贵州水族人群线粒体DNA序列多态分析

研究了我国贵州水族64个个体线粒体DNA控制区第一高变区序列的变异情况,在所测定的495bp序列中,共检测到73个点存在变异,界定了48种不同的单倍型,单倍型的系统发育分析表明水族中存在一些古老的单倍型类型,并且其中的几个单倍型在欧亚人群中也存在分布,来自群体历史动态分析估算的水族群体扩张时间距今约6万年,这些结果提示水族是一个古老的民族群体,结合前期测定的广西壮族以及其他报道的民族人群的比较分析发现,水族在总体上表现出与壮族相近,但又不同于壮族这一典型的南方民族群体。     

Postmortem Miscoding Lesions in Sequence Analysis of Human Ancient Mitochondrial DNA

Genetic miscoding lesions can cause inaccuracies during the interpretation of ancient DNA sequence data. In this study, genetic miscoding lesions were identified and assessed by cloning and direct sequencing of degraded, amplified mitochondrial DNA (mtDNA) extracted from human remains. Forty-two individuals, comprising nine collections from five geographic locations, were analyzed for the presence of DNA damage that can affect the generation of a correct mtDNA profile. In agreement with previous studies, high levels (56.5% of all damage sites) of proposed hydrolytic damage products were observed. Among these, type 2 transitions (cytosine → thymine or guanine → adenine), which are highly indicative of hydrolytic deamination, were observed in 50% of all misincorporations that occurred. In addition to hydrolytic damage products, oxidative damage products were also observed in this study and were responsible for approximately 43.5% of all misincorporations. This level of misincorporation is in contrast to previous studies characterizing miscoding lesions from the analysis of bone and teeth, where few to no oxidative damage products were observed. Of all the oxidative damage products found in this study, type 2 transversions (cytosine → adenine/guanine → thymine or cytosine → guanine/guanine → cytosine), which are commonly formed through the generation of 8-hydroxyguanine, accounted for 30.3% of all genetic miscoding lesions observed. This study identifies the previously unreported presence of oxidative DNA damage and proposes that damage to degraded DNA templates is highly specific in type, correlating with the geographic location and the taphonomic conditions of the depositional environment from which the remains are recovered. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

塔克拉玛干沙漠腹地隔离人群线粒体DNA序列多态性分析

对新疆塔克拉玛干沙漠腹地的75名克里雅人线粒体DNA的高可变I区的15996—16401的片段进行了序列分析,在所测定的75个个体中,共检测到68个位点存在变异,界定了71种不同的单倍型。克里雅人群的核昔酸变异度和平均核苷酸差异都介于所报道的东方人群和西方人群之间。根据Neighbor-joining法构建系统发育树,发现中亚的各人群均处于东方人群的亚洲谱系和西方人群的欧洲谱系之间,并且克里雅与新疆维吾尔和境外维吾尔之间的遗传距离最近,表明他们之间有很密切的亲缘关系。     

广西壮族人群线粒体ＤＮＡ序列遗传多态性分析

测定了８３例广西壮族人的线粒体ＤＮＡ（mtDNA）第一高变区５１５bp的序列,并从这一母系遗传标记的角度对壮族人群的起源和群体的遗传结构进行了探讨,在所测定的８３个个体中,共检测到６６个单倍型,包括７１个多态位点,显示了高度的遗传多样性,对单倍型的系统发育分析表明：壮族人群内部存在一定的分化,不同地区的壮族人群在mtＤＮＡ遗传变异上有一定的差异,其中柳州和河池地区的个体有一半以上属于聚类簇Ⅱ,而南宁和百色地区的个体属于聚类族Ⅰ的较多,结合已相关报道的人群比较分析显示,壮族和我国北方民族群体亲缘关系较远,而与南方的少数民族及南方汉族亲缘关系较近,壮族人群中单倍型辐射型体分布,系统树中壮族人群与其他人群聚类先后次序都表明壮族是一个较为典型的南方群体。     

人癌细胞线粒体DNA控制区序列特征分析

为了探讨癌细胞mtDNA控制区序列的变化特征, 采用PCR产物限制性片段长度多态性(PCR-RFLP)分析与直接测序相结合的方法,对比分析6株人癌细胞系、 6例癌患者及4例健康成人白细胞mtDNA控制区序列。发现第16519位T→C、16 534位A→G、46位T→G和49位A→C突变, 在癌细胞系和癌患者白细胞mtDNA中分别占50%(3/6)和33.3%(2/6), 健康成人白细胞mtDNA中未见此类型突变;第16 278位C→T突变,在癌细胞系mtDNA中占50%(3/6),显著高于正常人群mtDNA中此位点的多态性变异。表明癌细胞和癌患者白细胞mtDNA重链复制起点及其 相邻D环区的特征性突变可能与细胞癌变/或癌的易感性有关。 Abstract：　To explore the sequence feature of mitochondrial DNA(mtDNA) control region in human carcinoma cells, polymerase chain reaction-restriction fragment length polymorphism(PCR-RFLP) and direct sequence techniques were used to analyze the sequence of mtDNA control region of 6 human carcinoma cell lines versus white blood cells which from 6 tumor patients and 4 normal adults. The T to C mutation at np 16 519, A to G mutation at np 16 534, T to G mutation at np 46, and A to C mutation at np 49 was found in 50% (3/6 cases) of carcinoma cell lines and in 33.3%(2/6 cases) of tumor patients, but it was not found in normal adults. The C to T mutation at np 16 278 was found in 50%(3/6 cases) of carcinoma cell lines, it was significantly higher than that of the polymorphism of normal population. These findings suggest that the typical mutation in the starting area of heavy-strand replication and the first half of D-loop region might probably be associated with carcinogenesis or susceptibility of carcinoma.     

人癌细胞线粒体DNA控制区序列特征分析

为了探讨癌细胞mtDNA控制区序列的变化特征, 采用PCR产物限制性片段长度多态性(PCR-RFLP)分析与直接测序相结合的方法,对比分析6株人癌细胞系、 6例癌患者及4例健康成人白细胞mtDNA控制区序列。发现第16519位T→C、16 534位A→G、46位T→G和49位A→C突变, 在癌细胞系和癌患者白细胞mtDNA中分别占50%(3/6)和33.3%(2/6), 健康成人白细胞mtDNA中未见此类型突变;第16 278位C→T突变,在癌细胞系mtDNA中占50%(3/6),显著高于正常人群mtDNA中此位点的多态性变异。表明癌细胞和癌患者白细胞mtDNA重链复制起点及其 相邻D环区的特征性突变可能与细胞癌变/或癌的易感性有关。 Abstract：　To explore the sequence feature of mitochondrial DNA(mtDNA) control region in human carcinoma cells, polymerase chain reaction-restriction fragment length polymorphism(PCR-RFLP) and direct sequence techniques were used to analyze the sequence of mtDNA control region of 6 human carcinoma cell lines versus white blood cells which from 6 tumor patients and 4 normal adults. The T to C mutation at np 16 519, A to G mutation at np 16 534, T to G mutation at np 46, and A to C mutation at np 49 was found in 50% (3/6 cases) of carcinoma cell lines and in 33.3%(2/6 cases) of tumor patients, but it was not found in normal adults. The C to T mutation at np 16 278 was found in 50%(3/6 cases) of carcinoma cell lines, it was significantly higher than that of the polymorphism of normal population. These findings suggest that the typical mutation in the starting area of heavy-strand replication and the first half of D-loop region might probably be associated with carcinogenesis or susceptibility of carcinoma.     

Complete Genomic Sequence of the Human Retinoblastoma Susceptibility Gene

A 180,388-bp contig encompassing the human retinoblastoma gene was sequenced in its entirety. Partial analysis of the sequence revealed (1) a high (A + T)/(G + C) ratio and a high density of Line-1 (L1) repeat sequences, suggesting that the locus maps to G-bands 13q14.12 or 13q14.2; (2) Alu repeats that are asymmetrically oriented over a region extending 87 kb; (3) an overabundance of non-Alu-associated poly(A) tracts 10 bp or larger oriented in the antisense rather than the sense direction (36 vs 6); (4) an Alu sequence nested within an L1 repeat, indicating that the expansion of L1 repeats predates at least some of the Alu expansions; (5) at least three newly discovered microsatellite polymorphisms, one of which was subsequently found to be identical to a polymorphism in a microsatellite-based linkage map of the human genome published by another group; and (6) the basis of previously discovered intragenic RFLPs. This sequence should enhance studies of this locus and of the organization of the human genome.     

汉坦病毒的基因分型及其序列分析

为了探讨从核苷酸水平耐汉坦病毒进行分型,设计两对型特异性引物,采用反转录和聚合酶链式反应（ＲＴ－ＰＣＲ）,对亚太地区１８株汉坦病毒进行了扩增鉴定,并对其中７株汉坦病毒的ＰＣＲ产物进行了测序分析。ＰＣＲ的分型结果表明,Ⅰ型引物只能扩增血清Ⅰ型病毒的ｃＤＮＡ;Ⅱ型引物也只能扩增血清Ⅱ型病毒,其间无交叉反应。采用巢式ＰＣＲ和限制性内切酶验证了ＰＣＲ产物的特异性。序列分析结果表明,Ｒ３６Ｍ片段Ｇ１区的核苷酸序列与血清Ⅰ型病毒代表株７６－１１８的同源性为７８．４％,而与血清Ⅱ型病毒Ｒ２２的同源性为６８．１％;Ｒ３６与汉坦病毒序列同源性的成对比较结果也表明,Ｒ３６与血清Ⅰ型病毒的同源性均高于血清Ⅱ型病毒;Ｌｅａｋｅｙ虽然能被Ⅱ型引物扩增,但其序列与血清Ⅱ型病毒Ｒ２２的同源性仅为４４．９％,故不属于血清Ⅱ型病毒。上述研究结果表明,反转录聚合酶链反应能对多数汉坦病毒准确分型,但最终结果尚有赖于序列分析。     

Human-Specific HERV-K Insertion Causes Genomic Variations in the Human Genome

Human endogenous retroviruses (HERV) sequences account for about 8% of the human genome. Through comparative genomics and literature mining, we identified a total of 29 human-specific HERV-K insertions. We characterized them focusing on their structure and flanking sequence. The results showed that four of the human-specific HERV-K insertions deleted human genomic sequences via non-classical insertion mechanisms. Interestingly, two of the human-specific HERV-K insertion loci contained two HERV-K internals and three LTR elements, a pattern which could be explained by LTR-LTR ectopic recombination or template switching. In addition, we conducted a polymorphic test and observed that twelve out of the 29 elements are polymorphic in the human population. In conclusion, human-specific HERV-K elements have inserted into human genome since the divergence of human and chimpanzee, causing human genomic changes. Thus, we believe that human-specific HERV-K activity has contributed to the genomic divergence between humans and chimpanzees, as well as within the human population.     

在酿酒酵母中研究人类mOGT基质导向序列的功能

人类线粒体N-乙酰氨基葡萄糖转移酶(mitochondrial O-GlcNAc transferse,m OGT)通过其N端基质导向序列(matrix targeting sequence,MTS)定位于线粒体内膜上,m OGT的过表达将导致细胞的凋亡。蛋白质的O-Glc NAc修饰存在于绝大多数真核生物中,而酿酒酵母唯独缺乏该修饰途径。对人体m OGT过表达引起酿酒酵母生长缺陷的机制进行分析,期望利用该模型研究m OGT导致的细胞凋亡调控机制。在酿酒酵母细胞中表达人体m OGT及其截短序列发现:m OGT对酿酒酵母的生长抑制及细胞中的定位依赖于N端全长的MTS序列; MTS序列在酿酒酵母中共表达导致线粒体融合。MTS序列过表达的酿酒酵母菌可以作为研究m OGT引起细胞凋亡的模式细胞。     

Sequence Variation in the tRNA Genes of Human Mitochondrial DNA

Recent analyses have shown that nonsynonymous variation in human mitochondrial DNA (mtDNA) contains nonneutral variants, suggesting the presence of mildly deleterious mutations. Many of the disease-causing mutations in mtDNA occur in the genes encoding the tRNAs. Nucleotide sequence variation in these genes has not been studied in human populations, nor have the structural consequences of nucleotide substitutions in tRNA molecules been examined. We therefore determined the nucleotide sequences of the 22 tRNA genes in the mtDNA of 477 Finns and, also, obtained 435 European sequences from the MitoKor database. No differences in population polymorphism indices were found between the two data sets. We assessed selective constraints against various tRNA domains by comparing allele frequencies between these domains and the synonymous and nonsynonymous sites, respectively. All tRNA domains except the variable loop were more conserved than synonymous sites, and T stem and D stem were more conserved than the respective loops. We also analyzed the energetic consequences of the 96 polymorphisms recovered in the two data sets or in the Mitomap database. The minimum free energy (ΔG) was calculated using the free energy rules as implemented in mfold version 3.1. The ΔG’s were normally distributed among the 22 wild-type tRNA genes, whereas the 96 polymorphic tRNAs departed significantly from a normal distribution. The largest differences in ΔG between the wild-type and the polymorphic tRNAs in the Finnish population tended to be in the polymorphisms that were present at low frequencies. Allele frequency distributions and minimum free energy calculations both suggested that some polymorphisms in tRNA genes are nonneutral.Reviewing Editor: Dr. Rüdiger Cerff     

蝰科(Viperidae)蝮亚科(Crotalinae)线粒体12S rRBA基因序列分析及其系统发育

对蝮亚科（蛇岛蝮Ｇｌｏｙｄｉｕｓ ｓｈｅｄａｏｅｎｓｉｓ Ｚｈａｏ、黑眉蝮Ｇｌｏｙｄｉｕｓ ｓａｘａｔｉｌｉｓ Ｅｍｅｌｉａｎｏｖ、乌苏 里蝮Ｇｌｏｙｄｉｕｓ ｕｓｓｕｒｒｉｅｎｓｉｓ　 Ｅｍｅｌｉａｎｏｖ、　竹叶青Ｔｒｉｍｅｒｅｓｕｒｕｓ 　ｓｔｅｊｎｅｇｅｒｉ 　Ｓｃｈｍｉｄｔ和分别来自不 同地区的尖吻蝮Ｄｅｉｎａｇｋｉｓｔｒｏｄｏｎ　ａｃｕｔｕｓ　Ｇｕｅｎｔｈｅｒ、短尾蝮Ｇｌｏｙｄｉｕｓ　ｂｒｅｖｉｃａｕｄｕｓ　Ｓｔｅｊｎｅｇｅｒ各 两条）６种蛇共８个个体测定、分析了约３７０ｂｐ线粒体１２Ｓ ｒＲＮＡ基因序列;以游蛇科链蛇属半 棱鳞链蛇Ｄｉｎｏｄｏｎ ｓｅｍｉｃａｒｉｎａｔｕｓ　序列为外群构建分子系统树。分子数据结果支持尖吻蝮形态 学的属级分类地位;提示蛇岛蝮位于黑眉蝮的蛇岛亚种分类地位,同时探讨了蛇岛蝮的起源 问题;并提示短尾蝮和乌苏里蝮同位于种级分类地位。     

人类线粒体DNA变异的检测方法和思路

基于线粒体DNA（mtDNA)的研究对于人群源流迁移、线粒体相关疾病病因的探讨和法医鉴定等具有重意义,就检测人线粒体突变的一些常用方法,如RFLP、SSO和控制区测序等作一小结和归纳,并重点介绍目前mtDNA突变的筛选方法和思路,另外,还总结了近年来对人mtDNA方面的研究结果,对世界人群中主要单倍型类群（haplogroup)特征变异位点和相应的酶切检测引物作了归纳。     

轮状病毒RNA基因图谱变异分析

用聚丙烯酰胺凝胶电泳法对轮状病毒(RV)标准株进行RNA基因图谱分析,发现人轮状病毒(HRV)Wa株在MA104细胞上连续传5代后,其RNA基因图谱由原来的4:2:3:2模式变为4:3:3:2模式。继续分别在CV-1、MA104细胞上传代后,基因图谱进一步演变为4:3:4:2模式。经比较电泳、病毒空斑纯化,初步证实RVWa有变异林存在。目前尚未见RV标准株变异的报告,有待进一步研究。同时对多个RV标准株及1939年南京市区婴幼儿秋季腹泻的RV流行株进行基因图谱分析,发现不同的人RV及牛RV标准株有相同的基因图谱;不同实验室来源的同一标准株有不同的基因图谱。此外,尚证实HRV是1989年南京市区婴幼儿秋季腹泻的主要病源,总检出率45.1％,电泳长型为流行优势株89.2％。本文尚对RV RNA基因图谱变异的原因及意义进行了讨论。     

Mitochondrial Intronic Open Reading Frames in Podospora: Mobility and Consecutive Exonic Sequence Variations

The mitochondrial genome of 23 wild-type strains belonging to three different species of the filamentous fungus Podospora was examined. Among the 15 optional sequences identified are two intronic reading frames, nad1-i4-orf1 and cox1-i7-orf2. We show that the presence of these sequences was strictly correlated with tightly clustered nucleotide substitutions in the adjacent exon. This correlation applies to the presence or absence of closely related open reading frames (ORFs), found at the same genetic locations, in all the Pyrenomycete genera examined. The recent gain of these optional ORFs in the evolution of the genus Podospora probably account for such sequence differences. In the homoplasmic progeny from heteroplasmons constructed between Podospora strains differing by the presence of these optional ORFs, nad1-i4-orf1 and cox1-i7-orf2 appeared highly invasive. Sequence comparisons in the nad1-i4 intron of various strains of the Pyrenomycete family led us to propose a scenario of its evolution that includes several events of loss and gain of intronic ORFs. These results strongly reinforce the idea that group I intronic ORFs are mobile elements and that their transfer, and comcomitant modification of the adjacent exon, could participate in the modular evolution of mitochondrial genomes.     

蝰科(Viperidae)蝮亚科(Crotalinae)线粒体12S rRNA基因序列分析及其系统发育

对蝮亚科(蛇岛蝮Gloydius shedaoensis Zhao、黑眉蝮Gloydius saxatilis Emelianov、乌苏里蝮Gloydius ussurriensis Emelianov、竹叶青Trimeresurus stejnegeri Schmidt 和分别来自不同地区的尖吻蝮Deinagkistrodon acutus Guenther、短尾蝮Gloydius brevicaudus Stejneger各两条)6种蛇共8个个体测定、分析了约370bp线粒体12S rRNA基因序列,以游蛇科链蛇属半棱鳞链蛇Dinodon semicarinatus 序列为外群构建分子系统树.分子数据结果支持尖吻蝮形态学的属级分类地位;提示蛇岛蝮位于黑眉蝮的蛇岛亚种分类地位,同时探讨了蛇岛蝮的起源问题;并提示短尾蝮和乌苏里蝮同位于种级分类地位.     

Complete Genomic Sequence Analysis of a New SV40 Isolate

The genome of a new SV40 strain (SV-IMB) isolated from a rhesus monkey was completely sequenced and compared with other isolates. The results showed that the whole genome contains 5246bp, and the average identity of SV-IMB was 98.1% as compared to other SV40 isolates. Its regulatory region is composed of a complete enhancer and a defective enhancer. Amino acid changes occurred to some extent in both the large T antigen (T-Ag) and VP1 region. The findings demonstrate that the SV-IMB is a new SV40 isolate.     

猪传染性胃肠炎病毒的分离鉴定及全基因组序列分析

采用ST细胞培养,免疫荧光、理化试验、中和试验、电镜观察等方法,从四川疑似猪腹泻病料中分离到1株猪传染性胃肠炎病毒,命名为SC-Y.分离株在ST细胞上盲传至第8代时可出现稳定的细胞病变,病毒滴度TCID50为10-3.664/0.05m1,中和指数为52.应用长链RT-PCR技术成功地扩增出了覆盖SC-Y株全长基因组的5个片段,通过BioEdit软件对测序结果进行拼接,确认SC-Y株基因组全长28 590bp,包括7个开放阅读框,基因组5非编码区长315nt,3'端非编码区长277nt.TGEV基因组系统进化树显示,SC-Y株与美国Purdue株可能来源于共同的祖先.     

基于数字PCR的不同品种鸭组织中线粒体与核DNA拷贝数差异研究

肉和肉制品是人类生活的重要营养来源,但近年来肉制品中发生的掺假使假事件屡见不鲜,使得肉品的质量安全问题已经成为全世界关注的热点话题。以核酸为目标的动物源鉴定是当前普遍使用的方法。在核酸检测中,常用线粒体基因或核基因作为靶标,缺乏统一标准。以绍兴鸭和北京鸭等不同品种及生鲜组织(鸭血、鸭胸肉、鸭肝、鸭皮、鸭心和鸭腿肉)为实验材料,提取DNA后利用微滴式数字PCR开展线粒体和核DNA拷贝数的比较研究,以两者拷贝数及其比值的变异系数为判定依据。结果显示,核DNA的拷贝数在不同品种鸭组织间相对稳定,且变异系数小于线粒体DNA,表明核DNA是开展鸭肉制品掺假定量检测的最适DNA来源。鸭腿肉中线粒体/核DNA拷贝数比值的变异系数最小,表明线粒体DNA作为靶基因的鸭肉掺假比例定量检测时,鸭腿肉来源的肉制品是最佳选择。     

社鼠（Niviventer confucianus）线粒体基因组全序列分析

社鼠（Niviventer confucianus）属于啮齿目（Rodentia）、鼠科（Muridae）、白腹鼠属（Niviventer）,关于该物种的分子系统学研究极少。为获取社鼠线粒体基因组全序列,提取其基因组总DNA,参照近缘物种线粒体基因组全序列设计34对特异性引物,利用PCR扩增全部片段后进行测序,之后对其基因组组成及结构特点进行了初步分析。结果表明,社鼠线粒体基因组全序列长16 281 bp（GenBank收录号：KJ152220）,包含22个tRNA基因、13个蛋白质编码基因、2个rRNA基因和1个非编码控制区;基因组核苷酸组成为34.0%A、28.6%T、24.9%C、12.5%G。将所得序列与社鼠近缘物种（川西白腹鼠、小家鼠、褐家鼠）的线粒体全基因组进行比较,结果显示,四个物种的线粒体基因组虽然在基因组大小、部分tRNA二级结构、部分蛋白质编码基因的起始或终止密码子及控制区长度和碱基组成上有差异,但基因组结构和序列特征方面都具有较高的相似性。四个物种线粒体全基因组间的遗传距离显示,社鼠与川西白腹鼠距离最近,而与小家鼠距离最远。该研究为利用线粒体全基因组信息进行啮齿类分子系统学研究提供了有价值的资料。