Kuo C L, Campbell J L
Mol Cell Biol. 1983 Oct;3(10):1730-7. doi: 10.1128/mcb.3.10.1730-1737.1983.
The CDC8 gene, whose product is required for DNA replication in Saccharomyces cerevisiae, has been isolated on recombinant plasmids. The yeast vector YCp50 bearing the yeast ARS1, CEN4, and URA3 sequences, to provide for replication, stability, and selection, respectively, was used to prepare a recombinant plasmid pool containing the entire yeast genome. Plasmids capable of complementing the temperature-sensitive cdc8-1 mutation were isolated by transformation of a cdc8-1 mutant and selection for clones able to grow at the nonpermissive temperature. The entire complementing activity is carried on a 0.75-kilobase fragment, as revealed by deletion mapping. This fragment lies 1 kilobase downstream from the well-characterized sup4 gene, a gene known to be genetically linked to CDC8, thus confirming that the cloned gene corresponds to the chromosomal CDC8 gene. Two additional recombinant plasmids that complement the cdc8-1 mutation but that do not contain the 0.75-kilobase fragment or any flanking DNA were also identified in this study. These plasmids may contain genes that compensate for the lack of CDC8 gene product.
CDC8基因的产物是酿酒酵母DNA复制所必需的，该基因已通过重组质粒分离出来。携带酵母ARS1、CEN4和URA3序列的酵母载体YCp50，分别用于提供复制、稳定性和筛选功能，被用来制备一个包含整个酵母基因组的重组质粒库。通过转化cdc8 - 1突变体并选择能够在非允许温度下生长的克隆，分离出能够互补温度敏感型cdc8 - 1突变的质粒。缺失图谱分析表明，整个互补活性由一个0.75千碱基的片段承担。该片段位于特征明确的sup4基因下游1千碱基处，sup4基因已知与CDC8存在遗传连锁，从而证实克隆的基因对应于染色体上的CDC8基因。在本研究中还鉴定出另外两个重组质粒，它们能够互补cdc8 - 1突变，但不包含0.75千碱基的片段或任何侧翼DNA。这些质粒可能含有补偿CDC8基因产物缺失的基因。
