Cottrelle P, Thiele D, Price V L, Memet S, Micouin J Y, Marck C, Buhler J M, Sentenac A, Fromageot P
J Biol Chem. 1985 Mar 10;260(5):3090-6.
One gene coding for yeast cytoplasmic elongation factor 1 alpha (EF-1 alpha) was isolated by colony hybridization using a cDNA probe prepared from purified EF-1 alpha mRNA. A recombinant plasmid, pLB1, with a 6-kilobase yeast DNA insert, was found by hybrid selection and translation experiments to carry the entire gene. The nucleotide sequence of the gene with its 5'- and 3'-flanking regions was determined. The 5' and 3' ends of EF-1 alpha mRNA were localized by the S1 nuclease mapping technique. The cloned gene, called TEF1, encodes a protein of 458 amino acids (Mr = 50,071) in a single, uninterrupted reading frame. The amino acid sequence shows a strong homology with several domains of Artemia salina EF-1 alpha cytoplasmic factor, as evidenced by diagonal dot matrix analysis. Protein sequence homology is comparatively much lower with the yeast mitochondrial elongation factor. S1 nuclease mapping of the mRNA, hybridization analysis of chromosomal DNA using intragenic or extragenic DNA probes, and gene disruption experiments demonstrated the existence of two genes coding for the cytoplasmic elongation factor EF-1 alpha/haploid genome. The presence of an intact chromosomal TEF1 gene is not essential for growth of haploid yeast cells.
利用从纯化的酵母细胞质延伸因子1α（EF-1α）mRNA制备的cDNA探针，通过菌落杂交分离出一个编码该因子的基因。通过杂交筛选和翻译实验发现，带有6千碱基酵母DNA插入片段的重组质粒pLB1携带了完整的基因。测定了该基因及其5'和3'侧翼区域的核苷酸序列。利用S1核酸酶图谱技术确定了EF-1α mRNA的5'和3'末端。克隆的基因TEF1在单一的连续阅读框中编码一个由458个氨基酸组成的蛋白质（Mr = 50,071）。对角线点阵分析表明，该氨基酸序列与卤虫EF-1α细胞质因子的几个结构域具有很强的同源性。与酵母线粒体延伸因子的蛋白质序列同源性相对较低。对mRNA进行S1核酸酶图谱分析、使用基因内或基因外DNA探针进行染色体DNA杂交分析以及基因破坏实验表明，单倍体基因组中存在两个编码细胞质延伸因子EF-1α的基因。完整的染色体TEF1基因的存在对于单倍体酵母细胞的生长不是必需的。
