The 5.8S ribosomal RNA gene of Trypanosoma brucei: structural and transcriptional studies

Abstract

To further investigate the process of discontinuous transcription in trypanosomes, the 5.8S rRNA gene, present in the trypanosome genome as part of the multicopy rRNA gene cluster, has been cloned, sequenced, chromosomally mapped, and used in transcriptional studies. The gene's sequence confirms its identity and indicates that it is less conserved evolutionarily than the trypanosome 5S rRNA gene previously described by our laboratory (6). Examination of the chromosomal locations of the gene by pulsed-field gradient gel electrophoresis shows that the 5.8S rRNA genes occur on at least four differently-sized chromosomes in T. b. rhodesiense and at least three differently-sized chromosomes in T. b. brucei. The 5.8S transcript was analyzed by a run-off transcription assay using isolated nuclei. These studies strongly suggest that the 5.8S rRNA gene is transcribed spliced leader RNA and by a different RNA polymerase than either the spliced leader or 5S rRNA transcripts. Transcription of the trypanosome 5.8S rRNA is insensitive to very high levels of alpha-amanitin, a feature of the 5.8S rRNA in higher eukaryotes.