Homes for Orphan Genes
Federal and industrial scientists racing to decipher the human genome have already decoded an estimated 70,000 of the 100,000 genes that make up the human genetic blueprint. They hope to have the sequences of all of them by 2003.
But knowing the sequence of a gene and understanding what it does are two vastly different things. Of those 70,000 genes that have been deciphered, researchers know the function of only about 10,000. The others are called orphan genes, their precise parentage and purpose still mysterious.
Geneticists and molecular biologists around the world have donned the cloak of Sherlock Holmes to deduce the working of these orphan genes, a process called functional genomics. But that process is more difficult, labor-intensive and expensive than simply puzzling out the gene’s sequence, and every success represents years of hard work.
Today, two groups of researchers report the discovery that one orphan gene is the blueprint for the binding site of a peptide hormone called urotensin-II. The hormone-receptor pair, they say, is a powerful system that constricts blood vessels more strongly than any other natural product and may be a major contributor to high blood pressure.
The discovery by teams from UC Irvine and SmithKline Beecham Pharmaceuticals in King of Prussia, Pa., could lead to new, more selective drugs to control hypertension. But it also illustrates the methods used in the recent discoveries of genes for narcolepsy, for a brain receptor that plays a key role in regulating appetite and for another that controls anxiety.
“This is really cutting-edge technology,” said Alvin J. Glasky, president and chief scientific officer of Neotherapeutics Inc. in Irvine, which sponsored the UC Irvine research. “You can do some very creative things with it.”
But the opportunity to do those creative things relies on some old-fashioned, brute-force molecular biology techniques. Translating results from the Human Genome Project into clinical applications is a major undertaking.
Nothing but Hints
Although an architect’s blueprint clearly shows what is being constructed, the blueprint for a gene gives only hints. Geneticists can compare a newly discovered genetic blueprint to others that are already known and say that it is probably the plan for a receptor, an enzyme or a genetic on-off switch.
But they can’t look at the gene for a receptor and say, “This binds to dopamine” or insulin or some other hormone. And unless they can say that, the gene is of little use.
“An orphan receptor has no value,” said neuropharmacologist Olivier Civelli of UC Irvine, whose laboratory specializes in finding the functions of orphan receptors.
In his lab, about 10 researchers have been working with genes from the so-called GPCR (G-protein-coupled receptor) family. “These have been shown to bind to all kinds of small molecules in the brain,” he said.
So far, the genome project has identified about 1,000 genes that seem to belong to the GPCR family. At least 700 of them represent odor receptors that help the olfactory system identify scents. An additional 160 have already been characterized, leaving about 140 orphan genes. Civelli’s lab and many others are trying to ferret out their secrets.
The team selected 20 to 25 of the orphan genes that instinct and experience suggested might be important and went on a fishing expedition.
Using genetic engineering techniques, they inserted a gene into laboratory-grown kidney or ovary cells, which do not ordinarily bind to chemicals found in the brain. In this case, the gene was for something called the GPR14 receptor. The gene triggers the production of multiple copies of the receptor, which then stud the surface of the kidney or ovary cells. The cells are then attached to a solid instrument so they can be readily manipulated.
“We then go to a slaughterhouse and get some brains,” which are ground up into a thin gruel, Civelli said. Researchers dip the cells into the brain material, fishing for anything that binds to the receptor. If something does bind, it triggers a chemical change in the kidney or ovary cells that can be monitored by the researchers.
The cells are then used to purify the chemical--called a ligand--that bonds to the GPR14 receptors. The researchers might, for example, wash the brain tissue with an organic solvent, thereby removing any chemicals that are more soluble in the solvent than in water, and check to see which solution contains the ligand.
‘It Knocked Our Socks Off’
Further purification steps are carried out in the same fashion, until the researchers have isolated pure ligand. The challenge then is to figure out what the ligand-receptor combination does. “You never know where the biology is going to take you,” said Eliot Ohlstein of SmithKline Beecham. “You have to be alert to any clues the biology gives you.”
The new receptor reported in today’s issue of the journal Nature by the SmithKline team and in the October edition of Nature Cell Biology by Civelli’s group was paired with its ligand in this fashion. When the teams purified the ligand, they found it had the same structure as an orphan peptide called urotensin-II.
Urotensin-II was discovered in 1985 in the spinal cord of fish, but researchers had only a vague idea of what it does. Armed with the new receptor, Ohlstein and his colleagues at SmithKline put the combination through a large variety of biological tests.
“It had very potent vasoconstrictor [blood-vessel-narrowing] activity,” he said. “It turns out to be the most potent one yet identified. It knocked our socks off.”
Ohlstein said that he had spent the last 10 years working on another naturally occurring vasoconstrictor called endothelin and that SmithKline has developed experimental drugs that block its effects. Urotensin-II may be even more important, he said.
The team has found that there are large amounts of urotensin-II in the atherosclerotic plaque of people with blocked blood vessels. “It’s unclear what it is doing, but we know it is there,” he said. “I’m really happy to get this information published so that other researchers can begin studying it and evaluate the biology. Now that we have this unique pairing of an orphan receptor and a ligand without a receptor, the challenge is to figure out what it is doing. That’s what really drives us.”
What Does a Gene Do?
The Human Genome Project is yielding the seqences of many genes whose functions are not yet known. Identifying what these orphan genes do is a laborious, expensive project.
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Source: Olivier Civelli, UC Irvine
