Horinouchi S, Suzuki H, Beppu T
J Bacteriol. 1986 Oct;168(1):257-69. doi: 10.1128/jb.168.1.257-269.1986.
The nucleotide sequence of afsB from Streptomyces coelicolor A3(2), a pleiotropic gene which positively controls the biosynthesis of A-factor and the pigmented antibiotics actinorhodin and undecylprodigiosin in S. coelicolor A3(2) and "Streptomyces lividans," was determined. The determinant of the afsB gene, which includes the putative AfsB protein consisting of 243 amino acids, was mapped functionally by tests for A-factor and pigment production in "S. lividans" and S. coelicolor A3(2) after introduction of recombinant plasmids containing various restriction endonuclease fragments on the vector plasmids pIJ41 and pIJ702. The putative AfsB protein contains two regions separated by 167 residues which resemble conserved domains of known DNA-binding proteins. High-resolution nuclease S1 protection mapping revealed that the afsB mRNA, approximately 1,300 base pairs (bp) long, which was determined by Northern blot hybridization, had its start point 340 bp upstream of the putative methionine start codon. The Northern hybridization experiment also suggested that the afsB gene was constitutively transcribed throughout growth. Also shown by the Northern hybridization was the presence of an unidentified gene with an extraordinary amount of 880-bp mRNA located downstream from afsB. Dot hybridization with the brown pigment production genes, possibly involved in polyketide biosynthesis, as the probe suggested that the afsB gene did not stimulate transcription of the pigment production genes. In Southern blot DNA-DNA hybridization analysis with the afsB sequence as the probe, sequences exhibiting various degrees of homology were found in several Streptomyces spp. A DNA sequence showing strong homology to the afsB in Streptomyces griseus FT-1, a high streptomycin producer, behaved like an extrachromosomal element, homologous to the afsA gene, a structural gene for A-factor biosynthesis.
天蓝色链霉菌A3(2)的afsB基因的核苷酸序列已被确定，afsB是一个多效基因，它正向调控天蓝色链霉菌A3(2)和“变铅青链霉菌”中A因子以及色素抗生素放线紫红素和十一烷基灵菌红素的生物合成。通过将含有各种限制酶片段的重组质粒导入载体质粒pIJ41和pIJ702后，在“变铅青链霉菌”和天蓝色链霉菌A3(2)中检测A因子和色素产生，对afsB基因的决定簇进行了功能定位，该决定簇包括由243个氨基酸组成的假定AfsB蛋白。假定的AfsB蛋白包含两个区域，中间相隔167个残基，类似于已知DNA结合蛋白的保守结构域。高分辨率核酸酶S1保护图谱显示，通过Northern印迹杂交确定的约1300个碱基对（bp）长的afsB mRNA，其起始点在假定的甲硫氨酸起始密码子上游340 bp处。Northern杂交实验还表明，afsB基因在整个生长过程中组成型转录。Northern杂交还显示，在afsB下游存在一个未鉴定的基因，其mRNA长度为880 bp，数量异常。以可能参与聚酮生物合成的棕色色素产生基因作为探针进行点杂交，结果表明afsB基因不刺激色素产生基因的转录。以afsB序列为探针进行Southern印迹DNA-DNA杂交分析，在几种链霉菌属中发现了具有不同程度同源性的序列。在高产链霉素的灰色链霉菌FT-1中，一个与afsB具有高度同源性的DNA序列表现为一种与afsA基因同源的染色体外元件，afsA基因是A因子生物合成的结构基因。
