A detailed mutational analysis of the VSG gene expression site promoter.
The African trypanosome Trypanosoma brucei is a protozoan parasite that causes the disease African sleeping sickness. The parasite avoids the host's immune response by the process of antigenic variation, or by sequentially expressing antigenically different cell-surface coat proteins. These proteins, called variant surface glycoproteins (VSGs), are expressed from a specific locus, the VSG gene expression site (ES). In an attempt to understand expression of VSG genes, we expanded on earlier investigations of the promoter that controls the large VSG gene expression site transcription unit. We studied VSG ES promoter function both in transient transfection assays, and after stable integration at a chromosomal locus. Analysis of closely spaced deletion mutants showed that the minimum VSG ES promoter fragment that gives full activity is extremely small, and mapped precisely to a fragment that contains no more than -67 bp 5' to the putative transcription initiation site. The promoter lacked an upstream control element, or UCE, an element found at the PARP promoter, and at most eukaryotic Pol I promoters. Furthermore, linker scanning mutagenesis demonstrated that the VSG ES promoter contains at least two essential regulatory elements, including sequences within the region -67/-60 and the region -35/-20, both numbered relative to the initiation site. An altered promoter with mutated nucleotides surrounding the transcription initiation site still directed wild-type levels of expression. In this study, the results were similar for both insect and bloodstream form trypanosomes, suggesting that the same basic machinery for expression from the VSG ES promoter is found in both stages of the parasite.
Pham
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Department of Microbiology, Columbia College of Physicians and Surgeons, New York, NY 10032, USA.
Qi
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Gottesdiener
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D000818:Animals; D001483:Base Sequence; D004252:DNA Mutational Analysis; D016054:DNA, Protozoan / Q000235:genetics*; D005786:Gene Expression Regulation; D017125:Genes, Protozoan; D017930:Genes, Reporter; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D011401:Promoter Regions, Genetic; D015800:Protozoan Proteins; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis; D012045:Regulatory Sequences, Nucleic Acid; D016415:Sequence Alignment; D017384:Sequence Deletion; D012689:Sequence Homology, Nucleic Acid; D014158:Transcription, Genetic; D014162:Transfection; D014346:Trypanosoma brucei brucei / Q000235:genetics* / Q000254:growth & development; D014643:Variant Surface Glycoproteins, Trypanosoma / Q000235:genetics*
D016054:DNA, Protozoan; D015800:Protozoan Proteins; D011993:Recombinant Fusion Proteins; D014643:Variant Surface Glycoproteins, Trypanosoma