Volloch V, Schweitzer B, Zhang X, Rits S
Boston Biomedical Research Institute, MA 02114.
Proc Natl Acad Sci U S A. 1991 Dec 1;88(23):10671-5. doi: 10.1073/pnas.88.23.10671.
A substantial amount of cytochrome oxidase subunit III (COIII) mRNA continues to be synthesized de novo in Trypanosoma brucei in the presence of actinomycin D, presumably by a DNA-independent transcription process. We describe the identification of negative-strand COIII RNA molecules, characterization of their termini, and the detection of RNA-dependent RNA polymerase activity. Three lines of evidence for the existence of negative-strand COIII RNA are presented: (i) hybridization with oligonucleotide probes with the same polarity as mRNA after preliminary enrichment for putative negative-strand RNA by affinity purification; (ii) cloning and sequencing of negative-strand complements for the unedited, edited, and partially edited COIII RNA; and (iii) exact correspondence of the terminal sequences of the putative negative-strand RNA molecules to the ends of COIII RNA. The presence of negative-strand complements of COIII RNA is consistent with the notion that a significant amount of mRNA in T. brucei is synthesized by an RNA-dependent RNA polymerase with negative-strand RNA as an intermediate template.
在放线菌素D存在的情况下，布氏锥虫中仍有大量细胞色素氧化酶亚基III（COIII）mRNA持续从头合成，推测这是通过一种不依赖DNA的转录过程实现的。我们描述了负链COIII RNA分子的鉴定、其末端的特征以及RNA依赖性RNA聚合酶活性的检测。提出了三条支持负链COIII RNA存在的证据：（i）在通过亲和纯化初步富集假定的负链RNA后，与极性与mRNA相同的寡核苷酸探针杂交；（ii）对未编辑、已编辑和部分编辑的COIII RNA的负链互补序列进行克隆和测序；（iii）假定的负链RNA分子的末端序列与COIII RNA的末端精确对应。COIII RNA负链互补序列的存在与以下观点一致，即布氏锥虫中的大量mRNA是由以负链RNA为中间模板的RNA依赖性RNA聚合酶合成的。
