Testing for Selection on Synonymous Sites in Plant Mitochondrial DNA: The Role of Codon Bias and RNA Editing

Since plant mitochondrial genomes exhibit some of the slowest known synonymous substitution rates, it is generally believed that they experience exceptionally low mutation rates. However, the use of synonymous substitution rates to infer mutation rates depends on the implicit assumption that synonymous sites are evolving neutrally (or nearly so). To assess the validity of this assumption in plant mitochondrial genomes, we examined coding sequence for footprints of selection acting at synonymous sites. We found that synonymous sites exhibit an AT rich and pyrimidine skewed nucleotide composition compared to both non-synonymous sites and non-coding regions. We also found some evidence for selection associated with both biased codon usage and conservation of regulatory sequences involved in mRNA processing, although some of these findings are subject to alternative non-adaptive interpretations. Regardless, the inferred strength of selection appears too weak to account for the variation in substitution rates between the mitochondrial genomes of plants and other multicellular eukaryotes. Therefore, these results are consistent with the interpretation that plant mitochondrial genomes experience a substantially lower mutation rate rather than increased functional constraints acting on synonymous sites. Nevertheless, there are important nucleotide composition patterns (particularly the differences between synonymous sites and non-coding DNA) that remain largely unexplained.     

In Vitro Selection of Integration Host Factor Binding Sites

Integration host factor (IHF) is a bacterial protein that binds and severely bends a specific DNA target. IHF binding sites are approximately 30 to 35 bp long and are apparently divided into two domains. While the 3' domain is conserved, the 5' domain is degenerate but is typically AT rich. As a result of physical constraints that IHF must impose on DNA in order to bind, it is believed that this 5' domain must possess structural characteristics conducive for both binding and bending with little regard for specific contacts between the protein and the DNA. We have examined the sequence requirements of the 5' binding domain of the IHF binding target. Using a SELEX procedure, we randomized and selected variants of a natural IHF site. We then analyzed these variants to determine how the 5' binding domain affects the structure, affinity, and function of an IHF-DNA complex in a native system. Despite finding individual sequences that varied over 100-fold in affinity for IHF, we found no apparent correlation between affinity and function.     

Non-Silent Story on Synonymous Sites in Voltage-Gated Ion Channel Genes

Synonymous mutations are usually referred to as “silent”, but increasing evidence shows that they are not neutral in a wide range of organisms. We looked into the relationship between synonymous codon usage bias and residue importance of voltage-gated ion channel proteins in mice, rats, and humans. We tested whether translationally optimal codons are associated with transmembrane or channel-forming regions, i.e., the sites that are particularly likely to be involved in the closing and opening of an ion channel. Our hypothesis is that translationally optimal codons are preferred at the sites within transmembrane domains or channel-forming regions in voltage-gated ion channel genes to avoid mistranslation-induced protein misfolding or loss-of-function. Using the Mantel-Haenszel procedure, which applies to categorical data, we found that translationally optimal codons are more likely to be used at transmembrane residues and the residues involved in channel-forming. We also found that the conservation level at synonymous sites in the transmembrane region is significantly higher than that in the non-transmembrane region. This study provides evidence that synonymous sites in voltage-gated ion channel genes are not neutral. Silent mutations at channel-related sites may lead to dysfunction of the ion channel.     

Sequence Analysis of Fis Binding Sites Obtained by in Vitro Selection

Abstract

To study the specificity of the FIS protein binding to DNA, we have isolated new binding sites by in vitro selection, analyzed their binding and bending by gel electro-phoresis and compared the results to predictions based on sequence analysis of the known natural sites.     

Recent Selection on Synonymous Codon Usage in Drosophila

Evidence from a variety of sources indicates that selection has influenced synonymous codon usage in Drosophila. It has generally been difficult, however, to distinguish selection that acted in the distant past from ongoing selection. However, under a neutral model, polymorphisms usually reflect more recent mutations than fixed differences between species and may, therefore, be useful for inferring recent selection. If the ancestral state is preferred, selection should shift the frequency distribution of derived states/site toward lower values; if the ancestral is unpreferred, selection should increase the number of derived states/site. Polymorphisms were classified as ancestrally preferred or unpreferred for several genes of D. simulans and D. melanogaster. A computer simulation of coalescence was employed to derive the expected frequency distributions of derived states/site under various modifications of the Wright–Fisher neutral model, and distributions of test statistics (t and Mann–Whitney U) were derived by appropriate sampling. One-tailed tests were applied to transformed frequency data to assess whether the two frequency distributions deviated from neutral expectations in the direction predicted by selection on codon usage. Several genes from D. simulans appear to be subject to recent selection on synonymous codons, including one gene with low codon bias, esterase-6. Selection may also be acting in D. melanogaster. Received: 15 April 1998 / Accepted: 13 May 1999     

Studying Patterns of Recent Evolution at Synonymous Sites and Intronic Sites in Drosophila melanogaster

Most previous studies of the evolution of codon usage bias (CUB) and intronic GC content (iGC) in Drosophila melanogaster were based on between-species comparisons, reflecting long-term evolutionary events. However, a complete picture of the evolution of CUB and iGC cannot be drawn without knowledge of their more recent evolutionary history. Here, we used a polymorphism dataset collected from Zimbabwe to study patterns of the recent evolution of CUB and iGC. Analyzing coding and intronic data jointly with a model which can simultaneously estimate selection, mutational, and demographic parameters, we have found that: (1) natural selection is probably acting on synonymous codons; (2) a constant population size model seems to be sufficient to explain most of the observed synonymous polymorphism patterns; (3) GC is favored over AT in introns. In agreement with the long-term evolutionary patterns, ongoing selection acting on X-linked synonymous codons is stronger than that acting on autosomal codons. The selective differences between preferred and unpreferred codons tend to be greater than the differences between GC and AT in introns, suggesting that natural selection, not just biased gene conversion, may have influenced the evolution of CUB. Interestingly, evidence for non-equilibrium evolution comes exclusively from the intronic data. However, three different models, an equilibrium model with two classes of selected sites and two non-equilibrium models with changes in either population size or mutational parameters, fit the intronic data equally well. These results show that using inadequate selection (or demographic) models can result in incorrect estimates of demographic (or selection) parameters.     

Evidence of Conformational Selection Driving the Formation of Ligand Binding Sites in Protein-Protein Interfaces

Many protein-protein interactions (PPIs) are compelling targets for drug discovery, and in a number of cases can be disrupted by small molecules. The main goal of this study is to examine the mechanism of binding site formation in the interface region of proteins that are PPI targets by comparing ligand-free and ligand-bound structures. To avoid any potential bias, we focus on ensembles of ligand-free protein conformations obtained by nuclear magnetic resonance (NMR) techniques and deposited in the Protein Data Bank, rather than on ensembles specifically generated for this study. The measures used for structure comparison are based on detecting binding hot spots, i.e., protein regions that are major contributors to the binding free energy. The main tool of the analysis is computational solvent mapping, which explores the surface of proteins by docking a large number of small “probe” molecules. Although we consider conformational ensembles obtained by NMR techniques, the analysis is independent of the method used for generating the structures. Finding the energetically most important regions, mapping can identify binding site residues using ligand-free models based on NMR data. In addition, the method selects conformations that are similar to some peptide-bound or ligand-bound structure in terms of the properties of the binding site. This agrees with the conformational selection model of molecular recognition, which assumes such pre-existing conformations. The analysis also shows the maximum level of similarity between unbound and bound states that is achieved without any influence from a ligand. Further shift toward the bound structure assumes protein-peptide or protein-ligand interactions, either selecting higher energy conformations that are not part of the NMR ensemble, or leading to induced fit. Thus, forming the sites in protein-protein interfaces that bind peptides and can be targeted by small ligands always includes conformational selection, although other recognition mechanisms may also be involved.     

Interfacial Binding Sites for Cholesterol on G Protein-Coupled Receptors

A docking procedure is described that allows the transmembrane surface of a G protein-coupled receptor (GPCR) to be swept rapidly for potential binding sites for cholesterol at the bilayer interfaces on the two sides of the membrane. The procedure matches 89% of the cholesterols resolved in published GPCR crystal structures, when cholesterols likely to be crystal packing artifacts are excluded. Docking poses are shown to form distinct clusters on the protein surface, the clusters corresponding to “greasy hollows” between protein ridges. Docking poses depend on the angle of tilt of the GPCR in the surrounding lipid bilayer. It is suggested that thermal motion could alter the optimal binding pose for a cholesterol molecule, with the range of binding poses within a cluster providing a guide to the range of thermal motions likely for a cholesterol within a binding site.     

Estimating Selection Intensity on Synonymous Codon Usage in a Nonequilibrium Population

Codon usage bias is the nonrandom use of synonymous codons for the same amino acid. Most population genetic models of codon usage evolution assume that the population is at mutation–selection–drift equilibrium. Natural populations, however, frequently deviate from equilibrium, often because of recent demographic changes. Here, we construct a matrix model that includes the effects of a recent change in population size on estimates of selection on preferred vs. unpreferred codons. Our results suggest that patterns of synonymous polymorphisms affecting codon usage can be quite erratic after such a change; statistical methods that fail to take demographic effects into account can then give incorrect estimates of important parameters. We propose a new method that can accurately estimate both demographic and codon usage parameters. The method also provides a simple way of testing for the effects of covariates such as gene length and level of gene expression on the intensity of selection, which we apply to a large Drosophila melanogaster polymorphism data set. Our analyses of twofold degenerate codons reveal that (i) selection acts in favor of preferred codons, (ii) there is mutational bias in favor of unpreferred codons, (iii) shorter genes and genes with higher expression levels are under stronger selection, and (iv) there is little evidence for a recent change in population size in the Zimbabwe population of D. melanogaster.CODONS specifying the same amino acid are called synonymous codons. These are often used nonrandomly, with some codons appearing more frequently than others. This biased usage of synonymous codons has been found in many organisms such as Drosophila, yeast, and bacteria (Ikemura 1985; Duret and Mouchiroud 1999; Hershberg and Petrov 2008). Conventionally, synonymous codons for a given amino acid are divided into two classes: preferred and unpreferred codons (Ikemura 1985; Akashi 1994; Duret and Mouchiroud 1999). Several observations indicate that codon usage is affected by natural selection. First, in species with codon usage bias, preferred codons generally correspond to the most abundant tRNA species (Ikemura 1981). Second, highly expressed genes usually have higher codon usage bias than genes with low expression (Sharp and Li 1986; Duret and Mouchiroud 1999; Hey and Kliman 2002). Third, the synonymous substitution rate of a gene has been shown to be negatively correlated with its degree of codon usage bias (Sharp and Li 1986; Bierne and Eyre-Walker 2006). The most commonly cited explanations of the apparent fitness differences between preferred and unpreferred codons are selection for translation efficiency, translational accuracy, and mRNA stability (Ikemura 1985; Eyre-Walker and Bulmer 1993; Akashi 1994; Drummond et al. 2005). Recently, it has been proposed that exon splicing also affects codon usage bias (Warnecke and Hurst 2007).From a population genetics perspective, the extent of codon usage bias is ultimately a product of the joint effects of mutation, selection, genetic drift, recombination, and demographic history. The Li–Bulmer model of drift, selection, and reversible mutation between preferred and unpreferred codons at a site is the most widely used model (Li 1987; Bulmer 1991; McVean and Charlesworth 1999). Applications of this model generally assume that the population is at mutation–selection–drift equilibrium. However, empirical studies have suggested that changes in the strengths of various driving forces may not be unusual. For example, in Drosophila melanogaster, there is evidence that the population size (Li and Stephan 2006; Thornton and Andolfatto 2006; Keightley and Eyre-Walker 2007; Stephan and Li 2007), recombinational landscape (Takano-Shimizu 1999), and mutational process (Takano-Shimizu 2001; Kern and Begun 2005) may have changed significantly over the species'' evolutionary history.Such changes cause departures from equilibrium. Theoretical models show that it takes a very long time, proportional to the reciprocal of the mutation rate, for the population to approach a new equilibrium state (Tachida 2000; Comeron and Kreitman 2002). Before reaching equilibrium, the population often shows counterintuitive patterns of evolution (Eyre-Walker 1997; Takano-Shimizu 1999, 2001; Comeron and Kreitman 2002; Comeron and Guthrie 2005; Charlesworth and Eyre-Walker 2007). Despite these theoretical results, details of the patterns of polymorphism and substitution rates following a recent change in population size, and their effects on estimates of strength of selection, have not been determined.The above findings point to the importance of incorporating nonequilibrium factors into the study of codon usage bias. To this end, we extend the Li–Bulmer model to allow population size to vary over time, by representing the evolutionary process by a transition matrix. By analyzing this matrix model, we show that a recent change in population size can result in erratic patterns of codon usage and that methods failing to take into account these demographic effects can give false estimates of the intensity of selection.To solve these problems, we propose a new method, which does not require polarizing ancestral vs. derived states using outgroup data (cf. Cutter and Charlesworth 2006), but requires only knowledge of preferred vs. unpreferred states defined by patterns of codon usage. We use information on both polymorphic and fixed sites, which enables both mutational bias and the strength of selection to be estimated, in contrast to previous methods that use information on polymorphisms alone. Simulations indicate that this method can accurately estimate both demographic and codon usage parameters and can distinguish between selection and demography. We use the new method to analyze a large D. melanogaster polymorphism data set (Shapiro et al. 2007) and find evidence for natural selection on synonymous codons. We use our approach to show that genes with shorter coding sequences and higher levels of expression are under significantly stronger selection than longer genes with lower expression.     

Potent and Selective Ligands for Adenosine Binding Sites

Abstract

A number of selective ligands for the different binding sites of adenosine have been synthesized and tested in several pharmacological models. The aim of these synthetic efforts is both to improve the knowledge of structure-activity relationships in the adenosine-related biological systems and to develop drugs from some of these molecules.     

转录因子结合位点生物信息学研究进展

By using genome in situ hybridization (GISH) on root somatic chromosomes of allotetraploid derived from the cross Gossypium arboreum × G. bickii with genomic DNA (gDNA) of G. bickii as a probe, two sets of chromosomes, consisting of 26 chromosomes each, were easily distinguished from each other by their distinctive hybridization signals. GISH analysis directly proved that the hybrid G.arboreum×G. bickii is an allotetraploid amphiploid. The karyotype formula of the species was 2n = 4x = 52 = 46m (4sat) + 6sm (4sat). We identified four pairs of satellites with two pairs in each sub-genome. FISH analysis using 45S rDNA as a probe showed that the cross G. arboreum×G. bickii contained 14 NORs. At least five pairs of chromosomes in the G sub-genome showed double hybridization (red and blue) in their long arms, which indicates that chromatin introgression from the A sub-genome had occurred.     

Quantifying Selection against Synonymous Mutations in HIV-1 env Evolution

Intrapatient evolution of human immunodeficiency virus type 1 (HIV-1) is driven by the adaptive immune system resulting in rapid change of HIV-1 proteins. When cytotoxic CD8⁺ T cells or neutralizing antibodies target a new epitope, the virus often escapes via nonsynonymous mutations that impair recognition. Synonymous mutations do not affect this interplay and are often assumed to be neutral. We test this assumption by tracking synonymous mutations in longitudinal intrapatient data from the C2-V5 part of the env gene. We find that most synonymous variants are lost even though they often reach high frequencies in the viral population, suggesting a cost to the virus. Using published data from SHAPE (selective 2′-hydroxyl acylation analyzed by primer extension) assays, we find that synonymous mutations that disrupt base pairs in RNA stems flanking the variable loops of gp120 are more likely to be lost than other synonymous changes: these RNA hairpins might be important for HIV-1. Computational modeling indicates that, to be consistent with the data, a large fraction of synonymous mutations in this genomic region need to be deleterious with a cost on the order of 0.002 per day. This weak selection against synonymous substitutions does not result in a strong pattern of conservation in cross-sectional data but slows down the rate of evolution considerably. Our findings are consistent with the notion that large-scale patterns of RNA structure are functionally relevant, whereas the precise base pairing pattern is not.     

Opiate Binding Sites in Bovine Adrenal Medulla

Abstract

Previous studies using a variety of opiate ligands have suggested the existence of several subclasses of opiate receptors in crude membrane fractions of rat brain, and a similar diversity in bovine adrenal medulla. To examine the receptor profile of bovine adrenal medulla in detail we have studied the binding of classical ligands for mu (μ), delta (δ) and kappa (k) opiate receptors. [³H]naloxone ([³H]NAL), [³H] morphine ([³H]MOR), [³H]D-Ala²-D-Leu⁵-enkephalin ([³H]DAL) and [³H]ethyl-ketocyclazocine ([³H]EKCZ) were used as tracers; unlabeled competitors were NAL, MOR, DAL and ketocyclazocine (KCZ). In adrenal medulla [³H]NAL was specifically bound with a hierarchy of displacement NAL > MOR > KCZ ? DAL. No specific binding of [³H]DAL or [³H]EKCZ was found; for [³H]MOR very low levels of binding were seen, with no displacement by NAL or DAL, inconsistent displacement by KCZ and substantial displacement by MOR with an ED₅₀ of 1.5 nM. In parallel studies rat brain membranes bound each labeled ligand with affinity and specificity consistent with previously published reports. Identical results were obtained in membranes from both tissues prepared with a preincubation step including 100 mM Na⁺, suggesting that the results were not influenced by occupation of binding sites by endogenous ligands. We interpret these data as supporting the existence of opiate receptors of the μ subtype in bovine adrenal medulla. We find, however, no evidence of δ or k sites in this tissue.     

miRNA与其靶mRNA的相互作用：绑定位点的质量与数量特征的整合计算分析

miRNAs通过完全或不完全的碱基互补绑定到信使RNA(mRNA)上,通过抑制翻译或者直接导致mRNA降解的方式来调节靶基因的表达．为了研究miRNAs在转录水平上面的调控作用,两种人类基因组中组织特异的miRNAs(miR-1和miR-124)被转染到HeLa细胞中,微阵列(microarray)分析转染前后细胞中各基因mRNA表达水平变化情况的结果表明：动物基因组中靶基因与miRNAs不完全的碱基互补也会导致mRNA的直接降解．通过分析实验得到的mRNA表达水平变化数据,发现这相同miRNA的不同靶基因mRNA表达水平的下调倍数有着明显的差别,推测这些靶基因mRNA序列本身存在某些影响其受调节程度的因素．为此,提取和分析这些靶基因mRNA的序列特征,通过对这些序列特征与mRNA表达水平下调数据进行统计相关分析,最终发现,miRNA靶基因受调节的程度与以下几个因素相关联：mRNA序列中miRNA靶位点的个数,靶位点与miRNA序列碱基互补的程度,以及绑定后形成二级结构的稳定程度(即最低自由能的大小)．在此基础上,初步建立起一个多因子作用下的miRNA 靶基因mRNA表达水平下调程度模型,分析表明：该模型在一定程度上可以反映了部分序列特征对于miRNA靶基因mRNA表达水平下调程度的影响．