Downregulation of Trypanosoma brucei VSG expression site promoters on circular bacterial artificial chromosomes.

Abstract

Trypanosoma brucei has about 20 telomeric variant surface glycoprotein (VSG) gene expression sites (ESs), which are downregulated in the insect form. We investigated the transcriptional behaviour of ES promoters on bacterial artificial chromosomes (BACs) containing two different ESs and their flanking regions on fragments of about 140kb. Four different BACs containing either the 221 or the VO2 ES were introduced into insect form T. brucei. The BACs replicated as circular episomes as shown using pulsed field gel (PFG) analysis of DNA exposed to increasing doses of gamma radiation, and digestion with Dam methylation-sensitive restriction enzymes. BAC copy number per cell varied from about 3 for the 221 ES BACs to about 15 for the VO2 ES BACs. Increasing drug selection pressure on the VO2 BAC T. brucei transformants resulted in amplification to about 80 BACs per cell. Although BACs were maintained in the absence of drug selection for at least 56 days, copy number fell and there was no evidence for centromere activity. ES promoters on small plasmid episomes introduced into insect form T. brucei in transient transfections are derepressed. In contrast, ES promoters on large BAC episomes are downregulated both on the original ES BACs, and on ES BACs selected for a drug marker driven by a rDNA promoter fused to the BAC vector. This indicates that downregulation of ES promoters in insect form T. brucei is influenced by genomic context, but does not necessitate proximity to a chromosome end.