Johnson S R, Romig W R
Mol Gen Genet. 1979 Feb 16;170(1):93-101. doi: 10.1007/BF00268584.
Improved Vibrio cholerae donors were constructed by introducing the ampicillin transposon, Tn1, into both the conjugative plasmid, P, and the bacterial chromosome to provide "portable regions of homology." The resulting Tfr (Transposon-facilitated recombination) donors transferred genes at high frequency from origins specified by the chromosomally inserted Tn1 copies. Tn1 was transposed into the chromosome from a deleted P::Tn1 vector, which was eliminated from the cells by superinfection with a thermosensitive P::Tn9 (chloramphenicol) mutant plasmid. After eliminating the thermosensitive plasmid, the chromosomally resistant isolates were converted into donors with a P::Tn1 conjugative plasmid. Tfr donors were also obtained by isolating Tn1 insertion mutations in a gene for thymine biosynthesis. Chromosomal sites of Tn1 relative to bacterial genes were determined by measuring gene transfer frequencies and genetic linkage. In one case, linkage of the amp gene to the chromosomal genes that defined its location was demonstrated. Chromosomal transfer by Tfr donors was reversed by isolating P::Tn1 plasmids that contained Tn1 inserted in the opposite orientation.
通过将氨苄青霉素转座子Tn1导入接合质粒P和细菌染色体中，构建了改良的霍乱弧菌供体，以提供“可移动的同源区域”。所得的Tfr（转座子促进重组）供体从染色体插入的Tn1拷贝指定的起始位点高频转移基因。Tn1从缺失的P::Tn1载体转座到染色体上，通过用温度敏感的P::Tn9（氯霉素）突变体质粒进行超感染，该载体从细胞中消除。消除温度敏感质粒后，将染色体抗性分离株转化为带有P::Tn1接合质粒的供体。通过分离胸腺嘧啶生物合成基因中的Tn1插入突变也获得了Tfr供体。通过测量基因转移频率和遗传连锁来确定Tn1相对于细菌基因的染色体位点。在一个案例中，证明了氨苄基因与定义其位置的染色体基因之间的连锁。通过分离含有以相反方向插入Tn1的P::Tn1质粒，逆转了Tfr供体的染色体转移。
