XP10 基因體的純系化

Abstract

I 、中文摘要 XP10 是一種以Xanthomonas oryzae為寄主的溶菌性噬菌體，由過去的研究報告中瞭解到X p 10 感染寄主後，會誘導產生具有抗 rifampicin 能力的 RNA 聚合?（ RNA polymerase ) ，並且初步知道此由噬菌體誘導產生的 RNA 聚合?之一般特性。為了，希望經由純系（ clone ）之方法找到此 RNA 聚合?之基因，先將 Xp10 基因體藉著 pBR 322 質體轉入大腸菌中，以利更深入的研究。 本論文除了對 X p 10 DNA 之特性稍做分析外，並以核酸限制疏（ restriction endonulease ) Bam HI將Xp10 DNA 切成不同大小之片段，藉著 pBR 322 貢邊的攜帶，轉入大腸菌CS 412 中後，以抗生素挑選（(antibiotic selection ), 結果可以得到每 ug DNA 中有 6 , 800 個轉形菌株（ trans - formants ）其中約 20 ％的轉形菌株為對青黴素具抗性但對四氯環素為敏感性(ampicillin resistant, tetracycline ）。將這些 AprTcS 轉形菌株中的質體抽出，做進一步之分析比較，發現比原來的 pBR 322 質體還大的約佔 80 ％。而以 SalI切Xp10 DNA 所做出的 Apr 轉形菌株，在每 ug DNA 中共有 1 , 800 個，其中 30％ 為 AprTcS 。這些 AprTcs 的菌株再經最後篩選之結果有 82 ％的菌株是為純系（ clones ) 。 經由生化方法分析證明的確有 xP10 之不同 DNA 片段進入大腸菌 cs412 中，使得接合質體（ hybrid plasmid ）比原來之 pBR322 質體還大。同時，在洋菜膠電泳（ Agarose gel electrophoresis ）中比較純系質體 DNA 的大小，以及用 8 段 BamHI 之 X p 10 DNA 片段與 11 段 SalI之 Xp10 DNA 片段，和被純系進去的 X p 10 DNA 片段相比結果，得知，在所有純系中找到的 X p 10 DNA 之 BamHI 片段及SalI工片段，各有 5 段，根據初步估計其分子量結果知道大約在 0 . 5 kb 至 9 . 0kb 之間的 DNA 片段均可被純系進去，而大於 9 . 0kb 之片段則未找到有純系的。就 BamHI 言在分子量為 12 . 9kb 到 0 . 8kb 的 8 段 DNA 中以 4 . 0 kb 到 4 . 5kb 的 DNA 片段被純系之機會最大，約佔 35 % ，而其他片段被純系的機會均差不多約為 15－20 %。另外，在SalI的 11 個片段（分子量在 18 .9kb－0 . 4 kb 間之） DNA 中以 0 . 7 －1 . 2kb 的片段被純系進去之百分比最高，達 63 ％。而其餘被純系進去的片段之比例約為 10 － 15 % ，或者更低。

II. ABSTRACT Xpl0 phage is a lytic bacteriophage isolated from Xanthomonas oryzae. In previous studies, it has been shown that this phage induces a phage specific RNA polymerase with a molecular weight of about 66,000. In order to clone this gene, the transfer of Xpl0 genome into Escherichia coli with plasmid pBR322 was firstly studied. The DNA isolated from Xpl0 phage was digested with restriction endonuclease BamHI or SalI, the fragments in the digestion mixture were ligated with plasmid pBR322, and used to transform E. coli CS412. The transformants were selected with drug resistant markers located in plasmid. When the DNA fragments from BamHI digestion were ligated with pBR322 and used to transform E. coli CS412, 6,800 ampicillin resistant trans— formants per ug of Xpl0 DNA was obtained. Among these about twenty percent of them were sensitive to tetracycline. However, under the same condition, when the fragments were prepared by SalI digestion, only 1,800 ampicillin resistant were obtained among these about thirty percent of them were also tetracycline sensitive. The size of the plasmids isolated from these trans— formants were analyzed with agarose gel electrophoresis. Most of them were bigger than pBR322. It suggests that all these plasmids carried additional DNA fragments. The hybrid plasmids obtained from these transformants were further digested with BamHI and Sail respectively, and the digestion partern were analyzed with agarose gel electrophoresis. Five insertional DNA fragments from Xpl0 DNA were identified. It was found that the DNA fragments between 0.5 to 9.0 Kb was cloned, and no fragment bigger than 9.0 Kb was cloned.