Gene disruption by biolistic transformation in serotype D strains of Cryptococcus neoformans
Gene disruption by biolistic transformation in serotype D strains of Cryptococcus neoformans
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DOI：
10.1006/fgbi.1999.1180
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发表时间：
2000-02-01
影响因子：
3
通讯作者：
Heitman, J
中科院分区：
文献类型：
--
作者：
Davidson, RC;Cruz, MC;Heitman, J
新型隐球菌是一种机会致病真菌，具有明确的性周期和成熟的遗传和分子方法。已经开发了两种不同的转化系统，并且已经通过同源重组破坏了许多基因。然而，通过这些方法实现的同源重组的频率在A和D血清型菌株之间显著不同。在血清型D菌株中通过电穿孔的转化导致频率为1/1000至1/100，000的同源重组，而通过基因枪方法的转化导致血清型A菌株中基因破坏的频率在2 - 50%之间。我们发现同源重组的基因破坏可以在同源血清型D菌株系列中通过生物射弹转化实现，频率为1 - 4%。通过这种方法，我们已经很容易地破坏了编码MAPK同源物（CPK 1），钙调神经磷酸酶A催化亚基（CNA 1）和G蛋白α亚基（GPA 1）的基因。通过物理和遗传学方法，我们表明，这些突变的结果从有针对性的重组事件没有异位整合。由于遗传方法可以应用于同类血清型D菌株，我们的观察代表了分子方法的重大进展，以了解这一重要的人类病原体的生理和毒力。(C)北京大学出版社.
Cryptococcus neoformans is an opportunistic fungal pathogen with a defined sexual cycle and well-developed genetic and molecular approaches. Two different transformation systems have been developed, and a number of genes have been disrupted by homologous recombination, However, the frequency of homologous recombination achieved by these approaches has differed dramatically between strains of the A and D serotypes, Transformation by electroporation in serotype D strains results in homologous recombination at frequencies of 1/1000 to 1/100,000, whereas transformation by the biolistic method has resulted in gene disruption at frequencies between 2 and 50% in serotype A strains. We find that gene disruption by homologous recombination can be achieved in the congenic serotype D strain series by biolistic transformation with frequencies of similar to 1 to 4%, By this approach, we have readily disrupted the genes encoding a MAPK homolog (CPK1), the calcineurin A catalytic subunit (CNA1), and a G protein alpha subunit (GPA1). By physical and genetic methods, we show that these mutations result from targeted recombination events without ectopic integrations. Because genetic approaches can be applied in the congenic serotype D strains, our observations represent a significant advance in molecular approaches to understand the physiology and virulence of this important human pathogen. (C) 2000 Academic Press.