Polyploidy in halophilic Archaea: Evolutionary advantages and molecular regulation.

嗜盐古生菌是多倍体的，主染色体包含几个复制来源。我们将以模式物种Haloferax Volcanii为例，利用两个复制起点oriC1和oriC2来研究拷贝数调控的分子细节，这两个复制起点分别非常强和弱。我们已经建立了嗜盐古菌人工染色体(HACs)作为一种理想的工具来研究复制子拷贝数的确定及其生长阶段依赖的调控所涉及的元件。将生成仅包含核心起始点C1或C2、相邻基因以及核心起始点和延伸起始点的突变版本的各种HAC。对HAC拷贝数的影响将被量化。此外，标签版本的起源识别蛋白(ORCS)和邻近的基因产品将被用来解开复制起始处的相互作用网络。最近，我们已经产生了不同倍性水平的火山嗜血杆菌同基因菌株。这些菌株将第一次能够分别通过竞争生长和竞争生存实验来量化多倍体的进化优势，这些实验分别是颜色编码细胞的混合。许多基因组拷贝在应激和突变条件下的生存、长期生存、全球基因调控等方面的优势将被阐述。此外，还将确定到目前为止尚未确定的属的几个额外的嗜盐古生菌物种的倍性水平。

Halophilic archaea are polyploid and the major chromosome contains several origins of replication. We will use the model species Haloferax volcanii to study the molecular details of copy number regulation using the two replication origins oriC1 and oriC2, which are very especially strong and weak, respectively. We have established Haloarchaeal Artificial Chromosomes (HACs) as an ideal tool to study the elements involved in the determination of replicon copy numbers and its growth phase-dependent regulation. Various HACs will be generated containing only the core origins C1 or C2, also adjacent genes, as well as mutated versions of core origins and extended origins. The effects on the HAC copy numbers will be quantified. In addition, tagged versions of the Origin Recognition Proteins (ORCs) and adjacent gene products will be used to unravel interaction networks at replication origins. Recently we have generated isogenic strains of H. volcanii with different ploidy levels. For the first time these strains will enable to quantify the evolutionary advantages of polyploidy using competitive growth and competitive survival experiments, respectively, of mixtures of color-coded cells. The advantages of many genome copies for the survival of stressful and mutagenic conditions, for long-term survival, for global gene regulation etc. will be addressed. Furthermore, the ploidy levels of several additional haloarchaeal species of until now uncharacterized genera will be determined.