RNA polymerase I can mediate expression of CAT and neo protein-coding genes in Trypanosoma brucei.
We show that the ribosomal RNA (rRNA) promoter can efficiently direct expression of protein-coding genes in the parasitic protozoan Trypanosoma brucei. The rRNA promoter was characterized by: (i) point mutations at the rRNA transcription initiation site which completely abolished its promoter function in transient CAT transformation assays; (ii) the alpha-amanitin resistance of transcription of rRNA promoter-neomycin phosphotransferase (neo) genes in stably transformed trypanosomes; and (iii) the nucleolar location of neo RNA, synthesized under the control of the rRNA promoter. The rRNA promoter-derived CAT mRNA required a 3' splice acceptor site and the neo mRNA was trans-spliced and polyadenylated. In situ hybridization revealed neo RNA at the nucleolus in stably transformed trypanosomes in which rRNA promoter-neo constructs were integrated either at a rRNA locus or at a locus for the procyclic acidic repetitive protein (PARP) coding genes. We postulate that trans-splicing, by uncoupling the requirement for transcription of protein-coding genes by RNA polymerase II, allows RNA polymerase I mediated protein-coding gene transcription, presumably because a 5' cap can be transferred to the pre-mRNA by trans-splicing.
Department of Genetics and Development, Columbia University, New York, NY 10032.
G
G
Rudenko
H M
HM
Chung
V P
VP
Pham
L H
LH
Van der Ploeg
D000546:Amanitins / Q000494:pharmacology; D000818:Animals; D001483:Base Sequence; D015152:Blotting, Northern; D015139:Blotting, Southern; D015500:Chloramphenicol O-Acetyltransferase / Q000235:genetics*; D016054:DNA, Protozoan / Q000235:genetics; D008969:Molecular Sequence Data; D009154:Mutation; D009693:Nucleic Acid Hybridization; D010957:Plasmids; D011401:Promoter Regions, Genetic; D012318:RNA Polymerase I / Q000235:genetics*; D016053:RNA, Protozoan / Q000032:analysis; D015183:Restriction Mapping; D014158:Transcription, Genetic / Q000187:drug effects; D014346:Trypanosoma brucei brucei / Q000235:genetics*
D000546:Amanitins; D016054:DNA, Protozoan; D016053:RNA, Protozoan; D015500:Chloramphenicol O-Acetyltransferase; D012318:RNA Polymerase I