Posas F, Casamayor A, Morral N, Ariño J
Departament de Bioquímica i Biologia Molecular, Facultat de Veterinària, Universitat Autònoma de Barcelona, Spain.
J Biol Chem. 1992 Jun 15;267(17):11734-40.
DNA fragments containing structural characteristics found in Ser/Thr protein phosphatases were amplified by polymerase chain reaction from yeast genome. Amplification was carried out by using degenerate oligonucleotides encoding conserved sequences found in type 1, 2A, and 2B phosphatases. A 215-base pair amplification fragment was used to screen a size-selected library, and a positive clone was isolated and sequenced. Nucleotide sequencing revealed a 2076-base pair open reading frame encoding a 692-amino acid protein. The carboxyl half of the protein is structurally related to type 1 phosphatases and virtually identical with the sequence reported as PPZ1, whereas the amino-terminal half of the protein is unrelated to sequences found in other protein phosphatases. This region is very rich in Ser residues and presents a high number of basic amino acids. Therefore, the gene product, on the basis of its unique structure, would represent a novel class of protein phosphatase. The gene, which is located at chromosome XIII, is transcribed as a mRNA of about 2.7 kilobases, and the amount of message has been found to increase 3- to 4-fold during the culture. The product of the gene PPZ1 was identified by immunoblot analysis of cell extracts as a 75-kDa protein, and the amount of immunoreactive protein was increased in cells carrying multiple copies of the gene. Disruption of the gene resulted in viable cells, with no detectable phenotypic change, indicating that the gene is not essential for growth.
通过聚合酶链反应从酵母基因组中扩增出含有丝氨酸/苏氨酸蛋白磷酸酶结构特征的DNA片段。扩增是使用编码在1型、2A型和2B型磷酸酶中发现的保守序列的简并寡核苷酸进行的。一个215碱基对的扩增片段用于筛选大小选择文库，并分离出一个阳性克隆并进行测序。核苷酸测序揭示了一个2076碱基对的开放阅读框，编码一个692个氨基酸的蛋白质。该蛋白质的羧基末端一半在结构上与1型磷酸酶相关，并且与报道的PPZ1序列几乎相同，而该蛋白质的氨基末端一半与其他蛋白质磷酸酶中的序列无关。该区域富含丝氨酸残基，并且有大量碱性氨基酸。因此，基于其独特的结构，该基因产物将代表一类新型的蛋白质磷酸酶。该基因位于第十三号染色体上，转录为约2.7千碱基的mRNA，并且发现在培养过程中信息的量增加3至4倍。通过对细胞提取物的免疫印迹分析将基因PPZ1的产物鉴定为一种75 kDa的蛋白质，并且在携带该基因多个拷贝的细胞中免疫反应性蛋白质的量增加。该基因的破坏产生了存活细胞，没有可检测到的表型变化，表明该基因对于生长不是必需的。
