辽东湾斑海豹(Phoca largha)线粒体D-loop区异质型研究初探

采用PCR技术和DNA克隆测序技术,随机测定了3头斑海豹mtDNA控制区(D-loop区)csn-3上、下游1000bp左右的序列,每头斑海豹任选14个克隆菌斑进行测序,结果所得序列均无重复,得到42个单倍型.结合GenBank已发表的斑海豹mtDNA控制区序列(Phoca largha,AM181031),通过Clusta1X1.83、MEGA3.1和FastPCRv3.6等生物信息学软件进行序列比对,发现我国珍稀保护动物斑海豹个体内线粒体DNA(mtDNA)的控制区CSB-3之后存在异质型现象,且存在数目不等的串联重复序列.从分子水平进行了不同类型重复序列变化规律的研究,初探了mtDNA控制区异质型在斑海豹中的存在情况.     

鸮形目两种鸟类线粒体基因组全序列测定与比较研究

利用Long-PCR和Primer Walking结合克隆测序法对短耳鸮和长耳鸮线粒体基因组进行了全序列测定. 结果表明: 短耳鸮mtDNA序列全长为18858 bp, 长耳鸮mtDNA全长为18493 bp, 其中短耳鸮mtDNA是目前已知最长的鸟类线粒体基因组. 两种鸮类的基因组结构和基因排列顺序与家鸡相同, 无假控制区, 在ND3基因174位点都存在一个额外插入的胞苷酸(C). 控制区序列异常增大是造成这两种鸟类mtDNA增大的主要原因, 短耳鸮控制区长度为3288 bp, 长耳鸮为2926 bp, 这是目前已知的脊椎动物线粒体基因组中仅次于盲鳗的最大的控制区. 在其控制区3′端存在大量的串联重复序列, 分析发现这两种鸮类的重复序列和Mt5调控元件有较高的序列相似性, 且能形成多重的茎环二级结构, 这表明该重复序列可能具有一定的生理功能, 影响线粒体基因组的复制或转录表达, 从而使相应物种具有更大的选择优势, 以适应环境和生存竞争.     

动物线粒体基因组变异研究进展

动物mtDNA大多是共价闭合的环状双链分子,一般由2个非编码区和37个编码基因组成,不同动物线粒体基因组大小变异明显.孑遗疟虫(Plasmodium reichenowi)的线粒体基因组最小,仅为5966bp;领鞭毛虫(Monosiga brevicollis)的最大,达76568bp.动物线粒体基因组大小变异的原因主要有:控制区串联重复元件的变异;基因重复;基因重叠与基因间隔区大小的差异;基因缺失和增加.     

三种小型猪线粒体DNA控制区的比较研究

目的分析五指山小型猪、巴马小型猪和贵州香猪线粒体DNA控制区碱基序列,比较研究不同猪种的遗传标志。方法应用PCR技术分别对这三种小型猪的血液总DNA样品中线粒体DNA D-loop区进行扩增,测序比对。结果猪的线粒体DNA D-loop区分3个区域。Ⅰ区（靠近5′端区域）704 bp,五指山小型猪在此区共有6个变异位点,通过6个变异位点中归纳出3个单倍体,而巴马小型猪在此区有9个变异位点,通过9个变异位点归纳出4个单倍体,贵州香猪在此区共有6个变异位点,通过6个变异位点归纳出3个单倍体。     

三种小型猪线粒体DNA控制区的比较研究

目的分析五指山小型猪、巴马小型猪和贵州香猪线粒体DNA控制区碱基序列,比较研究不同猪种的遗传标志。方法应用PCR技术分别对这三种小型猪的血液总DNA样品中线粒体DNA D-loop区进行扩增,测序比对。结果猪的线粒体DNA D-loop区分三个区域。I区（靠近5’端区域）704bp,五指山小型猪在此区共有6个变异位点,通过6个变异位点中归纳出3个单倍体,而巴马小型猪在此区有9个变异位点,通过9个变异位点归纳出4个单倍体,贵州香猪在此区共有6个变异位点,通过6个变异位点归纳出3个单倍体。Ⅱ区（串联重复序列区）,五指山小型猪、巴马小型猪和贵州香猪序列相同。Ⅲ区（靠近3’端区域）三种小型猪的序列几乎相同。结论五指山小型猪、巴马小型猪和贵州香猪三种小型猪之间线粒体DNA碱基序列变异位点较少,五指山小型猪和巴马小型猪亲缘关系较近。     

口腔鳞状细胞癌线粒体DNA的HVRⅢ区突变的研究（英文）

目的：检测口腔鳞状细胞癌患者线粒体DNA复制控制区（mtDNA D-loop）高变Ⅲ区（hypervariable regionⅢ,HVRⅢ）的突变情况,并探讨其意义。方法：以口腔鳞状细胞癌患者癌旁组织及正常组织作为对照,对7例口腔鳞状细胞癌组织样本的mtDNA D-loop HVRⅢ区进行PCR扩增和测序分析。结果：在7例患者的癌组织、癌旁组织、正常组织样本中共发现72个（56种）核苷酸改变,其中51个（26种）为核苷酸多态性改变;3个肿瘤组织样本中共发现21个突变,其中16个位于HVRⅢ区范围内;癌旁组织及正常组织未发现突变;口腔鳞状细胞癌的mtDNA D-loop HVRⅢ区突变率为42.9%（3/7）。结论：mtDNA D-loop HVRⅢ区的变异可能与口腔鳞状细胞癌的易感性有一定的联系;本研究为寻找新的肿瘤基因诊断和肿瘤遗传易感性的标志物提供了依据。     

动物线粒体基因组研究进展

对动物线粒体分子生物学的最新研究进展进行了较详细的阐述.从线粒体基因组(mtDNA)的研究背景出发,重点介绍了动物线粒体基因组的组成和结构特点,以及目前动物mtDNA与核基因组的关系、线粒体基因的遗传、起源和进化研究中的热点问题.     

北京鸭线粒体基因组全序列测定和分析

线粒体DNA作为遗传标记,已在家鸡(Gallus gallus)和家鹅(Anser anser)的研究中取得了重大进展,而对家鸭(Anas platyrhychos domesticus)的研究却很少.本研究参照近源物种线粒体基因组序列设计15对引物,通过PCR扩增、测序、拼接,获得北京鸭(A.platyrhychos)线粒体基因组全序列,初步分析其特点和各基因的定位.结果显示,北京鸭线粒体基因组全长16 604 bp,碱基组成为29.19%A、22.20%T、15.80%G、32.81%C,包含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(D-loop),基因组成及排列顺序与其他鸟类相似.基于线粒体D-loop区全序列,用N-J法构建了7种雁形目鸟类系统进化树,结果表明,北京鸭与绿头鸭(A.platyrhychos)系统进化关系较近.     

野牦牛线粒体基因组序列测定及其系统进化

野牦牛属高寒地区的特有物种,是我国最珍贵的野生动物遗传资源之一,已被列为国家一级重点保护动物。对野牦牛mtDNA进行全序列测定和结构分析,并基于线粒体基因组序列对其系统发生进行了探讨。结果表明:(1)野牦牛线粒体基因组全序列的大小为16 322 bp,整个基因组由37个编码基因和D-loop区组成;22个tRNA基因序列长度为1 524 bp、2个RNA基因序列长度为2 528 bp、13个编码蛋白基因序列长度为11420 bp、D-loop区长度为892 bp。基因组中无间隔序列,基因间排列紧密,基因内无内含子。(2)野牦牛具有较丰富的遗传多样性。(3)分子系统发生关系显示牦牛为牛亚科中的一个独立属,即牦牛属(Poephagus),牦牛属包括家牦牛(Poephagus grunniens)和野牦牛(Poephagus mutus)2个种。野牦牛线粒体基因组全序列的获得和结构解析对研究牦牛的起源、演化和分类,以及野牦牛遗传资源的保护、开发和利用均具有重要的理论和实际意义。     

西藏小型猪线粒体D-loop区及微卫星多态性的遗传学分析

目的通过分析西藏小型猪线粒体控制区(D-loop区)及微卫星位点的遗传多态性,检测西藏小型猪的遗传背景,从而为其作为实验动物提供分子生物学方面的可靠依据。方法利用特异性引物对西藏小型猪的线粒体D-loop区及10个具有多态性的微卫星位点进行扩增,割胶纯化并对线粒体D-loop区进行测序,另外采用聚丙烯酰胺凝胶电泳的方法分离微卫星位点的等位基因。结果西藏小型猪线粒体D-loop区全序列没有多态性,微卫星位点则具有高度的遗传多态性和杂合度,分别为0.584和0.573。结论西藏小型猪线粒体基因组无多态性,证明其在母系进化和遗传上与其他猪种较为一致,本实验所用的西藏小型猪生长于一个封闭的环境,导致其微卫星位点遗传多态性的中低度水平。     

Genetic Diversity of the Mongolian Gazelle <Emphasis Type="Italic">Procapra guttorosa</Emphasis> Pallas, 1777

The mitochondrial DNA D-loop hypervariable fragment sequence polymorphism was examined in 27 the Mongolian gazelles from Mongolia, Russia, and China. Intraspecific polymorphism of the D-loop fragment examined was demonstrated. All haplotypes described were unique. The average nucleotide diversity (π) for the mtDNA fragment investigated constituted 5.85 ± 2.92%. A relatively high number of insertions and deletions was observed. In particular, a haplotype with the 77-bp insertion was described. The data obtained point to high genetic diversity of Mongolian populations. There was no correlation between the distribution of haplotypes examined and geographical location of the animal tissue sampling sites.     

Genetic diversity of Mongolian gazelle Procapra guttorosa Pallas, 1777

The mitochondrial DNA D-loop hypervariable fragment sequence polymorphism was examined in 27 Mongolian gazelles from Mongolia, Russia, and China. Intraspecific polymorphism of the D-loop fragment examined was demonstrated. All haplotypes described were unique. The average nucleotide diversity (pi) for the mtDNA fragment investigated constituted 5.85 +/- 2.92%. A relatively high number of insertions and deletions was observed. In particular, a haplotype with the 77-bp insertion was described. The data obtained point to high genetic diversity of Mongolian populations. There was no correlation between the distribution of haplotypes examined and geographical location of the animal tissue sampling sites.     

Genetic diversity of moose (Alces alces L.) in Eurasia

Polymorphism of nucleotide sequence of D-loop fragment of the mitochondrial DNA was studied in 20 moose from several local populations on the territory of Eurasia. Three main haplotype variants of D-loop were detected by molecular phylogenetic method, which formed three clusters named European, Asian and American. Intraspecies variation in the length of HVSI of D-loop of the mitochondrial DNA of moose was revealed. In the Far Eastern and Yakutian moose, haplotypes with a 75-bp deletion were found, which were most similar with haplotypes (also with the deletion), earlier observed in North American moose [1]. The highest diversity of the haplotypes of mitochondrial DNA is characteristic of Yakutia and the Far East (where three haplotype variants were found), which demonstrates the probable role of the region as the center of the species or as the region of ancient population mixture. The geographic region might be considered as a probable source of ancient moose migrations from Asia to America, basing on the data of distribution of mitochondrial haplotypes of D-loop and alleles of MhcAlal-DRB1. Divergence of nucleotide sequences of haplotypes with the 75-bp deletion (forming the American cluster on the phylogenetic tree) was the lowest (0.4%), which evidences respectively recent origin of the group of haplotypes. In Europe, only haplotypes of mitochondrial DNA referred to European variant were observed. Basing on analysis of variation of nucleotide sequences of D-loop, exon 4 of kappa-Casein and exon 2 of MhcALal-DRB1, we demonstrated that Eurasian moose studied belong to the unique species, which has probably passed through a bottle neck. The time of the origin of modern diversity of D-loop haplotypes of the species was estimated as 0.075-0.15 Myr ago.     

Genetic Diversity of Moose (Alces alces L.) in Eurasia

Polymorphism of nucleotide sequence of D-loop fragment of the mitochondrial DNA was studied in 20 moose from several local populations on the territory of Eurasia. Three main haplotype variants of D-loop were detected by molecular phylogenetic method, which formed three clusters named European, Asian, and American. Intraspecies variation in the length of HVSI of D-loop of the mitochondrial DNA of moose was revealed. In the Far Eastern and Yakutian moose, haplotypes with a 75-bp deletion were found, which were most similar with haplotypes (also with the deletion), earlier observed in North American moose [1]. The highest diversity of the haplotypes of mitochondrial DNA is characteristic of Yakutia and the Far East (where three haplotype variants were found), which demonstrates the probable role of the region as the center of the species origin or as the region of ancient population mixture. The geographic region might be considered as a probable source of ancient moose migrations from Asia to America, basing on the data of distribution of mitochondrial haplotypes of D-loop and alleles of MhcAlal-DRB1. Divergence of nucleotide sequences of haplotypes with the 75-bp deletion (forming the American cluster on the phylogenetic tree) was the lowest (0.4%), which evidences respectively recent origin of the group of haplotypes. In Europe, only haplotypes of mitochondrial DNA referred to European variant were observed. Basing on analysis of variation of nucleotide sequences of D-loop, exon 4 of -Casein and exon 2 of MhcAlal-DRB1, we demonstrated that Eurasian moose studied belong to the unique species, which has probably passed through a bottle neck. The time of the origin of modern diversity of D-loop haplotypes of the species was estimated as 0.075–0.15 Myr ago.     

基于线粒体DNA D-loop区序列的我国灰鹅遗传多样性和起源分析

为了探究我国灰鹅品种的遗传多样性和系统进化,运用DNA测序技术测定了我国13个灰鹅品种mtDNAD-loop区521bp序列。结果表明:这13个灰鹅品种521bpD-loop区序列的T、C、A和G碱基含量分别为23.8%、29.0%、32.3%和15.0%;平均单倍型多样度和核苷酸多样度分别为0.19245和0.00036;13个灰鹅种间变异大于种内变异,且均未出现群体扩张现象;共享单倍型和系统进化树分析表明,伊犁鹅起源于灰雁(Anser anser),其余12个灰鹅品种起源于鸿雁(A.cygnoides)。     

Mitochondrial D-loop sequence variation among the 16 maternal lines of the Lipizzan horse breed

Mitochondrial DNA from 49 Lipizzan horses representing 16 maternal lines from the original stud at Lipica was used for SSCP analysis and DNA sequencing. The SSCP analysis of the 444 bp long fragment of the D-loop region extending from the tRNA(Pro) gene to the central conserved sequence block revealed three distinct groups of SSCP patterns. Both ends of the D-loop region (378 bp and 310 bp), which are considered as the most variable regions within the mammalian mitochondrial DNA, were sequenced. According to 49 polymorphic sites identified within the both parts of the D-loop region, the 16 maternal lines were grouped into 13 distinct mitochondrial haplotypes. The minimal difference between two different haplotype DNA sequences was one nucleotide and the maximal 24 nucleotides. The inheritance of mitochondrial haplotypes was stable and no sequence variation potentially attributable to mutation within maternal line was observed. Considerable DNA sequence similarity of Lipizzan mitochondrial haplotypes with the haplotypes from other breeds was observed. Phylogenetic analysis of the sequence data revealed a dendrogram with three separated branches, supporting the historical data about the multiple origin of the Lipizzan breed.     

High frequency of mitochondrial DNA D-loop mutations in Ewing’s sarcoma

Somatic mutations and polymorphisms in the noncoding displacement (D)-loop of mitochondrial DNA (mtDNA) are present in a variety of human cancers. To investigate whether Ewing’s sarcoma (EWS) harbors genetic alterations within the D-loop region and their potential association with EWS carcinogenesis, we analyzed and compared the complete mtDNA D-loop sequences from 17 pairs of tumor tissues and corresponding peripheral blood samples using the direct DNA sequencing method. Our results revealed that 12 of the 17 EWS tumor specimens (70.6%) carried 19 somatic mutations in the D-loop of mtDNA, including 11 single-base substitutions, 3 insertions and 5 deletions. Among the tested 17 patients, we screened a total of 40 germline polymorphisms including one novel sequence variant in the D-loop fragment. Most of these identified mutations and germline variations were clustered within two hypervariable segments (HVS1 and HVS2) as well as the homopolymeric C stretch between nucleotide position 303 and 309. In addition, there was no significant correlation between mtDNA D-loop mutations and various clinicopathological factors of EWS. In conclusion, our study reports for the first time that mtDNA D-loop mutations occur at a high frequency in EWS. These data provide evidence of mtDNA alterations’ possible involvement in the initiation and/or progression of this rare malignancy.     

Genetic characterization of horse bone excavated from the Kwakji archaeological site,Jeju, Korea

We determined the nucleotide sequences of the hypervariable D-loop region of mitochondrial DNA (mtDNA) from horse bone (humerus, A.D. 700 to A.D. 800) that was excavated from the Kwakji archaeological site, Jeju, Korea. We compared them with ones from extant horses. We designed three pairs of oligonucleotide primers from the tRNA-Thr and tRNA-Phe gene regions of mtDNA that are highly conserved among many other animal species. We cloned 232, 336, and 644 bp from the horse bone in order to determine the mtDNA D-loop sequence. The sequence was 1,124 bp long; the middle contained 19 tandem repeats of an 8-bp sequence (TGTGCACC) that is specific to equines. The mtDNA D-loop region contained each base (total number, percentage of total) as follows: A (317, 28.20%), C (336, 29.89%), G (169, 15.04%), and T (302, 26.87%). This sequence, like those of other horse populations, was AT rich. Sequence divergence was the lowest (1.71%) between the ancient horse bone and that of the Thoroughbred horse 1. The neighbor-joining and strict consensus tree of three of the most parsimonious trees also suggested that the ancient bone was considerably unrelated to native Jeju horses. The molecular phylogenetic characteristics of the horse bone that was excavated from the Kwakji archaeological site (Jeju, Korea) showed that some horse breeds may have existed on Jeju Island, Korea before Mongolian horses were introduced. The horse bone that was excavated from the Kwakji archaeological site may aid future research on the origin and ancestry of native Jeju horses.     

Nuclear integrations of mitochondrial DNA in gorillas

Great ape systematics, particularly at the species level and below, is currently under debate, due in part to the recent influx of molecular data. The phylogenies of previously published mitochondrial control region (or D-loop) DNA sequences in gorillas show deep splits within West African gorillas (Gorilla gorilla gorilla), and very high levels of nucleotide diversity in this subspecies. Here we demonstrate that several previously reported D-loop haplotypes from West African gorillas are in all likelihood nuclear integrations of mitochondrial DNA. Revised estimates of the amount and pattern of mitochondrial DNA diversity in gorillas are provided, revealing two reciprocally monophyletic and highly divergent groups of gorillas, concurrent with their geographic distribution.