| dc.description.abstract | Biological processes, such as the cell-division, differentiation and development,
are driven by changes in gene expression. It is therefore important to examine the
molecular processes involved in the differentiation and development of trypanosomes
since the products of developmentally regulated genes which control these processes
present potential targets for intervention and control of trypanosomiasis. Expression of
developmentally expressed genes in the trypanosome however, is significantly influenced
by alterations in the external environment, such as temperature and nutrients. The
different developmental stages of Trypanosoma congolense are propagated in vitro under
different conditions, and current systems can generate sufficient numbers for molecular
and biochemical studies. This material is central to current studies at ILRAD on parasite
differentiation and proliferation. However, in vitro conditions for the propagation of the
different life cycle stages differ substantially from the in vivo conditions, and it is
important to determine whether differentially expressed sequences identified from in vitro
generated material also show the same expression profiles in vivo.
To determine whether differences do occur between the in vivo and in vitro
generated trypanosomes, procyclic forms from T.congolense IL1180 were isolated from
infected tsetse flies and culture. Total RNA was purified from both populations of
parasites. Enrichment of polytA)" RNA from both populations was carried out followed
by first strand cDNA synthesis in the presence of reverse transcriptase and oligo(dT). A
portion of both cDNAs was used a~the template inPCR reactions designed to specifically
amplify cDNA of trypanosome origin with primers which exploit the conserved 5' splicedleader
and 3' poly(A) sequences found on alltrypanosome mRNAs characterised to date.
Following removal of these primers and dNTPs, the resultant double-stranded cDNAs
were used as templates in a new technique developed at ILiAD termed "random
amplified differentially expressed sequences". The RADES-PCR analysis was carried out
with 10-mer arbitrary primers. The majority of primers generated fingerprints which were
similar or identical for both cDNA templates.
To ensure that the RADES-PCR products were of trypanosome rather than tsetse
fly origin, Southern blots of the products were probed with total labelled trypanosome
genomic DNA. Further confirmation of the specificity of the RADES-PCR products
specifically amplified from in vitro or in vivo derived material was achieved through
probing of Southern blots with labelled cDNA from the two sources. Some ofthe
differentially amplified products were cloned and sequenced. One of the in vivo-specific
products was of particular interest, since it displayed a significantly higher level of
expression in the in vivo generated parasites, but did not display differential expression
between the different in vitro generated life cycle stages, or between in vivo derived
bloodstream forms and the life cycle stages. Sequence analysis of the cloned product
revealed an apparently bi-functional gene product with significant homology in one
domain to the eukaryotic L19 ribosomal gene product and in another domain to
proteases, particularly serine proteases. An in vitro-specific product was also sequenced,
but no homologue was found in the current DNA sequence databases and expression of
this product was difficult to detect both in vitro and in vivo.
In conclusion, use of the RADES-PCR method has demonstrated that differences
in gene expression between in vivo and in vitro parasites do occur. The method was
particularly useful in these studies, since the in vivo derived material contained
predominantly host material, yet the cDNA sequences of trypanosome origin were easily
and selectively amplified. | en |
