Analysis of Metacyclic Telomeres in Trypanosoma brucei

Genetics, Parasitology

Shiels, Paul Gerard

University:University of Glasgow

关键词: Genetics, Parasitology;   

Others  :  http://theses.gla.ac.uk/78109/1/11007396.pdf

来源: University of Glasgow

【 摘 要 】

Trypanosoma brucei spp. are the agents responsible for African sleeping sickness in man and Nagana in cattle. The organisms have the ability to evade the host's immune system by antigenic variation of their surface coat. The surface coat of the infective forms is composed of a single molecular species, the variant surface glycoprotein (VSG). Each specific VSG is encoded by a separate gene, expression of which occurs in a loose hierarchical order. T. brucei has the coding capacity for approximately 10e3 VSG genes, which are found either in chromosomal clusters or at telomeric loci; it is only from the latter that the gene may be expressed. Telomeric expression sites (ESs) utilized during bloodstream infection are complex, typically between 40 - 60 Kb long, containing several non-VSG expression site associated genes (ESAGs) and preceded by a long barren region devoid of restriction sites. Transcription of telomeric ESs is insensitive to the toxin alpha-amanitin and transcription of a given ES appears to be mutually exclusive of other ESs in vivo. By virtue of their location, chromosomal internal VSG genes need to be transposed to a telomere for expression. The transposition event is duplicative and produces an expression linked copy of the gene. Telomeric VSG genes, however, can be expressed either by duplicative transposition or reciprocal recombination to an active ES, or by in situ activation. The metacyclic stage of the trypanosome life cycle is the first to express VSG genes. The metacyclic form utilizes a highly predictable subset of VSG genes (M-VSGs), comprising only 1-2% of the entire VSG gene repertoire, which appear to be expressed by a distinct and dominant mechanism to that employed during chronic bloodstream infection. Direct molecular analysis of M-VSG gene expression is precluded by the paucity of metacyclic forms in the salivary exudate of the tsetse fly vector and the transient nature of this developmental stage which cannot be cultured in vitro. M-VSG gene expression, however, is still extant in the host bloodstream in the first few days following fly bite. Analysis during this period is compounded by the polyclonal origin of the M-VSG genes expressed by individual organisms and instability of VSG expression. The work described in this thesis focuses on attempts to clone an M-VSG gene telomere and to gain insight into the predictability and stability of the M-VSG repertoire, by analysis of the telomere in a model trypanosome line which circumvents some of the problems associated with previous systems. Cloning and analysis of the BC telomeres for the M-VSG genes for GUTat 7.1 and ILTat 1.22 revealed that each had a remarkably small barren region and shared no sequence homology with ESs used in chronic bloodstream infection, apart from the 70 bp repeat sequence constituting the barren region 5'of the VSG gene. Transcriptional analysis of the ILTat 1.22 metacyclic ES, utilising the model line of trypanosomes expressing the gene in situ, revealed that the ES is extremely short in comparison to bloodstream ESs, extending no more than 3.5 - 4 Kb 5' of the VSG gene. One other region of the ILTat 1.22 BC and GUTat 7.1-2 BC telomeres appeared to be transcriptionally active. This comprised a genomic repetitive element which also was transcribed in procyclic tiypanosomes. The structural individuality of these telomeres was proposed as underlying the stability and physical distinction of the M- VSG repertoire. This hypothesis is supported by an epidemiological analysis of the ILTat 1.22 BC telomere over a 24 year period and spanning diverse epidemic foci of infection. Within Kenyan epidemic foci this telomere is present unaltered in all the stocks investigated over the the period examined and suggests that spread of the disease in East Africa is principally by mechanical transmission; this has important consequences for tackling the disease at source. Cloning and analysis of these telomeres now facilitates characterisation of metacyclic ES control elements and comparisons with other M-ES to be made.