Sclafani R A, Patterson M, Rosamond J, Fangman W L
Department of Genetics, University of Washington, Seattle 98195.
Mol Cell Biol. 1988 Jan;8(1):293-300. doi: 10.1128/mcb.8.1.293-300.1988.
The product of the CDC7 gene of Saccharomyces cerevisiae is known to be required in the mitotic cell cycle for the initiation of DNA replication. We show that changes in transcript levels do not account for this stage-specific function, since the steady-state mRNA concentration remains constant at 1 copy per cell throughout the cell cycle. By measuring the cell division capacity of a cdc7::URA3 mutant after loss of a single-copy plasmid containing the CDC7 gene, we show that the CDC7 protein is present in at least 200-fold excess of the amount required for a single cell division. These results appear to exclude periodic transcription or translation as a means by which CDC7 function is regulated. In contrast, the CDC7 protein is known to be dispensable for meiotic S phase, but is required for synaptonemal complex formation and recombination. We found that the CDC7 transcript level does vary during meiosis, reaching a maximum near the time at which recombination occurs. Meiotic spores containing a cdc7 null allele germinate but fail to complete cell division. Apparently the excess CDC7 product present in mitotic cells is physically excluded from the spores (or becomes inactivated) and must be produced de novo after germination. The cdc7-1 allele had previously been shown to confer a reduction in the rate of induced mutation. We show that the cloned wild-type CDC7 gene not only complements this defect, but that when the CDC7 gene is on a multiple copy plasmid, induced mutagenesis is increased. Therefore, in contrast to the excess CDC7 activity for cell division, the level of activity for some error-prone repair process may be normally limiting.
已知酿酒酵母CDC7基因的产物在有丝分裂细胞周期中是DNA复制起始所必需的。我们发现转录水平的变化并不能解释该阶段特异性功能,因为在整个细胞周期中,稳态mRNA浓度保持恒定,每个细胞为1个拷贝。通过测量含有CDC7基因的单拷贝质粒丢失后cdc7::URA3突变体的细胞分裂能力,我们发现CDC7蛋白的存在量比单次细胞分裂所需量至少过量200倍。这些结果似乎排除了周期性转录或翻译作为调节CDC7功能的一种方式。相反,已知CDC7蛋白在减数分裂S期是可有可无的,但在联会复合体形成和重组中是必需的。我们发现CDC7转录水平在减数分裂过程中确实会发生变化,在重组发生的时间附近达到最大值。含有cdc7无效等位基因的减数分裂孢子能够萌发,但无法完成细胞分裂。显然,有丝分裂细胞中存在的过量CDC7产物在物理上被排除在孢子之外(或变得失活),并且必须在萌发后重新产生。先前已表明cdc7-1等位基因会导致诱导突变率降低。我们发现克隆的野生型CDC7基因不仅能弥补这一缺陷,而且当CDC7基因位于多拷贝质粒上时,诱导诱变会增加。因此,与细胞分裂所需的过量CDC7活性相反,某些易出错修复过程的活性水平在正常情况下可能是有限的。