An improved counterselectable marker system for mycobacterial recombination using galK and 2-deoxy-galactose.
An improved counterselectable marker system for mycobacterial recombination using galK and 2-deoxy-galactose.
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DOI：
10.1016/j.gene.2010.09.005
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发表时间：
2011-01-01
期刊：
影响因子：
3.5
通讯作者：
Glickman MS
中科院分区：
文献类型：
--
作者：
Barkan D;Stallings CL;Glickman MS
反选择标记是遗传学中的有力工具，因为它们允许选择遗传标记的丢失而不是其存在。在分枝杆菌中，广泛使用的反向选择标记是编码莱万蔗糖酶（sacB）的基因，其赋予对蔗糖的敏感性，但频繁的自发失活使其使用复杂化。在这里，我们表明，E。大肠杆菌半乳糖激酶基因（galK）可作为耻垢分枝杆菌和结核分枝杆菌的反向选择标记。表达E. coli galK，而不是假定的M.结核菌galK，赋予敏感性2-脱氧半乳糖（2-DOG）在两个M。smegalgae和M.结核我们测试了E. coligalK作为分枝杆菌重组中的反向选择标记，单独和与sacB组合。我们发现，0.5%2-DOG有效地选择了已经丢失galK标记的重组体，其中galK丢失/galK突变失活的比率为约1：4。当我们组合galK和sacB作为双反选择标记并在0.2%2-DOG/5%蔗糖上选择双标记缺失时，98.6-100%的蔗糖/2-DOG抗性克隆经历了重组，表明两个标记的突变失活频率低于重组频率。这些结果建立了一种新的用于分枝杆菌的反向选择标记系统，可以缩短在分枝杆菌中产生未标记突变的时间。smegalgae和M.结核
Counterselectable markers are powerful tools in genetics because they allow selection for loss of a genetic marker rather than its presence. In mycobacteria, a widely used counterselectable marker is the gene encoding levan sucrase (sacB), which confers sensitivity to sucrose, but frequent spontaneous inactivation complicates its use. Here we show that the E. coli galactokinase gene (galK) can be used as a counterselectable marker in both Mycobacterium smegmatis and Mycobacterium tuberculosis. Expression of E. coli galK, but not the putative M. tuberculosis galK, conferred sensitivity to 2-deoxy-galactose (2-DOG) in both M. smegmatis and M. tuberculosis. We tested the utility of E. coli galK as a counterselectable marker in mycobacterial recombination, both alone and in combination with sacB. We found that 0.5% 2-DOG effectively selected recombinants that had lost the galK marker with the ratio of galK loss/galK mutational inactivation of approximately 1:4. When we combined galK and sacB as dual counterselectable markers and selected for dual marker loss on 0.2% 2-DOG/5% sucrose, 98.6–100% of sucrose/2-DOG resistant clones had undergone recombination, indicating that the frequency of mutational inactivation of both markers was lower than the recombination frequency. These results establish a new counterselectable marker system for use in mycobacteria that can shorten the time to generate unmarked mutations in M. smegmatis and M. tuberculosis.
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影响因子：
4.4
作者：
Sung, CK;Li, H;Morrison, DA
通讯作者：
Morrison, DA
影响因子：
14.9
作者：
Warming S;Costantino N;Court DL;Jenkins NA;Copeland NG
通讯作者：
Copeland NG
影响因子：
16.8
作者：
Gong, CL;Bongiorno, P;Glickman, MS
通讯作者：
Glickman, MS
影响因子：
3.6
作者：
SANDER, P;MEIER, A;BOTTGER, EC
通讯作者：
BOTTGER, EC
DOI：
10.1007/bf00328706
发表时间：
1984-01-01
期刊：
MOLECULAR AND GENERAL GENETICS
影响因子：
--
作者：
BOHMAN, K;RUUSALA, T;KURLAND, CG
通讯作者：
KURLAND, CG