犬科的线粒体细胞色素b DNA序列及其分子系统学研究

通过对犬科的赤狐、蓝狐、貉和狼４种的线粒体细胞色素ｂ约３７２ｂｐＤＮＡ片段序列分析,结合ＧｅｎＢａｎｋ中狗、西门豺和非洲野犬３种的该区段ＤＮＡ序列的比较,共发现１１３个核苷酸位点存在变异（约３０％）。ＮＪ法构建的分子系统树显示,非洲野犬最先从犬科动物中分化出来;犬属的我狼、狗和西门豺等３种为系统树上独立的一支,且其分歧的时间较赤狐、蓝狐和貉早;赤狐和蓝狐具有较近的亲缘关系。上述结果与形态的观点基本     

Phylogenetic Relationships of the Macaques (Cercopithecidae: Macaca), Inferred from Mitochondrial DNA Sequences

To study the phylogenetic relationships of the macaques, five gene fragments were sequenced from 40 individuals of eight species: Macaca mulatta, M. cyclopis, M. fascicularis, M. arctoides, M. assamensis, M. thibetana, M. silenus, and M. leonina. In addition, sequences of M. sylvanus were obtained from Genbank. A baboon was used as the outgroup. The phylogenetic trees were constructed using maximum-parsimony and Bayesian methods. Because five gene fragments were from the mitochondrial genome and were inherited as a single entity without recombination, we combined the five genes into a single analysis. The parsimony bootstrap proportions we obtained were higher than those from earlier studies based on the combined mtDNA dataset. Excluding M. arctoides, our results are generally consistent with the classification of Delson (1980). Our phylogenetic analyses agree with earlier studies suggesting that the mitochondrial lineages of M. arctoides share a close evolutionary relationship with the mitochondrial lineages of the fascicularis group of macaques (and M. fascicularis, specifically). M. mulatta (with respect to M. cyclopis), M. assamensis assamensis (with respect to M. thibetana), and M. leonina (with respect to M. silenus) are paraphyletic based on our analysis of mitochondrial genes.     

Quick Identification and Localization of CpG Islands in Large Genomic Fragments by Partial Digestion with Hpa II and Hha I

More than 50% of mammalian genes are associated with CpG islandsand thus they serve as a good gene marker. We have devised asimple method to scan large pieces of native or cloned genomicDNA for CpG islands. The method is based on the presence ofmultiple Hpa II and Hha I sites in CpG islands, at a frequency30 times higher than in the rest of the genome. The steps includecomplete digestion of DNA with a rare-cutting restriction endonuclease(to produce large fragments with defined ends), partial digestionwith Hpa II and Hha I, and subsequent Southern hybridizationwith an end probe. This identifies a CpG island as a clusterof sub-bands and, based on their electrophoretic mobility, onecan immediately locate the island relative to the ends. Formany vectors, universal probes flanking the cloning site areavailable, enabling the simultaneous analysis of a large numberof samples. We demonstrated the usefulness of the method byanalyzing known CpG islands in native genomic DNA and lambda,cosmid and P1 clones, and by isolating two novel transcribedislands from anonymous cosmid clones. Our method is quick, inexpensive,and can detect CpG islands with few or even no rare-cutter sites.     

A human CpG island randomly inserted into a plant genome is protected from methylation

In vertebrate genomes the dinucleotide CpG is heavily methylated, except in CpG islands, which are normally unmethylated. It is not clear why the CpG islands are such poor substrates for DNA methyltransferase. Plant genomes display methylation, but otherwise the genomes of plants and animals represent two very divergent evolutionary lines. To gain a further understanding of the resistance of CpG islands to methylation, we introduced a human CpG island from the proteasome-like subunit I gene into the genome of the plant Arabidopsis thaliana. Our results show that prevention of methylation is an intrinsic property of CpG islands, recognized even if a human CpG island is transferred to a plant genome. Two different parts of the human CpG island – the promoter region/ first exon and exon2–4 – both displayed resistance against methylation, but the promoter/ exon1 construct seemed to be most resistant. In contrast, certain sites in a plant CpG-rich region used as a control transgene were always methylated. The frequency of silencing of the adjacent nptII (Km^R) gene in the human CpG constructs was lower than observed for the plant CpG-rich region. These results have implications for understanding DNA methylation, and for construction of vectors that will reduce transgene silencing.     

The distribution of the dinucleotide CpG and cytosine methylation in the vitellogenin gene family

Summary Sequence data from regions of five vertebrate vitellogenin genes were used to examine the frequency, distribution, and mutability of the dinucleotide CpG, the preferred modification site for eukaryotic DNA methyltransferases. The observed level of the CpG dinucleotide in all five genes was markedly lower than that expected from the known mononucleotide frequencies. CpG suppression was greater in introns than in exons. CpG-containing codons were found to be avoided in the vitellogenin genes, but not completely despite the redundancy of the genetic code. Frequency and distribution patterns of this dinucleotide varied dramatically among these otherwise closely related genes. Dense clusters of CpG dinucleotides tended to appear in regions of either functional or structural interest (e.g., in the transposon-like Vi-element ofXenopus) and these clusters contained 5-methylcytosine (5 mC). 5 mC is known to undergo deamination to form thymidine, but the extent to which this transition occurs in the heavily methylated genomes of vertebrates and its contribution to CpG suppression are still unclear. Sequence comparison of the methylated vitellogenin gene regions identified CT and GA substitutions that were found to occur at relatively high frequencies. The predicted products of CpG deamination, TpG and CpA, were elevated. These findings are consistent with the view that CpG distribution and methylation are interdependent and that deamination of 5 mC plays an important role in promoting evolutionary change at the nucleotide sequence level.     

Comparative Genomic Study Reveals a Transition from TA Richness in Invertebrates to GC Richness in Vertebrates at CpG Flanking Sites： An Indication for Context-Dependent Mutagenicity of Methylated CpG Sites

Vertebrate genomes are characterized with CpG deficiency, particularly for GCpoor regions. The GC content-related CpG deficiency is probably caused by context-dependent deamination of methylated CpG sites. This hypothesis was examined in this study by comparing nucleotide frequencies at CpG flanking positions among invertebrate and vertebrate genomes. The finding is a transition of nucleotide preference of 5＇ T to 5＇ A at the invertebrate-vertebrate boundary, indicating that a large number of CpG sites with 5＇ Ts were depleted because of global DNA methylation developed in vertebrates. At genome level, we investigated CpG observed/expected （obs/exp） values in 500 bp fragments, and found that higher CpG obs/exp value is shown in GC-poor regions of invertebrate genomes （except sea urchin） but in GC-rich sequences of vertebrate genomes. We next compared GC content at CpG flanking positions with genomic average, showing that the GC content is lower than the average in invertebrate genomes, but higher than that in vertebrate genomes. These results indicate that although 5＇ T and 5＇ A are different in inducing deamination of methylated CpG sites, GC content is even more important in affecting the deamination rate. In all the tests, the results of sea urchin are similar to vertebrates perhaps due to its fractional DNA methylation. CpG deficiency is therefore suggested to be mainly a result of high mutation rates of methylated CpG sites in GC-poor regions.     

Features and trend of loss of promoter-associated CpG islands in the human and mouse genomes

CpG islands (CGIs) are often considered as gene markers, but the number of CGIs varies among mammalian genomes that have similar numbers of genes. In this study, we investigated the distribution of CGIs in the promoter regions of 3,197 human-mouse orthologous gene pairs and found that the mouse genome has notably fewer CGIs in the promoter regions and less pronounced CGI characteristics than does the human genome. We further inferred CGI's ancestral state using the dog genome as a reference and examined the nucleotide substitution pattern and the mutational direction in the conserved regions of human and mouse CGIs. The results reveal many losses of CGIs in both genomes but the loss rate in the mouse lineage is two to four times the rate in the human lineage. We found an intriguing feature of CGI loss, namely that the loss of a CGI usually starts from erosion at the both edges and gradually moves towards the center. We found functional bias in the genes that have lost promoter-associated CGIs in the human or mouse lineage. Finally, our analysis indicates that the association of CGIs with housekeeping genes is not as strong as previously estimated. Our study provides a detailed view of the evolution of promoter-associated CGIs in the human and mouse genomes and our findings are helpful for understanding the evolution of mammalian genomes and the role of CGIs in gene function.     

荧光定量PCR精确检测人胚胎干细胞线粒体DNA拷贝数方法的建立

建立一种精确定量人胚胎干细胞线粒体DNA拷贝数的方法。构建包含线粒体DNANDl和核单拷贝基β-globin基因序列的重组质粒作为标准品;收集无饲养层培养体系下人胚胎干细胞DNA样本,结合2个单独的Taqman探针实时荧光定量PCR对待测样本中线粒体NDl和核β-globin基因分别进行定量,从而对人胚胎干细胞线粒体DNA的含量进行了精确定量。结果提示,人胚胎干细胞线粒体DNA的平均拷贝数／细胞为1321±228。研究表明,该技术可对人胚胎干细胞线粒体DNA拷贝数进行准确的测定,为研究培养条件对人胚胎干细胞线粒体DNA拷贝数的影响及优化体外培养条件奠定了基础。     

Polymorphism of the Noncoding Region of the Mitochondrial Genome in Three Kazakh Populations Inhabiting Different Areas of Kazakhstan and in DNA Samples from Ancient Populations of the Kazakhstani Altai

The polymorphism of the major noncoding region of mitochondrial DNA (mtDNA D loop, 528 bp) has been studied in samples from three modern Kazakh populations (from Almaty, the Semipalatinsk Region, and the Altai Mountains) and in DNA samples of ancient human populations of the Kazakhstani Altai. Polymerase chain reaction (PCR) and restriction fragment length polymorphism (RFLP) analysis for 13 restriction sites, including BamHI, EcoRV, Sau3AI (one site each), KpnI (two sites), HaeIII (three sites), and RsaI (five sites) were used. The frequency distributions of all sites have been determined. The gene diversity (h) and the genetic distances between different Kazakh populations and other populations of the world have been calculated. The RFLP analysis of the mtDNA control region of fossil samples has been performed similarly to the analysis of modern mtDNA samples. Two fossil mtDNA samples from burial mound 11 are monomorphic with respect to all restriction sites analyzed.     

The Effect of the Nucleotide Context on Induction of Mutations in Hypervariable Segment 1 of Human Mitochondrial DNA

The distribution of unstable nucleotide positions with a higher frequency of homoplastic mutations was analyzed in hypervariable segment 1 (HVS1) of the major noncoding region of human mtDNA. Three motifs (GTAC, ACCC, CCTC) proved to be associated with a higher rate of point substitutions at unstable positions. The motifs were often arranged in direct, including tandem, repeats. Motifs CCTC and ACCC were found in extended poly(C) tracts, which form direct repeats associated with deletions and tandem duplications. The results suggested that the inconstancy of the human mitochondrial genome is to a great extent determined by context-dependent mutations.     

线粒体DNA拷贝量降低诱发人支气管上皮细胞钙信号失调

采用溴化乙锭（EtBr）诱导线粒体DNA（mitochondrial DNA,mtDNA）拷贝量降低的人支气管上皮细胞株（ρ-HBE）;Real—timePCR与共聚焦成像表明,经EtBr诱导60d并挑取的单克隆细胞株,其mtDNA拷贝量下降为正常细胞的24％,成功构建了ρ-HBE。与母本细胞相比,ρ-HBE群体倍增时间延长,生长速度减慢。流式细胞术检测细胞线粒体膜电位（△ψm）下降,以Fura-2标记胞浆内游离钙,ρ-HBE[Ca2＋]i升高;线粒体解耦联剂FccP刺激细胞后,激光共聚焦扫描显微镜动态监测单个活细胞[Ca2＋]i变化,发现[ca2＋]i水平波动幅度小。提示mtDNA拷贝数降低可导致细胞内钙信号调节紊乱。     

Role of CpG context and content in evolutionary signatures of brain DNA methylation

DNA methylation is essential in brain function and behavior; therefore, understanding the role of DNA methylation in brain-based disorders begins with the study of DNA methylation profiles in normal brain. Determining the patterns and scale of methylation conservation and alteration in an evolutionary context enables the design of focused but effective methylation studies of disease states. We applied an enzymatic-based approach, Methylation Mapping Analysis by Paired-end Sequencing (Methyl-MAPS), which utilizes second-generation sequencing technology to provide an unbiased representation of genome-wide DNA methylation profiles of human and mouse brains. In this large-scale study, we assayed CpG methylation in cerebral cortex of neurologically and psychiatrically normal human postmortem specimens, as well as mouse forebrain specimens. Cross-species human-mouse DNA methylation conservation analysis shows that DNA methylation is not correlated with sequence conservation. Instead, greater DNA methylation conservation is correlated with increasing CpG density. In addition to CpG density, these data show that genomic context is a critical factor in DNA methylation conservation and alteration signatures throughout mammalian brain evolution. We identify key genomic features that can be targeted for identification of epigenetic loci that may be developmentally and evolutionarily conserved and wherein aberrations in DNA methylation patterns can confer risk for disease.     

Analysis of Mutation Mechanisms in Human Mitochondrial DNA

The cause of the high variability of human mitochondrial DNA (mtDNA) remains largely unknown. Three mechanisms of mutagenesis that might account for the generation of nucleotide substitutions in mtDNA have been analyzed: deamination of DNA nitrous bases caused by deamination agents, tautomeric proton migration in nitrous bases, and the hydrolysis of the glycoside bond between the nitrous base and carbohydrate residue in nucleotides against the background of the free-radical damage of DNA polymerase γ. Quantum chemical calculations demonstrated that the hydrolysis of the N-glycoside bond is the most probable mechanism; it is especially prominent in the H strand, which remains free during mtDNA replication for a relatively long time. It has also been found that hydrolytic deamination of adenine in single-stranded regions of the H strand is a possible cause of the high frequency of T → C transitions in the mutation spectra of the L-chain of the major mtDNA noncoding region.     

The role of vicariance vs. dispersal in shaping genetic patterns in ocellated lizard species in the western Mediterranean

The schism between North Africa and Southern Europe caused by the opening of the Strait of Gibraltar and the consequent refilling of the Mediterranean basin at the end of Messinian salinity crisis (MSC), 5.33 million years ago, has been advocated as the main event shaping biogeographical patterns in the western Mediterranean as exemplified by the distribution of species and subspecies and genetic variation within the ocellated lizard group. To reassess the role of the MSC, partial sequences of three mitochondrial DNA genes (cytochrome b , 12S and 16S ribosomal RNA) and two nuclear genes (β-fibrinogen and C-mos) from species of the ocellated lizard group were analysed. Three alternative hypotheses were tested: that divergence was initiated (i) by post-MSC vicariance as the basin filled, (ii) when separate populations established either side of the strait by pre-MSC overseas dispersal, and (iii) by post-MSC overseas dispersal. The pattern and level of divergence detected clearly refute the post-MSC vicariance hypothesis, and support a model of divergence initiated by earlier overseas dispersal. Indeed, our best estimate is that the basal Euro-African divergence predates the MSC event by several million years. The estimated divergence times among the populations in former Miocene Mediterranean islands, the current Betic and Rifian mountains, from adjacent mainland populations suggest overseas dispersal for the former and overland dispersal, or perhaps vicariance, for the latter. These results suggest that the MSC may have played a much less important role in shaping the current western Mediterranean biogeographical patterns than might have been anticipated from the dramatic nature of the episode.     

Molecular Phylogeny of the Chipmunks Inferred from Mitochondrial Cytochrome b and Cytochrome Oxidase II Gene Sequences

There are currently 25 recognized species of the chipmunk genus Tamias. In this study we sequenced the complete mitochondrial cytochrome b (cyt b) gene of 23 Tamias species. We analyzed the cyt b sequence and then analyzed a combined data set of cyt b along with a previous data set of cytochrome oxidase subunit II (COII) sequence. Maximum-likelihood was used to further test the fit of models of evolution to the cyt b data. Other sciurid cyt b sequence was added to examine the evolution of Tamias in the context of other sciurids. Relationships among Tamias species are discussed, particularly the possibility of a current sorting event among taxa of the southwestern United States and the extreme divergences among the three subgenera (Neotamias, Eutamias, and Tamias).     

The Effect of the Gene Encoding EcoRII DNA Methyltransferase on the Phenotype of Transgenic Tumor Cell Lines of Nicotiana tabacumand the Methylation Level of CpNpG Sequences in Their Genome

Several tumor cell lines were obtained by transforming Nicotiana tabacumplants with the recombinant Ti plasmid comprising the gene encoding EcoRII DNA methyltransferase (M·EcoRII) and subjected to analysis. The transformed lines differed in their morphology, growth dependence on hormones, and nopaline-synthesizing capacity. Southern blot-hybridization showed that the M·EcoRII gene was present in the cells of all transformed lines. However, genome analysis using polymerase chain reaction with the oligonucleotide primers recognizing 5"-ends of the M·EcoRII gene did not exhibit the full-length copies of the gene. Lower methylation of CpNpG sequences characteristic of all transformed cells could result from the disturbance of one of several plant DNA methyltransferase genes following its homologous recombination with the M·EcoRII gene.