A HYPHAL-SPECIFIC CHITIN SYNTHASE GENE (CHS2) IS NOT ESSENTIAL FOR GROWTH, DIMORPHISM, OR VIRULENCE OF CANDIDA-ALBICANS
A HYPHAL-SPECIFIC CHITIN SYNTHASE GENE (CHS2) IS NOT ESSENTIAL FOR GROWTH, DIMORPHISM, OR VIRULENCE OF CANDIDA-ALBICANS
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DOI：
10.1073/pnas.91.13.6216
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发表时间：
1994-06-21
影响因子：
11.1
通讯作者：
KINSMAN, OS
中科院分区：
文献类型：
--
作者：
GOW, NAR;ROBBINS, PW;KINSMAN, OS
在二型真菌白色念珠菌中，CHS 2基因编码优先以菌丝形式表达的几丁质合成酶。在该二倍体无性真菌中CHS 2的基因破坏通过对酵母属描述的“ura-blaster”方案实现[Alani，E.，曹湖，加-地和Kleckner，N.（1987）Genetics 116，541-545]。这涉及通过用URA 3作为单一选择标记的整合转化连续破坏多个等位基因。在破坏第一个CHS 2等位基因后，通过重复的hisG序列之间的顺式重组回收Ura（-）表型，所述hisG序列将URA 3标记物储存在破坏盒中，然后再次用于破坏另外的CHS 2等位基因。这种基因破坏的方法非常适合于这种遗传上不稳定的人类病原体的突变分析。需要三轮破坏，表明菌株SGY 243是CHS 2基因座的三倍体。得到的纯合Delta chs 2：：hisG无效突变体是活的，并产生具有正常形态的芽管。在含血清的培养基中，芽管的形成比亲本菌株更慢，并且与亲本菌株的芽管相比，萌发细胞的几丁质含量减少了40%。酵母形式的几丁质含量不受影响。在正常和免疫抑制小鼠中检测时，chs 3无效突变体的原养型菌株的毒力没有显著减弱。
In the dimorphic fungus Candida albicans, the CHS2 gene encodes a chitin synthase that is expressed preferentially in the hyphal form. Gene disruption of CHS2 in this diploid asexual fungus was achieved by the ''ura-blaster'' protocol described for Saccharomyces [Alani, E., Cao, L. and Kleckner, N. (1987) Genetics 116, 541-545]. This involves the sequential disruption of multiple alleles by integrative transformation with URA3 as a single selectable marker. After disrupting the first CHS2 allele, the Ura(-) phenotype was recovered through cis recombination between repeated hisG sequences that Banked the URA3 marker in the disruption cassette, which was then used again to disrupt further CHS2 alleles. This method of gene disruption is well suited to the mutational analysis of this genetically recalcitrant human pathogen. Three rounds of disruption were required, suggesting that the strain SGY243 is triploid for the CHS2 locus. The resulting homozygous Delta chs2::hisG null mutants were viable and made germ tubes with a normal morphology. The germ tubes were formed more slowly than parental strains in serum-containing medium and the germinating cells had a 40% reduction in their chitin content compared to germ tubes of the parent strain. The chitin content of the yeast form was not affected. A prototrophic strain of the chs3 null mutant was not attenuated significantly in its virulence when tested in normal and immunosuppressed mice.