Bioinformatics Highways

Using DNA chips to obtain information about biological systems is only the first step. "The information delivered by the chips is so complex that effective interpretation requires elaborate data analysis and organization," explains Horn-Saban. Researchers at the Institute hope to address this challenge through computer and web-based technologies. They are working to link DNA results to GeneCards, an on-line database and software tool developed at the Institute that provides fast and convenient access to updated genetic information. Once a gene is identified from the DNA chip printout, the novel bioinformatics interface will provide a direct link to relevant information, including the proteins it encodes, cellular functions, diseases caused by its mutations, and other web sites. Another project, led by Dr. Naama Barkai of the Institute's Molecular Genetics Department, is aimed at using mathematics to interpret the function of highly complex gene expression patterns.

Having completed a working draft of the human genome, widely hailed as one of the most significant intellectual achievements of all time, one might think that those involved would head home for a well-earned vacation.

Far from it. They've rolled up their sleeves and are moving on with added speed. We now know how genes perform the cell's work, instructing it to string together different amino acids to create, say, a protein triggering blood clotting following injury, or a stomach enzyme to aid in food digestion. The next challenge is to better understand these gene products.

Proteins serve as the body's primary component and the basis of all enzymatic reactions, so the slightest change in their structure or function can lead to disease, even death. Being there -- in the right place, at the right time, and in the proper amount, is what the protein story is all about.

It's also about folding. The primary property influencing a protein's function is its 3-D structure. Consisting of one long chain, or several, the protein molecule is generally coiled or folded. Any damage to this structure can impair a protein's biological properties -- which is why, for instance, heat-wave temperatures of over 50^o C can be life threatening.

But proteins are "born" unfolded. When first produced by the ribosomal factory (which implements genetically encoded instructions arriving from the cell nucleus), proteins emerge as straight, unfurled strings. They must fold into their correct form to become functional. How does the "young" protein know how to do this? Do "folding instructions" arrive with the genetic package, or do these tabula rasa proteins receive some help from nearby friends?

Back in the 1980s Drs. John Ellis and Costa Georgopoulos (then at the University of Warwick and the University of Utah, respectively) discovered that proteins, in fact, have molecular mentors. Dubbed "protein chaperones," they walk the newborn protein through its first folding steps and provide a safe environment in which to do so. Understanding these chaperones, themselves proteins, is the main interest of Prof. Amnon Horovitz of the Weizmann Institute's Structural Biology Department. Horovitz studies the GroEL protein found in E. coli bacteria -- a molecule containing two back-to-back rings, with a cavity at each end. Folding takes place within these cavities with the help of an additional protein called GroES, which serves as a "lid," covering the cavities' exits to prevent the newborn proteins from falling out while folding.

It turns out that the chaperone molecule has two basic states. Initially, newborn proteins can easily enter the chaperone and attach themselves to its cavity walls. At this point, the chaperone molecule undergoes a dramatic structural transformation. Its cavity walls reject the newborn proteins, heaving them into the center where they can fold safely.

Horovitz: "In the past, people believed that proteins folded into their characteristic structure to attain the lowest possible energy level, just as a ball placed on a mountaintop rolls to the bottom. In other words, existing in the folded as opposed to the unfolded state requires far less energy. But what if there is a series of mountains with valleys in between? The ball might reach a relatively high valley, where it will stay put unless 'freed' by an earthquake. From a protein's perspective, this 'earthquake' can be any of a number of environmental changes, such as temperature changes or the presence of a solvent. Both can cause protein misfolding, resulting in disease." For instance, Creutzfeldt-Jakob disease, popularly known as mad cow disease, is caused by misfolded proteins that are otherwise normal.

To discover how chaperones prevent protein misfolding, Prof. Horovitz is collaborating with Dr. Gilad Haran of the Chemical Physics Department. In standard experiments, billions of molecules are examined simultaneously; but Haran examines them one by one. This means that experimental results are not merely an average of different molecules, which might differ in their conformation or environment, but a representation of the full molecular variety of the process under examination.

To study the chaperone, Haran tags it with a fluorescent molecule that emits light when activated by a laser. When the chaperone protein shifts from one state to the other, it affects the fluorescent molecule's properties. In turn, a microscope equipped with sensitive light detectors picks up these changes, enabling the scientists to track the chaperone's transition cycles and obtain a better understanding of the folding processes it hosts.

But Horovitz and Haran want an even closer look. To obtain this, they plan to fasten two fluorescent molecules onto different sites of an unfolded protein. Since the wavelengths emitted by these molecules and the relative intensity of their emission are dependent on their distance from each other, the researchers believe that this approach may offer an intimate glimpse into the chaperone during protein folding.

Achieving a better understanding of how proteins fold is the next greatest challenge in what promises to be biology's century. The Human Genome Project was only the beginning -- there's still a great deal of unfurling ahead.

Time was when highly trained and motivated teams, often urged on by patriotic fervor, would set out to explore vast geographical unknowns. Mount Everest, the Poles, and the moon were checked off one by one. The human genome project, say many, is this legacy's high-tech equivalent -- biology's moon landing.

Fittingly perhaps, while the scenery and dimensions have changed, the rush to uncover the genetic blueprint of humanity was shaped by competition. Despite the extraordinary cooperation marking the decade of work by thousands of researchers across the globe, efforts were also fueled by rivalry, with the privately owned Celera Genomics pitted against the international, publicly funded Human Genome Project, as well as the latter's individual chromosome sequencing teams, all racing to become number one.

Nobody could catch 22. It ran ahead of the pack marking the first human chromosome to be sequenced, with 21 hot on its heels. Then, on June 26, the match ended in a draw, as researchers at the public consortium and Celera jointly announced that they had completed a rough draft of the entire human genome.

With its 3.1 billion base pairs neatly laid out in cookbook fashion, what can we, its bearers, come to expect? New therapies or diagnostic tests warning of a predisposition to disease coupled with preventive measures? Enhanced prevention of genetic disorders such as Down syndrome, which currently affects up to one in 700 newborns?

With time, these scenarios will most likely mature to varying degrees. However, as scientists are quick to point out, it will probably be decades before the full benefits are realized. The players have been introduced, but now, in order to follow and direct the plot, we have to understand how a gene interacts with its fellow genes to form and maintain the human body, what goes wrong in disease, and what role environmental factors play * in other words, the actual gene dance.

Here's an in-depth look at one of the contributions made by Weizmann Institute scientists to the international Human Genome Project.

21 Down & Counting

Chromosome 21 is now fully sequenced. Having crossed the finish line following a genetic research trek spanning four continents and more than 20 years, the international consortium charged with decoding 21's wares has surprising news: in contrast to previous estimates, the human genome may contain only 40,000 genes. That's only roughly twice the amount found in Drosophila melanogaster -- the common fruit fly.

Though checking in as the smallest of all human chromosomes, 21 has more than delivered. Recently appearing in Nature, its decoded sequence suggests that we may have to revise our basic understanding of ourselves -- specifically, the unique design principles underlying the human genome that set us apart from other organisms despite considerable genetic overlap.

Likewise, while the chromosome was found to contain only 225 genes -- just one-fourth the estimate based on its size -- these genes are among the most intriguing to researchers. They hold the key to further understanding Down syndrome and the other leading genetic disorders that are often part of its package. Besides mental retardation, Down syndrome patients are much more prone to develop acute myeloid leukemia and diabetes, frequently exhibit congenital heart disease and immune deficiencies, and are often diagnosed with Alzheimer's disease by the time they are 35. And the chromosome's unraveling began right here, in Rehovot's Weizmann Institute of Science, over 20 years ago.

"The most common culprits behind genetic diseases are mutated genes unable to perform their protein production jobs. Yet in Down syndrome, the genes are perfectly normal; they are simply overexpressed," says Prof. Yoram Groner of the Institute's Molecular Genetics Department. According to Groner, scientists had known since 1959 that Down syndrome is primarily caused by the inheritance of three rather than two copies of chromosome 21. However, little had been done to probe the molecular origins of the disease. Which is why, equipped with the rudimentary gene isolation and cloning techniques available in the early 1980s, Groner and his departmental team set out to examine how an extra copy of otherwise normal genes can produce the patchwork of abnormalities manifest in Down syndrome.

Candidate Genes Raise Eyebrows

The SOD1 gene, encoding an enzyme that protects the cell from naturally occurring toxic oxygen radicals, turned out to be a central player. Groner's team zeroed in on SOD1, becoming the first to clone a chromosome 21 gene. When this human gene was introduced into a colony of mouse cells, it triggered a leading symptom of Alzheimer's disease, significantly reducing the cells' ability to bind neurotransmitters -- the chemical messages vital to neural communication. Later, transgenic mice containing the SOD1 gene exhibited muscle weakness caused by deterioration of the neuromuscular junction, another symptom common to Down syndrome patients. "The muscle defect was particularly intriguing because many Down syndrome patients have abnormally large tongues, and the tongue is largely a muscle," says Groner.
 

Examining tongue muscle from patients who underwent cosmetic surgery, Groner's team, working with Dr. Rena Yarom of Jerusalem's Hadassah Hospital, found that the defect in mice and in Down syndrome patients was identical. These findings led Groner to suggest that Down syndrome and its common tag-along diseases might actually be caused by select "candidate genes." When overexpressed, these genes send the cells' machinery off track, causing an overproduction of proteins that subsequently impairs organ development and function. "The candidate gene concept raised quite a few eyebrows at the time, since the idea that specific genes could have such far-reaching effects presented a considerable psychological barrier," says Groner.

Genes and Humans Play Ball

Teamwork, on both a gene-to-gene and human-to-human level, also entered the equation. Groner's team showed that imbalanced SOD1 levels expose the cell to oxidative stress, which enhances its vulnerability to other chromosome 21 genes, when these are overexpressed. Take APP for instance. This gene encodes a protein shown to drown neuronal cells in plaque, causing the neuronal degeneration found in Alzheimer's. Then, in chain reaction style, another gene, called BACE-2, enhances APP breakdown, further increasing the neuronal damage.

According to Groner, the gene teamwork was mirrored by strong cooperation between members of the Chromosome 21 Consortium. BACE-2, for example, was discovered by the Spanish team. However, here too, as in the genome project, human nature intervened. "The consortium operated by dividing the chromosomal sequencing among different teams, much like sections on a road map," says Groner. As when building a tunnel, the teams had to plan their route and course of action with great care to ensure that they would meet up." The different culture and mentality of the Japanese and German teams, who were allocated neighboring genome sections, apparently caused some planning difficulties. "Unfortunately, this meant bad news for us," he adds, smiling, "since we were involved in sequencing a gene that sits right in the middle of this junction, which is believed to cause leukemia."

Old Genes Learn New Tricks

One of the most striking findings is that chromosome 21 contains only 225 genes, far less than expected. This led the consortium to suggest that the entire human genome may contain only 40,000 genes -- roughly double the amount of a fly or an earthworm. Potentially chair-squirming numbers -- after all, although we can't fly, we're still somewhat more complex.

A possible explanation is that nature employs an ingenious recycling strategy. "Why invent new genes, when the old ones have a proven track record?" Groner points out. "Mounting evidence suggests that instead of requiring a linear increase in gene numbers along the evolutionary path, nature primarily invents new functions for old players, rearranging their combined cross-talk to increase organism complexity."

 

What's Gone Right, Not Wrong

Not Why, But How