對rifampicin敏感及抗性之水稻白葉枯病菌菌株之核醣核酸聚合?之比較

Abstract

水稻白葉枯病原菌(Xanthomonas oryzae)對rifampicin敏感與具抗性的RNA聚合?被純化出，以比較對rifampicin抗性不同的兩酵素，在結構上與性質上的異同。 純化步驟如下：收集菌體，以超音波震碎器打破細胞壁，在低溫下用PEG將RNA聚合?與DNA一起沉澱，再用高鹽將酵素與DNA離心溶離，上層液中的RNA聚合?經DEAE-cellulose, Bio-Gel A 1.5m, DNA-cellulose column色析。得到純化倍數273倍的對rifampicin具抗性的RNA聚合?與純化倍數250倍的對rifampicin敏感的RNA聚合?。 純化出來的?素，經SDS gel electrophoresis的分析，敏感型的酵素有β(β')，α，σ三條蛋白帶，而抗rifampicin的RNA聚合酣在相當σ部位的蛋白帶，僅餘微量。各次單位的分子量分別為β(β')(155,000)，σ(93,000)，α(38,000)。Densitometer掃瞄膠體的A600吸光值，由吸光?面積除以分子量可知敏感型RNA聚合?的次單位摩爾比值β(β')：α：σ為1:1:0.4，對rifampicin具抗性的RNA聚合?的摩爾比值則為1:1:0.08。 二酵素合成RNA都需要ATP，CTP，GTP，Mg2+，DNA範本，Mn2+可取代Mg2+。加熱變性的DNA不適做為此二酵素的範本，Calf thymus DNA的範本性質最好。Hg2+，Heparin，Ethedium bromide，Congo red，Actinomycin D等抑制劑對二酵素皆有抑制性，但彼此受抑制的相對性不一。1.77μg/m1的rifampicin對敏感型的酵素有70％的抑制，對rifampicin具抗性的RNA聚合?則只有19％的抑制。50μg/m1的streptovaricin，對敏感型有85％的抑制，對變異株則僅抑制21％。兩酵素皆以28?37℃為最適宜的反應溫度，RNA的合成隨時間延長而增加，於反應開始後，愈晚加入rifampicin，所合成的RNA量愈多。

The DNA-dependent RNA polymerases from rifampicin sensitive and resistant strains of X. oryzae were purified and their subunits as well as other properties were compared. The purification procedure involved: disruption of the bacterial cell with sonicator, polyethyleneglycol precipitation, chromatography on DEAE-cellulose. The purity after purification is 273 fold for rifampicin resistant RNA polymerase and 250 fold for rifampicin sensitive RNA polymerase. Based on SDS-gel electrophoresis, the subunits and molecular weight of the two enzymes were compared. Three bands corresponding to β(β'), α, σ, appeared on the sensitive RNA polymerase gel, and molar ratio was 1:1:0.4, but was tracing on the resistant RNA polymerase gel, and molar ratio β(β'): α: σ was 1:1:0.08. ATP, CTP, GTP, Mg2+ DNA were essential for the activities of two enzymes, Mg2+ could be replaced by Mn+. Heat-denatured DNAs were not suitable for the two enzymes as templates, but native calf-thymus DNAs were the best templates for the two enzymes. Hg2+ heparin, ethedium bromide, congo red, actinomycin Dinhibited the two enzymes, but not equally quantitively. l.67μg/ml rifampicin inhibited the rifampicin sensitive RNA polymerase by 70% activity, but just 1% inhibition to the rifampicin resistant RNA polymerase. Also 50μg/ml streptovaricin inhibited the rifampicin sensitive RNA polymerase by 85% and 21% inhibition to resistant RNA polymerase. 28-37℃were optimal temperature. Depending on the time increasing, the synthesis of RNA is increasing.