Estimation of the Amount of DNA Polymorphism When the Neutral Mutation Rate Varies among Sites

Knowing the amount of DNA polymorphism is essential to understand the mechanism of maintaining DNA polymorphism in a natural population. The amount of DNA polymorphism can be measured by the average number of nucleotide differences per site (π), the proportion of segregating (polymorphic) site (s) and the minimum number of mutations per site (s*). Since the latter two quantities depend on the sample size, θ is often used as a measure of the amount of DNA polymorphism, where θ = 4Nμ, N is the effective population size and μ is the neutral mutation rate per site per generation. It is known that θ estimated from π, s and s* under the infinite site model can be biased when the mutation rate varies among sites. We have therefore developed new methods for estimating θ under the finite site model. Using computer simulations, it has been shown that the new methods give almost unbiased estimates even when the mutation rate varies among sites substantially. Furthermore, we have also developed new statistics for testing neutrality by modifying Tajima's D statistic. Computer simulations suggest that the new test statistics can be used even when the mutation rate varies among sites.     

The Amount of DNA Polymorphism Maintained in a Finite Population When the Neutral Mutation Rate Varies among Sites

The expectations of the average number of nucleotide differences per site (π), the proportion of segregating site (s), the minimum number of mutations per site (s*) and some other quantities were derived under the finite site models with and without rate variation among sites, where the finite site models include Jukes and Cantor's model, the equal-input model and Kimura's model. As a model of rate variation, the gamma distribution was used. The results indicate that if distribution parameter α is small, the effect of rate variation on these quantities are substantial, so that the estimates of θ based on the infinite site model are substantially underestimated, where θ = 4Nv, N is the effective population size and v is the mutation rate per site per generation. New methods for estimating θ are also presented, which are based on the finite site models with and without rate variation. Using these methods, underestimation can be corrected.     

Statistical Versus Biological Hypothesis Testing: Response to Steidl

ABSTRACT In spite of the wide use and acceptance of information theoretic approaches in the wildlife sciences, debate continues on the correct use and interpretation of Akaike's Information Criterion as compared to frequentist methods. Misunderstandings as to the fundamental nature of such comparisons continue. Here we agree with Steidl's argument about situation-specific use of each approach. However, Steidl did not make clear the distinction between statistical and biological hypotheses. Certainly model selection is not statistical, or null, hypothesis testing; importantly, it represents a more effective means to test among competing biological, or research, hypotheses. Employed correctly, it leads to superior strength of inference and reduces the risk that favorite hypotheses are uncritically accepted.     

Properties of Statistical Tests of Neutrality for DNA Polymorphism Data

A class of statistical tests based on molecular polymorphism data is studied to determine size and power properties. The class includes TAJIMA''s D statistic as well as the D* and F* tests proposed by FU and LI. A new method of constructing critical values for these tests is described. Simulations indicate that TAJIMA''s test is generally most powerful against the alternative hypotheses of selective sweep, population bottleneck, and population subdivision, among tests within this class. However, even TAJIMA''s test can detect a selective sweep or bottleneck only if it has occurred within a specific interval of time in the recent past or population subdivision only when it has persisted for a very long time. For greatest power against the particular alternatives studied here, it is better to sequence more alleles than more sites.     

Statistical Significance of Mutation Frequencies,and the Power of Statistical Testing,Using the Poisson Distribution

The Poisson distribution may be employed to test whether mutation frequencies differ from control frequencies. This paper describes how this testing procedure may be used for either one-tailed or two-tailed hypotheses. It is also shown how the power of the statistical test can be calculated, the power being the probability of correctly concluding the null hypothesis to be false.     

A Simple Method for Testing Monthly Statistical Rates for Normalization

The spectral analysis of birth statistics, expressed in monthly rates, shows a strong periodic component even if a single child is born every day of the year. This is, of course, due to the different number of days per month. Most human or animal statistics as well as agricultural and biometeorological variables are only available as monthly, and not daily rates or values. If spectral components of time series are of interest, it is necessary to normalize monthly rates. This necessity has been a pre-analytical routine for quite some time, and various methods of normalization are described in the literature (1,3,7): the rates are either expressed as the daily mean within a month or as rates of normalized months with 31, 30 or 30.4 days (the latter results in a 365-day year). Each of the different methods leads to the same result, if the analysis can be based on relative deviations from the mean.

In the majority of the statistical material, the chosen method of normalization is clearly stated. But if a study tries to cover many different statistical populations and as many years as possible, the statistical material is often based on several (partly secondary) sources and photocopied extracts from large volumes. This can lead to confusion whether the used statistics contain normalized or crude original monthly rates.     

An Algorithm for Testing the Efficient Market Hypothesis

The objective of this research is to examine the efficiency of EUR/USD market through the application of a trading system. The system uses a genetic algorithm based on technical analysis indicators such as Exponential Moving Average (EMA), Moving Average Convergence Divergence (MACD), Relative Strength Index (RSI) and Filter that gives buying and selling recommendations to investors. The algorithm optimizes the strategies by dynamically searching for parameters that improve profitability in the training period. The best sets of rules are then applied on the testing period. The results show inconsistency in finding a set of trading rules that performs well in both periods. Strategies that achieve very good returns in the training period show difficulty in returning positive results in the testing period, this being consistent with the efficient market hypothesis (EMH).     

Testing the Sulfotransferase Molecular Pore Hypothesis

Human cytosolic sulfotransferases (SULTs) regulate the activities of hundreds of signaling metabolites via transfer of the sulfuryl moiety (-SO₃) from activated sulfate (3′-phosphoadenosine 5′-phosphosulfate) to the hydroxyls and primary amines of xeno- and endobiotics. How SULTs select substrates from the scores of competing ligands present in a cytosolic milieu is an important issue in the field. Selectivity appears to be sterically controlled by a molecular pore that opens and closes in response to nucleotide binding. This point of view is fostered by structures showing nucleotide-dependent pore closure and the fact that nucleotide binding induces an isomerization that restricts access to the acceptor-binding pocket. Molecular dynamics models underscore the importance of pore isomerization in selectivity and predict that specific molecular linkages stabilize the closed pore in response to nucleotide binding. To test the pore model, these linkages were disrupted in SULT2A1 via mutagenesis, and the effects on selectivity were determined. The mutations uncoupled nucleotide binding from selectivity and produced enzymes that no longer discriminated between large and small substrates. The mutations did not affect the affinity or turnover of small substrates but resulted in a 183-fold gain in catalytic efficiently toward large substrates. Models predict that an 11-residue “flap” covering the acceptor-binding pocket can open and admit large substrates when nucleotide is bound; a mutant structure demonstrated that this is so. In summary, the model was shown to be a robust, accurate predictor of SULT structure and selectivity whose general features will likely apply to other members of the SULT family.     

Simple Methods for Testing the Molecular Evolutionary Clock Hypothesis

Simple statistical methods for testing the molecular evolutionary clock hypothesis are developed which can be applied to both nucleotide and amino acid sequences. These methods are based on the chi-square test and are applicable even when the pattern of substitution rates is unknown and/or the substitution rate varies among different sites. Furthermore, some of the methods can be applied even when the outgroup is unknown. Using computer simulations, these methods were compared with the likelihood ratio test and the relative rate test. The results indicate that the powers of the present methods are similar to those of the likelihood ratio test and the relative rate test, in spite of the fact that the latter two tests assume that the pattern of substitution rates follows a certain model and that the substitution rate is the same among different sites, while such assumptions are not necessary to apply the present methods. Therefore, the present methods might be useful.     

DNA Polymorphism Detectable by Restriction Endonucleases

Data on DNA polymorphisms detected by restriction endonucleases are rapidly accumulating. With the aim of analyzing these data, several different measures of nucleon (DNA segment) diversity within and between populations are proposed, and statistical methods for estimating these quantities are developed. These statistical methods are applicable to both nuclear and nonnuclear DNAs. When evolutionary change of nucleons occurs mainly by mutation and genetic drift, all the measures can be expressed in terms of the product of mutation rate per nucleon and effective population size. A method for estimating nucleotide diversity from nucleon diversity is also presented under certain assumptions. It is shown that DNA divergence between two populations can be studied either by the average number of restriction site differences or by the average number of nucleotide differences. In either case, a large number of different restriction enzymes should be used for studying phylogenetic relationships among related organisms, since the effect of stochastic factors on these quantities is very large. The statistical methods developed have been applied to data of Shah and Langley on mitochondrial (mt)DNA from Drosophila melanogaster, simulans and virilis. This application has suggested that the evolutionary change of mtDNA in higher animals occurs mainly by nucleotide substitution rather than by deletion and insertion. The evolutionary distances among the three species have also been estimated.     

外源DAN导入黄瓜引起变异的研究

利用花粉管通道技术,以抗黄瓜霜霉病的“苦瓜”“津四”黄瓜为供体,用“长春密刺”为受体,进行外源DNA直接导入,获得突变体,并得到遗传。     

基于新一代测序技术的mtDNA突变检测方法学的建立

目的:大量研究证实线粒体DNA(mtDNA)突变与肿瘤发生及进展密切相关,但使用传统测序方法难以高通量、高精确度的检测mtDNA突变,为此本研究建立了基于新一代测序技术的mtDNA突变检测方法.方法:提取肝癌患者癌、癌旁组织以及外周血细胞总DNA,利用PCR技术对线粒体基因组进行富集并对PCR产物进行平末端、粘性末端连接或对PCR引物进行氨基修饰,构建mtDNA测序文库.经Illumina HiSeq 2000平台测序后利用生物信息学方法与人类mtDNA参考序列进行比对,并进行测序数据分析.结果:通过对不同质量基因组DNA进行评估后,发现三对引物法适用于大部分DNA样本的mtDNA富集.进一步我们发现PCR引物的氨基修饰可显著提高测序数据覆盖均一性,降低测序成本.结论:本研究利用新一代测序技术通过对线粒体DNA富集方法以及测序覆盖度均一性进行优化,建立了一套灵敏、特异、高通量的mtDNA突变检测策略,为mtDNA突变与疾病研究提供了新方法.     

Statistical Methods for Analyzing Drosophila Germline Mutation Rates

Most studies of mutation rates implicitly assume that they remain constant throughout development of the germline. However, researchers recently used a novel statistical framework to reveal that mutation rates differ dramatically during sperm development in Drosophila melanogaster. Here a general framework is described for the inference of germline mutation patterns, generated from either mutation screening experiments or DNA sequence polymorphism data, that enables analysis of more than two mutations per family. The inference is made more rigorous and flexible by providing a better approximation of the probabilities of patterns of mutations and an improved coalescent algorithm within a single host with realistic assumptions. The properties of the inference framework, both the estimation and the hypothesis testing, were investigated by simulation. The refined inference framework is shown to provide (1) nearly unbiased maximum-likelihood estimates of mutation rates and (2) robust hypothesis testing using the standard asymptotic distribution of the likelihood-ratio tests. It is readily applicable to data sets in which multiple mutations in the same family are common.     

Some Statistical Improvements for Estimating Population Size and Mutation Rate from Segregating Sites in DNA Sequences

In population genetics, under a neutral Wright-Fisher model, the scaling parameter straight theta=4Nmu represents twice the average number of new mutants per generation. The effective population size is N and mu is the mutation rate per sequence per generation. Watterson proposed a consistent estimator of this parameter based on the number of segregating sites in a sample of nucleotide sequences. We study the distribution of the Watterson estimator. Enlarging the size of the sample, we asymptotically set a Central Limit Theorem for the Watterson estimator. This exhibits asymptotic normality with a slow rate of convergence. We then prove the asymptotic efficiency of this estimator. In the second part, we illustrate the slow rate of convergence found in the Central Limit Theorem. To this end, by studying the confidence intervals, we show that the asymptotic Gaussian distribution is not a good approximation for the Watterson estimator.     

等位基因多态性群体遗传结构的多元非线性分析方法

长期以来,对于多维基因多态性数据的多元统计分析,如计算遗传距离时昕用的聚类分析、分析群体遗传结构时所用的主成分分析、因子分析和典型相关分析等,一直应用为无约束条件数据而设计的经典多元线性分析方法,并没有注意基因多态性数据的“闭合效应”所带来的问题。从分析基因多态性数据的分布和结构特征入手,文中指出了基因多态性分布具有“闭合数据”的特点,分析了由于“闭合效应”的影响,经典多元线性方法用于群体遗传结构分析昕面临的困难。根据成分数据统计分析的理论和方法,提出了基因多态性群体遗传结构的多元非线性分析基本方法。并以主成分分析为例,通过实例比较和分析了经典线性主成分分析和“对数比”非线性主成分分析的结果,证明“对数比”非线性主成分分析方法是研究基因多态性群体遗传结构的良好方法,具有特异、灵敏等优点,其结果符合群体遗传学规律。     

A Retrospective Approach to Testing the DNA Barcoding Method

A decade ago, DNA barcoding was proposed as a standardised method for identifying existing species and speeding the discovery of new species. Yet, despite its numerous successes across a range of taxa, its frequent failures have brought into question its accuracy as a short-cut taxonomic method. We use a retrospective approach, applying the method to the classification of New Zealand skinks as it stood in 1977 (primarily based upon morphological characters), and compare it to the current taxonomy reached using both morphological and molecular approaches. For the 1977 dataset, DNA barcoding had moderate-high success in identifying specimens (78-98%), and correctly flagging specimens that have since been confirmed as distinct taxa (77-100%). But most matching methods failed to detect the species complexes that were present in 1977. For the current dataset, there was moderate-high success in identifying specimens (53-99%). For both datasets, the capacity to discover new species was dependent on the methodological approach used. Species delimitation in New Zealand skinks was hindered by the absence of either a local or global barcoding gap, a result of recent speciation events and hybridisation. Whilst DNA barcoding is potentially useful for specimen identification and species discovery in New Zealand skinks, its error rate could hinder the progress of documenting biodiversity in this group. We suggest that integrated taxonomic approaches are more effective at discovering and describing biodiversity.     

Testing the Balanced Electrostatic Interaction Hypothesis of Hepatitis B Virus DNA Synthesis by Using an In Vivo Charge Rebalance Approach

Previously, a charge balance hypothesis was proposed to explain hepatitis B virus (HBV) capsid stability, assembly, RNA encapsidation, and DNA replication. This hypothesis emphasized the importance of a balanced electrostatic interaction between the positive charge from the arginine-rich domain (ARD) of the core protein (HBc) and the negative charge from the encapsidated nucleic acid. It remains unclear if any of the negative charge involved in this electrostatic interaction could come from the HBc protein per se, in addition to the encapsidated nucleic acid. HBc ARD IV mutant 173GG and ARD II mutant 173RR/R157A/R158A are arginine deficient and replication defective. Not surprisingly, the replication defect of ARD IV mutant 173GG can be rescued by restoring positively charged amino acids at the adjacent positions 174 and 175. However, most interestingly, it can be at least partially rescued by reducing negatively charged residues in the assembly domain, such as by glutamic acid-to-alanine (E-to-A) substitutions at position 46 or 117 and to a much lesser extent at position 113. Similar results were obtained for ARD II mutant 173RR/R157A/R158A. These amino acids are located on the inner surfaces of HBc icosahedral particles, and their acidic side chains point toward the capsid interior. For HBV DNA synthesis, the relative amount of positive versus negative charge in the electrostatic interactions is more important than the absolute amount of positive or negative charge. These results support the concept that balanced electrostatic interaction is important during the viral life cycle.Human hepatitis B virus (HBV) is an important human pathogen (5, 27, 34) that can replicate via an RNA intermediate (31, 33). Wild-type (WT) HBV core protein (HBc) is 183 amino acids long (adr and ayw subtypes) and consists of two distinct domains connected by a hinge region. The assembly domain spans amino acids 1 to 140, and the arginine-rich domain (ARD) spans amino acids 150 to 183. The ARD of HBc 150-183 is not required for capsid assembly in Escherichia coli (4, 8, 10, 21, 35). During nucleocapsid (capsid) formation, the HBc protein assembles into an icosahedral particle via a dimer intermediate (32). The ARD of HBc is known to be capable of binding to nucleic acids (12, 25). Serine phosphorylation at the C terminus of HBc is known to be important for RNA encapsidation, DNA synthesis, and virion secretion (2, 11, 14, 15, 17, 19, 24, 26, 39, 40). To date, there is no structural information available for the C terminus of HBc in capsids (32). The 4-helix bundle structure of HBc capsids is based on a C-terminally truncated capsid protein, HBc149 (6, 7, 36). Our research progress in the study of HBV biology has been hampered due to the lack of structural information about the HBc C-terminal tail, which plays an important regulatory role throughout the life cycle of HBV.Recently, we proposed a hypothesis that “charge balance” could be important for HBV capsid stability, assembly, RNA encapsidation, and DNA replication (17). This hypothesis postulates that many important viral activities could be influenced by the electrostatic interaction between the positive charge of the basic amino acid-rich domains of a nucleocapsid protein and the negative charge of the nucleocapsid-associated nucleic acids (17). Previously, we and others demonstrated that a mutant HBc 164, which lacks a total of eight arginine residues at the C terminus, can package both the nonspliced 3.5-kb pregenomic RNA (pgRNA) and the 2.2-kb spliced RNA (14, 17). However, pgRNA encapsidated by HBc 164 is RNase sensitive, while the encapsidated 2.2-kb spliced RNA is RNase resistant. The pgRNA and spliced RNA encapsidated by the full-length wild-type HBc 183 is also RNase resistant (14, 17). This result provided us with the first clue to invoke the previously coined “charge balance hypothesis” of RNA encapsidation and capsid stability (17). In subsequent studies, we added arginines back to the truncated HBc 164 gradually and noted that core particle-associated viral DNA also increased gradually in both size and intensity. This result provided us with the second clue to expand the previous hypothesis from RNA encapsidation to include viral DNA synthesis (17). Instead of totally nonquantitative or nonspecific binding between nucleic acids and a basic protein, our electrostatic-interaction hypothesis entails a stoichiometry-like concept between basic residues and their binding partner of nucleic acids. One of the reasons for such a demand for a more quantitative charge-charge interaction could be related to the putative intersubunit positive-charge repulsion built into the context of an icosahedral particle (22).We and others demonstrated previously that truncated HBc 173 (also denoted HBc 173RR in Fig. ​Fig.1)1) is sufficient for a WT-like DNA phenotype (17, 20). Mutant 173GG, containing two substitutions from arginine (R) to glycine (G) at codons 172 and 173 of HBc 173, exhibited a shorter-than-full-length HBV DNA phenotype, suggesting the importance of sufficient positive charge for viral DNA synthesis (17).Open in a separate windowFIG. 1.Arginine-deficient HBc mutants SVC173GG and SVC173/R157A/R158A exhibited a short DNA phenotype by complementation and Southern blot analysis. (A) To test the balanced electrostatic-interaction hypothesis, we engineered various HBc mutants with different arginine contents. The experimental approach is illustrated in the cartoon. (B) Amino acid sequence comparisons among the WT and HBc mutants. The hyphens represent amino acid sequences identical to that of the parental WT HBc. Roman numbers I to IV indicate the four different ARDs at the C terminus of HBc. The names SVC 173RR and SVC173 are used interchangeably in this paper. ARD IV mutant 173GG contains two arginine-to-glycine substitutions at positions 172 and 173, while ARD II mutant SVC173/R157A/R158A contains two arginine-to-alanine substitutions at positions 157 and 158. To further test the balanced electrostatic-interaction hypothesis, we restored arginines at the new positions 174 and 175 in mutant 175GGRR. (C, top) Plasmid 1903, an HBV genomic dimer containing an ablated core AUG initiation codon, was cotransfected with WT or various mutant core expression plasmids into Huh7 cells. HBV core-associated DNAs were analyzed by Southern blot analysis. The positive control mutant SVC173RR was WT-like in viral DNA synthesis (17, 20). More mature RC DNA of HBV was almost undetectable in mutants 173GG and 173/R157A/R158A, even after very long exposure to X-ray film. The replication defect of mutant 173GG could be rescued in mutant 175GGRR. The asterisk highlights the defect in synthesizing full-length viral DNA. In contrast, the black dots in lane SVC175GGRR highlight the functional rescue of full-length HBV DNA synthesis. SS, ssDNA replicative intermediate. (Bottom) Capsids collected from transfected cell lysates were measured by Western blot analysis using rabbit anti-core antibody.To test further the effect of electrostatic interaction on viral DNA synthesis, we asked if one could restore the DNA replication defect of the arginine-deficient ARD IV mutant 173GG or ARD II mutant 173RR/R157A/R158A by reducing their negative charges at specific positions. Indeed, our results showed that a single substitution from glutamic acid to alanine (E-to-A) at position 46 or 117, and to a lesser extent at position 113, could restore the WT-like DNA replication in the context of ARD IV mutant 173GG. Similarly, in the context of ARD II mutant 173RR/R157A/R158A, both E46A and E117A could rescue the full-length single-stranded DNA (ssDNA) and relaxed-circle (RC) DNA syntheses.Using an in vitro capsid assembly/disassembly assay, we demonstrated that the capsid stability of both wild-type full-length HBc 183 and truncated HBc 173 capsid particles from E. coli are dependent on the presence of encapsidated RNA (22). Upon treatment with micrococcal nuclease, encapsidated RNAs were digested and lost, leading to capsid disassembly. In this study, we demonstrated that mutant 173GG maintained capsid stability upon the loss of encapsidated RNA, probably due to the loss of intersubunit positive charge repulsion at ARD IV. Interestingly, when mutation E117A was introduced into the context of mutant 173GG, the capsid stability of mutant capsids E117A/173GG was once again dependent on the encapsidated RNA in a manner similar to that of its parental mutant, 173. Further studies also revealed that the truncated HBc mutant 172, like mutant 173, is sufficient for HBV DNA replication. Taken together, these results lend strong support to the balanced electrostatic-interaction hypothesis of HBV DNA replication.