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사람의 배양세포에 있어 자외선 및 MMS 에 의한 비동시 (非同時) DNA 합성과 염색체 이상에 미치는 감기상사체의 (監基相似體) 영향
엄경일,박상대 ( Kyung Il Um,Sang Dai Park ) 한국유전학회 1981 Genes & Genomics Vol.3 No.1
The effects of base analogs on UV-light and MMS-induced unscheduled DNA synthesis and chromosome aberrations were investigated in human cells and the results obtained were as follows: BUdR and IUdR were found to be both potent sensitizers enhancing UV-induced unscheduled DNA synthesis and chromosome aberrations. The sensitization effects of these base analogs were different in these two different repair processes. BUdR and IUdR did not seem to act as sensitizers for both unscheduled DNA synthesis and chromosome aberrations induced by MMS. These results suggest that excision repair may not be related to chromosome aberrations in human cells.
DNA 상해와 회복기작에 미치는 환경성 돌연변이원의 영향
엄경일,채기수,이주혜,정정남,이천복 한국유전학회 1986 Genes & Genomics Vol.8 No.4
The effects of aphidicolin, an inhibitor of DNA polymerase α, on ethyl methanesulfonate (EMS)- or bleomycin (BLM)-induced DNA repair were investigated in Chinese hamster ovary (CHO) cells. The amount of unscheduled DNA synthesis induced by EMS or BLM was increased according to dose increment and decreased with incubation time after treatment, and the damage almost seemed to be recovered during 4 hour incubation. The EMS-induced DNA single-strand breaks showed dose dependent increment and were increased according to EMS containing time. BLM also showed dose dependent increment on DNA single-strand breaks. On the other hand, it was shown that aphidicolin inhibited excision repair induced by EMS, and thus, the post-treatment with aphidicolin induced the higher degree of unscheduled DNA synthesis and the more accumulation of DNA single-strand breaks than the cells post-incubated without aphidicolin. However, aphidicolin did not inhibit excision repair induced by BLM, and thus, the cells post-incubated with or without aphidicolin for 1 hour had the same value in the amount of unscheduled DNA synthesis and of DNA single-strand breaks. These results suggested that the participation of DNA polymerase α in DNA repair induced by EMS or BLM was dependent on different types of damage.
환경성 유해요인이 유전물질과 세포활성에 미치는 영향 III. 포유동물세포에서 돌연변이원에 의한 DNA 상해의 회복에 미치는 DNA 중합효소저해제의 영향
엄경일,선우양일,이천복,신은주 한국환경성돌연변이발암원학회 1988 한국환경성돌연변이·발암원학회지 Vol.8 No.1
본 연구는 Ethyl methanesulfonato(EMS) 혹은 Bleomycin(BLM)에 의해 유발된 DNA상해의 회복에 미치는 DNA 종합효소 $\alpha$ 저해제인 Aphidicolin(APC)과 DNA 종합효소 $\beta$의 저해제인 2`, 3`-dideoxythymididine 5`-triphosphate(ddTTP)의 영향을 조사하기 위하여 Chinese hamster ovary(CHO)-Kl 세포를 재료로 비주기성 DNA 합성법과 알칼리유출법 및 스칼리 자당구배침강법으로 수행하여 얻은 결과는 다음과 같다. APC와 ddTTP는 EMS에 의해 유발된 DNA 상해의 회복을 저해하여 APC 혹은 ddTTP를 처리하지 않고 배양한 실험군 보다 비주기성 DNA 합성율과 DNA 단사 절단율이 증가되었다. 한편 BLM에 의해 유발된 DNA 상해의 회복에서는 ddTTP를 처리했을 경우에만 저해되었다. 즉 BLM 처리 후 ddTTP를 후처리한 실험군의 비주기성 DNA 합성율과 DNA단사 절단율은 ddTTP를 처리하지 않은 군보다 증가되었고, BLM 처리 후 APC를 후처리할 경우에 비주기성 DNA 합성율과 DNA 단사 절단율은 APC를 처리하지 않은 군과 유사하였다. 이상의 결과들에서 EMS에 의해 유발된 DNA 상해의 회복에는 DNA 중합효소 $\alpha$, $\beta$양자가 관여하나 BLM에 의해 유발된 DNA 상해의 회복에는 중합효소 $\beta$가 관여하는 것으로 추측된다. The effects of aphidicolin (APC), an inhibitor of DNA polymerase alpha, or 2', 3'-dideoxythymidine 5'-triphosphate (ddTTP), an inhibitor of DNA polymerase beta, on the repair of DNA damage induced by ethyl methanesulfonate (EMS) or bleomycin (BLM) were investigated in Chinese hamster ovary (CHO)-K1 cells. Three assays were employed in this study: unscheduled DNA synthesis, alkaline elution and alkaline sucrose gradient sedimentation. It was shown that APC or ddTTP inhibited DNA induced by EMS, and thus, the post-treatment with APC or ddTTP following EMS treatment was resulted in the more amount of unscheduled DNA synthesis, and the more accumulation of DNA single-stand breaks than the cells post-incubated without APC or ddTTP. While, in the BLM induced DNA repair, only ddTTP inhibited DNA repair induced by BLM. And thus, the groups post-incubated with or without APC after BLM treatment had the same value in the amount of unscheduled DNA synthesis and of DNA single-strand breaks, while post-treatment with ddTTP was resulted in the increased amount of unscheduled DNA synthesis and the increased DNA sin -strand breaks than the group without ddTTP. These results suggested that both of DNA polymerase $\alpha$ and $\beta$ participated in the repair of DNA damage induced by EMS, but in BLM-induced DNA repair, polymerase $\beta$ participated.ipated.
HeLa 細胞에서 Bleomycin에 의해 誘發된 DNA 回復合成에 미치는 沮害劑의 影響
嚴慶一 東亞大學校 敎育大學院 1981 敎育大學院 論文集 Vol.7 No.-
HeLa S_3 세포를 재료로 DNA 회복합성에 미치는 Bleomycin(BLM)의 농도에 따른 효과와 저해제[5-aminouracil(AU), actinomycin D(AMD), caffeine (Caf), 5-fluorodeoxyuridine (FUdR)]들 각각의 단독효과, 그리고 BLM에 의한 DNA 합성 및 회복합성에 미치는 저해제들의 영향을 ^3H-thymidine 처리에 의한 자기방사법으로 조사한 결과는 다음과 같다. 1. BLM 단독 처리인 경우 BLM에 의해 유발되는 DNA 회복합성율은 1.0㎍/㎖의 농도까 농도의 증가에 따라 증가한다. 세포당 평균 grain 수로 정량 분석한 grain 수도 농도의 증가에 따라 1.0㎍/㎖의 농도까지 증가한다. 2. 저해제들을 단독으로 각각 처리할 경우 DNA 회복합성에 영향을 주지 않는다. 3. BLM과 저해제를 복합 처리한 모든 군의 DNA 회복합성율은 BLM 단독 처리군에 대해 변화가 없다. 그러나 BLM과 AU, BLM과 FUdR을 처리한 군에서는 DNA 합성이 억제되었다. 이상의 결과들은 본 실혐 조건 하에서 BLM에 의해 유발된 DNA 회복합성에 저해제들이 영향을 미치지 않는 것으로 추측된다. The present study has been undertaken to determine the effects of inhibitors [5-amin-ouracil (AU), actinomycin D (AMD), caffeine (Caf) and 5-fluorodeoxyuridine (FUdR)] on bleomycin (BLM) induced DNA synthesis was measured by autoradiography. The results obtained were as follows: 1. In the BLM treated group, the induced DNA repair synthesis by BLM was shown to in direct proportion upto 1.0 ㎍/㎖. The quantitative analysis of unscheduled DNA synthesis as determined by the average number of grains per cell was also shown that the grains were increased directly proportional to dose increased upto 1.0 ㎍/㎖. 2. The single treatment with inhibitors did not affect DNA repair synthesis. 3. The percentage of DNA repair synthesized cells did not significantly change in all BLM plus inhibitors as compared with the corresponding single BLM, but the normal DNA synthesis by BLM plus AU or FUdR was slightly suppressed. The above results suggested that these inhibitors did not seem to induce the BLM induced DNA repair synthesis under this experimental condition.
전기천공법에 의한 Bacillus brevis P176-2의 형질전환
채기수,엄경일 東亞大學校附設基礎科學硏究所 1993 基礎科學硏究論文集 Vol.10 No.1
균체외단백질 생성균 Bacillus brevis P176-2 균주를 pUB110 plasmid DNA로 전기천공법에 의한 형질전환을 시도하여 본 방법의 유효성과 최적조건을 검토하였다. 본 실험에 사용한 전기천공 장치로 전장강도 12.5kV/㎝, capacitor 용량 7.3㎌의 조건하에서 고전압 pulse를 세포에 가했을 때 가장 높은 형질전환율(1.26× 10⁴/㎍ plasmid DNA)을 나타내었으며 세포 생존율은 10% 정도이었다. 세포의 생육도가 ??에서 약 1.2정도로 증식하였을 때 세포를 수확하여 형질전환 시키는 것이 가장 좋았으며 그 이상의 생육도에서는 세포의 생존율은 증가되었지만 형질전환율은 낮아졌다. 전강강도 12.5kV/㎝, capacitor용량 7.3㎌ 조건의 pulse를 세포에 2회 연속적으로 가할 때 최대의 형절전환율(2.40× 10⁴/㎍ plasmid DNA)을 보였다. 세포의 생존과 pulus의 지속시간에 영향을 주는 electroporation burrer는 7 mM HEPES(pH 7.4), 272mM sucrose 및 1mM MgCl₂을 함유하는 buffer를 사용하여 세포를 수확하고 현탁하여 형질전환할 때 형질전환율과 세포 생존율이 가장 높았다. 세포현탁액에 첨가되는 plasmid DNA의 농도가 10㎍/ml일 때 까지는 농도에 비례하여 형질전환율이 높아졌으나 그 상의 농도에서는 비례하지 않았다. The optimum conditions and mechanisms for the plasmid-mediated genetic transformation of intact cells of bacillus brevis P176-2, an extracellular protein producing bacterium, by electroporation were investigated. It was found that pUB110 plasmid DNA can be introduced into intact bacterial cells by electroporation. The frequency of transformation by this electroporation system depended upon the initial electric field strength, the capacity of the clectric discharge capacitor, growth stage, number of successive pulses and composition of electroporation buffer. It was effective for transformation that cells were harvested, washed and resuspended with HSM[7mM HEPES(pH 7.4), 272mM sucrose, 1mM MgCl₂] electroporation buffer when cell growth was attained to 1.2 at ?? A maximum frequency of transformation of 2.40× 10⁴ transformants per ㎍ plasmid DNA was obtained by two succesive pulses with an initial electric field strength of 12.5kV/㎝ and with a capacitance of 7.3㎌. Key words : Bacillus breves, pUB110, plamid DNA, transformation, electroporation.