| dc.description.abstract | Chromosome rearrangements that occur in trypanosomes appear to affect mainly the
medium-sized chromosomes. A possibility exists that non-duplicative activation of telomeric VSG genes might involve chromosome rearrangements occurring several kilobase pairs
upstream of the affected VSG gene, thus remaining undetectable by standard gel
electrophoresis and southern blot hybridizations with VSG gene probes. Since trypanosomes
do not condense their chromosomes at any stage of cell division, this hypothesis cannot be
tested by cytological hybridization. Some of the size alterations in chromosomes occur when
the trypanosomes are repeatedly passaged, cyclically transmitted through tsetse flies or
grown in rodents in the presence of trypanocidal drugs. In some instances, the alterations in
the sizes of some chromosomes may relate to a changed phenotype of the parasite. Pulsed
field gradient gel electrophoresis (PFGE) of chromosome-sized DNA molecules of lower
eukaryotic organisms is an effective way of characterizing such organisms. It has been used
to characterize many different isolates of parasitic protozoa including Plasmodium
falciparum, Leishmania spp., Trypanosoma spp., Theileria spp. and Toxoplasma. When a
particular chromosome rearranges in such organisms, it is impossible to identify the
rearranged chromosome in the absence of a marker specific for the chromosome.
Short oligonucleotide primers (1O-mers) of arbitrary nucleotide sequences have been
used in the polymerase chain reaction (PCR) to generate genomic fingerprints that facilitate
the characterization and differentiation of various organisms and for physical mapping of loci
which contain genes responsible for identifiable phenotypes.
It was proposed that the use of such primers to amplify purified individual
chromosomes may generate fingerprints which are characteristic of each chromosome. To
test this hypothesis, T congolense clone IL 1180 was used because of its sensitivity to
trypanocides and clear pedigree. PFGE conditions were optimized which best separates the
four medium-sized chromosomes, whose sizes are: 340 kb, 360 kb, 400 kb and 500 kb. The
four chromosomes thus separated were individually purified from agarose gel slices and then
peR-amplified with the random primers. The fingerprints of DNA fragments generated were
resolved by standard agarose gel electrophoresis. The majority of primers used generated
reproducible fingerprints, some of which were polymorphic for the different chromosomes
used.
In this way, a fragment of DNA was identified which hybridizes only to one of the
four chromosomes from which it was amplified by a random primer. Thus, taking this
approach, it is feasible to generate chromosome-specific hybridization markers. | en |
