植物线粒体DNA质料研究进展

本文列出了已发现的高等植物中的线粒体ＤＮＡ质粒,按分子形状分为线粒体环状ＤＮＡ质粒和线粒体线状ＤＮＡ质粒,环状线粒体ＤＮＡ质粒的特征是分子较小,序列中有正向／反向重复序列,ＯＲＦ一般较小。线状线粒体ＤＮＡ质粒的特征是分子较大,末端有重复序列,５’端与蛋白质共价结合,有较长的ＯＲＦ。还分别介绍了它们的复制机制、转录和起源。质粒间及质粒及核基因组、线粒体基因组、叶绿体基因组的同源性也作了介绍。最后,综     

植物线粒体与细胞质雄性不育研究进展

本文从能量代谢与植物细胞质雄性不育(CMS)、线粒体的结构和数量与CMS、线粒体DNA多态性与CMS、线粒体基因转录与CMS、线粒体多肽差异与CMS几个方面介绍了植物线粒体与CMS的关系。并介绍了与CMS相关的线粒体基因研究进展并对CMS形成的分子机制进行了探讨。     

植物线粒体与细胞质雄性不育研究进展

本文从能量代谢与植物细胞质雄性不育(CMS)、线粒体的结构和数量与CMS、线粒体DNA多态性与CMS、线粒体基因转录与CMS、线粒体多肽差异与CMS几个方面介绍了植物线粒体与CMS的关系。并介绍了与CMS相关的线粒体基因研究进展并对CMS形成的分子机制进行了探讨。     

水稻线粒体DNA的提取与分析

为了研究水稻细胞质雄性不育的分子基础,我们比较了各种提取线粒体ＤＮＡ（ｍｔＤＮＡ）的方法,并提出了一些改进措施。以丛广４１Ａ、丛广４１Ｂ和杂种一代广优青为材料,对所提取的材料ｍｔＤ－ＮＡ进行了紫外扫描、ＯＤ值测定、电泳、酶切等分析,结果表明,以新鲜材料进行不连续蔗糖密度梯度超速离心对提取高纯度的线粒体ＤＮＡ效果较好。     

线粒体DNA及其应用

线粒体DNA是在真核生物中普遍存在的一种核外遗传物质,具有分子小、结构简单、进化速度快、母性遗传、无组织特异性等特点。本综述了线粒体DNA在人类遗传疾病、动植物的亲缘关系、种群分化、群体多样性及植物细胞质雄性不育等研究领域中的应用情况。     

小麦K型及V型细胞质雄性不育系线粒体DNA的比较分析

采用ＲＦＬＰ和ＲＡＰＤ技术对具有相同核遗传背景的小麦Ｋ型不育系Ｋ１４９Ａ,Ｖ型不育系Ｖ１４９Ａ及相应保持系１４９Ｂ的线粒体ＤＮＡ进行了比较分析。结果表明它们之间线粒体ＤＮＡ的结构显著不同,ａｔｐＡ,ａｔｐ９,ｃｏｘⅡ,ｃｏｂ等线粒体功能基因有组织结构上的差异。     

3种小麦细胞质雄性不育系及其杂种线粒体DNA的RFLP分析

对细胞质分别来源于提莫菲维（Ｔ．ｔｉｍｏｔｈｅｅｖｉｉ）,粘果山羊草（Ａｅ．ｋｏｔｓｃｈｙｉ）,偏凸山羊草（Ａｅ．ｖｅｎｙｒｉｃｏｓａ）的３种普通小麦的雄性不育系,相应保持系和恢复系及其上的ｍｔＤＮＡ用１２个线粒体基因探针进行了ＲＦＬＰ分析,结果为：⑴Ｔ、Ｋ、Ｖ型不育系的ｍｔＤＮＡ在组织结构上存在显著差异;⑵Ｔ、Ｋ、Ｖ不育系的ｍｔＤＮＡ与共同的保持系间显著不同,失测ｍｔＤＮＡ与小麦ｃｍｓ有关;⑶在     

高等植物细胞质雄性不育分子机理的研究进展

从线粒体DNA、叶绿体DNA和线粒体质粒DNA方面较详细地阐述了高等植物细胞质雄性不育的分子机理及最新进展;探讨了细胞核DNA和细胞质DNA之间的相互关系。     

红莲型细胞质雄性不育水稻线粒体DNA的AP-PCR分析

为了研究红莲型细胞质雄性不育与线粒体基因组的关系。以水稻红莲型粤泰细胞质雄性不育系A和保持系B及杂种一代F1为材料。应用AP-PCR分析,用10个单引物对其线粒体DNA进行扩增。实验结果表明,不同的引物在3种材料间均有不同程度的差异。为红莲型细胞质雄性不育分子机理的研究提供了线索;此外,在引物6F1的扩增图谱中找到一条在YTA和F1中特异的带TAF6F2,Sounthern分析TAF6F2不育胞质的特异性,可能与红莲型水稻细胞质雄性 不育性状的形成有关。     

植物细胞质雄性不育分子机理研究进展

本文从线粒体基因组、线粒体基因、线粒体转录 RNA、 线粒体蛋白、转基因植物以及花粉败育机理六个方面详细介绍了植物细胞质雄性不育分子生物学研究的技术和方法。综述了植物细胞质雄性不育分子机理研究的进展,并对植物细胞质雄性不育分子机理的前景作了展望。     

动物线粒体DNA的分子生物学研究进展

动物线粒体ＤＮＡ的分子生物学研究进展张方米志勇（中国科学院发育生物学研究所北京１０００８０）绝大多数的真核生物中都含有线粒体（ｍｉ－ｔｏｃｈｒｏｄｒｉａ,ｍｔ）这种细胞器,它自身携带ＤＮＡ,可自我复制、表达,并有核基因编码的蛋白质和酶从细胞质输入线粒体,共同完成生物氧化的生理功能。通过多年来对各种生物线粒体基因组结构和功能的研究,人们推测线粒体起源于紫色光合细菌,这种细菌入侵真核生物,与真核...     

线粒体DNA遗传特性的研究新进展

结合传统的线粒体DNA遗传特性理论与该领域的最新研究成果,从mtDNA的结构、组织特异性、母系遗传、进化速率及其与核基因组的相互关系等方面做了简要综述。同时,对今后的研究工作提出了一些建议。     

Plasmid DNA purification

The demand for efficient production methods of plasmid DNA (pDNA) has increased vastly in response to rapid advances in the use of pDNA in gene therapy and in vaccines since the advantageous safety concerns associated with non-viral over viral vectors.A prerequisite for the success of plasmid-based therapies is the development of cost-effective and generic production processes of pDNA. However, to satisfy strict regulatory guidelines, the material must be available as highly purified, homogeneous preparations of supercoiled circular covalently closed (ccc) pDNA. Large-scale production of pDNA for therapeutic use is a relatively new field in bioprocessing. The shift from small-scale plasmid production for cell transfection to large-scale production sets new constraints on the bacterial fermentation, processing of bacterial lysate and final purification and formulation of the plasmid DNA. The choice of bacterial strain used for plasmid cultivation affects the plasmid yield, the proportion of different isoforms and the amount of endotoxins in the starting material. The choice of bacterial strain will be greatly influenced by the production and purification procedures of pDNA. Master and working cell banks need to be characterised and established. Alkaline lysis of the bacteria damages the pDNA, resulting in a reduced recovery of ccc pDNA and an increase in partially denaturated ccc pDNA and open circular (oc) forms. Shear stress in these processes needs to be tightly controlled, and buffer composition and pH need to be optimised. To obtain a homogeneous plasmid DNA preparation, different pDNA purification strategies aim at capturing ccc pDNA and eliminating the oc isoform. A highly purified final product corresponding to the stringent recommendations set forth by health and regulatory authorities can be achieved by (i). different chromatography techniques integrated with ultra/diafiltration to achieve optimal purification results; (ii). the formulation of the final pDNA product, that requires a detailed study of the plasmid structure; and (iii). the development of sensitive analytical methods to detect different impurities (proteins, RNA, chromosomal DNA, and endotoxins). We present here a revue of the whole process to obtain such a plasmid DNA, and report an example of RNAse-free purification of ccc pDNA that could be used for gene therapy.     

Plasmid DNA vaccines

DNA vaccination is a novel approach for inducing an immune response. Purified plasmid DNA containing an antigen’s coding sequences and the necessary regulatory elements to expres them is introduced into the tissue via intramuscular injection or particle bombardment. Once the DNA reaches the tissue, the antigen is expressed in enough quantity to induce a potent and specific immune response and to confer protection against further infections. The effectiveness of DNA vaccines against viruses, parasites, and cancer cells has been demonstrated in numerous animal models. This new approach comes as an aid for the prevention of infectious diseases for which the conventional vaccines have failed. DNA vaccine research is providing new insights into some of the basic immunological mechanisms of vaccination such as antigen presentation, the role of effector cells, and immunoregulatory factors. In addition, DNA vaccines may enable us to manipulate the immune system in situations where the response to agents is inappropriate or ineffective. The study of the potential deleterious effects of DNA vaccines is furthering our knowledge regarding the relationship between bacterial DNA and the immune system, as well as its potential application for the study of neonatal tolerance and autoimmunity.     

Plasmid DNA vaccination

Plasmid DNA vaccination against tuberculosis is a very powerful and easy method for the induction of strong humoral responses, CD4+ mediated secretion of Th1 cytokines and CD8+ mediated CTL activity in mice. Tuberculosis DNA vaccines have not been assessed so far in humans, and clinical trials with DNA in general have been somewhat disappointing. However, numerous studies have reported on the potent priming capacity of plasmid DNA for Th1 and CD8+ mediated immune responses, which can be boosted subsequently by recombinant protein or recombinant pox-viruses. With respect to tuberculosis, prime/boost regimens with Mycobacterium bovis BCG vaccine are particularly promising and warrant further analysis.     

Direct Transfer of Plasmid DNA from Intact Yeast Spheroplasts into Plant Protoplasts

We developed a polyethylene glycol (PEG)-mediated direct DNAtransfer method from intact Saccharomyces cerevisiae spheroplastsinto Arabidopsis thaliana protoplasts. To monitor the DNA transferfrom yeast to plant cells, ß-glucuronidase (GUS) reportergene in which a plant intron was inserted was used as a reporter.This intron-GUS reporter gene on a 2µm-based plasmid vectorwas not expressed in yeast transformants, while it expressedGUS activity when the plasmid DNA was introduced into plantcells. When a mixture of 1 x 10⁸ of S. cerevisiae spheroplastsharboring the plasmid and 2 x 10⁶ of A. thaliana protoplastswas treated with PEG and high pH-high Ca²⁺ solution (0.4 M mannitol,50 mM CaCl₂, 50 mM glycine-NaOH pH 10.5), GUS activity was detectedin the extract of the plant cells after a three-day culture.The GUS activity was higher than that of a reconstitution experimentin which the mixture of 1 x 10⁸ of S. cerevisiae spheroplastswhich did not carry the reporter gene, 2 x 10⁶ of A. thalianaprotoplasts and the same amount of the reporter plasmid DNAas that contained in 1 x 10⁸ of S. cerevisiae spheroplasts,was treated with PEG and high pH-high Ca²⁺ solution. Moreover,the GUS gene expression was resistant to micrococcal nucleasetreatment before and during PEG treatment. From these results,we concluded that plasmid DNA can be directly transferred fromintact yeast spheroplasts to plant protoplasts by a nuclease-resistantprocess, possibly by the cell fusion. ²Deceased on September 15, 1992.     

Plasmid DNA transduction in Bacillus subtilis

Transduction of Bacillus subtilis pUB110 plasmid by AR9 phage is described. Some aspects of this process are studied. Plasmid transduction depended on multiplicity of infection similar to cases of chromosomal markers transduction, though optimal multiplicity of infection was achieved using low number of phage particles. No cotransduction of plasmid and chromosomal markers was demonstrated. The transduction frequencies of plasmid and chromosomal markers increased after UV irradiation of phage suspensions within the range of definite doses.     

Plasmid Loss in Agaricus bitorquis without Alterations in Homologous Mitochondrial Sequences

Robison, M. M., and Horgen, P. A. (1993). Plasmid loss in Agaricus bitorquis without alterations in homologous mitochondrial sequences. Experimental Mycology , 18, 82-86. An isolate of the basidiomycete Agaricus bitorquis , a common edible lawn mushroom, contains two linear mitochondrial plasmids. With the intent of creating a plasmidless (or "cured") version of this isolate for future studies on a plasmid-homologous mitochondrial sequence, mycelium was grown in the presence of 50 μg/ml ethidium bromide. Regenerates were recovered that had lost only the larger plasmid or both plasmids. No obvious effects on mycelial or mushroom phenotype, associated with plasmid loss, were observed. Restriction fragment length polymorphism analysis of mitochondrial DNAs from 23 regenerates indicated that no apparent deletions or rearrangements of the mitochondrial DNA, particularly the plasmid-homologous mitochondrial sequence, occurred as a result of plasmid loss or ethidium bromide treatment.