線狀噬菌體Cf-11基因體插入寄主染色體之型式

Abstract

中文摘要 為找出噬菌體Cf-11基因體上之附著位置(attachment site； att site)，首先必須定出其基因體上核酸限制?之物理圖譜(physical map)；將Cf-11感染柑橘潰瘍原細菌(Xanthom-onas campestris pv. citri 品系xw47)寄主，並純化其在細胞內所形成之RF DNA，以8種限制?：EcoRI、Bam HI、Hind III、Pvu II、Bgl II、Xho I、Kpn I、Sst II切割，分別定出其在RF DNA上之物理圖譜。經分析結果得知EcoRI、Bam HI、Kpn I、Sst II祇有一個剪切位置，Hind III、Pvu II、Xho I有二個位置，Bgl II有三個位置，而Xba I則無任何剪切位置，並發現此確實與另一線狀噬菌體Cf(24,42)之物理圖譜一樣，因此二者為十分相似之同類線狀噬菌體(24)。進一步將經Cf-11感染後之細菌菌株染色體以限制?剪切，藉洋菜膠電泳(agarose gel electrophoresis)分開DNA片段，並以Southern blotting方法將DNA轉移(transfer)至Gene Screen TM membrane上，然後用〔α-32P〕TTP標記(label)之RF DNA當探針(probe)，作DNA-DNA雜交(hybridization)試驗及自動放射顯影(autoradiography)。將結果與用相同核酸限制?剪切之RF DNA片段對照組作比較，知：以Xho I剪切時，RF DNA可得5.85kb和1.30kb兩條感光帶，而細菌染色體則見5.85kb和另兩條分別為9.20kb與1.07kb之感光帶；以Bgl II剪切時，RF DNA可得5.13kb、1.89kb、0.47kb三條感光帶，而細菌染色體則見1.89kb、0.47kb和另兩條分別為7.70kb與6.63kb之感光帶；以Bgl II和Xho I兩種酵素一同剪切時，RF DNA可得4.22kb、1.89kb、1.10kb、0.19kb四條感光帶，而細菌染色體則見4.22kb、1.89kb、0.19kb和另兩條分別為8.34kb與1.15kb之感光帶；以Sst II和Xho I兩種酵素一同剪切時，RF DNA可得5.85kb、0.75kb、0.54kb三條感光帶，而細菌染色體則見5.85kb、0.75kb和另兩條分別為3.29kb與2.24kb之感光帶；以Kpn I和Xho I兩種酵素一同剪切時，RF DNA可得5.85kb、0.70kb、0.59kb三條感光帶，而細菌染色體則見5.85kb、0.70kb和另兩條分別為2.35kb與1.12kb之感光帶。因此得知RF DNA上之附著位置位於一約350bp的SstII-Bgl II DNA片段上，若以圖譜上唯一之EcoRI位置當作零點〔zero point)，則位在距此零點約67.4％和71.8％之間的DNA片段中。最後以逢機取樣方式進一步分析，得知經噬菌體Cf-11感染後之細菌菌株染色體上之噬菌體原(prophage)插入寄主DNA之附著位置可能一定。

Summary To localize the attachment site of bacteriophage Cf-11 genome, a physical map of the phage was constructed firstly. The RF DNAs in cells of bacterium Xanthomonas campestris pv. citri strain xw47 that had been infected with with Cf-11 were isolated, purified, and digested with restriction endonucleases EcoRI, BamHI, Hindlil, PvuII, BglII, XhoI, KpnI, and SstII. Respectively, EcoRI, BamHI, KpnI, and SstII has only one cleavage site on the RF DNA; HindIII, PvuII, and XhoI each has two sites; BglII has three sites, whereas XbaI has none. According to the results, it was found that the restriction map of Cf-11 is identical to that of another filamentous phage Cf. The chromosomal DNAs of Cf-11 infected host cells were digested with the restriction enzymes, fractioned by agarose gel electrophoresis, and transfered to Gene Screen TM membrane by Southern blotting method. Using the 332P-TTP labelled RF DNA probes to carry out DNA-DNA hybridization and autoradiography, and campared the results with the RF DNAs that had been digested with the same enzymes, it was shown: (1) with XhoI, the RF DNAs get two bands of 5.85 kb and 1.30 kb fragments, while 5.85 kb, 9.20 kb, and 1.07 kb bands in the bacterial chromosomes; (2) with BglII, the RF DNAs get three bands of 5.13 kb, 1.89 kb, and 0.47 kb fragments, while 1.89 kb, 0.47 kb, 7.70 kb, and 6.63 kb bands in the bacterial chromosomes; (3) with BglII and XhoI simultaneously, the RF DNAs get four bands of 4.22 kb, 1.89 kb, 1.10 kb, and 0.19 kb fragments, while 4.22 kb, 1.89 kb, 0.19 kb, 8.34 kb, 1.15 kb bands in the bacterial chromosomes; (4) with SstII and XhoI simultaneously, the RF DNA5 get three bands of 5.85 kb, 0.75 kb, 0.54 kb fragments, while 5.85 kb, 0.75 kb, 3.29 kb, and 2.24 kb bands in the bacterial chromosomes; (5) with KpnI and XhoI simultaneously, the RF DNAs get three bands of 5.85 kb, 0.70 kb, and 0.59 kb fragments, while 5.85 kb, 0.70 kb, 2.35 kb, and 1.12 kb bands in the bacterial chromosomes. So after the careful resolution, it was obtained that the attachment site on Cf-11 genome is located in an about 350 bp DNA fragment between the SstII and the BglII sites, that is 67.4%-71.8% from the zero point of the single EcoRI site on the map. Further analysis, when with random selection, suggested that the attachment sites on Cf-11 genome and the host chromosome may be persistent.