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  • 學位論文

篩選白色念珠菌短時間給予抗真菌藥後染色體丟失的頻率

Screening of Chromosome I Loss by Short-Term Treatment of Fluconazole in Candida albicans

指導教授 : 林哲堂

摘要


The opportunistic fungus C. albicans has been considered a diploid organism with no haploid state. However, recent studies have shown that under stress it goes through “concerted chromosome loss” mechanism, and has a viable haploid state in which it can efficiently restore its original diploid form. Therefore, the approach of this study is to obtain the rate and frequency of haploid isolates through concerted chromosome loss, specifically chromosome I, of diploid reference strains SC5314 and a new strain CG5, derivative from CAI4. At the same time, use a specific physiologically relevant stress, antifungal drug stress, with Fluconazole. Subsequently, select small morphologically distinct colonies in the inhibition zone by repeated single-cell separation between them. Then, analyze the DNA content by Flow Cytometry to test for the formation of haploid isolates and use PCR to test for the absence or presence of Gal1 or Ura3 marker in Chr1 in strain CG5. The study revealed that C. albicans undergoes a ‘concerted chromosome loss’ mechanism through which it gives rise to loss of chromosome 1 at a frequency of 1.6% to 16.6% in CG5, and in SC5314 at a frequency of 0.6%. The present study also employed Sytox Green flow cytometry analysis to show that the DNA content of the new FluR derivatives had decreased compared to that of the parental strain. This suggests that haploids are detected rarely because of their lower fitness granting them less chance to persist in the population. In addition, a new method for getting haploid isolates was developed by screening loss of chromosome 1 at the Gal1 gene locus.

並列摘要


The opportunistic fungus C. albicans has been considered a diploid organism with no haploid state. However, recent studies have shown that under stress it goes through “concerted chromosome loss” mechanism, and has a viable haploid state in which it can efficiently restore its original diploid form. Therefore, the approach of this study is to obtain the rate and frequency of haploid isolates through concerted chromosome loss, specifically chromosome I, of diploid reference strains SC5314 and a new strain CG5, derivative from CAI4. At the same time, use a specific physiologically relevant stress, antifungal drug stress, with Fluconazole. Subsequently, select small morphologically distinct colonies in the inhibition zone by repeated single-cell separation between them. Then, analyze the DNA content by Flow Cytometry to test for the formation of haploid isolates and use PCR to test for the absence or presence of Gal1 or Ura3 marker in Chr1 in strain CG5. The study revealed that C. albicans undergoes a ‘concerted chromosome loss’ mechanism through which it gives rise to loss of chromosome 1 at a frequency of 1.6% to 16.6% in CG5, and in SC5314 at a frequency of 0.6%. The present study also employed Sytox Green flow cytometry analysis to show that the DNA content of the new FluR derivatives had decreased compared to that of the parental strain. This suggests that haploids are detected rarely because of their lower fitness granting them less chance to persist in the population. In addition, a new method for getting haploid isolates was developed by screening loss of chromosome 1 at the Gal1 gene locus.

參考文獻


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