The normal gene for P-gp (left) and a triple mutant (right) make the same amount of protein in the cell. But the protein's structure and function are altered.

C. Kimchi-Sarfaty et al.

The Sound of a Silent Mutation

Another dogma in cell biology seems about to be toppled: If a mutation in a gene doesn't change the basic sequence of building blocks, then it has no effect. Chava Kimchi-Sarfaty of the U.S. Food and Drug Administration in Bethesda, Maryland, and colleagues report online this week in Science that such "silent mutations" can, under certain circumstances, determine how well a final protein performs--an "extremely provocative" result, says cell biologist William Skach of Oregon Health & Science University in Portland.

Silent mutations occur when the change of a single DNA nucleotide within a protein-coding portion of a gene does not affect the sequence of amino acids that make up the gene's protein. That's possible because proteins are encoded by "triplets" of nucleotides, each responsible for adding a particular amino acid to the protein chain. A change in one nucleotide, however, doesn't always change the triplet's meaning; the mutated triplet may still add the same amino acid. And when the amino acids of a protein stay the same, researchers believed, so do its structure and function.

But every once in a while, data crop up that don't make sense; for example, a gene called multidrug resistance-1 (MDR-1) has been found to frequently have a particular silent mutation in human cancer cells. MDR-1 produces P-gp, a protein that pumps chemotherapy drugs out of cancer cells, thus making the drugs useless. Researchers wondered why the silent mutation, called C3435T, showed up much more frequently than expected for a change that doesn't have an effect on the cancer cells' survival.

Kimchi-Sarfaty's team made cell lines that had either the normal MDR-1 gene, a version with the C3435T mutation, versions with either of two other mutations known to occur sometimes along with C3435T (one of them silent as well, the other nonsilent but without an effect on protein function), and versions with various combinations of two or three of the mutations.

They found that the mutations individually appeared to have no effect: The P-gp proteins encoded by each gene variant were just as proficient at pumping drugs out of cells. But cells with the MDR-1 gene containing the C3435T mutation plus one or two of the other two mutations did a much better job of ridding cancer cells of the drugs, allowing the cells to live another day.

How is this possible if the variant P-gp has the same string of amino acids as the normal one? To the researchers' surprise, a biochemical test suggested that the mutant P-gp has a slightly different three-dimensional shape. Perhaps, Kimchi-Sarfaty says, the silent mutations reside in nucleotide triplets that cells don't use very often, which could slow down the cell's proteinmaking machinery. Like designs made with Silly String spraying out at different velocities, the folding of an amino acid chain into a 3D structure is somewhat speed-dependent, and slower production could cause the protein to take an altered final form. The cell might be able to compensate for one silent mutation but not for multiple rarely used triplets.

The idea that silent mutations might have such effects is "an entirely new concept," Skach says. His prediction: More researchers will start listening to what silent mutations have to say.