Yankovsky N K, Bukanov N O, Gritzenko V V, Evtushenkov A N, Debabov V G
Institute for Genetics and Selection of Industrial Microorganisms, Moscow, U.S.S.R.
Gene. 1989 Sep 30;81(2):211-8. doi: 10.1016/0378-1119(89)90181-9.
Erwinia chrysanthemi ENA49 structural and regulatory ptl genes, coding for pectate lyase (Ptl) were cloned in Escherichia coli cells. Phage vector lambda L47.1 and phasmid vector lambda pMYF131 were used for constructing libraries of BamHI and EcoRI fragments, respectively, of Er. chrysanthemi chromosomal DNA. Among the 1,100 hybrid clones containing BamHI Er. chrysanthemi DNA fragments and 11,000 hybrid clones containing EcoRI fragments, six and 45 clones, respectively, were identified as having pectolytic activity. Two different structural genes, designated ptlA and ptlB, have been subcloned on multi-copy plasmids. Genes ptlA and ptlB are located side by side on the chromosome of Er. chrysanthemi and transcribe in the same direction. Each of the genes has its own promoter. Southern-blot hybridization analysis showed that the cloned ptl genes shared practically no homology and each of the genes was represented by a single copy on the Er. chrysanthemi chromosome. Other ptl genes capable of expression in E. coli cells were not found in the gene libraries. Negative regulation of the ptlA gene expression by a cloned gene called ptlR was shown. To screen the gene library for the ptlR gene, a specific genetic system was devised. The genes studied are located within an EcoRI chromosomal DNA fragment of 7.3 kb in the order: ptlA-ptlB-ptlR.
将编码果胶酸裂解酶（Ptl）的菊欧文氏菌ENA49的结构和调控ptl基因克隆到大肠杆菌细胞中。分别使用噬菌体载体λL47.1和噬菌粒载体λpMYF131构建菊欧文氏菌染色体DNA的BamHI和EcoRI片段文库。在含有菊欧文氏菌BamHI DNA片段的1100个杂交克隆和含有EcoRI片段的11000个杂交克隆中，分别鉴定出6个和45个具有果胶分解活性的克隆。两个不同的结构基因，命名为ptlA和ptlB，已被亚克隆到多拷贝质粒上。ptlA和ptlB基因在菊欧文氏菌染色体上并排排列，并以相同方向转录。每个基因都有自己的启动子。Southern杂交分析表明，克隆的ptl基因几乎没有同源性，并且每个基因在菊欧文氏菌染色体上都以单拷贝形式存在。在基因文库中未发现其他能够在大肠杆菌细胞中表达的ptl基因。研究表明，一个名为ptlR的克隆基因对ptlA基因表达具有负调控作用。为了在基因文库中筛选ptlR基因，设计了一个特定的遗传系统。所研究的基因按ptlA-ptlB-ptlR的顺序位于一个7.3 kb的EcoRI染色体DNA片段内。
