Tsuchiya E, Uno M, Kiguchi A, Masuoka K, Kanemori Y, Okabe S, Mikayawa T
Department of Fermentation Technology, Faculty of Engineering, Hiroshima University, Japan.
EMBO J. 1992 Nov;11(11):4017-26. doi: 10.1002/j.1460-2075.1992.tb05495.x.
We have cloned the gene NPS1 (nuclear protein of Saccharomyces) which encodes a nuclear protein of mol. wt 156 735 Daltons (1359 amino acids) essential for cell growth. NPS1 contains a 2 kb sequence that is highly homologous to the S. cerevisiae SNF2/GAM1 gene known as a transcriptional regulator for multiple genes. However, the NPS1 gene was found to have a distinct function from SNF2/GAM1. The growth of the cells carrying a nps1 delta :: URA3 deletion allele and galactose-inducible NPS1 on a plasmid was arrested under NPS1-repressed conditions with a cell cycle arrest phenotype, being arrested at the large-bud stage with a single nucleus that had a DNA content of G2/M phase. When the arrested cells were further incubated under NPS1-repressed conditions, re-replication of DNA occurred in some of the arrested cells without passage through mitosis. In the predicted amino acid sequence of NPS1, sequences homologous to the catalytic domain of protein kinases were found. We constructed a mutation which results in the substitution of a highly conserved lysine residue (Lys792) in the presumed ATP-binding site of this kinase-like domain with a glutamic acid codon. The mutant gene failed to rescue the growth defect caused by NPS1 disruption, suggesting that Lys792 is essential for the function of NPS1.
我们克隆了基因NPS1（酿酒酵母核蛋白），它编码一种对细胞生长至关重要的分子量为156735道尔顿（1359个氨基酸）的核蛋白。NPS1包含一段2 kb的序列，该序列与酿酒酵母的SNF2/GAM1基因高度同源，SNF2/GAM1基因是多个基因的转录调节因子。然而，发现NPS1基因具有与SNF2/GAM1不同的功能。携带nps1 delta::URA3缺失等位基因且在质粒上带有半乳糖诱导型NPS1的细胞，在NPS1抑制条件下生长停滞，并呈现细胞周期停滞表型，停滞在大芽阶段，细胞核单个，DNA含量处于G2/M期。当停滞的细胞在NPS1抑制条件下进一步培养时，一些停滞的细胞在未经过有丝分裂的情况下发生了DNA再复制。在NPS1的预测氨基酸序列中，发现了与蛋白激酶催化结构域同源的序列。我们构建了一个突变体，该突变体导致在这个类激酶结构域假定的ATP结合位点中一个高度保守的赖氨酸残基（Lys792）被谷氨酸密码子取代。突变基因未能挽救由NPS1破坏引起的生长缺陷，这表明Lys792对NPS1的功能至关重要。
