VSG gene expression site control in insect form Trypanosoma brucei.

Abstract

When the African trypanosome Trypanosoma brucei is taken up from mammals by a tse‐tse fly, it replaces its variant surface glycoprotein (VSG) coat by a procyclin coat. Transcription of VSG genes stops in the fly, but transcription of sequences derived from the promoter area of the VSG expression site(s) remains high. Whether this is due to continuing high activity of one promoter or to low activity of many promoters was unclear. We have used the small differences between the sequences of different expression sites to show that multiple expression site promoters are active in insect form trypanosomes. This is confirmed by the low expression of single copy marker genes introduced into the transcribed area. However, if the expression site promoter is removed from the genomic location of the expression site and inserted in the non‐transcribed spacer of the ribosomal DNA (rDNA), it is derepressed. Derepression of transcription can also be accomplished by replacing the promoter of an expression site by an rDNA promoter. We conclude that the down‐regulation of VSG gene expression site promoters in insect form trypanosomes is affected by both the DNA sequence of the promoter and the genomic context in which it resides.

Identification of the Glucosyltransferase That Converts Hydroxymethyluracil to Base J in the Trypanosomatid Genome

“…Re-expression of HA-tagged JGT-To allow ectopic expression of HA-JGT fusion, we utilized a modified ptub-phleo construct (22) that contains the enhanced green fluorescent protein ORF cloned between the tubulin flanking sequences. A 2012-bp PCR product corresponding to the T. brucei JGT ORF was PCR-amplified using the sense primer CCTGCAGGATG-GCTTACCCATATGATGTTCCAGATTACGCTGGAG-GTCCAAGTGAGGGGAAG (the SbfI site is underlined and sequence coding for the HA tag is shown in boldface) and the antisense primer GGCGCGCCTTAGTCTGCCTGCGAC-CCTCC (the AscI site is underlined) and cloned into the ptub-GFP vector digested with SbfI and AscI.…”

Bloodstream Form-specific Up-regulation of Silent VSG Expression Sites and Procyclin in Trypanosoma brucei after Inhibition of DNA Synthesis or DNA Damage

“…The insect form T. brucei transformants (RPX1-1, ESX1-1, ESX1-2, and rDES1-1) were described previously (23) and were maintained on 25 g ml Ϫ1 hygromycin. Transfection Constructs-The 221GP1 construct has a puromycin resistance gene excised from construct pBS-Pur (gift of Isabel Roditi, University of Berne) (29) and inserted between tubulin intergenic regions containing splice and polyadenylation sites (23). The GFP gene is eGFP (Clontech) flanked upstream by the tubulin 3Ј splice site present on a tubulin intergenic region, and downstream by the 221 VSG untranslated region and polyadenylation signal.…”

“…In contrast, in insect form T. brucei all ESs appear to be down-regulated to a great extent, although not as tightly as in the bloodstream form (23). Silencing is mechanistically different, as it is promoter sequence-specific and appears to involve repressed chromatin (15).…”

Functional Analysis of the Trypanosomal AAA ProteinTbVCP with trans-Dominant ATP Hydrolysis Mutants

“…Viable transformants grew out in 2 weeks and were maintained initially in conditioned media, and then in Cunningham's medium. In this system homologous integration is targeted into the nontranscribed ribosomal RNA spacer, a transcriptionally silent region of the trypanosome genome (37). For induction of mutant TbVCP expression, tetracycline was added to log phase cultures at a concentration of 0.5 g/ml.…”