A new dominant selectable marker for use in Cryptococcus neoformans
A new dominant selectable marker for use in Cryptococcus neoformans
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DOI：
10.1080/714030997
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发表时间：
2001-02-01
期刊：
影响因子：
2.9
通讯作者：
Cox, GM
中科院分区：
文献类型：
--
作者：
McDade, HC;Cox, GM
新型隐球菌是研究真菌感染的分子发病机制的一个很好的模型系统，只有一个占主导地位的可选择的市场，可用于这种生物体的转化，我们想开发另一个。我们发现不同的C.新生儿对氨基糖苷类抗生素诺尔丝菌素非常敏感，对这种药物的自发耐药性肯定是极其罕见的事件。对诺尔丝菌素的抗性是由来自诺尔斯链霉菌（Streptomyces ces noursei）的诺尔丝菌素乙酰基转移酶基因（nat 1）的产物赋予的。为了在C.我们通过用来自C. neoformans的启动子序列驱动nat 1的表达来创建融合构建体。neoformans actin基因。血清型A C的基因枪转化。具有该构建体的新型菌株H99和血清型D菌株JEC 21分别产生约1000个转化体μ g（-1）DNA和20个转化体μ g（-1）DNA的转化效率。使用来自一些H99转化体的DNA进行Southern印迹，这证实了所有抗性分离株都具有以随机方式整合在基因组内的构建体。努尔丝菌素抗性转化子对潮霉素B没有交叉抗性，潮霉素B是另一种用作C.新人类诺尔丝菌素抗性作为第二个主要的可选择市场的发展将有助于这一重要病原真菌的未来分子研究。
Cryptococcus neoformans is an excellent model system for studies on the molecular pathogenesis of fungal infections, There is only one dominant selectable market that can be used in the transformation of this organism, and we wanted to develop another. We found that various strains of C. neoformans are very sensitive to the aminoglycoside antibiotic nourseothricin, and that spontaneous resistance to this drug must be an extremely rare event. Resistance to nourseothricin is conferred by the product of the nourseothricin acetylltransferase gene (nat1) from Streptomyces,ces noursei. In order to express this gene in C. neoformans, we created a fusion construct by driving expression of nat1 with the promoter sequence from a C. neoformans actin gene. Biolistic transformation of the serotype A C. neoformans strain H99 and the serotype D strain JEC21 with this construct resulted in transformation efficiencies of approximately 1000 transformants mug(-1) of DNA and 20 transformants mug(-1) of DNA, respectively. Southern blots were performed using DNA from some of the H99 transformants, and this confirmed that all of the resistant isolates had the construct integrated in a random fashion within the genome. There was no cross-resistance of the nourseothricin-resistant transformants to hygromycin B, which is the other antibiotic used as a dominant selection marker in C. neoformans. The development of nourseothricin resistance as a second dominant selectable market will be helpful in future molecular studies on this important pathogenic fungus.