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A team of scientists at the University of Chicago has developed a non-invasive laboratory technique that allows them to instantly map when genes are switching off and on in a living bacterium as it becomes exposed to antibiotics and other changes in its environment.
"The standard assumption has been that DNA encoding for proteins was the sole actor to control the blueprint for all of life," said Philippe Cluzel, an Assistant Professor in Physics at the University of Chicago. "After announcing the completion of the human genome, biologists have realized that the DNA sequence wasn't enough to explain the observed complexity of biological function."
Cluzel, a biophysicist, co-authored the PNAS article with Thuc Le, Sebastien Harlepp, Calin Guet, Kimberly Dittmar, Thierry Emonet and Tao Pan, all of the University of Chicago. Their research was funded by the National Institutes of Health and the University of Chicago's Materials Research Center for Science and Engineering and the Institute for Biophysical Dynamics.
Cluzel compares the unaccounted-for complexity of biological function today to the situation 20 years ago in astronomy, when scientists realized that the celestial objects visible to them did not contain enough gravity to explain the motion of galaxies. "They proposed the hypothesis that the motion of galaxies is affected by the presence of invisible matter called 'dark matter,'" Cluzel said.
In recent years, astronomers have rushed to design experiments that might help them determine what dark matter is made of. "RNA molecules could be called the 'dark matter' of biology," Cluzel said.
Biologists have long known that RNA serves as an important intermediary between DNA and the factories throughout the cell that produce proteins. Some biologists have recently begun to identify families of RNA molecules that also play a major role in determining when and how genes are turned on and off, but they have lacked the ability to monitor action of these molecules as it happens within a single living cell.
Now Cluzel and his associates, using a technique called fluorescence correlation spectroscopy (FCS), have shown how to monitor the link between RNA synthesis and promoters-small pieces of DNA that turn genes on and off-in a living cell.
"Because RNA molecules are intrinsically unstable, it has been extremely challenging to measure and characterize the mode of action of RNA molecules within a living cell," Cluzel said.
The techniques previously used to study RNA required killing and breaking up a cell in order to extract an RNA molecule. With those techniques, "one cannot detect in real time the underlying dynamics of RNA synthesis associated with cellular activities," he said. But the scientists in his laboratory have found a way around this with a technique called fluorescence correlation spectroscopy (FCS).
First, they use an RNA molecule that will bind to a protein called MS2. This MS2 protein also is fused to a green fluorescent protein that a laser can detect within a microscopically small volume.
In the absence of RNA, the MS2 fused to the fluorescent protein moves rapidly. But when bound to RNA, the fused proteins move more slowly. From the speed of motion, "we can infer the concentration of RNA present inside the detection volume and a single living cell," said Thuc Le, a graduate student in Biochemistry & Molecular Biology and the lead author of the PNAS paper.
This new technique will now facilitate research that may reveal how RNA molecules, like proteins, turn genes on and off, Cluzel said.
Source: University of Chicago. June 2005.
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