Categorization and identification of the genes downstream of EFG1 and/or CPH1 in Candida albicans

• Main Author: 郭大榮
• Other Authors: 楊昀良
• Format: Others
• Language: zh-TW
• Published: 2002
• Online Access: http://ndltd.ncl.edu.tw/handle/02747762455653318107

碩士 === 國立交通大學 === 生物科技研究所 === 90 === Candida albicans is commonly found as a commensal in the human population, causing infection in immunocompromised patients. Many of the antifungal drugs have undesirable side effects and their widespread usages have led to the development of drug-resistant isolates. C.albicans is dimorphic and capable of growth in filamentous form and in yeast form. Filamentous growth is believed to be an important step in the invasion and pathogenesis process(43). A mutant strain lacking the transcription factors Efg1 and Cph1 is extremely defective in filamentous growth in YPD plus goat serum and also avirulence in a mouse model(32). Hence, they are involved in both morphology and virulence pathways. Genes regulated by Efg1 and Cph1 may be new antifungal drug targets of C.albicans. Previous research in the laboratory has used the method of suppression subtractive hybridization(SSH) for identifying virulence genes by screening for difference in the expression between the wild type strain(SC5314) and the cph1/cph1 efg1/efg1 double mutant strain(HLC54) in C.albicans. If a gene has higher expression level in the wild-type strain than in the cph1/cph1 efg1/efg1 double mutant strain, this gene is downstream of the CPH1 and EFG1 and is positively regulated by them. The gene may involve in filamentous growth and may even be a virulence gene. 991 clones have come out of the SSH screening. EFG1 is the most abundant one. Restriction mapping was used to sort those clones into groups to reduce the number of samples for sequencing. After comparing those sequences with the databases of Stanford's Current Assembly of Candida albicans Sequence and NCBI, the sequences are classified by functions. Totally, there are 52 known genes and 48 unknown genes. I pick 10 samples, including ASH1、HEM13、HWP1、KEL2、RCK2、TCA2、TPS2、UBR1、URA3 and YMR128, to determine the expression level of mRNA. Five of them, HWP1、URA3、HEM13、KEL2 and YMR128, match the expected results. Their gene expression levels are higher in the wild type than in the cph1/cph1 efg1/efg1 double mutant. The SSH can be used for other organisms whose gene chips are not yet available.