June 26, 2000

READING THE BOOK OF LIFE

Data From Genome Project Transforming Biology Research

By KENNETH CHANG

	Johnson & Johnson, top; University of California, San Diego, bottom
	One segment of a 16-segment DNA chip, shown at approximately 20 times its actual size of 7.5 by 9.5 millimeters. Each circle represents a gene. Bottom, Dr. Charles S. Zuker and his collaborators used a map of the genes to uncover secrets about bitterness receptors.
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any substances leave a bitter taste in the mouth. But how? That's one of the questions that biologists are answering with data from the Human Genome Project.

The effort to decode the biological instruction set of humans turned into a race between a private company, Celera Corporation of Rockville, Md., and a public consortium of universities. But participants on both sides are now minimizing the occasionally acrimonious competition and pointing to the ways that data from the project is already transforming biology research.

"It gets a lot of attention, but it's a colossal distraction," said Dr. Eric S. Lander, director of the Whitehead Center for Genome Research in Cambridge, Mass., a major participant in the consortium. Emphasis on the race is diverting attention from the important contributions genome data are already making, he said.

Even incomplete, the databases of DNA sequences are a treasure trove for researchers, providing answers in a few minutes at a computer terminal rather than after months of laborious, expensive laboratory experiments. For pharmaceutical companies, that speeds the development of new drugs with several promising compounds already undergoing human clinical trials.

For university researchers, that opens up areas of inquiry that would previously not have been worth the time and effort.

"You have democratized gene discovery," said Dr. Charles S. Zuker, a professor of biology and neurosciences at University of California in San Diego and an investigator at the university's Howard Hughes Medical Institute.

The DNA sequence put together by the consortium now covers about 90 percent of the 3 billion "letters" contained in the genetic blueprint of a human being, with new sequences added nightly. Celera says its map is 99 percent complete.

The remaining gaps are important, Dr. Zuker said, only in the unlikely event that a research target falls in one of the gaps. That was not the case when Dr. Zuker and his collaborators looked for the bitter-tasting genes.

Bitterness is often a warning to not eat a harmful substance, but the chemical details of how some molecules latch onto taste buds and dispatch a message of distaste to the brain are not well understood.

Two facts scientists already knew: bitterness receptors probably belonged to a family of molecules known as G protein-coupled receptors, and the location of the gene that produces one of these receptors lay along a section of Chromosome 5.

The structure of G protein-coupled receptors is distinctive: they thread up and down through the cell membrane, crossing seven times.

"Then we went to this wonderful resource of databases of human sequences," Dr. Zuker said, and searched for genes that produced proteins with membrane-crossing signature.

Focusing on the section of Chromosome 5, several hundred thousand base pairs long, the researchers got lucky.

"Bingo, there was one," Dr. Zuker said.

Since many different substances taste bitter, the researchers reasoned, there should be a family of bitterness receptors. So they queried the database to see if it could find similar DNA sequences elsewhere in the genome.

"Bingo," Dr. Zuker said, "we found there was a whole set of human genes that were related to this one member."

The computer has not completely replaced the laboratory. With the genes identified, the researchers still needed to confirm that cells with the genes responded to bitter substances, but the genome databases reduced to half a year what would once have taken years.

Genetic research consists of two phases: finding the gene, then understanding how the gene works. Before the advent of the genome databases, "The hard labor was involved in the first phase," Dr. Zuker said, "while really, ideally where you want to put your effort is into understanding the function of the gene. This will allow you to concentrate your effort into what is going to have the greatest impact in biomedical research."

When researchers at SmithKline Beecham P.L.C., a pharmaceutical manufacturer, went looking for an enzyme that sliced up a protein into toxic fragments within the brains of Alzheimer's patients, they too turned to a computer.

Scientists knew of the enzyme's existence from the protein-slicing damage it left behind, but had not identified the culprit, which they named beta-secretase. The search for beta-secretase has been littered with claims of discovery that were disputed, then discarded.

The SmithKline Beecham researchers suspected that beta-secretase belonged to a family of proteins known as aspartic proteases. They also knew that the enzyme acted near the membranes of brain cells.

So they plumbed computer databases of human DNA sequences, looking for the encoding instructions of aspartic proteases that also had a telltale structure which weaved the protein through cell walls.

The search turned up two candidate genes. One was on Chromosome 21, the chromosome duplicated in people with Down syndrome and who suffer similar tangles of protein in their brains.

The other gene, on Chromosome 19, turned out to be the one they were seeking. It encoded an enzyme that matched the profile of beta-secretase, producing the expected protein fragments.

The identification of beta-secretase could lead to drugs to thwart its activities that would be similar to the protease inhibitors that block H.I.V. from reproducing.

The SmithKline Beecham researchers, one of four teams that independently reported the discovery last fall, found the gene and enzyme "pretty quickly," said Dr. Colin Dingwall, assistant director of molecular neurobiology at the company's laboratory in Harlow, England. "We were just searching databases, and it turned up."

By contrast, researchers at Amgen Inc., a biotechnology company in Thousand Oaks, Calif., needed two years of tedious experiments to find the same beta-secretase gene, first locating a string of about 100 genes containing the one they wanted, then homing in on the gene through elimination. When Amgen began its work in 1997, the genome database was still almost empty.

"The rate of change is absolutely incredible," said Dr. Gillian R. Woollett, associate vice president for biologics and biotechnology of the Pharmaceutical Research and Manufacturers of America, a trade organization. "It's actually changing the way drug development is even conceived."

Human Genome Sciences Inc. of Rockville, Md., takes an unusual approach, looking for proteins, like insulin and human growth hormone, that can be used as drugs. "I call it the most natural form of medicine," said Dr. William A. Haseltine, the company's chairman and chief executive.

The company now has four drug candidates that have reached the clinical trial stage, all derived from the company's proprietary gene database.

The company announced on Friday that the Food and Drug Administration had approved human tests of B-lymphocyte stimulator, or BLyS, a protein that spurs the production of antibodies. BLyS may prove helpful to patients with immune system disorders that leave them vulnerable to infections.

Genomes of other creatures can provide clues as well. At the Whitehead Institute, Dr. Richard A. Young, professor of biology, can track how the genes of the tuberculosis bacterium turn on and off as it infects human cells.

"In war, which is what this is, if you could monitor the progress of both sides by monitoring all communications that are going on on both sides -- from the smallest military unit up to the supreme commander -- you'd have a very good picture of the different tactics that are being employed," Dr. Young said. "The results suggest a potential clinical therapy for turbuculosis."

PathoGenesis Corporation, a pharmaceutical company in Seattle, along with the Cystic Fibrosis Foundation and the University of Washington Genome Center, sequenced the genome of the bacterium Pseudomonas aeruginosa, hoping to find weaknesses that could lead to new antibiotics.

Here, too, the human genome proved useful, offering clues about whether a potential antibiotic might also be toxic to people. "Do humans also have that gene?" said Dr. A. Bruce Montgomery, PathoGenesis's executive vice president of research and development. "If so, that's not a good drug target. We're looking for something the bacteria has and we don't."

The wealth of genome information has also led drug researchers to a new, seemingly backwards, approach: instead of choosing a disease, finding a gene defect associated with the disease and then trying to devise a drug to counteract the defect, some researchers now look for genes that appear amenable to drug treatment and then try to figure out what diseases those drugs might cure.

The weakness in the traditional strategy is that finding the gene is often a dead end, with no easy way to mend or bypass the defect.

Johnson & Johnson, one of the companies that have adopted this new strategy, surveyed the 100 top-selling drugs and found that most were aimed at a handful of types of proteins. The company's scientists reasoned that other members of these protein families might also prove to be highly profitable targets.

"They have the right pedigree," said Dr. Michael R. Jackson, senior director for drug discovery of Johnson & Johnson. "Certain families have a tremendous track record. You'd be crazy not to take note of that."

The company's researchers culled about 5,000 promising genes by delving into the gene database put together by Incyte Pharmaceuticals Inc. of Palo Alto, Calif., even though Incyte's database, unlike the work of Celera and the public consortium of universities, includes only the 3 percent to 4 percent of the genome that encodes proteins.

The Johnson & Johnson scientists knew they could probably build drugs aimed at the proteins made by the genes they selected, but for the most part, they had no idea what these proteins did, or even where in the body they were found.

To learn more about the genes' where and why, they created a "DNA chip" with samples of the 5,000 genes. The chip tests which genes are turned on within a tissue sample. By compiling a vast library of information about the genes, the scientists hope to deduce their functions.

The efforts have already yielded one major find: a third receptor for histamine, a chemical released in the body in response to stress, inflammation and allergies.

The two known histamine receptors had already inspired hugely successful drugs. H-1 is the target of the anti-allergy medicines Benadryl and Claritin. The H-2 receptor, which regulates the release of stomach acid, is the target of Tagamet.

Experiments in 1983 suggested the existence of third, undiscovered histamine receptor. "Since then, there's been quite a bit of work trying to explore what exactly is the H-3 receptor, what are its functions, whether it is a potential drug target," said Dr. Tim Lovenberg of the R. W. Johnson Pharmaceutical Research Institute in La Jolla, Calif.

A year and a half ago, Dr. Lovenberg was skimming through the results of a search through the Incyte database when one piece caught his eye.

"We just happened to spot a little tiny snip," Dr. Lovenberg said. "It kind of clued us in it might be a novel receptor."

Some experimental work showed that the gene fragment was switched on in the brain. Then, looking in a database of genes active in the thalamus part of the brain, Dr. Lovenberg's team found the rest of the DNA sequence. "When we looked at the rest of the sequence, you could tell it was a receptor," he said. "It was, at that point, very exciting."

They then tested what chemicals cells with the gene responded to. "It only responded to histamine," Dr. Lovenberg said. "That was the end of the story there."

Johnson & Johnson has already developed a compound to block the H-3 receptor, causing the thalamus to increase its production of histamines. That is believed to heighten wakefulness -- the reverse of the drowsiness side effect of some antihistamines -- but the researchers are not yet focusing on any specific medical conditions. "The plan is to do some experimental clinical work based on what we think the role of an H-3 antagonist would do," Dr. Lovenberg said.

The compound is still far from becoming a drug on the market, but the 18 months between the spotting of the gene snippet and the creation of a candidate H-3 blocker is far shorter than the usual development time of several years.

With the nearly complete DNA sequence, scientists can also begin to consider the interplay between genes. Dr. Lawrence B. Salkoff, a professor of neurobiology and genetics at Washington University in St. Louis, is mapping out the genes that create channels that let potassium atoms into and out of cells.

"These are like the transistors of the brain," Dr. Salkoff said. "These are little switches that open and close and allow a current to flow into or out of a cell," producing the electrical activity that drives the brain and muscles.

Dr. Salkoff is now looking at small worms known as nematodes, but the findings appear to apply to higher animals, too. The 80 potassium channel genes in the nematode fall into the same 8 types as in mammals. "What we conclude by this is that these genes evolved into their present form in a common ancestor between worms and man," Dr. Salkoff said.

Before the genome sequences, those comparisons would not have been possible.

"Scientists always look where the light is good," said Dr. Lander of the Whitehead Institute. "The genome project has turned on the lights on most of the genome. It's worth looking now."