Candida albicans DBF4 gene with two heterologous alleles is involved in hyphae-suppression and is inducibly duplicated by URA3-blaster

• Main Authors: Ting, 簡莛
• Other Authors: 謝家慶
• Format: Others
• Language: zh-TW
• Published: 2010
• Online Access: http://ndltd.ncl.edu.tw/handle/35388603846979599251

碩士 === 中山醫學大學 === 生物醫學科學學系碩士班 === 98 === Candida albicans is an opportunistic fungal pathogen that causes both superficial and systemic infections in immunocompromised patients. Study of C. albicans has been hampered because it is a natural diploid without complete sexual cycle and possesses an unconventional codon usage. Candida albicans is a polymorphic fungus with three major growth forms, termed yeast, pseudohyphae and hyphae. The ability to change morphology from the yeast form to a filamentous form is thought to be important for the pathogenicity of C. albicans. We have been interested in studying C. albicans genes involved in the G1-S transition of the cell cycle for their being important in morphogenesis that is associated with pathogenicity. Among those, genes encoding C. albicans equivalence of Cdc7-Dbf4 kinase complex (the Dbf4-dependent kinase, DDK), originally identified in Saccharomyces cerevisiae, are evolutionarily conserved and regulate the initiation and progression of DNA replication at least partly through phosphorylation of minichromosomal maintenance (MCM) proteins in all eukaryotes being studied. In recent studies, the DDK is thought to be an essential target inactivated by the S-phase checkpoint machinery that inhibits DNA replication. Structurally, Dbf4 protein of C. albicans is highly homologous to that of S. cerevisiae. Three small stretches of protein motifs, named Dbf4-motif-N, Dbf4-motif-M and Dbf4-motif-C, are present in all the known Dbf4 molecules from various eukaryotes. Dbf4-motif -N shows some similarity to the domain I of BRCT, which has been shown to be present in wide varieties of repair and DNA damage/replication checkpoint protein. Dbf4-motif-M and Dbf4-motif-C are shown to be sufficient for interaction and activation with catalytic subunit. Previously, we have found that depletion of C. albicans CDC7 leads to cells with filaments. To establish the function relationship between C. albicans CDC7 (CaCDC7) and DBF4 (CaDBF4), we made a C. albicans strain, CaDBF4-02, with one CaDBF4 allele deleted and the other controlled by MET3 promoter that is methionine and cysteine repressible. Unexpectedly, CaDBF4 depleted strain remained in yeast form. Staggeringly, Southern blotting analysis of strain CaDBF4-02 revealed presence of three copies of CaDBF4 in C. albicans genome. Therefore, we constructed another C. albicans strain CaDBF4-0 from strain CaDBF4-02 in which the third DBF4 copy was knocked out. Similar to that of CaCDC7 depleted strain, we observed that the strain emerged hyphal form when the MET3 promoter was repressed. However, we were unable to obtain homozygous CaDBF4 null mutant. Hence, it is likely that CaDBF4 might be an essential gene. In addition, we constructed another C. albicans strain CaDBF4-05, with a plasmid integrated into the RP10 locus of the genome of strain CaDBF4-04 possessing an endogenous CaDBF4 whose expression is repressible by the control of MET3 promoter, which carries a copy of exogenous DBF4 capable of being expressed constitutively under the ACT1 promoter. We observed that cells of the strain CaDBF4-05 grew from yeast to the hyphal form by the expression of exogenous CaDBF4 when the endogenous CaDBF4 was repressed. Furthermore, constitutively expressing CaCDC7 in the strain CaDBF4-04 repressing CaDBF4 expression could not suppress hyphal growth. Therefore, we confirmed that CaDBF4, together with CaCDC7, is a negative regulator of hyphal formation. From the above observations, we suggested presence of interaction between CaDBF4 and CaCDC7 in C. albicans, although functionally divergent from other eukaryotes. Interestingly, during the experimental process, we noted that C. albicans DBF4 gene with two heterologous alleles and the other allelic heterogeneity also exists in the C. albicans genome. Importantly, we determined that the third copy of DBF4 is inducibly duplicated by URA3-blaster.