线粒体ND4-ND4L基因在黑腹果蝇种组中的进化特征

本实验对黑腹果蝇种组(melanogaster species group)中8个种亚组33个样品两个线粒体基因ND4和ND4L进行了测序,并分析了ND4基因的序列差异和碱基替换特点,发现近缘物种中存在很明显的转换倾向,而在远缘物种中由于重复替换导致转换数处于饱和状态,我们的实验数据证实了线粒体基因较核基因有较快的进化速度。最后根据D．melanogaster与D．yakuba的遗传距离推算了8个种亚组的分化时间,ananassae种亚组最先分化,然后依次是montium,melanogaster,ficsphila,eugracilis,elegans,suzukii和takahashii最后分化。     

以ND4L和ND4基因为标记探讨黑腹果蝇种组的系统发育关系

多年来的形态学、染色体组学以及DNA序列几个方面的研究均没有很好地阐明黑腹果蝇种组内的系统发育关系。本实验测定了33个样品的ND4和31个样品的ND4L基因序列,以D．obscuroides为外群,用最大简约法和Bayesian法分别构建进化树。结果表明两种方法构建的拓扑结构一致,而且大部分支系的支持率较高。整个黑腹果蝇种组分成三大谱系：1）montium种亚组;2）ananssae种亚组;3）Oriental种亚组（melanogaster、ficsphila、eugracilis、elegans、suzukii、takahashii）。montium是最早分化的种亚组。在第三谱系中,melanogaster分化得最早;然后依次是ficsphila,eugracilis,elegans;suzukii与takahashii为姐妹种亚组,最后分化。     

基于ND4和ND5基因序列分析的鳅超科鱼类系统发育关系

ND4和ND5是线粒体基因组中编码NADH脱氢酶亚基4和亚基5的两个蛋白质编码基因.该研究以鳅超科鱼类为研究对象,新测定了10个物种的ND4和ND5基因全序列以及中间的3个tRNA基因共212 bp的序列,结合从GenBank 下载的15个物种的15条序列进行序列比较和系统发育关系分析.结果显示:鳅超科鱼类ND4基因全长1380～1387 bp,以ATG为起始密码子,终止密码子为不完全终止信号;ND5基因全长1821～1839bp,同样起始密码子为ATG,终止密码子为TAA或TAG;ND4和ND5基因之间插入了3个tRNA基因,分别编码携带组氨酸、丝氨酸、亮氨酸的tRNA.ND4和ND5基因(包含3个tRNA基因)中A、T、G、C的平均含量分别为30.4%、27.3%、14.2%、28.1%,A+T(57.7%)的含量高于G+C(42.3%)的含量.转换与颠换比(Ti/Tv)平均值为1.586.选取斑马鱼和鲤鱼作为外类群,采用最大简约法(MP)、最大似然法(ML)和贝叶斯推断法(BI)进行系统发育树的重建.三种方法的系统发育分析结果都显示:花鳅亚科、条鳅亚科、沙鳅亚科、平鳍鳅科及Vaillantellidae分别构成单系;它们的系统发育关系为:(Vaillantellidae+(沙鳅亚科+(花鳅亚科+(条鳅亚科+平鳍鳅科).这与线粒体全基因组和某些核基因(如RAG1基因)的研究结果类似,且支持率较高,表明ND4和ND5基因用于鳅超科鱼类的系统发育分析是可行的;但是该研究的结果有别于其他线粒体基因的分析结果,如基于cytb和D-loop基因进行的系统发育分析表明,条鳅亚科和花鳅亚科聚为姐妹群,再和平鳍鳅科聚在一起.这种差异可能是由于使用的基因长度差异造成的,长度越长,信息量越大,所反映的系统发育结果可能更加接近真实情况.     

象甲科线粒体基因组特征及系统发育分析

昆虫线粒体基因组广泛应用于系统发育关系的重新建立、分子进化、谱系地理学及物种诊断等领域。为揭示象甲科昆虫线粒体全基因组序列的主要结构特征,探究其系统发育相关信息,为进化遗传学研究和分子标记选取等提供参考依据,本研究利用比较基因组学和生物信息学方法,对NCBI上已公布的35种象甲科物种线粒体全基因组序列进行了分析。结果显示:(1)象甲科tRNA基因存在排序及数目异常情况,不同物种中蛋白质编码基因和2种rRNAs排列相同,线粒体全基因组具有明显AT偏向;(2)COX1、ATP6、ND5、ND4、ND4L和ND1基因除标准三联密码子外,还存在特殊的起始密码子AAT、TTG和GTG;(3)13种蛋白质编码基因的进化速率顺序为COX3ATP8ND2ND5ND1ND4ND6ND4LND3ATP6CytbCOX1COX2;(4)13个蛋白编码基因和rRNAs基因中,ND5、rrnL、ND4和ND2基因变异位点数较高,可作为备选的分子标记;(5)各亚科的系统发育关系可能为(((小蠹亚科Scolytinae+长小蠹亚科Platypodinae)+(隐喙象亚科Cryptorhynchinae+魔喙象亚科Molytinae+象虫亚科Curculioninae)+((孢喙象亚科Cyclominae+粗喙象亚科Entiminae)+(隐颏象亚科Dryophthorinae+长小蠹亚科))),为象甲科的系统发育分析有提供参考。     

中国狼（Canis lupus chanco）线粒体全基因组序列分析

参照近缘物种线粒体全基因组序列,设计14 对特异引物,采用PCR产物直接测序法测得中国狼线粒体基因组全序列,并分析其基因组特点和各基因的定位.用pDRAW32软件,预测12种限制性酶的酶切图谱.结果表明,中国狼线粒体基因组全长16 774 bp, 包括13 个蛋白质编码基因、2 个rRNA 基因、22 个tRNA 基因和1个非编码控制区.除ND2、 ND3和ND5 基因以ATA作为起始密码子外,其它基因的起始密码子均为ATG.除COXⅢ、 ND4、 ND3基因的终止密码子分别为不完全的T,T,TA;ND2,COXⅡ,Cytb基因的终止密码子分别是TAG、TAG、AGA外;其余基因均以TAA作为终止密码子,而欧亚狼和狗COXⅡ基因则以TAA终止.基于近缘哺乳动物15种近缘物种的线粒体基因组的12S rRNA 和 16S rRNA 基因全序列,用邻近法、最大简约法和最大似然法构建系统进化树,系统进化关系与传统的系统分类基本一致.并在已有文献的基础上,探讨了中国狼的进化地位.     

线粒体假基因用作分子化石推算两个榕小蜂姐妹种的分化时间（英文）

线粒体假基因(nuclear mitochondrial pseudogenes, NUMTs)是指由生物体的线粒体基因组转移至核基因组内的DNA片段。由于其独立进化的特点, NUMTs在用于系统发育分析时是一把双刃剑。我们用基于PCR扩增的方法研究了对叶榕Ficus hispida上两姐妹种榕小蜂Philotrypesis pilosa和Philotrypesis sp.中起源于线粒体Nad1 12S片段的NUMTs。该两姐妹种榕小蜂由同域物种形成过程产生, 它们生活在同一生态环境里（即同一榕果内）, 因此可以用作很好的模型来研究在相同生态环境里物种的行为学及遗传学细微差异的进化。这些深入研究都依赖于对两个物种分化时间的正确估算。通过对所获取的NUMTs进行进化分析, 我们发现： 1）这些NUMTs都是最近引入核基因组事件; 2）NUMTs引入事件发生在物种分化之前。由于这些NUMTs引入核内时间尚短, 其碱基替换速率与线粒体基因相似, 而节肢动物线粒体基因的平均碱基替换速率约为2.3×10^-8 替换/位点·年。根据这些进化历史特征可帮助我们将这两个姐妹种榕小蜂的分化时间追溯至0.40-0.48百万年以前。结果提示, 一些线粒体假基因可以很好的用作分子化石来推断一些重要进化事件如物种形成。     

吸虫类线粒体基因组的特征及系统发生分析

本研究分析了吸虫纲下单殖亚纲的13个物种和复殖亚纲的37个物种,共50个物种的线粒体全基因组序列,旨在揭示吸虫类线粒体基因组的主要基本特征,探究吸虫纲物种的系统发生关系。吸虫类的线粒体基因组具有明显的A+T含量偏向性。吸虫类的12个蛋白质编码基因和两个核糖体RNA基因的变异程度很大,其变异比例在48.8%到92.0%之间。根据线粒体基因组的12个蛋白质编码基因和两个核糖体RNA基因的差异位点分析,建议采用ND5、CYTB和ND4基因作为16S、12S和COⅠ基因的辅助分子标记。吸虫类各物种的线粒体基因组出现了大量的基因重排现象,除鸮形目裂体科和多后盘目外,其余物种均遵循类似的基因排列顺序。线粒体基因组的系统发育结果强烈支持将单殖亚纲提升到单殖纲,并支持将目前分类地位未定的腹盘科野牛平腹吸虫归入斜睾目同盘总科。本研究将为吸虫类线粒体基因组的研究、分子进化和分子诊断等方面提供诸多信息,并在今后的实践应用中起到一定的指导作用。     

孟加拉笛鲷线粒体基因组序列结构及其进化

采用Long-PCR扩增线粒体全基因组方法得到了孟加拉笛鲷线粒体基因组全序列.序列分析结果表明,孟加拉笛鲷线粒体基因组序列全长16 511 bp,共有13个编码蛋白质基因、22个tRNA基因、2个rRNA基因和1个D-loop区.在编码蛋白质基因中,除COⅠ是以GTG作为起始密码子外,其它均是以ATG起始,NDⅠ、COⅡ、ND3以TAG作为终止密码子,而ND4、Cyt b则以不完全的T为终止密码子,其余8个蛋白质基因的终止密码子均为TAA.孟加拉笛鲷线粒体基因组各基因长度、位置与典型的脊椎动物相似,其编码蛋白质基因和rRNA基因与其它硬骨鱼类具有很高的同源性.基于14种笛鲷线粒体区段COⅠ、COⅡ和Cyt b基因的全序列合并成的一个组合数据集构建系统进化树,显示孟加拉笛鲷与四带笛鲷关系最为密切.     

基于线粒体基因组的鼠兔高海拔适应机制探讨

鼠兔隶属于兔型目,大多生活在海拔较高的地区,是研究哺乳动物对高寒缺氧环境适应机制的理想模型。线粒体通过氧化磷酸化产生能量,在机体的氧气利用和能量代谢中发挥重要作用。本研究测定了柯氏鼠兔线粒体全基因,然后分析了5种分布于较高海拔的鼠兔与6种较低海拔的兔科物种的线粒体基因组,并探讨了鼠兔高海拔适应时线粒体基因的作用。研究表明相比于较低海拔物种,鼠兔积累了更多的非同义突变,并且在鼠兔的线粒体编码基因的NADH2、NADH4L、NADH4、NADH5、NADH6、Cytb上均检测到正选择信号。ND2、ND4和ND5亚基的正选择位点位于非跨膜区,鼠兔的ND4L、ND6亚基的正选择位点位于TM区,但并不在与质子转运相关的关键区域。NADH脱氢酶亚基可能通过氨基酸变异干扰了质子转运过程的有效性。Cytb上的正选择信号位于跨膜螺旋H,氨基酸的变异可能影响到泛醇的结合与转移,进而改变了能量代谢。     

中国特有果蝇 Drosophila curviceps 种亚组在Drosophila immigrans种组中的种系发生地位

果蝇immigrans种组中的curviceps种亚组是1992年新建立的中国特有果蝇类群。该种亚组中的物种主要分布在中国大陆和台湾。目前除了形态学水平的研究外,还没有其他证据支持建立该种亚组的合理性及其起源和种系发生地位。为了在DNA分子水平上探讨果蝇curviceps种亚组在果蝇immigrans种组中的种系发生地位,从而为今后更深入地研究中国特有果蝇,甚至为果蝇亚属的进化遗传学提供理论依据,测定了immigrans种组5个种亚组（nasuta、immigrans、hypocausta、quadrilineata、curviceps)中12个代表物种的rDNA的ITS1和部分Adh基因的序列。其中ITS1序列的长度为513－587bp,共有191个信息位点;Adh基因片段的长度在714－747bp之间,共99个信息位点。考虑到单个分子提供的信息较少,将两个分子的序列综合起来,组成一个较长的复合序列。分别根据ITS1,Adh和两个分子的复合序列排比（Alignment)结果,和最大简约法和邻接法构建分子系统树,其中根据复合序列构建的系统树与形态学研究结果最为一致。分子树显示curviceps种亚组的特种确定单独形成一个分枝,为种亚组级的分类阶元,支持了形态学将其建立为一个新种亚组。根据Kimura距离,估算了复合分子的替换速率约为每百万年1．48％,进而计算出5个种亚组的分 歧年代。结合各物种的地理分布,推测了immigrans种组的进化历史：curviceps种亚组与quadrilineata种亚组的亲缘关系最近,主要分布在中国南部的温带地区。它们之间的分歧时间大约为3．4百万年,是最年轻的两个种亚组。主要分布在苏门答腊及附近的热带地区的hypocausta种亚组的物种是最早分化出来的,与其他种亚组的分歧时间约为9．2百万年。该结果与形态学和生物地理学研究相吻合。值得一提是的,目前归属仍存在争议的物种D.neohypocausta,在分子系统树中与hypocausta种亚组的物种相距较远,而与immiagrasn种亚组的关系较近,但分枝置信度较低（＜50％）。由于还缺乏其他方面的证据,因此D.neohypocausta的归属有待今后的研究来作定论。     

Catabolism of 4-Hydroxyacids and 4-Hydroxynonenal via 4-Hydroxy-4-phosphoacyl-CoAs

4-Hydroxyacids are products of ubiquitously occurring lipid peroxidation (C₉, C₆) or drugs of abuse (C₄, C₅). We investigated the catabolism of these compounds using a combination of metabolomics and mass isotopomer analysis. Livers were perfused with various concentrations of unlabeled and labeled saturated 4-hydroxyacids (C₄ to C₁₁) or 4-hydroxynonenal. All the compounds tested form a new class of acyl-CoA esters, 4-hydroxy-4-phosphoacyl-CoAs, characterized by liquid chromatography-tandem mass spectrometry, accurate mass spectrometry, and ³¹P-NMR. All 4-hydroxyacids with five or more carbons are metabolized by two new pathways. The first and major pathway, which involves 4-hydroxy-4-phosphoacyl-CoAs, leads in six steps to the isomerization of 4-hydroxyacyl-CoA to 3-hydroxyacyl-CoAs. The latter are intermediates of physiological β-oxidation. The second and minor pathway involves a sequence of β-oxidation, α-oxidation, and β-oxidation steps. In mice deficient in succinic semialdehyde dehydrogenase, high plasma concentrations of 4-hydroxybutyrate result in high concentrations of 4-hydroxy-4-phospho-butyryl-CoA in brain and liver. The high concentration of 4-hydroxy-4-phospho-butyryl-CoA may be related to the cerebral dysfunction of subjects ingesting 4-hydroxybutyrate and to the mental retardation of patients with 4-hydroxybutyric aciduria. Our data illustrate the potential of the combination of metabolomics and mass isotopomer analysis for pathway discovery.     

Leukotriene B4, C4, D4 and E4 inactivation by hydroxyl radicals

Leukotriene B4 chemotactic activity and leukotriene C4, D4 and E4 slow reacting substance activity were rapidly decreased by hydroxyl radicals generated by two different iron-supplemented acetaldehyde-xanthine oxidase systems. At low Fe2+, leukotriene inactivation was inhibited by catalase, superoxide dismutase, mannitol and ethanol, suggesting involvement of hydroxyl radicals generated by the iron-catalyzed interaction of superoxide and H2O2 (Haber-Weiss reaction). Leukotriene inactivation increased at high Fe2+ concentrations, but was no longer inhibitable by superoxide dismutase, suggesting that inactivation resulted from a direct interaction between H2O2 and Fe2+ to form hydroxyl radicals (Fenton reaction). The inactivation of leukotrienes by hydroxyl radicals suggests that oxygen metabolites generated by phagocytes may play a role in modulating leukotriene activity.     

Glutathione transferase A4-4 resists adduction by 4-hydroxynonenal

4-Hydroxy-2-trans-nonenal (HNE) is a lipid peroxidation product that contributes to the pathophysiology of several diseases with components of oxidative stress. The electrophilic nature of HNE results in covalent adduct formation with proteins, fatty acids and DNA. However, it remains unclear whether enzymes that metabolize HNE avoid inactivation by it. Glutathione transferase A4-4 (GST A4-4) plays a significant role in the elimination of HNE by conjugating it with glutathione (GSH), with catalytic activity toward HNE that is dramatically higher than the homologous GST A1-1 or distantly related GSTs. To determine whether enzymes that metabolize HNE resist its covalent adduction, the rates of adduction of these GST isoforms were compared and the functional effects of adduction on catalytic properties were determined. Although GST A4-4 and GST A1-1 have striking structural similarity, GST A4-4 was insensitive to adduction by HNE under conditions that yield modest adduction of GST A1-1 and extensive adduction of GST P1-1. Furthermore, adduction of GST P1-1 by HNE eliminated its activity toward the substrates 1-chloro-2,4-dinitrobenzene (CDNB) and toward HNE itself. HNE effects on GST A4-4 and A1-1 were less significant. The results indicate that enzymes that metabolize HNE may have evolved structurally to resist covalent adduction by it.     

A short path to synthesis of 4-deoxy-4-fluoroglucosaminides: methylumbelliferyl N-acetyl-4-deoxy-4-fluoro-beta-D-glucosaminide

A convenient method of synthesis of 1,6-anhydro-4-deoxy-2-O-tosyl-4-fluoro-beta-D-glucopyranose by fusion of 1,6;3,4-dianhydro-2-O-tosyl-beta-D-galactopyranose with 2,4,6-trimethylpyridinium fluoride was found. By successive action of ammonia, methyl trifluoroacetate, and acetic anhydride, the resulting compound was transformed into 1,6-anhydro-3-O-acetyl-2,4-dideoxy-2-trifluoroacetamido-4-fluoro-beta-D-glucopyranose, which was converted into 3,6-di-O-acetyl-2,4-dideoxy-2-trifluoroacetamido-4-fluoro-alpha-D-glucopyranosyl fluoride by the reaction with HF/Py. The resulting fluoride was further used as a glycosyl donor in the synthesis of methylumbelliferyl N-acetyl-4-deoxy-4-fluoro-beta-D-glucosaminide.     

Hydration pattern of A4T4 and T4A4 DNA: a molecular dynamics study

Hydration pattern and energetics of 'A-tract' containing duplexes have been studied using molecular dynamics on 12-mer self-complementary sequences 5'-d(GCA4T4GC)-3' and 5'-d(CGT4A4CG)-3'. The structural features for the simulated duplexes showed correlation with the corresponding experimental structures. Analysis of the hydration pattern confirmed that water network around the simulated duplexes is more conformation specific rather than sequence specific. The calculated heat capacity change upon duplex formation showed that the process is entropically driven for both the sequences. Furthermore, the theoretical free energy estimates calculated using MMPBSA approach showed a higher net electrostatic contribution for A4T4 duplex formation than for T4A4, however, energetically both the duplexes are observed to be equally stable.     

Human C4 haplotypes with duplicated C4A or C4B

In the course of study of families for the sixth chromosome markers HLA-A, C, B, D/DR, BF, and C2, the two loci for C4, C4A, and C4B, and glyoxalase I, we encountered five examples of probable duplication of one or the other of the two loci for C4. In one of these, both parents and one sib expressed two different structural genes for C4B, one sib expressed one, and one sib expressed none, suggesting that two C4B alleles were carried on a single haplotype: HLA-A2, B7, DR3, BFS1, C2C, C4A2, C4B1, C4B2, GLO1. In a second case, two siblings inherited C4B*1 and C4B*2 from one parent and C4B*Q0 from the other. This duplication appeared on the chromosome as HLA-AW33, B14, DR1, BFS, C2C, C4A2, C4B1, C4B2, GLO2. In a third, very large family with 3 generations, a duplication of the C4B locus occurred which was followed in 2 generations. In one individual, there were three C4B alleles and two C4A alleles. One of the C4B alleles had a hemolytically active product with electrophoretic mobility near C4B2 and was designated C4B*22. It segregated with C4B1 in the family studied. The complete haplotype was HLA-A11, CW1, BW56, DR5, BFS, C2C, C4A3, C4B22, C4B1, GLO2. In another family with 12 siblings, one parent and eight children expressed two C4A alleles on the haplotype HLA-AW30, BW38, DR1, BFF, C2C, C4A3, C4A2, C4BQ0, GLO1.(ABSTRACT TRUNCATED AT 250 WORDS)     

Genetics of complement C4. Two homoduplication haplotypes C4S C4S and C4F C4F in a family

Summary A family in which two homoduplicated C4 haplotypes (or supergenes) segregate is described. One haplotype C4F ^* 3 C4F ^*2.2 is composed of two C4F alleles and the other C4S ^* 5.1 C4S ^*1 of two C4S alleles. The C4F duplication haplotype is a partial inhibitor of the Rodgers antigen, and judged from our family and population material, it seems to be rather frequent and associated with HLAB ^*35, Bf ^* F, and HLAD/DR ^*1. The C4S duplication haplotype is Rg(a-) and is not identified in individuals without another S, Ch(a+) variant.This work was supported by grant No 12-1727 from the Danish Medical Research Council     

Non-cross-reactivity of antibodies to murine LDH-C4 with LDH-A4 and LDH-B4

The induction of infertility by immunization with the sperm-specific lactate dehydrogenase, LDH-C4, suggests its use in a contraceptive vaccine. Development of an immunological contraceptive for human use, however, requires that there be no cross-reactions with somatic tissues. We have demonstrated, using enzyme-linked immunoabsorbence, solid-phase radioimmunoassay, and competitive inhibition radioimmunoassay, that antisera to LDH-C4 is specific and does not cross-react with the somatic isozymes, LDH-A4 and LDH-B4.     

Structure and stability of sodium and potassium complexes of dT4G4 and dT4G4T.

The ends of eukaryotic chromosomes contain specialized structures that include DNA with multiple tandem repeats of simple sequences containing clusters of G on one strand, together with proteins which synthesize and bind to these sequences. The unit repeat in the protozoan Oxytricha with the cluster dT4G4 can form structures containing tetrads of guanine residues, referred to G4 DNA, in the presence of metal ions such as Na+ or K+. We show here that, in the presence of Na+, dT4G4 forms a tetramer with parallel strands by means of a UV cross-linking assay. In the presence of K+, two further interactions are observed: at low temperature, higher order complexes are formed, provided the 3' end of the strand is G; a single 3'T inhibits this association in dT4G4T. At high temperature, these complexes dissociate, leading to a tetramer with a different ordered structure that melts only at very high temperatures. These results suggest that the cohesive properties of DNA containing G clusters might depend on associative interactions driven by a free 3'G terminus in the presence of K+, as well as by connecting antiparallel G hairpins as has been postulated.