| Summary: | 碩士 === 國立陽明大學 === 遺傳學研究所 === 86 === Tn4811是一個位於鏈黴菌Streptomyces lividans染色體末端，長5.4-kb的轉位子。它似乎可編譯出五個蛋白質：氧化還原酵素和它的轉譯調節蛋白及三個與轉仕相關的蛋白。Tn4811位在染色體上的位置相當符合複製性轉位發生於兩個線形DNA間的模式。
為了偵測Tn4811的複裂性轉位，我們設計了兩個方法來偵測「共嵌體」的存在。首先，利用「交配表現」的方法，以含有環狀接合質體SCP2和pLUS729(一個不會自我驅動也不會被驅動的質體，帶有Tn4811及tsr基因)的Streptomyce coelicolor 1190(hisAl uraAl strAl)，與不含任何質體的S.lividansTK64(pro-2 str-6)進行接合生殖。挑選His+ Ura+ Tsr的接受株(轉接合株)，分析pLUS729與 SCP2的存在。當這兩個質體同時存在於轉接合株內，是因為pLUS729藉由共嵌體形式而傳送到接受株中。我以Southern轉漬雜配分析轉接合株的質體時，以Tn4811為探針，發現一個大於23-kb的訊號，但其並非源自於共嵌體，而有待進一步的分析。
另外，我將pLUS729和一個溫度敏感質體pGM3(帶有aphl基因)一起送入S. lividans TK64中，培養於39℃以去除pGM3。挑選Tsr及Nmr的菌株。這些菌株內含的質體應該是由pLUS729和pGM3共同形成的共嵌體。而在我所挑選的兩株Tsr Nmr菌株中，我無法分離到它門的質體DNA。
我亦藉由挑選S. coelicolor [pLUS729]的melC回復表現，來偵測Tn4811 的保留性轉位。挑選到的四個melC回復株中，其中三株的質體與plJ702相 似，另一株則同時含有兩個質體，分別與pIJ702及pLUS729相似。而利用Tn-4811作為探針的Southern轉漬雜配分析中，發現其染色體上有一個380-kb的訊號，可能來自於由pLUS729轉仕列染色體的Tn4811。
Tn4811 is a 5.4-kb transposon located near the termini of the Streptomyces lividans chromosome. It encodes five proteins: an oxidoreductase and its transcription regulator, and three putative transposition-related proteins. The terminal location of Tn4811 is consistant with the model for replicative transposition between two linear replicons.
To test whether Tn4811 transposes replicatively, we employed two strategies to detect its obligated cointegrate intermediates. Firstly, Streptomyces coelicolor 1190 (hisAl uraAl strAl) which harbors a conjugative circular plasmid SCP2 and pLUS729 (a non-conjugative and non-mobolizable circular plasmid containing Tn4811 and tsr) was mated with plasmidless S. lividans TK64 (pro-2 str-6). Thiostrepton-resistant (Tsr), His+ Ura+ transconjugants of S. lividans were selected and checked for the presence of pLUS729 and SCP2. The appearance of both plasmids in the recipient would indicate the transmission of pLUS729 via cointegrate formation. In the transconjugants, we found a signal of Southern hybridization using a probe for Tn4811 is larger than 23.0-kb. The signal is not from cointegrate intermediate and other possible DNA topological forms and should be study more.
Secondly, we placed pLUS729 and a temperture-sensitive plasmid, pGM3, that contained aphl ( neomycin-resistant; Nmr) in S. lividans TK64, and incubated the culture at 39℃ to cure pGM3. Tsr and Nmrr colonies were selected and checke for the presence of possible cointegrates formed by two plasmids. But we could not isolate the plasmid DNA in two putative survival strains at 39℃.
To test whether Tn4811 transposes conservatively, we selected the melC revertant in S. coelicolor with pLUS729. Four putative revertants were analyzed. Three contain plasmids like pIJ702. Another contains both pIJ702-like and pLUS729-like plasmids. For Tn4811 probing, we could find a 380-kb signal in chromosome indicating that Tn4811 may be transposed from pLUS729.
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