The promoter for a variant surface glycoprotein gene expression site in Trypanosoma brucei

• Published in: The EMBO journal 1990-09, Vol.9 (9), p.2791-2801
• Main Authors: Zomerdijk, J. C., Ouellette, M., Asbroek, A. L., Kieft, R., Bommer, A. M., Clayton, C. E., Borst, P.
• Format: Article
• Language: English
• Subjects: Animals; Base Sequence; Biological and medical sciences; Cell Nucleus - metabolism; Chloramphenicol O-Acetyltransferase - genetics; Chloramphenicol O-Acetyltransferase - metabolism; Cloning, Molecular; DNA - genetics; DNA - isolation & purification; Fundamental and applied biological sciences. Psychology; Gene Library; Molecular and cellular biology; Molecular genetics; Molecular Sequence Data; Promoter Regions, Genetic; Restriction Mapping; RNA - genetics; RNA - isolation & purification; Transcription, Genetic; Transcription. Transcription factor. Splicing. Rna processing; Trypanosoma brucei brucei - genetics; Trypanosoma brucei brucei - immunology; Variant Surface Glycoproteins, Trypanosoma - genetics
• ISSN: 0261-4189; 1460-2075
• DOI: 10.1002/j.1460-2075.1990.tb07467.x

The variant‐specific surface glycoprotein (VSG) gene 221 of Trypanosoma brucei is transcribed as part of a 60 kb expression site (ES). We have identified the promoter controlling this multigene transcription unit by the use of 221 chromosome‐enriched DNA libraries and VSG gene 221 expression site specific transcripts. The start of transcription was determined by hybridization and RNase protection analysis of nascent RNA. The 5′ ends of the major transcripts coming from the initiation region map at nucleotide sequences that do not strongly resemble rRNA transcriptional starts even though the transcripts are synthesized by an RNA polymerase highly resistant to alpha‐amanitin. The cloned VSG gene 221 ES transcription initiation region promotes high CAT gene expression, when reintroduced by electroporation into T. brucei. We show that the activity of this expression site is controlled at or near transcription initiation in bloodstream trypanosomes. The 221 ES is inactivated without any sequence alteration within 1.4 kb of the transcription start site. This excludes mechanisms of promoter inactivation involving DNA rearrangements in the vicinity of the transcription start site, e.g. promoter inversion or conversion.