Simpson A M, Simpson L
Mol Biochem Parasitol. 1980 Dec;2(2):93-108. doi: 10.1016/0166-6851(80)90034-1.
Kinetoplast DNA (kDNA) and kinetoplast RNA (kRNA) were isolated from bloodstream and procyclic culture forms of two clonal strains of Trypanosoma brucei. No differences were observed in kDNA (maxicircle) restriction profiles between bloodstream or procyclic culture forms of the same strain. Some differences were observed in kDNA maxicircle restriction sites between the two strains. Buoyant density analysis of Pst I digested kDNA showed the release of a minor low density band representing unit length linearized maxicircle DNA. Pst I or Bam H1-linearized maxicircle DNA was isolated by the Hoechst 33258 dye--CsCl method and a restriction enzyme map of the maxicircle was constructed. Closed monomeric minicircles released from kDNA networks by sonication sedimented with a molecular size of around 1100 base pairs. A substantial minor length heterogeneity was evident in acrylamide gel electrophoresis of once cut minicircles. Several minicircle sequence classes and two Hind III maxicircle fragments representing approx. 50% of the maxicircle were cloned in the bacterial plasmid, pBR322, in Escherichia coli. A purified kinetoplast-mitochondrion fraction was isolated from procyclic culture forms by the Renografin flotation method. The major kRNA components were two small RNAs which comigrated with Leishmania tarentolae 9 and 12 S kRNAs in denaturing gels. These RNAs hybridized to the maxicircle component of the kDNA, specifically to the smaller cloned Hind III maxicircle fragment. This cloned fragment had substantial sequence homology with the cloned maxicircle fragment from L. tarentolae which contains the 9 and 12 S RNA genes, implying an evolutionary conservation of the 9 and 12 S gene sequences. Identical kRnAs were observed in cultured bloodstream forms of T. brucei.
从布氏锥虫两个克隆株的血流型和前循环型培养物中分离出线粒体DNA（kDNA）和线粒体RNA（kRNA）。同一菌株的血流型或前循环型培养物之间，kDNA（大环）限制性图谱未观察到差异。两株之间的kDNA大环限制性位点存在一些差异。对经Pst I消化的kDNA进行浮力密度分析，结果显示释放出一条较小的低密度带，代表单位长度线性化的大环DNA。采用Hoechst 33258染料-CsCl法分离出Pst I或Bam H1线性化的大环DNA，并构建了大环的限制性酶切图谱。通过超声处理从kDNA网络中释放出的闭合单体小环，其沉降分子大小约为1100个碱基对。在一次切割小环的丙烯酰胺凝胶电泳中，明显存在大量较小的长度异质性。几种小环序列类别以及代表约50%大环的两个Hind III大环片段被克隆到大肠杆菌的细菌质粒pBR322中。通过泛影葡胺浮选法从前循环型培养物中分离出纯化的线粒体-线粒体部分。主要的kRNA成分是两种小RNA，它们在变性凝胶中与大利什曼原虫9和12 S kRNA迁移位置相同。这些RNA与kDNA的大环成分杂交，特别是与较小的克隆Hind III大环片段杂交。该克隆片段与来自大利什曼原虫的包含9和12 S RNA基因的克隆大环片段具有大量序列同源性，这意味着9和12 S基因序列具有进化保守性。在布氏锥虫的培养血流型中观察到相同的kRNA。
