In a car, when the radiator springs a leak, the car engine heats up dangerously. You stop the car, fill the radiator with coolant, and drive the "sick" car to the repair shop to plug the leak or replace the radiator. The leaking radiator is like an altered, or mutated, gene. By learning that a defective radiator can make the car dangerously hot, you find out that a properly functioning radiator keeps the car engine cool enough to ensure normal operation.
Similarly, ORNL biologists "break" genes in mice to find out their normal roles in a healthy mouse. "We induce mutations in mouse genes and study the resulting disease state in mice so we can determine what the normal versions of the genes do in the body," says Ed Michaud, a senior research biologist in ORNL's Life Sciences Division (LSD). One way that mutations are created is to expose male mice to ethylnitrosourea (ENU), a powerful chemical mutagen discovered by ORNL's Bill Russell in 1979 that alters a single base pair in a gene. Another way is to use recombinant DNA technology or ENU to alter a gene in embryonic stem cells later inserted into mouse embryos. Or an altered gene can be inserted into a fertilized egg, which is implanted in a female mouse and brought to birth as a "transgenic" mouse.
"Because genes operate in complex interacting networks, the mutation of one gene often results in an alteration of other genes in the same network," Michaud says. "Therefore, we are interested not only in determining the functions of individual genes but also how these genes interact with each other and with the environment."
Michaud and other ORNL biologists are studying complex biological systems in mice in collaboration with ORNL researchers using microarrays, mass spectrometers, and bioinformaticsthe discipline in which large amounts of data are sorted into intuitive databases, analyzed, and presented in an understandable form. The first complex biological system he has focused on is the network of genes that affect the development and functioning of the skin. In 1992 Michaud and fellow ORNL biologists identified and cloned the mouse agouti gene, which plays a role in the development of skin and hair pigment. He subsequently identified and cloned mutant forms of the agouti gene that cause obesity, diabetes, and skin cancer in the mouse. The mouse agouti gene has a counterpart in the human genome.
Mice and humans each have some 35,000 genes. These genes are distributed among chromosomes, which are long strands of DNA packed in the nucleus of each cell whose job is to determine and transmit hereditary characteristics. The human has 23 pairs of chromosomes and the mouse has 20 pairs of chromosomes.
"We are now interested in genes scattered among all chromosomes of the mouse genome that affect skin," Michaud says. "The skin is a highly metabolic organ with the largest surface area in the body. The skin has many important protective and defensive functions, such as regulating water loss and body temperature and defending the body against chemical and biological agents in the environment. Because the skin comes into direct contact with the external environment, it is ideally suited for studying genetic and environmental interactions. Mutations affecting the skin are also easy to observe and to study throughout the life of the mouse.
"We look at how disease affects animals under different conditions. We are interested in determining how certain genetic mutations make animals more sensitive to environmental toxins. The Department of Energy is interested in the effects of environmental exposures on mice because mice and humans have a similar genetic makeup, and the information from mice can be used to better understand human health risks."
Michaud and his colleagues are studying an ORNL mouse mutant that was born with a disease called epidermal dysplasia. Mice with this disease lose their hair, have thickened skin, and are more susceptible to getting skin cancer, observed as tumors on the skin.
"We are interested in seeing which genes are altered in mice with epidermal dysplasia because that information will then point to the normal role that these genes play in the development and functioning of the skin," Michaud says. "Like all organs in the body, the health of the skin is dependent upon the well-orchestrated interactions between complex networks of genes and exposure to numerous environmental variables."
Michaud gives an example of a genetic network. "Suppose that gene A regulates genes B, C, and D," he says. "What happens to these three genes if A is knocked out or mutated?" It's a little like making a hole in the oil pan of a car and then driving it. Eventually, the lack of lubrication of all the mechanical parts will cause the car to grind to a halt. The car breaks down because the motor seizes up, but the hole in the oil pan is the main problem. Determining these types of interactions between genes and the environment gives Michaud and colleagues a better understanding of how the skin protects the body.
Michaud suspects that epidermal dysplasia in the ORNL mice is due partly to a mutated transcription factor gene, whose job is to regulate the function of many other genes. To find out what genes this transcription factor is responsible for, he and his colleagues had to determine which genes are turned on normally when the transcription factor gene is working. Many of these genes would likely have a known function in the skin, such as DNA repair, cell growth, cell differentiation, and programmed cell death. If the transcription factor is knocked out, the expression of some of those genes will be altered.
To obtain this information, mouse geneticists Brynn Jones, Bem Culiat, and Ed Michaud turned to Mitch Doktycz, Peter Hoyt, and their colleagues in LSD who design microarrays, or gene chips, and use them to perform analyses of gene expression patterns (See Gene Chip Engineers.) A microarray allows a comparison between genes expressed by a normal organism and genes expressed by a mutant organism or an organism exposed to an environmental toxin. When a gene is turned on, a specific DNA segment corresponding to this gene is copied into a messenger RNA (mRNA) molecule, which is chemically very similar to DNA. This shorter-lived RNA copy moves from the cell's nucleus to the cytoplasm where its codeits sequence of DNA bases, or nucleotidesis translated, causing specific amino acids to be strung together in a specific order to form proteins.
To determine which genes are being expressed at any given time for an important cellular activity, scientists collect the mRNA molecules transcribed in a cell or tissue at that moment. In the laboratory, those RNA messages are reverse transcribed to form more stable complementary DNA (cDNA) molecules. These cDNA samples are prepared from tissues in which biologists want to examine differences in gene expression, such as in skin from mutant mice and normal mice. To detect changes in gene expression, the two cDNA samples are labeled with fluorescent dyes (one for each sample) and then allowed to bind with their complementary DNA templates on a gene chip. The gene chips are glass microscope slides that are spotted with DNA sequences from many hundreds or thousands of different genes in an orderly array. The slides are then exposed to laser light of two different wavelengths and the ratio of fluorescent intensities is measured for each gene. If a gene on the chip is expressed at a high level in the skin of the mutant mouse compared with the normal mouse, the dye used for the mutant sample will shine brighter than the dye used for the normal sample, and this difference can be quantified.
Computers are used to keep track of information for each gene on the microarray. Scientists need to know many variables related to the microarray experiments, including the location of each gene on the array, the DNA sequence and identity of each gene, the mouse tissue that the labeled samples came from, the hybridization conditions, the fluorescent ratios for each gene, and the analysis of the data. At ORNL researchers in LSD's Computational Biology SectionJay Snoddy, Denise Schmoyer, and Sergey Petrovare writing the computer programs to handle the data as part of the development of ORNL's Genosensor Information Management System (GIMS).
For the analysis of genes from normal mice and mouse models of skin and hair disease, Jones, Culiat, and Michaud designed microarrays containing about 500 mouse genes that were arrayed in triplicate on glass slides. These gene chips were used to determine which genes in the skin of the mice with epidermal dysplasia have altered expression.
As a result of this work, these ORNL investigators made some important discoveries. "We found 30 different genes with altered expressionthat is, the levels of mRNAs produced by these genes were different in the mutant mice from those in the normal mice," Michaud says. "We found six altered keratin genes, which are responsible for scaffolding in the skin. We also found four programmed cell death genes and two genes involved in the handling of calcium that were altered in the mutant mouse. These genes are important in the normal development and renewal of the skin."
Several other genes had unknown functions, but the gene chip study indicates that these genes play a role in skin and hair production and function. More biology experiments are being done to verify that these genes have a function related to skin and hair.
Michaud noted that the mice may now be used to determine the effects of various environmental agents on gene networks in the skin. "The skin often protects us from low doses of environmental agents such as ultraviolet radiation, microbes, and chemicals," he says. "Mice with mutations in skin genes, or humans with natural genetic variation, may be at increased risk from these same exposures, and the gene chips can help us to uncover the relevant genes."
The structure of the proteins produced by the expressed genesboth the normal and mutant onesis determined by LSD's Gerry Bunick using X-ray crystallography. Doktycz's group is also planning to develop a protein microarray to pinpoint protein-DNA interactions.
"In our study of complex systems biology, we are focusing on genetic variation in the skin and increased susceptibility to disease from environmental exposures," Michaud says, "but this approach for determining which genes are interacting can be applied to the study of any organ, ranging from the heart to the pancreas to the nervous system and brain. For example, we have a collaboration with Russ Knapp, leader of the Nuclear Medicine Group in LSD, in which we are examining gene expression patterns in diabetic mice treated with new insulin-sensitizing drugs."
An important goal of complex systems biology is to understand the functions and interactions of the estimated 35,000 human genes, of which very few are known. Fortunately, in this post-genomic era, new analytical tools, including mutagenesis techniques, gene chips, and supercomputers, are available to help scientists find meaning in the rough drafts of DNA sequences that have been completed for the mouse and human.
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