A New ColE1-Like Plasmid Group Revealed by Comparative Analysis of the Replication Proficient Fragments of Vibrionaceae Plasmids

( Li Pan ),( P. C. Leung ),( Ji Dong Gu ) 한국미생물 · 생명공학회 2010 Journal of microbiology and biotechnology Vol.20 No.8

Plasmids play important roles in horizontal gene transfer among Vibrionaceae, but surprisingly little is known about their replication and incompatibility systems. In this study, we successfully developed a bioinformatics-assisted strategy of experimental identification of seven Vibrio plasmid replicons. Comparative sequences analysis of the seven Vibrio plasmid replicons obtained in this study together with eight published Vibrionaceae plasmid sequences revealed replication-participating elements involved in the ColE1 mode of replication initiation and regulation. Like plasmid ColE1, these Vibrionaceae plasmids encode two RNA species (the primer RNA and the antisense RNA) for replication initiation and regulation, and as a result, the 15 Vibrionaceae plasmids were designated as ColE1-like Vibrionaceae (CLV) plasmids. Two subgroups were obtained for the 15 CLV plasmids, based on comparison of replicon organization and phylogenetic analysis of replication regions. Coexistence of CLV plasmids were demonstrated by direct sequencing analysis and Southern hybridization, strongly suggesting that the incompatibility of CLV plasmids is determined mainly by the RNA I species like the ColE1-like plasmids. Sequences resembling the conserved Xer recombination sites were also identified on the CLV plasmids, indicating that the CLV plasmids probably use the host site-specific recombination system for multimer resolution like that used by ColE1-like plasmids. All the results indicated that the 15 plasmids form a new ColE1-like group, providing a basis for the rapid characterization and classification of Vibrionaceae plasmids.

가금에서 분리한 Salmonella속 균의 항균물질에 대한 감수성 및 plasmid profile

김원용,장영효,박경윤,김철중,신광순,박용하,Kim, Won-yong,Chang, Young-hyo,Park, Kyoung-yoon,Kim, Chul-joong,Shin, Kwang-soon,Park, Yong-ha 대한수의학회 1995 大韓獸醫學會誌 Vol.35 No.3

In this study, we aim to find the presence of virulence-related plasmid in Salmonella isolates from poultry, and the difference between S pullorum and S gallinarum on the plasmid profile and antibiotics resistance. We used seventeen isolates of Salmonella spp that were isolated from poultry. Thirteen isolates, S typhimurium(ST), S pullorum(SP) and S gallinarum(SG), contained virulence-related plasmids. These are 95Kd plasmid in ST and 85Kd plasmid in SP and SG. Three(1/4 of ST, 1/1 of SE, and 1/9 of SP) isolates have no detectable plasmids. The isolates of ST have relatively variable plasmid profile but the isolates of S pullorum except No 12(additional 3.0Kb plasmid) have common 85K6, 8.1Kb, 4.0Kb and 2.3Kb plasmid and two of three isolates of S gallinarum have common 85Kb, 4.0Kb and 2.3Kb plasmid but the rest has only 85Kb plasmid. Interestingly, all of the isolates of SP have 8.1Kb plasmid, and same size of plasmid is also found in one of ST isolates. All of the isolates have the resistance to penicillin, chloramphenicol, rifampicin, streptomycin, sulfamethazine and some isolates show the resistance to ampicillin and tetracycline. There is no relatedness between plasmid profile and antibiotics resistance and no differences between SP and SG in antibiotics resistance. Therefore further differentiation of each isolates by restriction enzyme assay and, if possible, charaterization of each plasmid, especially, 8.1Kb plasmid in SP and ST, may be necessary.

Plasmid profiling of multi-drug resistant Vibrio sp. isolated from influent and effluent water samples of fish farms in Jeju, South Korea

파루크 아딜,운노타쯔야,Farooq, Adeel,Unno, Tatsuya The Microbiological Society of Korea 2018 미생물학회지 Vol.54 No.1

The objective of this study was to investigate the plasmid profiling of multi-drug resistant (MDR) Vibrio in influent (inflow) and effluent (discharged) water samples of fish farms in Jeju, South Korea. MDR isolates identified through disc diffusion susceptibility tests, were subjected to plasmid profiling. One hundred fifty Vibrio isolates were obtained from each influent and effluent water sample. All MDR isolates were subjected to plasmid profiling. Greater number of bacteria were enumerated from effluents (61%) comparing to influents (39%). High incidence of neomycin, sulfamethoxazole, amoxicillin and oxytetracycline resistance was observed among the isolates, which was higher in effluent samples. In contrast, Vibrio isolates were more susceptible to florfenicol, chloramphenicol, ciprofloxacin, and nalidixic acid. Among 99 (influent 39 and effluent 60) MDR isolates, a total of 58 (influent 38 and effluent 20) were found to bear plasmids ranging from 1.7 kb to >10 kb and showed 19 different antibiograms according to the size of plasmids. MDR isolates showed six and four distinct plasmid profiles in influent and effluent, respectively. Effluent samples contained more plasmid-carrying MDR Vibrio isolates with more diverse plasmid profiles and antibiograms, suggesting that fish farm tanks may serve as a reservoir of antibiotic resistance genes. The presence of plasmid-carrying MDR Vibrio isolates in fish farm effluent water may contribute to the dissemination of antibiotic resistance genes to the environments, which ultimately poses threat to human health. 이 실험은 제주도내 양식장으로부터 유입수와 방출수의 다제내성(MDR)을 가진 Vibrio 균의 plasmid profiling을 위해 진행하였다. Plasmid profiling을 위해 사용한 다제내성을 가진 균주는 디스크 확산법을 통해 확인하였고, 유입수와 방출수으로부터 각각 150개의 Vibrio 균주를 분리하였다. 모든 다제내성 균주를 대상으로 plasmid profiling을 실시하였으며, 유입수와 비교하여 방출수에서 많은Vibrio 균이 열거되었다(유입수 39%, 방출수 61%). 방출수에서는 neomycin, sulfamethoxazole, amoxicillin 및 oxytetracycline의 내성을 가진 균주가 유의적으로 많은 것으로 확인되었고, 대조적으로 Vibrio 균주는 florfenicol, chloramphenicol, ciprofloxacin 및 nalidixic acid에 더 민감한 것으로 확인이 되었다. 99종의 다제내성 균주(유입수 39종과 방출수 60종) 중에서 총 58종(유입수 38종과 방출수 20종)이 1.7 kb에서 10 kb 이상의 플라스미드를 가지고 있는 것을 확인하였으며 플라스미드 크기마다 19가지의 다른 다제내성 패턴을 보였다. 6종의 유입수와 4종의 방출수에서 다제내성 균주는 특이적인 plasmid profile이 확인되었다. 방출수 샘플은 보다 많은 플라스미드를 가진 다제내성 Vibrio 균주와 다양한 plasmid profile들과 다제내성 패턴을 가지고 있었고 이는 양식장의 저장탱크가 항생제내성 유전자의 저장소 역할을 할 수 있다는 점을 시사한다. 양식장의 방출수에서 plasmid를 가진 다제내성 Vibrio 균주의 존재는 항생제 내성 유전자의 전파에 기여할 수 있으며 이로 인해 인간의 건강을 위협할 수 있다.

국내 분리 다제내성 장티프스균의 Plasmid 및 β-lactamase의 특성에 관한 연구

유정식,신영학,오경수,이점규,김기상 국립보건연구원 1999 국립보건원보 Vol.36 No.-

Glucoamylase 유전자 STA1이 포함된 재조합 Plasmid를 갖는 Saccharomyces diastaticus 의 Glucoamylase 생산성에 미치는 Plasmid 안정성의 영향

Ahn, Jong-Seog,Hwang, In-Kyu,Jeong, Min-Sun,Mheen, Tae-Ick 한국미생물 · 생명공학회 1989 한국미생물·생명공학회지 Vol.17 No.6

For the purpose of improving glucoamylase productivity of Saccharomyces diastaticus, useful yeast in direct ethanol fermentation of starch, the effects of growth rate on the plasmid stability and glucoamylase productivity of S. diastaticus harboring recombinant plasmid pYES 18 containing glucoamylase gene STA 1 were investigated. In a selective medium, the recombinant plasmids were maintained stably at constant level but glucoamylase productivity was very low. On the other hand, in the complex medium containing starch, growth rate of the cell was stimulated by the supplementation of glucose and plasmid stability was improved by growth stimulation. We can conclude that glucoamylase productivity of S. diastaticus harboring the recombinant plasmid was increased as the maintaining of high plasmid stability in the cell. 전분질의 에탄을 직접발효에 유용한 Saccharomyces diastaticus의 glucoamylase 생산성 향상을 위하여 glucoamylase 유전자 STA1을 함유한 재조합 plasmid pYES 18을 갖는 S. diastaticus의 형질전환 균주에서 성장속도가 plasmid의 안정성에 미치는 영향과 plasmid 안정성에 따른 glucoamylase 생산성을 조사하였다. 최소 선택배지에서는 plasmid가 일정한 수준에서 안정하게 유지되었으나, 균체의 증식과 glucoamylase 생산성은 매우 미약하였다. Starch가 포함된 완전배지에서는 glucose를 첨가해 줌으로써 생육속도를 촉진시킬 수 있었으며 생육속도의 촉진에 의해 plasmid 안정성이 증가되었고 이에 따라 glucoamylase 생산성이 향상됨을 알았다.

새로운 conjugation 방법을 응용한 R plasmid 함유 어병세균의 분리와 양식장 내성균의 현황 분석

유민호(Min Ho Yoo),정준범(Joon Beom Jeong),김은희(Eun Heui Kim),이형호(Hyoung Ho Lee),정현도(Hyun Do Jeong) 한국수산과학회 2002 한국수산과학회지 Vol.35 No.2

R plasmid의 분포와 새로운 conjugation 방법 개발을 위하여, 한국의 남해안과 동해안의 양식현장의 어류질병관련 세균으로부터 항균제 다제 내성균을 분리하였다. 분리균 134종에서 10균주가 chloramphenicol, tetracycline, streptomycin, ampicillin, colistin, nalidixic acid, oxolinic acid and kanamycin 등에 대한 다제내성의 특성을 가지고 있었으며 이중 V. damsela JE1 한 균주는 chloramphenicol과 tetracycline에 대한 내성전달 특성을 갖는 R plasmid와 ampicillin과 kanamycin에 대한 유전자를 chromosome 등에 함유하고 있었다. 분리된 어병세균의 다제내성 특성이 R plasmid에 기인하는 것인지의 확인은 다양한 장내세균의 선택배지로 최근 개발된 CC 배지에서 각기 다른 균주들이 나타내는 증식여부 또는 집락 색깔의 차이를 이용하여 donor, recipient 그리고 transconjugant를 구별할 수 있는 방법을 적용하였다. Conjugation의 최적화를 위해 여러 가지 조건을 비교한 결과, V. damsela JE1의 R plasmid의 전달은 filter mating법을 사용 하는 것이 broth mating법보다 약 100배 이상의 높은 성공률을 보여 주었으며 이때 사용온도는 30℃ 이상, mating시간은 24시간에서 가장 높은 전달 빈도를 나타내었다. R plasmid를 함유한 V. damsela JE1이 분리된 4개월후, 동일 양식장의 동일 그룹의 넙치 장내세균에서 분리한 다제내성균 Spsingomonas sp. 3균주는 V. damsela JE1에서 분리된 것과 같은 크기의 R plasmid를 함유하고 있었으며, 그 특성 또한 V. damsela JE1의 R plasmid와 마찬가지로 다제 내성 중 Cm과 Tc에 대한 유전자만이 recipient로 전달되어 장내 세균총이 R plasmid의 reservoir로서의 역할을 하고 있음을 확인하였다. To develop a new method of conjugation and to determine the distribution of R plasimds, we isolated multi-drug resistant strains from fish pathogenic bacteria in the farms of south and east seacoasts of Korea. Out of the 134 isolates examined, 10 showed resistance to chloramphenicol, tetracycline, streptomycin, ampicillin, colistin, nalidixic acid, oxolinic acid and kanamycin. One out of 10 multi-drug resistance bacteria, Vibrio damsela JE1 (V. damsela JE1), contained transferable R plasmid of chloramphenicol-tetracycline resistance genes and other nucleic acids encoding ampicillin and kanamycin resistance. The presence of the R plasmid was confirmed by conjugation using the chromocult medium (CC) as a selective and differential medium for transconjugants with identification based on the growth or colors of the colonies. The frequency of R plasmid transfer with filter mating method was come out much higher than that of broth mating method and appeared to be dependent upon the mating time and temperature. The optimum conditions for filter mating method were found to be 30℃ and 24hrs as mating temperature and period, respectively. Moreover, donor cells with R plasmid, both isolate and standard bacteria, were shown to have an ability to transfer the plasmid against Escherichia cotli K-12 HB101 (E. coli HB101) and Edwardsiella tarda (E. tarda) RE14 at fairly high frequencies. Finally, we isolated 3 isolates of Sphingomonas sp., carrying R plasmid from 12 multi-drug resistant bacteria in normal microflora of the flounder (Paralichthys oliraceus) group used for the isolation of V. damsela JE1 four months before. The same size and gene transfer charateristics of R plasimds with those of V. damsela JE1 confirmed that normal microflora have the reservoir activity for R plasmid in natural aquatic environment

동물에서 분리된 Salmonella균의 병원성 관련 Plasmid에 관한 연구

최원필,정석찬,Choi, Won-pil,Jung, Suk-chan 대한수의학회 1992 大韓獸醫學會誌 Vol.32 No.3

This paper dealt with plasmid DNA profile in 98 Salmonella(S) isolated from pigs and cattle sources in Taegu, Gyeongbook and Gyeongnam during the period from 1984 to 1987. Also we were studied for restriction enzyme analysis of the plasmid DNA, and mouse infection, Sereny test and normal setum resistance test in guinea pig for S typhimurium and S enteritidis harbored or cured 60 megadalton(Md) plasmid and 36 Md plasmid, respectively. Of the 13 Salmonella isolated from cattle, 7 Salmonella harbored one or more plasmids and molecular sizes of the large plasmids were 60 Md for S typhimurium and 36 Md for S enteritidis. Of the 85 Salmonella isolated from pigs, 47 Salmonella were confirmed as being one or more plasmids, and all the S typimurium stains harbored 60 Md plasmid. In enzyme digestion with 8 types of restriction endonuclease for 60 Md plasmid DNA of S typhimurium, cleavage patterns were varied to enzymes, and the DNA was segmented into 4 to 15 fragments. In restriction enzyme analysis of 36 Md plasmid DNA obtained from four strains of S. enteritidis, the DNA showed the same cleavage patterns obtained with Eco RI, Hind III and Bam H I, and was segmented into 3 to 5 fragments. In virulence for mice by measuring the 50% lethal dose ($LD_{50}$), the $LD_{50}$ values obtained for 60 Md virulence-associated plasmid harbored strains of S typhimurium and 36 Md virulence-associated plasmid of S enteritidis were up to $10^4$-fold lower than the values obtained for the plasmid-cured strains of the same serotype. Only the plasmid harbored strains were resistant to the bactericidal activity of 90% guinea pig serum, and only they gave positive responses in sereny test. We suggested that their plasmid DNA might be associated with virulence for mice.

대장균이 보유한 Plasmid 유래 Tetracycline A, B 내성결정기의 분포

이유철,김팔동,김정민,이제철,이상화,설성용,조동택 慶北大學校 醫科大學 1994 慶北醫大誌 Vol.35 No.1

목적 : 현재까지 13가지 정도가 알려져 있는 Tc 내성 유전자의 대장균내의 분포를 조사하여 두면 균의 생활상 뿐아니라 발병 및 확산 경로의 추적에 지표가 결 것이다. 대상 및 방법 : 입원한 환자의 소변에서 분리한 대장균 유래 4개의 Plasmid와 건강인의 대변에서 분리한 대장균 유래 22개의 plasmid 등 총 26개의 tetracycline(Tc) 내성 plasmid를 대상으로 tetracycline A, B 내성 유전자(tet A, tet B)의 분포를 조사하였다. 결과 : EcoRI에 의한 절편들에 tet A probe를 사용한 hybridization의 결과, 5.6kb의 공통된 절편과 9.5kb의 절편에서 강한 hybridization band를 나타내는 2종류였다. tet B probe를 사용한 결과 소변에서 분리한 대장균 유래의 plasmid들에서 나타난 2.8kb 및 3.6kb 크기의 tet B 내성 유전자의 절편은 대변유래의 plasmid들에서는 관찰할 수 없었다. tet B 내성 유전자를 가진 plasmid들의 Tc 및 minocycline에 대한 최소발육저지농도가 tet A 내성 유전자를 가진 plasmid들 보다 높았다. 결론 : 건강인의 대변에서 분리한 대장균이 보유한 plasmid는 tet A를 보유한 것이 15개, tet B가 5개, tet A와 tet B의 두가지를 보유한 것이 2개로 다른 보고자들의 결과와는 달랐으며, 입원한 환자의 소변에서 분리한 대장균 유리 4개의 plasmid는 모두 tet B를 보유하고 있다. Using restriction endonuclease analysis and DNA-hybridization techniques with the tet A and let B resistant determinant DNA probes, we have examined the frequency of these determinants among 26 tetracy-cline(Tc) resistant plasm13s. All the 4 plasmids from 4 strains of Escherichia coli isolated from urine hybridized only with the tet B probe. Of the 22 plasmids from 22 strains of Escherichia coli isolated from healthy stool, 15(68%) plasmids hybridized with the tet A, 5(23%) plasmids with the tet B, and 2(9%) plasmids with the two probes, tet A and tet B. The tet A probe hybridized with 5.6kb DNA fragments of 6 plasmids and 9.5kb of 2 plasmids from fecal isolates. The hybridization pattern_with the tet B probe showed more complicated than that with tet A probe. The tet B probe hybridized with 2.8kb DNA fragments of all 4 plasmids and hybridized with 3.6kb fragments of 3 plasmids from urinary isolates. But the 2.8kb and 3.6kb DNA fragments were not founded in the EcoRI treated plasmids from fecal isolates. The transconjugant Escherichia coli strains harboring the plasmids with the tel B resistant determinants showed higher level of resistance to Tc and minocycline than those with the tet A.

내재형 Plasmid pBL1이 제거된 Brevibacterium lactofermentum 개발과 형질전환

이규남,민본홍,윤기홍 한국산업미생물학회 1995 한국미생물·생명공학회지 Vol.23 No.2

코리네형 아미노산 생산균을 위한 plasmid vectors의 개발에 이용되어 온 내재형 cryptic alsmid pBL1을 Brevibacterium lactofermentum으로부터 제거 하였다. B. lactofermentum에서는 복제할 수 없으나 ㎞^r 유전자를 지니고 있는 plasmid pEM1으로 제한 효소 Sau3AI에 의해 절단된 pBL1의 DNA fragments를 도입시킨 pEM1 유도체를 제조한 후 이를 Escherichia coli로부터 B. lactofermentum으로 conjugal transfer 시킴으로써 ㎞에 대해 내성을 갖는 B. lacto-fermentum transconjugant를 얻었다. ㎞^r transconjugant에는 pBL1과 pEM1 유도체간에 상동성 재조합으로 생겨난 plasmid pEB14가 존재하였으며 이것은 pEM1으로부터 유래된 ㎞^r 유전자를 지니고 있는 것으로 확인되었다. B. lactofermentum transconjugant의 ㎞^r 표현형질을 선별인자로 이용하여 ㎞에 대한 내성과 함께 pEB14이 동시에 상실된 균주를 선별함으로써 최종적으로 pBL1-free B. lactofermentum 균주가 제조되었다. plasmid pBL1이 존재하는 균과 상실된 균을 숙주균으로 사용하여 plasmid DNA에 의한 형질전환 효율과 형질전환체에서 plasmid 안정성을 비교한 결과 pBL1-free strain이 원래의 균주보다 유전자조작을 위한 숙주균으로서 우수하였다. 또한 pBL1이 존재하지 않으므로 pBL1-free strain에서 외래 plasmid DNAs가 간편하게 분리 및 분석될 수 있다. An endogenous cryptic plasmid, pBL1, which has been used to construct plasmid vectors for coryneform bacteria producing amino acids, was eliminated from Brevibacterium lactofermentum. The pBL1 was partially digested with Sau3AI and the resulting DNA fragments were subcloned into a suicide vector pEM1 which contains a kanamycin-resistant (㎞^r) gene. Km^r B. lactofermentum transconjugants were obtained by conjugal transfer of the pEM1 derivatives containing pBL1 DNA fragments from Escherichia coli into B. lactofermentum. Transconjugants were obtained by conjugal transfer of the pEM1 derivatives containing pBL1 DNA fragments from Escherichia coli into B. lactofermentum. A ㎞^r transconjugant was analyzed to contain a plasmid pEB14, which occurred in vivo by homologous recombination between pBL1 and the conjugal-transferred plasmid. The pEB14 including the pEM1-derived ㎞^r gene was found to be lost concomitantly with ㎞^r phenotype, resulting in the construction of a pBL1-free strain of B. lactofermentum. Based on transformation efficiencies and palsmid stability, the resultant pBL1-free strain is more useful than wild strain as a host cell for genetic manipulation. It could be concluded that foreign plasmid DNAs are efficiently isolated and analyzed from the pBL1-free strain because of the absence of endogenous pBL1 plasmid.

다제 내성 대장균에 있어서 Plasmid DNA의 Curing 현상에 관한 연구

김원섭 충북대학교 의과대학 충북대학교 의학연구소 1999 忠北醫大學術誌 Vol.9 No.1

연구 목적: 많은 종류의 박테리아에는 고유의 유전자를 지니는 염색체외에도 플래즈미드(plasmid)라는 유전인자 물질이 광범위하게 존재한다. 이 플래즈미드는 항생제 내성, 살균 물질 생산, 병원성 독소 생산 등의 기능과 관련된 유전인자를 지니고 있으나, 인위적으로 조합된 플래즈미드의 경우는 매우 불안정하다고 알려져 있다. 이에 냉동과 실온을 반복한 뒤 플래즈미드가 소실되는지의 여부와 항생제 내성 발현 여부를 조사하여, 자연적인 curing의 가능성에 대하여 고찰하였다. 재료 및 방법: -70℃에 보관한 균주를 실온에 방치 한 뒤 다시금 -70℃에 보관하는 과정을 3회 이상 반복 시행한 뒤 플래즈미드의 소실 여부를 관찰하였다. 결 과: E. coli 7A에서 추출되었던 플래즈미드는 여러 개의 각기 다른 크기의 compatible plasmid로서 항생제 내성인자가 공통적으로 존재하였을 가능성을 시사해 주었다. 결론: 자연적인 curing현상은 transposon 연구에 이용되기도 하지만 균주에 들어 있는 플래즈미드의 예상치 못한 소실을 초래할 수 있다. 이런 경우는 -70℃에 보관하였던 균주를 계대 배양하지 않고 자주 실온에 녹여 재 사용하는 경우 발생할 수 있는 것으로 보인다. 따라서 이러한 플래즈미드 curing현상을 방지하기 위해서는 high copy-number플래즈미드를 지닌 균주를 장기간 보관할 경우 일단 실온에서 녹인 균주는 반드시 게대 배양하여 새롭게 보관하여야 할 것으로 사료된다. 그리고 내성인자의 보존유무는 전기영동을 통해 직접 플래즈미드를 확인하는 것이 바람직할 것으로 사료된다. Purpose : Extrachromosomal genetic elements (plasmids) are distributed widespread in both gram-negative and gram-positive bacteria. They provide an important role to the flexibility of the organism's response to changes in environment. The widely studied plasmid-borne characteristics are drug-resistance, production of antimicrobial proteins, toxins, formation of sex pilli, etc. Plasmids are very fluid in their make-up; the presence of transposons leads to gene rearrangement by the process of transposition. The characteristic feature of plasmids is their instability. The plasmid-borne features are often lost from a population at a higher frequence than would be expected from the normal process of mutation. There are three distinct causes involved; plasmid integrity, partitioning at cell division, and differential growth rates. High-copy number plasmids such as naturally occurring R-factor and genetically engineered plasmids are structurally unstable. Therefore, it is possible for them to be cured naturally. Materials and Methods : In this study, naturally occurred curing was investigated in the multiple drug-resistant E. coli. These isolates were stored at -70℃ and were taken out to at room temperature. This procedure was repeated more than 3 times. The loss of plasmids was identified in these isolates by gel electrophoresis. Results : The level of ampicillin resistance in plasmid-cured isolates was lowered as compared to the original level, but was increased more of less after subculture with ampicillin induction. Among these isolates, the plasmid DNA of E. coli 7A was found to show five bands before curing and to be lost after curing on the agarose gel electrophoresis. The size of original bands were 4.0 Mdal, 3.5 Mdal, 3.0 Mdal, 1.4 Mdal, and 0.8 Mdal. The 1.4 Mdal and 0.8 Mdal-sized bands were thought to be identical but other bands were thought to be different ones by analysis of these plasmid bands which had been electroeluted and digested with HindⅢ. These bands might be compatible plasmids with common characteristics. With these results, it's assumed that the drug-resistant gene element of E. coli 7A may be trandposition-related or may be positioned on the chromosomal DNA. And in order to maintain high copy number plasmid such as recombinant DNA, it seems to be necessary to subculture with continuous antibiotic selective pressure.