DNA REPLICATION STUDIES BY A NEW METHOD FOR ISOLATION OF CELL MEMBRANE-DNA COMPLEXES
DNA REPLICATION STUDIES BY A NEW METHOD FOR ISOLATION OF CELL MEMBRANE-DNA COMPLEXES
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DOI：
10.1101/sqb.1968.033.01.079
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发表时间：
1968-01-01
期刊：
影响因子：
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通讯作者：
SCHAECHTER, M
中科院分区：
文献类型：
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作者：
EARHART, CF;TREMBLAY, GY;SCHAECHTER, M
细菌细胞膜似乎在DNA复制（Ganesan和Lederberg，1965; Smith和Hanawalt，1967）和分离（Jacob等人，1963; Lark，1966）。通过对薄片进行电子显微镜检查（Ryter，1968）和通过蔗糖梯度对裂解物进行沉降（Ganesan和Lederberg，1965; Smith和Hanawalt，1967），在各种细菌中发现了DNA和细胞膜之间的物理连接。制备伪影对这些方法所得结果的影响无法确定评估。我们已经发现了一种分离膜-DNA复合物的方法，该方法主要不依赖于这两种细胞组分的沉降特性（Tremblay，戈伊，M. Daniels和M. Schaechter，1969）。相反，。该方法利用了DNA所附着的细胞膜部分粘附于晶体疏水表面的能力。晶体是通过混合镁离子和洗涤剂Sarkosyl（月桂酰肌氨酸钠）形成的。将这些晶体与温和制备的裂解物混合后，大部分细胞DNA与晶体结合，并可在通过蔗糖梯度低速离心后作为条带（下文称为M-带）回收。该方法已适用于可容易转化为原生质球的微生物（例如巨大芽孢杆菌）和需要特殊操作才能使其变脆的微生物（例如大肠杆菌）。
The bacterial cell membrane seems to have a direct role in DNA replication (Ganesan and Lederberg, 1965; Smith and Hanawalt, 1967) and segregation (Jacob et al., 1963; Lark, 1966). A physical connection between DNA and the cell membrane has been found in various bacteria by electron microscopic examination of thin sections (Ryter, 1968) and by sedimentation of lysates through sucrose gradients (Ganesan and Lederberg, 1965; Smith and Hanawalt, 1967). The influence of artifacts of preparation on the results obtained by these methods cannot be assessed with certainty. We have discovered a method for the isolation of membrane-DNA complexes which does not depend primarily on the sedimentation properties of these two cell components (Tremblay, GY, M. Daniels, and M. Schaechter, 1969). Instead,. the method exploits the ability of the portions of the cell membrane to which DNA is attached to adhere to hydrophobic surfaces of crystals. The crystals are formed by mixing magnesium ions and the detergent Sarkosyl (sodium lauroyl sarcosinate). After these crystals are mixed with gently prepared lysates, most of the cells' DNA is associated with the crystals and can be recovered as a band (hereafter termed the M-band) after low speed centrifugation through a sucrose gradient. The method has been adapted for use with organisms that can be readily converted to spheroplasts (eg, Bacillus megaterium) and with organisms which require special manipulations to be rendered fragile (eg, Escherichia coli).