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"We're attempting to create mutations that destroy innate
immunity and in this way, to identify all of the genes involved
in innate immunity," Beutler says, "or at least as many as
we can."
The mutagenesis involves attacking genomes with ethylnitrosourea
and looking for defective innate immune function phenotypes.
Treatment with ethylnitrosourea creates about 10,000 to 30,000
randomly distributed mutations in the germline, mostly A–T
or A–G substitutions. About 1.6 percent of these mutations
fall within coding regions, and about three quarters of these
result in an amino acid change. Thus in every separate mutant
genome, there are between 100 and 300 mutations that create
a coding change.
By examining several thousand such genomes, one can get very
deep coverage of the entire genetic content of a species—hundreds
of thousands of mutations are induced in all, with at least
one mutation in almost every gene and several mutations in
some of the larger genes. Then, Beutler says, one simply looks
for phenotypes that are of interest. In his case, these include
instances of immunodeficiency.
"Then if we find an individual that is immunocompromised,"
says Beutler, "we can go back and positionally clone the critical
gene that we have hit."
Though this may sound routine, there are many more failures
than successes, and the competition can be fierce, especially
when the mapping and sequencing steps take years, as they
did in the case of Tlr4. Beutler described the long battle
in a historical narrative recently published in the Journal
of Endotoxin Research. Of the race to clone Lps,
he wrote:
"On occasion, we rejoiced, feeling that our goal was within
sight, only to have our hopes dashed within days. Ever present
was the fear that, upon opening the next issue of Nature,
we would read that the gene had been cloned elsewhere, as
a gambler fears the cards of those arrayed against him."
Bringing it Home to TSRI
After the successful cloning effort, many new questions
related to endotoxin signaling immediately presented themselves,
and Beutler continues to study this protein and its relatives,
hopeful that a full understanding of the events that occur
in signaling may emerge. By making chimeras of human Tlr-4
and mouse Tlr-4 proteins, he and his coworkers hope to pinpoint
the part of the protein that actually "touches" the endotoxin
molecule, leading to cell activation.
Other follow-up work has already panned out. A standard gene
searching program called Basic Local Alignment Search Tool
(BLAST) has revealed three novel Toll-like receptors in mammals,
which were then independently cloned in 2000 by the Beutler
and Ulevitch laboratories. These molecules were dubbed TLRs
7, 8, and 9. TLR10—which may prove to be the "last" TLR—was
cloned shortly thereafter in the Ulevitch lab. The binding
specificity of most of the TLRs remains unknown. "And," Beutler
says, "everybody is interested to learn about the structure
of these proteins."
Beutler calls the process of positional cloning 'addictive.'
"The methods used in hunting genes are compelling, and I will
continue to use them whenever possible," says Beutler. "The
power of a mutation to disclose function is tremendous, and
provided that one begins with a clear phenotype, the genes
that are ultimately identified will most certainly be relevant
to the biological question that one has in mind."
There is something in biology referred to as the phenotype
gap—the discrepancy between the 30,000 to 40,000 genes
we believe are present in the human genome and the mere total
of 5,000 distinguishable traits that have been identified
through studies of inherited diseases and knockout mutations
produced by gene targeting. The inference to be made is that
the critical function of most genes remains unknown. This
applies in the field of immunology as in many other fields.
The precise question of which genes serve innate immunity
is one of several lines of inquiry Beutler brought with him
when he came to TSRI last year. He is also interested in discovering
why the placenta is not rejected during pregnancy as any "normal"
tissue allograft would be. Since half of a fetus's genetic
material hails from its father, the fetus is sufficiently
non-self to cause an immune reaction. But in most cases, it
does not.
Beutler is trying to create a model in which semi-allogeneic
pregnancy (in which the fetus is derived from genetically
different parents) is not tolerated, but syngeneic pregnancy
(in which the fetus is genetically identical to both parents)
is tolerated. "We are asking how it happened that placenta
could arise in evolution, although when it did, there was
already a very good adaptive immune system," Beutler says.
For Beutler, coming to TSRI was a definite homecoming. Raised
in southern California and very familiar with San Diego from
having attended the University of California at San Diego,
he also joins his father, Ernest Beutler, who is chair of
the Department of Molecular and Experimental Medicine.
"I love it here," he says. "The scientific environment at
Scripps is superb, my colleagues here are wonderfully supportive,
and not least, I always wanted to come back to California,
having missed it ever since I left 25 years ago."
Beutler got his start in science while working in his father's
laboratory as a teenager. "My father worked on red cell enzymes
at the time, and my first project involved assaying glutathione
peroxidase activity in human erythrocytes," says Beutler.
"He was a fine teacher, who gave me an in-depth introduction
to science at a young age."
When asked his opinion, Ernest Beutler smiled widely and
nodded in approval. "I'm glad he's here," he said.
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Alignment of TIR sequences from the ten human Toll-like receptors.
The TIR is the most conserved portion of members of the Toll-like
receptor family. Different amino acid residues are shown in
different color in this picture for clarity, with colors grouped
according to physico-chemical properties. For example, hydrophobic
residues L and V are both colored pink. The sequences are
optimally aligned with the inclusion of gaps where required.
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