Del Pozo L, Abarca D, Hoenicka J, Lmenez A
Centro de Biología Molecular U.A.M./C.S.I.C. Universidad Autónoma de Madrid, Spain.
Eur J Biochem. 1993 Apr 15;213(2):849-57. doi: 10.1111/j.1432-1033.1993.tb17828.x.
Two genes (SCR1 and SCR2) encoding natural cycloheximide resistance in the budding yeast Schwanniomyces occidentalis have been cloned by expression in Saccharomyces cerevisiae. Both genes determine resistance to the inhibitory action of cycloheximide on the ribosome, SCR1 and SCR2 are present as single copies in Schwanniomyces occidentalis, where they map on chromosomes II and V, respectively. The nucleotide sequence of SCR2 contains an open reading frame of 321 nucleotides which is interrupted by an intron of 452 nucleotides. It encodes a polypeptide of 106 amino acids of molecular mass 12.25 kDa and pI 11.19. The deduced amino acid sequence shows a high degree of similarity to the L41 protein of the 60S ribosomal subunit from several eukaryotic organisms. The intron and the 5' non-coding region of SCR2 possess conserved elements which are typical of yeast ribosomal protein genes. A single amino acid change determines the resistance or sensitive phenotype to cycloheximide of the 80S ribosome since replacement of Gln56 in L41 from Schwanniomyces with Pro, by site-directed mutagenesis, confers cycloheximide sensitivity. SCR2 may serve as a practical yeast cloning marker if integrated in a multicopy plasmid.
通过在酿酒酵母中表达，克隆出了在西方许旺酵母中编码天然放线菌酮抗性的两个基因（SCR1和SCR2）。这两个基因都决定了对放线菌酮对核糖体抑制作用的抗性，SCR1和SCR2在西方许旺酵母中以单拷贝形式存在，它们分别定位在第二条和第五条染色体上。SCR2的核苷酸序列包含一个321个核苷酸的开放阅读框，该阅读框被一个452个核苷酸的内含子打断。它编码一个106个氨基酸的多肽，分子量为12.25 kDa，pI为11.19。推导的氨基酸序列与几种真核生物60S核糖体亚基的L41蛋白具有高度相似性。SCR2的内含子和5'非编码区具有酵母核糖体蛋白基因典型的保守元件。单个氨基酸的变化决定了80S核糖体对放线菌酮的抗性或敏感表型，因为通过定点诱变将西方许旺酵母L41中的Gln56替换为Pro会赋予放线菌酮敏感性。如果整合到多拷贝质粒中，SCR2可作为一种实用的酵母克隆标记。
