Yoon H J, Campbell J L
Braun Laboratories, California Institute of Technology, Pasadena 91125.
Proc Natl Acad Sci U S A. 1991 May 1;88(9):3574-8. doi: 10.1073/pnas.88.9.3574.
The CDC7 protein of Saccharomyces cerevisiae may be involved in the G1/S-phase transition and/or in the initiation of mitotic DNA synthesis. The CDC7 gene has two in-frame AUG codons as possible translation start sites, which would produce 58- and 56-kDa proteins, respectively. Both p58 and p56 derived from recombinant plasmids complement the temperature-sensitive growth defect of the cdc7-1 allele. To determine the biochemical function of the CDC7 protein, the CDC7 gene was cloned and polyclonal antibodies were produced against the CDC7 protein. CDC7 immune complexes prepared from yeast with these antibodies phosphorylate histone H1. Kinase activity is thermolabile in strains carrying the cdc7-1 temperature-sensitive mutant allele and is elevated greater than 10-fold in strains carrying plasmids overexpressing either p56 or p58, confirming that the kinase in the immunoprecipitates is the CDC7 gene product. In addition, we show that CDC7 is a phosphoprotein itself. Indirect immunofluorescence and biochemical fractionation show that the CDC7 protein is present at relatively high concentrations in the nucleus compared with the cytoplasm, suggesting that nuclear proteins may be substrates for the CDC7 protein.
酿酒酵母的CDC7蛋白可能参与G1/S期转换和/或有丝分裂DNA合成的起始。CDC7基因有两个符合读框的AUG密码子作为可能的翻译起始位点，分别会产生58 kDa和56 kDa的蛋白质。源自重组质粒的p58和p56均能弥补cdc7-1等位基因的温度敏感型生长缺陷。为确定CDC7蛋白的生化功能，克隆了CDC7基因并制备了针对CDC7蛋白的多克隆抗体。用这些抗体从酵母中制备的CDC7免疫复合物可使组蛋白H1磷酸化。在携带cdc7-1温度敏感型突变等位基因的菌株中，激酶活性不耐热，而在携带过表达p56或p58质粒的菌株中，激酶活性升高超过10倍，这证实免疫沉淀中的激酶是CDC7基因产物。此外，我们发现CDC7本身就是一种磷蛋白。间接免疫荧光和生化分级分离表明，与细胞质相比，CDC7蛋白在细胞核中的浓度相对较高，这表明核蛋白可能是CDC7蛋白的底物。
