By David N. Leff

As the poet wrote, "Things are not what they seem." Including some primates:

* Flying lemurs (Cynocephalus variegatus) are not lemurs, but colugos. What's more, they don't fly; they glide. And they're not primates but dermoptera.

* African bush babies (Galago crassicaudatus) live in trees, not bushes. And once they grow up, they're not babies.

* Red howler monkeys (Alouatta seniculus) don't howl; they roar.

* And humankind (Homo sapiens) is not all that sapient — defined as "having great wisdom and discernment."

The mammalian species listed above are four of nine, eight primates plus the domestic dog (Canis familiaris), that had portions of their genome sequences compared to measure their evolutionary distance from H. sapiens. The following four primates, all closer than the preceding four to the human species, are: chimpanzee (Pan troglodytes), gorilla (Gorilla gorilla), orangutan (Pongo pygmaeus) and Rhesus monkey (Macaca mulatta).

"The study marks the first time that DNA microchips have been used to sequence nucleotides from nonhuman primates," said biochemist and molecular geneticist Joseph Hacia. He is first author of a paper in the upcoming February issue of Nature Genetics, which reports this feat. Its title: "Evolutionary sequence comparisons using high-density oligonucleotide arrays."

That article's senior author is molecular geneticist Francis Collins, who heads the National Institutes of Health's National Human Genome Research Institute (NHGRI), and the Human Genome Project, in Bethesda, Md.

What the team compared in the various primate genomes was their nucleotide sequences corresponding to the human familial breast cancer gene, BRCA-1.

They began with a chip the size of a thumbnail paved with some 48,000 DNA fragments that added up to the 3.4 kilobases of exon 11 on the BRCA-1 gene. This segment represents the lion's share of that gene's total 5.2 kilobases.

"Our primary goal in this work," Hacia told BioWorld Today, "is a continuing project in mutation analysis. In this particular study, we tested primate genomic sequences just as proof of principle, to see how well the chip would perform in sequences that were very diverse."

Evolutionary divergence was the key element in their comparative analysis:

Mammal Millions Of Years Nucleotide
Since Divergence Identity
From Human Detected
Human 100%
Chimpanzee 5.9 99.2%
Gorilla 7.2 99.1%
Orangutan 13.9 98.2%
Rhesus monkey 25.0 95.9%
Red howler 35.0 92.6%
African bush baby 55.0 85.0%
Flying lemur 80-90 87.2%
Dog 80-90 83.5%

"There's a lot of basic science interest," Hacia observed, "in being able to know, on a biochemical level, the differences in DNA sequences between humans and our closest relatives on the evolutionary tree, chimps, gorillas and orangutans. Those percentages," he pointed out, "relate to the BRCA-1 gene, not the entire genome. Genome-wide, chimps are 98.5 percent identical to humans.

"That divergent 1.5 percent of sequences," he explained, "are going to be scattered on the genome. The key will be to find the differences, and then figure out which are the critical ones."

In that long-term quest, chip technology will play a part, Hacia went on, "but you can't use a chip to decipher the entire primate genome. The chip is useful only for areas that are non-repetitive or not complex."

Chips will take their place alongside the current dideoxy technology by which the human genome is being sequenced. "What we want to do, he said, "is use the investment in dideoxy sequencing, which is a relatively expensive endeavor, but extremely accurate — it's really the gold standard — and use that information from a human to see if in a much cheaper and quicker fashion, namely by chip, we could get the equivalent information from a chimp or a gorilla."

To acquire the starting DNA information from their panel of eight primates and a dog, Collins and his crew began by thawing samples from a deep-freeze zoo of mammalian blood.

As Hacia's assistant technician, Keith Edgemon, told BioWorld Today, "First we amplified the BRCA-1 exon by PCR. Next, we transcribed the gene sequence from DNA to RNA, added a fluorescent label, then hybridized this RNA, in solution, to the chip for four hours." (See BioWorld Today, May 24, 1994, p. 1, and Dec. 2, 1996, p. 1.)

This entire project, Hacia noted, is a collaborative effort with Affymetrix Inc., of Santa Clara, Calif., which designs and delivers the chips to NHGRI specifications.

Besides the primate project, their partnership also encompasses the institute's long-term pursuit of human gene mutations. This involves not only BRCA-1 but BRCA-2, and the gene for ataxia telangiectasia, an inherited, multisystem disease.

"I'm pretty enthused," Hacia said, "that eventually this chip technology will be scaled up and used for a primate whole-genome scan. As soon as we determine the sequence of an organism through conventional dideoxy means, then we can get the sequence of any other organism that's very highly conserved, that is, carry identical stretches of DNA.

"So if you have another organism — other mammals, closely related microbes, even insects — that are 99 percent conserved, you get more bang for your buck."

He sees such comparisons going down from species to subspecies, "so perhaps when we compared everything together, we could get a regulatory element out of it." *