Pritchard A E, Vasil M L
J Bacteriol. 1986 Jul;167(1):291-8. doi: 10.1128/jb.167.1.291-298.1986.
A 3.3-kilobase-pair fragment of Pseudomonas aeruginosa DNA containing the phospholipase C (heat-labile hemolysin) gene was sequenced, and the location of the gene was determined. The gene product contains at its NH2 terminus a 38-amino acid sequence which structurally resembles the signal peptides of other secreted proteins but is unusually long and positively charged (6+). The location of the translation start codon was determined by constructing a series of plasmids in which the promoter of a transcription vector was ligated to Pseudomonas DNA containing deletions at the 5' end of the gene. The plasmids were used to transform Escherichia coli, and the resulting clones were assayed for hemolysin activity. In addition, sizes of truncated proteins produced by mutants with translation terminators introduced at specific sites were analyzed in E. coli maxicells. The gene is transcribed, starting just upstream of the hemolysin gene, as an mRNA of approximately 2,800 bases. Analysis of the nucleotide sequence, analysis of mutants in maxicells, and transcriptional studies indicate that the hemolysin is part of an operon composed of two genes. Phosphate regulation of the operon is at the transcriptional level. The location of the 5' end of the transcript was determined by S1 mapping.
对包含磷脂酶C（热不稳定溶血素）基因的铜绿假单胞菌DNA的一个3.3千碱基对片段进行了测序，并确定了该基因的位置。该基因产物在其氨基末端含有一个38个氨基酸的序列，其结构类似于其他分泌蛋白的信号肽，但异常长且带正电荷（6个正电荷）。通过构建一系列质粒来确定翻译起始密码子的位置，在这些质粒中，转录载体的启动子与在基因5'端含有缺失的铜绿假单胞菌DNA连接。这些质粒用于转化大肠杆菌，并对所得克隆进行溶血素活性检测。此外，在大肠杆菌大细胞中分析了在特定位点引入翻译终止子的突变体产生的截短蛋白的大小。该基因从溶血素基因上游开始转录，形成一个约2800个碱基的mRNA。核苷酸序列分析、大细胞中的突变体分析以及转录研究表明，溶血素是由两个基因组成的操纵子的一部分。该操纵子的磷酸盐调节在转录水平。转录本5'端的位置通过S1图谱分析确定。
