O'Farrell's Gels Blazed New Pathways
Patrick O'Farrell
Every so often, a biomedical scientist invents clever research techniques that spread like wildfire among fellow explorers of cells and molecules. The technique leads to fantastic discoveries never before possible. But the inventor's feat usually remains unsung outside the realms of science.
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What is a gel and how does it work? Two-dimensional gel electrophoresis involves making a thin "gel" between two glass plates by pouring a solution that attains the consistency of a gelatin. The researcher applies a tiny amount of a protein preparation extracted from cells midway down the left edge of the square gel. Proteins move up or down according to how acidic they are. Next, the researcher chemically treats the gel to unfold the proteins and add negative electrical charges to them. The researcher then applies an electrical field across the gel from left to right. All the "denatured" proteins, which are now highly charged electrically, move across the gel, with the smaller ones moving faster. Proteins end up all over the gel, separated from one another by weight and acidity. |
As for O'Farrell - and for countless others who have used the experimental protocols he developed - his invention opened a new window on the biochemical pathways that underlie health and disease.
In 1974, genetic approaches were limited and slow. But researchers could cause genetic mutations in single-cell organisms or in fruit flies. Scientists could detect these mutations not just by noting changes in behavior or appearance, but also by observing the absence of certain proteins or signs of their alteration in sample preparations. Ready For Prime Time
Researchers could go much further after O'Farrell overhauled a new technique - two-dimensional gel electrophoresis - and made it ready for prime time. Scientists could now observe changes in the amounts of other cellular proteins that were associated with particular mutations. Through a well-designed series of experiments, they could figure out how individual proteins are acted on by other proteins to switch genes and protein production on and off - the mechanisms underlying cellular dramas of birth, growth and death, not to mention everyday cellular housekeeping. "Back then, being able to identify every gene was not something one could remotely think about," recalls O'Farrell. "We had hardly identified anything in mammalian cells." Previously, only the most common proteins from cells could be detected easily. But with his 2-D gels, O'Farrell was separating more than 1,000 proteins from one another. To use the technique, a researcher applies a protein preparation made from cell extracts to a gel that is sandwiched flat between two square glass plates. The gel and an electrical current applied to the gel cause the proteins to separate from one another according to their weight and acidity. Radioactive tags (fluorescent tags, nowadays) allow for easy imaging of the protein spots. With O'Farrell's invention, it became possible to see proteins that are not abundant, but still very important to the cell. But three decades ago, doubters at first declined to publish the paper he submitted describing the technique. In the meantime, the youthful O'Farrell kept up a busy schedule teaching senior scientists and laboratory leaders how the technique worked while he labored to finish experiments to complete work on his own doctoral thesis. He soon came to UCSF as a postdoctoral fellow, but demand for his tutorials remained strong. "No one was used to experimental systems where one looked at so much," recalls O'Farrell, who has been on the UCSF faculty since 1979. People asked, 'How can you deal with so many spots?'" Today, genetic researchers use powerful computers to help them collect and interpret the huge amounts of data that can now be generated during a single experiment. Thanks to the Human Genome Project, all genes are mapped. But scientists are still figuring out how genes are switched on and off in a coordinated way - a result of the actions of the proteins they encode. Continuing Interest in Pathways
O'Farrell has remained steadfast in his interest in understanding of how biochemical pathways operate. He uses a similar strategy - disable one molecule at a time, and see what happens - but he uses different tools. He doesn't think he has "run a 2-D gel" since 1979. Recently, O'Farrell has set ambitious goals for the intensive use of another technique, called RNA interference. Several years ago, researchers discovered that genes can be switched off by double-stranded RNA that matches DNA sequences in the gene. RNA is a nucleic acid that forms chains with a slightly different molecular backbone than DNA. This recently discovered role for double-stranded RNA differs profoundly from the long-understood role of single-stranded RNA in translating the genetic code from DNA into production of the encoded protein. The functions of various naturally occurring double-stranded RNAs still are being investigated. But in the meantime, researchers now can make short, double-stranded RNA molecules to match the genetic code of any gene, and can thereby turn off specific genes at will. RNA Interference
Two years ago, O'Farrell and one of his graduate students, Edan Foley, used RNA interference to silence 7,000 genes - one at a time - to better define the biochemical pathways that mobilize the first line of immune system defenses in organisms ranging from worms and flies to humans. RNA interference is new, but three decades after O'Farrell first made it useful, two-dimensional gel electrophoresis also remains a popular way to approach biological challenges. Now the technique often is used for different purposes. Researchers routinely run gels as a starting point to prepare more purified protein samples to feed into a more elaborate protein separation device called a mass spectrophotometer. A field of "proteomics" has sprung up, with vigorous efforts devoted to cataloging proteins and their physical associations with other proteins. O'Farrell looks back on the development of his classic laboratory protocol as almost another lifetime. "Some two-dimensional approaches had already been tried," he says. "I was just pretty stubborn about getting it to work well." Even by today's standards, there is still quite a bit of valuable information in those protein spots. Photo by Majed