Biology Division researcher Gerry Bunick and his colleagues are taking advantage of the absence of gravity to obtain premium quality crystals of a DNA-protein complex, which in turn will allow them to better study the structure of the genetic material in the cell nucleus.
NASA officials have given researchers at the Department of Energy (DOE) lab the go-ahead to place their experiment on a Space Shuttle mission planned for Sept. 28. The ORNL experiment will attempt to grow the DNA-protein crystals in microgravity, first on the shuttle and then several months later on the Russian Mir Space Station.
Those crystals will contain nucleosomes- which are an important constituent of chromosomes-that Bunick and his colleagues will then study by X-ray diffraction. As Bunick related, it's a complicated experiment on a complicated subject, but it's well suited to the scientific capabilities of NASA and DOE, not to mention the United States and Russia.
The complexity of DNA structures makes analysis difficult. X-ray diffraction is the best means for analyzing the fundamental structural component of chromosomes, although the overwhelming variety in the makeup of the genetic blueprint means that researchers must perform some fairly intensive manipulation.
"We have developed a DNA sequence of 146 base pairs that can be produced in large quantities from bacterial cultures," Bunick said. "The pure, defined sequence DNA is then mixed with proteins to form 'reconstituted' nucleosomes in which the DNA sequences and positions are all identical. Because they are alike, we can perform the diffraction at a much higher resolution."
The 146-base-pair sequence is a stride forward in itself, but they've taken it one step beyond that. "We made a DNA palindrome - the sequence reads the same forward and backward," Bunick said. "This makes the nucleosomes in the crystal that much more homogeneous and further improves the diffraction."
Funding for this research is provided by DOE's Office of Health and Environmental Research and the National Institutes of Health.
In the absence of gravity, Bunick said, the crystals that contain the nucleosomes will probably grow better, and that's important. The force of gravity on these crystals, however, causes imperfections.
"Gravity is the driving force that causes the crystals of the protein and DNA complex to grow faster-so fast that they often grow hollow, and that's not a desirable quality for X-ray diffraction," Bunick explained. "Other experiments have shown that in microgravity, crystals tend to grow more slowly, controlled by diffusion alone. When they work, the results usually are slower growth and more perfect crystals, which is exactly what we want."
Shortly before shuttle mission STS-73 is to lift off on Sept. 28, the researchers will start the crystal growth so that there will be time to form crystals during the two-week mission. In anticipation of any delays in liftoff, Bunick plans to have extra experiments set up in sequence. Starting the experiment too early would yield crystals subjected to gravity's effects; so the timing is critical. That promises to be a hectic process, at least until things get off the ground. A payload specialist will tend to the experiment aboard the shuttle.
The mission will test conditions and allow fine-tuning of the experiments in microgravity so that on a later shuttle flight, which will dock with the Russian Mir space station, the nucleosome crystals will have about three months to grow. The duration of the stay on Mir will relieve the pressure of timing the experiments, Bunick hopes.
Once Bunick, Joel Harp, and colleagues have their crystals, which are large enough to see with the naked eye, they'll set to work doing preliminary assessments in their Biology Division lab. More thorough analyses will probably be performed at the synchrotron light source at DOE's Brookhaven National Laboratory.
In the meantime, Bunick and Harp are working on other aspects of this complex experiment, including a method to protect the crystals from the X-rays' harsh effects. "We're developing a way to cool the crystals so that the X-ray bombardment will not damage the sample," Bunick said. "The technique is called flash-cooling."
The microgravity crystallization experiment is the first biological experiment from ORNL to go into space. Bunick believes the experiment was selected because the research promises to provide much insight into how the structure of life's building blocks determines how they function. "And," he said, "it is also a great example of how DOE and NASA, two government agencies, can combine their resources to accomplish fundamental biological research."
ORNL, one of DOE's multiprogram national research and development facilities, is managed by Lockheed Martin Energy Systems, which also manages the Oak Ridge K-25 Site and the Oak Ridge Y-12 Plant.
You can learn more about this research and many other exciting projects by visiting ORNL Oct. 21 during its Community Day event. Many of the lab's facilities will be open to the public that day. For additional information, call ORNL Public Affairs, 865-574-4160.