Multifunctional centromere binding factor 1 is essential for chromosome segregation in the human pathogenic yeast Candida glabrata
Multifunctional centromere binding factor 1 is essential for chromosome segregation in the human pathogenic yeast Candida glabrata
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DOI：
10.1128/mcb.21.15.4875-4888.2001
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发表时间：
2001-08-01
影响因子：
5.3
通讯作者：
Carbon, J
中科院分区：
文献类型：
--
作者：
Stoyan, T;Gloeckner, G;Carbon, J
通过功能互补酿酒酵母 cbf1 缺失突变体的蛋氨酸生物合成缺陷，克隆了光滑念珠菌的 CBF1（着丝粒结合因子 1）基因。 C. glabrata 编码的蛋白质 CgCbf1 包含碱性螺旋-环-螺旋亮氨酸拉链结构域，并且具有与其他芽殖酵母 Cbf1 蛋白质相似的特征。 CgCbf1p 在体外以高亲和力 (0.9 x 10(9) M-1) 结合着丝粒 DNA 元件 I (CDEI) 序列 GTCACATG。带移实验揭示了 CgCEN DNA 上的蛋白质-DNA 复合物模式与已知的酿酒酵母不同。我们使用新开发的菌落分区测定法检查了改变 CDEI 结合位点对 CEN 质粒分离的影响。 CDEI 结合位点的内部删除仅导致质粒丢失率增加五倍，这表明 Cbf1p 与着丝粒 DNA 的直接结合并不是完整功能所必需的。然而，CDEI 左侧序列的额外删除导致质粒丢失率增加 70 倍。事实证明，CBF1 基因的缺失对于光滑 C.glabrata 是致命的。含有 CBF1 基因的光滑 C.glabrata 细胞在关闭启动子 (tetO-ScHOP) 的影响下在活性药物多西环素存在下培养 5 小时后停止生长。捕获细胞的 DAPI（4',6'-二脒基-2-苯基吲哚）染色显示，单核、2C DNA 含量和短纺锤体的大芽细胞数量显着增加，表明细胞周期的 G(2)/M 转变存在缺陷。因此，我们得出结论，Cbf1p 是光滑 C. glabrata 染色体分离所必需的。
The CBF1 (centromere binding factor 1) gene of Candida glabrata was cloned by functional complementation of the methionine biosynthesis defect of a Saccharomyces cerevisiae cbf1 deletion mutant. The C. glabrata-coded protein, CgCbf1, contains a basic-helix-loop-helix leucine zipper domain and has features similar to those of other budding yeast Cbf1 proteins. CgCbf1p binds in vitro to the centromere DNA element I (CDEI) sequence GTCACATG with high affinity (0.9 x 10(9) M-1). Bandshift experiments revealed a pattern of protein-DNA complexes on CgCEN DNA different from that known for S. cerevisiae. We examined the effect of altering the CDEI binding site on CEN plasmid segregation, using a newly developed colony-sectoring assay. Internal deletion of the CDEI binding site led only to a fivefold increase in rates of plasmid loss, indicating that direct binding of Cbf1p to the centromere DNA is not required for full function. Additional deletion of sequences to the left of CDEI, however, led to a 70-fold increase in plasmid loss rates. Deletion of the CBF1 gene proved to be lethal in C.glabrata. C.glabrata cells containing the CBF1 gene under the influence of a shutdown promoter (tetO-ScHOP) arrested their growth after 5 h of cultivation in the presence of the reactive drug doxycycline. DAPI (4 ' ,6 ' -diamidino-2-phenylindole) staining of the arrested cells revealed a significant increase in the number of large-budded cells with single nuclei, 2C DNA content, and short spindles, indicating a defect in the G(2)/M transition of the cell cycle. Thus, we conclude that Cbf1p is required for chromosome segregation in C. glabrata.