Selection for activation of a new variant surface glycoprotein gene expression site in Trypanosoma brucei can result in deletion of the old one.
The African trypanosome Trypanosoma brucei expresses the active variant surface glycoprotein (VSG) gene in a telomeric VSG gene expression site. We have generated trypanosomes with a neomycin resistance gene inserted behind an active VSG gene expression site promoter, and a hygromycin resistance gene behind a silent one. By alternating drug selection, we could select for trypanosomes that had switched between the two marked VSG gene expression sites. Surprisingly, trypanosomes that had activated a new VSG gene expression site had often lost the old one. Using polymerase chain reaction (PCR), we screened large numbers of switched trypanosomes and found that sequences lost invariably included the drug marker near the promoter, as well as the telomeric VSG gene many tens of kilobases away. We postulate that stable activation of a new expression site requires silencing of the old one. If silencing does not occur at a sufficient rate by normal switch-off, stable activation of the new site can only occur if the old site is lost in random deletion events. The fact that we pick up these normally infrequent deletions, indicates that inactivation of the old VSG expression site could be rate limiting during switching in our strain of T. brucei.
Department of Molecular Biology, The Netherlands Cancer Institute, Amsterdam. gloria.rudenko@zoo.ox.ac.uk
G
G
Rudenko
I
I
Chaves
A
A
Dirks-Mulder
P
P
Borst
D000818:Animals; D000900:Anti-Bacterial Agents / Q000494:pharmacology; D000940:Antigenic Variation; D002934:Cinnamates; D004351:Drug Resistance / Q000235:genetics; D016521:Electrophoresis, Gel, Pulsed-Field; D005786:Gene Expression Regulation; D015321:Gene Rearrangement; D017125:Genes, Protozoan; D006921:Hygromycin B / Q000031:analogs & derivatives / Q000494:pharmacology; D009355:Neomycin / Q000494:pharmacology; D011401:Promoter Regions, Genetic; D015183:Restriction Mapping; D016615:Telomere; D014158:Transcription, Genetic; D014346:Trypanosoma brucei brucei / Q000187:drug effects / Q000235:genetics* / Q000254:growth & development; D014643:Variant Surface Glycoproteins, Trypanosoma / Q000235:genetics*
D000900:Anti-Bacterial Agents; D002934:Cinnamates; D014643:Variant Surface Glycoproteins, Trypanosoma; D006921:Hygromycin B; C026273:hygromycin A; D009355:Neomycin