For their Chaucer analysis, Barbrook, Howe, and co-workers employed the same computer techniques used by evolutionary biologists to identify the most primitive organisms. They entered each of the Prologue's 850 lines from all 58 versions into the computer and searched for textual similarities and differences. Features common to most sources presumably reflected the original text, while variations that arose from copying errors or deliberate changes were used to construct a kind of genealogy of the manuscripts.
The British team found that 44 manuscripts fell neatly into 5 groups, evidently descended from 5 different copying sources. (The remaining 14 showed more extreme deviations and were eliminated from consideration.) One group of 11 manuscripts proved crucial, for it lay much closer to the presumed original than the others. Surprisingly, these 11 texts had received comparatively little study from literary scholars. “In time, this may lead editors to produce a radically different text of The Canterbury Tales,” Barbrook and colleagues concluded.
Computer-aided analysis of several dozen hand-copied manuscripts of Geoffrey Chaucer's Canterbury Tales points to several distinct groups of texts, denoted by letters. Each manuscript is represented as a line on this diagram; the length of the lines corresponds to the amount of deviation from a presumed primary source. Of these, the groups marked “O” appear to be closest to Chaucer's lost original (after Barbrook et al., 1998).
Molecular phylogenists adopt the same analytical approach. Their “texts” consist of strands of genetic molecules—DNA or its close cousin, RNA—with long sequences of the genetic letters. Thanks to new, rapid sequencing techniques, numerous genomes have been documented, including more than a hundred genomes of various microbes. Phylogenists use powerful computer algorithms to compare their genetic texts.
The universally acknowledged pioneer of molecular phylogeny is University of Illinois geneticist Carl Woese, a shy and retiring maverick who had to wait many years for widespread scientific acceptance of his ideas. In a landmark 1977 article in the Proceedings of the National Academy of Sciences, “Phylogenetic structure of the prokaryotic domain: The primary kingdoms,” Woese and Illinois colleague George Fox uprooted what had become the firmly established tree of life. Prior to the “Woesian revolution,” most biologists recognized five kingdoms of living organisms: animals, plants, fungi, protoctists (complex single-celled organisms with a membrane-bounded nucleus), and bacteria (metabolically diverse organisms that lack a nucleus). Based on his laborious phylogenetic analyses (which were much more difficult in the 1970s than they are today), Woese realized that animals, plants, fungi, and eukaryotes are remarkably similar in their biochemical characteristics and so constitute just one domain of life, the Eukarya. Prokaryotes, on the other hand, displayed astonishing chemical diversity and fell into two distinct kingdoms, which he called Bacteria and Archaea. Bacteria were well known for their role in causing disease, but the Archaea, that as prokaryotes resemble other bacteria in many ways, constituted an unrecognized group of microbes. So contentious was this view of life that it took almost two decades to gain general acceptance.
In retrospect, the lateness of Woese's discovery should not seem too surprising. Most species in the two microbial kingdoms, Bacteria and Archaea, occur as nondescript microscopic spheres or rods that are virtually impossible to distinguish, even with sophisticated chemical and physiological tests. Consequently, previous workers lumped them all together. Only after the application of molecular phylogeny did the profound differences become obvious.
Woese's initial intention was to unravel aspects of evolution—to establish a top-down family tree of life and infer the complex history of branchings from parent to daughter species. This evolutionary pursuit led quickly to insights regarding the nature of the last common ancestor. He proposed, for example, that Archaea and Bacteria arose long before Eukarya.
Other discoveries followed. Many of the most primitive microbes are extremophiles that thrive at elevated temperature. To many researchers, Woese's discoveries thus lent credence to the idea of a deep, hot emergence of life. Other scientists, however, quickly took issue with that conclusion. Deep microbes might well have been the only survivors following a massive asteroid impact and thus, by default, became the last common ancestors of life on Earth today. But those heat-loving microbes might well have evolved from strains of earlier, surface-dwelling cells.
Recent studies point to other intriguing possible characteristics of the presumed last common ancestor. For example, many of the most primitive Archaea are autotrophs, organisms that synthesize their own biomolecules. However, many origin specialists, Stanley Miller and his colleagues among them, have long argued that the first cells must have been heterotrophs, which scavenge molecules from their environment. Once again, the controversy cannot be resolved by phylogenetic studies, for deep-dwelling autotrophs that evolved from surface heterotrophs might have been the sole survivors of a globe-sterilizing event.
Carl Woese compared the genetic sequence of many different organisms, especially microbes, to construct a phylogenetic tree of life. He found three distinct branches of life, including the previously unrecognized Archaea. The majority of the most deeply rooted organisms are extremophiles living at high temperature (after Pace, 1997).
Additional complexity arises from the tendency of microbes to swap sections of DNA. Remarkably, throughout the history of life, cells have picked up chunks of DNA from completely different species to form new, hybrid genomes. Thus the idealized view of an unbroken branching history of DNA evolution from parent to daughter is invalid. There is no single “last common ancestor.”
In spite of these uncertainties, molecular phylogeny provides invaluable information regarding the shared biochemical heritage of all cells—information that continues to inform origins research. Two observations stand out. First, all cells employ RNA to carry genetic information and assemble proteins. The earliest genetic mechanism thus points to an ancient “RNA World” (see Chapter 16). Second, at the heart of every cell's chemical machinery lies a simple metabolic strategy called the citric acid cycle, which is powered by simple chemical reactions. Any successful model of life's emergence must thus account for the origin of this cycle (see Chapter 15).
An important conclusion from these phylogenetic studies is that Earth's earliest cells were not so different from those of today. They probably relied on similar, though simpler, biochemical pathways. Those similarities provide a clear focus for experimental studies of emergent biochemistry.
The greatest challenge in understanding life's emergence lies in finding mechanisms by which just the right combination of smaller molecules was selected, concentrated, and organized into the larger macromolecular structures like RNA and in self-replicating cycles of molecules like the citric acid cycle. But regardless of how much organic stuff was made, the primordial ocean—with an estimated volume greater than 320 cubic miles—formed a hopelessly dilute soup in which it would have been all but impossible for the right combination of molecules to bump into one another and make anything useful in the chemical path to life. Complex emergent systems require a minimum concentration of interacting agents. Many scientists have therefore settled on an obvious solution: Focus on surfaces, where molecules tend to concentrate.
Interesting chemistry takes place on surfaces where two different materials meet and molecules often congregate. The surface of the ocean, where air meets water, is one such promising place. Perhaps a primordial “oil slick” concentrated organic molecules. Evaporating tidal pools, where rock and water meet and cycles of evaporation concentrate stranded chemicals, represent another appealing location for origin-of-life chemistry. Deep within the crust and in hydrothermal volcanic zones, mineral surfaces may have played a similar role, selecting, concentrating, and organizing molecules on their periodic, crystalline surfaces. Whatever the mise-en-scène, a surface seems a logical site for life's origin.
But just suppose a collection of organic molecules could organize themselves in such a
way that they provided their own surface? Now that would be a trick worth learning!
11
Isolation
The self-assembly process seems to defy our intuitive expectation from the laws of physics that everything on average becomes more disordered.
David Deamer, 2003
Water provides the universal medium for life. All known cells are mostly water on the inside, and most are surrounded by water on the outside. That aquatic lifestyle poses a problem, however, because water is one of the best solvents. You don't want your body's cells to dissolve every time you take a bath. Life had to develop an insoluble protective membrane, but what chemical to use?
Lipid molecules, which feature hydrocarbon chains (a row of carbon atoms surrounded by hydrogen atoms), provide the perfect answer to the problem. Lipids, including various fats, oils, and waxes, are characterized by their insolubility in water—oil and water don't mix. The special phospholipid molecules that form most modern cell membranes are no exception. Each of these molecules is shaped something like a tiny bobby pin, with two long hydrocarbon chains of atoms attached to a rounded end. The two exposed hydrocarbon chains are hydrophobic (“water hating”), so most of the elongated molecule is water-repellent. By contrast, the rounded end incorporates a phosphate group (phosphorus and oxygen atoms); that hydrophilic (“water loving”) end attracts water. Such molecules, with both hydrophobic and hydrophilic regions, are called amphiphiles.
SELF-ORGANIZATION
When placed in water, amphiphilic lipids deal with their love-hate relationship in a remarkable way. All natural systems tend to rearrange themselves to reduce their total energy content: A tightly stretched elastic band snaps, a precariously perched boulder tumbles to the valley below, a firecracker explodes. By the same token, a solution of lipid molecules searches for a state of lower energy in which only the hydrophilic phosphate ends contact water. In the early 1960s, Alec Bangham, a biophysicist from Cambridge, England, discovered that lipids that were extracted from egg yolk and immersed in water spontaneously organized themselves into tiny spheres—structures now known as vesicles.
The energy-reducing strategy employed by the molecules that form cell membranes is nothing short of magical. Millions of individual amphiphilic molecules quickly clump together, forming a smooth, flexible double layer of lipids—a lipid bilayer. The resilient lipid bilayer provides a simple and elegant solution to the phospholipid's ambivalence toward water. All of the hydrophobic chains of atoms point toward the middle of the structure, well away from water, while the hydrophilic phosphate ends all wound up on the outside of the cell facing the wet environment or on the inside facing the water-based contents of the cell. This arrangement accomplishes the vital functions of holding the cell together while separating its inside from the outside.
Life has perfected this task of separating the inside from the outside, but could such an emergent, self-organizing process have arisen naturally in the lifeless prebiotic soup? The answer, once again, is to be found in the laboratory. Some amazing experiments have been performed by the Swiss biochemist Pier Luigi Luisi, who has spent decades studying lipid self-organization.
Not only can Luisi and co-workers form vesicles with ease, but they also demonstrate that these structures can grow, gradually incorporating new lipid molecules from solution. They've also shown that vesicles are autocatalytic—that is, they can act as templates that trigger the formation of more vesicles. And, under the proper circumstances, vesicles can even divide—a kind of self-replication.
These intriguing emergent behaviors have led Luisi to propose the “Lipid World” scenario for life's origin. In this conceptually simple model, prebiotic lipids formed abundantly on Earth and in space. Once in solution, these lipids self-organized into cell-like vesicles that captured a primitive genetic molecule, some early, simpler version of DNA or RNA. Now the Swiss team has set its sights on incorporating self-replicating pieces of RNA into self-replicating vesicles, perhaps even to make the first synthetic life-form. It hasn't happened yet, but the chemical pieces are close to falling into place.
Cell membranes are formed from amphiphiles, which are elongated molecules that have both water-attracting and water-repellent ends (A). When placed in water, these molecules self-organize into a bilayer (B), which can form a spherical vesicle (C).
MEMBRANES FROM SPACE
In the relatively brief history of origins research, a mere handful of experiments may be counted as classics. Louis Pasteur's refutation of spontaneous generation and Stanley Miller's electric-spark synthesis experiments have achieved that status, as has the novel vacuum-chamber research of Lou Allamandola and his colleagues at NASA Ames. Their results changed the way we think about life's origins. The same high regard is accorded David Deamer's remarkable discoveries of lipid self-organization in spaceborne molecules.
For almost three decades, Dave Deamer has been a popular professor of biochemistry in the University of California system. Lean, bright-eyed, with a neat graying beard and dark-rimmed glasses, he delivers lectures in a gentle and reassuring voice, like a scientific Mister Rogers. Listening to his low-key delivery, you might not guess that he is one of the world's most respected experts on the origin of life. [Plate 6]
Deamer caught the origins bug in 1975, when he took a sabbatical from the Davis campus of the University of California and went to study lipids with Alec Bangham at Cambridge. Their work revealed that the size and resilience of vesicles depends on the size and shape of the dissolved lipid molecules. In the course of these investigations, they realized that vesicles might have provided the first sheltering environment for life. If lipids existed in the early oceans, then prebiotic vesicles may have been abundant.
Deamer returned to Davis and continued this line of research, which led to his most famous experiment. That work, completed in 1988, focused on carbon-based molecules extracted from the Murchison meteorite. Ever since it landed in the Melbourne cow field in 1969, origins scientists all around the world had been bargaining and pleading for a piece of the prize. Deamer's precious 90-gram Murchison fragment, about the size of a walnut, arrived from the Field Museum in Chicago. Dave and his collaborator, chemist Richard Pashley of the Australian National University, went straight to work. Their focus was the lipids, essential biomolecules but ones that did not seem to be produced in sufficient abundance by Miller's spark process. Perhaps, they thought, carbonaceous chondrites provided those necessary raw materials for life's membranes.
Deamer and Pashley ran their sample through a series of chemical steps to break apart the dense black meteoric mass into chemically distinct fractions—steps that in some ways mimicked millennia of chemical weathering processes on the primitive Earth. Whatever molecules they found were thus likely to have occurred on the prebiotic Earth, as well. First they pulverized a portion of the meteorite into fine black powder. Their straightforward procedure involved grinding the rock while it was submerged in a liquid mixture of water, alcohol, and chloroform. These solvents don't affect the crystalline minerals that form the bulk of the Murchison, but they do dissolve different suites of interesting organic molecules. After several minutes of grinding, Deamer and Pashley poured their fine-grained slurry into a test tube, placed it into a centrifuge, and let it spin.
In the centrifuge, the pulverized meteorite solution rapidly separated into three fractions. A small pile of dense mineral fragments settled to the bottom of the tube, to be set aside just in case more studies were required. On top was a layer of water–alcohol solution, which dissolved and concentrated amino acids, sugars, and a variety of other water-soluble organic species. This fraction, too, was set aside. In the middle was a layer of chloroform, an effective solvent for any lipids the meteorite might hold. They found that the chloroform fraction had extracted more than a tenth of a percent of the meteorite fragment's mass—a surprisingly high concentration of tantalizing organic species.
Further separation was performed using chromatography. Following muc
h the same protocols as Stanley Miller had employed decades earlier to separate his amino acids, Deamer and Pashley evaporated a portion of their chloroform sample to reveal a yellowish-brown concentrated solution. They placed a drop of this concentrate on the corner of a 4 × 4-inch glass plate that had been coated with a soft, porous white powder (an effective replacement for the older-style chromatography paper). They used ether, a colorless strong-smelling solvent, for the first chromatographic stage, stretching the dried dot into a streak. Then they rotated the plate 90 degrees and used chloroform to spread the streak into a distinctive two-dimensional array of compounds.
Viewed in daylight, the dried glass plate was unimpressive, with only the original brownish spot and a few faint yellowish areas nearby. But Deamer knew that many otherwise invisible compounds fluoresce brightly under “black light.” When he darkened the room and shone an ultraviolet lamp on the plate, he was delighted to see a rich display of colors sweeping across it in a broad arc.
Deamer and Pashley identified a half-dozen distinct fluorescent regions, each with a different, as yet unknown suite of ancient cosmic organic molecules. They meticulously outlined each area by scratching the soft, powdery white surface; then they scraped off and collected powder from each of those areas into test tubes. A quick wash in chloroform was all that it took to recover the precious suites of Murchison molecules.
Genesis: The Scientific Quest for Life's Origin Page 17