Mitochondrial DNA sequence variations and systematics of the genus Distoechodon (Teleostei : Cyprinidae)

In the present paper, nucleotide sequences (925-929 bases) of the mitochondrial D-loop region and complete cytochrome b gene (1140 bases) were determined and analysed to investigate the systematic status of the genus Distoechodon . CSB1, CSB2, CSB3, CSB-D and ETAS were successfully identified in the D-loop region. The sequence variations among different samples suggest that Distoechodon compressus is a valid species and has its distribution in Taiwan, and that D. tumirostris multispinnis does not seem to be a valid species.

线粒体 DNA 相关疾病的系统发育学研究

线粒体是细胞内提供能量的细胞器，并负责调节细胞的程序化死亡。因遗传 缺陷引起的线粒体功能障碍会导致ATP 合成障碍、能量产生不足而出现一系列 病症。线粒体DNA 相关疾病目前日益受到广泛的关注，然而在线粒体DNA 相 关疾病领域常用的病例对照法容易受到遗传背景、群体分层、数据质量等方面因 素的影响以致经常得到假阳性结果。系统发育分析方法有助于解决这些方面的问 题，因此我们以此分析方法开展了如下工作： 首先我们对线粒体DNA C1494T 突变及其所属单倍型类群和氨基糖苷类药 物性耳聋之间的关联进行了研究。之前有研究报道了两个中国氨基糖苷类药物性 耳聋的家系，经过对先征者的线粒体DNA 全序列测定在这两个家系中都发现了 C1494T 突变。我们采用系统发育方法分析这两个家系先征者的全序列后，发现 这两个个体都属于线粒体单倍型A。巧合的是，在我们之前的研究中，在一个来 自武汉的汉族样本WH6980 中也有C1494T 突变，而且该个体同样也属于单倍型 类群A。这不由得使人想到：C1494T 突变可能是单倍型类群A 中一个分支的界 定位点，或者，受母系遗传背景的影响，该突变偏好于在A 类群中发生。那么 很有可能单倍型类群A对能够引发氨基糖苷类药物性耳聋的C1494T 突变的发生 有促进作用。 为了验证这个假设，我们从三个省份随机选取了553 个正常个体来检测 C1494T 突变，以调查该突变在普通人群中的发生频率。另外，我们从1823 个中 国人样本中筛选出属于单倍型类群A 的111 个体，在这111 个个体中检测C1494T 突变，以调查该突变是否为单倍型类群A 特有的变异位点。我们的结果表明： 在553 个随机样本群中没有检测到C1494T 突变，这说明该突变是一个稀有的突 变，在正常人群中发生的频率极低。另外，在111 个属于单倍型类群A 的样本 群中，我们也没有检测到C1494T 突变的存在，这说明该突变并非单倍型类群A 特有的变异位点。经过对带有C1494T 突变的全序列进行综合的系统发育学分析 表明，这三条序列的C1494T 突变应是来源于同一次突变事件。同时，根据序列 之间共享变异位点的状况推断，两个耳聋家系具有很近的母系亲缘关系，我们推 测这两个家系的C1494T 突变是来源于一位共同的近期母系祖先。综合这些结果 表明，C1494T 突变的发生与单倍型类群即母系遗传背景没有相关性，是属于在人群中频率极低的散发性突变。 我们又将系统发育分析的方法应用于肿瘤相关的线粒体DNA 突变的研究 中。线粒体在细胞的自由基产生以及细胞凋亡中扮演重要角色；有研究报道线粒 体功能的缺陷能导致癌症的发生，同时又有很多报道指出癌变组织的线粒体基因 组存在异常。为探讨癌组织中线粒体基因组的变异情况及其在癌症的发生和发展 中所扮演的角色，我们选取乳腺癌早期患者为研究对象。 近年来，有大量的研究都报导了在癌组织中存在高频率高密度的线粒体 DNA 体细胞突变，并认为这些突变在肿瘤发生过程中可能具有功能相关性。但 这些研究存在着不可回避的问题，总结来讲归为三个方面：①数据质量差：很多 前面报道的突变数据，经系统发育分析，发现存在不少因样本交叉污染所造成的 假阳性突变。②所测片断太短。大多数研究只是检测了D-loop 区，只占了整个 基因组的约十六分之一，很难全面反映问题。③对照设置有问题，许多研究进行 简单的case-control 分析，忽略了不同个体间母系遗传背景的差异，导致大量假 阳性突变的产生。 针对以上问题的存在，我们基于系统发育分析的方法在中国的乳腺癌病人中 开展了一项研究。分别取得10 例乳腺癌早期患者的癌组织、癌旁组织、以及远 端正常组织；提取了总DNA，对每一份组织样品进行线粒体全基因组测序。这 样做有两个研究目的：第一是调查在严格的质量控制手段下，排除交叉污染所造 成的假阳性突变后，在癌症病人中是否仍然能观察到高频率高密度的线粒体 DNA 体细胞突变。第二个目的是调查中国乳腺癌患者线粒体DNA 体细胞突变情 况，为乳腺癌早期诊断提供有效的信息。 为了避免过去部分研究中出现的问题，我们采取了相应的措施。首先是在严 格的质量控制下，我们对同一个病人的三种不同组织，即癌组织，癌旁组织，正 常组织中的线粒体基因组进行全序列测定的方式来检测体细胞突变。同时将所测 得线粒体全基因组序列进行系统发育的分析；结果表明属于同一个病人的不同组 织都能忠实的聚到一支，而且每一条全序列都完整的带有所属单倍型特有的界定 位点，不存在任何样本交叉或者污染的情况；这样进一步确证了我们数据的可靠 性。同时经过这样的比较，可以非常清晰的筛选出在某一种组织类型中所发生的 体细胞突变。来自10 例病人的癌组织，癌旁组织和正常组织的29 条（7#患者远端正常组 织没有得到）线粒体基因组全序列中，我们只检测到了两个体细胞突变（T2275C 和A8601G）。这两个突变经PCR 克隆试验的验证，在远端正常组织中均不存在， 是真实的体细胞突变。突变A8601G 同时出现在3#患者的癌组织和癌旁组织中； 提示线粒体DNA 的体细胞突变可能先于细胞的癌变，这可能对乳腺癌的早期临 床诊断具有一定的意义。突变T2275C 只出现在6#患者的癌组织中，该突变位于 16S rRNA 基因非常保守的位点上，我们对该突变位点潜在的生物学作用作了进 一步的讨论。 在我们研究的病例中体细胞突变的比率要远远低于先前的报道；造成差异的 主要原因可能是因为我们采取了严格的质量控制手段。综合我们的研究结果表 明，先前所报道的乳腺癌组织中的线粒体基因体细胞突变的频率存在被高估的现 象。目前线粒体突变与肿瘤发生的关系还有待于更深入的研究，同时我们呼吁本 领域的研究要加强数据质量的可靠性。我们的研究表明，系统发育的分析方法在 线粒体DNA 相关疾病的研究中是有效的，因此我们推荐在线粒体DNA 相关疾 病的研究中进行推广。

藏鸡和姬鼠属(Apodemus )系统进化中的比较基因组学研究

在过去的十几年时间里，利用不同的分子标记促进了分子生态学和群 体遗传学的较大发展，其中线粒体DNA 和微卫星DNA 分析在分子生态学 研究中得到了最为广泛的应用，多核基因分子标记也越来越受到人们的关 注。本文采用了比较基因组学方法来研究了藏鸡(Tibetan Chickens)和姬鼠 属(Apodemus)的系统进化，对藏鸡的研究主要是为了解决藏鸡的系统进化 地位及其线粒体基因组的进化特征；对姬鼠属的研究目的在于发展建立一 种崭新的研究野生动物系统进化和生物地理的比较基因组学手段。主要结 果描述如下： (1)藏鸡(Tibetan Chickens)线粒体全基因组序列的测序和分析 通过利用PCR 扩增，测序，拼接，获得藏鸡线粒体全基因组序列并进 行数据分析处理。藏鸡线粒体全基因组序列全长16783bp，共有13 个蛋白 质编码基因、2 个rRNA 基因、22 个tRNA 基因和1 个D-loop 区。模拟电 子酶切结果显示，藏鸡DraI 酶的酶切结果和其他家鸡及红原鸡的酶切结果 都相同。基于D-loop 区全序列和13 个蛋白质编码基因序列，采用N-J 算 法与原鸡属4 个种，3 个亚种和3 个家鸡品系构建系统进化树：初步确定 藏鸡起源于红原鸡，与家鸡中的来航鸡、白洛克鸡亲缘关系最近，但是藏 鸡的进化与来航鸡、白洛克鸡这两个家鸡品系又显得相对独立。推测可能 原因是藏鸡的祖先在进入高原以后处于相对封闭的环境，从而形成了独特 群体遗传特性。 (2)姬鼠属(Apodemus)系统进化中的比较基因组学研究 本文中我们利用比较基因组学的研究方法寻找Exon-Primer-Intron- Crossing(EPIC)座位，并在中国四川省姬鼠属3 个种18 个个体中进行检验。 其方法是：通过比较人和小家鼠基因组，选择其中的外显子高度保守的单 拷贝基因，然后在500-1500bp 长度内含子的两端利用外显子序列设计了引 物，再进行PCR 扩增和克隆分析。通过PCR 扩增，我们在102 对引物选 择了6 对引物在18 个姬鼠属个体中进行PCR 产物克隆和测序。通过和 Cyt-b 相比较，在6 个座位当中，有5 个座位构建的系统进化树和Cyt-b 构建的系统进化树的拓扑结构高度一致，其中4 个座位可以很好的区分地 理不同的种群。通过计算核酸多样性，6 个座位的得到的结果都很接近， 说明6 个座位的突变率没有太大的差别。由此可见，我们利用比较基因组学的方法寻找EPIC 座位用于系统发育和群体遗传学的研究是可行的，通 过利用模式物种的基因组信息来研究野生非模式物种的系统发育和群体 遗传学将会提供前所未有的数据量和分辨率。

哀牢髭蟾（Vibrissaphora ailaonica）和红瘰疣螈(Tylototriton verrucosus)的分子系统地理学研究

本研究用线粒体细胞色素b 基因全序列和ND4 基因序列探讨了哀牢髭蟾 （Vibrissaphora ailaonica）分子亲缘地理学和保护遗传学，利用线粒体细胞色素b、 控制区全序列和部分12SrRNA 基因序列研究了分布于中国的红瘰疣螈 （Tylototriton verrucosus）分子亲缘地理学和保护遗传学。 哀牢髭蟾为生活于高海拔生境（大约2000-2600m）的濒危蛙类。各种致危因 素引起了关于对小的、隔离种群长期存活的关注，然而迄今没有关于该物种遗传 多样性的报道。本论文首次研究了分布于中国云南省的哀牢髭蟾的亲缘地理学和 保护遗传学。对采自于9 个种群81 个个体，我们应用线粒体mtDNA cyt b 和 ND4 基因共计1990bp, 获得了51 个单倍型。系统发育分析显示三个分化较深且互为单 系的族群，大致相应于分别被元江和藤条江河谷分开的三个地理单元（元江以东、 金平和藤条江以西地理单元），暗示了长期的地理隔离分化。分子变异等级分析 （AMOVA）显示遗传变异主要为三个地理单元之间（81.68％）和种群内的个体之 间（11.26%）。三个地理单元显著的地理分布暗示在空间和时间上的隔离，这与中 国西南地区在晚中新世到上新世的哀牢山的造山运动和隆升所引起的重要的气候 和古地质变化时间相一致。我们推测低海拔干热河谷可能是三个地理单元之间基 因交流受限的主要生态障碍。根据上述研究结果，我们建议对这三个遗传分化显 著的地理单元，元江以东地区、金平地区、藤条江以西地区作为独立的管理单元 分别加以保护。 本研究通过测定中国横断山区的红瘰疣螈（Tylototriton verrucosus）17个采集 点的123个红瘰疣螈标本的线粒体mtDNA Cyt b、D-loop和12SrRNA三个基因片段 (2347 bp)序列，首次研究了红瘰疣螈在横断山区的群体遗传结构和分子系统地理格 局，并讨论了T. shanjing的物种有效性。基于123个样品定义的49个单倍型的贝叶 斯和NJ系统发育分析表明：(1) T. verrucosus和T. shanjing均未各自构成单系，而是 共同构成一个单系群;(2) 横断山区的红瘰疣螈分为3个线粒体DNA地理单元，即滇 东南、滇中-滇西和片马地理单元，并且地理单元间不存在共享单倍型，说明红瘰 疣螈具有明显的系统地理分布格局。AMOVA分析同样表明3个地理单元之间存在 显著差异，并且分子变异主要发生在3个地理单元间（62.4%）。T. shanjing与T.verrucosus的mtDNA cyt b 序列差异平均值仅为1.1％,明显小于它们与两外群(贵 州疣螈和大凉疣螈)及外群间的遗传距离(6.5-9.9%)。因此，根据T. verrucosus和 T.shanjing的遗传差异以及系统发育分析结果都不支持T. shanjing的物种地位,T. shanjing为T. verrucosus的同物异名，并建议恢复T. verrucosus的中文名红瘰疣螈。 基于上述结果，我们建议将分布于滇东南、滇中－滇西、片马地区的红瘰疣螈作 为三个独立的管理单元分别加以保护。

Probing lanthanide ions binding on tRNA(Phe) by H-1 NMR

The structure of phenylalanine transfer ribonucleic acid (tRNA(Phe)) in solution was explored by H-1 NMR spectroscopy to evaluate the effect of lanthanide ion on the structural and conformational change. It was found that La3+ ions possess specific effects on the imino proton region of the H-1 NMR spectra for yeast tRNA(Phe). The dependence of the imino proton spectra of yeast tRNA(Phe) as a function of La3+ concentration was examined, and the results suggest that the tertiary base pair G(15). C-48, which is located in the terminal in the augmented dihydrouridine helix (D-helix), was markedly affected by La3+ (shifted to downfield by as much as 0.35). Base pair U-8. A(14) in yeast tRNA(Phe), which are stacked on G(15). C-48, was also affected by added La3+ when 1 similar to 2 Mg2+ were also present. Another imino proton that may be affected by La3+ in yeast tRNA(Phe) is that of the tertiary base pair G(19). C-56. The assignment of this resonance in yeast tRNA(Phe) is tentative since it is located in the region of highly overlapping resonances beween 12.6 and 12.2. This base pair helps to anchor the D-loop to the T Psi C loop. The binding of La3+ caused conformational change of tRNA, which is responsible for shifts to upfield or downfield in H-1 NMR spectra.

Effect of lanthanum ions on tRNA(Phe) structure: Imino proton NMR spectroscopy

The effect of lanthanum ions on the structural and conformational change of yeast tRNA(Phe) was studied by H-1 NMR. The results suggest that the tertiary base pair (G-15)(C-48), which was located in the terminal in the augmented dihydrouridine helix (D-helix), was markedly affected by adding La3+ and shifted 0.33 downfield. Based pair (U-8)(A-14), which is associated with a tertiary interaction, links the base of the acceptor stem to the D-stem and anchors the elbow of the L structure, shifted 0.20 upfield. Another imino proton that may be affected by La3+ in tRNA(Phe) is the tertiary base pair (G-19)(C-56). The assignment of this resonance is tentative since it is located in the region of highly overlapping resonances between 12.6 and 12.2. This base pair helps to anchor the D-loop to the T psi C loop.

高原鼢鼠线粒体谱系地理学和遗传多样性

高原鼢鼠是一类地下独居啮齿动物,为青藏高原特有种之一.为研究该物种的谱系地理学和遗传多样性,本文测定了采自青藏高原东部3个地理种群8个小种群共37个个体的线粒体D-loop区序列变异.在长度为627 bp的序列中,共发现50个变异位点,定义了26种单倍型.该物种的单倍型多样性(Haplotype diversity,H)较高和核苷酸多样性(Nucleotide diversity,πn)较低.谱系分析得到3个稳定的分支,分别与采集的地理种群相吻合:同一地理种群内单倍型之间遗传差异小,而不同地理来源的单倍型之间存在较大区别.距离隔离分析表明高原鼢鼠的遗传分化与地理距离呈正相关.AMOVA分析同样表明地理种群之间存在显著差异:地理种群间变异占遗传变异的80.45%.高原鼢鼠的这种遗传结构特点可能主要是由于第四纪气候变迁、该物种稳定的地下生活环境和有限的迁移能力造成的

Origin of mitochondrial DNA diversity of domestic yaks

Background: The domestication of plants and animals was extremely important anthropologically. Previous studies have revealed a general tendency for populations of livestock species to include deeply divergent maternal lineages, indicating that they were domesticated in multiple, independent events from genetically discrete wild populations. However, in water buffalo, there are suggestions that a similar deep maternal bifurcation may have originated from a single population. These hypotheses have rarely been rigorously tested because of a lack of sufficient wild samples. To investigate the origin of the domestic yak (Poephagus grunnies), we analyzed 637 bp of maternal inherited mtDNA from 13 wild yaks (including eight wild yaks from a small population in west Qinghai) and 250 domesticated yaks from major herding regions.Results: The domestic yak populations had two deeply divergent phylogenetic groups with a divergence time of > 100,000 yrs BP. We here show that haplotypes clustering with two deeply divergent maternal lineages in domesticated yaks occur in a single, small, wild population. This finding suggests that all domestic yaks are derived from a single wild gene pool. However, there is no clear correlation of the mtDNA phylogenetic clades and the 10 morphological types of sampled yaks indicating that the latter diversified recently. Relatively high diversity was found in Qinghai and Tibet around the current wild distribution, in accordance with previous suggestions that the earliest domestications occurred in this region. Conventional molecular clock estimation led to an unrealistic early dating of the start of the domestication. However, Bayesian estimation of the coalescence time allowing a relaxation of the mutation rateConclusion: The information gathered here and the previous studies of other animals show that the demographic histories of domestication of livestock species were highly diverse despite the common general feature of deeply divergent maternal lineages. The results further suggest that domestication of local wild prey ungulate animals was a common occurrence during the development of human civilization following the postglacial colonization in different locations of the world, including the high, arid Qinghai-Tibetan Plateau.

7-Amino actinomycin D bound to single-stranded hairpin DNA enhanced by loop sequence-dependent luminescent Eu3+ and Tb3+ binding

Lanthanide Eu3+ and Tb3+ ions have been widely used in luminescent resonance energy transfer (LRET) for bioassays to study metal binding microenvironments. We report here that Eu3+ or Tb3+ can increase the binding affinity of antitumor antibiotic drug agent, 7-amino actinomycin D (7AACTD), binding to 5'-GT/TG-5' or 5'-GA/AG-5' mismatched stem region of the single-stranded hairpin DNA. Further studies indicate that the effect of Eu3+ or Tb3+ on 7AACTD binding is related to DNA loop sequence. Our results will provide new insights into how metal ions can enhance antitumor agents binding to their targets.

A Fast-Locking Phase-Locked Loop Using a Seven-State Phase Frequency Detector

A seven-state phase frequency detector (S.S PFD) is proposed for fast-locking charge pump based phase-locked loops (CPPLLs) in this paper. The locking time of the PLL can be significantly reduced by using the seven-state PFD to inject more current into the loop filter. In this stage, the bandwidth of the PLL is increased or decreased to track the phase difference of the reference signal and the feedback signal. The proposed architecture is realized in a standard 0.35 mu m 2P4M CMOS process with a 3.3V supply voltage. The locking time of the proposed PLL is 1.102 mu s compared with the 2.347 mu s of the PLL based on continuous-time PFD and the 3.298 mu s of the PLL based on the pass-transistor tri-state PFD. There are 53.05% and 66.59% reductions of the locking time. The simulation results and the comparison with other PLLs demonstrate that the proposed seven-state PFD is effective to reduce locking time.

Mud mass transport due to waves based on an empirical rheology model featured by hysteresis loop

A vertical 2-D water-mud numerical model is developed for estimating the rate of mud mass transport under wave action. A nonlinear semi-empirical rheology model featured by remarkable hysteresis loops in the relationships of the shear stress versus both the shear strain and the rate of shear strain of mud is applied to this water mud model. A logarithmic grid in the vertical direction is employed for numerical treatment, which increases the resolution of the flow in the neighborhood of both sides of the interface. Model verifications are given through comparisons between the calculated and the measured mud mass transport velocities as well as wave height changes. (C) 2006 Elsevier Ltd. All rights reserved.

三峡永久船闸高边坡开挖三维离散元数值模拟:3-D Simulation of the Excavation of High Steep Slope of Three-Gorges Permanent Lock by Distinct Element Method

采用面一面接触的三维离散元刚性块体模型,从实测节理面中取出其中的三组,按照其倾向、倾角和节理间距将三峡永久船闸未开挖的区域划分为10~5个离散单元,通过施加力边界条件,给出了与实测初始地应力场接近的数值模拟结果；然后,分4步模拟了永久船闸的开挖过程。计算结果表明：开挖过程会引起节理面出现张开趋势,个别岩体还会沿着节理面滑移。岩体位移的不对称现象较为自然地说明了由节理引起的岩体各向异性特征。

Perturbational Finite Volume Method For The Solution of 2-D Navier-Stokes Equations on Unstructured and Structured Colocated Meshes

Based on the first-order upwind and second-order central type of finite volume( UFV and CFV) scheme, upwind and central type of perturbation finite volume ( UPFV and CPFV) schemes of the Navier-Stokes equations were developed. In PFV method, the mass fluxes of across the cell faces of the control volume (CV) were expanded into power series of the grid spacing and the coefficients of the power series were determined by means of the conservation equation itself. The UPFV and CPFV scheme respectively uses the same nodes and expressions as those of the normal first-order upwind and second-order central scheme, which is apt to programming. The results of numerical experiments about the flow in a lid-driven cavity and the problem of transport of a scalar quantity in a known velocity field show that compared to the first-order UFV and second-order CFV schemes, upwind PFV scheme is higher accuracy and resolution, especially better robustness. The numerical computation to flow in a lid-driven cavity shows that the under-relaxation factor can be arbitrarily selected ranging from 0.3 to 0. 8 and convergence perform excellent with Reynolds number variation from 102 to 104.

High Aspect Ratio (L/D) Riser Viv Prediction Using Wake Oscillator Model

A two-dimensional (2-D) vortex-induced vibration (VIV) prediction model for high aspect ratio (LID) riser subjected to uniform and sheared flow is studied in this paper. The nonlinear structure equations are considered. The near wake dynamics describing the fluctuating nature of vortex shedding is modeled using classical van der Pol equation. A new approach was applied to calibrate the empirical parameters in the wake oscillator model. Compared the predicted results with the experimental data and computational fluid dynamic (CFD) results. Good agreements are observed. It can be concluded that the present model can be used as simple computational tool in predicting some aspects of VIV of long flexible structures. (C) 2008 Elsevier Ltd. All rights reserved.

Stress intensity factor histories of a steadily propagating crack under 3-D combined mode traction

Elastodynamic stress intensity factor histories of an unbounded solid containing a semi-infinite plane crack that propagates at a constant velocity under 3-D time-independent combined mode loading are considered. The fundamental solution, which is the response of point loading, is obtained. Then, stress intensity factor histories of a general loading system are written out in terms of superposition integrals. The methods used here are the Laplace transform methods in conjunction with the Wiener-Hopf technique.