Modified bases characterized in intact DNA by mass-analyzed ion kinetic energy spectrometry

Pyrolysis of DNA into a chemical ionization source yields protonated bases and other base-containing ions. Kinetic energy spectra allow the characterization of the bases 5-methylcytosine and 1-methyladenine from underivatized salmon sperm DNA. Isomeric bases are distinguishable with this technique.     

The Study of Relativity between the DNA Conformation and the Canceration

The conformation of DNA in the granuloblast of the human leukaemia(HL) is also different from that of the normal human (HN) was determined by the fluorescence titration.These results show the view that DNA conformation variety is related with the canceration of human cell.     

The energetic basis of specificity in the Eco RI endonuclease--DNA interaction

High sequence selectivity in DNA-protein interactions was analyzed by measuring discrimination by Eco RI endonuclease between the recognition site GAATTC and systematically altered DNA sites. Base analogue substitutions that preserve the sequence-dependent conformational motif of the GAATTC site permit deletion of single sites of protein-base contact at a cost of +1 to +2 kcal/mol. However, the introduction of any one incorrect natural base pair costs +6 to +13 kcal/mol in transition state interaction energy, the resultant of the following interdependent factors: deletion of one or two hydrogen bonds between the protein and a purine base; unfavourable steric apposition between a group on the protein and an incorrectly placed functional group on a base; disruption of a pyrimidine contact with the protein; loss of some crucial interactions between protein and DNA phosphates; and an increased energetic cost of attaining the required DNA conformation in the transition state complex. Eco RI endonuclease thus achieves stringent discrimination by both "direct readout" (protein-base contracts) and "indirect readout" (protein-phosphate contacts and DNA conformation) of the DNA sequence.     

思茅松毛虫6龄幼虫肠道细菌的ARDRA分析与鉴定

从自然种群的思茅松毛虫(Dendrolimus kikuchii Matsumura)6龄幼虫肠道环境中分离、纯化、培养获得了6株细菌.以细菌基因组DNA为模板进行PCR扩增,用2种双酶组合Hae Ⅲ和Hin d Ⅲ、HinfⅠ和TaqⅠ对PCR产物进行核糖体DNA扩增片段限制性内切酶分析(ARDRA),得到了3个操作分类单元(OTUs).对每个操作分类单元(OTU)的代表菌株进行16 S rDNA序列测定,序列分析结果表明,分离到的6株肠道细菌中,6N01、6N02、6N03、6N04和6N05属于芽孢杆菌属(Bacillus sp.),6N06属于克雷伯氏杆菌属(Klebsiella sp.).     

Single strands, triple strands, and kinks in H-DNA

A naturally occurring (dT-dC)18:(dA-dG)18 repeat in the H conformation of DNA was shown to contain single-stranded nucleotides in the center of the TC18 repeat and on one half of the AG18 repeat. These results support the model that H-DNA is a structure containing both triple-stranded and single-stranded regions. The stability of this structure was affected by both pH and the degree of negative supercoiling: at pH 7.6 to 7.7, a high level of supercoiling was needed to keep about half of the molecules in the H conformation; at pH 6 and pH 5, normal levels of supercoiling supported H-DNA; and at pH 4, no supercoiling was required. At mildly alkaline pH, the TC/AG18 repeat assumed a novel conformation called J-DNA that differed from both the B and H forms. A three-dimensional model for the structure of H-DNA is proposed that accounts both for the single-strandedness of the nucleotides and for the influence of supercoiling on H-DNA formation. This model predicts and evidence is presented that H-DNA introduces a sharp kink in the DNA. Moreover, the angle of this kink appears not to be fixed, so that H-DNA is also a hinged-DNA.     

Recombinant antibodies to the small GTPase Rab6 as conformation sensors

Here we report an approach, based on antibody phage display, to generate molecular conformation sensors. Recombinant antibodies specific to the guanosine triphosphate (GTP)-bound conformation of the small guanosine triphosphatase (GTPase) Rab6, a regulator of membrane traffic, were generated and used to locate Rab6.GTP in fixed cells, and, after green fluorescent protein (GFP) tagging and intracellular expression, to follow Rab6.GTP in vivo. Rab6 was in its GTP-bound conformation on the Golgi apparatus and transport intermediates, and the geometry of transport intermediates was modulated by Rab6 activity. More generally, the same approach could be applied to other molecules that can be locked in a particular conformation in vitro.     

Iron(II) EDTA used to measure the helical twist along any DNA molecule

A new method has been devised to measure the number of base pairs per helical turn along any DNA molecule in solution. A DNA restriction fragment is adsorbed onto crystalline calcium phosphate, fragmented by reaction with iron(II) EDTA, and subjected to electrophoresis on a denaturing polyacrylamide gel. A modulated cutting pattern results, which gives directly the helical periodicity of the DNA molecule. A 150-base pair sequence directly upstream of the thymidine kinase gene of the type 1 herpes simplex virus was found to have an overall helical twist of 10.5 base pairs per turn, which is characteristic of the B conformation of DNA. In addition, purines 3' to pyrimidines showed lower than expected reactivity toward the iron cutting reagent, which is evidence for sequence-dependent variability in DNA conformation.     

1-methyladenine biosynthesis in starfish ovary: action of gonad-stimulating hormone in methylation

Gonad-stimulating substance, a hormonal peptide released from the nervous system of starfishes, acts on the ovary to produce 1-methyladenine, an inducer of oocyte maturation. Addition of methionine to the incubation mixture of ovarian fragments and gonad-stimulating substance enhanced the production of 1-methyladenine, whereas ethionine inhibited it. Incubation of ovarian tissue with methionine alone failed to produce 1-methyladenine. Use of a radioactive label showed that methionine is a methyl donor in the biosynthesis of 1-methyladenine, suggesting that gonad-stimulating substance is involved in the methylation process.     

Structural basis for DNA damage-dependent poly(ADP-ribosyl)ation by human PARP-1

Poly(ADP-ribose) polymerase-1 (PARP-1) (ADP, adenosine diphosphate) has a modular domain architecture that couples DNA damage detection to poly(ADP-ribosyl)ation activity through a poorly understood mechanism. Here, we report the crystal structure of a DNA double-strand break in complex with human PARP-1 domains essential for activation (Zn1, Zn3, WGR-CAT). PARP-1 engages DNA as a monomer, and the interaction with DNA damage organizes PARP-1 domains into a collapsed conformation that can explain the strong preference for automodification. The Zn1, Zn3, and WGR domains collectively bind to DNA, forming a network of interdomain contacts that links the DNA damage interface to the catalytic domain (CAT). The DNA damage-induced conformation of PARP-1 results in structural distortions that destabilize the CAT. Our results suggest that an increase in CAT protein dynamics underlies the DNA-dependent activation mechanism of PARP-1.     

Valinomycin crystal structure determination by direct methods

The conformation of an uncomplexed form of the antibiotic valinomycin (C(54)N(6)O(18)H(90)) has been determined by direct methods including a novel technique for strong enantiomorph discrimination via the calculation and systematic analysis of cosine invariants of a special type. The intramolecular hydrogen bonding scheme and the isopropyl group stereochemistry of uncomplexed valinomycin are compatible with interpretations of spectral measurements for the complexed and uncomplexed molecule in solution but are different from any previously proposed structure. The simple conformational change of a hydrogen bond shift, which could be induced by the process of potassium ion complexing, transforms the uncomplexed into the complexed structure.     

The DNA binding arm of lambda repressor: critical contacts from a flexible region.

Segments of protein that do not adopt a well-ordered conformation in the absence of DNA can still contribute to site-specific recognition of DNA. The first six residues (NH2-Ser1-Thr2-Lys3-Lys4-Lys5-Pro6-) of phage lambda repressor are flexible but are important for site-specific binding. Low-temperature x-ray crystallography and codondirected saturation mutagenesis were used to study the role of this segment. All of the functional sequences have the form [X]1-[X]2-[Lys or Arg]3-[Lys]4-[Lys or Arg]5-[X]6. A high-resolution (1.8 angstrom) crystal structure shows that Lys3 and Lys4 each make multiple hydrogen bonds with guanines and that Lys5 interacts with the phosphate backbone. The symmetry of the complex breaks down near the center of the site, and these results suggest a revision in the traditional alignment of the six lambda operator sites.     

Z-DNA forms without an alternating purine-pyrimidine sequence in solution

Nuclear magnetic resonance spectra (proton and phosphorus-31) and ultraviolet absorption spectra of the DNA decamer d(br5CGbr5CGATbr5CGbr5CG), in which the central two adenine-thymine base pairs are out of order with the rest of the purine-pyrimidine alternation sequence, indicate that under appropriate solvent conditions (high salt and methanol) the molecule undergoes a structural transition from a right-handed B-DNA conformation to a left-handed Z-DNA conformation. Measurements of the two-dimensional nuclear Overhauser effect on the decamer indicate that all of the guanines as well as the two equivalent thymines adopt the syn conformation.     

The collapse of free polymer chains in a network

The conformation of linear polymer chains trapped in a matrix of cross-linked polymer has been measured by neutron scattering. Three regimes were found depending on the length of the linear chain, Nl, with respect to the mesh size of the network, N(c). When N(c) > Nl, the radius of gyration, R(g), of the linear chain is the same as that observed in the uncrosslinked melt. When N(c) < Nl, R(g) shrinks according to the scaling relation R(g)(-1) approximately N(c)(-1) that has been predicted for isolated polymer chains trapped in a field of random obstacles. When N(c) < Nl the linear chains are observed to segregate.     

N~+介导对水稻幼苗中性蛋白水解酶表达的影响

利用蛋白质复性电泳技术,研究了低能N+注入介导玉米DNA的水稻幼苗叶片和根系中性蛋白水解酶的表达。用能量为25 keV、剂量为3.0×1017N+/cm2的氮离子束对水稻豫粳6号种胚进行照射处理,照射的种子于质量浓度为100μg/mL玉米基因组DNA介导液中进行转化处理,其他处理同时进行,结果表明,各处理均引起水稻幼苗叶片和根系中性蛋白水解酶种类及活性差异表达,叶片与根系中均检测出多条差异酶带,离子束介导玉米DNA的处理中性蛋白水解酶带变化活跃,有新酶带表达,也有部分酶带缺失。说明低能离子束介导远缘大分子DNA引起水稻幼苗蛋白质表达的差异。     

DNA分子标记在动物种群遗传结构研究中的应用

简述了DNA分子标记包括限制性片段长度多态性(RFLP)、随机扩增多态DNA(RAPD)、微卫星标记、小卫星标记、扩增片段长度多态性(AFLP)、单链构象多态性(SSCP)及单核苷酸多态性(SNP)的原理及特点,并对分子标记技术在动物群体遗传结构研究中的部分成果做了概述。     

贵州地方猪品种脂蛋白脂酶基因第8内含子的多态性研究

以可乐猪、萝卜猪和香猪3个贵州地方猪种和欧洲猪种为材料,采用PCR-SSCP技术对脂蛋白脂酶（Lip-oprotein lipase,LPL）基因第8内含子的多态性进行了研究。以特异性PCR从样品基因组中扩增出163bp的DNA片段,经非变性聚丙烯酰胺凝胶电泳,从两个猪群体的LPL基因第8内含子中都检测到了AA基因型和AB基因型,未检测到BB基因型;欧洲猪品种和贵州地方猪品种均以AB基因型占优势;与欧洲猪品种相比,贵州地方猪品种的B等位基因频率明显较高,A等位基因频率则较低。     

Parasitism and pathogenicity of Radopholus similis to Ipomoea aquatica,Basella rubra and Cucurbita moschata and genetic diversity of different populations

Ten populations of Radopholus similis from different ornamental hosts were tested for their parasitism and pathogenicity to water spinach(Ipomoea aquatic), malabar spinach(Basella rubra), and squash(Cucurbita moschata) in pots. The results showed all three plants were new hosts of R. similis. Growth parameters of plants inoculated with nematodes were significantly lower than those of healthy control plants. All R. similis populations were pathogenic to the three plants, but pathogenicity differed among populations from different hosts. The same R. similis populations also showed different pathogenic effects in the three different plants. Rad N5 population from Anthurium andraeanum had the highest pathogenicity to the three studied plants. Rad N1 from A. andraeanum had the lowest pathogenicity to squash and Rad N7 from Chrysalidocarpus lutesens had the lowest pathogenicity to water spinach and malabar spinach. R. similis is usually associated with root tissues, but here we report that it could be found to move and feed in the stem bases of all three studied plants. Sequence and phylogenetic analyses of DNA markers of the 18 S r RNA, 28 S r RNA, ITS r RNA, and mitochondrial DNA gene sequences of ten R. similis populations revealed significant genetic diversity. Rad N5 and Rad N6 populations from anthurium showed a close genetic relationship and could be distinguished from other populations by PCR-RFLP. At the same time, Rad N5 and Rad N6 populations were the most pathogenic to three studied plants. These results confirm the e xistence of large biological variability and molecular diversity among R. similis populations from the same or different hosts, and these characteristics are related to pathogenic variability.     

The structure of an infectious P22 virion shows the signal for headful DNA packaging

Bacteriophages, herpesviruses, and other large double-stranded DNA (dsDNA) viruses contain molecular machines that pump DNA into preassembled procapsids, generating internal capsid pressures exceeding, by 10-fold, that of bottled champagne. A 17 angstrom resolution asymmetric reconstruction of the infectious P22 virion reveals that tightly spooled DNA about the portal dodecamer forces a conformation that is significantly different from that observed in isolated portals assembled from ectopically expressed protein. We propose that the tight dsDNA spooling activates the switch that signals the headful chromosome packing density to the particle exterior.