonic stem cells into differentiated cardiomyocytes, skeletal muscle, neuronal, epithelial and vascular smooth muscle cells. During differentiation, tissue-specific genes, proteins, ion channels, receptors and action potentials were expressed in a developmentally controlled pattern. This pattern closely recapitulates the developmental pattern during embryogenesis in the living organism. In vitro, the controlled developmental pattern was found to be influenced by differentiation and growth factor molecules or by xenobiotics. Furthermore, the differentiation system has been used for genetic analyses by ‘gain of function’ and ‘loss of function’ approaches in vitro. This revised version was published online in July 2006 with corrections to the Cover Date.     

The mitochondrial control region of Cervidae: evolutionary patterns and phylogenetic content

The mitochondrial control region (CR) sequence, also known as the D- loop, has been determined for six Cervidae (Artiodactyla, Ruminantia): the red and fallow deers (subfamily Cervinae), the brocket deer and two roe deers (subfamily Odocoileinae), and the Chinese water deer (Hydropotinae). These new sequences have been aligned with available cervid and bovid orthologues. Comparative analyses indicate that the 5'- peripheral domain exhibits a 75-bp length polymorphism near sequences associated with the termination of the H-strand replication. The New World Odocoileinae possess the longest cervid CR due to the presence of an additional 47-bp tandem repeat, located in the 3'-peripheral domain, downstream of the initiation site for H-strand replication (OH) and the first conserved sequence block (CSB-1). This insertion represents a duplication spanning the OH to CSB-1 region and constitutes an exclusive synapomorphy for New World Odocoileinae. Phylogenetic analyses of the complete CR support the paraphyly of antlered deers due to the nesting of the antlerless Hydropotes within Odocoileinae. Capreolus is the closest relative of Hydropotes, and the divergence of this Old World Odocoileinae clade may have occurred between 8.7 and 10.4 MYA. The conserved central domain of CR can be aligned across ungulates and indicates the Pecora monophyly, their close association with cetaceans, and the earlier emergence of suiformes.      

A novel approach for predicting the evolutionary time of acquisition of foreign genes by bacteria: Application of codon usage analyses

Lysogenic bacteriophages are considered as a major player for the introduction of foreign genes into bacterial strains. At the time of introduction foreign genes do not fit well into the translation system of the recipient host bacterium as they tend to retain the characteristics of the donor bacterium from which they have been transferred. Consequently foreign genes are poorly transcribed at the early phase of their evolution within the host bacterium. This is largely due to the difference in the codon usage pattern between the horizontally transferred genes and the host bacterium. In this study we present detailed analyses of various parameters of the codon usages such as codon adaptation index (CAI), mean difference (MD) of the relative adaptiveness, synonymous substitution rate (SSR) of six different phage encoded toxin genes (cholera toxin, shiga toxin, diphtheria toxin, neurotoxin C1, enterotoxin type A and cytotoxin), and proposed conceptual relationship between the evolutionary time of acquisition of the foreign genes and the selected set of parameters of the codon usage. On the basis of the observed data we hypothesize that CAI, MD and SSR of the phage encoded toxin genes are correlated with the evolutionary time of their acquisition, and have developed a novel approach based on the analyses of these parameters, which can be used to predict the evolutionary time of their acquisition by the corresponding host bacterium.