title: Expression site activation in Trypanosoma brucei with three marked variant surface glycoprotein gene expression sites.
abstract: The genes for the Variant Surface Glycoprotein (VSG) of Trypanosoma brucei are transcribed in telomeric expression sites (ESs). There are about 20 different ESs per trypanosome nucleus. Usually, only one is active at a time, but trypanosomes can switch the ES that is active at a low rate (<10(-5) per cell per generation). To study activation and silencing of ESs, we have generated a line of T. brucei 427 with three ESs marked with a different drug resistance gene. We show that a selection with any combination of two of these drugs leads to an unstable double-resistant phenotype in which the two ESs containing the corresponding marker genes switch backward and forward at a very high rate (>10(-1) per cell per generation). Unstable triple-resistant trypanosomes were not obtained. We conclude that the unstable rapid-switching state is a natural intermediate in ES switching. It only involves two ESs, whereas the other ESs are not expressed. Furthermore, we show that "inactive" ESs can exist at several different stable levels of activation. Whereas, a "silent" ES shows a low level of expression of promoter proximal sequences, the level of activation can be reversibly increased, leading to partially activated ESs.
authors.affiliation: Department of Molecular Biology and Centre of Biomedical Genetics, The Netherlands Cancer Institute, Plesmanlaan 121, 1066 CX Amsterdam, The Netherlands.
authors.forename: Sebastian
authors.initials: S
authors.lastname: Ulbert
authors.forename: Inês
authors.initials: I
authors.lastname: Chaves
authors.forename: Piet
authors.initials: P
authors.lastname: Borst
mesh_terms: D000818:Animals; D001483:Base Sequence; D002934:Cinnamates; D019175:DNA Methylation / Q000187:drug effects; D018432:Drug Resistance, Multiple / Q000235:genetics; D005786:Gene Expression Regulation / Q000187:drug effects; D017930:Genes, Reporter / Q000235:genetics; D006921:Hygromycin B / Q000031:analogs & derivatives* / Q000494:pharmacology; D020128:Inhibitory Concentration 50; D008769:Methylnitronitrosoguanidine / Q000494:pharmacology; D008969:Molecular Sequence Data; D016296:Mutagenesis; D009355:Neomycin / Q000494:pharmacology; D010692:Phleomycins / Q000494:pharmacology; D011401:Promoter Regions, Genetic / Q000235:genetics; D013997:Time Factors; D014346:Trypanosoma brucei brucei / Q000187:drug effects / Q000235:genetics*; D014643:Variant Surface Glycoproteins, Trypanosoma / Q000235:genetics*
chemical_list: D002934:Cinnamates; D010692:Phleomycins; D014643:Variant Surface Glycoproteins, Trypanosoma; D008769:Methylnitronitrosoguanidine; D006921:Hygromycin B; C026273:hygromycin A; D009355:Neomycin