Growing Crystals in Space|
Mice and Kidney Disease
Mouse Mutants and Drugs
Rhenium and Cancer
New Light on Photosynthesis
Re-engineering a Plant Enzyme
Mass Spectrometry and Biomolecules
Cystic Fibrosis Gene Screening
nergy heats, powers, and lights our homes and businesses, but it has a dark side. By-products of its development, production, and use can include radiation, potentially toxic chemical emissions, and greenhouse gases. For more than 50 years, ORNL researchers have studied the effects of these by-products on the health of present and future generations of living organisms. In studying biological processes, we have acquired basic knowledge that has been used in human medical treatment and in monitoring and restoring the environment.
Our investigations of the mammalian genome, especially mutations in mice, have helped scientists establish human radiation-exposure limits, find safer treatments for cancer, understand genetic disorders, and design mouse models for a variety of human diseases. Through genetic engineering, researchers are systematically manipulating (1) the genome of mice to locate genes responsible for abnormal and normal functions, and (2) protein structure of an essential life-giving plant enzyme as an avenue for increasing crop yields and decreasing atmospheric carbon dioxide, a greenhouse gas. For the future, we will focus on gaining a thorough understanding of gene-protein function in the search for medical solutions to prevent and treat disease and improve the nation's health.
Growing DNA-Protein Crystals in Space
On October 20, 1995, for the first time an ORNL experiment blasted off aboard the U.S. Space Shuttle Columbia, although the scheduled liftoff was September 28. The idea of the experiment was to grow near-perfect crystals of chromosome building blocks called nucleosomes by capitalizing on the absence of gravity in outer space. It is believed such crystals will grow more slowly, making them defect-free, solid instead of hollow, more uniformly shaped, and larger than nucleosome crystals grown on earth.
Gerard Bunick observes nucleosome crystals in a device designed for growing crystals in space. His DNA-protein crystals were grown in zero gravity on the orbiting Russian Mir space laboratory. Photograph by Tom Cerniglio
Because of the launch delay, most ORNL crystals had already started growing in special vials in earth's gravity. Fortunately, however, a single crystal in a highly acidic environment did not begin important growth until after the shuttle launch. Because it nucleated in space, it came back looking chunky, like a piece of thick chalk, while the other crystals resemble pencils.
The two-week experiment was regarded as a shakedown for the five-month-long crystal-growing experiments on the Russian Mir space station. These experiments were started after March 22, 1996, when the U.S. Space Shuttle Atlantis docked with the orbiting space station. From the 1995 experience the researchers learned about problems in the crystal-growing chambers, so the chambers were redesigned and new ones were built for the 1996 experiments.
Researchers from ORNL's Biology Division and other labs will peer into these near-perfect crystals using X-ray diffraction tools at ORNL and the National Synchrotron Light Source at DOE's Brookhaven National Laboratory. To protect the crystals from the damaging radiation of X rays so they may be adequately studied, ORNL researchers perfected "flash cooling," using liquid nitrogen, which works well with small crystals; they are now developing the technique to make it work for large crystals.
The goal is to obtain a detailed picture of the nucleosome's protein core and the DNA surrounding it. The nucleosome is the fundamental building block of the chromosome, a unit in a cell's nucleus that carries strands of DNA, genetic material that determines an organism's characteristics. Careful selection of the DNA base sequence in the nucleosomes allows the growth of highly ordered crystals, enabling a better view of the arrangement of atoms. The information may help scientists decipher the three-dimensional structure of a chromosome in the quest to unravel the genetic code. It is hoped that information from outer space will make understanding of earth-bound chromosomes closer to crystal clear.
Funding for this research is provided by DOE's Office of Health and Environmental Research, the National Institutes of Health, and the National Aeronautics and Space Administration.
Rescuing Mice from Kidney Disease
ORNL geneticists are on a search-and-rescue mission. They are searching for genes in mice that cause diseases like those that afflict humans. Part of their search involves rescuing the mice born with a specific disease to trace the effects of defective genes.
Take polycystic kidney disease (PKD), a condition that can cause high blood pressure, infections, and premature death in children. The genetic disease is known to cause kidney and liver lesions that are linked to localized cell proliferation. Because kidney lesions can lead to organ failure and death, children suffering from PKD are often given kidney transplants. Although kidney transplants markedly improve longevity, it's uncertain whether PKD patients might develop and die prematurely from complications associated with liver lesions.
Rick Woychik (left) and Brad Yoder examine a mouse whose kidney they rescued from polycystic kidney disease. Photograph by Tom Cerniglio.
Using genetic engineering, we produced mice born with PKD and later rescued the kidney but not the liver from the destructive effects of the disease. To produce the PKD mutation in mice, we inserted a cloned fragment of DNA into a gene to disrupt its expression. This "insertional mutagenesis" allowed us to locate, clone, and characterize the corresponding normal version of the gene that was not functioning properly in the mutant animals. Then, we reintroduced a specially modified version of the cloned gene into the mutant animals. This version rescued the kidney, but the liver lesion remained.
In the meantime, we are letting these partially cured mice age to see if they live the normal lifespan of two years. If they die prematurely, we will autopsy them to determine whether liver cancer or some other condition associated with the liver lesion eventually proves to be fatal. We will not give up the search because many are in need of rescue.
This research was supported by DOE's Office of Health and Environmental Research, Health Effects Research Program, and by the National Institutes of Health.
Mouse Mutants Important for Developing Drugs
Because mice and men possess a high degree of genetic similarity, researchers have used ORNL's unique archive of mouse strains to set radiation standards, assess health risks of chemicals, study developmental abnormalities, and clone defective genes related to human disease. Now, biotechnology firms are interested in using clonable genes from our mouse mutants to develop disease-fighting drugs. The pharmaceutical segment of the biotechnology industry is already grossing billions of dollars in sales from only a few drugs developed by the use of cloned genes.
Under a cooperative research and development agreement, we are now working with Darwin Molecular Corporation to identify mutant mice that have defective immune systems and to clone genes that are responsible for diseases. Human disorders involving malfunctioning immune systems include asthma, cancer, diabetes, multiple sclerosis, rheumatoid arthritis, and systemic lupus. Cloning genes allows us to study their function and identify the origin of immune diseases, while pharmacological manipulation of pathways from gene to disease determines strategies for immunotherapy and drug development.
Lisa Stubbs examines an inhabitant of ORNL's treasured Mouse House. Photograph by Tom Cerniglio.
Mutations are produced in mice by exposing mature male mice to radiation or a mutagenic chemical like acrylamide. The treated males are mated with untreated females. The male progeny are raised to maturity and tested for fertility. We are interested in screening the offspring of treated males that are semi-sterile, that produce half as many young as normal males. The reason: a relatively high proportion of their surviving progeny will carry observable genetic defects. Mutant mice that have immunological dysfunction will die early, develop tumors early, or have skin defects. We send tissue from these mice to Darwin.
Mutations spring from the induction of reciprocal translocations in germ cells (developmental precursors of sperm cells) of treated male mice. Translocations arise from breaks in chromosomes that lead to exchanges in and rearrangements of chromosomal parts. Because genes are located on chromosomes, genes can be broken in reciprocal translocations. When we see a mutation, disease, or defect, we suspect a broken gene. By looking at sizes and positions of stained bands of chromosomes in the light microscope, we can identify rearranged chromosomes and locate the breaksthe starting point for cloning to make copies of defective, or broken, genes. Our unique collection of clonable mouse mutants is a national treasure, and biotechnology companies are eager to help us exploit it.
Funding for our collection of mouse chromosomal mutants and research has come through DOE's Office of Health and Environmental Research and the National Institute of Environmental Health Sciences.
Rhenium Drugs May Treat Cancer Effects
In prostate, lung, and other cancers, the pain can go literally all the way to the bone. As original tumor cells spread, secondary tumors grow in the bone, causing inflammation and exerting excruciating pressure on nerves. Safe, effective, and inexpensive relief may be at hand in the form of a by-product of a tungsten radioisotope from ORNL's High Flux Isotope Reactor.
An agent labeled with rhenium-188 from ORNL's tungsten-188/rhenium-188 generator shows good uptake in a patient's skeletal lesions for treatment of bone pain from metastasized prostate cancer. This study was conducted by the Nuclear Medicine Department of Kent and Canterbury Hospital in England.
Rhenium-188, a tungsten-188 decay product that appears in ORNL-designed, soon-to-be commercialized radioisotope generators, may become the treatment of choice for cancer-induced bone pain. The energy of rhenium's beta radiation reduces inflammation and shrinks tumors, thus relieving bone pain. Rhenium can be attached to phosphorus compounds that tend to concentrate in bones.
This therapy is expected to be considerably less costly than using strontium-89, rhenium-186, and other radioisotopes currently used to treat bone pain. If studies show this prediction to be true, rhenium-188 treatment could help reduce health care costs.
In a 1995 cooperative study by nuclear medicine collaborators at Canterbury and Kent Hospital in Canterbury, England, early tests showed excellent uptake of a rhenium [Re(V)-188 DMSA] complex in skeletal lesions in patients with metastases from prostate and bronchial cancer. Because these initial studies also showed low liver and kidney uptake, therapeutic studies using larger amounts of the rhenium agent are under way to assess the agent's effectiveness in relieving cancer-induced bone pain.
Rhenium also has potential for treating other types of tumors. In a cooperative research and development agreement between ORNL and RhoMed, Inc., of Albuquerque, New Mexico, tumor-targeting agents radiolabeled with therapeutic rhenium radioisotopes are being evaluated for their effectiveness in treating tumors in research animals. The rhenium is attached to peptides, which are formed from amino acids, the building blocks of proteins. These peptides imitate the action of somatostatin, which binds to receptors on cell surfaces (somewhat like a thistle sticking to cloth). Because tumor cells also have receptors for somatostatin, peptides can be used to deliver radioisotopes to tumors like a guided missile carrying a warhead to a target.
Using rhenium-188 from the ORNL generator, RhoMed has developed a simple "kit" for on-demand labeling of its RC-160 peptide for cancer radioimmunotherapy. Peptides radiolabeled with rhenium were injected into mice having experimental tumors grown from implanted human prostate, small-cell carcinoma, and breast cancer cells. The mice were scanned to determine where the radioisotope concentrated, and results showed the tumors retained the radioactivity a long time. In addition, peptides labeled with rhenium-188 achieved a greater reduction of tumors than did rhenium-186 (produced in HFIR), demonstrating greater effectiveness of higher-energy beta particles. While no improvement was observed in untreated animals, the rhenium-188 peptide significantly shrank or eradicated tumors in treated animals.
This method offers a new approach for treatment of tumors in specific tissues, helping ORNL achieve a nuclear medicine goal. Twinkling atoms from ORNL radioisotopes may offer yet another answer to cancer.
The collaborative research with investigators at Canterbury, England, is supported by DOE's Office of Health and Environmental Research. The research with RhoMed, Inc., is supported by funds from OHER through ORNL's Small CRADA Program.
New Light on Photosynthesis
The classic theory of photosynthesis, published in dozens of textbooks, states that two structurally different components of plant membranes react with sunlight to start building plant tissue. That, however, is not the only route to photosynthesis. In studying the molecular mechanism of how green plants convert light energy into chemical energy, we found that photosynthesis can be performed with only one light reaction; two light reactions (sunlight reacting with two plant components) are not necessarily required, as the standard model predicts. When exposed to sunlight, one of these components (Photosystem II) can do it allsplit water molecules to get electrons to take up airborne carbon dioxide, providing energy for plant tissue synthesis and oxygen for us. It had been thought that Photosystem I also was required to convert atmospheric carbon dioxide to plant tissue.
It has long been assumed that green plants evolved from photosynthetic bacteria similar to those existing today. One bacterial type, which contains iron and sulfur, is similar in structure to Photosystem I; another type, which resembles an iron-free hemoglobin, is similar to Photosystem II. But neither acts like Photosystem II, which uniquely breaks down water to produce the oxygen that sustains the animal kingdom.
However, we found that a mutant algal strain, possessing Photosystem II only, may be a model for the "missing link" of photosynthesis. At high light intensities, our mutant performs photosynthesis more stably under anaerobic conditions than in an environment containing oxygen. This is a clue to the missing link because it is generally believed that the earth's primordial atmosphere did not contain as much oxygen as today's atmosphere. One of our theories is that the hemoglobin-like group of bacteria initially developed an oxygen-producing, water-splitting capability. However, when oxygen became a major component (21%) of the earth's atmosphere by cumulative photosynthesis, this "missing" strain added a second light reaction. According to our theory, photosynthesis evolved into a two- rather than a one-light-reaction process to protect against the combined effects of high light intensities and high oxygen concentrations.
|This depiction of two plant membranes (circles) shows that photosynthesis can occur with one light reaction, rather than the two explained by the classic theory of photosynthesis.|
Our discovery (published in the August 3, 1995, issue of Nature) suggests that the maximum thermodynamic conversion efficiency of light energy into chemical energy potentially can be doubled from about 10% to 20%a gain that could dramatically improve the productivity of food crops and biomass energy plantations, if the plants can be made to use all levels of solar energy. If that happens, the textbooks will have to be revised again.
The research was supported by DOE's Offices of Basic Energy Sciences and Energy Efficiency and Renewable Energy, the Pittsburgh Energy Technology Center, and the National Science Foundation.
Re-engineering a Plant Enzyme
If the world's most abundant protein, Rubisco, could be re-engineered, growth rates of crops and forests could double, providing more food and fuel. Lush biomass would take more carbon dioxide (CO2) from the air, controlling the greenhouse effect.
Since the 1970s, we have focused on understanding Rubisco, the CO2-fixation enzyme present in all photosynthetic organisms. It catalyzes the initial reaction (a carboxylation) necessary to convert atmospheric CO2 into carbohydrates and other complex biomolecules needed to sustain plant life. Unfortunately, by a quirk of nature, Rubisco can mistake the much more abundant atmospheric oxygen (O2) for CO2. The ensuing oxygenation reaction leads to degradation, rather than biosynthesis, of carbohydrates. As a result, energy is wasted and plant growth is restricted.
We have genetically redesigned Rubisco to learn how it distinguishes CO2 from O2 and how it catalyzes the two competing reactions. Our recent successes using protein engineering suggest the feasibility of altering the Rubisco enzyme to improve its efficiency.
A key to improving the efficiency of the Rubisco plant enzyme may be to alter flexible loops in its structure (shown in this computer graphics representation) that are crucial to stabilization of reaction intermediates and their processing to normal reaction products. Models are based on atomic coordinates of the activated enzyme from tobacco (Schreuder et al., 1993, Proc. Natl. Acad. Sci. USA 90, 9968-9972) and are displayed using the program MOLSCRIPT (Kraulis, 1991, J. Appl. Crystallogr. 24, 946950).
The sequence of chemical bases in a gene instructs cells to assemble a protein from specific building blocks (20 different amino acids) in a certain order. Protein engineering involves manipulating a gene's base sequence to systematically alter a protein's amino acid sequence.
Through protein engineering, we instructed cells to produce an altered Rubisco in which one of the enzyme's 475 amino acid residues was replaced with a different amino acid. Then using protein chemistry, we modified that substitute amino acid to make it more like the original onea modest structural change. Our tests showed that the novel Rubisco displays an altered ratio of carboxylation-oxygenation activity.
We also found a possible mechanism for crippling the enzyme's oxygen activity and enhancing its carboxylation. In many enzyme-catalyzed reactions, unstable chemical intermediates are formed along the road to final products. We identified mobile Rubisco "loops" that lock these intermediates into place so they don't decompose into nonproductive side products. If the protein's structure could be altered to selectively destabilize intermediates of oxygenation, this undesirable reaction might be undermined.
Our studies of Rubisco have greatly enhanced understanding of enzyme mechanisms in general. Because enzymes underpin all chemical reactions in all living organisms and are also the target of most therapeutic drugs, our research is benefiting society beyond the goal of attaining the biomass yield of dreams.
The research has been supported by DOE, Office of Health and Environmental Research.
Mass Spectrometry Analyzes Biological Molecules
Once used only for lightweight, volatile molecules, mass spectrometry is becoming a heavyweight among tools that analyze bulky biological molecules. Recent revolutionary changes in technologies that introduce and sort molecules allow elucidation of the molecular weight, structural characteristics, and chemical-base sequences of heavier molecules such as DNA and proteins. ORNL has been one of the world's leaders in exploiting these remarkably flexible technologies for biological research. Through our research, we better understand how they work and how to adapt them to an ever-increasing range of analytical problems.
|Bob Hettich and Michelle Buchanan prepare to analyze modified DNA oligomers on ORNL's 3-tesla Fourier transform ion cyclotron resonance (FTICR) mass spectrometer. A 7-tesla instrument that was to be installed in mid-1996 will substantially enhance achievable mass range and mass resolution for analysis of biomolecules. Photograph by Curtis Boles|
One new technique for introducing ions to the spectrometer is electrospray ionization (ES). It overcomes the problem of liberating nonvolatile biological molecules from solutions for study as a gas by mass spectrometry. In ES, after flowing through a thin capillary, biological molecules dissolved in a solvent are sprayed into a fine mist. These droplets are pulled through the pinhole entrance of a quadrupole ion trap mass spectrometer. There ions emerge from the droplets as naked gaseous ions that are detected. Our research showed that the ES process works like an electrochemical cell, suggesting ways to expand the ionization technique's range of applicability. Using a quadrupole ion trap, we have also developed new ways to probe the structure of trapped ES-generated gaseous biomolecules, including novel ion-ion reactions.
Another technique for generating ions from high-mass biological molecules is matrix-assisted laser desorption ionization (MALDI). Here, compounds such as DNA oligomers are mixed with a chemical (matrix) that absorbs ultraviolet laser light. In the mass spectrometer, a laser is used to vaporize the matrix molecules, which carry the ionized biomolecules into the mass analyzer.
At ORNL, two types of mass analyzers detect MALDI-generated ionstime-of-flight (TOF) and Fourier transform ion cyclotron resonance (FTICR) mass spectrometers. Our current FTICR mass spectrometer, which has a 3-tesla superconducting magnet, introduces ions at one end by MALDI and at the other by ES ionization. We are expecting delivery soon of a 7-tesla magnet, which has increased mass range and performance.
We have recently developed ways to detect amplified DNA for use in molecular diagnostics. Using mass spectrometry, we have distinguished among nucleic acids of different sizes, making it an important tool in diagnosing disease, analyzing bacteria in the environment, screening consumer products for contamination, and characterizing evidence that could be used in court.
Using MALDI TOF, we have identified a segment of DNA that is specific for the bacterium that causes Legionnaire's disease. We have also developed the capability to detect DNA fragments with a three-base deletion, characteristic of individuals who have cystic fibrosis. Evaluation of this capability has shown it can be used to screen individuals for cystic fibrosis.
We continue to explore new ways to detect and structurally analyze smaller quantities of biological molecules at higher sensitivities, while minimizing the need for extensive sample preparation. With these advances, mass spectrometry is emerging as a standout among tools for analyzing complex biological molecules.
DOE's Offices of Basic Energy Sciences and Health and Environmental Research and ORNL's Laboratory Directed Research and Development Fund supported this work.
New Technique for Cystic Fibrosis Gene Screening
Cystic fibrosis (CF) is an inherited, fatal disease in which mucus buildup promotes digestive disorders and bacterial infections in the lungs. CF patients must take heavy doses of antibiotics and often die young.
Because each person with CF is the child of parents who both carry defective forms (alleles) of a particular gene, there is interest in large-scale screening to let people know their chances of having a child with CF.
We have developed a new technique that could be used to rapidly screen many people for a specific defect in a gene on chromosome 7 that causes 70% of all CF cases. The defect is the absence of three base pairs of DNA in both alleleles that control production of CFTR, a protein that prevents mucus buildup. CF carriers have a single defective allele that may be passed on to their offspring. CF patients have two defective alleles.
Steve Allman (left) and Kai Tang are part of the team that developed the laser mass spectrometer for detection of genetic defects that can lead to cystic fibrosis.
Our CF gene screening technique uses laser mass spectrometry, marking the first time that mass spectometry has been used to diagnose a genetic disease by DNA analysis. The technique screens in minutes, not hours, making it ten times faster than conventional gel electrophoresis. Also, it does not use toxic chemical or radioactive materials, which require costly methods of disposal.
For the experiment, researchers from ORNL and the University of Tennessee Medical Center (UTMC) extracted DNA from human hair samples and isolated and copied millions of times the part of each CF gene that would contain the known defect if present.
After receiving 30 droplet samples for a blind test, the researchers mixed each DNA segment with a chemical that absorbs ultraviolet laser light and vaporized the mixture with a laser. The electrically charged DNA segments formed in the vapor were separated by size in a time-of-flight mass spectrometer; the defective alleles that lack three DNA base pairs are smaller and lighter and, therefore, travel faster than the heavier, normal gene segments. ORNL's identifications of the 30 samples agreed completely with the results of conventional analyses, showing the technique was 100% accurate in distinguishing between normal and defective CF genes.
The research was supported by ORNL's Laboratory Directed Research & Development Fund and by DOE's Office of Health and Environmental Research.