A groundbreaking comparison of human and Neandertal genomes reveals astonishingly few differences in the DNA that codes for proteins.

A groundbreaking comparison of human and Neandertal genomes reveals “astonishingly few” differences in the DNA that codes for proteins, says Howard Hughes Medical Institute (HHMI) investigator Gregory J. Hannon, one of the lead investigators on the study, which is part of the larger Neandertal Genome Project.

Hannon’s group collaborated on the new research with Svante Pääbo, a biologist at the Max Planck Institute in Germany and leader of the Neandertal Genome Project. Hannon, Paabo and their teams examined the DNA that encodes 14,000 proteins, and found that just 88 DNA letters—or nucleotides—differ between humans and Neandertals, the thick-bodied hominids that inhabited a huge swath of Europe and the Middle East until some 30,000 years ago. Hannon and colleagues report their findings in the May 7, 2010, issue of the journal Science.

Viewed from the level of the proteome, there are not big differences between us and them.

Gregory J. Hannon

In the same issue of Science, Pääbo and a large international team of scientists, including HHMI investigator Evan Eichler at the University of Washington, are publishing the completed genome of a different Neandertal individual—all 3 billion letters of the hominid’s DNA. That study, too, found that relatively few changes have been incorporated into the human genome in the last few hundred thousand years of human evolution. The completion of the Neandertal genome, pieced together over the last decade by Pääbo and colleagues from DNA isolated from Neandertal bones found in a cave in Croatia, should lead to a new understanding of the evolutionary relationship between humans and Neandertals.

In their analysis, Hannon’s team focused on the small percentage of human and Neandertal DNA that actually encodes proteins. This gave them a picture of what kind of variation exists between the complete sets of proteins—the proteomes—produced by the cells of each of the hominids. “Viewed from the level of the proteome, there are not big differences between us and them (Neandertals),” says Hannon, also a professor at Cold Spring Harbor Laboratory (CSHL) in New York. “That was shocking to see.”

The study was made possible by a new technique invented by Hannon, his postdoctoral fellow, Emily Hodges, and their CSHL colleagues that permitted the scientists to isolate and sequence targeted stretches of DNA. The method helped the researchers overcome genetic contamination of Neandertal DNA samples, a major obstacle that prevented researchers from extracting pure snippets of Neandertal DNA from ancient bones. In the study, Hannon’s team analyzed DNA from a 49,000-year-old Neandertal bone flake that had been found in 2006 in a Spanish cave called El Sidrón. Some 99.8 percent of the DNA extracted from the bone was not Neandertal, but originated instead from bacteria, fungus, and “whatever else crawled in there,” says Hannon.

An RNA biologist by trade, Hannon was drawn into the project when Pääbo, who is perhaps the world’s foremost expert on ancient DNA, approached him to work on another study. Hannon told Pääbo about his new DNA sequencing technology and suggested it might be useful for studying ancient DNA. Pääbo was skeptical at first. To demonstrate the power of the new sequencing strategy, Hannon and his colleagues ran pilot tests on human genetic material that had been mixed with DNA from papayas and worms. Those tests, Hannon says, were quite effective is showing how hominid DNA might be isolated and purified from a mélange of genetic detritus. Impressed, Pääbo agreed to a collaboration.

Pääbo’s team provided Hannon’s group with immortalized samples of Neandertal DNA, and the groups worked together to capture and analyze the critical areas. “It was a terrific collaboration with investigators moving back and for the between Cold Spring Harbor and Leipzig,” says Hannon. Hannon also approached his employer, the Howard Hughes Medical Institute for additional support for the project, which was a significant divergence from the research on RNA interference that is the main focus of Hannon’s lab.

The new sequencing technique, which Hannon calls “DNA capture and resequencing,” relies on DNA microarrays, glass slides spotted with tens of thousands of fragments of DNA. The complete human and Neandertal genomes are quite large, but DNA capture allows Hannon to isolate for sequencing only the relevant segments. In this case, his group chose to study the DNA that actually encodes proteins.

For this study, Hannon and Pääbo wanted to answer the question of where along the evolutionary line Neandertals fit between chimps and humans. So their team first compared the published sequences of the human and chimp genomes and identified all of the protein-coding differences between the two species. This process highlighted about 14,000 genes that have changed in humans sometime since their evolutionary split with chimpanzees approximately 6.5 million years ago.

Working with Agilent, a commercial DNA microarray manufacturer, Hannon then produced DNA microarrays spotted with fragments from all 14,000 human genes identified in their early analysis. Next, the teams amplified the mixture of assorted DNA found in the Neandertal bone and washed that genetic soup over the microarray chips. Because Neandertals are closely related to chimps and humans—far more so than the microbes whose DNA comprised the bulk of the sample—this enabled the team to “capture” Neandertal genes that were closely related to the genes they had placed on the microarray. Fragments of DNA that roughly matched the 14,000 genes stuck to the chip, while the rest of the DNA—all of the microbial contaminants, as well as unwanted portions of the Neandertal genome—was washed away.

The team then sequenced the Neandertal DNA that stuck to the chip and compared those sequences to the DNA sequences of the same genes from humans. After completing the analysis, the groups identified just 88 proteins that differed by at least one amino acid between humans and Neandertals.

A rigorous series of control experiments confirmed that contamination from modern human DNA, which might have come from laboratory workers, did not influence the results.

Pääbo has been spearheading an international collaboration to study the Neandertal genome since the mid-1990s. Previously, Pääbo and his team suggested that humans and Neandertals began diverging from a common ancestor about 800,000 years ago. By 300,000 years ago, that separation was complete, although other recent research on Neandertal DNA suggests that humans and Neandertals interbred.

Hannon says that follow-up work could focus on whether the protein differences identified in the study lead to changes in how the proteins function. “Many of the protein differences we identified may be neutral, and not have any effect on how the protein works,” says Hannon. “while other changes may be adaptive and responsible for differences between Neandertals and modern humans.”

Hannon adds that now that “capture and sequencing” has proved its worth with Neandertals, it could be used to study ancient, contaminated DNA from other species. In fact, he has begun collaborating with others to study ancient mammoth DNA.

“We played a small part in this historic event,” says Hannon. “I feel very privileged to contribute to it, to be one piece of this very long history of trying to understand what makes Neandertals and modern humans different, to help answer what it means to be human.”

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