McCoy J M, Khorana H G
J Biol Chem. 1983 Jul 10;258(13):8456-61.
A fusion between the genes for bacteriorhodopsin and beta-galactosidase was constructed on a multicopy plasmid, pXB/Gal 101. The fusion gene, containing the bacteriorhodopsin gene fused upstream from the beta-galactosidase gene, was under the control of tandem lipoprotein and lac gene promoters. When expressed in Escherichia coli the fusion protein retained beta-galactosidase activity. Mutations in the fusion gene were produced by passage of pXB/Gal 101 through the E. coli mutator strain mut D5. Amber mutations were then selected by examining the loss of the lac+ phenotype imparted by the fusion protein to lac- E. coli cells. Amber mutations occurring within the bacteriorhodopsin gene were localized by replacing the beta-galactosidase region of each mutant plasmid with a beta-galactosidase region which was known to be unmutated. Precise localization of the mutations was achieved first by sizing the prematurely terminated peptides produced by the mutant plasmids in in vitro coupled transcription-translation reactions, and secondly by DNA sequence analysis. Six amber mutants in the gene for bacteriorhodopsin were characterized in this way. One of these was a transversion mutation at a lysine codon; the other five were all transition mutations at tryptophan codons, codons 10, 12, 80, 86, and 137 of the bacteriorhodopsin sequence.
在多拷贝质粒pXB/Gal 101上构建了细菌视紫红质基因与β-半乳糖苷酶基因的融合体。融合基因包含与β-半乳糖苷酶基因上游融合的细菌视紫红质基因，受串联脂蛋白和乳糖基因启动子的控制。当在大肠杆菌中表达时，融合蛋白保留了β-半乳糖苷酶活性。通过使pXB/Gal 101通过大肠杆菌诱变菌株mut D5产生融合基因中的突变。然后通过检查融合蛋白赋予lac-E. coli细胞的lac+表型的丧失来选择琥珀突变。通过用已知未突变的β-半乳糖苷酶区域替换每个突变体质粒的β-半乳糖苷酶区域，定位细菌视紫红质基因内发生的琥珀突变。首先通过在体外偶联转录-翻译反应中测定突变体质粒产生的过早终止肽的大小，其次通过DNA序列分析，实现突变的精确定位。以这种方式鉴定了细菌视紫红质基因中的六个琥珀突变体。其中一个是赖氨酸密码子处的颠换突变；其他五个都是色氨酸密码子处的转换突变，即细菌视紫红质序列的第10、12、80、86和137位密码子。
