Horinouchi S, Nishiyama M, Suzuki H, Kumada Y, Beppu T
J Antibiot (Tokyo). 1985 May;38(5):636-41. doi: 10.7164/antibiotics.38.636.
By cleavage with restriction endonucleases and cloning the resultant fragments, the A-factor determinant cloned from streptomycin-producing Streptomyces bikiniensis (Horinouchi et al., J. Bacteriol. 158: 481 approximately 487, 1984) was narrowed down to a 1.1-kilobase (kb) fragment. A hybrid multicopy plasmid (pAFB15) carrying the 1.1-kb fragment conferred A-factor production in a large quantity to A-factor-deficient mutants of S. bikiniensis and Streptomyces griseus as well as afsA mutants of Streptomyces coelicolor A3(2). A transcriptional control signal in the 1.1-kb fragment was identified by using a promoter-probe vector pARC 1. Plasmid pAFB15 also caused A-factor production with a marked gene dosage effect in four different Streptomyces strains which originally had no ability to produce A-factor and no DNA sequence homologous to the S. bikiniensis A-factor determinant, suggesting that precursors of A-factor are common metabolites in streptomycetes and that acquisition of only a single key enzyme encoded by the 1.1-kb fragment is sufficient for Streptomyces strains to synthesize A-factor.
通过用限制性内切核酸酶切割并克隆所得片段，从产生链霉素的比基尼链霉菌（Horinouchi等人，《细菌学杂志》158:481 - 487，1984年）克隆的A因子决定簇被缩小到一个1.1千碱基（kb）的片段。携带1.1 kb片段的杂种多拷贝质粒（pAFB15）能使比基尼链霉菌和灰色链霉菌的A因子缺陷型突变体以及天蓝色链霉菌A3(2)的afsA突变体大量产生A因子。通过使用启动子探针载体pARC 1鉴定了1.1 kb片段中的转录控制信号。质粒pAFB15在四种原本无能力产生A因子且没有与比基尼链霉菌A因子决定簇同源DNA序列的不同链霉菌菌株中也引起了A因子的产生，并具有明显的基因剂量效应，这表明A因子的前体是链霉菌中的常见代谢物，并且仅获得由1.1 kb片段编码的单一关键酶就足以使链霉菌菌株合成A因子。
