The research, available in the online edition of Nature, may help scientists understand whether bits of RNA called microRNAs act as protective mechanisms in healthy development not just by strategically turning off gene activity, but by making sure it stays turned off.

More specifically, researchers report linking a specific microRNA - miR 196 - to limb development, a finding that may be useful in understanding birth defects.

Until about five years ago, genetic researchers focused on DNA, which contains all the genetic instructions for the human body, and RNA, which translates DNA's message into proteins - the building blocks of cells, organs and all of the various systems of the body.

Unnoticed next to the main ingredients, microRNAs were considered to be "junk" DNA, leftovers from millions of years of evolution. More recently, this genetic material is suspected to be part of an intricate mechanism that helps repress about one-third of our 25,000 genes. It has been linked to diabetes, hepatitis C, leukemia, lymphoma and breast cancer.

But only now have microRNAs been connected to actual growth processes.

"We found miR 196 expressed only in the hindlimbs of mice, not the forelimbs - in other words, the feet but not the hands," said Brian Harfe, Ph.D., an assistant professor of molecular genetics and microbiology in the College of Medicine and a member of the UF Genetics Institute. "In developmental biology, there has always been debate about why forelimbs are different from hindlimbs. We now think this microRNA is regulating something important in the hindlimbs but not in the forelimbs."

Scientists do not know exactly what is happening, but they think miR 196 acts as a protective mechanism in the hindlimbs in the event normal gene transcription goes awry.

"A large body of evidence indicates this new class of regulators is not something to turn things off in the first place, but a fail-safe," said Clifford Tabin, Ph.D., a professor of genetics at Harvard Medical School and senior author of the research. "You don't want cells in a hindlimb seeing cells that should only be in a forelimb - it would create a defective limb. So you not only want to shut the faucet tight on the wrong cells, you want to shove a towel into it, too, to really make sure the wrong thing doesn't leak out. One way of doing that is with microRNA."

Researchers looked at gene activity in chicken embryos and in mice, finding miR-196 silences a chemical important for transferring information from DNA to RNA within a cell - a transcription factor.

"It's turning off a transcription factor in the hindlimb that is important for forelimb development," Harfe said. "But it still doesn't explain why a hindlimb is a hindlimb and a forelimb is a forelimb."

The next step in the research is to observe limb development in mice engineered to not express miR 196.

"The authors have shown a role for miR 196 in limb development," said John Fallon, the University of Wisconsin's Harland Winfield Mossman professor of anatomy. "People talked about ways microRNA may have a role in embryonic development, and this work is a solid contribution that supports that idea. Researchers have also been looking for differences between gene expression in forelimbs and hindlimbs, with little success. This paper suggests there is a new mechanism to control the fidelity of protein expression in the limbs through microRNA expression. That is a hypothesis that people in the field will have to test, but it is strongly supported by their research."

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Bushman and his team made cells that were depleted of LEDGF and found that integration was less frequent in transcription units and in genes regulated by LEDGF. "This implies that LEDGF is part of the machinery that helps dictate the placement of retroviral integration sites within chromosomes," says Bushman.

Bushman notes that finding that LEDGF is part of the cellular apparatus necessary for HIV replication is important to the field of gene therapy. Controlling where gene-therapy vehicles insert in the human genome could help make the delivery of new therapeutic sequences safer. The new findings about LEDGF suggest that engineered tethering interactions might some day allow control over integration site selection during gene therapy. According to Bushman, this finding is of particular importance in light of recent cases where integration of gene-therapy vectors near cancer genes contributed to the development of leukemia in gene-therapy patients.

"This is first example of a cellular factor that's a clear player in target site selection," says Bushman. "This isn't engineering yet, but it's a key piece of information on the way."

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