Schu P, Suarez Rendueles P, Wolf D H
Institut für Biochemie der Universität Stuttgart, Federal Republic of Germany.
Eur J Biochem. 1991 Apr 10;197(1):1-7. doi: 10.1111/j.1432-1033.1991.tb15874.x.
The gene for proteinase yscB inhibitor I2B (PBI2) from Saccharomyces cerevisiae was isolated by oligonucleotide screening of a genomic DNA library, and was sequenced. The gene codes for a single protein of 75 amino acids. In contrast to the published amino acid sequence [Maier, K., Müller, H., Tesch, R., Trolp, T., Witt, I. & Holzer, H. (1979) J. Biol. Chem. 254, 12,555-12,561] the DNA sequence revealed a valine instead of a leucine at position 33 (32 of the mature protein). Therefore the primary sequences of the isoinhibitors I2B of S. cerevisiae and I1B of Saccharomyces carlsbergensis differ only at position 34 (glutamic acid/lysine). The open reading frame of PBI2 was replaced in vitro by the URA3 gene and a I2B null mutant of S. cerevisiae was constructed by gene replacement. The mutation resulted in an elevation of the protein degradation rate by 50% when grown under nutritional stress compared to the isogenic wild type. Growth and viability of the cells was not significantly affected by the absence of I2B.
通过对基因组DNA文库进行寡核苷酸筛选,分离出了来自酿酒酵母的蛋白酶yscB抑制剂I2B(PBI2)基因,并进行了测序。该基因编码一种由75个氨基酸组成的单一蛋白质。与已发表的氨基酸序列[迈尔,K.,米勒,H.,特施,R.,特罗尔普,T.,维特,I.和霍尔泽,H.(1979年)《生物化学杂志》254卷,12555 - 12561页]相比,DNA序列显示在第33位(成熟蛋白的第32位)是缬氨酸而非亮氨酸。因此,酿酒酵母的I2B同型抑制剂和卡尔斯伯酵母的I1B同型抑制剂的一级序列仅在第34位(谷氨酸/赖氨酸)不同。PBI2的开放阅读框在体外被URA3基因取代,并通过基因替换构建了酿酒酵母的I2B缺失突变体。与同基因野生型相比,在营养胁迫下生长时,该突变导致蛋白质降解速率提高了50%。I2B的缺失对细胞的生长和活力没有显著影响。