하폐수의 자연환경에서 R plasmid와 재조합 유전자의 전이특성( I ) -$Km^rCm^r$유전자의 클로닝-

Transfer of R plasmids of Bacterial Isolates and Their Cloned R Genes in Natural Wastewater Environments (I) -Cloning of $Km^rCm^r$Gene-

항생물질 내성유전자를 유전자 조작기법으로 재조합시켜 만든 세균에서 그 유전자들이 전이되는 특성을 하폐수의 자연환경에서 연구하기 위하여, 자연계로부터 분리한 Gram 음성세균 중에서 kanamycin (Km), chloramphenicol (Cm), streptomycin (Sm), sulfadiazine(Su)에 내성을 띄는 균주로 DK1을 선발하였다. DK1 균주는 4개의 plasmid를 가지고 있으나 그 중 크기가 약 68kb인 pDK101 plasmid에 Km$^{${\gamma}$}$ Cm$^{${\gamma}$}$ 유전자를 가지고 있었다. 이 pDK101 plasmid에는 EcoRI site가 16개, PstI site가 32개, SalI site가 6개씩 있었다. pDK101 plasmid 와 vector로 사용한 pKT230을 E. coli으로 처리하여 얻은 절편들로부터 Km$^{${\gamma}$}$Cm$^{${\gamma}$}$ 유전자를 가지는 pDT309와 pDTS29 등의 chimeric piasmid를 제조하였다. 이들을 다시 E. coli C600과 E. coli HB101 에 형질전환 시킴으로써, DKC601, DKC602, DKH 102 그리고 DKH103 등의 재조합 균주를 얻었다. 이들 재조합 균주에서는 모두 Km$^{${\gamma}$}$Cm$^{${\gamma}$}$유전자가 잘 발현되었으며, 그 중 DKC601과 DKH103에서는 각각 pDT529와 pDT309의 chimeric plasmid가 포함되어 있는 것이 전기영동 방법으로 명확히 확인되었다.

In order to study the transfer of antibiotics resistance genes of the genetically cloned bacteria in water environments, DK1 strain, which is resistant to kanamycin (Km), chloramphenicol (Cm), streptomycin (Sm), and sulfadiazine (Su), was selected from the Gram-negative bacterial isolates from wastewater. One of 4 plasmids harboured in the DK1 strain was found to possess Km$^{${\gamma}$}$Cm$^{${\gamma}$}$ gene and be about 68 kb in size, and it was designated as pDK101. The plasmid of pDK101 was also found to have 16, 32, and 6 restriction sites for EcoRI. .PstI, and SalI, respectively. From the digestion fragments of pDK101 plasmid and pKT230 used as a vector by EcoRI restriction endonuclease, pDT309 and pDT529 were constructed as chimeric plasmids which possess Km$^{${\gamma}$}$Cm$^{${\gamma}$}$ gene and are 30.9 and 52.9 kb in size, respectively. When the chimeric plasmids were trasformed into E. coli C600 or E. coli HB101, transformants of DKC601, DKC602, DKH102, and DKH103 were obtained as cloned bacterial cells. The Km$^{${\gamma}$}$Cm$^{${\gamma}$}$ genes were well expressed in those cloned cells and the chimeric plasmids were clearly detected in the cloned cells of DKC601 and DKH103.