猪线粒体DNA多态性与中国地方猪种起源分化的关系

用24种限制性内切酶分析了我国21个具代表性地方猪品种、1个引进品种和2个来自中国和越南的野生近缘种mtDNA的RFLP。结果表明:在74个个体中检出的32种限制性态型可归结成7种单倍型,其间的差异主要来源于少数几个限制性位点的点突变;地方猪种4种单倍型间的平均遗传距离为0.143％,遗传多态程度(π值)仅为0.007％,说明遗传多样性非常贫乏,提示中国地方猪种可能起源于一个野猪亚种。     

Versatile method employing basic techniques of genetic engineering to study the ability of low-molecular-weight compounds to bind covalently with DNA in cell-free systems

Numerous antitumor and carcinogenic compounds and free radicals are able to modify DNA by forming covalent bonds, mainly with nucleophilic centers in nucleobases. Such a binding is usually of utmost importance for the biological outcome. The level of DNA adducts formed by a given agent is in most cases extremely low; hence their detection is very difficult. Here we propose a simple approach, exploiting techniques widely used in genetic engineering, to demonstrate and characterize the covalent modification of a DNA fragment by any low-molecular-weight compound of interest in a cell-free system. The specifically designed, several-hundred-base-pairs-long double-stranded deoxyoligonucleotide (PCR amplified)--subject to modification--includes two restriction sites: one containing only GC base pairs recognized by restriction endonuclease MspI and the other including only AT base pairs recognized by restriction endonuclease Tru1I. The covalent modification of the restriction sites abolishes their recognition and thus cleavage by the endonucleases applied. The formation of DNA adducts is induced by incubating the oligonucleotide with increasing concentrations of a studied compound, in the appropriate activating system if required. Then, the modified oligonucleotide is submitted to digestion by the above-mentioned restriction endonucleases and the DNA fragments are separated by polyacrylamide gel electrophoresis. The inhibition of cleavage indicates the occurrence of covalent modification of the restriction site(s) while simultaneously pointing at the kind of base pairs involved in DNA adduct formation. The validation of the method was performed for two DNA binding antitumor compounds, cisplatin and CC-1065, which form adducts preferentially with guanine and adenine, respectively.     

In vitro interaction between calf thymus DNA and Escherichia coli LPS in the presence of divalent cation Ca2+

With increasing addition of Escherichia coli LPS to calf thymus DNA, both dissolved in CaCl2, absorption maxima of DNA at 260 nm decreased gradually with the appearance of isosbastic points at both ends of spectra, which implied some binding between DNA and LPS. Hill plot of absorbance data showed that the binding interaction was positive cooperative in nature. For any fixed concentration of DNA and LPS, extent of interaction increased as concentration of CaCl2 was raised from 1.0 to 100 mM, signifying the electrostatic nature of the interaction, mediated through Ca2+ ion. Stepwise addition of EDTA, a chelating agent for divalent cations, to DNA-LPS bound complex gradually reversed the spectral shift with increase in absorbance at 260 nm, which implied opening up of the complex, that is, reversible nature of the interaction. Circular dichroism spectral changes of DNA by the addition of LPS indicated partial transition of DNA from B to A form. Isothermal titration calorimetric (ITC) study showed that the DNA-LPS binding was an exothermic and enthalpy-driven phenomenon. Moreover, in the presence of 100 mM CaCl2, binding constant of the interaction was found to be 2.6 x 10(4) M(-1) and 3.1 x 10(4) M(-1) from the analysis of Hill plot and ITC result, respectively. DNA-melting study showed that the LPS binding had increased the melting temperature of DNA, indicating more stabilization of DNA double helix. The binding of LPS to DNA made the complex resistant to digestion with endonucleases EcoRI and DNase I.     

Applying the stochastic difference equation to DNA conformational transitions: a study of B-Z and B-A DNA transitions

Despite the existence of numerous models to account for the B-Z DNA transition, experimenters have not yet arrived at a conclusive answer to the structural and dynamical features of the B-Z transition. By applying the stochastic difference equation to simulate the B-Z DNA transition, we have shown that the stretched intermediate model of the B-Z transition is more probable than other B-Z transition models such as the Harvey model. This is accomplished by comparing potential energy profiles of various B-Z DNA transition models and calculating relative probabilities based on the stochastic difference equation with respect to length (SDEL) formalism. The results garnered in this article allow for new approaches in determining the structural transition of B-DNA to Z-DNA experimentally. We have also simulated the B-A DNA transition using the stochastic difference equation. Unlike the B-Z DNA transition, the mechanism for the B-A DNA transition is well established. The variation in the pseudorotation angle during the transition is in good agreement with experimental results. Qualitative features of the simulated B-A transition also agree well with experimental data. The SDEL approach is thus a suitable numerical technique to compute long-time molecular dynamics trajectory for DNA molecules.     

Rapid and prolonged stalling of human DNA topoisomerase I in UVA-irradiated genomic areas

DNA topoisomerase I appears to be involved in DNA damage and repair in a complex manner. The enzyme is required for DNA maintenance and repair, but it may also damage DNA through its covalently DNA-bound, catalytic intermediate. The latter mechanism plays a role in tumor cell killing by camptothecins, but seems also involved in oxidative cell killing and certain stages of apoptosis. Stalling and/or suicidal DNA cleavage of topoisomerase I adjacent to nicks and modified DNA bases has been demonstrated in vitro. Here, we investigate the enzyme's interactions with UVA-induced DNA lesions inside living cells. We irradiated cells expressing GFP-tagged topoisomerase I with an UVA laser focused through a confocal microscope at confined areas of the nuclei. At irradiated sites, topoisomerase I accumulated within seconds, and accumulation lasted for more than 90 min. This effect was apparently due to reduced mobility, although the enzyme was not immobilized at the irradiated nuclear sites. Similar observations were made with mutant versions of topoisomerase I lacking the active site tyrosine or the N-terminal domain, but not with the N-terminal domain alone. Thus, accumulation of topoisomerase I at UVA-modified DNA sites is most likely due to non-covalent binding to damaged DNA, and not suicidal cleavage of such lesions. The rapid onset of accumulation suggests that topoisomerase I functions in this context as a component of DNA damage recognition and/or a cofactor of fast DNA-repair processes. However, the prolonged duration of accumulation suggests that it is also involved in more long-termed processes.     

Capillary electrophoresis is a sensitive monitor of the hairpin-random coil transition in DNA oligomers

Capillary electrophoresis (CE) has been used to characterize the hairpin-random coil transition of four octamers in the GCxxxxGC minihairpin family, where xxxx is GAAA, TTTC, TTTT, or AAAA. The transition can be monitored by CE because differences in the frictional coefficients of the hairpin and coil forms of each octamer lead to a difference of approximately 9% in the free solution mobilities of the two conformations. The GAAA octamer is unusually stable, with a melting temperature of 65 degrees C. The TTTT octamer forms a minihairpin with a melting temperature of 29 degrees C, the TTTC octamer has a melting temperature of 16 degrees C, and the AAAA octamer has a melting temperature below 0 degrees C. The thermal transitions of the TTTT, TTTC, and AAAA octamers are well fitted by a structure prediction algorithm; however, the GAAA minihairpin is considerably more stable than predicted. The melting temperature of the GAAA minihairpin is reduced to 47 degrees C in aqueous buffers containing 7.2M urea and to 33 degrees C in buffers containing 7.2M urea plus 40% (v/v) formamide. The combined results indicate that CE is a sensitive technique for monitoring conformational transitions in small DNA molecules.     

Solution conformational study of nociceptin an(d its 1-13 and 1-11 fragments using circular dichroism and two-dimensional NMR in conjunction with theoretical conformational analysis.

Conformational studies of nociceptin (NC-NH2), its fully active fragment, NC(1-13)-NH2, and two significantly less potent fragments, NC(1-13)-OH and NC(1-11)-OH, were conducted in water and TFE solutions by the employment of circular dichroism, and in DMSO-d6 by 2DNMR spectroscopy in conjunction with theoretical conformational analysis. The conformations of all thepeptides studied were calculated taking two approaches. The first assumes multiconformational equilibrium of the peptide studied, which is characterized by a set of conformations (and their statistical weight values)obtained from a global conformational analysis using three methods: the electrostatically driven Monte-Carlo (EDMC) with the ECEPP/3 force field, the simulated annealing (SA) protocols in the AMBER and CHARMM force fields. The second approach incorporates the interproton distance and dihedral angle constraints into the starting conformation. Calculations were performed using the distance geometry and SA protocol in the CHARMM force field implemented in the X-PLOR program. The CD experiments indicated that for the active peptides, hydrophobic solvents induced a significantly higher (compared with those remaining)content order, probably a helical structure. Unfortunately, as a result of the conformational flexibility of thepeptides, the analysis of conformations obtained with both approaches and different force fields did not alllow the selection of any structural elements of the NC peptides that might be connected with their bioactivity. The only common element found in most conformations of the active peptides was a helical character of fragment 8-13, which allowed the side chains of basic amino acid residues to be exposed to the outside of the molecule and probably to interact with the ORL1 receptor.     

A short GC‐rich palindrome of human mannose receptor gene coding region displays a conformational switch

Conformational switching in DNA is fundamental to biological processes. The structural status of a palindromic GC‐rich dodecamer DNA sequence, integral part of human MRC2 coding region, and a related sequence of opposite polarity from human FDX1 gene were characterized and compared. UV‐melting, circular dichroism, and gel electrophoresis experiments demonstrated the formation of intermolecular structures. Although stability and molecularity of both the oligomeric structures were found to be almost identical, their secondary structures differed remarkably as A1 MRC2 sequence showed A‐like and B‐like DNA conformation, whereas the A2 FDX1 sequence exhibited only the A‐like signatures. The study is relevant for understanding structural polymorphism at genomic locations depending on DNA sequence and solution environment. © 2012 Wiley Periodicals, Inc. Biopolymers 97:950–962, 2012.     

Singular value decomposition of 3-D DNA melting curves reveals complexity in the melting process

The thermal denaturation of synthetic deoxypolynucleotides of defined sequence was studied by a three dimensional melting technique in which complete UV absorbance spectra were recorded as a function of temperature. The results of such an experiment defined a surface bounded by absorbance, wavelength, and temperature. A matrix of the experimental data was built, and analyzed by the method of singular value decomposition (SVD). SVD provides a rigorous, model-free analytical tool for evaluating the number of significant spectral species required to account for the changes in UV absorbance accompany-ing the duplex – to – single strand transition. For all of the polynucleotides studied (Poly dA – Poly dT; [Poly (dAdT)]₂; Poly dG – Poly dC; [Poly(dGdC)]₂), SVD indicated the existence of at least 4 – 5 significant spectral species. The DNA melting transition for even these simple repeating sequences cannot, therefore, be a simple two-state process. The basis spectra obtained by SVD analysis were found to be unique for each polynucleotide studied. Differential scanning calorimetry was used to obtain model free estimates for the enthalpy of melting for the polynucleotides studied, with results in good agreement with previously published values. Received: 16 April 1997 / Accepted: 9 July 1997     

基因组编辑技术中供体DNA类型及选择

基因组编辑技术能够实现基因组的精确修饰和改造,是后基因组时代研究基因功能和遗传信息的主要手段。传统的基因打靶技术通过低效率的细胞自发同源重组实现目的基因的定点修饰。真核细胞中DNA双链断裂介导的同源重组效率远高于自发同源重组,利用人工核酸内切酶特异性地在基因组靶序列处引入双链断裂,通过提供适当形式的、含有一定长度同源臂的供体DNA,能够实现相对高效的基因组靶向编辑。本文系统总结了环状质粒、线性化质粒、聚合酶链式反应产物及单链寡聚脱氧核苷酸4种类型的供体DNA在基因组精确编辑研究中的应用及候选原则,以期为以后相关研究中供体DNA的选择、设计提供参考和借鉴。     

Exploring the antioxidant property of bioflavonoid quercetin in preventing DNA glycation: a calorimetric and spectroscopic study

Reducing sugars for example glucose, fructose, etc., and their phosphate derivatives non-enzymatically glycate biological macromolecules (e.g., proteins, DNA and lipids) and is related to the production of free radicals. Here we present a novel study, using differential scanning calorimetry (DSC) along with UV/Vis absorption and photon correlation spectroscopy (PCS), on normal and glycated human placenta DNA and have explored the antioxidant property of the naturally occurring polyhydroxy flavone quercetin (3,3',4',5,7-pentahydroxyflavone) in preventing the glycation. The decrease in the absorption intensity of DNA in presence of sugars clearly indicates the existence of sugar molecules between the two bases of a base pair in the duplex DNA molecule. Variations were perceptible in the PCS relaxation profiles of normal and glycated DNA. The melting temperature of placenta DNA was decreased when glycated suggesting a decrease in the structural stability of the double-stranded glycated DNA. Our DSC and PCS data showed, for the first time, that the dramatic changes in the structural properties of glycated DNA can be prevented to a significant extent by adding quercetin. This study provides valuable insights regarding the structure, function, and dynamics of normal and glycated DNA molecules, underlying the manifestation of free radical mediated diseases, and their prevention using therapeutically active naturally occurring flavonoid quercetin.     

Intramolecular and intermolecular guanine quadruplexes of DNA in aqueous salt and ethanol solutions

DNA guanine quadruplexes are all based on stacks of guanine tetrads, but they can be of many types differing by mutual strand orientation, topology, position and structure of loops, and the number of DNA molecules constituting their structure. Here we have studied a series of nine DNA fragments (G(3)Xn)(3)G(3), where X = A, C or T, and n = 1, 2 or 3, to find how the particular bases and their numbers enable folding of the molecule into quadruplex and what type of quadruplex is formed. We show that any single base between G(3) blocks gives rise to only four-molecular parallel-stranded quadruplexes in water solutions. In contrast to previous models, even two Ts in potential loops lead to tetramolecular parallel quadruplexes and only three consecutive Ts lead to an intramolecular quadruplex, which is antiparallel. Adenines make the DNA less prone to quadruplex formation. (G(3)A(2))(3)G(3) folds into an intramolecular antiparallel quadruplex. The same is true with (G(3)A(3))(3)G(3) but only in KCl. In NaCl or LiCl, (G(3)A(3))(3)G(3) prefers to generate homoduplexes. Cytosine still more interferes with the quadruplex, which only is generated by (G(3)C)(3)G(3), whereas (G(3)C(2))(3)G(3) and (G(3)C(3))(3)G(3) generate hairpins and/or homoduplexes. Ethanol is a more potent DNA guanine quadruplex inducer than are ions in water solutions. It promotes intramolecular folding and parallel orientation of quadruplex strands, which rather corresponds to quadruplex structures observed in crystals.     

Thermodynamic stability of ribonuclease A in alkylurea solutions and preferential solvation changes accompanying its thermal denaturation: a calorimetric and spectroscopic study

The effect of methylurea, N,N'-dimethylurea, ethylurea, and butylurea as well as guanidine hydrochloride (GuHCl), urea and pH on the thermal stability, structural properties, and preferential solvation changes accompanying the thermal unfolding of ribonuclease A (RNase A) has been investigated by differential scanning calorimetry (DSC), UV, and circular dichroism (CD) spectroscopy. The results show that the thermal stability of RNase A decreases with increasing concentration of denaturants and the size of the hydrophobic group substituted on the urea molecule. From CD measurements in the near- and far-UV range, it has been observed that the tertiary structure of RNase A melts at about 3 degrees C lower temperature than its secondary structure, which means that the hierarchy in structural building blocks exists for RNase A even at conditions at which according to DSC and UV measurements the RNase A unfolding can be interpreted in terms of a two-state approximation. The far-UV CD spectra also show that the final denatured states of RNase A at high temperatures in the presence of different denaturants including 4.5 M GuHCl are similar to each other but different from the one obtained in 4.5 M GuHCl at 25 degrees C. The concentration dependence of the preferential solvation change delta r23, expressed as the number of cosolvent molecules entering or leaving the solvation shell of the protein upon denaturation and calculated from DSC data, shows the same relative denaturation efficiency of alkylureas as other methods.     

Aggregation and conformational transition in aqueous solution of a bombolitin III analogue containing a photoreactive side-chain group

The peptide toxin bombolitin III [B(III)], originally isolated from bumblebee venom, has been shown to undergo a concentration-dependent conformational change from a random structure to an α-helix induced by aggregation. The aggregation process and the consequent folding results from a delicate balance of electrostatic and hydrophobic interactions. The conformational change is strongly dependent on pH and salt concentration. In order to gain insight on the structure of the aggregates, and in particular, on the aggregation number and relative orientation of helices in the molecular complexes, the following analogue of bombolitin III was designed and synthesized: Ile-Lys-Bpa-Met-Asp-Ile-Leu-Ala-Lys-Leu-Gly-Lys-Val-Leu-Ala-His-Val-NH₂ Bpa³-B(III) where Bpa is benzoylphenylalanine. Bpa³-B(III) aggregates were investigated by CD and nmr techniques. The observed nuclear Overhauser effect pattern accounts for an antiparallel orientation of two distinct helices. The Bpa side chain allows for the photoinduced cross reaction with any aliphatic proton in spatial proximity. After irradiation, the reaction mixture was analyzed by high performance liquid chromatography and electrospray mass spectrometry. The results confirmed the presence of dimeric and trimeric complexes of bombolitin III formed upon interhelix cross-linking. © 1997 John Wiley & Sons, Inc. Biopoly 42: 147–156, 1997     

The effect of temperature and lipid on the conformational transition of gramicidin A in lipid vesicles

The present study investigated the effect of temperature and lipid/peptide molar ratio on the conformational changes of the membrane peptide gramicidin A from a double-stranded helix to a single-stranded helical dimmer in 1,2-dimyristoyl-glycerol-3-phosphochloine (DMPC) vesicles. Tryptophan fluorescence spectroscopy results suggested that the conformational transition fitted a three-state (two-step) "folding" model. Rate constants, k(1) and k(2), were determined for each of the two steps. Since k(1) and k(2) increased with an increase in temperature, we hypothesized that the process corresponded to the breakage and formation of the backbone hydrogen bonds. The k(1) was from 10 to 45 folds faster than k(2), except for lipid/peptide molar ratios above 89.21, where k(2) increased rapidly. At molar ratios below 89.21, k(2) was insensitive to changes in lipid concentration. To account for this phenomenon, we proposed that while the driving interaction at high molar ratios is between the indole rings of the tryptophan residues and the lipid head groups, at low molar ratios there may be an intermolecular interaction between the tryptophan residues that causes gramicidin A to form an organized aggregated network. This aggregated network, caused by the tryptophan-tryptophan interaction, may be the main effect responsible for the slow down of the conformation change.     

Cloning in a cosmid vector of complete 37 kb and 25 kb ribosomal DNA repeat units from the chicken

DNA fragments of up to 40 kb containing rRNA-coding sequences have been isolated from a chicken liver DNA library prepared in the cosmid pHC79. Characterization of the cloned DNA by R-loop and restriction mapping has shown that there are two size classes of repeat unit, one of 37 kb and one of 25 kb, the larger of which is a family of units which vary slightly in size. These two classes were shown to be present in the DNA of a single chicken. The size of the internal transcribed spacer in the chicken was measured to be 4.4 kb from analysis of R-loops and heteroduplexes between chicken and Xenopus laevis rDNAs. No introns were observed in either the 18 S or the 28 S coding sequences. The number of copies of the chicken rDNA unit was measured by titration against the cloned sequences to be 202±51 per haploid genome.     

Sequence diversity of the control region of mitochondrial DNA in Tuscany and its implications for the peopling of Europe

The control region of mitochondrial DNA has been widely studied in various human populations. This paper reports sequence data for hypervariable segments 1 and 2 of the control region from a population from southern Tuscany (Italy). The results confirm the high variability of the control region, with 43 different haplotypes in 49 individuals sampled. The comparison of this set of data with other European populations allows the reconstruction of the population history of Tuscany. Independent approaches, such as the estimation of haplotype diversity, mean pairwise differences, genetic distances and discriminant analysis, place the Tuscan sample in an intermediate position between sequences from culturally or geographically isolated regions of Europe (Sardinia, the Basque Country, Britain) and those from the Middle East. In spite of the remarkable genetic homogeneity in Europe, a degree of variability is shown by local European populations and homogeneity increases with the relative isolation of the population. The pattern of mitochondrial variation in Tuscany indicates the persistence of an ancient European component subsequently enriched by migrational waves, possibly from the Middle East. © 1996 Wiley-Liss, Inc.     

Arabidopsis thaliana AtPOLK encodes a DinB-like DNA polymerase that extends mispaired primer termini and is highly expressed in a variety of tissues

Cell survival after DNA damage depends on specialized DNA polymerases able to perform DNA synthesis on imperfect templates. Most of these enzymes belong to the recently discovered Y-family of DNA polymerases, none of which has been previously described in plants. We report here the isolation, functional characterization and expression analysis of a plant representative of the Y-family. This polymerase, which we have termed AtPolkappa, is a homolog of Escherichia coli pol IV and human pol kappa, and thus belongs to the DinB subfamily. We purified AtPolkappa and found a template-directed DNA polymerase, endowed with limited processivity that is able to extend primer-terminal mispairs. The activity and processivity of AtPolkappa are enhanced markedly upon deletion of 193 amino acids (aa) from its carboxy (C)-terminal domain. Loss of this region also affects the nucleotide selectivity of the enzyme, leading to the incorporation of both dCTP and dTTP opposite A in the template. We detected three cDNA forms, which result from the alternative splicing of AtPOLK mRNA and have distinct patterns of expression in different plant organs. Histochemical localization of beta-glucuronidase (GUS) activity in transgenic plants revealed that the AtPOLK promoter is active in endoreduplicating cells, suggesting a possible role during consecutive DNA replication cycles in the absence of mitosis.     

A possible mechanism for the dynamics of transition between polymerase and exonuclease sites in a high-fidelity DNA polymerase

The fidelity of DNA synthesis by DNA polymerase is significantly increased by a mechanism of proofreading that is performed at the exonuclease active site separate from the polymerase active site. Thus, the transition of DNA between the two active sites is an important activity of DNA polymerase. Here, based on our proposed model, the rates of DNA transition between the two active sites are theoretically studied. With the relevant parameters, which are determined from the available crystal structure and other experimental data, the calculated transfer rate of correctly base-paired DNA from the polymerase to exonuclease sites and the transfer rate after incorporation of a mismatched base are in good agreement with the available experimental data. The transfer rates in the presence of two and three mismatched bases are also consistent with the previous experimental data. In addition, the calculated transfer rate from the exonuclease to polymerase sites has a large value even with the high binding affinity of 3′-5′ ssDNA for the exonuclease site, which is also consistent with the available experimental value. Moreover, we also give some predictive results for the transfer rate of DNA containing only A:T base pairs and that of DNA containing only G:C base pairs.