Transient activity assays of the Trypanosoma brucei variant surface glycoprotein gene promoter: control of gene expression at the posttranscriptional level.

Abstract

The putative promoter of the variant surface glycoprotein (VSG) gene of Trypanosoma brucei was cloned into a plasmid containing the chloramphenicol acetyltransferase (CAT) gene. After electroporation into trypanosomes, this construct directed the expression of the CAT reporter gene. The essential region for promoter activity was found to reside within 88 bp upstream of the putative transcription start site. Transcription of the CAT construct occurred at approximately the same level in both bloodstream and procyclic forms and was resistant to alpha-amanitin. However, CAT expression appeared to be modulated in the two forms of the parasite. Sequences 3' to the gene seemed to be important in this respect, as CAT activity in bloodstream forms was readily detectable only when the 3' region of a VSG cDNA was placed downstream of the CAT gene. Two separate VSG gene promoter sequences, both cloned from T. brucei AnTat 1.3A, were equally able to direct CAT expression, which suggests that there are a number of potential VSG gene promoters in the genome, although usually only one expression site is fully active at any one time.

Transcription is initiated on silent variant surface glycoprotein expression sites despite monoallelic expression in Trypanosoma brucei

“…Thus, VSG ESs are active when transfected as episomes in cells, indicating that several promoters can be active simultaneously (38). This conclusion is also true in procyclic forms, where the ES promoters are normally not active (38). The very simple structure and ribosomal nature of the ES promoters (39,40) is in keeping with the absence of regulatory sequences.…”

“…37). Thus, VSG ESs are active when transfected as episomes in cells, indicating that several promoters can be active simultaneously (38). This conclusion is also true in procyclic forms, where the ES promoters are normally not active (38).…”

Control of gene expression in Trypanosomatidae

“…Several groups have shown that, although VSG mRNAs are not detected in procyclic forms, transcription initiation does occur at a low level from many, if not all, ES promoters (27)(28)(29). This is consistent with the observation of CAT or luciferase expression under the control of VSG gene promoters cloned in episomal vectors in transiently transfected procyclic parasites (29,30). However, when VSG promoters were further analyzed within their chromosomal context, instead of plasmid vectors, it was found that the transcription elongation step is subjected to stage-specific regulation, i.e., the active VSG gene is fully transcribed in bloodstream parasites but most transcriptional activity stops within 1 kb of the VSG promoter in procyclics (27)(28)(29).…”

In vivo import of firefly luciferase into the glycosomes of Trypanosoma brucei and mutational analysis of the C-terminal targeting signal.

“…Transient transformation of trypanosomes with foreign DNA (Clayton et al, 1990;Rudenko et al, 1990;Jefferies et al, 1991) or from a T. brucei receptor RNA gene (Rudenko et al, 1991). The neo gene could also be stably expressed without the addition of a promoter element by inserting it into an actively transcribed region, such as the tubulin or calmodulin locus (ten Asbroek et al, 1990;Eid and Sollner-Webb, 1991).…”