title: Development of a highly efficient gene targeting system induced by transient repression of YKU80 expression in Candida glabrata.
abstract: In the pathogenic yeast Candida glabrata, gene targeting to generate knockouts and "knockins" is a potentially powerful method for the analysis of gene function. Its importance increased after the C. glabrata genome sequence project, but progress in the field is hampered by inefficient mechanisms for gene targeting. With the use of 40-bp homologous flanking DNA, no gene targeting was identified. To address this issue, YKU80 was disrupted, leading to an increase in targeting efficiency of 5.1% using 40-bp flanking homologous DNA. To harness the beneficial effects of YKU80 inactivation on gene targeting frequency without incurring any negative effects, such as synthetic sickness or lethality, we developed a new system whereby the expression of YKU80 was restored following a transient knockdown of expression during transformation. Strains used for this new system carried a SAT1 flipper in the YKU80 promoter region, which was used to repress expression during transformation but was spontaneously excised from the locus after the transformation. By using this strain, DNA damage induced by methyl methane sulfonate, H(2)O(2), UV irradiation, and hydroxyurea before and during gene targeting was evaluated and the mutation rate of URA3 was determined. No significant effects of the SAT1 flipper on these processes have been identified. After the SAT1 flipper is excised, a 34-bp FLP recombination target sequence is left in the promoter region. However, the levels of mRNA transcription were restored and no difference in the survival ratio in vivo compared to that with the YKU80 wild-type strain was identified.
authors.affiliation: Research Center for Pathogenic Fungi and Microbial Toxicoses, Chiba University, 1-8-1 Inohana, Chiba 260-8673, Japan.
authors.forename: Keigo
authors.initials: K
authors.lastname: Ueno
authors.forename: Jun
authors.initials: J
authors.lastname: Uno
authors.forename: Hironobu
authors.initials: H
authors.lastname: Nakayama
authors.forename: Kaname
authors.initials: K
authors.lastname: Sasamoto
authors.forename: Yuzuru
authors.initials: Y
authors.lastname: Mikami
authors.forename: Hiroji
authors.initials: H
authors.lastname: Chibana
mesh_terms: D000217:Acyltransferases / Q000235:genetics / Q000378:metabolism; D041221:Candida glabrata / Q000235:genetics*; D004249:DNA Damage; D004268:DNA-Binding Proteins / Q000235:genetics / Q000378:metabolism; D005656:Fungal Proteins / Q000235:genetics / Q000378:metabolism; D015870:Gene Expression; D018390:Gene Targeting / Q000379:methods*; D009154:Mutation; D011401:Promoter Regions, Genetic; D029701:Saccharomyces cerevisiae Proteins / Q000235:genetics / Q000378:metabolism; D014158:Transcription, Genetic
chemical_list: D004268:DNA-Binding Proteins; D005656:Fungal Proteins; D029701:Saccharomyces cerevisiae Proteins; C520770:YKU80 protein, S cerevisiae; C081647:high affinity DNA-binding factor, S cerevisiae; D000217:Acyltransferases