Characterization of the minimal replicon of pHM300 and independent copy number control of major and minor chromosomes of Haloferax mediterranei
Characterization of the minimal replicon of pHM300 and independent copy number control of major and minor chromosomes of Haloferax mediterranei
复制标题
pHM300 最小复制子的表征以及 Haloferax mediterranei 主要和次要染色体的独立拷贝数控制。
DOI：
10.1111/1574-6968.12052
复制
发表时间：
2013-02-01
影响因子：
2.1
通讯作者：
Xiang, Hua
中科院分区：
文献类型：
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作者：
Liu, Xiaoqing;Miao, Di;Xiang, Hua
微小染色体在盐古菌中很常见，但关于主、次染色体之间的复制协调机制却知之甚少。在这项研究中，我们分析了pHM 300的复制，一个321，908-bp的微型染色体，它编码的多功能代谢途径在地中海盐藻。预测pHM 300的复制起点在cdc 6 K和tbp 4基因之间的699-bp的基因间区域中，并且通过测定其在Haloarcula hispanica中自主复制的能力来确定最小复制子，所述最小复制子由富含AT的区域和邻近的cdc 6 K基因组成，所述富含AT的区域两侧是推定的起点识别盒（ORB）。Southern杂交分析表明，从对数生长期早期到稳定期中期，pHM 300与染色体的比值逐渐增加。然后通过实时PCR评估这些次要和主要染色体的拷贝数，并显示两者在稳定期均减少。但主染色体拷贝数的下降比pHM 300的下降早一些，且幅度更大，说明微染色体pHM 300的拷贝数控制与主染色体的拷贝数控制无关。
The presence of minichromosomes is very common in haloarchaea, but little is known about the coordination of replication between the major and minor chromosomes. In this study, we analyzed the replication of pHM300, a 321,908-bp minichromosome, which encodes versatile metabolism pathways in Haloferax mediterranei. The replication origin of pHM300 was predicted in the 699-bp intergenic region between the cdc6K and tbp4 gene, and the minimal replicon, consisting of an AT-rich region flanked by putative origin recognition boxes (ORBs) and the adjacent cdc6K gene, was determined by assaying for its ability to replicate autonomously in Haloarcula hispanica. Southern blot analysis indicated that the ratio of pHM300 to chromosome increased from the early exponential to middle stationary phase. The copy numbers of these minor and major chromosomes were then evaluated by real-time PCR and showed that both decreased in stationary phase. However, the decrease in the copy number of the major chromosome was a little earlier and much greater than that of pHM300, revealing that the copy number control of the minichromosome pHM300 is independent from that of the major chromosome in H. mediterranei.