Elizbarashvili D A, Baĭmukhanova S K, Egorov I A, Tarasov V A
Genetika. 1989 Feb;25(2):207-13.
Functionally active genetic determinant of alpha-hemolysin was cloned. Hemolytic plasmid pHly195 was used as a donor of the determinant and pBR322 plasmid served as recipient. Cloning was done with a help of HindIII restriction endonuclease. The recombinant plasmid obtained represents pBR322 plasmid with the built-in fragment of 7.4 kb containing genes of functionally active determinant of alpha-hemolysin. Restriction map was constructed using HindIII, EcoRI, BamHI and SalI restriction endonucleases. Insertional mutagenesis was carried out with the help of the Tn1000 transposon. Plasmid DNAs were isolated from insertional mutants of Hly- phenotype and treated with EcoRI, SalI and BamHI. On the basis of the sizes of restriction fragments of the mutant plasmid DNAs localization and orientation of insertions of Tn1000 into the cloned determinant of alpha-hemolysin were determined.
α-溶血素功能活性基因决定簇被克隆。溶血性质粒pHly195用作该决定簇的供体，pBR322质粒用作受体。克隆借助HindIII限制性内切酶完成。获得的重组质粒是带有一个7.4 kb内置片段的pBR322质粒，该片段包含α-溶血素功能活性决定簇的基因。使用HindIII、EcoRI、BamHI和SalI限制性内切酶构建了限制酶图谱。借助Tn1000转座子进行插入诱变。从Hly-表型的插入突变体中分离出质粒DNA，并用EcoRI、SalI和BamHI处理。根据突变体质粒DNA限制片段的大小，确定了Tn1000插入克隆的α-溶血素决定簇的位置和方向。
