THE search for genes--just as for gold--may be long and arduous, but the rewards are great. Genes and the proteins they produce hold the keys that unlock the mysteries of genetic diseases and allow the development of gene and drug therapies. The worldwide Human Genome Project has the ultimate goals of finding all the genes in the DNA sequence, developing tools for using this information to study human biology and medicine, and improving human health.
The task of locating a particular gene in the human genome can be more daunting than searching for a vein of precious ore underground. For perspective, the human body has 100 trillion cells, each of which contains 23 pairs of chromosomes. Each chromosome carries a complete set of DNA. If the DNA of one cell, which contains about three billion nucleic acid units or "base pairs," were formed into a single continuous strand, it would stretch six feet long. (The four chemical "bases"--adenine, thymine, guanine, and cytosine--bind together to form base pairs that are the building blocks of DNA.) About 3 percent of this DNA forms working genes. The task facing gene hunters is to search through the "genetic junk" of the human genome to find that one string of DNA that comprises the gene in question.
It's a task that makes digging for gold in a mountain of dirt and rock look easy.

The Search for a Kidney Disease Gene
For many years, researchers from the Karolinska Institute in Stockholm, Sweden, and the University of Oulu in Finland had been seeking the gene for congenital nephrotic syndrome, an inherited kidney disease that causes massive amounts of proteins to be excreted by the kidneys. The disorder, which occurs primarily in families of Finnish origin, develops shortly after birth and usually causes death within a year. The only alternative for this progressive disease is a kidney transplant.
By 1993, the researchers, led by medical chemistry professor Karl Tryggvason from Karolinska Institute's Department of Medical Biochemistry and Biophysics, had narrowed their search to chromosome 19. Because Lawrence Livermore is well known for its mapping and sequencing of chromosome 19, Tryggvason contacted Livermore biomedical scientist Anne Olsen for assistance.
"Other laboratories and institutions are sequencing pieces of 19, but we are the only one addressing the entire chromosome," Olsen explained. (For more information about the Laboratory's work in DNA sequencing, see S&TR, November 1996, pp. 24­26, and July/August 1997, pp. 18­20.)
When the European researchers contacted Livermore, they knew where the gene resided on the genetic linkage map, but not on the physical map of chromosome 19. (See the box on p. 25.) In 1993, the physical map of chromosome 19 was less developed than it is today. Olsen and other biomedical scientists worked for more than a year to complete a physical map of the genetic region in question, providing the European team with well-characterized DNA fragments or clones. The collaborators used those cloned pieces of chromosome 19 to further narrow down the site of the fatal gene, tracing it to an area containing 150,000 base pairs.






Narrowing Down the Search
At this point, the teams had gone as far as they could go with physical mapping, and it was time to sequence the individual base pairs to determine their exact order on the chromosome. Jane Lamerdin and Paula McCready led another Lawrence Livermore team that sequenced the bases using the Laboratory's high-throughput sequencing machines. The Finnish collaborators used the clones provided by Livermore for biological analysis and located 11 likely genes in the candidate region.
Of those genes, they finally narrowed it down to one. That particular gene was mutated in the families carrying the disease, and the protein associated with the gene was well-expressed in the kidneys.





"Even though our part is done, the story is just beginning," said Olsen. "Since the main symptom of this disease--protein excreted in the urine--appears in other conditions, this work may offer insights into other kidney ailments as well."
The breakthrough was announced in March 1998 when a paper on the research appeared in the journal Molecular Cell (March 1998, pp. 575­582). This announcement came hot on the heels of another genetic discovery involving a rare hereditary susceptibility to a variety of cancers--the Peutz­Jeghers syndrome. Pinpointing the location of the Peutz­Jeghers gene took only one year.
"The difference in time indicates how far we've come with mapping and sequencing techniques and technologies over the past few years," Olsen said. "When Karl Tryggvason first contacted us about the kidney disease gene, we didn't have a highly developed physical map for that region. Three years later, when we were asked to collaborate on the search for the Peutz­Jeghers gene, our map was much better developed, and that search went more quickly. Better, more detailed maps mean the search for genes will only accelerate in the future."
--Ann Parker




A Primer on Maps, Markers, and Sequencing

A genome map describes the order of genes or other markers and the spacing between them on each chromosome. Human genome maps are constructed on several different scales or levels of resolution. At the coarsest resolution are genetic linkage maps, constructed by observing how frequently two markers are inherited together. These maps depict the relative chromosomal locations of DNA markers (genes and other identifiable DNA sequences) by their patterns of inheritance. Two markers near each other on the same chromosome tend to be passed together from parent to child. During the normal production of sperm and egg cells, DNA strands occasionally break and rejoin in different places on the same chromosome or on the other copy of the same chromosome. This process-- called meiotic recombination--can result in the separation of two markers originally on the same chromosome. The closer the markers are to each other, the more tightly linked they are, making it less likely a recombination event will separate them. Recombination frequency thus provides an estimate of the distance between two markers.
The value of the genetic map is that an inherited disease can be located on the map by following the inheritance of a DNA marker, even though the responsible gene is not yet identified.
Physical maps, in contrast, provide a finer resolution of the absolute location of a gene. A physical map lays out the order of all the base pairs on a chromosome. The ultimate physical map of the human genome is the complete DNA sequence, or the determination of all base pairs on each chromosome.
For more information about basic genetics as well as mapping and sequencing techniques, see the U.S. Department of Energy's "Primer on Molecular Genetics" on the World Wide Web at http://www.ornl.gov/TechResources/Human_Genome/publicat/primer/intro.html




Key Words: chromosome 19, congenital nephrotic syndrome, DNA clones, DNA mapping and sequencing, Human Genome Project, kidney disease, Peutz­Jeghers syndrome.

For further information contact Anne Olsen (925) 423-4927 (olsen2@llnl.gov).


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