从植物细胞核分离大分子量核DNA

研究了从植物中分离百万碱基对级大分子量核DNA的方法。该方法利用差速离心分离植物细胞核,经低熔点琼脂糖块或低熔点琼脂糖微珠包埋,蛋白酶K原位裂解后制备大分子量核DNA。结果表明,选择不同生长时期的材料和不同的包埋细胞核方式对大分子量核DNA的制备有很大的影响,由黄化苗或幼嫩的绿叶为材料分离细胞核,进行胶块包埋是制备大分子量核DNA的最佳条件。利用该法获得的DNA分子量在200kb－5．7Mb之间,主要集中在2．2～5．7Mb之间;每一胶块DNAE量为18～20μg。与包埋原生质体制备大分子量核DNA的方法相比,该方法获得的DNA纯度较高,去除了大部分细胞器DNA的污染;易于被限制性内切酶部分和完全消化,其消化结果具可重复性。该方法操作简单、适用植物种类广泛,用该方法从水稻（OryzasativaL．）、苹果（MaluspumilaMill．）、大豆（Glycinemax（L．）Merr．）、玉米（ZeamaysL．）等多种植物材料中成功地制备了大分子量核DNA。该方法制备的核DNA适用于植物的脉冲交变电泳基因组分析和构建人工细菌染色体文库和人工酵母染色体文库。     

Regulation of Macronuclear DNA Content in Tetrahymena thermophila*†

Synopsis.
Unequal macronuclear division in Tetrahymena thermophila introduces variance into G1 macronuclei; unless eliminated such variance would result in continuous variation in DNA content. Analysis of G1 and G2 macronuclear variances reveals that the added variance is eliminated by action on the extremes of macronuclear DNA content. In this model (Model II), macronuclei with small amounts of DNA have an additional complete S phase, while those with large amounts of DNA skip S. From available data, chromatin extrusion is shown not to contribute significantly, if at all, to the elimination of variance. Computer simulations utilizing haploid subunits indicate that model II predictions apply reasonably well to experimental data in terms of coefficients of variation, mean DNA content, and frequency of additional and skipped S phases. The simulations reveal also that within certain constraints, particularly the thresholds for additional and skipped S phases, macronuclear assortment is unaffected by Model II regulation. The relationships between Model II and other aspects of the cell cycle are briefly discussed.     

Properties of Purified L-Ornithine Decarboxylase (EC 4.1.1.17) from Tetrahymena thermophila

Ornithine decarboxylase (ornithine carboxy lyase; EC 4.1.1.17) (ODC) from Tetrahymena thermophila was purified 6,300 fold employing fractionated ammonium sulfate precipitation, gel permeation chromatography on Sephadex G-150, ion exchange chromatography on DEAE-Sepharose CL-6B, and preparative isoelectric focussing. The product obtained in 24% yield was a preparation of the specific activity of 10,200 nmol CO₂mdh^-1mdmg^-1. The purified enzyme was rather stable at 37°C (14% loss of activity within 1 h). The molecular and catalytic properties of this enzyme were investigated. The isoelectric point was 5.7 and the molecular weight (MW) was estimated to be 68,000 under nondenaturing conditions. The pH optimum was between 6.0 and 7.0, the K_m for the substrate L-ornithine was 0.11 mM, and the K_m for the cofactor pyridoxal 5-phosphate was 0.12 μM; the product of ODC catalysis, putrescine, was a poor inhibitor with an estimated K_i of about 10 mM. The enzyme was inhibited competitively by D-ornithine with a K_i of 1.6 mM and by α-difluoromethylornithine with a K_i of 0.15 mM. The latter one, an enzyme activated irreversible inhibitor of mammalian ODC, inactivated the enzyme from T. thermophila at high concentrations with a half life time of 14 min. Other basic amino acids, e.g. L-lysine, L-arginine, and L-histidine, were neither substrates nor inhibitors of the enzyme, as were the diamines 1,3-diaminopropanol and cadaverine, the polyamines spermidine and spermine and the cosubstrate analogues pyridoxal and pyridoxamine-5-phosphate. Polyanions were activators of the enzyme: The half maximal ODC stimulating concentrations were 2.2 μgmdml^-1 for RNA, 6.1 μgmdml^-1 for DNA, and 0.25 μgmdml^-1 for heparin. These results indicate that ODC from T. thermophila shares several properties with ODC preparations from other organisms but in some respects, especially in activator and inhibitor specificity, there are some special qualities unique to this particular protozoan enzyme.     

Isolation of Sperm Vesicles from Adult Male Mayflies and Other Insects to Prepare High Molecular Weight Genomic DNA Samples

We describe here a simple and efficient protocol for genomic DNA isolation from adult males of insects: e.g., Ephemeroptera, Odonata, Orthoptera and Dictyoptera. To minimize contamination of external DNA source, the sperm vesicles were isolated from male individuals from which high molecular weight genomic DNA was extracted. According to this protocol, the genomic DNA samples obtained were high quality (intact), and abundant enough for genotyping analyses and molecular cloning. The protocol reported here enables us to process a huge number of individuals at a time with escaping from cross-contamination, and thus it is quite useful for conducting genetic studies at least in some species of insects. The large yield of high molecular weight DNA from single individual may be advantageous for non PCR-based experiments. As a case study of the protocol, partial coding sequences of histone H3 and EF-1α genes are determined for some insects with PCR-amplified DNA fragments.     

Extraction of High Molecular Weight Genomic DNA from Soils and Sediments

The soil microbiome is a vast and relatively unexplored reservoir of genomic diversity and metabolic innovation that is intimately associated with nutrient and energy flow within terrestrial ecosystems. Cultivation-independent environmental genomic, also known as metagenomic, approaches promise unprecedented access to this genetic information with respect to pathway reconstruction and functional screening for high value therapeutic and biomass conversion processes. However, the soil microbiome still remains a challenge largely due to the difficulty in obtaining high molecular weight DNA of sufficient quality for large insert library production. Here we introduce a protocol for extracting high molecular weight, microbial community genomic DNA from soils and sediments. The quality of isolated genomic DNA is ideal for constructing large insert environmental genomic libraries for downstream sequencing and screening applications. The procedure starts with cell lysis. Cell walls and membranes of microbes are lysed by both mechanical (grinding) and chemical forces (β-mercaptoethanol). Genomic DNA is then isolated using extraction buffer, chloroform-isoamyl alcohol and isopropyl alcohol. The buffers employed for the lysis and extraction steps include guanidine isothiocyanate and hexadecyltrimethylammonium bromide (CTAB) to preserve the integrity of the high molecular weight genomic DNA. Depending on your downstream application, the isolated genomic DNA can be further purified using cesium chloride (CsCl) gradient ultracentrifugation, which reduces impurities including humic acids. The first procedure, extraction, takes approximately 8 hours, excluding DNA quantification step. The CsCl gradient ultracentrifugation, is a two days process. During the entire procedure, genomic DNA should be treated gently to prevent shearing, avoid severe vortexing, and repetitive harsh pipetting.Download video file.^{(129M, mp4)}     

从石蜡包埋组织中获取高质量基因组DNA的方法改进

从石蜡包埋组织中制备基因组DNA(gDNA)难度大、获取样本量很少.传统方法为二甲苯脱蜡法.为了简化操作,获得更高质量的gDNA,我们采用水浴法替代二甲苯进行脱蜡,亲和层析纯化石蜡包埋肝细胞癌(HCC)组织标本中基因组DNA,以纯化gDNA为模板PCR扩增看家基因.结果显示改良水浴法提取的gDNA质量、数量均显著高于传统二甲苯脱蜡法,PCR产物量也得到显著提高.与传统方法相比,改良水浴法简单、快捷,可以更有效的回收石蜡包埋组织中的gDNA.     

Development of oxidovanadium and oxido-peroxido vanadium-based artificial DNA nucleases via multi spectroscopic investigations and theoretical simulation of DNA binding

Benzimidazole is a neutral ligand which is often used to synthesize bioactive compounds. Two transition metal benzimidazole-based complexes, namely, vanadium (IV) dioxido complex (complex 1) and vanadium (V) oxido-peroxido complex (complex 2) with tridentate benzimidazole ligand, 2,6-di (1H-benzo[d]imidazol-2-yl) pyridine (Byim) have been designed with the intention of developing potential DNA nuclease. Different studies involving biochemical and biophysical techniques along with molecular docking suggest that both the complexes interact with DNA, while the mode of binding is intercalation. The complexes were further used for DNA cleavage activity. Both of them were found to have substantial DNA nuclease activity, but complex 2 was more potent than complex 1 in exhibiting such activity.     

A Simple and Rapid Extraction of High Molecular Weight Chromosomal DNA from Bacillus subtilis Protoplasts for Cosmid Cloning and Interspecific Transformation

After conversion of Bacillus subtilis vegetative cells to protoplasts, a simple and rapid method for extracting high-molecular-weight chromosomal DNA was devised with the inclusion of bovine serum albumin and phenol-chloroform treatments. The DNA sample thus prepared was the size of 100-450 kb and could be used for cosmid cloning and interspecific transformation.     

Structural and biochemical characterization of CRN-5 and Rrp46: An exosome component participating in apoptotic DNA degradation

Rrp46 was first identified as a protein component of the eukaryotic exosome, a protein complex involved in 3′ processing of RNA during RNA turnover and surveillance. The Rrp46 homolog, CRN-5, was subsequently characterized as a cell death-related nuclease, participating in DNA fragmentation during apoptosis in Caenorhabditis elegans. Here we report the crystal structures of CRN-5 and rice Rrp46 (oRrp46) at a resolution of 3.9 Å and 2.0 Å, respectively. We found that recombinant human Rrp46 (hRrp46), oRrp46, and CRN-5 are homodimers, and that endogenous hRrp46 and oRrp46 also form homodimers in a cellular environment, in addition to their association with a protein complex. Dimeric oRrp46 had both phosphorolytic RNase and hydrolytic DNase activities, whereas hRrp46 and CRN-5 bound to DNA without detectable nuclease activity. Site-directed mutagenesis in oRrp46 abolished either its DNase (E160Q) or RNase (K75E/Q76E) activities, confirming the critical importance of these residues in catalysis or substrate binding. Moreover, CRN-5 directly interacted with the apoptotic nuclease CRN-4 and enhanced the DNase activity of CRN-4, suggesting that CRN-5 cooperates with CRN-4 in apoptotic DNA degradation. Taken together all these results strongly suggest that Rrp46 forms a homodimer separately from exosome complexes and, depending on species, is either a structural or catalytic component of the machinery that cleaves DNA during apoptosis.     

Probable somatic DNA rearrangements in mating type determination in Tetrahymena thermophila: A review and a model

Experimental data on mating type determination in T. thermophila, collected by Nanney, Allen, and their collaborators over a period of 25 years, are reinterpreted in the light of our current understanding of macronuclear genetics. A strong case is developed supporting the idea that mating type determination involves the developmental alteration of somatic DNA that occurs regularly in developing macronuclei in conjugating pairs. A. testable DNA deletion/splicing model is developed that although based on a few simple, plausible assumptions, explains the observations remarkably well. The model is in (at least) superficial analogy to the mechanism that must be involved to explain the somatic differentiation and alteration of DNA sequences that ultimately constitute an expressed vertebrate immunoglobulin gene. Because of the genetic, biochemical, and micromanipulative versatility of Tetrahymena, it may well turn out to be a uniquely suitable microbial eukaryotic experimental system for the study of developmental alterations of somatic DNA.     

Improved Yield of High Molecular Weight DNA Coincides with Increased Microbial Diversity Access from Iron Oxide Cemented Sub-Surface Clay Environments

Despite over three decades of progress, extraction of high molecular weight (HMW) DNA from high clay soils or iron oxide cemented clay has remained challenging. HMW DNA is desirable for next generation sequencing as it yields the most comprehensive coverage. Several DNA extraction procedures were compared from samples that exhibit strong nucleic acid adsorption. pH manipulation or use of alternative ion solutions offered no improvement in nucleic acid recovery. Lysis by liquid N₂ grinding in concentrated guanidine followed by concentrated sodium phosphate extraction supported HMW DNA recovery from clays high in iron oxides. DNA recovered using 1 M sodium phosphate buffer (PB) as a competitive desorptive wash was 15.22±2.33 µg DNA/g clay, with most DNA consisting of >20 Kb fragments, compared to 2.46±0.25 µg DNA/g clay with the Powerlyzer system (MoBio). Increasing PB concentration in the lysis reagent coincided with increasing DNA fragment length during initial extraction. Rarefaction plots of 16S rRNA (V1–V3 region) pyrosequencing from A-horizon and clay soils showed an ∼80% and ∼400% larger accessed diversity compared to the Powerlyzer soil DNA system, respectively. The observed diversity from the Firmicutes showed the strongest increase with >3-fold more operational taxonomic units (OTU) recovered.     

Purification and Characterization of a Novel Chemorepellent Receptor from Tetrahymena thermophila

Chemosensory transduction and adaptation are important aspects of signal transduction mechanisms in many cell types, ranging from prokaryotes to differentiated tissues such as neurons. The eukaryotic ciliated protozoan, Tetrahymena thermophila, is capable of responding to both chemoattractants (O'Neill et al., 1985; Leick, 1992; Kohidai, Karsa & Csaba, 1994, 1995) and chemorepellents (Francis & Hennessey, 1995; Kuruvilla, Kim & Hennessey, 1997). An example of a nontoxic, depolarizing chemorepellent in Tetrahymena is extracellular lysozyme (Francis & Hennessey, 1995; Hennessey, Kim & Satir, 1995). Lysozyme is an effective chemorepellent at micromolar concentrations, binds to a single class of externally facing membrane receptors and prolonged exposure (10 min) produces specific chemosensory adaptation (Kuruvilla et al., 1997). We now show that this lysozyme response is initiated by a depolarizing chemoreceptor potential in Tetrahymena and we have purified the membrane lysozyme receptor by affinity chromatography of solubilized Tetrahymena membrane proteins. The solubilized, purified protein is 42 kD and it exhibits saturable, high affinity lysozyme binding. Polyclonal antibodies raised against this 42 kD receptor block the in vivo lysozyme chemoresponse. This is not only the first time that a chemoreceptor potential has been recorded from Tetrahymena but also the first time that a chemorepellent receptor has been purified from any unicellular eukaryote. Received: 28 July 1997/Revised: 14 November 1997     

Characterization and properties of cholesterol desaturases from the ciliate Tetrahymena thermophila

Live Tetrahymena thermophila transforms exogenous cholesterol into 7,22-bis, dehydrocholesterol (DHC) by desaturation at positions C7(8) and C22(23) of the cholesterol moiety. In this first report on expression, isolation, characterization, and reconstitution of Tetrahymena's cholesterol desaturases in cell-free extracts, we describe conditions for increasing the expression of both desaturases based on the addition of specific sterols to the culture medium. Reactions performed in vitro, with isolated microsomes, yield only the mono-unsaturated derivatives, 7-DHC and/or 22-DHC. However, selectivity towards one product can be improved with the addition of specific compounds: beta-mercaptoethanol inhibited C22(23) desaturase activity completely, while ethanol selectively increased this activity. Detergent-solubilized microsomes showed no desaturase activity, but partial restoration could be achieved with addition of dilauroyl-phosphatidylcholine liposomes (25%). Both cholesterol desaturases require molecular oxygen and cytochrome b(5). NADH or NADPH can serve as reduced cofactors, albeit with different efficiency, delivered by reductases present in the microsomal fraction. Azide and cyanide, but not azole compounds, inhibited these desaturases, suggesting a key role for cytochrome b(5) in these reactions.     

Surface trapping and AFM detection of DNA topological intermediates generated from an oxidative chemical nuclease

Direct observation of DNA topological intermediates generated from a 'chemical nuclease' treatment has been made by atomic force microscopy (AFM). The intermediates were trapped at the mica-water interface and imaging was carried out in the dynamic force mode. Complete conversion from supercoiled circular state to relaxed circular/linear state has been observed over a time scale of 8 min. Implication of such studies in complementing gel electrophoresis data has been predicted.     

Critical Parameters for Efficient Sonication and Improved Chromatin Immunoprecipitation of High Molecular Weight Proteins

Solubilization of cross-linked cells followed by chromatin shearing is essential for successful chromatin immunoprecipitation (ChIP). However, this task, typically accomplished by ultrasound treatment, may often become a pitfall of the process, due to inconsistent results obtained between different experiments under seemingly identical conditions. To address this issue we systematically studied ultrasound-mediated cell lysis and chromatin shearing, identified critical parameters of the process and formulated a generic strategy for rational optimization of ultrasound treatment. We also demonstrated that whereas ultrasound treatment required to shear chromatin to within a range of 100–400 bp typically degrades large proteins, a combination of brief sonication and benzonase digestion allows for the generation of similarly sized chromatin fragments while preserving the integrity of associated proteins. This approach should drastically improve ChIP efficiency for this class of proteins.     

A Method for Isolation of Chloroplast DNA and Mitochondrial DNA from Sunflower

We present a method for isolation of chloroplast and mitochondrial DNA from sunflower seedlings. The protocol includes: organelle isolation, deoxyribonuclease treatment, lysis, deproteinisation and a final DNA purification with sodium dodecyl sulphate and potassium acetate. The organelle DNA yield is 5–10 micrograms per gram of tissue and the DNA is fully restrictable. The technique is inexpensive and appropriate for the isolation of multiple samples of organelle DNA from a small amount of tissue.     

一种从动物组织中提取高质量总RNA的方法

RNA提取技术是分子生物学研究中经常应用的最重要的实验技术。简要介绍了一种高纯度、高产量的从动物组织中提取总RNA的方法．该方法具有实用性强、重复性好的特点。提取的RNA无DNA等污染物,并且其产量、纯度完全能满足分子克隆和基因表达研究的需要。利用此方法提取牛组织的总RNA,进行了NRDR基因在牛组织中的表达分布研究。     

一种高效提取猕猴桃DNA和RNA的方法

在总核酸提取方法(PS法)的基础上,经多次实践改进,得出一种以高盐低pH的HAc-NaAc缓冲体系提取总核酸的简便方法,可以从富含多糖、多酚时猕猴桃叶片和花蕾中提取同时含有DNA和RNA的总核酸.所得的总核酸在LiCl溶液中选择性沉淀RNA,从而有效地分离出DNA和RNA样品.紫外分光光度法和琼脂糖凝胶电泳分析表明,所提取的DNA和RNA具有较高的纯度和完整性.通过样品DNA的PCR和样品RNA的RT-PCR,认为所提取的DNA样品和RNA样品能够满足分子生物学试验的基本要求.     

一种经济快速提取丝状真菌基因组DNA的方法

以螺旋木霉(Trichoderma SpiraleXX)、小克银汉霉(Cunninghamella phaeospora MK)和卵形孢球托霉(Gongronella butleri XT)3种丝状真菌为材料,采用改进的CTAB法提取基因组DNA.方法改进后无需液氮、聚乙烯砒咯烷酮(PVP)和NaAc等试剂,过程简洁,且所需菌体量少,提取的DNA纯度较好,适用于一次微量提取多个样品的基因组DNA.此方法得到的基因组DNA可用于PCR扩增.