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

Abstract

African trypanosomes survive the immune defense of their hosts by regularly changing their antigenic coat made of variant surface glycoprotein (VSG). The Trypanosoma brucei genome contains more than 1,000 VSG genes. To be expressed, a given VSG gene must be located in one of 15 telomeric regions termed "VSG expression sites" (ESs), each of which contains a polycistronic transcription unit that includes ES-associated genes. Only one ES is fully active at a time, so only one VSG gene is transcribed per cell. Although this monoallelic expression is controlled at the transcriptional level, the precise molecular mechanism for this control is not understood. Here we report that in single cells transcription is initiated on several ESs simultaneously, indicating that the monoallelic control is not determined only at transcription initiation, but also at further control steps such as transcription elongation or RNA processing.

Keywords: antigenic variation; single cell RT-PCR; sleeping sickness.

Fig. 1.

Schematic of the single-cell RT-PCR experiment. (A) Ten different bloodstream 328-114–derived cell lines transfected with the same plasmid (p2T7¹⁷⁷) backbone containing different inserts were mixed. Serial dilution was performed on this population to obtain one cell (Trypanosome:T) per3 µL (suspension A). (B) One microliter of suspension A was added to each well of a 96-well plate. Each well contained 100 µL of culture medium. The suspension was cultivated for 7 d. (C) One microliter of suspension A also was added to each of five Eppendorf tubes, which were frozen and stored before the single-cell PCR or RT-PCR experiment was performed. (D) After 1 wk, all the positive wells were subjected to DNA extraction, and PCR was performed using primers binding to the plasmid backbone to amplify the different inserts. (E) After the validity of the serial dilution experiment in the 96-well plate (step D) was confirmed, RT-PCR was performed on the Eppendorf tubes containing an average of one trypanosome per three tubes using primers located (as shown at the beginning of a typical ES) and amplifying the beginning (nucleotide +1 to nucleotide +149) of the ES (PES1) or ESAG6/7 (PES6/7).