RODENTS and people may not appear to be closely related, but consider this the next time you look in a mirror: the genes of human beings and mice are 85 percent identical. This similarity is one of the reasons Lawrence Livermore scientists are studying mice. At Livermore's Human Genome Center, biomedical scientist Lisa Stubbs is leading a team that is studying the mouse genome to better understand the functions of human genes. Comparative genomics-analyzing and comparing the genetic material of different species-is viewed by bioscientists as an important method for studying evolution, the functions of genes (what they do and why), and inherited diseases.

Hunting Down Damaged Chromosomes
The mice used by Stubbs and her team were initially bred at the experimental mouse genetics facility at the Oak Ridge National Laboratory, one of the largest of such facilities in the world. It was originally established to conduct genetic risk assessment and toxicology studies. The mice brought to Livermore comprised some 130 unstudied mouse family lines, descendants of mice that were exposed years ago to radiation or chemicals for the purpose of studying the genetic effects of these agents. The offspring of the original mice were normal, although they were carriers of a mutation. But some of the descendants of those offspring, inheriting two copies of a mutated gene, showed anomalies (phenotypes) that are visible signs of the genetic mutations. Stubbs brought the carrier mice to Livermore in 1997 to identify inherited traits associated with the mouse lines and to find the genes disturbed by each mutation. "We look for phenotypes such as deafness, movement disorders, limb deformities, obesity, and susceptibilities to cancer," says Stubbs. If the trait is passed down to successive generations of a single mouse line, the team knows it is genetic.






 To find out which gene is responsible, a researcher takes a small snip of skin from a mouse tail and grows the skin cells in a petri dish. Chromosomes from those cells are then spread on a microscope slide. In the laboratory, the researchers look for one particular kind of mutation called a translocation, which involves obvious changes in chromosome structure. Because the chromosomes are visibly disrupted, researchers can easily map the position of the mutated gene using only a simple light microscope. For example, the figure at right shows two mouse chromosomes-2 and 14-where such a translocation has occurred. "We knew immediately that the gene responsible for the trait would be found on one of those two chromosomes," Stubbs explains.
The researchers then use a procedure called fluorescent in situ hybridization (FISH), a technique for painting chromosomes with a fluorescent dye, to pinpoint the gene's location. They label a gene from a normal chromosome 2 with the fluorescent dye and add it to a slide containing the mutant mouse chromosomes. The labeled gene probe recognizes DNA sequences spread over the slide that are identical to its own and binds to the chromosome at that site. "In this way," Stubbs says, "we can map any gene relative to the translocation and `trap' the mutated gene in a small, well-defined region."
The researchers repeat the process for other genes on chromosome 2 until they have narrowed down the "breakpoints," that is, the end pieces of the two broken and rejoined chromosome segments. Once they zero in on the chromosome section involved, they search the DNA sequence of that region to identify the genes that have been disrupted by the chromosome break.
"By the time we go to the DNA sequence and begin searching out individual genes," Stubbs says, "we know exactly which spot on the chromosome we must deal with." She continues, "When an organism is exposed to radiation or chemicals, we don't know ahead of time which genes will be affected. It's random, like potluck. All genes `do something,' but some genes are less important than others-their activities affect our development or our health in very subtle ways. If such genes are mutated, we see no visible effects. Others genes, such as those that predispose someone to cancer or obesity, are essential, and their mutations have very obvious impact. All of our mice have mutations. But by focusing specifically on those with visible abnormalities, we are aiming at those genes that play the most important roles in maintaining health."


Of Human Genes and the Department of Energy

Humans have 23 pairs of chromosomes that are made up of DNA (deoxyribonucleic acid), chemical characters arrayed in a particular order in a chain. The chromosomes contain the three billion characters that make up the human genome.
Sequencing is the work of determining the exact order of four individual chemical building blocks that form DNA. These four chemical bases-commonly abbreviated as A, G, C, and T-bind together to create base pairs of DNA molecules. After researchers sequence a piece of DNA, they search for the special strings of sequences that form genes.
The Department of Energy's Joint Genome Institute (JGI) combines the work of Lawrence Livermore, Lawrence Berkeley, and Los Alamos national laboratories. JGI operates around the clock as researchers work to determine the sequence of the information-carrying units that comprise the DNA of three human chromosomes-5, 16, and 19-as part of the international effort to decipher the human genetic code. The purpose is to understand the genetic basis of life. This understanding, in turn, will enable us to understand and attack the root causes of hereditary disorders and susceptibility to diseases such as cancer, heart disease, stroke, diabetes, schizophrenia, Alzheimer's disease, and many others. Because comparison to genomes of other, distant species such as the mouse aids in the discovery and analysis of genes embedded in the human sequence, the JGI will also contribute significantly to sequencing of the mouse genome. That sequencing is part of an international effort slated to proceed in earnest as the human sequence nears completion.

Tracking a Cancer Gene
One case the team researched involves a particular kind of stomach cancer known as adenocarcinoma. In some cases, this cancer begins with the Helicobacter pylori bacteria commonly found in human stomachs. Although many people carry these bacteria, only some develop chronic gastritis-a painful inflammation of the stomach lining. If the bacterial infection is left untreated, many of the infected will develop a low-grade stomach lymphoma, which can progress with time. A small but significant percentage of lymphoma patients will eventually develop adenocarcinoma if the bacterial infection persists long enough. "We know," says Stubbs, "that whether or not one develops gastritis and cancer is determined at least in part by genetic predisposition. However, no one knows which genes encode these predisposing factors."
The stomach cancer issue arose when Stubbs identified a family of mice susceptible to gastritis, lymphoma, and adenocarcinoma. "This mouse mutant family develops a disease that looks precisely like what has been documented in susceptible, Helicobacter-infected people," says Stubbs. "These mice therefore provide a unique model for tracing the elusive genetic susceptibility factors."
The first thing the researchers did was to raise this mouse family in an environment without Helicobacter or other known pathogenic bacteria. "Although mice that have been exposed to bacteria quickly develop lymphoma," she says, "these pathogen-free animals did not. However, they did exhibit precancerous changes in cells of the stomach lining. We are observing some of those animals over time to see if the precancerous changes will progress to adenocarcinoma. Our hypothesis is that the genetic defect carried by these mice makes them susceptible to infection. But because they develop precancerous lesions without bacteria, the mutation must also mimic the effects of bacterial infection in some unknown but important way."
Using the FISH technique, they tracked down a mutation in a gene that produces a component of the mucus normally coating the intestinal lining. The mutant mice turn this gene on in the wrong location-the stomach-and have the properties of their stomach linings altered in significant ways. "It may be," says Stubbs, "that people who have a defect in the corresponding human gene may also have a higher susceptibility to this kind of stomach cancer. We hope to verify this possibility in future research."



Similar but Different

Imagine taking human chromosomes, shattering them into pieces of varying lengths, and putting them back together in a different order. "That's what mouse chromosomes look like," says Lisa Stubbs, a Lawrence Livermore bioresearcher. For instance, as the figure shows, almost all of the long arm of human chromosome 19 is related to mouse chromosome 7. That is, the two chromosomal regions contain human and mouse versions of the same genes, organized roughly in the same order. In contrast, the short arm of human chromosome 19 comprises nine segments, each containing 20 to 100 genes and corresponding to different mouse chromosomes. Despite this scrambling of the genetic material, gene content and order within each mouse and human segment mirrors the other quite closely. Humans have this genetic material contained in 23 pairs of chromosomes, whereas mice have 20 pairs. But this difference reflects organization of genes and not their relative numbers
Humans and mice also have about the same number of genes-now estimated to be approximately 140,000-and with some exceptions, each human gene has a clear and quite similar counterpart in the mouse. Those rare exceptions may prove to be quite important to the differences between humans and mice and must be understood more fully. For example, mice have some members of the cytochrome gene family that encode proteins needed to metabolize toxins, which humans do not possess. This genetic difference is reflected in the fact that mice and humans deal with certain toxins differently. Likewise, humans carry a gene encoding a protein called Apo(a) that plays an important role in developing atherosclerosis. Normal mice do not have the gene and never exhibit the symptoms of this deadly cardiovascular disease.
However, these species-specific genes altogether account for roughly 1 percent or less of the two gene sets and do not determine all the differences between humans and mice. The major differences between the species arise from the wide variation in the coding sequences of the counterpart genes. When the average 15 percent difference in mouse and human protein coding sequences is multiplied by 140,000 genes, the overall genetic difference is quite significant.
Not all genes are indispensable, and many of the differences found when mouse and human genes are compared have little effect on our biology. Many living organisms, including humans, carry single-gene changes and chromosomal defects such as translocations (swapped bits) and deletions (missing bits). These changes can mean nothing, or everything. "Consider," says Stubbs, "that because of the large coding capacity and complexity of the genome, a mere 15 percent difference in genes gives you a completely different organism-a human instead of a mouse. But turning the tables around, it is also quite remarkable that humans and mice are as genetically similar as we actually are."

For the Future: Playing Off the Strengths
On the one hand, much less sequencing has been completed for the mouse than for the human genome. On the other hand, a wealth of knowledge exists regarding the inheritance of genetic traits in mice.
Every family has some undesirable hereditary trait-whether near-sightedness, obesity, asthma, allergies, high cholesterol, or other ailments. In such cases, says Stubbs, it's almost impossible to see the pertinent genetic signal over the general noise of random-and often unimportant-changes that distinguish our highly varied genomes. Humans are a heterogeneous population with a huge amount of genetic variation-some important, some not. Which change is the one that accounts for the asthma, the obesity? "Mutant mice will help us find out," says Stubbs. "We can study groups of mice that are genetically identical, like identical twins, except that one mouse carries a mutation in a single, unknown gene. Because the background is stable, we can follow the activities of that single gene with good certainty. We study the pathology of the mutant mouse, track down the gene involved, and then identify its human counterpart to study whether that gene plays a role in human diseases with similar pathology [see box above for comparison of mouse and human genes]. Knowing which gene is responsible for a health disorder is the first step toward designing treatments to alleviate the condition."
The mouse is a powerful genetic model that is being used worldwide for gene-function studies. As new information about human genes is developed at the Department of Energy's Joint Genome Institute and through the international sequencing effort, there will be more and more new genes to study and understand. And as more and more health-related genes-such as those for obesity, deafness, and developmental disorders-are discovered first in the mouse, the power of studying a human gene through its mouse counterpart is increasingly obvious. Stubbs sums it up this way: "It's very exciting to be working at the center of the next wave of genome research."
-Ann Parker

Key Words: adenocarcinoma, chromosome, comparative genetics, DNA, fluorescent in situ hybridization (FISH), gene, Human Genome Center, Joint Genome Institute, translocation.

For further information contact Lisa Stubbs (925) 422-8473 (stubbs5@llnl.gov).

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