大趾鼠耳蝠线粒体DNA控制区结构及变异

采用PCR产物直接测序法首次测定大趾鼠耳蝠(Myotis macrodactylus)10个个体的线粒体DNA(mtDNA)控制区全序列,并进行了结构和变异分析。结果表明,大趾鼠耳蝠的控制区结构与其他哺乳动物相似,可分为一个中央保守区(包括F、E、D、C、B元件)和两个外围结构域:延伸的终止结合序列区(包括ETAS1和ETAS2元件)和保守序列区(包括CSB1、CSB2和CSB3元件),其中最为保守的是中央保守区(核苷酸变异度为1.8%)。大趾鼠耳蝠控制区核苷酸全序列具有丰富的长度多态性(1778～2048bp),主要是由在碱基组成、重复数目和排列方式上异质的串联重复序列造成的。在ETAS内发现了TACAT及其反向互补序列ATGTA,支持滑移错配模式(slipped mispairing model)。本研究为该物种的进一步研究和保护提供基础遗传数据。     

猪线粒体控制区微卫星(mtMs)变异及其分布特征

猪线粒体DNA长度因D-loop中mtMs序列重复数变异而不同。为阐明猪mtMs变异及其在不同群体中的分布特征,本研究对4个藏猪群体、八眉猪、烟台黑猪及3个引入猪群体共164个个体mtDNA D-loop全序进行测定,并与GenBank中发表的相关序列进行比对,分析了其中的重复单元核苷酸变异、重复次数及其在各群体间的分布规律。结果表明,因重复单元“5’-TGCGTACACG-3’”第2~4和10位上碱基变异,猪mtMs形成了以其为核心序列的多种重复单元(R^A^R^G)组成的复合结构,重复数介于3~47之间。单一重复(R^A)组成的mtMs结构在各群体中表现出分布优势,而大多数复合结构(R^AR^B)分布在国外选育群体中。藏猪的mtMs复合结构多达8个,R^AR^CR^E为其特有,另有5个与八眉猪共享。中国野猪和韩国地方猪种也有其特有mtMs结构。丰富的mtMs变异和特有结构与长期适应进化有关,本研究为地方猪种遗传资源及适应性研究提供了标记工具和理论基础。     

鲤鱼线粒体tRNA~(Cys)基因和轻链复制起始区的结构分析

我们测定了鲤鱼线粒体半胱氮酸tRNA 基因和轻链(L 链)复制起始区的核苷酸序列,绘制了半胱氨酸tRNA 三叶草形的二级结构以及L 链复制区的茎环结构。通过五种脊椎动物tRNA~(cya)基因的核苷酸序列分析发现,鲤鱼线粒体tRNA~(cya)基因有许多不同于细胞质tRNA~(cya)基因的不寻常的结构特点。鲤鱼线粒体L 链复制起始区含有36个碱基,复制起始区茎环结构中的茎含有11对碱基,而环则是由14个碱基组成。同其它10种脊椎动物L-链复制起始区的核苷酸序列比较发现,鲤鱼茎环结构中的茎序列是非常保守的,而环的序列及环的长度则变化较大。茎环结构可能在轻链复制中起着重要的作用。     

圆斑星鲽及相关种类线粒体DNA控制区结构分析

采用PCR产物直接测序法测定了圆斑星鲽(Verasper variegatus)的24个个体的线粒体控制区(Control region)核苷酸全序列, 并进行了结构分析。结果表明, 圆斑星鲽线粒体控制区核苷酸全序列具有长度多态性, 得到4种长度单元型, 主要表现为控制区中的串联重复序列的长度不同。对鲽形目鱼类如鲽科的条斑星鲽(Verasper moseri)、黄盖鲽(Limanda feruginea)、马舌鲽 (Reinhardtius hippoglossoides), 美洲拟庸鲽(Heppoglossoides platessoides )和鲆科的牙鲆(Paralichthys olivaceus)以及鳎科的欧洲鳎(Solea solea)、塞内加尔鳎(S. senegalensis)和沙鳎(S. lascari)的控制区的比较研究发现, 鲽形目鱼类的线粒体控制区均存在相似的结构, 即线粒体控制区可分为终止相关序列区(ETAS)、中央保守区(包括CSB-A、CSB-B、CSB-C、CSB-D、CSB-E、CSB-F)以及保守序列区(CSB1、CSB2、CSB3)和重复序列区(Repeat region)4个区域。通过与脊椎动物各个纲线粒体控制区序列的比较分析, 发现只有鲽形目(包括鲆、鲽类和鳎类)鱼类和两栖纲的无尾类在CSB-3之后存在相似的串联重复序列。     

都柳江南方白甲鱼群体mtDNA D-loop序列及遗传多样性分析

为探究都柳江流域南方白甲鱼种群种质资源现状。本研究以采集自都柳江的40尾南方白甲鱼个体为实验样本,使用mtDNA D-loop分子标记技术,得到了长度近700 bp的南方白甲鱼D-loop序列。经分析序列中碱基A、T的含量高于碱基C、G,识别了序列的保守序列区、终止序列区和中央保守区以及相关的核心序列。都柳江南方白甲鱼群体D-loop序列有变异点13个,变异类型包括缺失、插入、转换和颠换。多态位点11个,其中单突变点和简约信息点分别有3个和8个。碱基转换颠换比为4.5∶1,变异尚未达饱和。确定了15个单倍体型。都柳江南方白甲鱼群体D-loop序列单倍型多样性度和核苷酸多样性指数分别为0.877 7和0.003 3。都柳江南方白甲鱼群体近期可能存在扩张现象,但其核苷酸多样性较低,需要开展其种质资源研究和保护。本研究从分子水平上为都柳江南方白甲鱼种质资源的评价、开发和利用提供基础。     

猪POU1F1基因部分序列变异和同源性分析

对长白、杜洛克、约克夏、姜曲海、梅山和香猪等六个猪种的POU1F1基因第四、第五和第六外显子分别进行PCR扩增,并对含有第四、第六外显子的PCR产物和含有第五外显子的克隆产物进行测序。结果表明：六个猪种中,POU1F1基因的第四外显子存在碱基突变,为T→C。对该序列进行Nla Ⅲ 酶消化,产生两种不同的基因型(GG和HH);而第五和第六外显子则高度保守,未发现任何突变。将人POU1F1基因第四外显子、POU同源区核苷酸编码序列和氨基酸序列,分别与猪、小鼠、牛的POU1F1基因相应的核苷酸序列和氨基酸序列进行同源性比较,结果发现：人与猪、小鼠、牛的POU1F1基因第四外显子的核苷酸同源性分别高达93.9%、86.7%、92.1%,而由第四外显子编码的部分POU特异区的氨基酸序列则完全一致;人与猪、小鼠、牛POU同源区的核苷酸同源性分别为91.4%、85.1%、87.9%,氨基酸同源性分别为96.6%、94.8%、90.2%。这说明在哺乳动物中,其POU1F1蛋白的POU同源区和由第四外显子编码的POU特异区部分是高度保守的;猪可作为实验动物,建立人类相关疾病模型,为医学研究提供参考依据。     

荒漠昆虫小胸鳖甲转录组微卫星的生物信息学分析

位于蛋白质编码区及非编码区(可转录区)的微卫星是常用的一种功能分子标记。本研究以荒漠拟步甲科昆虫小胸鳖甲(Microdera punctipennis)为材料,从其转录组数据库中筛选出由1~6个碱基单元组成的简单序列重复进行分析,并对其微卫星的丰度和特征进行描述。研究显示,小胸鳖甲转录组中微卫星的分布频率为7.94%。其中,单碱基重复序列占微卫星的47.17%,是最丰富的重复序列;其次为3碱基重复序列,占39.81%,而2、4、5、6碱基重复序列分别占10.94%、0.90%、0.88%和0.29%。将小胸鳖甲转录本与同为拟步甲科的赤拟谷盗(Tribolium castaneum)编码序列进行比对,分析同源序列微卫星基元(motif)发现,二者蛋白质编码区微卫星具有高度物种特异性。对小胸鳖甲低温响应转录本和总转录本进行微卫星基元和不同长度微卫星重复次数的比较发现,它们之间没有显著性差异。对含有微卫星的冷响应基因进行基因本体论(gene ontology,GO)分析,发现主要富集到生物学过程、分子功能和细胞组分3个类群。     

务川黑牛mtDNA遗传变异及起源进化分析

目的:测定务川黑牛mtDNA D-loop区全序列,以了解其遗传背景。方法:采用PCR直接测序方法测定务川黑牛mtD-NA D-loop区全序列。结果:32个个体D-loop区的全序列中,四种碱基A、T、C和G含量分别为:33.1%、28.4%、25.0%和13.5%,A+T平均含量61.5%。经过序列比对,共检测到57个变异位点,占所测mtDNA序列总长的6.26%,其中转换54个,颠换3个;产生17种单倍型,其中6种是普通牛单倍型,11种是瘤牛单倍型;平均核苷酸差异为22.623,单倍型多样度0.901±0.039,核苷酸多样度0.0249±0.00147。结论:务川黑牛这一品种遗传多样性非常丰富,据此构建的NJ进化树显示务川黑牛含有普通牛和瘤牛的血统。     

动物mtDNA控制区及保守与异质

本文通过文献综述,对动物线粒体DNA控制区进行了阐述.从线粒体控制区（control region）基因组的研究出发,重点介绍了动物线粒体控制区基因组结构特点.主要结论：由于碱基替换、插入和缺失以及重复序列数目的变异致使D-loop成为mtDNA中变异最多的区域,但突变和结构重排并不是发生在整个D-loop区域,而是在高变区;大多研究集中在mtDNA D-loop保守区和异质方面：对D-loop序列分析,能较好地阐明动物的起源,在动物亲缘关系鉴定、系统进化和物种形成方式的研究等领域具有广阔的研究和应用前景.     

三倍体湘云鲫线粒体DNA序列变异性分析

对26尾三倍体湘云鲫的线粒体tRNA-Thr基因、tRNA-Pro基因和部分控制区的核苷酸序列进行了测定,获得26条长度为837—839 bp的同源基因序列,共发现65个多态性核苷酸变异位点,多态位点比例为0.077,定义了8种单元型。在湘云鲫8种单元型中确认了DNA复制终止相关的序列TAS、中央保守区序列(CSB-F、CSB-E和CSB-D)和保守序列CSB1,8种单元型含有3—5个TAS序列。在65个变异位点中,大部分序列变异为转换,8种单元型之间的序列差异在0.1%—6.3%之间。该研究为三倍体湘云鲫的繁殖和遗传改良提供了一些有价值的信息。     

DNA sequence variation in the mitochondrial control region of red-backed voles (Clethrionomys)

The complete mitochondrial DNA (mtDNA) control region was sequenced for 71 individuals from five species of the rodent genus Clethrionomys both to understand patterns of variation and to explore the existence of previously described domains and other elements. Among species, the control region ranged from 942 to 971 bp in length. Our data were compatible with the proposal of three domains (extended terminal associated sequences [ETAS], central, conserved sequence blocks [CSB]) within the control region. The most conserved region in the control region was the central domain (12% of nucleotide positions variable), whereas in the ETAS and CSB domains, 22% and 40% of nucleotide positions were variable, respectively. Tandem repeats were encountered only in the ETAS domain of Clethrionomys rufocanus. This tandem repeat found in C. rufocanus was 24 bp in length and was located at the 5' end of the control region. Only two of the proposed CSB and ETAS elements appeared to be supported by our data; however, a "CSB1-like" element was also documented in the ETAS domain.     

Evolution of Repeated Sequence Arrays in the D-Loop Region of Bat Mitochondrial DNA

Analysis of mitochondrial DNA control region sequences from 41 species of bats representing 11 families revealed that repeated sequence arrays near the tRNA-Pro gene are present in all vespertilionine bats. Across 18 species tandem repeats varied in size from 78 to 85 bp and contained two to nine repeats. Heteroplasmy ranged from 15% to 63%. Fewer repeats among heteroplasmic than homoplasmic individuals in a species with up to nine repeats indicates selection may act against long arrays. A lower limit of two repeats and more repeats among heteroplasmic than homoplasmic individuals in two species with few repeats suggests length mutations are biased. Significant regressions of heteroplasmy, θ and π, on repeat number further suggest that repeat duplication rate increases with repeat number. Comparison of vespertilionine bat consensus repeats to mammal control region sequences revealed that tandem repeats of similar size, sequence and number also occur in shrews, cats and bighorn sheep. The presence of two conserved protein-binding sequences in all repeat units indicates that convergent evolution has occurred by duplication of functional units. We speculate that D-loop region tandem repeats may provide signal redundancy and a primitive repair mechanism in the event of somatic mutations to these binding sites.     

Lineage Specificity of the Evolutionary Dynamics of the mtDNA D-Loop Region in Rodents

This paper reports an intraorder study on the D-loop-containing region of the mitochondrial DNA in rodents. A complete multialignment of this region is not feasible with the exception of some conserved regions. The comparative analysis of 25 complete rodent sequences from 23 species plus one lagomorph has revealed that only the central domain (CD), a conserved region of about 80 bp in the extended termination-associated sequences (ETAS) domain, adjacent to the CD, the ETAS1, and conserved sequence block (CSB) 1 blocks are present in all rodent species, whereas the presence of CSB2 and CSB3 is erratic within the order. We have also found a conserved region of 90 bp located between tRNAPro and ETAS1 present in fat dormouse, squirrel, guinea pig, and rabbit. Repeated sequences are present in both the ETAS and the CSB domain, but the repeats differ in length, copy number, and base composition in different species. The potential use of the D-loop for evolutionary studies has been investigated; the presence/absence of conserved blocks and/or repeated sequences cannot be used as a reliable phylogenetic marker, since in some cases they may be shared by distantly related organisms but not by close ones, while in other ones a relationship between tree topology and presence/absence of such motifs is observed. Better results can be obtained by the use of the CD, which, however, due to its reduced size, when used for tracing a phylogenetic tree, shows some nodes with low statistical support. Received: 26 February 2001 / Accepted: 6 June 2001     

齐口裂腹鱼线粒体DNA控制区结构分析

利用直接测序法对齐口裂腹鱼(Schizothorax prenanti)线粒体DNA控制区进行了测序,并对其序列结构进行了分析.结果表明,齐口裂腹鱼线粒体控制区碱基组成中碱基A和T的含A明显高于G和C的含量,所有类型碱基组成中碱基G的含量最低,这与其他硬骨鱼类控制区碱基组成一致.通过与哺乳类和鲤形目鱼类控制区序列进行对...     

南蝠线粒体DNA控制区结构及贵州7个种群遗传多样性的分析研究

采用PCR技术获得了贵州7个南蝠(Ia io)自然种群42个个体的线粒体DNA控制区全序列,长度为1256～1340 bp.对控制区结构进行分析,识别了其延伸的终止结合序列区(包括ETAS1和ETAS2元件)、中央保守区(包括F、E、D、C、B元件)和保守序列区(包括CSB1、CSB2和CSB3元件);同时,在延伸的终止结合序列区还发现了若干能形成发夹结构的主体序列TACAT—ATGTA.在7个自然种群42个个体中共定义了16个单倍型.遗传多样性分析表明:贵州南蝠种群具有较高的单倍型多样性(h=0.945)和中等的核苷酸多样性(π=0.012).基因流、AMOVA和系统进化树分析表明贵州这7个南蝠自然群体间没有发生遗传分化.     

五种鲟鱼线粒体控制区异质性和系统发育分析

利用保守引物得到五种鲟鱼的线粒体DNA（mtDNA）控制区（D-loop）全长,长度在795~813 bp。序列中包括了CBS（conserved sequence block）和TAS（termination-associated sequence）区域。利用最大似然法、最大简约法和贝叶斯法构建了系统发育树,发育树分成两枝,呈现明显的生物地理分布。分析表明,现有的鳇属鱼类不是单系群起源。五种鲟鱼D-loop序列都存在长度和数目不等串联重复序列,长度在78~82 bp之间,重复序列拷贝数在4~6次不等,因此造成了mtDNA广泛的异质性现象。不同种类的重复序列单元十分相似,达氏鳇和史氏鲟重复序列单元相似度为82.93%,西伯利亚鲟和俄罗斯鲟重复序列单元相似度为90.59%。在串联重复序列后是一段不完全重复序列。通过与已有同种的重复序列比对发现不同鲟鱼重复序列相同,不同地理区域相同物种的重复序列可能发生过分子内重组。这些表明重复序列在鲟鱼进化上具有相关意义,推测重复序列可能产生在种分化前,重组发生在种分化后。     

Inter-species sequence diversity in the replication initiation region of Paramecium mitochondrial DNA

Mitochondrial DNA from Paramecium aurelia is a linear molecule. Replication is initiated at a unique cross-linked molecular terminus. During replication dimer length molecules, consisting of two head-to-head monomers, are generated. We have cloned the head-to-head dimer initiation region from five different species and several stocks (or races) within species and determined its DNA sequence. For all species, this dimer initiation region consists of a central non-palindromic sequence containing almost exclusively A and T, arranged in an array of direct tandem repeats. In an intra-species comparison, the sequences of the repeat units are relatively homogeneous; inter-species comparisons, however, show diversity except for a conserved "Goldberg-Hogness box", T-A-T-A-A-A-T-A. The size of a repeat unit and the number of repeats within a molecule can vary over a wide range, even in an intra-species comparison. Because of these wide inter-species variations observed, it is likely that the function of this region imposes few constraints on the sequence other than its high A + T content and possibly a Goldberg-Hogness box. The array of direct tandem repeats may have arisen from unequal recombination or crossover within this region. Adjacent to the non-palindromic region is a transcribed sequence which is highly conserved for all species and presumably represents a gene coding region.     

Sequence simplicity and evolution of the 3′ untranslated region of the histone H1° Gene

The H1° gene has a long 3′ untranslated region (3′UTR) of 1,125 nucleotides in the rat and 1,310 in humans. Analysis of the sequences shows that they have features of simple DNA that suggest involvement of replication slippage in their evolution. These features include the length imbalance between the rat and human sequences; the abundance of single-base repeats, two-base runs and other simple motifs clustered along the sequence; and the presence of single-base repeat length polymorphisms in the rat and mouse sequences. Pairwise comparisons show numerous short insertions/deletions, often flanked by direct repeats. In addition, a proportion of short insertions/deletions results from length differences in conserved single-base repeats. Quantification of the sequence simplicity shows that simple sequences have been more actively incorporated in the human lineage than in the rodent lineage. The combination of insertions/deletions and nucleotide substitutions along the sequence gives rise to three main regions of homology: a highly variable central region flanked by more conserved regions nearest the coding region and the polyA addition site. Correspondence to: P. Suau     

Sequence and organization of the complete mitochondrial genomes of spotted halibut (Verasper variegatus) and barfin flounder (Verasper moseri).

In this work, the mitochondrial genomes for spotted halibut (Verasper variegatus) and barfin flounder (Verasper moseri) were completely sequenced. The entire mitochondrial genome sequences of the spotted halibut and barfin flounder were 17,273 and 17,588 bp in length, respectively. The organization of the two mitochondrial genomes was similar to those reported from other fish mitochondrial genomes containing 37 genes (2 rRNAs, 22 tRNAs and 13 protein-coding genes) and two non-coding regions (control region (CR) and WANCY region). In the CR, the termination associated sequence (ETAS), six central conserved block (CSB-A,B,C,D,E,F), three conserved sequence blocks (CSB1-3) and a region of 61-bp tandem repeat cluster at the end of CSB-3 were identified by similarity comparison with fishes and other vertebrates. The tandem repeat sequences show polymorphism among the different individuals of the two species. The complete mitochondrial genomes of spotted halibut and barfin flounder should be useful for evolutionary studies of flatfishes and other vertebrate species.