噬菌體XP10基因體轉錄之研究

Abstract

中文摘要 為瞭解噬菌體 X p 10 感染寄主後在寄主細胞中轉錄的情形，曾在生體及試管的條件下，探求其轉錄的產物－ mRNA ，並進一步的利用 Southern 的方法探求其基因轉錄的安排。在生體條件的試驗中，將生體內的 X p 10 mRNA 以“ H － uridine 標幟，經 hot phenol 及酒精萃取，以 polyacrylamide 凝膠電泳分析法及凝膠放射活性測定法分析，得知其早期基因之轉錄產物可以產生 5 種 mRNA ，其中有一種會迅速被分解；中、晚期基因之轉錄開始於感染後 10 － 20 分鐘之間，可以產生 6 種 mRNA 。其中似乎尚有早期基因的 mRNA ，其原因可能是由於早期基因轉錄產物中的一個負責抑止寄主 RNA 聚合?之抑制劑作用不完全，或是新誘導的轉錄系統亦有轉錄早期基因的能力。 利用感染菌中純化出來的噬菌體 RNA 聚合?及感染前、後之寄主 RNA聚合?，於試管中進行轉錄，用凝膠放射活性測定法分析，發現感染後的寄主 RNA 聚合酪活性很低，原寄主 RNA 聚合?可產生 4 種，而噬菌體所誘導的 RNA 聚合?產生 3 種 mRNA 。此結果與生體條件下做出來的不甚相同。這種差異可能很正常，因為在生體條件下有更複雜的調節系統存在，而在試管條件下作用較單純，故一定會有所差別。為進一步瞭解基因表現的安排，另用 Southern 的 DNA 和 RNA 雜交分析法，將 X p l0 感染細胞內用 32P 標幟的 m R NA 與 nitrocellulose membrane 上經各種不同核酸鑑識?切成的 X p 1 0 DNA 片段進行雜交，因在方法上尚有待改善之處，故未得良好結果。

In order to understand the transcription Xpl0 phage on one in infected ce11, the transcription products—mRNA crc ctudicd with gel electrophoresis, and the gene arrange— lent of Xpl0 genome were also explored with Southern blotting nalysis. In vivo study phage infected cells were fed with H-uridine, and mRNA was extracted with phenol and precipitated with ethanol, and the labeled mRNA were analyzed with gel electro) horesis. For early gene 3 species of mRNA was detected, among these, one mRNA was rather unstable. For late gene 6 species of mRNA were obtained. Among these, early gene transcripts were also detected. In vitro study the purified phage - specific RNA polymerase was used and Xpl0 DNA used as template. For the comparison purified host RNA polymerase and modified host RNA polymerase were also used. Phage induced RNA polymerase produced 5 species of mRNA, host RNA polymerase produced 4 species of mRNA, and modified host RNA polymerase produced nothing. Based on the size of mRNA species the pattern of in vitro system was completely different from that of in vivo system. In order to explored the arrangement of gene expression, the Southern blotting analysis method was developed. The whole system seems to working well.