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School of Medicine researchers have reached a milestone in the history of genetics -- the completion of a high-resolution map of chromosome X. The map was published in this month's issue of Genome Research.
The map has 2,100 unique landmarks -- three times as many as any previous X chromosome map. If it were a road map from St. Louis to San Francisco, it would show a marker every mile.
The researchers also located hot spots for genes and detected a large region where the DNA remains intact as it passes from one generation to another.
The map is speeding the search for disease genes on X, which is associated with many inherited disorders. "And the completion of a map with this level of detail has made X one of the earliest chromosomes for DNA sequencing -- the next phase of the Human Genome Project," said David Schlessinger, Ph.D., director of the medical school's Center for Genetics in Medicine and principal investigator for the X project. Ramaiah Nagaraja, Ph.D., research instructor in molecular microbiology, is the paper's lead author.
Chromosome X determines gender -- women have two copies and men have one X and one Y. X's DNA is one long double helix -- 160 million nucleotide base pairs. On average, the new map has a landmark every 75,000 base pairs. The national goal for chromosome mapping is one landmark every 100,000 base pairs.
Whereas someone mapping a road could drive along the route and record landmarks in sequence, the researchers had a much more difficult task. They started with more than 5,000 fragments from seven different libraries of human DNA. They then identified unique landmarks on the fragments. If two pieces contained the same landmark, they knew these fragments must overlap. By painstakingly aligning all the pieces of DNA, they mapped the entire length of X.
A method for cloning large pieces of DNA made this jigsaw puzzle manageable. In the 1980s, David T. Burke, then a graduate student in the Washington University laboratory of Maynard V. Olson, Ph.D., invented the yeast artificial chromosome (YAC). As yeast cells divide, they copy the artificial chromosome over and over, generating sufficient DNA for analysis. "Because each YAC can contain hundreds of thousands of base pairs, a reasonable number of YACs fit along a chromosome," said Schlessinger, who also is a professor of molecular microbiology, of genetics and of medicine.
Finding features that could act as landmarks was another key development. In 1990, Olson and Eric D. Green, then a Washington University M.D./Ph.D. student, unveiled a strategy to use short, unique segments within YACs. These sequence-tagged sites could act as landmarks on chromosome maps the way highway exits and rest areas punctuate road maps, the researchers reasoned. "The cleverness of this system is that it automatically gives you the landmarks and the map at the same time," Schlessinger said.
The team also had to develop new software to order and store the vast amount of data. Philip P. Green, Ph.D., devised several programs, including SEGMAP, which has proved particularly valuable. Maynard Olson and Philip Green now are at the University of Washington.
The project's completion has permitted the first comparison between a physical map and a genetic map of a chromosome. Genetic maps are constructed by studying the passage of traits from one generation to another. The closer two genes are on a chromosome, the less likely they are to get separated as chromosomes swap genetic material during egg and sperm formation. Distances on genetic maps can differ greatly from those on physical maps, however, because some regions of chromosomes recombine more often than others.
The genetic map of X has a few hundred markers. When the researchers compared it with their map of X, they found an area in the middle of the genetic map that corresponds with a much longer stretch -- 17 million base pairs -- of the physical map. "So this region is uneventful on the genetic map, whereas it contains a whole bunch of markers on the physical map," Schlessinger said. "But we don't know why the X chromosome should have this large area of poor recombination."
The researchers also were able to determine how the chemical composition of X varies along the chromosome. "This gave us an early estimate of the relative density of genes across the chromosome," Schlessinger said.
The project enabled Schlessinger and colleagues to locate several disease genes as YACs containing the relevant regions of X became available. They found the gene for an overgrowth disorder called Simpson-Golabi-Behmel syndrome and a gene for ectodermal dysplasia, which impairs the development of hair follicles, teeth and sweat glands. They also were part of an international team that tracked down the gene for fragile X syndrome, the second most common cause of mental retardation. And they have mapped and are analyzing genes that prematurely halt ovarian function.
The X project began in 1987 after the invention of the YAC raised the possibility of large-scale human genome mapping. This prospect prompted the James S. McDonnell Foundation to establish the Center for Genetics in Medicine with a $1.8 million grant. "The foundation funding supported the pilot studies that proved our mapping techniques would work," Schlessinger said.
In 1990, the National Institute of Human Genome Research -- now the National Human Genome Research Institute -- made the medical school's facility one of the first four federally funded genome centers. The institute has supported the center with two consecutive four-year grants totaling $27.7 million. The grants funded the mapping of both X and chromosome 7. Eric Green began a high-resolution map of 7 at Washington University and completed it at the National Institutes of Health, but the data are not yet published.
Schlessinger's associates at Applied Biosystems Division, Perkin-Elmer, in San Francisco and at Washington University's Genome Sequencing Center now are sequencing portions of chromosome X, using materials and markers from the mapping project. "This will determine the entire nucleotide sequence of X and locate all the genes along the chromosome," Schlessinger said.
-- Linda Sage
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