Analysis of a variant surface glycoprotein gene expression site promoter of Trypanosoma brucei by remodelling the promoter region.
Trypanosoma brucei survives in the mammalian bloodstream by antigenic variation of its variant surface glycoprotein (VSG) coat. VSG genes are found in telomeric expression sites (ESs), and only one ES is fully transcribed at a time. The parasite changes its coat by either bringing another VSG gene into the active ES, or by switching on another ES and silencing the first. It has previously been shown that the promoter of an active ES can be replaced by a ribosomal promoter without affecting the function of the ES. This study has now analysed the conserved sequences flanking the ES promoter by deletion or replacement of these sequences in intact trypanosomes. The results show that the sequences 3' of the promoter and extending down to the first protein-coding gene, ESAG 7, are not required in the bloodstream-form parasite either for high-level transcription or for switching of the ES. Transformants in which the sequences 5' of the promoter extending up to simple-sequence 50-bp repeats had been removed were not obtained unless the 5' ES sequences were replaced with exogenous DNA, or unless the ES promoter was replaced by a ribosomal promoter, and even these transformants were rare. Transformants lacking the 5' ES sequences displayed a less complete transcriptional repression of silent ESs. These results indicate that the area 5' of an ES promoter is required for optimal functioning of an ES.
Division of Molecular Biology, The Netherlands Cancer Institute, Amsterdam.
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Blundell
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Borst
D000818:Animals; D015139:Blotting, Southern; D003001:Cloning, Molecular; D017124:Conserved Sequence; D005455:Fluorescent Antibody Technique; D015321:Gene Rearrangement; D008562:Membrane Glycoproteins / Q000235:genetics*; D051379:Mice; D016366:Open Reading Frames; D011401:Promoter Regions, Genetic; D015800:Protozoan Proteins / Q000235:genetics*; D051381:Rats; D014158:Transcription, Genetic; D014162:Transfection; D014346:Trypanosoma brucei brucei / Q000737:chemistry / Q000235:genetics*
D008562:Membrane Glycoproteins; D015800:Protozoan Proteins