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Watching a gene at workHow human proteins are made from DNA can be followed in real time. Scientists have viewed the expression of an individual gene inside a human cell. Knowledge of the real-time dynamics of gene expression may help researchers to explain variation among genetically identical cells and the molecular processes that lead to cancer.Traditionally, biochemists and cell biologists examined the time-averaged behaviour of thousands or millions of cells in order to understand how the information contained in genes is used to make proteins. Then, in the late 1990s, researchers developed a technique to tag genes so that they produce a fluorescent signal the moment they are transcribed into protein blueprints known as messenger RNA1. Researchers have imaged individual genes in bacteria and single-celled animals, and found that, rather than humming along at a constant rate as had been assumed, they seem to flicker on and off in bursts as they produce mRNA2. Until now, however, no one had applied the visualization technique to observing a single gene in mammalian cells. "This represents the continuing evolution of a technology that is going to revolutionize the way people think about biology," says Gordon Hager, a cell biologist at the National Cancer Institute in Bethesda, Maryland, who was not involved in the study. The chief problem with previous methods for visualizing transcription in mammalian cells is that these require researchers to blast cells with hundreds of copies of the specially tagged gene. Once inside the cell, the tagged genes are inserted into a cell's genome at random. Some regions of the genome are naturally transcribed into proteins at a high rate, whereas other regions are essentially silent. Overall, therefore, the process obscures the behaviour of specific genes. "In our system, the cell line has a target sequence in its genome and any sequence you send in will always go to that place," says senior author Yaron Shav-Tal, a cell biologist at Bar-Ilan University in Ramat Gan, Israel. "You can make different cell lines and not be worried about where the gene went in." Shav-Tal and his colleagues describe the technique online today in Nature Methods3. To test the method, they created two clones of a human embryonic kidney cell line with an engineered version of the gene cyclin D1, which controls the cell cycle. Both clones included a DNA sequence that allow a fluorescent protein expressed in the cell to bind to cyclin D1 RNA the moment it is transcribed. One clone depended on the gene's natural promoter — the binding site for the polymerase enzyme that transcribes DNA into mRNA — whereas the other was fused to a viral promoter known to overexpress genes by producing an abundance of mRNA. By visualizing the process at the level of a single gene, the researchers were able to work out the different mechanics of transcription between the human and viral promoter. The cells with the normal promoter shut down for about 20 minutes every 200 minutes, whereas the cells with the viral promoter remained active for a 10-hour stretch. More significantly, the latter group of cells recruited twice as many polymerase enzymes — about 14 — which crammed along the gene's length, all producing mRNA. The method will allow researchers to
investigate the mechanics of other promoters, as well as disparate
phenomena such as the pulsing of hormones produced by the endocrine
system. "This is a whole new outlook," says Shav-Tal. "People now know
that even if the whole population of cells is supposed to be identical,
each one has a different expression profile."
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