Characterisation of Genes Identified During a RADES-PCR Screen of Concanavalin A-Treated Procyclic Trypanosoma brucei rhodesiense

Lillico, Simon Geoffrey (1999) Characterisation of Genes Identified During a RADES-PCR Screen of Concanavalin A-Treated Procyclic Trypanosoma brucei rhodesiense. PhD thesis, University of Glasgow.

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Abstract

Previous studies demonstrated that procyclic Trypanosoma brucei could be induced to die by culturing cells in the presence of the lectin Concanavilin A, and that throughout the three day death process de novo gene expression could be detected. The current study utilised a differential display reverse transcription polymerase chain reaction (DDRT-PCR) technique known as randomly amplified differentially expressed sequences (RADES) PCR to identify cDNAs whose expression levels fluctuated during the time-course of death. 64 PCR products were detected, of which 27 were reamplified and sequenced. Database searches were conducted using the BLAST algorithm, identifying 17 significant matches with known genes in the database. 8 of these encoded novel T. brucei genes. Northern blot analysis was attempted in order to confirm expression patterns indicated by RADES-PCR. However, the data obtained was inconclusive due to lack of a marker constitutively expressed during cell death. Two genes identified during this study, QM and MOB1, were characterised further. The human QM gene was first identified as a transcript upregulated in a non- tumourigenic Wilms' tumour microcell hybrid relative to the parental cell line, and subsequent experiments suggested that the QM gene encoded a transcription factor. More recent evidence indicates that QM is actually a ribosomal protein associated peripherally with the 60S ribosomal subunit. During the current study Southern blot analysis was conducted, indicating the presence of 2 copies of the T. brucei QM gene, one of which was isolated from a genomic lambda library. Sequence analysis revealed 60% amino acid identity between the T. brucei QM and QM homologues from diverse eukaryotes. A recombinant epitope-tagged QM was inducibly expressed in procyclic T. brucei. Indirect immunofluorescence microscopy revealed nuclear exclusion and colocalisation with GPI8, a component of the transamidase complex responsible for glycosylphosphatidylinositol (GPI) anchor attachment which is hypothesised to localise to the endoplasmic reticulum. Tagged QM in cellular extracts was demonstrated to be insoluble following lysis in a 1% Triton X-100 buffer, suggesting an association with a large protein complex. Taken together these results suggest that the T. brucei QM is a ribosomal protein. MOB1 is an essential yeast gene required for completion of mitosis and maintenance of ploidy. While a number of interacting partners have been identified for MOB1, the function of this protein remains unknown. During the current study Southern blot analysis showed the presence of 2 copies of the T. brucei MOB1 gene within 12 kb of each other. Both of these genes were isolated from a single genomic X clone and were named MOB1-1 and MOB1-2. Sequence analysis revealed that while >96% of the amino acid sequence encoded by MOB1-1 was conserved in the MOB1-2 gene, the latter had a predicted N-terminal extension of 82 residues. Inducible expression of an antisense MOB1-1 mRNA in procyclic T. brucei resulted in a significant reduction in proliferation, indicating a role for MOB1 in cell cycle progression. A recombinant epitope-tagged MOB1-1 was inducibly expressed in procyclic T. brucei. Indirect immunofluorescence microscopic analysis revealed a homogenous distribution throughout the cell, allowing no insight into function. A recombinant MOB1-1 polypeptide was produced in E. coli and used to inoculate rabbits. Resultant antiserum detected a T. brucei protein of the predicted size of MOB1-1. This antiserum will prove invaluable for future studies, allowing the subcellular location of native MOB1-1 to be established, and purification of interacting partners through co-immunoprecipitation to be carried out.

Item Type: Thesis (PhD)
Qualification Level: Doctoral
Additional Information: Adviser: Susan Welburn
Keywords: Genetics
Date of Award: 1999
Depositing User: Enlighten Team
Unique ID: glathesis:1999-76352
Copyright: Copyright of this thesis is held by the author.
Date Deposited: 19 Nov 2019 15:22
Last Modified: 19 Nov 2019 15:22
URI: http://theses.gla.ac.uk/id/eprint/76352

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