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After years of relying solely on DNA sequencing, geochemists have finally developed methods for sequencing ancient proteins that may make the retrieval of genetic information from creatures that lived hundreds of thousands of years ago a realistic possibility. The advance will allow scientists to more reliably reconstruct phylogenic relationships between ancient organisms and their modern counterparts. Attempts to sequence DNA from fossils--some more successful than others--have been going on for more than a decade. Trace amounts of DNA can be extracted from fossils, amplified to a detectable signal using the polymerase chain reaction, and then sequenced. Sequencing of ancient DNA has been a boon to scientists hoping to track the evolution of modern humans, trace the migrations of animal populations over time, and determine the geographic origins of fossilized plants and animals. But the fragility of DNA--its phosphate backbone is easily hydrolyzed and enzymes that degrade it are hard to get rid of--makes sequencing of DNA from very old samples extremely difficult. The study of ancient DNA sequences from fossils is likely to be limited to samples less than 100,000 years old and perhaps much younger. Recently, however, plant DNA from 300,000- to 400,000-year-old sediments has been sequenced (C&EN, April 21, page 48). Still, the technical difficulty of distinguishing sample DNA from that of common contaminants such as fungi and bacteria calls into question the reliability of ancient DNA analysis. Scientists have long suspected that some proteins might outlast DNA in very old samples. Theoretical calculations suggest that proteins may survive for more than half a million years at ambient temperatures [The Biochemist, 24, 12 (2002)]. Ancient protein sequencing has proved difficult, however, leaving ancient DNA sequencing the only game in town. "But the development of methods for sequencing ancient proteins will allow scientists to trace evolutionary relationships even further back in time," noted Richard P. Evershed, an analytical chemistry professor at the University of Bristol, in the U.K. New advances in this area were highlighted in a symposium titled "Ancient Biomolecules: New Perspectives in Archaeology and Paleobiology" that Evershed helped to organize for the Division of Geochemistry at the American Chemical Society national meeting held last month in New Orleans. Most notable was work presented by geological sciences professor Peggy H. Ostrom of Michigan State University. She described efforts by her lab and the lab of Christina M. Nielsen-Marsh, a geochemist at the University of Newcastle upon Tyne, in England, to use matrix-assisted laser desorption ionization mass spectrometry (MALDI-MS) to sequence proteins from a bison bone more than 55,000 years old [Geology, 30, 1099 (2002)]. This provides the first definitive evidence that proteins can survive in fossils this old, Ostrom told C&EN. In fact, Ostrom and Nielsen-Marsh believe that the protein--osteocalcin--can be sequenced from even older samples. Ostrom presented unpublished data from Nielsen-Marsh's lab suggesting that osteocalcin can be sequenced from fossils more than 100,000 years old, and perhaps from bones that are half a million years old. OSTEOCALCIN is a small and abundant bone protein found only in vertebrates. Fungi, bacteria, plants, and other organisms that commonly contaminate ancient samples do not contain osteocalcin. The protein uses three g-carboxyglutamic acid residues to tether itself to the mineralized hydroxyapatite found in bone. It's this feature that allows the protein to survive the test of time, Ostrom said. Unlike most DNA procedures, Ostrom and Nielsen-Marsh's protein sequencing method requires only a few milligrams of bone and therefore doesn't destroy valuable fossils. After removing bone mineral and collagen from the sample, they separate the osteocalcin from the remaining proteins by several rounds of gravity column chromatography and high-performance liquid chromatography. They then use MALDI-MS to sequence the purified osteocalcin. Ancient protein sequencing will unlock a "treasure of untapped genetic information." Because the string of 13 amino acids at osteocalcin's N-terminal tail varies among species, the amino acid sequence of this region can be used to reconstruct evolutionary relationships. The osteocalcin sequence that Ostrom and Nielsen-Marsh gleaned from the 55,000-year-old Bison priscus bone is identical to that of the modern Bison bison. But the fossil sequence differs by only one amino acid from that of the modern domestic cow Bos taurus. Together, these observations suggest that domestic cows and modern and ancient bison have a common ancestor. Ostrom and Nielsen-Marsh are trying to sequence osteocalcin from even older bison bones in hopes of identifying the common forebear. The osteocalcin lifetimes that Nielsen-Marsh and Ostrom observe are significantly longer than those predicted from kinetic studies of pure osteocalcin in solution. "The key to the preservation of proteins in bone lies in these molecules' intimate relationship with bones' mineral components," Ostrom said. The team is now searching for other well-preserved bone proteins in fossils that may submit to sequencing. Ancient protein sequencing will unlock a "treasure of untapped genetic information," Ostrom told C&EN. But, she acknowledged, the genetic information content of protein sequences is not as rich as that gleaned from DNA. She and Nielsen-Marsh hope that their search for other well-preserved proteins will turn up ones boasting greater amino acid sequence variability--a feature that would yield more information for reconstructing phylogenic relationships. "Protein sequences may not change as rapidly as DNA sequences," Nielsen-Marsh said, "but because proteins are inherently more stable, they offer a unique opportunity to extend the search for biomolecules further back in time." That makes it possible that ancient protein sequencing might be archaeologists' and paleobiologists' most dangerous weapon yet. AGING GRACEFULLY The MALDI mass spectra of osteocalcin extracted from bones of various ages show a peak with a mass-to-charge ratio (5,590) identical to that of modern osteocalcin. COURTESY OF PEGGY OSTROM |
| UPDATE | 04.03 |
| AUTHOR | geological sciences professor Peggy H. Ostrom of Michigan State University. She described efforts by her lab and the lab of Christina M. Nielsen-Marsh, a geochemist at the University of Newcastle upon Tyne, in England |
| LITERATURE REF. | [Geology, 30, 1099 (2002)]. |
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