Identification of negative-strand complements to cytochrome oxidase subunit III RNA in Trypanosoma brucei.
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.
Volloch
V
V
Boston Biomedical Research Institute, MA 02114.
Schweitzer
B
B
Zhang
X
X
Rits
S
S
D000818:Animals; D001483:Base Sequence; D015152:Blotting, Northern; D003001:Cloning, Molecular; D004247:DNA / Q000235:genetics / Q000302:isolation & purification; D003609:Dactinomycin / Q000494:pharmacology; D003576:Electron Transport Complex IV / Q000235:genetics*; D046911:Macromolecular Substances; D008969:Molecular Sequence Data; D009690:Nucleic Acid Conformation; D009693:Nucleic Acid Hybridization; D009838:Oligodeoxyribonucleotides; D016133:Polymerase Chain Reaction / Q000379:methods; D012333:RNA, Messenger / Q000096:biosynthesis / Q000235:genetics* / Q000302:isolation & purification; D014158:Transcription, Genetic; D014346:Trypanosoma brucei brucei / Q000201:enzymology* / Q000235:genetics
D046911:Macromolecular Substances; D009838:Oligodeoxyribonucleotides; D012333:RNA, Messenger; D003609:Dactinomycin; D004247:DNA; D003576:Electron Transport Complex IV