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February 01, 1996
A New Blood Test for Chagas Disease
Several years ago, Larry Simpson was approached with a project that
intrigued him. A colleague had access to tissue from 2,500-year-old
Chilean mummies and he wanted Simpson to determine if the preserved
tissue showed signs of infection by Trypanosoma cruzi, the
parasite that causes Chagas disease.
Interested, Simpson told his colleague, Christian Orrega, that he
would modify the polymerase chain reaction (PCR) test in such a way
that it would tell them whether the mummies had been infected with
T. cruzi. One might say that Orrega's request was tailor-made
for Simpson, who had thought about employing modern science to settle a
longstanding debate about whether the legendary evolutionist Charles
Darwin had contracted Chagas disease while he was in South America on
the voyage of the Beagle.
“Minicircle DNA from Leishmania tarentolae.”
But as Simpson, who is currently in the Howard Hughes Medical
Institute at UCLA, and his graduate students, Nancy Sturm and Herbert
Avila, and his Brazilian collaborators, Wim Degrave, Otavio Thiemann
and Carlos Morel, began to adapt the PCR test, they immediately
realized how their twist on this powerful assay might transform the
current method of diagnosing Chagas disease. At the time, the "gold
standard" for diagnosis resembled an ancient torture ritual. The
infected person was required to endure a blood feeding by uninfected
"kissing bugs," insects that transmit Chagas disease. The bugs were
then taken back to the lab, and several weeks later were dissected and
examined for the presence of T. cruzi. If T. cruzi was
present in the dissected kissing bugs, the patient had been
infected.
As it turns out, the scientists were right because the PCR-based
assay for T. cruzi detection has revolutionized the diagnosis of
Chagas disease. It is rapid, portable and allows medical workers to
test people who are far from modern laboratories — something not
possible before. Simpson and his Brazilian collaborators continue to
make gradual improvements to the PCR test, but, unfortunately, in all
the hubbub, the group has had to put the Chilean mummy project on hold.
"We might get around to it sometime soon," Simpson said.
The work behind developing the Chagas disease blood test began many
years ago as a quest by Simpson and his colleague and wife, Agda, to
understand the unique structures of mitochondrial DNA in Leishmania
tarentolae, a trypanosomatid parasite that infects lizards.
Mitochondria are the energy-producing organelles within cells, and are
present in the trypanosomatids such as L. tarentolae and T.
cruzi. Such parasites are also called kinetoplastids because of the
presence of a small granule, or kinetoplast, at the base of the
flagellum when they were stained with certain dyes. "The kinetoplast
actually represents a portion of the single complex mitochondrion of
the cell that contains a huge compact mass of DNA," Simpson said.
The Simpsons found that the kinetoplast DNA of trypanosomatid
parasites consists of thousands of interlocking minicircles and a few
interlocking maxicircles which together form a single giant network of
DNA. No one knew the function of minicircle DNA when Simpson and Carlos
Morel of the Oswaldo Cruz Institute in Rio de Janeiro, Brazil found
that the kinetoplast DNA of various strains of T. cruzi differed
greatly in their DNA sequence. "This suggested to us," said Simpson,
"that minicircle DNA could be used to identify different strains of
T. cruzi and provide important epidemiological and perhaps even
clinical data about the spread of the parasite."
Simpson's laborious analysis of minicircle and maxicircle DNA in
T. cruzi and L. tarentolae led him and several other
investigators to a discovery that would come close to shaking the very
foundations of modern molecular biology. The central dogma of molecular
biology is that DNA makes RNA and RNA makes protein. In 1986,
scientists in Amsterdam found an instance of "RNA editing," where an
RNA transcript of the maxicircle DNA was modified within the coding
region by the insertion of uridine bases to form a translatable
message. (Uridine is one of the four bases in RNA.)
Simpson and his graduate student, Janet Shaw, together with Jean
Feagin and Ken Stuart at the Seattle Biomedical Research Institute,
soon discovered more dramatic examples of sequence modifications of
maxicircle transcripts in several trypanosomatid species, involving
both the addition and deletion of uridine residues. In essence, the
RNAs were being changed into genes that were not found in the DNA.
Simpson and Shaw used the term "cryptogenes" to describe these hidden
genes. The most astonishing examples of cryptogenes were discovered by
Feagin and Stuart in which hundreds of uridines were added at different
sites within the entire length of the RNA transcript, a phenomenon
known as "pan-editing." "It truly appeared as if 'new genes' were being
created from whole cloth," Simpson said.
As evidence for cryptogenes mounted, the Simpson and Stuart labs
began to publish their data. But since the matter seemed to be a
challenge to the central dogma, this raised much controversy.
Cryptogenes were not exactly derided by the scientific community, but
their existence was not met with instant acceptance, either, Simpson
said. "The data supporting cryptogenes was usually met with
astonishment and termed bizarre," he said.
The mystery of cryptogenes was finally explained in 1990 with the
discovery of a new class of RNA molecule by Simpson and his
postdoctoral fellows, Beat Blum and Norbert Bakalara, which they called
guide RNAs (gRNAs). The gRNAs were discovered during a computer search
of maxicircle sequences for short stretches of sequence that could pair
up with the known edited RNA sequences. The computer search turned up
seven short RNA sequences that were scattered throughout the
maxicircle. Simpson's team soon showed that these sequences were
transcribed into small RNAs of about 50 nucleotides in length that have
sequences at one end that allow the gRNAs to anchor to specific mRNA
sequences adjacent to the sites of uridine insertions. And the gRNAs
carried an additional length of uridine residues at the other end that
were not encoded in the DNA sequence.
"Guide RNAs had come to the rescue of the central dogma," Simpson
said. "Of course we were very excited by this discovery, but at the
same time a bit chagrined that the answer to the secret of editing was
not something completely new and earthshaking, but something that
obeyed the simple rules of base-pairing." The only novelty was that
guanine seemed to pair frequently with uridine when the gRNAs and
edited mRNAs interacted.
Simpson and Sturm and others next showed that most of the gRNAs in
the cell were encoded in the minicircle molecules, thus finally
providing a function for these mysterious molecules. This also neatly
explained the extensive sequence differences in the minicircle DNA
molecules, since each minicircle class encoded a different gRNA, and
many gRNAs were required for all the different editing events. In
recent work, Simpson and Avila have shown that the minicircle sequence
differences between different strains of T. cruzi are the result
of the accumulation of mutations that do not affect the editing
process.
The story was made even more complex when the Simpson lab developed
a biochemical model whereby the gRNA actually guides the insertion and
deletion of uridine residues at specific sites within the mRNA. Soon
thereafter, Simpson and Blum discovered yet another class of RNA
molecules which consisted of gRNAs chemically linked to mRNA fragments
at editing sites. This discovery led them to propose a model for
editing which was very similar to the way in which the so-called
introns or intervening sequences found in most genes in higher
organisms are removed from mRNAs in mitochondria in other organisms,
the phenomenon known as RNA splicing.
Soon after Simpson and Blum had discovered the existence of these
fused gRNA/mRNA molecules and developed the splicing model, but before
they had published their findings, Simpson was invited by Tom Cech,
a Hughes investigator at the University of Colorado, Boulder, to give a
talk in Colorado. To Simpson's surprise, Cech said that he too had
solved RNA editing and proceeded to draw on a board the entire splicing
model. "As happens frequently in science, when ideas are ripe, they
germinate simultaneously in several gardens," Simpson said.
Prior to the discovery of RNA editing and gRNAs, Simpson had thought
that the unique nature of kinetoplast DNA might one day allow it to
serve as a molecular target for identifying different strains of T.
cruzi and other trypanosomatid parasites. This has proven to be
true, and the PCR assay developed in the Simpson laboratory is in use
around the world — from the steamy Amazon jungle, where Chagas disease
is still a deadly threat, to metropolitan Los Angeles where infected
immigrants who are selling their blood for money are depositing Chagas
disease in blood banks.
Simpson has not given up on his original idea of settling the
question of Darwin's illness once and for all. "After all," he mused,
"Darwin is buried in a very accessible location — in Westminster Abbey
right next to Isaac Newton. And we only need a little piece."
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