Choi W J, Campbell J L, Kuo C L, Jong A Y
Department of Pediatrics and Microbiology, University of Southern California School of Medicine, Los Angeles 90033.
J Biol Chem. 1989 Sep 15;264(26):15593-9.
The yeast SOC8-1 gene was originally identified by partial complementation of cdc8 mutant strains. We have carried out Bal31 deletion analysis of the SOC8-1 gene to define the minimal size which is required for the complementation of the cdc8 mutation. When the SOC8-1 gene is cloned in a multicopy plasmid, it enables temperature-resistant growth in the cdc8 mutant strain, while the SOC8-1 gene in a single copy plasmid does not. Thus, its suppression of the cdc8 mutant is dosage dependent. The high copy number vector carrying the SOC8-1 gene can complement five different cdc8 alleles, indicating that the suppression is not allele specific. Since CDC8 encodes thymidylate kinase, cells bearing a high copy number plasmid containing SOC8-1 gene were tested for the ability to phosphorylate several nucleoside monophosphates, including UMP, GMP and dTMP. Significantly increased phosphorylation activity was observed, suggesting that SOC8-1 encodes a nucleotide kinase. Both restriction enzyme analysis of the SOC8-1 gene and partial purification of the overproduced kinase in SOC8-1 overproducing strains suggest that SOC8-1 may be allelic with URA6. Consistent with these results, both SOC8-1 and URA6 are located on chromosome XI. Thus, one possible suppression mechanism is that SOC8-1 may provide a trans-acting dTMP kinase activity, bypassing the cdc8 gene defect.
酵母SOC8-1基因最初是通过对cdc8突变菌株的部分互补鉴定出来的。我们对SOC8-1基因进行了Bal31缺失分析，以确定互补cdc8突变所需的最小大小。当SOC8-1基因克隆到多拷贝质粒中时，它能使cdc8突变菌株在高温下生长，而单拷贝质粒中的SOC8-1基因则不能。因此，它对cdc8突变的抑制是剂量依赖性的。携带SOC8-1基因的高拷贝数载体可以互补五种不同的cdc8等位基因，表明这种抑制不是等位基因特异性的。由于CDC8编码胸苷酸激酶，因此对携带含有SOC8-1基因的高拷贝数质粒的细胞进行了磷酸化几种核苷单磷酸（包括UMP、GMP和dTMP）能力的测试。观察到磷酸化活性显著增加，表明SOC8-1编码一种核苷酸激酶。对SOC8-1基因的限制性酶切分析以及SOC8-1高产菌株中过量产生的激酶的部分纯化均表明，SOC8-1可能与URA6等位。与这些结果一致，SOC8-1和URA6都位于第十一条染色体上。因此，一种可能的抑制机制是，SOC8-1可能提供一种反式作用的dTMP激酶活性，绕过cdc8基因缺陷。
