Volume 5, Issue 39 November 2008
Doing the DNA Shuffle
Mark Schlissel is Dean of Biological Sciences in the College of Letters and Science and a professor of immunology and pathogenesis. Image credit: UC Berkeley
In the battle against infection, the odds look stacked against us. We rely on antibodies to recognize and sound the alarm on potential invaders. Yet our cells come programmed with less than 30,000 genes, far fewer than the billions of foreign structures we might encounter.
Even so, says Mark Schlissel, a Berkeley professor of immunology and pathogenesis, "the immune system is capable of recognizing literally hundreds of millions of foreign chemical structures."
This phenomenal flexibility comes courtesy of a remarkable DNA shuffling system called V(D)J recombination. Just as riffling a deck of cards can produce an endless variety of poker hands, shuffling specialized DNA segments in developing immune cells can produce a different antibody structure nearly every time. During the reaction, enzymes select one of many available genetic versions for each variable antibody segment, snip out the unused portions, and stitch the chosen pieces back together. The resulting antibody travels to the surface of the immune cell, or lymphocyte, where it can recognize bacteria, viruses and toxins in the bloodstream.
The immune system produces a staggering variety of different antibodies using a process called V(D)J recombination. VH, DH, and JH gene segments are rearranged during lymphocyte development to generate new DNA blueprints for each antibody. Image credit: Mark Schlissel
"All of us have developing lymphocytes in our bone marrow shuffling these antibody genes around continuously, from the time we are in the womb," Schlissel says. Schlissel studies V(D)J recombination and its place in lymphocyte development. Understanding when this reaction occurs and how it is regulated will help scientists learn to treat leukemia, lymphoma, immune deficiencies and a wide array of autoimmune diseases.
V(D)J recombination occurs in two types of lymphocytes, B cells and T cells. The genes modified in each cell type are different, but the enzymes involved are the same. How does the recombinase determine which genes to work on in each cell? In a pivotal experiment, Schlissel isolated nuclei from B cells and T cells, and added recombinase enzymes to each. The enzymes rearranged T cell genes when given T cell nuclei, and B cell genes when given B cell nuclei. This indicated that the enzymes were responding to differences in the accessibility of various genes within the cells' DNA. Subsequent experiments have implicated structural variations in how the DNA is packaged into a structure called chromatin in different types of cells. Schlissel is fleshing out how these modifications affect recombinase behavior and influence the fate of a nascent lymphocyte.
The AMuLV virus infects early B cell precursors, arresting their development and causing leukemia. Inset: Treating leukemic cells with the chemotherapeutic agent Gleevec turns B cell recombinase genes back on, allowing the cells to mature. Image credit: Mark Schlissel
Gene shuffling may be an elegant solution to a thorny biological problem, but it also carries risks. "Many leukemias are caused by errors in this shuffling mechanism," Schlissel says. "Instead of shuffling two antibody genes together, they'll mistakenly take a potential cancer gene and shuffle it next to a piece of antibody gene."
To understand what can go wrong, Schlissel employs a virus that causes leukemia in mice. The virus has a gene that turns recombinase enzyme genes off, at the same time making lymphocytes malignant. Schlissel has not only identified the lymphocyte genes affected by the virus, but discovered that this mechanism contributes to how a chemotherapy drug for leukemia called Gleevec operates. "More than 90 percent of people with chronic myelogenous leukemia have a chromosomal translocation that involves this same gene," Schlissel says.
Every shuffle of DNA segments also carries the danger of producing an antibody that recognizes the body's own tissues. To avoid such potential autoimmune reactions, every new antibody undergoes self-tolerance testing in the bone marrow. If the antibody flunks, the recombinase returns to the nucleus for another round of gene rearrangement. Schlissel is tracking down the cellular signals that regulate this process. "There's a signal that notifies the nucleus to keep making recombination enzymes because a bad antibody has been made, as well as a signal that says you did a good job, shut off the system. We're tracking down both," Schlissel says.
Schlissel demonstrated that recombination enzymes recognize antibody genes in B cell nuclei and T cell receptor genes in T cell nuclei but both types of genes in purified DNA. Arrows indicate evidence of gene rearrangement where B=B cell nuclei, T = T cell nuclei, N = skin cell nuclei, and gen. DNA = purified DNA. Image credit: Mark Schlissel
Schlissel is now poised to begin a very different avenue in his career. This fall, he began a five-year appointment as Dean of Biological Sciences in the College of Letters and Science. As dean, he says, "I feel I can contribute to scientific success much more broadly, to help recruit and retain faculty across the entire landscape of modern biology. I plan to try to promote good science, make sure that our educational mission is given adequate attention and resources, and spend our limited financial resources wisely and responsibly."
Serving as dean is a full-time job in and of itself, but Schlissel is determined to retain his own research program as well. Here, Schlissel is following in the footsteps of his mentor Don Brown, who ran a prolific laboratory and served as director of the Carnegie Institute of Washington for many years. "He said his job was to make it so he was the only guy in the building that had to worry about anything other than science. I'd like to do the same."
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The Instruction Manual of the Genome
Michael Eisen is also a cofounder of the Public Library of Science (PLoS), a publisher of peer-reviewed science journals freely available on the internet, and was named a Howard Hughes Medical Institute investigator this spring. Photo credit: Noah Berger/AP, copyright Howard Hughes Medical Institute.
A squirrel, a squid and a spider appear as different as animals can be. Yet the building materials for each—a vast array of protein molecules—are by and large the same. So how can a squid have ultra-flexible tentacles while a spider has stiff, jointed limbs? It boils down to how those proteins are assembled. And the instruction manual for each body, like the code for each protein, is written within an organism's DNA.
"Many of the interesting and important differences between species and individuals arise from differences in the way genes are turned on and off," says Michael Eisen, a Berkeley professor of genetics, genomics and development. "Yet we remain largely ignorant of how such regulatory changes are encoded in DNA."
Eisen is learning to decipher the instruction manual of the genome. He combines bench-top experiments with computational biology to link genetic sequence variations with functional differences. "By characterizing the molecular machinery of gene regulation in detail, and coupling that with analyses of the genome sequences they're reading, we'll ultimately be able to understand what they do," he says.
Eisen is particularly interested in a platoon of proteins called transcription factors that control how DNA is read. Each transcription factor binds to a specific DNA sequence to influence when nearby genes are turned on and off.
Proteins called transcription factors influence how DNA is read. With the help of a special fluorescent marker, Eisen can see where certain transcription factors are active in fruit fly embryos. Image credit: courtesy Michael Eisen
Scientists are still deciphering how transcription factors work. Among the outstanding questions are why transcription factors alight on only a small fraction of possible regulatory sequences. This is partly because much of the genome is coiled around special packaging proteins. These affect how readily transcription factors bind. To complicate matters further, transcription factors may enhance or inhibit each other's binding affinity.
Eisen employs a transgenic system to reveal gene regulation in fruit flies at a glance. He inserts small chunks of DNA that activate genes in specific cell sets within developing fly embryos. Eisen joins these regulatory elements to a gene that tints cells blue when switched on. With this tool, he can identify every cell where the regulatory element is active.
Other species of Drosophila contain similar regulatory genes. Even though the specific sequences are different from those in D. melanogaster, they perform the same function.
Eisen is particularly interested in a platoon of proteins called transcription factors that control how DNA is read. Each transcription factor binds to a specific DNA sequence to influence when nearby genes are turned on and off.
Eisen hopes to use these gene segments in the same way that archeologists used the Rosetta Stone-to translate an unknown language. "It's like the same paragraph was written in all of these different languages," he says. "If we can gather different versions of these enhancers from different species, each of which has the same function but is written in a different way, we'll be able to figure out what they all have in common. We'll glean from that the heretofore elusive understanding of what actually creates these patterns of expression."
Eisen is identifying differences in transcription factor binding patterns among Drosophila fruit fly species. The study will demonstrate how gene regulation affects evolution. Image credit: courtesy Michael Eisen
Eisen will soon have plenty of enhancers to compare. He and other scientists plan to sequence the genomes of all 4,000 or so species in the Drosophila genus within the next five years.
Eisen will undertake a similar comparison among hundreds of D. melanogaster fly lineages, whose genomes are also being sequenced. For each transcription factor, he is likely to find groups of flies with different binding patterns. "We'll be able to identify what's different about the genomes of these groups, and deduce their functional effects. This is where we'll see the role that gene regulation really plays in evolution-by learning how these flies actually differ," Eisen says.
His work has major implications for the future of health care. Within the next ten years, most Americans will be able to afford to have their own personal genomes sequenced. The information could help doctors select better medications for their patients and avert potentially serious diseases. But between now and then, science has to figure out what the individual variations in each person's sequence mean.
"What limits our ability to utilize the genome sequences is figuring out what the genome says," Eisen says. "Our hope is that we'll be able to use what we learn from Drosophila and apply it broadly to the consequences of genetic variation in humans."
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Symposium to discuss genes and the "human condition"
The University of California, Berkeley, will host a free public symposium on Saturday, Nov. 15, to explore what human genes - and the genes of extinct human ancestors - can tell us about our history, the origin of language, susceptibility to disease and the origins of mental illness.
Titled "Humanity's Genes and the Human Condition," the symposium is meant to draw other disciplines, in particular the humanities, into a discussion of how recent technological breakthroughs in genome sequencing will affect our understanding of the "human condition," according to organizers Michael Botchan and Steve Martin, UC Berkeley professors of molecular and cell biology.
"Human genome information is now telling us about who we are, where we came from and where we're going," said Botchan. "What was once the purview of molecular biology now intersects with a broad range of disciplines, from anthropology and psychology to linguistics and the law."
The symposium, sponsored by Genentech, takes place from 9 a.m. to 5 p.m. in Berdahl Auditorium, 105 Stanley Hall.
Participant Sydney Brenner, who won the 2002 Nobel Prize in Physiology or Medicine, challenged scientists in his Nobel acceptance speech to study in detail "natural human genetic variation and its correlation with ...health and disease." Calling this "the major challenge in human biology and medicine in the next decade," Brenner urged development of the technology to make this possible.
These tools are now in place and improving at a rapid pace. Gene sequencing technology has advanced to the point where the price of sequencing a single human genome - all 20,000 to 25,000 genes - will soon be as low as $5,000. Private companies offering to sequence a customer's "personal genome" are vying for attention, and one company has posted portions of eight genomes on a new Personal Genome Project website that some day could include thousands of genomes.
While fruit flies and nematodes can tell scientists a lot about what genes do and how they work, Martin said, "Brenner has always believed that the proper study of mankind is man. That is where science and UC Berkeley research should be moving."
Edward Penhoet, former director of the Gordon and Betty Moore Foundation and former dean of UC Berkeley's School of Public Health, will open the symposium, while Princeton University geneticist David Botstein will deliver the keynote address. Brenner, who is an adjunct professor at both UC Berkeley and the Salk Institute in La Jolla, Calif., will provide closing remarks. The other main speakers are:
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