There are nearly countless possible sequences of bases that can make up a gene because genes are of different lengths, so the bases can appear in different sequence and numbers. These genes, these sections of DNA with various arrangements of different numbers of bases, provide the instructions for all the many, many protein molecules that are found in living things.
The way these instructions are read is a process described in every high school biology text, thanks to the several Nobel laureates that figured the whole thing out. The double-stranded spiral shape of DNA is pulled apart and the single strand of DNA is copied by the cell machinery to produce a mirror image not of DNA, but of a similar molecule called RNA, or ribonucleic acid. In RNA another base occupies the place of thymine. It is uracil, or U. The RNA molecule can then be read by cellular machines called ribosomes that translate G, C, A, and U into twenty different amino acids in the sequence and quantity specified by the genes. In this part of the code three bases code for an amino acid.
A code that can be read forward by the cell to produce a protein can be read backward today. This usually requires some sophisticated machinery and techniques involving mass spectrometry. The makeup of a substance can be determined by looking at the proportions of nitrogen and other chemicals in the tissue. Each protein has a distinctive profile.
Protein molecules have distinctive shapes, as well, that relate to the roles they play in bodily chemistry.
COLLAGEN
Collagen is a large, very strong molecule made of up fibrils, which are bundled together like multistrand rope in tissues. The long, ropelike molecules have great tensile strength and find their use in bone, tendons, cartilage—many tissues that need strength and some flex as well.
Collagen has also become one of the prime biochemical fossils. It can last over millions of years, as had been shown before Mary began her work. It can be extracted from fossil bones with great difficulty, and then it is possible to find out the distinctive sequence of amino acids in this version of collagen. Because substances like collagen evolve over time just like the shapes of limbs and the number of toes and the length of a beak. Bird collagen is subtly different from mammal collagen. And within mammals, or birds, collagen may undergo changes as well. By identifying different sequences in different collagen molecules and analyzing how long the changes may have taken to occur, scientists can have a new benchmark for evolutionary change. Just as the shape of a tooth may mark the change from one kind of mammal to another, so changes in collagen may one day be tracked to the transition from birds to dinosaurs. Or, the similarity of bird and dinosaur collagen may help attest to the strength of this evolutionary link.
When geochemists began to turn their attention to fossils as well as rock, they found that just as ancient rock contained the fossil bones of animals, the fossil bones contained their own microscopic histories. So, a few geochemists added the prefix
bio-
to their discipline and began to prospect within fossils in the way that most of my colleagues and I prospect in the Hell Creek or Two Medicine Formations.
One of these biogeochemists, and a pioneer in her field, is Peggy Ostrom at Michigan State University. “Molecules are fossils too,” she says, as a kind of challenge and manifesto. “They can persist over time. They have shapes and sizes. They’re beautiful, just like the bones. Indeed they have form and they have function.” And they have a wealth of information to provide about the history of life.
Peggy Ostrom has targeted a different protein, a component of bone called osteocalcin. She chose it partly because it is small, with only forty-nine amino acids. It is also found only in vertebrates, so there is no worry about contamination from bacteria or fungi. At a meeting of the American Association for the Advancement of Science a few years ago, Peggy explained how she goes about looking for ancient osteocalcin in fossil bone.
Starting with about twenty milligrams of bone, she chemically extracts the biochemicals in the bone from the mineral matrix and then crystallizes it. The next step is a mass spectrometer, which can detect different proteins.
There are various kinds of mass spectrometry, and the technique, despite the fact that it is mentioned frequently on television, still has an aura of magic about it. Take a smidgen of something, pop it into a machine, and then, presto, you know who killed Colonel Mustard because of the traces of cyanide powder on the butler’s gloves. The reality lacks magic, but not wonder. And although the techniques can be complicated, the essence of the process is simple. All elements, like hydrogen, oxygen, gold, and copper, have different masses because they are made up of different combinations of protons, neutrons, and electrons. Molecules made up of various combinations of atoms have different masses as well. In mass spectrometry a substance is vaporized and the gas molecules are hit with electrons, breaking them up into atoms. In the process the atoms are ionized, giving them a positive electrical charge. Then a detector sorts out the different masses and the data is fed into a computer to produce a graph that shows the makeup of the sample in terms of the different elements. Each molecule has a specific signature or chart, and even different versions, or isotopes, of the same molecule have different signatures.
Peggy Ostrom and her colleagues used mass spectrometry to investigate samples of fifty-five-thousand-year-old bison bone preserved in permafrost. They found indications of osteocalcin. Then they used enzymes to cut the protein they thought they had found into its component parts to get a “fingerprint” of the amino acids. She was able to do this to such a level of detail that she could identify a change in one amino acid in the osteocalcin found in the fossil bison and a modern cow. The fifth amino acid in the string of forty-nine was tryptophan in the bison and glycine in modern cattle.
She and her colleagues have also sequenced osteocalcin in the fossilized bone from an extinct horse found in Juniper Cave, Wyoming. The horse,
Equus scotti,
is dated to forty-two thousand years ago. Comparisons of the fossil molecule with modern horses and zebras showed that the osteocalcin molecule in horses had not changed, and comparisons between modern horses and zebras showed that no change had occurred in the molecule in the last million years.
So far, Peggy has gone back as far as half a million years to find osteocalcin in musk oxen fossils. Fossil molecules, she has said, are “beautiful, just like the bones, only the fact is, you can’t see them.” She continued, “These ancient proteins are windows into the past for us. We can now do genetic time travel. We can now, instead of looking at modern organisms to figure out how they’re related, we can go back in time and actually look at the real molecules,” thousands or hundreds of thousands of years old. What about sixty-eight million years old? That was the time Mary wanted to travel to with her bits of fossil from B. rex. Half a million years ago North America was filled with mammals, some we would find unrecognizable, but others that would seem familiar, like bison. Humans, in the taxonomic sense, which is to say, species in the genus
Homo,
had been around for at least a million years.
Homo erectus
had spread from Africa to Asia and Europe using stone tools, and perhaps using fire. A hominid described as an archaic form of
Homo sapiens
had appeared, somewhere between
Homo erectus
and anatomically modern humans, who appeared about two hundred thousand years ago.
It would be hundreds of thousands of years after the time of those fossil musk oxen before behaviorally modern humans left Africa (around fifty thousand years ago) and eventually took over the globe and started digging up fossils of its own ancestors. Still, five hundred thousand years ago, though not truly comprehensible as an expanse of time, leads us back to a moment in the life of the planet when the scene would at least have been comprehensible to us.
INTO DEEP TIME
Mary was trying to make a leap into deep time. And every difficulty in testing fossil bone increases with increasing age. What if the sample is contaminated, what if the tests are wrong, or inconclusive? When dealing with minuscule amounts of ancient fossil bone, one finds that the results of tests are often not as clear as they are with modern material. This fact becomes more and more problematic the farther back in time one goes.
When Mary set out to look for collagen, she attacked the problem on several levels with several different methods—the scanning electron microscope, the transmission electron microscope, atomic force microscopy, mass spectroscopy, and immunoassays. She and her collaborator, John Asara at Harvard, used all of these techniques to pin down what had first appeared to be collagen.
One of the reasons so many techniques were necessary was the minimal amount of organic material in the fossil bone. The traces of proteins were not at all easy to detect, and the threat of contamination or misreading was always present, so every avenue of investigation had to be pursued and every bit of evidence collected.
With the scanning electron microscope, coupled with X-ray scattering, she looked for indications that the substance was collagen. Then she did the same with the transmission electron microscope, which, however, requires samples that are only seventy nanometers thick. The transmission microscope, Mary said, “required that your sections be thin enough for electrons to pass through completely so the electrons are transmitted from one side to the other, which gives you an incredibly high resolution.”
“One thing we know about collagen, the major protein in bone, is that it’s cross-banded. It has to do with the molecular makeup of the collagen. You get a sixty-seven-nanometer repeat banding.
“So it’s diagnostic of collagen. If you’ve got a fibrous material with sixty-seven nanometer bands, you’ve got collagen.”
With the transmission electron microscope scientists can analyze the molecules much more closely by doing what is called elemental analysis. With a scanning electron microscope, Schweitzer says, you can tell that dinosaur bone has calcium and phosphate with some other traces. “What that can tell me is, yeah, it’s a carbonate mineral.”
“With the elemental analysis that I can do on a transmission electron microscope. I can tell you that it is one hundred percent fluorapatite. I can tell that it’s hydroxyapatite. I can tell you that it is biogenic hydroxyapatite.”
Schweitzer also uses atomic force microscopy, in which the researcher can literally push the probe of the microscope physically onto the material and get a quantitative measure of its elasticity. “We can see what the springiness of the material is, compare it with modern bone, and get at how much of the original functionality of the material is there.”
The process of gas chromatography requires its own experts to run the experiments. The equipment that Mary uses at North Carolina State—a gas chromatograph coupled to a mass spectrometer—has its own room and takes half a day just to calibrate. The whole process of testing a substance demands the marriage of the most advanced computer technology and expertise with skills that would have been familiar to an alchemist in medieval Europe.
The preparation of the sample is the kind of work that might have been done by Merlin. It begins with a small amount of powdered fossil that has been prepared by Mary’s lab. This is measured into a tin boat, a small open container of tin about half the size of a Chiclet that is shaped something like a gravy boat. The boat is pinched shut and this spitball-sized object, weight precisely determined, is dropped into the well of the chromatograph, at which point the temperature flashes to seventeen hundred degrees Fahrenheit, atomizes the substance at hand, and turns the elements into gases. Those gases are drawn though filters, and molecules of different size travel through the filters at different speeds. Sensors record the amount of the different elements present, and the finding can distinguish the relative abundance of carbon and nitrogen, so you have a carbon-nitrogen ratio, which tells you whether there may be a protein in the sample. It can’t prove that the ratio is the result of a protein, but it can detect ratios that exclude the presence of a protein.
Other tests are necessary, such as the immunoassay. Immunology is one of the most sophisticated modern tools for finding out what proteins are in any given substance.
It is based on the amazing ability of immune systems to respond to any sort of foreign invader—or antigen—and quickly create a designer defending molecule—an antibody. Immune system cells are able to shuffle genes like a deck of cards to keep coming up with new hands, except the hands are molecules of different shapes. Every foreign molecule has areas on it, parts of its shape, on which a protein can latch, in a lock-and-key fashion. When an invader enters the body, whether it’s dust or a virus or a bacteria, the immune-cell factories start churning out antibodies of various sorts.
“You can actually take your body and inject it with material from Mars that no human being has ever seen and your body will make anti-Mars antibodies that are specific because of the flexibility of the immune system.
“So we take our dinosaur tissues that we have demineralized. We embed them. We section them very thin, and then we hit them with an antiavian collagen antibody. . . . If the antibody sees something it recognizes, it binds to it. We use a second antibody that recognizes the first one that has bound to tissues, and that second one has been tagged with a fluorescent label. We then put the section under a fluorescent microscope, and if it lights up it’s there and if it doesn’t light up, there’s nothing there.”
In the April 2007 issue of
Science,
Mary and six other scientists, including myself and John M. Asara of Harvard Medical, reported finding collagen in B. rex. She offered multiple lines of evidence, including electron microscopy, antibody tests, and mass spectrometry.