Lott T, Ohta N, Newton A
Department of Molecular Biology, Princeton University, NJ 08544.
Mol Gen Genet. 1987 Dec;210(3):543-50. doi: 10.1007/BF00327210.
Two methods for determining the time of gene replication in Caulobacter crescentus using a temperature sensitive DNA synthesis mutant to synchronize chromosome replication are described. Swarmer cells, blocked before DNA initiation at 37 degrees C, initiate chromosome replication within 2 min after releasing the temperature block in 32P-orthophosphate medium, as indicated by the appearance of a small number of unique genomic DNA fragments. The time at which a given chromosome segment replicates was determined by isolating genomic DNA from cells labeled for progressively longer times during the S period of the cell cycle and hybridizing the probes to cloned C. crescentus genes. The time of replication of genetically mapped Tn5 insertions was determined by preparing DNA from the Tn5 insertion mutants that had been labeled with 32P in similar experiments and hybridizing it to lambda::Tn5 DNA. These results furnish the first correlation between the order of chromosome replication and the genetic map of C. crescentus. They also show that the times of replication and expression of the hook protein and the flagellin genes, which require DNA synthesis for their transcription, both occur near mid-S phase.
本文描述了两种利用温度敏感型DNA合成突变体来同步染色体复制，从而确定新月柄杆菌基因复制时间的方法。在37℃下DNA起始合成前被阻断的游动细胞，在32P-正磷酸盐培养基中解除温度阻断后2分钟内开始染色体复制，这可通过少量独特的基因组DNA片段的出现来表明。通过从细胞周期S期内标记时间逐渐延长的细胞中分离基因组DNA，并将探针与克隆的新月柄杆菌基因杂交，来确定给定染色体片段的复制时间。通过在类似实验中用32P标记的Tn5插入突变体中制备DNA，并将其与λ::Tn5 DNA杂交，来确定基因定位的Tn5插入片段的复制时间。这些结果首次建立了新月柄杆菌染色体复制顺序与遗传图谱之间的关联。它们还表明，钩蛋白和鞭毛蛋白基因的复制和表达时间，这两个基因转录需要DNA合成，都发生在S期中期附近。
