A new dominant selectable marker for use in Cryptococcus neoformans.
A new dominant selectable marker for use in Cryptococcus neoformans.
复制标题
用于新型隐球菌的新型显性选择标记。
DOI：
10.1080/mmy.39.1.151.154
复制
发表时间：
2001
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
新生隐球菌是研究真菌感染分子发病机制的理想模型.只有一个占主导地位的可选择的市场可以用于这种生物的转化，我们想开发另一个。我们发现，各种不同的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。诺尔斯链霉菌（Streptomyces noursei）的诺尔斯菌素乙酰转移酶基因（nat 1）的产物赋予了对诺尔斯菌素的抗性。在neoformans中，我们通过用来自aC的启动子序列驱动nat 1的表达来创建融合构建体。neoformansactin基因血清型AC的生物射弹转化。具有该构建体的新型菌株H99和血清型D菌株JEC21的转化效率分别为约1000个转化体μg− 1的DNA和20个转化体μg− 1的DNA。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 neoformansis 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 ofC. neoformansare 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 acetyltransferase gene (nat1) fromStreptomyces noursei.In order to express this gene inC. neoformans, we created a fusion construct by driving expression of nat1 with the promoter sequence from aC. neoformansactin gene. Biolistic transformation of the serotype AC. neoformansstrain H99 and the serotype D strain JEC21 with this construct resulted in transformation efficiencies of approximately 1000 transformants µg−1of DNA and 20 transformants µg−1of 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 crossresistance of the nourseothricin-resistant transformants to hygromycin B, which is the other antibiotic used as a dominant selection marker inC. neoformans. The development of nourseothricin resistance as a second dominant selectable market will be helpful in future molecular studies on this important pathogenic fungus.