家蚕核型多角体病毒gp64基因的定位及克隆

以苜蓿丫纹夜蛾核型多角体病毒ＡｃＭＮＰＶ的膜表面糖蛋白基因ｇｐ６４作为探针，对经不同内切酶酶切的家蚕核型多角体病毒基因组ＤＮＡ进行Ｓｏｕｔｈｅｒｎ杂交分析，发现ＢａｍＨⅠ酶切产生的４．２ｋｂ和７．６ｋｂ片段呈阳性杂交，经ＤＮＡ片段回收，将４．２ｋｂ片段进行了克隆     

福建两个蛋鸭品种和常见野鸭线粒体DNA（mtDNA）限制性酶？…

用７种限制性内切酶分析了蛋鸭,野鸭的ｍｔＤＮＡ限制性类型,并用双酶法构建了以上鸭类的ｍｔＤＮＡ限制性酶切图谱。结果表明,蛋鸭与野鸭在ｍｔＤＮＡ结构上发生了明显分歧,比较两种野鸭和金定鸭的图变,发现金定鸭与绿头鸭的亲缘关系较近。     

柳毒蛾核型多角体病毒内蒙株（LsNPV—IM）DNA的限制性 …

将柳毒蛾核型多角体悬液降解,降解产物经Ｔｒｉｓ－饱和酚和氯仿抽提,乙醇沉淀分离出ＬｓＮＰＶ－ＤＮＡ,用限制性内切酶ＰｓｔＩ,ＨｉｎｄⅢ,ＰｓｔＩ／ＨｉｎｄⅢ,ＨｉｎｄⅢ／ＥｃｏＲＩ和Ｐｓｔ／ＢａｍＨＩ单酶切或双酶切,经琼脂糖凝胶电泳并对其结果进行了分析,建立了酶切图谱。     

豌豆中GPAT基因编码cDNA的克隆及分析

参照国外报道的豌豆ＧＰＡＴ基因ｃＤＮＡ序列，设计并合成一对寡核苷酸引物，通过ＲＴＰＣＲ技术，从云南地方栽种的豌豆品种中分离到了ＧＰＡＴ基因的编码ｃＤＮＡ片段，并将其纯化后直接克隆到ｐＧＥＭＴ载体系统中，经ＮｃｏⅠ和ＮｏｔⅠ双酶切鉴定，所得的重组质粒中含有１３８０ｂｐ左右的片段．采用ＰｓｔⅠ，ＨｉｎｄⅢ两种限制性内切酶进行酶切，酶切图谱分析表明所克隆的豌豆ＧＰＡＴ基因编码ｃＤＮＡ的酶切图谱与国外所报道的该基因ｃＤＮＡ的酶切图谱一致，暗示了该基因在进行上具有一定的保守性．     

福建两个蛋鸭品种和常见野鸭线粒体DNA(mtDNA)限制性酶切图谱的比较

用７种限制性内切酶（ＢａｍＨⅠ、ＢｇｌⅡ、ＥｃｏＲⅠ、ＥｏｃＲⅤ、ＰｓｔⅠ、ＳａｃⅠ、ＸｂａⅠ）分析了蛋鸭（金定鸭、莆田黑鸭）、野鸭（斑嘴鸭、绿头鸭）的ｍｔＤＮＡ限制性类型,并用双酶法构建了以上鸭类的ｍｔＤＮＡ限制性酶切图谱．结果表明,蛋鸭与野鸭在ｍｔＤＮＡ结构上发生了明显分岐．比较两种野鸭和金定鸭的图谱,发现金定鸭与绿头鸭的亲缘关系较近．     

小灵猫华东亚种线粒体DNA限制性位点图

提纯了小灵猫华东亚种（ＶｉｖｅｒｒｉｃｕｌａｉｎｄｉｃａｐａｌｉｄａＧｒａｙ）的肝脏ｍｔＤＮＡ．用ＢａｍＨⅠ、ＢｇｌⅡ、ＥｃｏＲⅤ、ＨｐａⅠ、ＫｐｎⅠ、ＭｌｕⅠ、ＰｓｔⅠ、ＰｖｕⅡ、ＳａｃⅡ、ＳａｌⅠ和ＸｈｏⅠ等１１种识别６碱基对的限制性内切酶对小灵猫华东亚种ｍｔＤＮＡ进行消化分析，１１种限制性内切酶均有１～５个位点；根据单酶消化和双酶消化片段的数目和分子量，构建了小灵猫华东亚种ｍｔＤＮＡ限制性位点图     

蓖麻蚕蛹mtDNA的限制性内切酶图谱

采用碱变性法制备蓖麻蚕蛹ｍｔＤＮＡ，经九种限制性内切酶单酶和双酶酶解后，琼脂糖凝胶电泳检测酶切位点数和酶切片段长度。根据片段的大小确定消失片段与新生片段关系，通过对片段重叠、拼接、判断各片段邻近位置，从而构建了９种限制性内切酶、共２４个酶切位点蓖麻蚕蛹ｍｔＤＮＡ酶切图谱。     

中国锦铃虫核多角体病毒（HeliothisarmigeraNPV）VHA273毒株 …

ＶＨＡ２７３毒株原为ＭＮＰＶ型，经纯系中国棉铃虫扩增而得出约１０％的ＳＮＰＶ，高度纯化两型多角体，温和碱解后，酚法抽提ＳＮＰＶＤＮＡ和ＭＮＰＶＤＮＡ，限制性内切酶ＢｇｌⅡ，ＢａｍＨＩ，ＥｃｏＲＩ和ＨｉｎｄⅢ分别平行酶切两种ＤＮＡ，依次均得出１１，８，１４和１３条电泳带。     

红莲型水稻不育系与保持系线粒体DNA酶切分析

为研究红莲型水稻细胞质雄性不育的分子基础，我们提取纯化了红莲型水稻丛广４１Ａ和丛广４１Ｂ的线粒体ＤＮＡ，采用限制性内切酶酶切片段长度多态性（ＲＦＬＰ）方法比较了红莲型不育系和保持系线粒体ＤＮＡ酶切图谱，发现两者之间存在一定的差异，部分支持了细胞质雄性不育与线粒体ＤＮＡ有关的结论。     

小菜蛾颗粒体病毒DNA限制性酶切分析及基因组DNA片段克隆

小菜蛾颗粒体病毒ＤＮＡ用ＢａｍＨＩ和ＸｈｏＩ酶切，经０．７５％琼脂糖凝胶电泳，分别得到１２条带和８条带，平均分子量为１１３．０ｋｂ。用Ｓｕｐｅｒｃｏｓ１ Ｃｏｓｍｉｄ作载体，将Ｓａｕ３ＡＩ部分消化的ＰｘＧＶ－ＤＮＡ随机片段插入该载体的ＢａｍＨＩ酶切位点，转化于ＸＬ１－Ｂｌｕｅ受体菌，经Ａｍｐ平板筛选转子，挑出２５６个Ａｍｐ＾＋菌落，以＾３２Ｐ－ｄＣＴＰ标记的ＰｘＧＶ－ＤＡＮ为探针，斑点杂交筛选得到     

Single-molecule analysis of DNA uncoiling by a type II topoisomerase

Type II DNA topoisomerases are ubiquitous ATP-dependent enzymes capable of transporting a DNA through a transient double-strand break in a second DNA segment. This enables them to untangle DNA and relax the interwound supercoils (plectonemes) that arise in twisted DNA. In vivo, they are responsible for untangling replicated chromosomes and their absence at mitosis or meiosis ultimately causes cell death. Here we describe a micromanipulation experiment in which we follow in real time a single Drosophila melanogaster topoisomerase II acting on a linear DNA molecule which is mechanically stretched and supercoiled. By monitoring the DNA's extension in the presence of ATP, we directly observe the relaxation of two supercoils during a single catalytic turnover. By controlling the force pulling on the molecule, we determine the variation of the reaction rate with the applied stress. Finally, in the absence of ATP, we observe the damping of a DNA crossover by a single topoisomerase on at least two different timescales (configurations). These results show that single molecule experiments are a powerful new tool for the study of topoisomerases.     

Optical mapping of a rice BAC clone using restriction endonuclease and imaging with fluorescent microscopy at single molecule level

A method of constructing restriction map by optical mapping and single molecule fluorescent microscopy is described. DNA molecules were aligned and adsorbed on a glass coverslip surface by a modified “molecular combing” technique, and then the surface-immobilized DNAs were cleaved in situ with a restriction endonuclease. Individual DNA molecules digested by the endonuclease EcoRⅠ were observable with fluorescent microscopy. Using optical mapping, a physical map of a rice bacterial artificial chromosome clone was constructed. This method will facilitate genomic mapping and tracing the dynamic process in real time at a single molecule level with fluorescence microscopy.     

Processive translocation and DNA unwinding by individual RecBCD enzyme molecules

RecBCD enzyme is a processive DNA helicase and nuclease that participates in the repair of chromosomal DNA through homologous recombination. We have visualized directly the movement of individual RecBCD enzymes on single molecules of double-stranded DNA (dsDNA). Detection involves the optical trapping of solitary, fluorescently tagged dsDNA molecules that are attached to polystyrene beads, and their visualization by fluorescence microscopy. Both helicase translocation and DNA unwinding are monitored by the displacement of fluorescent dye from the DNA by the enzyme. Here we show that unwinding is both continuous and processive, occurring at a maximum rate of 972 +/- 172 base pairs per second (0.30 microm s(-1)), with as many as 42,300 base pairs of dsDNA unwound by a single RecBCD enzyme molecule. The mean behaviour of the individual RecBCD enzyme molecules corresponds to that observed in bulk solution.     

Selection in vitro of an RNA enzyme that specifically cleaves single-stranded DNA.

The discovery of RNA enzymes has, for the first time, provided a single molecule that has both genetic and catalytic properties. We have devised techniques for the mutation, selection and amplification of catalytic RNA, all of which can be performed rapidly in vitro. Here we describe how these techniques can be integrated and performed repeatedly within a single reaction vessel. This allows evolution experiments to be carried out in response to artificially imposed selection constraints. We worked with the Tetrahymena ribozyme, a self-splicing group I intron derived from the large ribosomal RNA precursor of Tetrahymena thermophila that catalyses sequence-specific phosphoester transfer reactions involving RNA substrates. It consists of 413 nucleotides, and assumes a well-defined secondary and tertiary structure responsible for its catalytic activity. We selected for variant forms of the enzyme that could best react with a DNA substrate. This led to the recovery of a mutant form of the enzyme that cleaves DNA more efficiently than the wild-type enzyme. The selected molecule represents the discovery of the first RNA enzyme known to cleave single-stranded DNA specifically.     

Single-molecule imaging of DNA pairing by RecA reveals a three-dimensional homology search

DNA breaks can be repaired with high fidelity by homologous recombination. A ubiquitous protein that is essential for this DNA template-directed repair is RecA. After resection of broken DNA to produce single-stranded DNA (ssDNA), RecA assembles on this ssDNA into a filament with the unique capacity to search and find DNA sequences in double-stranded DNA (dsDNA) that are homologous to the ssDNA. This homology search is vital to recombinational DNA repair, and results in homologous pairing and exchange of DNA strands. Homologous pairing involves DNA sequence-specific target location by the RecA-ssDNA complex. Despite decades of study, the mechanism of this enigmatic search process remains unknown. RecA is a DNA-dependent ATPase, but ATP hydrolysis is not required for DNA pairing and strand exchange, eliminating active search processes. Using dual optical trapping to manipulate DNA, and single-molecule fluorescence microscopy to image DNA pairing, we demonstrate that both the three-dimensional conformational state of the dsDNA target and the length of the homologous RecA-ssDNA filament have important roles in the homology search. We discovered that as the end-to-end distance of the target dsDNA molecule is increased, constraining the available three-dimensional (3D) conformations of the molecule, the rate of homologous pairing decreases. Conversely, when the length of the ssDNA in the nucleoprotein filament is increased, homology is found faster. We propose a model for the DNA homology search process termed 'intersegmental contact sampling', in which the intrinsic multivalent nature of the RecA nucleoprotein filament is used to search DNA sequence space within 3D domains of DNA, exploiting multiple weak contacts to rapidly search for homology. Our findings highlight the importance of the 3D conformational dynamics of DNA, reveal a previously unknown facet of the homology search, and provide insight into the mechanism of DNA target location by this member of a universal family of proteins.     

Cloned and expressed nitric oxide synthase structurally resembles cytochrome P-450 reductase.

Nitric oxide is a messenger molecule, mediating the effect of endothelium-derived relaxing factor in blood vessels and the cytotoxic actions of macrophages, and playing a part in neuronal communication in the brain. Cloning of a complementary DNA for brain nitric oxide synthase reveals recognition sites for NADPH, FAD, flavin mononucleotide and calmodulin as well as phosphorylation sites, indicating that the synthase is regulated by many different factors. The only known mammalian enzyme with close homology is cytochrome P-450 reductase.     

Chi-sequence recognition and DNA translocation by single RecBCD helicase/nuclease molecules

Major pathways of recombinational DNA repair in Escherichia coli require the RecBCD protein--a heterotrimeric, ATP-driven, DNA translocating motor enzyme. RecBCD combines a highly processive and exceptionally fast helicase (DNA-unwinding) activity with a strand-specific nuclease (DNA-cleaving) activity (refs 1, 2 and references therein). Recognition of the DNA sequence 'chi' (5'-GCTGGTGG-3') switches the polarity of DNA cleavage and stimulates recombination at nearby sequences in vivo. Here we attach microscopic polystyrene beads to biotin-tagged RecD protein subunits and use tethered-particle light microscopy to observe translocation of single RecBCD molecules (with a precision of up to approximately 30 nm at 2 Hz) and to examine the mechanism by which chi modifies enzyme activity. Observed translocation is unidirectional, with each molecule moving at a constant velocity corresponding to the population-average DNA unwinding rate. These observations place strong constraints on possible movement mechanisms. Bead release at chi is negligible, showing that the activity modification at chi does not require ejection of the RecD subunit from the enzyme as previously proposed; modification may occur through an unusual, pure conformational switch mechanism.     

A general method for site-directed mutagenesis in prokaryotes

The genetic analysis of genes from prokaryotic species for which experimental genetic systems have not yet been developed is often limited by the difficulty of producing mutations in those genes. We report here a general technique applicable to Gram-negative prokaryotes for site-directed mutagenesis of cloned DNA fragments which we have applied to the study of the symbiotic nitrogen fixation genes of Rhizobium meliloti. In particular, we mutagenized cloned R. meliloti restriction fragments in Escherichia coli with transposon Tn5 and then replaced the wild-type parental DNA sequences with the mutant DNA sequences in the R. meliloti genome. Using this method we show that an R. meliloti DNA restriction fragment, cloned previously on the basis of homology to Klebsiella pneumoniae nif genes, contains gene(s) essential for symbiotic nitrogen fixation. In addition, we use this method to construct a physical genetic map of a subset of the R. meliloti nif genes.     

从大肠杆菌中获得高浓度质粒DNA的方法

通过改进菌体的培养以及质粒小提方法,可以简便快捷地得到浓度很高的质粒DNA,经琼脂糖凝胶电泳、紫外分光光度法、酶切、PCR的验证,所提质粒可以应用于分子生物学的研究,特别是可以满足瞬时基因枪转化的要求。     

Translocation step size and mechanism of the RecBC DNA helicase

DNA helicases are ubiquitous enzymes that unwind double-stranded DNA. They are a diverse group of proteins that move in a linear fashion along a one-dimensional polymer lattice--DNA--by using a mechanism that couples nucleoside triphosphate hydrolysis to both translocation and double-stranded DNA unwinding to produce separate strands of DNA. The RecBC enzyme is a processive DNA helicase that functions in homologous recombination in Escherichia coli; it unwinds up to 6,250 base pairs per binding event and hydrolyses slightly more than one ATP molecule per base pair unwound. Here we show, by using a series of gapped oligonucleotide substrates, that this enzyme translocates along only one strand of duplex DNA in the 3'-->5' direction. The translocating enzyme will traverse, or 'step' across, single-stranded DNA gaps in defined steps that are 23 (+/-2) nucleotides in length. This step is much larger than the amount of double-stranded DNA that can be unwound using the free energy derived from hydrolysis of one molecule of ATP, implying that translocation and DNA unwinding are separate events. We propose that the RecBC enzyme both translocates and unwinds by a quantized, two-step, inchworm-like mechanism that may have parallels for translocation by other linear motor proteins.