They struck out. "We characterized every bloody gene present in that DNA and none of it was the gene" associated with CLL, Croce recalls. "I was very frustrated." So were his students and collaborators. "Oh, I burned the lives of a few people," Croce adds. One researcher quit science altogether to get a degree in business administration.
In 2001, Croce hired George Calin, a Romanian gastroenterologist, to take on the project everyone had grown to hate. "He had nothing worse in the lab," Calin jokes.
"Look," Croce told Calin, "the gene has to be there."
Around the same time, a new understanding of genetics was beginning to circulate. Oddly enough, it was facilitated by a mutant worm that was unable to lay eggs. The animal met a ghastly fate: hundreds of eggs hatched inside its body, causing it to burst open. Victor Ambros, a developmental biologist then at Harvard (now at the University of Massachusetts Medical School), was studying the mutation responsible for the worm's genetic defect. The worm, Caenorhabditis elegans, is a microscopic creature that geneticists love to study because it is easy to grow—it eats common bacteria—and is transparent, so all of its 900 or so cells can be observed as they develop. Curiously, as Ambros searched for the mutated gene, the section where it seemingly had to be became too small to contain a normal gene. "It became less and less clear that this piece of DNA could encode a protein," he says. "It was pretty astonishing."
Across the Charles River, at Massachusetts General Hospital, a molecular biologist named Gary Ruvkun was studying a different C. elegans mutant. Ambros and Ruvkun both suspected that the gene Ambros was looking for somehow controlled the gene that had gone awry in Ruvkun's worms. Working on a hunch, they decided to compare the two genes to see if they resembled each other.
"We e-mailed each other our sequences and we agreed to call in later on if we saw anything," Ambros recalls. "One of us called the other one and I said, 'Gary, you see it? And he said, 'Yes, I see it!'" They had found a perfect match—a stretch of DNA from Ambros' short genetic sequence identical to a section of Ruvkun's normal-size gene.
Ambros' gene was truly tiny, only 70 bases long, not 10,000 bases like other genes. Stranger still, the gene didn't make a protein, as other genes do. Instead, it made another kind of genetic material, which is now called microRNA. Traditional genes make RNA also, a molecule that is chemically similar to DNA, but that RNA is short-lived, serving as a mere messenger or intermediary in the construction of proteins. But this microRNA was the gene's end product, and it was no mere messenger.
MicroRNA, Ambros and Ruvkun realized, worked by an intriguing mechanism: it acted like a miniature strip of Velcro. Because the microRNA gene matched part of a traditional gene, the microRNA stuck to RNA produced by the traditional gene. In doing so, it blocked the other gene from producing protein.
It was a fascinating find, but the two scientists thought it was just an oddity until, seven years later in 2000, a researcher in Ruvkun's lab, Brenda Reinhart, found a second microRNA gene in the worm. "That told me that small RNAs were going to be more common than we expected," says developmental biologist Frank Slack, who helped with the discovery in Ruvkun's lab and is now at Yale.
The Ruvkun lab started looking for microRNA genes in other animals. As it happened, it was a great time to search for genetic anomalies. In 2001, scientists completed a draft of the entire sequence of human DNA, known as the human genome, and they were rapidly sequencing other genomes, including those of the mouse, mustard plant, fruit fly and malaria parasite. Some genomes were becoming available on Internet databases, and Ruvkun found the same microRNA gene from the C. elegans worm in fruit flies and human beings. Then he found the gene in mollusks, zebra fish and other species. Meanwhile, Ambros' group and others were finding dozens of additional microRNA genes.