噬菌體Xp10感染後寄主RNA聚合?及蛋白質合成之改變

Abstract

Xp10為水稻白葉枯病病原菌(Xanthomonas campestris pv. oryzae)之溶解性噬菌體。由Xp10感染後的菌體經35S-methionine做體內蛋白質標識實驗，得知寄主轉譯活性在Xp10感染後急速下降，且在感染期間菌體新合成之蛋白質大約可歸類成三群，分別為感染早期，中期及晚期新合成之蛋白群。為了進一步研究寄主如何因Xp10感染而導至其轉譯活性銳減的機制，則由Xp10感染前後之菌體分離出總RNA，以電泳分析之，再以寄主染色體DNA片段為探針(probe)，追蹤經Xp10感染後，寄主轉錄情形。RNA-DNA雜合實驗結果(Northern hybridization)顯示其轉錄情形並無顯著差異。 另外，已知經純化的RNA聚合?在Xp10感染後會遺失其σ次單位。為了避免繁複純化步驟所可能導致的人為結果，便採用免疫沉澱法，分析Xp10感染前後不同時間時，粗酵素萃取液中RNA聚合?次單位蛋白質的組成。結果發現，σ次單位在感染後三分鐘已有明顯的減少現象 並在約十分鐘內完全自全?(holoenzyme)上遺失。在分析免疫沉澱物之過程中發現，與全?一起沉澱下來的一些蛋白質會因Xp10感染而有所變異，其中有一大約11 kD之蛋白質在感染20分鐘後開始遞增，經勝?免疫轉印法證實此蛋白質非全?的降解?物，因而初步認定為全?的輔因數(cofactor)，其功能仍需作進一步的研究方能揭曉。

Xp10 is a virulent bacteriophage of Xanthomonas campestris pv. oryzae, a pathogen which causes leave blight in rice plants. Results of in vivo protein pulse labelling showed a rapid decrease in host translational activity upon Xp10 infection. Protein synthesis during the infection period were catagorized as early, middle, and late stage synthesis. Northern hybridization analysis of RNA isolated at various intervals after infection did not reveal any differences in the transcription of host genes. To monitor the compositional changes of host RNA polymerase in Xp10 infected cells, a one-step immunoprecipitation method was employed to isolate the enzyme. Results showed a rapid loss of σ subunit from the RNA polymerase on the onset of infection. At 3 minutes after infection, only a trace amount of the subunit was detected, whereas at 10 minutes infection, σ was completely lost from the enzyme. This coincides with the sharp decrease of the host RNA polymerase transcriptional actitvity reported earlier. Along with the loss of σ subunit, some putative binding proteins were also reduced to various extents. In contrast, increasing levels of several other polypeptides could be detected. A phage specific band of about 11 kD appeared after 20 minutes infection. Immunoanalysis showed that this 11 kD polypeptide was not a proteolytic fragment of any of the enzyme subunits, but its function is still unclear.