線狀噬菌體Cf的基因體插入寄主染色體之研究

Abstract

中文摘要 自台灣柑橘瘡瘍病病原細菌 xanthomonas campestris pV. Citri 所分離的溫和性噬菌體?XW116，以不連續梯度密度的氯化絕離心（ CSCI density gradient Centrifugation ）純化，經電子顯微鏡觀察發現其為線狀噬菌體。進一步與感染同一寄主的線狀噬菌體 C f 比較，發現無論在血清反應，噬菌體的核酸大小， RF ( replicative form ) DNA 的限制?樣式（ restriction pattern ）上均與噬菌體 C f 相同，且噬菌體?XW116的 DNA 可以和噬菌體 C f DNA 進行 DNA － DNA 雜交（hybridization ）。故將噬菌體?XW116歸入為柑橘療瘍病病原細菌的線狀噬菌體 C f 。又因噬菌體必?XW116感染寄主後可引起寄主的突變，將野生菌株、營養需求菌株、回復菌株、被噬菌體感染的寄主細菌的 DNA 抽出後，以限制?作用，其 DNA 片段經洋菜凝膠電泳分開，用 Southern blotting 方法轉移到 nitrocellulose membrane 上，再經由 nick translation 所得到的32P 標記的 RF DNA 當做探針（ probe ) ，利用自動放射顯影發現營養需求菌株的細菌 DNA 有 RF DNA 的插入，且營養需求菌株的 DNA 以 EcoRI 作用後可得二條感光帶， Hind III 作用後可得三條感光帶， Bgl II作用後可得二條感光帶，由感光帶出現的位置可推測 RF DNA 進入寄主 DNA 的斷裂點可能是位於 Bgl II作用後的 k 片段之末端。而且可推知 Bgl II的 h 帶， Hind III 的 s 帶的 DNA 片段可進入寄主染色體之中。

Summary A temperate phage XW1l6 has been isolated from Xanthomonas campestris pv. citri (Wu et al., 1981). Since the morphology of this phage has not been thoroughly characterized, the phage was purified and its morphology was observed with an electron microscope in this study. It is shown to be a filamentous phage which is very similar to phage Cf isolated by Hu (1972). A comparison of the two phages was, therefore, made. Based on the morphology of virion, serological properties, the size of phage DNA, and the result of DNA-DNA hybridization, these two phages were proved to be identical. Since the infection of XW116 induced high mutation rate of host cells (Huang, 1982) , the possibility of the integration of phage genome into host chromosome was investigated. The chromosome DNAs isolated from wild type, auxotrophs and revertant strains were cleavaged with restriction enzymes i.e., BglII, EccRI and HindIII. The DNA fragments were fractionated by agarose gel electrophoresis, and the integration of phage genome into host chromosome was analyzed by Southern blotting method. The integration event was found in auxotrophs but not in wild type and revert ant. From the hybridization pattern of Cf genome by three restriction enzymes, the site of integration of Cf genome was suggested.