The Mitochondrial Genome of Trypanosomes 
can be performed in vitro by incubation of syn- 
thetic RNAs with a mitochondrial extract and that 
this process is anchor-dependent. 
The work on RNA editing was supported by a 
grant from the National Institutes of Health. 
Evolutionary Considerations 
' The existence of split genes is not novel, but 
the existence of genes in which the RNA product 
of one gene contains information for the correc- 
tion of coding sequences within transcripts of the 
other gene is novel. If the transesterification 
model proves to be correct, this would suggest 
that RNA editing is on the same evolutionary 
pathway as RNA splicing and may in fact repre- 
sent a primitive type of trans-splicing with partial 
integration. The trypanosome type of RNA editing 
has not yet been reported in other organisms, but 
other types of modifications of the sequences of 
coding RNAs have been observed in organisms as 
diverse as plants and humans. C to U changes at 
specific sites occur in transcripts of several hu- 
man genes and also in transcripts from many plant 
mitochondrial and chloroplast genes. The deter- 
mination of site specificity for these multiple 
transitions is completely unknown. 
The evolution of RNA editing in the trypano- 
somes is interesting in itself. To investigate it, we 
plan to examine representatives of more-primi- 
tive kinetoplastid lineages. 
The Kinetoplast Genome as a Target 
for Disease Diagnosis 
We have shown that the kinetoplast DNA mini- 
circle molecule of Trypanosoma cruzi, the 
causal agent of Chagas' disease, is an appropriate 
multicopy target for detection and strain classifi- 
cation of the parasite in patients, animals, or in- 
sects. The minicircle of T. cruzi consists of four 
conserved regions and four variable regions. 
There are multiple minicircle sequence classes in 
a network, and minicircles in different strains are 
very polymorphic. Primers to the conserved re- 
gion were used for polymerase chain reaction 
(PGR) amplification of minicircle fragments 
from either the conserved region or the variable 
region. This method is being developed into a 
diagnostic procedure to detect small numbers of 
parasites in blood of chronically ill patients and 
to classify the strain of the parasite. 
We recently developed a method to recover 
blood from patients and preserve total DNA with- 
out refrigeration. The DNA is then cleaved with a 
chemical nuclease to release linearized minicir- 
cles, and the fragments are amplified with spe- 
cific primer sets. We are attempting to expand 
this diagnostic procedure by developing a multi- 
plex PCR-based assay for the detection of multi- 
ple blood-borne viral and parasitic disease 
agents. 
The kinetoplast DNA minicircle has also 
proved to be an appropriate target for the diagno- 
sis of other pathogenic kinetoplastid infections, 
such as those caused by Leishmania species. 
Grants from the World Health Organization 
and the Rockefeller Foundation supported this 
diagnostic work. 
RNA Editing as Possible Target 
for Intervention 
Whenever a parasite has a biochemical path- 
way that is unique to the parasite and not found in 
the human host, there is a potential for selective 
chemotherapy. As RNA editing is dissected bio- 
chemically, we plan to examine the possibility of 
inhibiting components of the editing machinery 
in the parasites without affecting the host. This 
may open up a new direction for selective che- 
motherapy of the many trypanosome-caused 
diseases. 
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