The Mitochondrial Genome of Trypanosomes 
Larry Simpson, Ph.D. — Investigator 
Dr. Simpson is also Professor of Cell and Molecular Biology at the University of California, Los Angeles. 
He received his B.A. degree in biology from Princeton University and his Ph.D. degree in molecular 
parasitology with William Trager at the Rockefeller University. His postdoctoral training was with Maurice 
Steinert at the Free University of Bruxelles. 
THE kinetoplastids represent a large group of 
parasitic protozoa that are tiie causal agents 
for a variety of human and animal diseases, in- 
cluding African sleeping sickness, Chagas' dis- 
ease, and leishmaniasis. There are no vaccines for 
any of these, and chemotherapies are either non- 
existent or inadequate. As cells, the kinetoplast- 
ids represent one of the most ancient lineages of 
the eukaryotic kingdom and thereby have many 
novel physical and biochemical features. Some 
species, such as Crithidia, are parasitic in only a 
single invertebrate host. Others, such as Leish- 
mania, Trypanosoma, and Pfoytomonas, are par- 
asitic in both an invertebrate and a vertebrate (or 
plant) host. 
Kinetoplast DNA 
The mitochondrial genome of these cells, 
which is known as kinetoplast DNA, consists of a 
single giant network containing approximately 
10,000 minicircles and 20-50 maxicircles, all 
linked together by catenation. The maxicircles 
comprise a subset of mitochondrial genes that en- 
codes two small rRNAs, three subunits of cy- 
tochrome oxidase, cytochrome b, four subunits 
of NADH dehydrogenase, and three yet unidenti- 
fied proteins. No tRNAs appear to be encoded by 
the mitochondrial genome and therefore must be 
imported into the organelle by some yet to be 
defined mechanism. The function of the minicir- 
cle was unknown until recently. 
RNA Editing 
RNA editing is a post-transcriptional process in 
which uridine (U) residues are inserted and de- 
leted from coding regions of the primary tran- 
scripts of several maxicircle "cryptogenes." The 
extent of editing varies from a few U residues to 
hundreds, at hundreds of sites throughout the en- 
tire mRNA (pan-editing) . The function of editing 
is to create translatable mRNAs encoding mito- 
chondrial proteins. 
The extent of editing of specific genes varies 
from species to species. For example, the NADH 
dehydrogenase subunit 7 mRNA is internal and 
5'-edited in Leishmania tarentolae but pan- 
edited in Trypanosoma brucei. We have re- 
cently shown that at least one G-rich intergenic 
region in both species encodes a transcript that is 
pan-edited to produce ribosomal protein SI 2 for 
the mitochondrial ribosome. It is likely that five 
other G-rich regions represent additional pan- 
edited cryptogenes, which would bring the total 
mitochondrial structural gene content in these 
cells to 17, of which 12 are cryptogenes. 
Mechanism of RNA Editing 
We discovered in 1990 that maxicircles also 
encode another class of RNAs — the guide RNAs 
(gRNAs) — which contain the necessary se- 
quence information for the edited genes. These 
are small RNAs, which can form perfect duplex 
hybrids with edited mRNAs, both within the 
edited region and 3' of the edited region, pro- 
vided G-U base pairs are allowed. The gRNAs also 
have nonencoded 3' oligo-[U] tails ranging in 
length from 5 to 28 nucleotides. We then discov- 
ered that gRNAs are also encoded in the minicir- 
cles, finally providing a genetic role for these 
enigmatic molecules. 
We have proposed two models for the involve- 
ment of gRNAs in editing. In both models the ini- 
tial interaction of the gRNA and the mRNA is the 
formation of an anchor duplex just downstream 
of the pre-edited region. The "enzyme cascade" 
model invokes an endonuclease that cleaves at 
the first mismatch, a terminal uridylyl transferase 
that adds a U to the 3'-hydroxyl, and an RNA ligase 
joining the two mRNA fragments. In the "transes- 
terification" model, the added U's are derived 
from the oligo-[U] tail of the gRNA by means of 
two successive transesterifications. In both mod- 
els the gRNA provides an internal guide sequence 
to specify the precise addition and deletion of U's 
at specific sites. 
A major goal is to obtain an in vitro system in 
which the entire editing process occurs in an ac- 
curate manner, thereby allowing a biochemical 
dissection and reconstitution of the underlying 
enzymatic machinery. We have recently shown 
that at least the initial step of RNA editing — the 
formation of gRNA-mRNA chimeric molecules — 
577 
