Control of Metacyclic VSG Gene Expression in the Life Cycle of Trypanosoma brucei

Wymer, Ben (1997) Control of Metacyclic VSG Gene Expression in the Life Cycle of Trypanosoma brucei. PhD thesis, University of Glasgow.

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Abstract

African trypanosomes evade the immune response in the bloodstream of mammalian hosts by antigenic variation, that is by continually switching their variant surface glycoprotein (VSG) coat on the cell surface. VSG genes are first expressed at the host- infective metacyclic stage in the salivary glands of the insect vector, the tsetse fly. At this stage each trypanosome expresses a single gene from a small specific subset of VSG genes (≤27 in Trypanosoma brucei rhodesiense). Unusually for trypanosomes the metacyclic VSG genes (M-VSG genes) are transcribed as short, monocistronic transcription units located at the ends (telomeres) of the largest set of trypanosome chromosomes. Previously, nuclear run-on analysis identified the promoter region of one metacyclic VSG gene, the ILTat 1.22 M-VSG gene, at a position 2.6-3.0 kb upstream of the VSG gene. The promoter region was shown to be active only at the metacyclic stage and silent at both the procyclic and bloodstream stages. This is the only gene analysed thus far in Kinetoplastida shown to be transcriptionally regulated: all other genes are, at least in part, under post- transcriptional control. In this study a cloned restriction fragment containing the 1.22 M-VSG gene promoter was placed upstream of a copy of a chloramphenicol acetyltransferase (CAT) reporter gene in a plasmid vector and used in transient transfection of trypanosomes. In transient transfection of bloodstream trypanosomes the putative 1.22 M-VSG gene promoter was highly active although, previous studies had shown the same fragment to be inactive in transient transfection of procyclic trypanosomes (Graham and Barry, 1995). In order to determine what sequences within the promoter region were necessary for the observed activity of the promoter, a series of deletion mutants were prepared across the promoter region in a 5' to 3' direction and 3' to 5' direction. These deletion mutants were cloned into a CAT reporter plasmid and promoter fragment activity was determined by transient transfection of bloodstream trypanosomes. The deletion analysis indicated a region of 96 bp was necessary to maintain frill promoter activity. In vivo promoter analysis had shown that the 1.22 M-VSG gene promoter was inactive in bloodstream trypanosomes and it was possible that promoter activity in the transient transfection experiments was due to loss of regulation imposed by its normal chromosomal context. To study the effect of chromosomal location on the 1.22 M-VSG gene promoter, experiments integrating reporter constructs into the bloodstream trypanosome genome were conducted. First, bloodstream trypanosomes of stock EATRO 795 (from which the putative promoter fragment of the 1.22 M-VSG gene was isolated) were adapted to axenic culture to allow transfection, cultivation and selection of stable transformants. Culture adapted bloodstream trypanosomes were phenotypically bloodstream forms which were highly virulent in mice and could differentiate, at least partially, to procyclic forms. For stable transformation two reporter constructs were used to integrate the promoter first to its endogenous expression telomere and second, to a chromosomal-internal position, the untranscribed spacer region of the ribosomal RNA gene transcription unit. In both cases the reporter construct contained a copy of the 1.22 M- VSG gene promoter directing transcription of a CAT gene. The promoter was flanked 5' by sequences homologous to the appropriate site of genomic integration. CAT gene expression was analysed in the cloned, transformed cells lines by CAT enzyme activity and Northern blot analysis. For the ribosomal untranscribed spacer region, nuclear run-on analysis showed that CAT gene transcription initiated at the 1.22 M-VSG gene promoter. The promoter was found to be up to 40 times more active at the ribosomal locus than when positioned at the 1.22 M-VSG gene expression telomere. Reduced activity of the 1.22 M- VSG gene promoter at the 1.22 M-VSG gene expression telomere suggests that one mechanism for down-regulation of the M-VSG gene promoter in bloodstream trypanosomes may be related to its telomere positioning.

Item Type: Thesis (PhD)
Qualification Level: Doctoral
Additional Information: Adviser: Sheila Graham
Keywords: Genetics, Parasitology
Date of Award: 1997
Depositing User: Enlighten Team
Unique ID: glathesis:1997-75222
Copyright: Copyright of this thesis is held by the author.
Date Deposited: 19 Nov 2019 21:43
Last Modified: 19 Nov 2019 21:43
URI: http://theses.gla.ac.uk/id/eprint/75222

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