Barnhart B J, Cox S H
J Virol. 1973 Jul;12(1):165-76. doi: 10.1128/JVI.12.1.165-176.1973.
DNA synthesis during transition from the lysogenic state to the lytic cycle and throughout the latter has been studied in Haemophilus influenzae BC200 (HP1c1). Following exposure to ultraviolet light, there is a 30-min delay in DNA synthesis after which there is a rapidly increasing rate of phage DNA synthesis. The phage genome is replicated without extensive utilization of segments or of breakdown products of the bacterial chromosome. The mode of phage DNA replication was investigated by zonal sedimentation of labeled DNA in 5 to 20% neutral and alkaline sucrose gradients. Tritiated thymidine, incorporated during a 2-min pulse given at 38 min, chases rapidly into DNA, sedimenting like linear DNA of approximately 2 x 10(8) daltons, and then, at the expense of label in this peak, chases into slower-sedimenting phage DNA (2 x 10(7) daltons). The fast-sedimenting, rapidly labeled DNA satisfies certain criteria for being a concatenated replicative intermediate. Observations in the electron microscope revealed linear concatemers in the faster-sedimenting material and circular phage-sized DNA in the slower-sedimenting DNA. When induced cells are gently lysed with lysozyme and Brij 58 to maintain DNA-membrane associations and sedimented in neutral sucrose over a cesium chloride shelf, the concatemer is found with the cell-membrane-wall complex. Membrane-associated label chases to membrane-free material sedimenting like deproteinized HP1c1 DNA. When membrane-associated DNA from the cesium chloride shelf is deproteinized and resedimented in neutral sucrose, the sedimentation profile reveals that sedimentation rates of labeled DNA from this complex are indicative of sizes ranging from 2 x 10(8) daltons down to phage-sized pieces of 2 to 3 x 10(7) daltons. A model is presented which places HP1c1-DNA replication on the cell membrane where a concatemer of phage DNA is synthesized and subsequently degraded to phage-equivalent DNA. Phage-equivalent DNA is then either released from the membrane for packaging or is packaged while still membrane associated. Thus, the cell membrane is not only the site of DNA replication during which phage DNA is synthesized in multiple phage-equivalent concatemers but it is also the site at which these concatemers are selectively reduced to phage-sized pieces.
在流感嗜血杆菌BC200（HP1c1）中，研究了从溶原状态转变为裂解周期以及整个裂解周期过程中的DNA合成。暴露于紫外线下后，DNA合成会延迟30分钟，之后噬菌体DNA合成速率迅速增加。噬菌体基因组在复制时，并未大量利用细菌染色体的片段或降解产物。通过在5%至20%的中性和碱性蔗糖梯度中对标记DNA进行区带沉降，研究了噬菌体DNA的复制模式。在38分钟时给予2分钟的脉冲标记期间掺入的氚化胸腺嘧啶，迅速掺入DNA中，沉降情况如同约2×10⁸道尔顿的线性DNA，然后，以该峰中的标记为代价，掺入沉降较慢的噬菌体DNA（2×10⁷道尔顿）。快速沉降、快速标记的DNA符合串联复制中间体的某些标准。电子显微镜观察显示，沉降较快的物质中有线性串联体，沉降较慢的DNA中有噬菌体大小的环状DNA。当用溶菌酶和Brij 58轻轻裂解诱导细胞以维持DNA与膜的结合，并在氯化铯梯度上的中性蔗糖中沉降时，串联体存在于细胞膜 - 细胞壁复合物中。与膜相关的标记物会转移到像脱蛋白的HP1c1 DNA一样沉降的无膜物质中。当来自氯化铯梯度的与膜相关的DNA脱蛋白后，在中性蔗糖中重新沉降，沉降图谱显示，来自该复合物的标记DNA的沉降速率表明其大小范围从2×10⁸道尔顿到2至3×10⁷道尔顿的噬菌体大小片段。本文提出了一个模型，该模型认为HP1c1 - DNA复制发生在细胞膜上，在那里合成噬菌体DNA的串联体，随后降解为噬菌体等效DNA。噬菌体等效DNA然后要么从膜上释放以进行包装，要么在仍与膜相关时进行包装。因此，细胞膜不仅是DNA复制的场所，在此期间噬菌体DNA以多个噬菌体等效串联体的形式合成，而且也是这些串联体被选择性地降解为噬菌体大小片段的场所。
