Identification of 142 single nucleotide polymorphisms in 41 candidate genes for rheumatoid arthritis in the Japanese population

Single-nucleotide polymorphisms (SNPs) can make an important contribution to our understanding of genetic backgrounds that may influence medical conditions and ethnic diversity. We undertook a systematic survey of genomic DNA for SNPs located not only in coding sequences but also in non-coding regions (e.g., introns and 5' flanking regions) of selected genes. Using DNA samples from 48 Japanese patients with rheumatoid arthritis (RA) as templates, we surveyed 41 genes that represent candidates for RA, screening a total of 104 kb of DNA (30 kb of coding sequences and 74 kb of non-coding DNA). Within this 104 kb of genomic sequences we identified 163 polymorphisms (1 per 638 bases on average), of which 142 were single-nucleotide substitutions and the remainder, insertions or deletions. Of the coding SNPs, 52% were non-synonymous substitutions, and non-conservative amino acid changes were observed in a quarter of those. Sixty-nine polymorphisms showed high frequencies for minor alleles (more than 15%) and 20 revealed low frequencies (<5%). Our results indicated a greater average distance between SNPs than others have reported, but this disparity may reflect the type of genes surveyed and/or the relative ethnic homogeneity of our test population.     

Patterns of DNA-Sequence Divergence Between <Emphasis Type="Italic">Drosophila miranda</Emphasis> and <Emphasis Type="Italic">D. pseudoobscura</Emphasis>

Contrary to the classical view, a large amount of non-coding DNA seems to be selectively constrained in Drosophila and other species. Here, using Drosophila miranda BAC sequences and the Drosophila pseudoobscura genome sequence, we aligned coding and non-coding sequences between D. pseudoobscura and D. miranda, and investigated their patterns of evolution. We found two patterns that have previously been observed in comparisons between Drosophila melanogaster and its relatives. First, there is a negative correlation between intron divergence and intron length, suggesting that longer non-coding sequences may contain more regulatory elements than shorter sequences. Our other main finding is a negative correlation between the rate of non-synonymous substitutions (d _N) and codon usage bias (F _op), showing that fast-evolving genes have a lower codon usage bias, consistent with strong positive selection interfering with weak selection for codon usage.     

Analysis of Genomic G + C Content, Codon Usage, Initiator Codon Context and Translation Termination Sites In Tetrahymena Thermophila

In recent years, the amount of molecular sequencing data from Tetrahymena thermophila has dramatically increased. We analyzed G + C content, codon usage, initiator codon context and stop codon sites in the extremely A + T rich genome of this ciliate. Average G + C content was 38% for protein coding regions, 21% for 5' non-coding sequences, 19% for 3' non-coding sequences, 15% for introns, 19% for micronuclear limited sequences and 17% for macronuclear retained sequences flanking micronuclear specific regions. The 75 available T. thermophila protein coding sequences favored codons ending in T and, where possible, avoided those with G in the third position. Highly expressed genes were relatively G + C-rich and exhibited an extremely biased pattern of codon usage while developmentally regulated genes were more A + T-rich and showed less codon usage bias. Regions immediately preceding Tetrahymena translation initiator codons were generally A-rich. For the 60 stop codons examined, the frequency of G in the end + 1 site was much higher than expected whereas C never occupied this position.     

Unusual codon bias occurring within insertion sequences in Escherichia coli

The large open reading frames of insertion sequences from Escherichia coli were examined for their spatial pattern of codon usage bias and distribution of rarely used codons. There is a bias in codon usage that is generally lower toward the terminal ends of the coding regions, which is reflected in the occurrence of an excess of nonpreferred codons in the 3 portions of the coding regions as compared with the 5 portions. In contrast, typical chromosomal genes have a lower codon usage bias toward the 5 ends of the coding regions. These results imply that the selective forces reflected in codon usage bias may differ according to position within the coding sequence. In addition, these constraints apparently differ in important ways between genes contained in insertion sequences and those in the chromosome.     

亚心型扁藻叶绿体psbA基因的克隆及序列分析

psbA基因是叶绿体基因组中一个重要的光调控基因,编码光和系统Ⅱ反应中心的D1蛋白。根据叶绿体基因组序列高度保守的特性,利用菜茵衣藻（Chlamydomonasreinhardtii）psbA基因的保守序列（基因登录号：HQ667991．1）设计引物,采用PCR步移的方法从亚心型扁藻（Platymonassubcordiformis）基因组DNA中克隆到psbA基因全长（基因登录号：KF528742）。序列分析表明,亚心型扁藻psbA基因全长1939bp,编码区长度为1062bp,推导编码353个氨基酸,包括4个赖氨酸残基。有效密码子数显示脚删基因具有明显的密码子偏好性,并且偏好使用以A／T结尾的密码子。相对同义密码子使用度表明25个密码子在编码使用时具有偏好性,其中20个密码子以A／T碱基结尾,占到80％。其终止密码子使用了TAG。     

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.     

Interaction of silent and replacement changes in eukaryotic coding sequences

We examined the codon usages in well-conserved and less-well-conserved regions of vertebrate protein genes and found them to be similar. Despite this similarity, there is a statistically significant decrease in codon bias in the less-well-conserved regions. Our analysis suggests that although those codon changes initially fixed under amino acid replacements tend to follow the overall codon usage pattern, they also reduce the bias in codon usage. This decrease in codon bias leads one to predict that the rate of change of synonymous codons should be greater in those regions that are less well conserved at the amino acid level than in the better-conserved regions. Our analysis supports this prediction. Furthermore, we demonstrate a significantly elevated rate of change of synonymous codons among the adjacent codons 5' to amino acid replacement positions. This provides further support for the idea that there are contextual constraints on the choice of synonymous codons in eukaryotes.     

Variable SNP density in aspartyl-protease genes of the malaria parasite Plasmodium falciparum

An analysis of the diversity of the aspartyl proteases of Plasmodium falciparum, known as plasmepsins (PMs), was completed in view of their possible role as drug targets. DNA sequence polymorphisms were identified in nine pm genes including their non-coding (introns and 5' flanking) sequences. All genes contained at least one single nucleotide polymorphism (SNP). Extensive microsatellite diversity was observed predominantly in non-coding sequences. All but one non-synonymous polymorphism (a conservative substitution) were mapped to the surface of the predicted protein, contradicting a possible role in enzymatic activity. The distribution of SNPs was found to be non-random among pm genes, with pm6 and pm10 having significantly higher SNP densities, suggesting they were under selection. For pm6 the majority of the SNPs were in introns and some of these may contribute to splice site variation. SNPs were found at a high density in both the coding and non-coding sequences of pm10. Recombination was important in generating additional diversity at this locus. Although direct selection for pm10 mutations could not be ruled out, the presence of balancing selection and a high density of SNPs in non-coding sequence led us to propose that another gene under selection may be influencing the diversity in the region. By sequencing short DNA tags in a 200 kb region flanking pm10 we show that a cluster of antigen genes, known to be under diversifying selection, may contribute to the observed diversity. We discuss the importance of diversity and local selection effects when choosing drug targets for intervention strategies.     

DNA variation in the metallothionein genes in wild rice Oryza rufipogon: relationship between DNA sequence polymorphism, codon bias and gene expression

This study examines the relationship between DNA sequence variation and level of gene expression in four metallothionein genes from wild rice Oryza rufipogon. The nucleotide diversity was 0.0028 to 0.0117 over the entire coding and non-coding region, and it was negatively correlated with gene expression for three type 2 metallothionein genes. In contrast, codon bias and percent of preferred codons correlated positively with gene expression. These results indicate that the intensity of natural selection depends on the level of gene expression, which in turn shapes the level of nucleotide polymorphism. In addition, significant linkage disequilibria were frequent between the metallothionein genes, although significance was not confirmed after multiple test correction. This result suggests that metallothionein genes expressed at different levels are epistatic with respect to fitness, and that gene expression is an important factor determining level of DNA polymorphism.     

An Evaluation of Measures of Synonymous Codon Usage Bias

Synonymous codons are not generally used at equal frequencies, and this trend is observed for most genes and organisms. Several methods have been proposed and used to estimate the degree of the nonrandom use of the different synonymous codons. The estimates obtained by these methods, however, show different levels of both precision and dispersion when coding regions of a finite number of codons are under analysis. Here, we present a study, based on computer simulation, of how the different methods proposed to evaluate the nonrandom use of synonymous codons are affected by the length of the coding region analyzed. The results show that some of these methods are heavily influenced by the number of codons and that the comparison of codon usage bias between coding regions of different lengths shows a methodological bias under different conditions of nonrandom use of synonymous codons. The study of the dispersion of the estimates obtained by the different methods gives, on the other hand, an indication of the methods to be applied to compare values of codon usage bias among coding regions of equivalent length. Received: 10 September 1997 / Accepted: 23 March 1998     

A method for measuring the non-random bias of a codon usage table

We describe a new statistical method for measuring bias in the codon usage table of a gene. The test is based on the multinomial and Poisson distributions. The method is used to scan DNA sequences and measure the strength of codon preference. For E. Coli we show that the strength of codon preference is related to levels of gene expression. The method can also be used to compare base triplet frequencies with those expected from the base composition. This second type of codon bias test is useful for distinguishing coding from non-coding regions.     

Does the 'non-coding' strand code?

The hypothesis that DNA strands complementary to the coding strand contain in phase coding sequences has been investigated. Statistical analysis of the 50 genes of bacteriophage T7 shows no significant correlation between patterns of codon usage on the coding and non-coding strands. In Bacillus and yeast genes the correlation observed is not different from that expected with random synonymous codon usage, while a high correlation seen in 52 E. coli genes can be explained in terms of an excess of RNY codons. A deficiency of UUA, CUA and UCA codons (complementary to termination) seems to be restricted to the E. coli genes, and may be due to low abundance of the relevant cognate tRNA species. Thus the analysis shows that the non-coding strand has the properties expected of a sequence complementary to a coding strand, with no indications that it encodes, or may have encoded, proteins.     

Interaction of silent and replacement changes in eukaryotic coding sequences

Summary We examined the codon usages in wellconserved and less-well-conserved regions of vertebrate protein genes and found them to be similar. Despite this similarity, there is a statistically significant decrease in codon bias in the less-well-conserved regions. Our analysis suggests that although those codon changes initially fixed under amino acid replacements tend to follow the overall codon usage pattern, they also reduce the bias in codon usage. This decrease in codon bias leads one to predict that the rate of change of synonymous codons should be greater in those regions that are less well conserved at the amino acid level than in the better-conserved regions. Our analysis supports this prediction. Furthermore, we demonstrate a significantly elevated rate of change of synonymous codons among the adjacent codons 5 to amino acid replacement positions. This provides further support for the idea that there are contextual constraints on the choice of synonymous codons in eukaryotes.     

A first insight into peach [Prunus persica (L.) Batsch] SNP variability

Three factors may have reduced the diversity at both individual gene and whole genome levels in cultivated peach: its self-compatible mating system, the narrow genetic basis of most commercial cultivars, and the recent strong selection towards agronomically interesting traits. Previous diversity analyses with markers such as simple sequence repeats (SSRs) have revealed low levels of genetic variability. Here, we sequenced 23 genome-wide distributed DNA fragments in 47 occidental peach varieties, also observing reduced variability levels. On average, there was one single nucleotide polymorphism (SNP) every 598 bp and one indel every 4,189 bp. As expected, variability was higher in non-coding than in coding regions (one SNP every 390 non-coding bp versus one in 1,850 bp in coding DNA). In general, SNPs were observed at relatively high frequency, mean minor allele frequency?=?0.225, meaning that a large proportion of the SNPs discovered by sequencing similar germplasm will be useful for other purposes, such as association mapping. The average heterozygosity of the varieties was 0.28, with a low correlation between SSR and SNP heterozygosity. The whole sequence of two candidate genes, a pectate lyase 1 candidate for fruit firmness (CGPAA2668) and a sucrose synthase 1 candidate for sugar content (CGPPB6189), in the 47 varieties revealed that they both may have suffered a process of balancing selection.     

Determinants of synonymous and nonsynonymous variability in three species of Drosophila

We estimated the intensity of selection on preferred codons in Drosophila pseudoobscura and D. miranda at X-linked and autosomal loci, using a published data set on sequence variability at 67 loci, by means of an improved method that takes account of demographic effects. We found evidence for stronger selection at X-linked loci, consistent with their higher levels of codon usage bias. The estimates of the strength of selection and mutational bias in favor of unpreferred codons were similar to those found in other species, after taking into account the fact that D. pseudoobscura showed evidence for a recent expansion in population size. We examined correlates of synonymous and nonsynonymous diversity in these species and found no evidence for effects of recurrent selective sweeps on nonsynonymous mutations, which is probably because this set of genes have much higher than average levels of selective constraints. There was evidence for correlated effects of levels of selective constraints on protein sequences and on codon usage, as expected under models of selection for translational accuracy. Our analysis of a published data set on D. melanogaster provided evidence for the effects of selective sweeps of nonsynonymous mutations on linked synonymous diversity, but only in the subset of loci that experienced the highest rates of nonsynonymous substitutions (about one-quarter of the total) and not at more slowly evolving loci. Our correlational analysis of this data set suggested that both selective constraints on protein sequences and recurrent selective sweeps affect the overall level of codon usage.     

沙枣叶绿体全基因组序列及其使用密码子偏性分析

该研究以2株野生沙枣(Elaeagnus angustifolia Linn.)嫩枝经温室水培后的嫩叶为材料,采用CTAB法分别提取总DNA,并利用第二代测序技术进行总DNA从头测序,组装后得到2株沙枣叶绿体基因组全序列,并详细分析了其蛋白质编码基因密码子使用的偏好性及其原因,为沙枣叶绿体基因工程和分子系统进化等研究奠定基础。结果显示:(1)组装得到沙枣叶绿体基因组序列全长150 546 bp,由长度为81 113 bp的长单拷贝(LSC)区域和25 494 bp的短单拷贝(SSC)区域,以及1对分隔开它们的长18 445 bp的反向重复序列(IRS)组成;注释共得到132个基因,包括86个蛋白编码基因、38个tRNA基因和8个rRNA基因。(2)沙枣叶绿体基因组蛋白编码基因密码子的第三位碱基GC含量(GC_3)为28.47%,明显低于整个叶绿体基因组GC含量(37%),也低于第一位(GC_1)和第二位(GC_2)碱基的GC含量,说明密码子对AT碱基结尾有偏好性;其中, UCU、CCU、UGU、GCU、CUU、GAU、UCA和UAA为最优密码子。(3)同义密码子相对使用频率(RSCU)分析发现,影响密码子使用模式的因素并不单一,密码子的偏好性受到突变、选择及其他因素的共同影响,并且自然选择表达引起的序列差异比突变对密码子偏好性的影响要显著;中性绘图分析、有效密码子数(ENC-plot)分析和奇偶偏好性(PR2-plot)分析表明,沙枣叶绿体基因组使用密码子的偏性受选择的影响更大。(4)通过最大似然法、最大简约法和贝叶斯方法对胡颓子科6个物种和1个枣的叶绿体基因序列构建系统发育树,与它们使用密码子偏性聚类的结果一致,表明叶绿体基因组使用密码子偏性与物种的亲缘关系相关。     

Synonymous codon bias and functional constraint on GC3-related DNA backbone dynamics in the prokaryotic nucleoid

While mRNA stability has been demonstrated to control rates of translation, generating both global and local synonymous codon biases in many unicellular organisms, this explanation cannot adequately explain why codon bias strongly tracks neighboring intergene GC content; suggesting that structural dynamics of DNA might also influence codon choice. Because minor groove width is highly governed by 3-base periodicity in GC, the existence of triplet-based codons might imply a functional role for the optimization of local DNA molecular dynamics via GC content at synonymous sites (≈GC3). We confirm a strong association between GC3-related intrinsic DNA flexibility and codon bias across 24 different prokaryotic multiple whole-genome alignments. We develop a novel test of natural selection targeting synonymous sites and demonstrate that GC3-related DNA backbone dynamics have been subject to moderate selective pressure, perhaps contributing to our observation that many genes possess extreme DNA backbone dynamics for their given protein space. This dual function of codons may impose universal functional constraints affecting the evolution of synonymous and non-synonymous sites. We propose that synonymous sites may have evolved as an ‘accessory’ during an early expansion of a primordial genetic code, allowing for multiplexed protein coding and structural dynamic information within the same molecular context.     

Variation in synonymous codon use and DNA polymorphism within the Drosophila genome

A strong negative correlation between the rate of amino-acid substitution and codon usage bias in Drosophila has been attributed to interference between positive selection at nonsynonymous sites and weak selection on codon usage. To further explore this possibility we have investigated polymorphism and divergence at three kinds of sites: synonymous, nonsynonymous and intronic in relation to codon bias in D. melanogaster and D. simulans. We confirmed that protein evolution is one of the main explicative parameters for interlocus codon bias variation (r(2) approximately 40%). However, intron or synonymous diversities, which could have been expected to be good indicators of local interference [here defined as the additional increase of drift due to selection on tightly linked sites, also called 'genetic draft' by Gillespie (2000)] did not covary significantly with codon bias or with protein evolution. Concurrently, levels of polymorphism were reduced in regions of low recombination rates whereas codon bias was not. Finally, while nonsynonymous diversities were very well correlated between species, neither synonymous nor intron diversities observed in D. melanogaster were correlated with those observed in D. simulans. All together, our results suggest that the selective constraint on the protein is a stable component of gene evolution while local interference is not. The pattern of variation in genetic draft along the genome therefore seems to be instable through evolutionary times and should therefore be considered as a minor determinant of codon bias variance. We argue that selective constraints for optimal codon usage are likely to be correlated with selective constraints on the protein, both between codons within a gene, as previously suggested, and also between genes within a genome.     

The Effect of Local Nucleotides on Synonymous Codon Usage in the Honeybee (<Emphasis Type="Italic">Apis mellifera</Emphasis> L.) Genome

Using all currently predicted coding regions in the honeybee genome, a novel form of synonymous codon bias is presented that affects the usage of particular codons dependent on the surrounding nucleotides in the coding region. Nucleotides at the third codon site are correlated, dependent on their weak (adenine [A] or thyamine [T]) versus strong (guanine [G] or cytosine [C]) status, to nucleotides on the first codon site which are dependent on their purine (A/G) versus pyrimidine (C/T) status. In particular, for adjacent third and first site nucleotides, weak–pyrimidine and strong–purine nucleotide combinations occur much more frequently than the underabundant weak–purine and strong–pyrimidine nucleotide combinations. Since a similar effect is also found in the noncoding regions, but is present for all adjacent nucleotides, this coding effect is most likely due to a genome-wide context-dependent mutation error correcting mechanism in combination with selective constraints on adjacent first and second nucleotide pairs within codons. The position-dependent relationship of synonymous codon usage is evidence for a novel form of codon position bias which utilizes the redundancy in the genetic code to minimize the effect of nucleotide mutations within coding regions. [Reviewing Editor: Dr. Brian Morton]