By Sean Henahan, Access Excellence

Using refined and expensive genetic techniques, U.S. and German researchers extracted mitochondrial DNA from Neanderthal bone. These studies showed that the Neanderthal 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 findings will cause of reconsideration of the current consensus that Neandertals became extinct only 30,000 years ago and co- existed for some time with modern  humans in Europe. The new research indicated that Neandertals and modern humans diverged genetically 500,000 to 600,000 years ago. While the two species may have lived at the same time, Neandertals did not contribute genetic material to modern humans, the researchers report.
 
The team analyzed bone from a Neanderthal specimen found in the eponymous valley. This is the first time researchers have been able to extract useful DNA fragments from such a specimen.

"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.

The researchers devised an innovative technique using overlapping short strands of DNA to obtain a mitochondrial DNA sequence of 378 base pairs. The researchers ran multiple extractions and amplifications. to ensure that errors caused by damaged DNA were not incorporated into the sequence and that modern human DNA did not contaminate the samples. At the same time the researchers 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 Neanderthal, the researchers prepared primers based on their extracted sample and ran them on numerous human DNA samples.

"The Neandertals primers did not amplify any human DNA," says Stoneking. "Most human primers would probably not work on Neanderthal DNA."

The researchers compared the Neanderthal sequence with 2,051 human sequences and 59 common chimpanzee sequences. They found that the differences in Neanderthal 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 Neanderthal 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 Neanderthal 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 Neanderthal 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 Neanderthal 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.

"I really looked for holes in the methodology, but I just couldn't find any. It seems to be an authentic sequence and certainly as far as I can tell the most rigorous ancient DNA study I've ever seen," says evolutionary biologist and ancient DNA researcher Blair Hedges of Penn State.

The researchers caution that the current results are derived from only one individual and note that DNA may be difficult to extract from other specimens. Even if  theNeanderthals did not contribute mitochondrial DNA to modern humans, it is still possible that they contributed other genes, they emphasize.

The research appears in the June 11, 1997 issue of Cell.