鲫鱼遗传多样性的初步研究

用１７种限制性内切酶对鲫属普通鲫鱼低背型,高背型,异育银鲫,日本白鲫及华南鲤的变种红鲤共１２４个个体的线粒体ＤＮＡ进行了ＲＦＬＰ分析,１４种酶具多态,共计４３种限制性态型,１１种单倍型。     

小麦胞质突变型雄性不育系85EA与其保持系线粒体DNA的比较研究

８５ＥＡ是通过电子束辐照获得的胞质突变型小麦不育要用ＲＦＬＰ和ＲＡＰＤ技术对８５ＥＡ及春保持系的线粒体ＤＮＡ进行了比较研究。ＲＦＬＰ分析表明８５ＥＡ线粒体基因组中ｃｏｘⅡ基因的位置结构与保持系发生了变化；ＲＡＰＤ分析中引物ＯＰＤ－１５扩增产物在不育系和保持系间有明显差异，不育系的扩增产物比保持系多１条分子量为０．６ｋｂ的特展览 要带，用Ｔ－ｅａｓｙ ｖｅｃｔｏｒ克隆该不育系特异条带并命名为ＯＰＤ－     

Strand scission in DNA by Gossypol and Cu(II): Role of Cu(I) and oxygen-free radicals

Gossypol, a polyphenolic binaphthyl dialdehyde found in cotton seeds, is a dietary mutagen and a potential male contraceptive. In the presence of Cu(II), gossypol caused breakage of supercoiled plasmid pBR322 DNA. The products were relaxed circles or a mixture of these and linear molecules. Other metal ions tested [Ni(II), Co(II), Mn(II), and Fe(II)] were ineffective or less effective in the DNA breakage reaction. In the case of gossypol-Cu(II) mediated cleavage, (Cu(I) was shown to be an essential intermediate by using the Cud) sequestering reagent bathocuproine. By using job plots, it was established that in the absence of DNA, eight Cu(II) ions can be reduced by one gossypol molecule. The involvement of active oxygen species, such as singlet oxygen and H₂O₂, was established by the inhibition of DNA breakage by catalase and by sodium azide. It was further shown that gossypol is capable of directly producing H₂O₂.     

The N-terminal Domain of the Drosophila Mitochondrial Replicative DNA Helicase Contains an Iron-Sulfur Cluster and Binds DNA

The metazoan mitochondrial DNA helicase is an integral part of the minimal mitochondrial replisome. It exhibits strong sequence homology with the bacteriophage T7 gene 4 protein primase-helicase (T7 gp4). Both proteins contain distinct N- and C-terminal domains separated by a flexible linker. The C-terminal domain catalyzes its characteristic DNA-dependent NTPase activity, and can unwind duplex DNA substrates independently of the N-terminal domain. Whereas the N-terminal domain in T7 gp4 contains a DNA primase activity, this function is lost in metazoan mtDNA helicase. Thus, although the functions of the C-terminal domain and the linker are partially understood, the role of the N-terminal region in the metazoan replicative mtDNA helicase remains elusive. Here, we show that the N-terminal domain of Drosophila melanogaster mtDNA helicase coordinates iron in a 2Fe-2S cluster that enhances protein stability in vitro. The N-terminal domain binds the cluster through conserved cysteine residues (Cys⁶⁸, Cys⁷¹, Cys¹⁰², and Cys¹⁰⁵) that are responsible for coordinating zinc in T7 gp4. Moreover, we show that the N-terminal domain binds both single- and double-stranded DNA oligomers, with an apparent K_d of ∼120 nm. These findings suggest a possible role for the N-terminal domain of metazoan mtDNA helicase in recruiting and binding DNA at the replication fork.     

Mitochondrial phylogeographies of five widespread Eurasian bird species

Five species of Eurasian birds displayed a range of mitochondrial DNA phylogeographic structures, including a single widespread lineage (common sandpiper), two geographically unsorted and closely related lineages (long-tailed tit), three partially overlapping closely related lineages (reed bunting), and two divergent geographically isolated lineages that rival species distinction (red-breasted flycatcher and skylark). Only the red-breasted flycatcher and the skylark displayed congruent phylogeographic structures. These five species represent different stages of diversification and speciation. There was little evidence that natural selection had influenced mitochondrial NADH dehydrogenase subunit 2 (ND2) sequences. In several instances, population growth was hypothesized, based on haplotype distributions within populations.     

Replication Intermediates of the Linear Mitochondrial DNA of Candida parapsilosis Suggest a Common Recombination Based Mechanism for Yeast Mitochondria

Variation in the topology of mitochondrial DNA (mtDNA) in eukaryotes evokes the question if differently structured DNAs are replicated by a common mechanism. RNA-primed DNA synthesis has been established as a mechanism for replicating the circular animal/mammalian mtDNA. In yeasts, circular mtDNA molecules were assumed to be templates for rolling circle DNA-replication. We recently showed that in Candida albicans, which has circular mapping mtDNA, recombination driven replication is a major mechanism for replicating a complex branched mtDNA network. Careful analyses of C. albicans-mtDNA did not reveal detectable amounts of circular DNA molecules. In the present study we addressed the question of how the unit sized linear mtDNA of Candida parapsilosis terminating at both ends with arrays of tandem repeats (mitochondrial telomeres) is replicated. Originally, we expected to find replication intermediates diagnostic of canonical bi-directional replication initiation at the centrally located bi-directional promoter region. However, we found that the linear mtDNA of Candida parapsilosis also employs recombination for replication initiation. The most striking findings were that the mitochondrial telomeres appear to be hot spots for recombination driven replication, and that stable RNA:DNA hybrids, with a potential role in mtDNA replication, are also present in the mtDNA preparations.     

苦荞谷胱甘肽转移酶(FtGST)基因的克隆与序列分析

采用RACE技术,从苦荞(Fagopyrum tatarium)中克隆得到一个谷胱甘肽转移酶(Glutathione S-transferase protein,FtGST)基因。序列分析表明,FtGST基因全长DNA序列和cDNA序列编码区分别为746 bp和666 bp,DNA序列含有一个长度为80 bp(342-421 bp)的内含子;开放阅读框(ORF)长666 bp,编码221个氨基酸。生物信息学分析表明,FtGST基因推导的蛋白质含有Tau家族典型的底物结合口袋、谷胱甘肽结合位点(G-site)和疏水性底物结合位点(H-site)氨基酸残基,表明FtGST为Tau家族蛋白。     

Voltage-dependent anion channel involved in the mitochondrial calcium cycle of cell lines carrying the mitochondrial DNA A4263G mutation

In this study, we investigated the effects of the voltage-dependent anion channel (VDAC) on the mitochondrial calcium cycle in cell lines carrying the mitochondrial DNA A4263G mutation. We established lymphoblastoid cell lines from three symptomatic individuals and one asymptomatic individual from the large Chinese Han family carrying the A4263G mutation; these were compared with three control cell lines. The mitochondrial Ca²⁺ concentration and membrane potential were detected by loading cells with Rhod-2 and JC-1, respectively. Confocal imagines showed the average Rhod-2 and JC-1 fluorescence levels of individuals carrying the tRNA^Ile A4263G mutation were lower than those of the control group (P < 0.05). The baseline Rhod-2 fluorescence in the control group increased after exposure to atractyloside (an opener of the adenine nucleotide translocator, P < 0.05), but no significant change was detected in the cell line harboring the A4263G mutation (P > 0.05). The baseline JC-1 fluorescence in both the mutated and control cell lines decreased after subsequent exposure to atractyloside (P < 0.05), whereas this effect of atractyloside was inhibited by Cyclosporin A (CsA, a VDAC blocker). We conclude that the mitochondrial VDAC is involved in both the increase of mitochondrial permeability to Ca²⁺ and the decrease of mitochondrial membrane potential in cell lines carrying the mtDNA A4263G mutation.     

Phylogenetic Analysis of Different Breeds of Domestic Chickens in Selected Area of Peninsular Malaysia Inferred from Partial Cytochrome b Gene Information and RAPD Markers

The present investigation was carried out in an attempt to study the phylogenetic analysis of different breeds of domestic chickens in Peninsular Malaysia inferred from partial cytochrome b gene information and random amplified polymorphic DNA (RAPD) markers. Phylogenetic analysis using both neighbor-joining (NJ) and maximum parsimony (MP) methods produced three clusters that encompassed Type-I village chickens, the red jungle fowl subspecies and the Japanese Chunky broilers. The phylogenetic analysis also revealed that majority of the Malaysian commercial chickens were randomly assembled with the Type-II village chickens. In RAPD assay, phylogenetic analysis using neighbor-joining produced six clusters that were completely distinguished based on the locality of chickens. High levels of genetic variations were observed among the village chickens, the commercial broilers, and between the commercial broilers and layer chickens. In this study, it was found that Type-I village chickens could be distinguished from the commercial chickens and Type-II village chickens at the position of the 27th nucleotide of the 351 bp cytochrome b gene. This study also revealed that RAPD markers were unable to differentiate the type of chickens, but it showed the effectiveness of RAPD in evaluating the genetic variation and the genetic relationships between chicken lines and populations.     

Molecular Mechanisms for the Variation of Mitochondrial Gene Content and Gene Arrangement Among Chigger Mites of the Genus Leptotrombidium (Acari: Acariformes)

The gene content of a mitochondrial (mt) genome, i.e., 37 genes and a large noncoding region (LNR), is usually conserved in Metazoa. The arrangement of these genes and the LNR is generally conserved at low taxonomic levels but varies substantially at high levels. We report here a variation in mt gene content and gene arrangement among chigger mites of the genus Leptotrombidium. We found previously that the mt genome of Leptotrombidium pallidum has an extra gene for large-subunit rRNA (rrnL), a pseudo-gene for small-subunit rRNA (PrrnS), and three extra LNRs, additional to the 37 genes and an LNR typical of Metazoa. Further, the arrangement of mt genes of L. pallidum differs drastically from that of the hypothetical ancestor of the arthropods. To find to what extent the novel gene content and gene arrangement occurred in Leptotrombidium, we sequenced the entire or partial mt genomes of three other species, L. akamushi, L. deliense, and L. fletcheri. These three species share the arrangement of all genes with L. pallidum, except trnQ (for tRNA-glutamine). Unlike L. pallidum, however, these three species do not have extra rrnL or PrrnS and have only one extra LNR. By comparison between Leptotrombidium species and the ancestor of the arthropods, we propose that (1) the type of mt genome present in L. pallidum evolved from the type present in the other three Leptotrombidium species, and (2) three molecular mechanisms were involved in the evolution of mt gene content and gene arrangement in Leptotrombidium species. [Reviewing Editor: Dr. Martin Kreitman]     

红麻不育系和保持系中cob基因的克隆与分析

开展红麻的CMS机理的研究有利于更好地利用其杂种优势。分别提取红麻CMS系和保持系线粒体DNA,Southern blot分析表明,cob基因的组织形式存在差异。根据不同物种cob基因的保守序列设计引物,在红麻CMS系和保持系中克隆到了cob基因,GenBank序列号分别为HM535786和HM535787。基因长度均为1179bp,编码392个氨基酸残基,分子量约为43kD。序列比较发现cob基因在CMS系和保持系同源性达99.8%,和其他物种中cob基因的同源性大于92.3%。研究结果为下一步克隆cob基因的侧翼序列,进而揭示红麻的CMS机理提供了很好的研究基础。     

Structure and Evolution of the Mitochondrial Control Region of Leaf Beetles (Coleoptera: Chrysomelidae): A Hierarchical Analysis of Nucleotide Sequence Variation

Abstract To assess the levels of variation at different evolutionary scales in the mitochondrial (mt) control region of leaf beetles, we sequenced and compared the full mt control region in two genera (Chrysomela and Gonioctena), in two species within a genus (Gonioctena olivacea and G. pallida), in individuals from distant populations of these species in Europe, and in individuals from populations separated by moderate (10- to 100-km) to short (<5-km) distances. In all individuals, a highly repetitive section consisting of the tandem repetition of 12 to 17 imperfect copies of a 107- to 159-bp-long core sequence was observed. This repetitive fragment accounts for roughly 50% of the full control-region length. The sequence variability among repeated elements within the control region of a given individual depends on the species considered: the variability within any G. olivacea individual is much higher than that within G. pallida individuals. Comparisons of the repeated elements, in a phylogenetic framework, within and among individuals of G. olivacea and G. pallida suggests that the repetitive section of the control region experienced recurrent duplications/deletions, leading to some degree of concerted evolution. Comparisons between Chrysomela and Gonioctena control regions revealed virtually no significant sequence similarity, except for two long stretches of A's and several [T(T)A(A)] repeats, all found in the control region of other insect orders. Our analyses allowed us to identify portions of the control region with enough variation for population genetic or phylogeographic studies.     

草鱼PAX7基因的克隆、序列分析及组织表达

配对盒基因7(Paired box 7 gene,PAX7)在神经嵴发育及原肠胚形成、肌肉自主更新与再生中扮演着重要角色,对细胞更新、分化、凋亡等起着十分重要的调控作用。参照Gen Bank中斑马鱼、线鳍电鳗和虹鳟等物种PAX7序列的保守区域设计简并引物,进行反转录PCR扩增,获得草鱼PAX7基因的部分序列,长645 bp,编码214个氨基酸,包含编码128个氨基酸的PAX7蛋白配对框(PD)。氨基酸序列比对结果发现,草鱼PAX7基因与其他物种具有高度的相似性,与斑马鱼、线鳍电鳗、日本青鳉、金头鲷、虹鳟、大西洋鲑、北极嘉鱼、人、野牦牛、褐家鼠和小鼠的PAX7氨基酸序列同源性可达90%-97%;各物种PAX7部分氨基酸序列的系统进化树结果显示,草鱼与斑马鱼、日本青鳉、北极嘉鱼、大西洋鲑、虹鳟、线鳍电鳗和金头鲷聚为一大支,小鼠、褐家鼠,野牦牛与人类另聚一支,所得结果符合物种传统分类进化地位。运用实时荧光定量PCR检测草鱼PAX7基因在各组织中的差异表达,结果显示PAX7基因在肌肉中表达量最高;其次是前肠和皮肤,在心、脑、肾和肝中仅少量表达,所得结果与该基因的功能相一致。     

两株真养产碱杆菌部分基因的克隆与序列分析

采用随机引物PCR技术从新建细胞培养室空气中获得两段长度414 bp及450 bp的片段.通过克隆测序及序列分析,结果表明,所测序列与真养产碱杆菌主要参考菌株的同源性分别高迭79%-83%及79%-84%,由其推导的氨基酸序列与真养产碱杆菌主要参考菌株的同源性高达86.4%-89.1%,由两分离菌株所获得基因片段推导的氨基酸序列之间的同源性高达98.1%,从而确定所分离两菌株为真养产碱杆菌不同亚型的菌株.     

中国棉铃虫核型多角体病毒DNA聚合酶基因的克隆和序列分析

应用谷实夜蛾核型多角体病毒（ＨｚＳＮＰＶ）ＤＮＡ聚合酶基因ＨｉｎｄⅢ／ＰｓｔⅠ３５９６ｂｐ片段作探针,经Ｓｏｕｒｔｈｅｒｎｂｌｏｔ杂交,克隆了中国棉铃虫核型多角体病毒（ＨａＳＮＰＶ）完整的ＤＮＡ聚合酶基因,大小约为３．４ｋｂ。限制性内切酶分析表明,ＨａＳＮＰＶＤＮＡ聚合酶基因限制性内切酶图谱与ＨｚＳＮＰＶ相似。用双脱氧链终止法测定该基因部分核苷酸序列（８０５ｂｐ）,推导出编码区２０６ａ。序列同源性比较显示,ＨａＳＮＰＶＤＮＡ聚合酶与ＨｚＳＮＰＶ之间具有高度的同源性;与ＬｄＭＮＰＶ、ＡｃＭＮＰＶ、ＢｍＳＮＰＶ、ＣｆＭＮＰＶ和ＯｐＭＮＰＶ也具有一定的同源性     

Molecular genetic evidence for the post-Pleistocene divergence of populations of the arctic-alpine ground beetle Amara alpina (Paykull) (Coleoptera: Carabidae)

Aim This study examines the hypothesis that the biogeographic history of a species is reflected in the distribution of molecular genetic diversity and the phylogenies of extant populations. Location Populations of arctic-alpine ground beetle Amara alpina were analysed from Beringia (Alaska and northernmost British Columbia), the Hudson Bay region, the northern Appalachian Mountains, and the central Rocky Mountains of North America. Methods Mitochondrial restriction site variation of specimens from twenty-two populations were assayed by using radioactively labelled mtDNA to probe Southern membranes containing restriction enzyme digested total DNA. Restriction sites were mapped and genetic distances were calculated by pairwise comparison of presence and absence of restriction sites. Genetic distances were used in a molecular analysis of variance and to construct a minimal spanning tree. Parsimony methods were used to investigate the phylogenetic relationships between the haplotypes. These results were compared to an existing model for postglacial dispersal based on fossil and modern occurrences of arctic-alpine beetles. Results Among the twenty-two populations, fifteen haplotypes were detected. Genetic variation within each of the four regions corresponded to that expected from the palaeontologically based model. Beringian populations were the most genetically diverse. In contrast, no restriction site variation was observed in populations from the Hudson Bay region. Intermediate amounts of variation were observed in alpine populations of the Rocky and Appalachian Mountains. Maximum parsimony and cluster analysis provide evidence that at least two ancestral haplotypes existed in the Southern refugium from which the Rocky and the Appalachian Mountains populations were founded. Main conclusions The genetic results are generally consistent with the palaeontologically based model. The diversity of Beringian populations is consistent with this region having been continuously inhabited by Amara alpina throughout the Pleistocene. The Hudson Bay region was not deglaciated until about 6000 years, and its populations have no restriction site variation. The molecular genetic data support the interpretation that the Hudson Bay region was colonized from Beringia based on the occurrence of the same haplotype in both regions.     

白鱼线粒体DNA控制区结构和种群遗传多样性分析

用特异性引物对白鱼(Anabarilius grahami)DNA进行PCR扩增,获得了白鱼线粒体DNA控制区基因全序列(930bp)。控制区T、C、A和G碱基组成为29.8%、22.5%、33.0%和14.7%。对照其他已报道的鱼类控制区结构,对白鱼控制区结构进行了分析,识别了其终止序列区、中央保守区和保守序列区,找到了终止相关的序列TAS以及保守序列(CSB-F、CSB-D、CSB-1、CSB-2、CSB-3)。同时运用DNA分析软件对白鱼一个驯养种群(中国科学院昆明动物研究所珍稀鱼类繁育中心)及两个自然地理种群(江川县明星鱼洞、江川县牛摩村)进行了遗传多样性分析。结果显示:两个自然种群存在较强基因交流,未出现遗传分化;人工驯养种群遗传多样性最高,种群复壮程度较好。     

人分裂细胞核抗原基因片段的筛选、测序及对启动子的分析

经６．６×１０５个克隆筛选,从装在λ噬菌体载体Ｃｈａｒｏｎ３０中的人基因库中筛选到了一个含人分裂细胞核抗原（ＰＣＮＡ）基因的克隆。经Ｓｏｕｔｈｅｒｎ杂交分析插入基因长约１４ｋｂ,有较长的５＇上游区,但３＇端缺少一部分。经亚克隆和测序已确定从５＇上游１２６３ｂｐ到３＇端与λ载体接点共４９６９ｂｐＰＣＮＡ基因片段的核苷酸序列。将ＰＣＮＡ基因启动子核苷酸序列与ＤＮＡ聚合酶α,拓扑异构酶Ⅱα,胸苷酸激酶基因的启动子进行比较有３０％以上同源性,具有“看家基因”特征。在转录起始点的５＇上游几百ｂｐ的范围内都有与ＣＡＴ,ＳＰ１,Ｅ２Ｆ,ＮＦＨＢ,Ｏｃｔ１和ＡＴＦ等转录因子的结合位点相似的核苷酸序列。