- 作者： 楊美桂; 余曉清; 李昭鋐
- 作者服務機構： 輔仁大學生物系; 美國印地安那大學病理系
- 中文摘要：
為探討DNA甲基?對轉移子Tn3轉移作用之影響，以質體pCHL161與pHS1::Tn3#5分別為Tn3之轉移之受體
與供體，進行轉移作用之分析。將兩質體同時送入不同之E. coli菌株內，以cointegrate之形成測定轉移頻率，發現E. coli
之dam基因對Tn3之轉移沒有影響，而Tn3在dcm+菌株較dcm-菌株容易轉移。將帶有EcoRII甲基?基因（EcoR II
甲基?與dcm甲基?有相同作用）之質體pR215送入dcm-菌株，則Tn3之轉移頻率有顯著提升。此結果顯示E. coli
dcm methylase參與Tn3之轉移作用。
至於transposase定否具甲基化的能力，則以pCHL161（含Tn3的38 bp尾端重複序列和Ap基因）與pLB101（提
供transposase）同時送入E. coli GM272（recA+ dam- dcm- hsd）後抽出之pCHL161，以辨認-CC(A/T)GG-的
二種限制?EcoR II和Apy I作用，可見明顯甲基化作用，其他限制?Bgl I 與 Ava II亦無法將pCHL161完全切割，
表示Tn3 transposase可將Bgl I 與Ava II之辨識序列進行部份甲基化，其甲基化情形與作用機制則有待進一步分析。l - 英文摘要： The effects of DNA methyltransferases on Tn3 transposition were investigated. The E. coli dam(deoxyadenosine methylase) gene was found to have no effect on Tn3 transposition. In contrast, Tn3 wasfound to transpose more frequently in dcm+ (deoxycytosine methylase) cells than in dcm- mutants. Whenthe EcoRII methylase gene was introduced into dcm- cells (E. coli strain GM208), the frequency of Tn3transposition in GM208 was dramatically increased. The EcoRII methylase recognizes and methylatesthe same sequence as does the dcm methylase. These results suggest that deoxycytosine methylase mod-ified DNA may be a preferred target for Tn3 transposition. Experiments were also performed to determinewhether the Tn3 transposase was involved in DNA modification. Plasmid DNA isolated from dcm- E. colicontaining the Tn3 transposase gene was susceptible to ApyI digestion but resistant to EcoRI digestion,suggesting that Tn3 transposase modified the dcm recognition sequence. In addition, restriction enzymesTaqI, AvaII, BgII and HpaII did not digest this DNA completely, suggesting that the recognition sequencesof TaqI, AvaII, BgII and HpaII were modified by Tn3 transposase to a certain degree. The type(s), theextent and mechanism(s) of this modification remain to be investigated.
- 中文關鍵字： E. coli; dcm methylase
- 英文關鍵字： --
