New Selectable Marker for Manipulating the Simple Genomes of Mycoplasma Species
New Selectable Marker for Manipulating the Simple Genomes of Mycoplasma Species
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DOI：
10.1128/aac.00388-09
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发表时间：
2009-10-01
影响因子：
4.9
通讯作者：
Merryman, Chuck
中科院分区：
文献类型：
--
作者：
Algire, Mikkel A.;Lartigue, Carole;Merryman, Chuck
在过去的几年中，在Mollicutes（可以在无菌培养中生长的最简单的细胞）的分子遗传学方面取得了重大进展。然而，在基因操作成为常规之前，仍然需要一些基本的分子工具。本文描述了一种新的基于白斑链霉菌嘌呤霉素- n-乙酰转移酶的显性选择标记的开发。purromycin是一种抗生素，它模拟氨基酰化trna的3'端，并附着在生长的蛋白质链的羧基端。这会阻止蛋白质合成。由于嘌呤霉素招募了细胞中所有不同trna使用的rRNA识别元件，因此不太可能通过简单的点突变获得自发的抗生素耐药性——这是现有支原体标记物的一个恼人问题。我们的密码子优化盒对我们迄今测试的所有五种支原体都具有明显的嘌呤霉素耐药性。抗性盒也被设计用于大肠杆菌，这简化了穿梭载体的构建，使产生支原体转化所需的大量DNA变得轻而易举。鉴于这些特点，我们期望嘌呤霉素标记物能够成为研究这些简单细胞和病原体的广泛适用的工具。
Over the past several years, significant advances have been made in the molecular genetics of the Mollicutes (the simplest cells that can be grown in axenic culture). Nevertheless, a number of basic molecular tools are still required before genetic manipulations become routine. Here we describe the development of a new dominant selectable marker based on the enzyme puromycin-N-acetyltransferase from Streptomyces alboniger. Puromycin is an antibiotic that mimics the 3'-terminal end of aminoacylated tRNAs and attaches to the carboxyl terminus of growing protein chains. This stops protein synthesis. Because puromycin conscripts rRNA recognition elements that are used by all of the various tRNAs in a cell, it is unlikely that spontaneous antibiotic resistance can be acquired via a simple point mutation-an annoying issue with existing mycoplasma markers. Our codon-optimized cassette confers pronounced puromycin resistance on all five of the mycoplasma species we have tested so far. The resistance cassette was also designed to function in Escherichia coli, which simplifies the construction of shuttle vectors and makes it trivial to produce the large quantities of DNA generally necessary for mycoplasma transformation. Due to these and other features, we expect the puromycin marker to be a widely applicable tool for studying these simple cells and pathogens.