植物叶绿体DNA

叶绿体是专营光合作用的细胞器。本世纪初已经证明叶斑现象是细胞质遗传的,因此认为叶绿体中很可能存在遗传物质,以后随着核酸检测技术的发展,予测叶绿体中存在DNA。1963年Sager和石田首先成功地从衣藻叶绿体中提取了DNA,接着证明叶绿体中也存在自身的转录,翻译系统。用以往经典遗传学方法很难适用于叶绿体DNA的遗传分析,因此几似没有进行。最近由于引入以重组DNA为主的新技术,叶绿体DNA的遗传分析才真正开始进行。本文主要是从DNA碱基顺序的水平讨论叶绿体基因的情况。     

叶绿体DNA及其在植物系统学研究中的应用

叶绿体ＤＮＡ及其在植物系统学研究中的应用黄瑶,李朝銮,马诚,吴乃虎（中国科学院成都生物研究所,成都,６１００４１）（中国科学院发育生物学研究所,北京,１０００８０）ＣＨＬＯＨＯＰＬＡＳＴＤＮＡＡＮＤＩＴＳＡＰＰＬＩＣＡＴＩＯＮＴＯＰＬＡＮＴＳＹＳＴＥ...     

叶绿体DNA的限制性内切酶谱分析在植物系统分类学中的应用

业已证明,叶绿体DNA为一共轭闭合环状分子。绝大多数植物叶绿体DNA均含有一对反向重复序列。对于所有已检测过的植物,叶绿体基因组所编码的基因数量、基因组成和基因排列是基本一致的。这表明叶绿体DNA具有进化上的保守性。由于其进化上的保守性,叶绿体DNA常为研究层次较高的分类单位间(属间、科间、目间、纲间等)进化关系和进化历史提供较准确的信息。1976年,Vedel等首先建立了叶绿体DNA的内切酶谱分析方法,并将其应用于植物系统分类学中。Vedel等用限制酶ECoRI酶切从豌豆     

植物叶绿体与线粒体基因组DNA特异性比较研究

研究不同植物间叶绿体和线粒体基因组DNA的差异,探讨其在法庭科学中的应用价值.根据叶绿体和线粒体基因组DNA核苷酸序列的特点,分别设计了一系列相应的引物,经PCR扩增后,电泳鉴别不同的植物.结果表明在设计的一系列引物中,叶绿体基因组DNA的PCR产物差异不大,鉴别效果不明显;而线粒体基因DNA的PCR产物差异大,鉴别效果明显.因此以线粒体DNA为模板进行PCR扩增,在不同植物间存在良好的差异性,适合于不同植物间的鉴别,在法庭科学中具有实际应用价值.     

水稻叶绿体DNA的提取和纯化

以籼稻品种珍讪97B为材料,采用溶液捣碎和不连续蔗糖梯度离心的方法提取了籼稻的叶绿体DNA,DNA经限制性内切酶酶解和琼脂糖胶电泳可以得到清晰的条带,来自蚕豆的核酮糖—1,5—二磷酸羧化氧合酶大亚基基因探针和23SrRNA基因探针可以与酶切条带杂交,由此确定了含这二种基因的BamHI酶切片段。     

油菜细胞质雄性不育系叶绿体DNA特异片段的分子克隆

采用高离子浓度缓冲液法分别提取油菜不育系及保持系的叶绿体DNA。经Sepharcse 4B柱层析纯化后,得到具有较高纯度的叶绿体DNA样品。将其分别用Eco RI、Bam HI、HimdHI、PstI和XhoI 5种限制性内切酶酶解,得到5种限制性内切酶图谱。其中除PstI图谱外,其它4种酶谱均显示出明显的两系间叶绿体DNA结构差异。以pBR 322为载体,分别克隆了不育系Bam HI图谱上的3个特异片段。得到的3个克隆,经克隆杂交及电泳分析后,证实分别带有上述3个目的片段。这些特异片段的特性及其与花粉育性的关系尚在研究中。     

植物叶绿体4.5S核糖体rRNA作为分子杂交探针的研究

我们提取纯化了芹菜,菠菜和蕃茄叶绿体核糖体4.5SRNA(4.5SrRNA)并在其5’端标记~(32)P,作为探针与菠菜,蕃茄和芹菜叶绿体DNA(ctDNA)进行分子杂交。结果不仅证明4.5SrRNA可作为公用分子杂交探针,而且也说明不同植物4.5SrRNA序列有相当大的同源性。     

茄科植物叶绿体基因组插入、缺失和核苷酸替代的发生方式及影响

摘要: 核苷酸替代和indels(插入、缺失统称)发生是进化的重要动力。以茄科植物为研究对象, 探讨茄属中番茄和马铃薯、烟草属中绒毛状烟草和普通烟草分化时叶绿体基因组indels和核苷酸替代的发生方式, 以及这两种突变对基因组造成的影响。结果显示: indels和核苷酸替代的发生都不是随意的。indels发生在A+T丰富的区域, 1 bp indels占据总数的30%以上, 大部分indels都为低于10 bp的较短片段。核苷酸替代表现出Ts(转换)/Tv(颠换)偏差, 但T→G, A→C颠换频率却明显增加。Ts/Tv比值出现种属特异性, 番茄和马铃薯比较时替代的Ts/Tv比值低于绒毛状烟草和普通烟草比较时Ts/Tv比值。不同物种替代的(A+T)/(G+C)比值有一定差异, 从而影响基因组的(G+C)%, 此比值的差异与形成物种的生长习性有一定的关系。     

基于叶绿体DNA的黄土高原特有植物蕤核的谱系地理学

该文利用母系遗传的叶绿体DNA片段(psbA-trnH,psbI-psbK和psbJ-petA)对黄土高原地区的特有植物蕤核(Prinsepia uniflora)进行谱系地理学研究,以揭示其现有的遗传结构和群体历史动态.结果表明:(1)蕤核自然种群总的遗传多样性较高(HT=0.796),而种群内的遗传多样性较低(HS...     

黑麦属种间叶绿体DNA的变异性和种系发生的关系

为了获得有关黑麦属种间关系、黑麦属与小麦属和山羊草属种系发牛关系的新资料,应用Ban HI等8种限制性核酸内切酶酶解黑麦属5个种的叶绿体DNA,进行琼脂糖凝胶电泳,分析其酶解图谱。结果表明,黑麦属种叶绿体基因组的大小与小麦属和山羊草属的叶绿体基因组非常相似。根据黑麦属5个种遗传距离的估算,并与小麦属和山羊草属已知叶绿体基因组间所观察到的遗传距离相比,黑麦属叶绿体基因组的分化很少;这些资料进一步证实黑麦属的近代起源;并表明黑麦属、小麦属和山羊草属间的亲缘关系是非常密切的。     

Redundancy in ribonucleotide excision repair: Competition,compensation, and cooperation

The survival of all living organisms is determined by their ability to reproduce, which in turn depends on accurate duplication of chromosomal DNA. In order to ensure the integrity of genome duplication, DNA polymerases are equipped with stringent mechanisms by which they select and insert correctly paired nucleotides with a deoxyribose sugar ring. However, this process is never 100% accurate. To fix occasional mistakes, cells have evolved highly sophisticated and often redundant mechanisms. A good example is mismatch repair (MMR), which corrects the majority of mispaired bases and which has been extensively studied for many years. On the contrary, pathways leading to the replacement of nucleotides with an incorrect sugar that is embedded in chromosomal DNA have only recently attracted significant attention. This review describes progress made during the last few years in understanding such pathways in both prokaryotes and eukaryotes. Genetic studies in Escherichia coli and Saccharomyces cerevisiae demonstrated that MMR has the capacity to replace errant ribonucleotides, but only when the base is mispaired. In contrast, the major evolutionarily conserved ribonucleotide repair pathway initiated by the ribonuclease activity of type 2 Rnase H has broad specificity. In yeast, this pathway also requires the concerted action of Fen1 and pol δ, while in bacteria it can be successfully completed by DNA polymerase I. Besides these main players, all organisms contain alternative enzymes able to accomplish the same tasks, although with differing efficiency and fidelity. Studies in bacteria have very recently demonstrated that isolated rNMPs can be removed from genomic DNA by error-free nucleotide excision repair (NER), while studies in yeast suggest the involvement of topoisomerase 1 in alternative mutagenic ribonucleotide processing. This review summarizes the most recent progress in understanding the ribonucleotide repair mechanisms in prokaryotes and eukaryotes.     

Structure of p22 headful packaging nuclease

Packaging of viral genomes into preformed procapsids requires the controlled and synchronized activity of an ATPase and a genome-processing nuclease, both located in the large terminase (L-terminase) subunit. In this paper, we have characterized the structure and regulation of bacteriophage P22 L-terminase (gp2). Limited proteolysis reveals a bipartite organization consisting of an N-terminal ATPase core flexibly connected to a C-terminal nuclease domain. The 2.02 Å crystal structure of P22 headful nuclease obtained by in-drop proteolysis of full-length L-terminase (FL-L-terminase) reveals a central seven-stranded β-sheet core that harbors two magnesium ions. Modeling studies with DNA suggest that the two ions are poised for two-metal ion-dependent catalysis, but the nuclease DNA binding surface is sterically hindered by a loop-helix (L₁-α₂) motif, which is incompatible with catalysis. Accordingly, the isolated nuclease is completely inactive in vitro, whereas it exhibits endonucleolytic activity in the context of FL-L-terminase. Deleting the autoinhibitory L₁-α₂ motif (or just the loop L₁) restores nuclease activity to a level comparable with FL-L-terminase. Together, these results suggest that the activity of P22 headful nuclease is regulated by intramolecular cross-talk with the N-terminal ATPase domain. This cross-talk allows for precise and controlled cleavage of DNA that is essential for genome packaging.     

Isolation and phenotypic characterization ofChlamydomonas reinhardtii mutants defective in chloroplast DNA segregation

Summary Each wild-typeChlamydomonas reinhardtii cell has one large chloroplast containing several nuclei (nucleoids). We used DNA insertional mutagenesis to isolate Chlamydomonas mutants which contain a single, large chloroplast (cp) nucleus and which we namedmoc (monokaryotic chloroplast). DAPI-fluorescence microscopy and microphotometry observations revealed thatmoc mutant cells only contain one cp-nucleus throughout the cell division cycle, and that unequal segregation of cpDNA occurred during cell division in themoc mutant. One cell with a large amount of cpDNA and another with a small amount of cpDNA were produced after the first cell division. Unequal segregation also occurred in the second cell division, producing one cell with a large amount (about 70 copies) of cpDNA and three other cells with a small amount (only 2–8 copies) of cpDNA. However, most individualmoc cells contained several dozen cpDNA copies 12 h after the completion of cell division, suggesting that cpDNA synthesis was activated immediately after chloroplast division. In contrast to the cpDNA, the mitochondrial (mt) DNA of themoc mutants was observed as tiny granules scattered throughout the entire cell. These segregated to each daughter cell equally during cell division. Electron-microscopic observation of the ultrastructure ofmoc mutants showed that a low-electron-density area, which was identified as the cp-nucleus by immunoelectron microscopy with anti-DNA antibody, existed near the pyrenoid. However, there were no other structural differences between the chloroplasts of wild-type cells andmoc mutants. The thylakoid membranes and pyrenoid were identical. Therefore, we propose that the novelmoc mutants are only defective in the dispersion and segregation of cpDNA. This strain should be useful to elucidate the mechanism for the segregation of cpDNA.Abbreviations DAPI 4,6-diamidino-2-phenylindole - VIMPCS video-intensified microscope photon-counting system     

Polymorphism of DNA conformation inside the bacteriophage capsid

Double-stranded DNA bacteriophage genomes are packaged into their icosahedral capsids at the highest densities known so far (about 50 % w:v). How the molecule is folded at such density and how its conformation changes upon ejection or packaging are fascinating questions still largely open. We review cryo-TEM analyses of DNA conformation inside partially filled capsids as a function of the physico-chemical environment (ions, osmotic pressure, temperature). We show that there exists a wide variety of DNA conformations. Strikingly, the different observed structures can be described by some of the different models proposed over the years for DNA organisation inside bacteriophage capsids: either spool-like structures with axial or concentric symmetries, or liquid crystalline structures characterised by a DNA homogeneous density. The relevance of these conformations for the understanding of DNA folding and unfolding upon ejection and packaging in vivo is discussed.     

Homologies to chloroplast DNA in the nuclear DNA of a number of Chenopod species

Summary Sequences homologous to chloroplast (ct)DNA have been found in nuclear DNA in five species of the Chenopodiaceae, extending the earlier observations of promiscuous DNA in Spinacia oleracea (Timmis and Scott 1983). Using the 7.7 kbp spinach ctDNA Pst I fragment as a hybridization probe, several separately located homologies to ctDNA were resolved in the nuclear DNA of Beta vulgaris, Chenopodium quinoa, and Enchylaena tomentosa. In Chenopodium album and Atriplex cinerea the major region of homology was to a nuclear Eco RI fragment (6 kbp) indistinguishable from that in ctDNA. These homologies may therefore involve larger tracts of ctDNA because the same restriction sites are apparently retained in the nucleus. This suggests that in these latter two species there is a contrasting, more homogeneous arrangement of ctDNA transpositions in the nucleus.     

Distribution of restriction site polymorphism within the chloroplast genome of the genus Glycine,subgenus Soja

Summary Restriction fragment length polymorphisms (RFLPs) have been used to detect intragenic sequence diversity in Glycine subgenus soja chloroplast DNA. The distribution of these RFLPs allow Glycine max and G. soja accessions to be grouped according to cytoplasmic genetic relatedness. DNA clones from mung bean chloroplast DNA were used to locate the RFLPs to specific regions of the chloroplast genome. In the course of the experiments, several previously unobserved RFLPs were also identified. At least six molecular changes were detected, including both restriction site loss or gain and insertion/deletion events. Three of the fragment polymorphisms detected are due to changes in the juncture region between one inverted repeat region and the large single-copy region. Probes detecting polymorphisms in three representative soybean genotypes were used to screen additional cultivars and Plant Introductions. The distribution of RFLP patterns in these accessions were consistent with the patterns of previously described cytoplasmic groupings, with the exception of one accession, which formed a new plastome group.