title: DNA rearrangements associated with multiple consecutive directed antigenic switches in Trypanosoma brucei.
abstract: Changes in variant surface glycoprotein (Vsg) expression allow Trypanosoma brucei to elude the immune response. The expressed vsg is always located at the telomeric end of a polycistronic transcription unit known as an expression site (ES). Although there are many ESs, only one is active at any particular time. The mechanisms regulating ES transcription and switching are unknown. Chromosome rearrangements within or upstream of the ES have been described to occur in occasional switch events, but no changes have been consistently associated with switching. We inserted the drug resistance genes neo and ble, conferring resistance to G418 and phleomycin, respectively, 1 kb downstream of "silent" ES promoters. This demonstrated that short-range transcription could be achieved from a silent ES promoter. From one initial transformant clone, panels of independent consecutive on-off-on switch clones were generated and analyzed. The first activation of the neo-targeted ES was always associated with deletion of the upstream tandem promoter in this ES, but no further rearrangements were detected in consecutive off-on switches of this ES. On the other hand, direct analysis of ES promoters showed that deletions and duplications occurred elsewhere. Activation of a ble-tagged 300-kb chromosome could not be achieved, but phleomycin-resistant clones could be obtained. One such clone arose from recombination between three ESs. Taken together, our experiments suggest that ES switching may occur after a period of chromosomal interactivity that may or may not leave tangible evidence in the form of detectable sequence changes.
authors.affiliation: Laboratory of Molecular Parasitology, The Rockefeller University, New York, New York 10021-6399, USA.
authors.forename: M
authors.initials: M
authors.lastname: Navarro
authors.forename: G A
authors.initials: GA
authors.lastname: Cross
mesh_terms: D000818:Animals; D001483:Base Sequence; D002874:Chromosome Mapping; D003001:Cloning, Molecular; D017931:DNA Primers; D016054:DNA, Protozoan / Q000235:genetics / Q000378:metabolism*; D005796:Genes; D017125:Genes, Protozoan; D005819:Genetic Markers; D008969:Molecular Sequence Data; D016133:Polymerase Chain Reaction; D011401:Promoter Regions, Genetic; D011994:Recombinant Proteins / Q000096:biosynthesis; D015183:Restriction Mapping; D014158:Transcription, Genetic; D014162:Transfection; D014346:Trypanosoma brucei brucei / Q000235:genetics* / Q000276:immunology*; D014643:Variant Surface Glycoproteins, Trypanosoma / Q000096:biosynthesis* / Q000235:genetics*
chemical_list: D017931:DNA Primers; D016054:DNA, Protozoan; D005819:Genetic Markers; D011994:Recombinant Proteins; D014643:Variant Surface Glycoproteins, Trypanosoma