7-11-97
University Park, Pa. -- A team of U.S. and German researchers has extracted mitochondrial DNA from Neandertal bone showing that the Neandertal DNA sequence falls outside the normal variation of modern humans.These results indicate that Neandertals did not contribute mitochondrial DNA to modern humans, says Dr. Mark Stoneking, associate professor of anthropology at Penn State.

Neandertals are not our ancestors.The research also reaffirms the origins of modern humans in Africa. Reporting in today's (July 11) issue of the journal Cell, the researchers detail their methods and the results of analysis of Neandertal mitochondrial DNA. The research team includes Matthias Krings, graduate student, and Dr. Svante Paabo, professor of zoology, University of Munich; Dr. Ann Stone, postdoctoral fellow, University of Arizona; Ralf W.

Schmitz and Heike Krainitzki of Rhineland Museum, Bonn, Germany; and Stoneking.Current theory holds that Neandertals became extinct only 30,000 years ago and co-existed with modern humans in Europe. The team, however, found that Neandertals and modern humans diverged genetically 500,000 to 600,000 years ago, suggesting that though they may have lived at the same time, Neandertals did not contribute genetic material to modern humans.Since 1991, an interdisciplinary project of the Rhineland Museum, headed by Schmitz, has focused on the Neandertal-type specimen. This specimen was found in 1856 near Dusseldorf, Germany.

As a part of this project, a sample was removed for DNA analysis.The ability to extract DNA from ancient bone is dependent on many factors, including preservation, temperature and humidity, says Stoneking, a faculty member in Penn State's College of the Liberal Arts.Paabo previously showed that even if extracting ancient DNA is possible, it tends to be damaged and degraded, yielding only short fragments. The researchers used a method of overlapping short strands of DNA to obtain a mitochondrial DNA sequence of 378 base pairs.

To ensure that errors caused by damaged DNA were not incorporated into the sequence and that modern human DNA did not contaminate the samples, the researchers ran multiple extractions and amplifications. They also sent a sample to Penn State's Anthropological Genetics Laboratory where Stone, then a Ph.D. candidate at Penn State, ran a parallel extraction and amplification of the DNA.To begin amplification, the researchers used two human primers -- small pieces of DNA that match the beginning of the sequence to be amplified.The first two human primers we chose worked, says Stoneking.

It turns out this was a lucky choice.To check that the amplified DNA was really Neandertal, the researchers prepared primers based on their extracted sample and ran them on numerous human DNA samples.The Neandertal primers did not amplify any human DNA, says Stoneking. Most human primers would probably not work on Neandertal DNA.The researchers compared the Neandertal sequence with 2,051 human sequences and 59 common chimpanzee sequences.

They found that the differences in Neandertal DNA occurred at sites where differences usually occur in both humans and chimps.The changes reflect the evolutionary pattern typical of mitochondrial DNA sequences of living humans and chimpanzees, not that of random damage or degradation, says Stoneking.When the researchers looked at the Neandertal sequence with respect to 994 human mitochondrial DNA lineages including Africans, Europeans, Asians, Native Americans, Australians and Pacific Islanders, they found the number of base pair differences between the Neandertal sequence and these groups was 27 or 28 for all groups.While Neandertals inhabited the same geographic region as contemporary Europeans, the observed differences between the Neandertal sequence and modern Europeans do not indicate a closer relationship to modern Europeans than to other contemporary human populations, says Stoneking. The researchers used phylogenetic tree reconstruction -- a method that uses mitochondrial DNA to place individual groups in relative relationship -- to check the results of their pair-wise DNA comparisons. The trees show that the Neandertal sequence branches before the divergence of the various human mitochondrial DNA lineages, but after the split from chimpanzees.

This phylogenetic tree also shows that the first three branches of humans are of African origin, with only the fourth branch showing non-African sequences.The branching pattern indicates that the ancestor of the mitochondrial DNA gene pool of contemporary humans lived in Africa, says Stoneking of Penn State.The researchers are confident with their results, but they caution that they are derived from only one individual. They also warn that DNA may be difficult to extract from other specimens.

While the results indicate that Neandertals did not contribute mitochondrial DNA to modern humans, it is still possible that they contributed other genes.**aem**
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