An archaeal chromosomal autonomously replicating sequence element from an extreme halophile, Halobactetium sp strain NRC-1
An archaeal chromosomal autonomously replicating sequence element from an extreme halophile, Halobactetium sp strain NRC-1
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DOI：
10.1128/jb.185.20.5959-5966.2003
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发表时间：
2003-10-01
影响因子：
3.2
通讯作者：
DasSarma, S
中科院分区：
文献类型：
--
作者：
Berquist, BR;DasSarma, S
本文报道了从极端嗜盐嗜盐菌菌株NRC-1的染色体中鉴定并首次克隆了一个自主复制的序列元件。通过克隆到一个非复制的质粒上，我们通过与orc7基因和宿主菌株的复制能力的关联来确定可能的复制来源。缺失分析表明，位于orc7基因翻译起始区上游750bp的序列，加上orc7基因和下游50bp的序列，足以使该质粒具有复制能力，这是通过表达一个编码甲氧诺林的可选择标记和转化后的质粒恢复来判断的。位于另外两个染色体携带的海洋古生菌矿石基因(ORC6和ORC8)附近的序列不能促进有效的自主复制。位于orc7上游750个碱基对的区域内有一个近乎完美的31个碱基对的反向重复序列，它位于189个碱基对的AT含量极高的区域(44%)的两侧。当反向重复序列的一个拷贝被删除时，质粒的复制能力就会丧失。此外，另外两个嗜盐细菌Haloarcula marismortui和Haloferax Volcanii基因组序列中orc7同源对数附近的反向重复结构是高度保守的。我们的结果表明，在嗜盐古生菌中，染色体复制起源与orc7同源物物理上有关，并且这一元件足以促进自主复制。我们讨论了与迄今为止在所有盐生古菌基因组中发现的大量ORCL-cdc6同源物有关的功能性哈罗考古起源的发现。
We report on the identification and first cloning of an autonomously replicating sequence element from the chromosome of an archaeon, the extreme halophille Halobacterium strain NRC-1 The putative replication origin was identified by association with the orc7 gene and replication ability in the host strain, demonstrated by cloning into a nonreplicating plasmid. Deletion analysis showed that sequences located up to 750 bp upstream of the orc7 gene translational start, plus the orc7 gene and 50 bp downstream, are sufficient to endow the plasmid with replication ability, as judged by expression of a plasmid-encoded mevinollin resistance selectable marker and plasmid recovery after transformation. Sequences located proximal to the two other chromosomally carried haloarchaeal ore genes (orc6 and orc8) are not able to promote efficient autonomous replication. Located within the 750-bp region upstream of orc7 is a nearly perfect inverted repeat of 31 bp, which flanks an extremely AT-rich (44%) stretch of 189 bp. The replication ability of the plasmid was lost when one copy of the inverted repeat was deleted. Additionally, the inverted repeat structure near orc7 homollogs in the genomic sequences of two other halophiles, Haloarcula marismortui and Haloferax volcanii, is highly conserved. Our results indicate that, in halophillic archaea, a chromosomal origin of replication is physically linked to orc7 homologs and that this element is sufficient to promote autonomous replication. We discuss the finding of a functional halloarchaeall origin in relation to the large number of orcl-cdc6 homologs identified in the genomes of all haloarchaea to date.