Voordouw G, Kam Z, Borochov N, Eisenberg H
Biophys Chem. 1978 May;8(2):171-89. doi: 10.1016/0301-4622(78)80008-8.
For the study of DNA conformations, conformational transitions, and DNA-protein interactions, covalently closed supercoiled ColE1-plasmid DNA has been purified from cultures of Escherichia coli harboring this plasmid and grown in the presence of chloramphenicol according to the method of D.B. Clewell [J. Bact. 110 (1972)667]. The open circular and linear forms of the plasmid were prepared by digestion of the covalently closed, supercoiled form with pancreatic deoxyribonuclease and EcoRI-restriction endonuclease, respectively. The linear form was found to be very homogeneous by electron microscopy and sedimenting boundary analysis. Its physical properties (s0 20,w=16.3 S,D0 20,W=1.98 X 10(-8) cm2 s-1 and [eta]=2605 ml g-1) have been carefully determined in 0.2 M NaCl, 0.002 M NaPO4 pH 7.0,0.002 M EDTA, at 23 degrees C. Combination of s0 20, w (obtained by quasielastic laser light scattering) gave Ms,D=4.39 x 10(6). This value is in reasonable agreement with the molecular weight from total intensity laser light scattering M=4.30 x 10(6). The covalently closed and open circular forms of the ColE1-plasmid are less homogeneous due to slight cross-contamination and the presence of small amounts of dimers in these preparations. The weight fractions of the various components as determined by boundary analysis or electron microscopy are given together with the average quantities obtained in the same solvent for the supercoiled form ((s0 20,w)w=25.4 S, (D0 20,w)z=2.89 x 10(-8) cm2 s-1, [eta]= 788 ML G-1,Ms,D=4.69 x 10(6) and Mw=4.59 x 10(6)) and the open circular form (s0 20, w)w=20.1 S, (D0 20,w)z=2.45 x 10(-8) cm2 s-1, [eta]=1421 ml g-1,Ms,D=4.37 x 10(6) and Mw=4.15 x 10(6)). Midpoint analysis of the sedimenting boundaries allows unambiguous determination of the sedimentation coefficients of these two forms: s0 20,w=24.5 S and s0 20,w=18.8 S, respectively. Also deduced from total intensity light scattering were radii of gyration Rg (103.5, 134.2 and 186 nm) and second virial coefficients A2 (0.7, 4.8 AND 5.4 x 10(-4) mole ml/g2) for the supercoiled, the open circular and linear forms, respectively. The data presented are discussed in relation to the conformational parameters for the three forms in solution.
为了研究DNA构象、构象转变以及DNA-蛋白质相互作用，共价闭合超螺旋ColE1质粒DNA已从携带该质粒并在氯霉素存在下培养的大肠杆菌培养物中，按照D.B. Clewell的方法[《细菌学杂志》110 (1972) 667]进行纯化。质粒的开环形式和线性形式分别通过用胰脱氧核糖核酸酶和EcoRI限制性内切酶消化共价闭合的超螺旋形式来制备。通过电子显微镜和沉降边界分析发现线性形式非常均一。其物理性质（s0 20,w = 16.3 S，D0 20,W = 1.98×10(-8) cm2 s-1，[η]= 2605 ml g-1）已在0.2 M NaCl、0.002 M NaPO4 pH 7.0、0.002 M EDTA中，于23℃下仔细测定。s0 20,w（通过准弹性激光光散射获得）的组合给出Ms,D = 4.39×10(6)。该值与总强度激光光散射得到的分子量M = 4.30×10(6)合理一致。由于这些制备物中存在轻微的交叉污染和少量二聚体，ColE1质粒的共价闭合形式和开环形式不太均一。通过边界分析或电子显微镜测定的各种组分的重量分数，以及在相同溶剂中对超螺旋形式（(s0 20,w)w = 25.4 S，(D0 20,w)z = 2.89×10(-8) cm2 s-1，[η]= 788 ML G-1，Ms,D = 4.69×10(6)，Mw = 4.59×10(6)）、开环形式（(s0 20,w)w = 20.1 S，(D0 20,w)z = 2.45×10(-8) cm2 s-1，[η]= 1421 ml g-1，Ms,D = 4.37×10(6)，Mw = 4.15×10(6)）获得的平均量一并给出。沉降边界的中点分析允许明确测定这两种形式的沉降系数：分别为s0 20,w = 24.5 S和s0 20,w = 18.8 S。从总强度光散射还推导出超螺旋、开环和线性形式的旋转半径Rg（103.5、134.2和186 nm）以及第二维里系数A2（0.7、4.8和5.4×10(-4) mole ml/g2）。所呈现的数据将结合溶液中三种形式的构象参数进行讨论。
