Ohi R, McCollum D, Hirani B, Den Haese G J, Zhang X, Burke J D, Turner K, Gould K L
Department of Cell Biology, School of Medicine, Vanderbilt University, Nashville, TN 37232.
EMBO J. 1994 Jan 15;13(2):471-83. doi: 10.1002/j.1460-2075.1994.tb06282.x.
The Schizosaccharomyces pombe cdc5+ gene was identified in the first screen for cell division cycle mutants in this yeast. The cdc5+ gene was reported to be required for nuclear division but because of its modest elongation and leaky nature at the non-permissive temperature, it was not investigated further. Here, we report the characterization of the single allele of this gene, cdc5-120, in more detail. The mutant arrests with a 2N DNA content and a single interphase nucleus. Further genetic analyses suggest that cdc5+ gene function is essential in the G2 phase of the cell cycle. We have cloned and sequenced the cdc5+ gene. The deduced protein sequence predicts that Cdc5 is an 87 kDa protein and contains a region sharing significant homology with the DNA binding domain of the Myb family of transcription factors. Deletion mapping of the cdc5+ gene has shown that the N-terminal 232 amino acids of the protein, which contain the Myb-related region, are sufficient to complement the cdc5ts strain. A cdc5 null mutant was generated by homologous recombination. Haploid cells lacking cdc5+ are inviable, indicating that cdc5+ is an essential gene. A fusion protein consisting of bacterial glutathione S-transferase joined in-frame to the N-terminal 127 amino acids of the Cdc5 protein is able to bind to DNA cellulose at low salt concentrations. This evidence suggests that cdc5+ might encode a transcription factor whose activity is required for cell cycle progression and growth during G2.
粟酒裂殖酵母的cdc5+基因是在对该酵母细胞分裂周期突变体的首次筛选中鉴定出来的。据报道,cdc5+基因是核分裂所必需的,但由于其在非允许温度下伸长适度且具有渗漏性,因此未作进一步研究。在此,我们更详细地报道了该基因的单一等位基因cdc5-120的特性。该突变体停滞在2N DNA含量和单个间期核的状态。进一步的遗传分析表明,cdc5+基因功能在细胞周期的G2期至关重要。我们已克隆并测序了cdc5+基因。推导的蛋白质序列预测Cdc5是一种87 kDa的蛋白质,并且包含一个与Myb家族转录因子的DNA结合结构域具有显著同源性的区域。cdc5+基因的缺失图谱分析表明,该蛋白质的N端232个氨基酸(包含Myb相关区域)足以互补cdc5ts菌株。通过同源重组产生了一个cdc5缺失突变体。缺乏cdc5+的单倍体细胞无法存活,表明cdc5+是一个必需基因。一种由细菌谷胱甘肽S-转移酶与Cdc5蛋白的N端127个氨基酸框内连接组成的融合蛋白能够在低盐浓度下与DNA纤维素结合。这一证据表明,cdc5+可能编码一种转录因子,其活性是细胞周期进程和G2期生长所必需的。