Improved in vitro packaging of coliphage lambda DNA: a one-strain system free from endogenous phage.
Improved in vitro packaging of coliphage lambda DNA: a one-strain system free from endogenous phage.
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改进的大肠杆菌噬菌体 lambda DNA 体外包装：一种不含内源噬菌体的单菌株系统。
DOI：
10.1016/0378-1119(85)90215-x
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发表时间：
1985
期刊：
影响因子：
3.5
通讯作者：
Stahl,FW
中科院分区：
文献类型：
--
作者：
Rosenberg,SM;Stahl,MM;Kobayashi,I;Stahl,FW
在先前用于λ DNA体外包装的系统中，λ DNA由包装组分以及添加的DNA产生。我们已经开发了一种新的遗传策略，在体外包装，绕过这个内源性噬菌体的问题。我们的系统采用了一种单一的细菌菌株，其λ原噬菌体编码所有的包装蛋白，但由于包装来源而被删除。单一溶原的粗提物：（i）几乎不含内源性噬菌体，（ii）有效包装添加的λ DNA，（iii）易于制备。使用thecos-体外包装系统，我们表明，包装的λ线性单体是一个二级反应，但包装从多联体制备的退火或连接是一级。我们的结论是，在ourcos−系统中，线性单体是体外包装的不良底物，但多联体包装效果良好。
In previous systems for in vitro packaging of λ DNA, phages are produced from the packaging components as well as from added DNA. We have developed a new genetic strategy for in vitro packaging that bypasses this endogenous phage problem. Our system employs a single bacterial strain whose λ prophage codes for all of the packaging proteins but is deleted forcos, the packaging origin. Crude extracts of the single lysogen: (i) are virtually free from endogenous phages, (ii) package added λ DNA efficiently and (iii) are easy to prepare. Using thecos−in vitro packaging system we show that packaging of λ linear monomers is a second-order reaction, but that packaging from concatemers prepared by annealing or ligation is first order. We conclude that in ourcos−system, linear monomers are a poor substrate for in vitro packaging but that packaging from concatemers works well.