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Abstract
Activation of the metacyclic variant antigen type 7 (MVAT7) variant surface glycoprotein (VSG) gene in bloodstream Trypanosoma brucei rhodesiense involves a duplicative transposition of the gene. The DNA transposition unit extends from a site approximately 3.0 kilobases upstream of the VSG gene through the coding region and includes a 73-base pair sequence that possesses promoter activity in transient transfections. This MVAT7 promoter has 80% identity to a previously characterized promoter for the MVAT4 VSG gene. Nuclear run-on assays demonstrate that the MVAT7 promoter is active in MVAT7 bloodstream organisms and that its transcript is synthesized by an RNA polymerase resistant to alpha-amanitin, consistent with previously published reports regarding VSG gene transcription. The transcription start site was identified by primer extension studies and a modified rapid amplification of cDNA ends protocol. Selective mutational analysis of the MVAT7 promoter showed that two conserved trinucleotide regions are important for full promoter function. This study demonstrates that the MVAT7 VSG gene is co-duplicated with its promoter and transcribed into a monocistronic precursor RNA.
Highlights
African trypanosomes are protozoan parasites that cause African trypanosomiasis, or sleeping sickness, in many tropical regions of Africa
Identification of the Upstream Crossover Site of the metacyclic variant antigen type 7 (MVAT7) variant surface glycoprotein (VSG) Gene Duplication—Our original characterization of the MVAT7 VSG gene was conducted on DNA and RNA from metacyclic trypanosomes and demonstrated that, during the metacyclic stage, it exists as a single-copy telomerelinked gene [4]
We noticed this phenomenon with the MVAT4 VSG gene [5], and in that case, we found that the expressed telomere-linked gene gave a weaker, more diffuse signal than did the silent telomere-linked donor gene because of heterogeneity in the length of the expressed telomere, a phenomenon observed by other investigators using trypanosomes from other serodemes [19]
Summary
African trypanosomes are protozoan parasites that cause African trypanosomiasis, or sleeping sickness, in many tropical regions of Africa. We characterized a promoter for the MVAT4 VSG gene of Trypanosoma brucei rhodesiense utilizing a bloodstream trypanosome clone that re-expresses MVAT4 VSG [5] In these bloodstream organisms, the telomere-linked MVAT4 VSG gene is activated in situ without a duplication event or other detectable DNA rearrangements. The telomere-linked MVAT4 VSG gene is activated in situ without a duplication event or other detectable DNA rearrangements This finding suggests that either (i) the promoter that functions in metacyclic organisms was reactivated in the bloodstream parasites or (ii) two promoters exist in this expression locus, one for expression at the metacyclic stage and one for the bloodstream stage. Another MVAT VSG gene, encoding MVAT5 VSG, was found to be activated via duplication to a conventional bloodstream expression site whose promoter is far upstream of the VSG gene [6]
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