Chapter 1:  The Evolution of the Cell

Simple Biological Molecules Can Form Under Prebiotic Conditions^{1, 2}

The conditions that existed on the earth in its first billion years are still a matter of dispute. Was the surface initially molten? Did the atmosphere contain ammonia, or methane? Everyone seems to agree, however, that the earth was a violent place with volcanic eruptions, lightning, and torrential rains. There was little if any free oxygen and no layer of ozone to absorb the ultraviolet radiation from the sun. The radiation, by its photochemical action, may have helped to keep the atmosphere rich in reactive molecules and far from chemical equilibrium.

Simple organic molecules (that is, molecules containing carbon) are likely to have been produced under such conditions. The best evidence for this comes from laboratory experiments. If mixtures of gases such as CO₂, CH₄, NH₃, and H₂ are heated with water and energized by electrical discharge or by ultraviolet radiation, they react to form small organic molecules - usually a rather small selection, each made in large amounts (Figure 1-1). Among these products are compounds, such as hydrogen cyanide (HCN) and formaldehyde (HCHO), that readily undergo further reactions in aqueous solution (Figure 1-2). Most important, representatives of most of the major classes of small organic molecules found in cells are generated, including amino acids, sugars, and the purines and pyrimidines required to make nucleotides.

Although such experiments cannot reproduce the early conditions on the earth exactly, they make it plain that the formation of organic molecules is surprisingly easy. And the developing earth had immense advantages over any human experimenter; it was very large and could produce a wide spectrum of conditions. But above all, it had much more time - tens to hundreds of millions of years. In such circumstances it seems very likely that, at some time and place, many of the simple organic molecules found in present-day cells accumulated in high concentrations.

Complex Chemical Systems Can Develop in an Environment That Is Far from Chemical Equilibrium

Simple organic molecules such as amino acids and nucleotides can associate to form polymers. One amino acid can join with another by forming a peptide bond, and two nucleotides can join together by a phosphodiester bond. The repetition of these reactions leads to linear polymers known as polypeptides and polynucleotides, respectively. In present-day living cells, large polypeptides - known as proteins - and polynucleotides - in the form of both ribonucleic acids (RNA) and deoxyribonucleic acids (DNA)are commonly viewed as the most important constituents. A restricted set of 20 amino acids constitute the universal building blocks of the proteins, while RNA and DNA molecules are constructed from just four types of nucleotides each. Although it is uncertain why these particular sets of monomers were selected for biosynthesis in preference to others that are chemically similar, we shall see that the chemical properties of the corresponding polymers suit them especially well for their specific roles in the cell.

The earliest polymers may have formed in any of several ways - for example, by the heating of dry organic compounds or by the catalytic activity of high concentrations of inorganic polyphosphates or other crude mineral catalysts. Under laboratory conditions the products of similar reactions are polymers of variable length and random sequence in which the particular amino acid or nucleotide added at any point depends mainly on chance (Figure 1-3). Once a polymer has formed, however, it can itself influence subsequent chemical reactions by acting as a catalyst.

The origin of life requires that in an assortment of such molecules there must have been some possessing, if only to a small extent, a crucial property: the ability to catalyze reactions that lead, directly or indirectly, to production of more molecules of the catalyst itself. Production of catalysts with this special self-promoting property would be favored, and the molecules most efficient in aiding their own production would divert raw materials from the production of other substances. In this way one can envisage the gradual development of an increasingly complex chemical system of organic monomers and polymers that function together to generate more molecules of the same types, fueled by a supply of simple raw materials in the environment. Such an autocatalytic system would have some of the properties we think of as characteristic of living matter: it would comprise a far from random selection of interacting molecules; it would tend to reproduce itself; it would compete with other systems dependent on the same feedstocks; and if deprived of its feedstocks or maintained at a wrong temperature that upsets the balance of reaction rates, it would decay toward chemical equilibrium and "die."

But what molecules could have had such autocatalytic properties? In present-day living cells the most versatile catalysts are polypeptides, composed of many different amino acids with chemically diverse side chains and, consequently, able to adopt diverse three-dimensional forms that bristle with reactive sites. But although polypeptides are versatile as catalysts, there is no known way in which one such molecule can reproduce itself by directly specifying the formation of another of precisely the same sequence.

Polynucleotides Are Capable of Directing Their Own Synthesis³

Polynucleotides have properties that contrast with those of polypeptides. They have more limited capabilities as catalysts, but they can directly guide the formation of exact copies of their own sequence. This capacity depends on complementary pairing of nucleotide subunits, which enables one polynucleotide to act as a template for the formation of another. In the simplest case a polymer composed of one nucleotide (for example, polycytidylic acid, or poly C) can line up the subunits required to make another polynucleotide (in this example, polyguanylic acid, or poly G) along its surface, thereby promoting their polymerization into poly G (Figure 1-4). Because C subunits preferentially bind G subunits, and vice versa, the poly-G molecule in turn can promote synthesis of more poly C.

Consider now a polynucleotide with a more complex sequence of subunits - specifically, a molecule of RNA strung together from four types of nucleotides, containing the bases uracil (U), adenine (A), cytosine (C), and guanine (G), arranged in some particular sequence. Because of complementary pairing between the bases A and U and between the bases G and C, this molecule, when added to a mixture of activated nucleotides under suitable conditions, will line them up for polymerization in a sequence complementary to its own. The resulting new RNA molecule will be rather like a mold of the original, with each A in the original corresponding to a U in the copy and so on. The sequence of nucleotides in the original RNA strand contains information that is, in essence, preserved in the newly formed complementary strands: a second round of copying, with the complementary strand as a template, restores the original sequence (Figure 1-5).

Such complementary templating mechanisms are elegantly simple, and they lie at the heart of information transfer processes in biological systems. Genetic information contained in every cell is encoded in the sequences of nucleotides in its polynucleotide molecules, and this information is passed on (inherited) from generation to generation by means of complementary base-pairing interactions.

Templating mechanisms, however, require additional catalysts to promote polymerization; without these the process is slow and inefficient and other, competing reactions prevent the formation of accurate replicas. Today, the catalytic functions that polymerize nucleotides are provided by highly specialized catalytic proteinsthat is, by enzymes. In the "prebiotic soup" primitive polypeptides might perhaps have provided some catalytic help. But molecules with the appropriate catalytic specificity would have remained rare unless the RNA itself were able somehow to reciprocate and favor their production. We shall come back to the reciprocal relationship between RNA synthesis and protein synthesis, which is crucially important in all living cells. But let us first consider what could be done with RNA itself, for RNA molecules can have a variety of catalytic properties, besides serving as templates for their own replication. In particular, an RNA molecule with an appropriate nucleotide sequence can act as catalyst for the accurate replication of another RNA molecule - the template - whose sequence can be arbitrary. The special versatility of RNA molecules is thought to have enabled them to play a central role in the origin of life.

Self-replicating Molecules Undergo Natural Selection^{3, 4}

RNA molecules are not just strings of symbols that carry information in an abstract way. They also have chemical personalities that affect their behavior. In particular, the specific sequence of nucleotides governs how the molecule folds up in solution. Just as the nucleotides in a polynucleotide can pair with free complementary nucleotides in their environment to form a new polymer, so they can pair with complementary nucleotide residues within the polymer itself. A sequence GGGG in one part of a polynucleotide chain can form a relatively strong association with a CCCC sequence in another region of the same molecule. Such associations produce complex three-dimensional patterns of folding, and the molecule as a whole takes on a specific shape that depends entirely on the sequence of its nucleotides (Figure 1-6).

The three-dimensional folded structure of a polynucleotide affects its stability, its actions on other molecules, and its ability to replicate, so that not all polynucleotide shapes will be equally successful in a replicating mixture. Moreover, errors inevitably occur in any copying process, and imperfect copies of the originals will be propagated. With repeated replication, therefore, new variant sequences of nucleotides will be continually generated. Thus, in laboratory studies, replicating systems of RNA molecules have been shown to undergo a form of natural selection in which different favorable sequences eventually predominate, depending on the exact conditions. Most important, RNA molecules can be selected for the ability to bind almost any other molecule specifically. This too has been shown, in experiments in vitro that begin with a preparation of short RNA molecules with random nucleotide sequences manufactured artificially. These are passed down a column packed with beads to which some chosen substance is bonded. RNA molecules that fail to bind to the chosen substance are washed through the column and discarded; those few that bind are retained and used as templates to direct production of multiple copies of their own sequences. This new RNA preparation, enriched in sequences that bind the chosen substance, is then used as the starting material for a repetition of the procedure. After several such cycles of selection and reproduction, the RNA is found to consist of multiple copies of a relatively small number of sequences, each of which binds the test substance quite specifically.

An RNA molecule therefore has two special characteristics: it carries information encoded in its nucleotide sequence that it can pass on by the process of replication, and it has a specific folded structure that enables it to interact selectively with other molecules and determines how it will respond to the ambient conditions. These two features - one informational, the other functional - are the two properties essential for evolution. The nucleotide sequence of an RNA molecule is analogous to the genotype - the hereditary information - of an organism. The folded three-dimensional structure is analogous to the phenotype - the expression of the genetic information on which natural selection operates.

Specialized RNA Molecules Can Catalyze Biochemical Reactions⁵

Natural selection depends on the environment, and for a replicating RNA molecule a critical component of the environment is the set of other RNA molecules in the mixture. Besides acting as templates for their own replication, these can catalyze the breakage and formation of covalent bonds between nucleotides. For example, some specialized RNA molecules can catalyze a change in other RNA molecules, cutting the nucleotide sequence at a particular point; and other types of RNA molecules spontaneously cut out a portion of their own nucleotide sequence and rejoin the cut ends (a process known as self-splicing). Each RNA-catalyzed reaction depends on a specific arrangement of atoms that forms on the surface of the catalytic RNA molecule (the ribozyme), causing particular chemical groups on one or more of its nucleotides to become highly reactive.

Certain catalytic activities would have had a cardinal importance in the primordial soup. Consider in particular an RNA molecule that helps to catalyze the process of templated polymerization, taking any given RNA molecule as template. (This ribozyme activity has been directly demonstrated in vitro, albeit in a rudimentary form.) Such a molecule, by acting on copies of itself, can replicate with heightened speed and efficiency (Figure 1-7A). At the same time, it can promote the replication of any other type of RNA molecules in its neighborhood (Figure 1-7B). Some of these may have catalytic actions that help or hinder the survival or replication of RNA in other ways. If beneficial effects are reciprocated, the different types of RNA molecules, specialized for different activities, may evolve into a cooperative system that replicates with unusually great efficiency.

Information Flows from Polynucleotides to Polypeptides⁶

There are strong suggestions, therefore, that between 3.5 and 4 billion years ago, somewhere on earth, self-replicating systems of RNA molecules, mixed with other organic molecules including simple polypeptides, began the process of evolution. Systems with different sets of polymers competed for the available precursor materials to construct copies of themselves, just as organisms now compete; success depended on the accuracy and the speed with which the copies were made and on the stability of those copies.

However, as we emphasized earlier, while the structure of polynucleotides is well suited for information storage and replication, their catalytic abilities are limited by comparison with those of polypeptides, and efficient replication of polynucleotides in modern cells is absolutely dependent on proteins. At the origin of life any polynucleotide that helped guide the synthesis of a useful polypeptide in its environment would have had a great advantage in the evolutionary struggle for survival.

But how could the information encoded in a polynucleotide specify the sequence of a polymer of a different type? Clearly, the polynucleotides must act as catalysts to join selected amino acids together. In present-day organisms a collaborative system of RNA molecules plays a central part in directing the synthesis of polypeptides - that is, protein synthesis - but the process is aided by other proteins synthesized previously. The biochemical machinery for protein synthesis is remarkably elaborate. One RNA molecule carries the genetic information for a particular polypeptide in the form of a code, while other RNA molecules act as adaptors, each binding a specific amino acid. These two types of RNA molecules form complementary base pairs with one another to enable sequences of nucleotides in the coding RNA molecule to direct the incorporation of specific amino acids held on the adaptor RNAs into a growing polypeptide chain. Precursors to these two types of RNA molecules presumably directed the first protein synthesis without the aid of proteins (Figure 1-7C).

Today, these events in the assembly of new proteins take place on the surface of ribosomes - complex particles composed of several large RNA molecules of yet another class, together with more than 50 different types of protein. In Chapter 5 we shall see that the ribosomal RNA in these particles plays a central catalytic role in the process of protein synthesis and forms more than 60% of the ribosome's mass. At least in evolutionary terms, it appears to be the fundamental component of the ribosome.

It seems likely, then, that RNA guided the primordial synthesis of proteins, perhaps in a clumsy and primitive fashion. In this way RNA was able to create tools - in the form of proteins - for more efficient biosynthesis, and some of these could have been put to use in the replication of RNA and in the process of tool production itself.

The synthesis of specific proteins under the guidance of RNA required the evolution of a code by which the polynucleotide sequence specifies the amino acid sequence that makes up the protein. This code - the genetic code - is spelled out in a "dictionary" of three-letter words: different triplets of nucleotides encode specific amino acids. The code seems to have been selected arbitrarily (subject to some constraints, perhaps); yet it is virtually the same in all living organisms. This strongly suggests that all present-day cells have descended from a single line of primitive cells that evolved the mechanism of protein synthesis.

Membranes Defined the First Cell⁷

One of the crucial events leading to the formation of the first cell must have been the development of an outer membrane. For example, the proteins synthesized under the control of a certain species of RNA would not facilitate reproduction of that species of RNA unless they remained in the neighborhood of the RNA; moreover, as long as these proteins were free to diffuse among the population of replicating RNA molecules, they could benefit equally any competing species of RNA that might be present. If a variant RNA arose that made a superior type of enzyme, the new enzyme could not contribute selectively to the survival of the variant RNA in its competition with its fellows. Selection of RNA molecules according to the quality of the proteins they generated could not occur efficiently until some form of compartment evolved to contain the proteins made by an RNA molecule and thereby make them available only to the RNA that had generated them (Figure 1-8).

The need for containment is easily fulfilled by another class of molecules that has the simple physicochemical property of being amphipathic, that is, consisting of one part that is hydrophobic (water insoluble) and another part that is hydrophilic (water soluble). When such molecules are placed in water, they aggregate, arranging their hydrophobic portions as much in contact with one another as possible and their hydrophilic portions in contact with the water. Amphipathic molecules of appropriate shape spontaneously aggregate to form bilayers, creating small closed vesicles whose aqueous contents are isolated from the external medium (Figure 1-9). The phenomenon can be demonstrated in a test tube by simply mixing phospholipids and water together: under appropriate conditions, small vesicles will form. All present-day cells are surrounded by a plasma membrane consisting of amphipathic molecules - mainly phospholipids - in this configuration; in cell membranes, the lipid bilayer also contains amphipathic proteins. In the electron microscope such membranes appear as sheets about 5 nm thick, with a distinctive three-layered appearance due to the tail-to-tail packing of the phospholipid molecules.

Presumably, the first membrane-bounded cells were formed by spontaneous assembly of phospholipid molecules from the prebiotic soup, enclosing a self-replicating mixture of RNA and other molecules. It is not clear at what point in the evolution of biological catalysts and protein synthesis this first occurred. In any case, once RNA molecules were sealed within a closed membrane, they could begin to evolve in earnest as carriers of genetic instructions: they could be selected not merely on the basis of their own structure, but also according to their effect on the other molecules in the same compartment. The nucleotide sequences of the RNA molecules could now be expressed in the character of a unitary living cell.

All Present-Day Cells Use DNA as Their Hereditary Material^{3, 6, 8}

The picture we have presented is, of course, speculative: there are no fossil records that trace the origins of the first cell. Nevertheless, there is persuasive evidence from present-day organisms and from experiments that the broad features of this evolutionary story are correct. The prebiotic synthesis of small molecules, the self-replication of catalytic RNA molecules, the translation of RNA sequences into amino acid sequences, and the assembly of lipid molecules to form membrane-bounded compartments - all presumably occurred to generate primitive cells 3.5 to 4 billion years ago.

It is useful to compare these early cells with the simplest and smallest present-day cells, the mycoplasmas. Mycoplasmas are small bacteria of a degenerate type that normally lead a parasitic existence in close association with animal or plant cells (Figure 1-10). Some have a diameter of about 0.3 mm and contain only enough nucleic acid to direct the synthesis of about 400 different proteins. Some of these proteins are enzymes, some are structural; some lie in the cell's interior, others are embedded in its membrane. Together they synthesize essential small molecules that are not available in the environment, redistribute the energy needed to drive biosynthetic reactions, and maintain appropriate conditions inside the cell.

The first cells on the earth were presumably less sophisticated than a mycoplasma and less efficient in reproducing themselves. There was, however, a more fundamental difference between these primitive cells and a mycoplasma, or indeed any other present-day cell: the hereditary information in all cells alive today is stored in DNA rather than in the RNA that is thought to have stored the hereditary information during the earliest stages of evolution. Both types of polynucleotides are found in present-day cells, but they function in a collaborative manner, each having evolved to perform specialized tasks. Small chemical differences fit the two kinds of molecules for distinct functions. DNA acts as the permanent repository of genetic information, and, unlike RNA, it is found in cells principally in a double-stranded form, composed of a pair of complementary polynucleotide molecules. This double-stranded structure makes DNA in cells more robust and stable than RNA; it also makes DNA relatively easy to replicate (as will be explained in Chapter 3) and permits a repair mechanism to operate that uses the intact strand as a template for the correction or repair of the associated damaged strand. DNA guides the synthesis of specific RNA molecules, again by the principle of complementary base-pairing, though now this pairing is between slightly different types of nucleotides. The resulting single-stranded RNA molecules then perform two primeval functions: they direct protein synthesis both as coding RNA molecules (messenger RNAs) and as RNA catalysts (ribosomal and other nonmessenger RNAs).

The suggestion, in short, is that RNA preceded DNA in evolution, having both genetic and catalytic properties; eventually, DNA took over the primary genetic function and proteins became the major catalysts, while RNA remained primarily as the intermediary connecting the two (Figure 1-11). With the advent of DNA cells were enabled to become more complex, for they could then carry and transmit an amount of genetic information greater than that which could be stably maintained in RNA molecules.

Summary

Living cells probably arose on earth about 3.5 billion years ago by spontaneous reactions between molecules in an environment that was far from chemical equilibrium. From our knowledge of present-day organisms and the molecules they contain, it seems likely that the development of the directly autocatalytic mechanisms fundamental to living systems began with the evolution of families of RNA molecules that could catalyze their own replication. With time, one of these families of cooperating RNA catalysts developed the ability to direct synthesis of polypeptides. Finally, as the accumulation of additional protein catalysts allowed more efficient and complex cells to evolve, the DNA double helix replaced RNA as a more stable molecule for storing the increased amounts of genetic information required by such cells.

Introduction

It is thought that all organisms living now on earth derive from a single primordial cell born more than 3 billion years ago. This cell, out-reproducing its competitors, took the lead in the process of cell division and evolution that eventually covered the earth with green, changed the composition of its atmosphere, and made it the home of intelligent life. The family resemblances among all organisms seem too strong to be explained in any other way. One important landmark along this evolutionary road occurred about 1.5 billion years ago, when there was a transition from small cells with relatively simple internal structures - the so-called procaryotic cells, which include the various types of bacteria - to a flourishing of larger and radically more complex eucaryotic cells such as are found in higher animals and plants.

Procaryotic Cells Are Structurally Simple but Biochemically Diverse¹⁰

Bacteria are the simplest organisms found in most natural environments. They are spherical or rod-shaped cells, commonly several micrometers in linear dimension (Figure 1-12). They often possess a tough protective coat, called a cell wall, beneath which a plasma membrane encloses a single cytoplasmic compartment containing DNA, RNA, proteins, and small molecules. In the electron microscope this cell interior appears as a matrix of varying texture without any obvious organized internal structure (see Figure 1-12B).

Bacteria are small and can replicate quickly, simply dividing in two by binary fission. When food is plentiful, "survival of the fittest" generally means survival of those that can divide the fastest. Under optimal conditions a single procaryotic cell can divide every 20 minutes and thereby give rise to 5 billion cells (approximately equal to the present human population on earth) in less than 11 hours. The ability to divide quickly enables populations of bacteria to adapt rapidly to changes in their environment. Under laboratory conditions, for example, a population of bacteria maintained in a large vat will evolve within a few weeks by spontaneous mutation and natural selection to utilize new types of sugar molecules as carbon sources.

In nature bacteria live in an enormous variety of ecological niches, and they show a corresponding richness in their underlying biochemical composition. Two distantly related groups can be recognized: the eubacteria, which are the commonly encountered forms that inhabit soil, water, and larger living organisms; and the archaebacteria, which are found in such incommodious environments as bogs, ocean depths, salt brines, and hot acid springs (Figure 1-13).

There are species of bacteria that can utilize virtually any type of organic molecule as food, including sugars, amino acids, fats, hydrocarbons, polypeptides, and polysaccharides. Some are even able to obtain their carbon atoms from CO₂ and their nitrogen atoms from N₂. Despite their relative simplicity, bacteria have existed for longer than any other organisms and still are the most abundant type of cell on earth.

Metabolic Reactions Evolve^{10, 11}

A bacterium growing in a salt solution containing a single type of carbon source, such as glucose, must carry out a large number of chemical reactions. Not only must it derive from the glucose the chemical energy needed for many vital processes, it must also use the carbon atoms of glucose to synthesize every type of organic molecule that the cell requires. These reactions are catalyzed by hundreds of enzymes working in reaction "chains" so that the product of one reaction is the substrate for the next; such enzymatic chains, called metabolic pathways, will be discussed in the following chapter.

Originally, when life began on earth, there was probably little need for such elaborate metabolic reactions. Cells with relatively simple chemistry could survive and grow on the molecules in their surroundings. But as evolution proceeded, competition for these limited natural resources would have become more intense. Organisms that had developed enzymes to manufacture useful organic molecules more efficiently and in new ways would have had a strong selective advantage. In this way the complement of enzymes possessed by cells is thought to have gradually increased, generating the metabolic pathways of present organisms. Two plausible ways in which a metabolic pathway could arise in evolution are illustrated in Figure 1-14.

If metabolic pathways evolved by the sequential addition of new enzymatic reactions to existing ones, the most ancient reactions should, like the oldest rings in a tree trunk, be closest to the center of the "metabolic tree," where the most fundamental of the basic molecular building blocks are synthesized. This position in metabolism is firmly occupied by the chemical processes that involve sugar phosphates, among which the most central of all is probably the sequence of reactions known as glycolysis, by which glucose can be degraded in the absence of oxygen (that is, anaerobically). The oldest metabolic pathways would have had to be anaerobic because there was no free oxygen in the atmosphere of the primitive earth. Glycolysis occurs in virtually every living cell and drives the formation of the compound adenosine triphosphate, or ATP, which is used by all cells as a versatile source of chemical energy. Certain thioester compounds play a fundamental role in the energy-transfer reactions of glycolysis and in a host of other basic biochemical processes in which two organic molecules (a thiol and a carboxylic acid) are joined by a high-energy bond involving sulfur (Figure 1-15). It has been argued that this simple but powerful chemical device is a relic of prebiotic processes, reflecting the reactions that occurred in the sulfurous, volcanic environment of the early earth, before even RNA had begun to evolve.

Linked to the core reactions of glycolysis are hundreds of other chemical processes. Some of these are responsible for the synthesis of small molecules, many of which in turn are utilized in further reactions to make the large polymers specific to the organism. Other reactions are used to degrade complex molecules, taken in as food, into simpler chemical units. One of the most striking features of these metabolic reactions is that they take place similarly in all kinds of organisms, suggesting an extremely ancient origin.

Evolutionary Relationships Can Be Deduced by Comparing DNA Sequences¹²

The enzymes that catalyze the fundamental metabolic reactions, while continuing to serve the same essential functions, have undergone progressive modifications as organisms have evolved into divergent forms. For this reason the amino acid sequence of the same type of enzyme in different living species provides a valuable indication of the evolutionary relationship between these species. The evidence obtained closely parallels that from other sources, such as the fossil record. An even richer source of information is locked in the living cell in the sequences of nucleotides in DNA, and modern methods of analysis allow these DNA sequences to be determined in large numbers and compared between species. Comparisons of highly conserved sequences, which have a central function and therefore change only slowly during evolution, can reveal relationships between organisms that diverged long ago (Figure 1-16), while very rapidly evolving sequences can be used to determine how more closely related species evolved. It is expected that continued application of these methods will enable the course of evolution to be followed with unprecedented accuracy.

Cyanobacteria Can Fix CO₂ and N₂ ¹³

As competition for the raw materials for organic syntheses intensified, a strong selective advantage would have been gained by any organisms able to utilize carbon and nitrogen atoms (in the form of CO₂ and N₂) directly from the atmosphere. But while they are abundantly available, CO₂ and N₂ are also very stable. It therefore requires a large amount of energy as well as a number of complicated chemical reactions to convert them to a usable form - that is, into organic molecules such as simple sugars.

In the case of CO₂ the major mechanism that evolved to achieve this transformation was photosynthesis, in which radiant energy captured from the sun drives the conversion of CO₂ into organic compounds. The interaction of sunlight with a pigment molecule, chlorophyll, excites an electron to a more highly energized state. As the electron drops back to a lower energy level, the energy it gives up drives chemical reactions that are facilitated and directed by protein molecules.

One of the first sunlight-driven reactions was probably the generation of "reducing power." The carbon and nitrogen atoms in atmospheric CO₂ and N₂ are in an oxidized and inert state. One way to make them more reactive, so that they participate in biosynthetic reactions, is to reduce them - that is, to give them a larger number of electrons. This is achieved in several steps. In the first step electrons are removed from a poor electron donor and transferred to a strong electron donor by chlorophyll in a reaction that is driven by sunlight. The strong electron donor is then used to reduce CO₂ or N₂. Comparison of the mechanisms of photosynthesis in various present-day bacteria suggests that one of the first sources of electrons was H₂S, from which the primary waste product would have been elemental sulfur. Later the more difficult but ultimately more rewarding process of obtaining electrons from H₂O was accomplished, and O₂ was released in large amounts as a waste product.

Cyanobacteria (also known as blue-green algae) are today a major route by which both carbon and nitrogen are converted into organic molecules and thus enter the biosphere. They include the most self-sufficient organisms that now exist. Able to "fix" both CO₂ and N₂ into organic molecules, they are, to a first approximation, able to live on water, air, and sunlight alone; the mechanisms by which they do this have probably remained essentially constant for several billion years. Together with other bacteria that have some of these capabilities, they created the conditions in which more complex types of organisms could evolve: once one set of organisms had succeeded in synthesizing the whole gamut of organic cell components from inorganic raw materials, other organisms could subsist by feeding on the primary synthesizers and on their products.

Bacteria Can Carry Out the Aerobic Oxidation of Food Molecules¹³

Many people today are justly concerned about the environmental consequences of human activities. But in the past other organisms have caused revolutionary changes in the earth's environment (although very much more slowly). Nowhere is this more apparent than in the composition of the earth's atmosphere, which through oxygen-releasing photosynthesis was transformed from a mixture containing practically no molecular oxygen to one in which oxygen constitutes 21% of the total (Figure 1-17).

Since oxygen is an extremely reactive chemical that can interact with most cytoplasmic constituents, it must have been toxic to many early organisms, just as it is to many present-day anaerobic bacteria. However, this reactivity also provides a source of chemical energy, and, not surprisingly, this has been exploited by organisms during the course of evolution. By using oxygen, organisms are able to oxidize more completely the molecules they ingest. For example, in the absence of oxygen glucose can be broken down only to lactic acid or ethanol, the end products of anaerobic glycolysis. But in the presence of oxygen glucose can be completely degraded to CO₂ and H₂O. In this way much more energy can be derived from each gram of glucose. The energy released in respiration - the aerobic oxidation of food molecules - is used to drive the synthesis of ATP in much the same way that photosynthetic organisms produce ATP from the energy of sunlight. In both processes there is a series of electron-transfer reactions that generates an H⁺ gradient between the outside and inside of a membrane-bounded compartment; the H⁺ gradient then serves to drive the synthesis of the ATP. Today, respiration is used by the great majority of organisms, including most procaryotes.

Eucaryotic Cells Contain Several Distinctive Organelles¹⁴

As molecular oxygen accumulated in the atmosphere, what happened to the remaining anaerobic organisms with which life had begun? In a world that was rich in oxygen, which they could not use, they were at a severe disadvantage. Some, no doubt, became extinct. Others either developed a capacity for respiration or found niches in which oxygen was largely absent, where they could continue an anaerobic way of life. Others became predators or parasites on aerobic cells. And some, it seems, hit upon a strategy for survival more cunning and vastly richer in implications for the future: they are believed to have formed an intimate association with an aerobic type of cell, living with it in symbiosis. This is the most plausible explanation for the metabolic organization of present-day cells of the eucaryotic type (Panel 1-1, pp. 18-19) with which this book will be chiefly concerned.

Eucaryotic cells, by definition and in contrast to procaryotic cells, have a nucleus (caryon in Greek), which contains most of the cell's DNA, enclosed by a double layer of membrane (Figure 1-18). The DNA is thereby kept in a compartment separate from the rest of the contents of the cell, the cytoplasm, where most of the cell's metabolic reactions occur. In the cytoplasm, moreover, many distinctive organelles can be recognized. Prominent among these are two types of small bodies, the chloroplasts and mitochondria (Figures 1-19 and 1-20). Each of these is enclosed in its own double layer of membrane, which is chemically different from the membranes surrounding the nucleus. Mitochondria are an almost universal feature of eucaryotic cells, whereas chloroplasts are found only in those eucaryotic cells that are capable of photosynthesis - that is, in plants but not in animals or fungi. Both organelles almost certainly have a symbiotic origin.

Eucaryotic Cells Depend on Mitochondria for Their Oxidative Metabolism¹⁵

Mitochondria show many similarities to free-living procaryotic organisms: for example, they often resemble bacteria in size and shape, they contain DNA, they make protein, and they reproduce by dividing in two. By breaking up eucaryotic cells and separating their component parts, it is possible to show that mitochondria are responsible for respiration and that this process occurs nowhere else in the eucaryotic cell. Without mitochondria the cells of animals and fungi would be anaerobic organisms, depending on the relatively inefficient and antique process of glycolysis for their energy. Many present-day bacteria respire like mitochondria, and it seems probable that eucaryotic cells are descendants of primitive anaerobic organisms that survived, in a world that had become rich in oxygen, by engulfing aerobic bacteria - keeping them in symbiosis for the sake of their capacity to consume atmospheric oxygen and produce energy. Certain present-day microorganisms offer strong evidence of the feasibility of such an evolutionary sequence. There are several hundred species of single-celled eucaryotes that resemble the hypothetical ancestral eucaryote in that they live in oxygen-poor conditions (in the guts of animals, for example) and lack mitochondria altogether. Comparative nucleotide sequence analyses have revealed that at least two groups of these organisms, the diplomonads and the microsporidia, diverged very early from the line leading to other eucaryotic cells (Figure 1-21). There is another eucaryote, the amoeba Pelomyxa palustris, that, while lacking mitochondria, nevertheless carries out oxidative metabolism by harboring aerobic bacteria in its cytoplasm in a permanent symbiotic relationship. Diplomonads and microsporidia, on the one hand, and Pelomyxa, on the other, therefore resemble two proposed stages in the evolution of eucaryotes such as ourselves.

Acquisition of mitochondria must have had many repercussions. The plasma membrane, for example, is heavily committed to energy metabolism in procaryotic cells but not in eucaryotic cells, where this crucial function has been relegated to the mitochondria. It seems likely that the separation of functions left the eucaryotic plasma membrane free to evolve important new features. In particular, because eucaryotic cells need not maintain a large H⁺ gradient across their plasma membrane, as required for ATP production in procaryotes, it became possible to use controlled changes in the ion permeability of the plasma membrane for cell-signaling purposes. Thus, a variety of ion channels appeared in the eucaryotic plasma membrane. Today, these channels mediate the elaborate electrical signaling processes in higher organisms - notably in the nervous system -and they control much of the behavior of single-celled free-living eucaryotes such as protozoa (see below).

Chloroplasts Are the Descendants of an Engulfed Procaryotic Cell¹⁶

Chloroplasts carry out photosynthesis in much the same way as procaryotic cyanobacteria, absorbing sunlight in the chlorophyll attached to their membranes. Some bear a close structural resemblance to the cyanobacteria, being similar in size and in the way that their chlorophyll-bearing membranes are stacked in layers (see Figure 1-20). Moreover, chloroplasts reproduce by dividing, and they contain DNA that is nearly indistinguishable in nucleotide sequence from portions of a bacterial chromosome. All this strongly suggests that chloroplasts share a common ancestry with cyanobacteria and evolved from procaryotes that made their home inside eucaryotic cells. These procaryotes performed photosynthesis for their hosts, who sheltered and nourished them. Symbiosis of photosynthetic cells with other cell types is, in fact, a common phenomenon, and some present-day eucaryotic cells contain authentic cyanobacteria (Figure 1-22).

Figure 1-23 shows the evolutionary origins of the eucaryotes according to the symbiotic theory. It must be stressed, however, that mitochondria and chloroplasts show important differences from, as well as similarities to, present-day aerobic bacteria and cyanobacteria. Their quantity of DNA is very small, for example, and most of the molecules from which they are constructed are synthesized elsewhere in the eucaryotic cell and imported into the organelle. Although there is good evidence that they originated as symbiotic bacteria, they have undergone large evolutionary changes and have become greatly dependent on - and subject to control by - their host cells.

The major existing eucaryotes have in common both mitochondria and a whole constellation of other features that distinguish them from procaryotes (Table 1-1). These function together to give eucaryotic cells a wealth of different capabilities, and it is debatable which of them evolved first. But the acquisition of mitochondria by an anaerobic eucaryotic cell must have been a crucial step in the success of the eucaryotes, providing them with the means to tap an abundant source of energy to drive all their complex activities.

Eucaryotic Cells Contain a Rich Array of Internal Membranes

Eucaryotic cells are usually much larger in volume than procaryotic cells, commonly by a factor of 1000 or more, and they carry a proportionately larger quantity of most cellular materials; for example, a human cell contains about 1000 times as much DNA as a typical bacterium. This large size creates problems. Since all the raw materials for the biosynthetic reactions occurring in the interior of a cell must ultimately enter and leave by passing through the plasma membrane covering its surface, and since the membrane is also the site of many important reactions, an increase in cell volume requires an increase in cell surface. But it is a fact of geometry that simply scaling up a structure increases the volume as the cube of the linear dimension while the surface area increases only as the square. Therefore, if the large eucaryotic cell is to keep as high a ratio of surface to volume as the procaryotic cell, it must supplement its surface area by means of convolutions, infoldings, and other elaborations of its membrane.

This probably explains in part the complex profusion of internal membranes that is a basic feature of all eucaryotic cells. Membranes surround the nucleus, the mitochondria, and (in plant cells) the chloroplasts. They form a labyrinthine compartment called the endoplasmic reticulum (Figure 1-24), where lipids and proteins of cell membranes, as well as materials destined for export from the cell, are synthesized. They also form stacks of flattened sacs constituting the Golgi apparatus (Figure 1-25), which is involved in the modification and transport of the molecules made in the endoplasmic reticulum. Membranes surround lysosomes, which contain stores of enzymes required for intracellular digestion and so prevent them from attacking the proteins and nucleic acids elsewhere in the cell. In the same way membranes surround peroxisomes, where dangerously reactive hydrogen peroxide is generated and degraded during the oxidation of various molecules by O₂. Membranes also form small vesicles and, in plants, a large liquid-filled vacuole. All these membrane-bounded structures correspond to distinct internal compartments within the cytoplasm. In a typical animal cell these compartments (or organelles) occupy nearly half the total cell volume. The remaining compartment of the cytoplasm, which includes everything other than the membrane-bounded organelles, is usually referred to as the cytosol.

All of the aforementioned membranous structures lie in the interior of the cell. How, then, can they help to solve the problem we posed at the outset and provide the cell with a surface area that is adequate to its large volume? The answer is that there is a continual exchange between the internal membrane-bounded compartments and the outside of the cell, achieved by endocytosis and exocytosis, processes unique to eucaryotic cells. In endocytosis portions of the external surface membrane invaginate and pinch off to form membrane-bounded cytoplasmic vesicles that contain both substances present in the external medium and molecules previously adsorbed on the cell surface. Very large particles or even entire foreign cells can be taken up by phagocytosis - a special form of endocytosis. Exocytosis is the reverse process, whereby membrane-bounded vesicles inside the cell fuse with the plasma membrane and release their contents into the external medium. In this way membranes surrounding compartments deep inside the cell serve to increase the effective surface area of the cell for exchanges of matter with the external world.

As we shall see in later chapters, the various membranes and membrane-bounded compartments in eucaryotic cells have become highly specialized - some for secretion, some for absorption, some for specific biosynthetic processes, and so on.

Eucaryotic Cells Have a Cytoskeleton

The larger a cell is, and the more elaborate and specialized its internal structures, the greater is its need to keep these structures in their proper places and to control their movements. All eucaryotic cells have an internal skeleton, the cytoskeleton, that gives the cell its shape, its capacity to move, and its ability to arrange its organelles and transport them from one part of the cell to another. The cytoskeleton is composed of a network of protein filaments, two of the most important of which are actin filaments (Figure 1-26) and microtubules. These two must date from a very early epoch in evolution since they are found almostunchanged in all eucaryotes. Both are involved in the generation of cellular movements. Actin filaments enable individual eucaryotic cells to crawl about, for example, and they participate in the contraction of muscle in animals; microtubules are the main structural and force-generating elements in cilia and flagella - the long projections on some cell surfaces that beat like whips and serve as instruments of propulsion.

Actin filaments and microtubules are also essential for the internal movements that occur in the cytoplasm of all eucaryotic cells. Thus microtubules in the form of a mitotic spindle are a vital part of the usual machinery for partitioning DNA equally between the two daughter cells when a eucaryotic cell divides. Without microtubules, therefore, the eucaryotic cell could not reproduce. In this and other examples movement by free diffusion would be either too slow or too haphazard to be useful. In fact, most of the organelles in a eucaryotic cell appear to be attached, directly or indirectly, to the cytoskeleton and, when they move, to be propelled along cytoskeletal tracks.

Protozoa Include the Most Complex Cells Known¹⁷

The complexity that can be achieved by a single eucaryotic cell is nowhere better illustrated than in the free-living, single-celled eucaryotes known as protists (Figure 1-27). These are evolutionarily diverse (see Figure 1-16) and exhibit a bewildering variety of different forms and behaviors: they can be photosynthetic or carnivorous, motile or sedentary. Their anatomy is often complex and includes such structures as sensory bristles, photoreceptors, flagella, leglike appendages, mouth parts, stinging darts, and musclelike contractile bundles. Although they are single cells, protists, especially the larger and more active types known as protozoa, can be as intricate and versatile as many multicellular organisms. This is particularly well illustrated by the group known as ciliates.

Didinium is a carnivorous ciliate. It has a globular body, about 150 mm in diameter, encircled by two fringes of cilia; its front end is flattened except for a single protrusion rather like a snout (Figure 1-28). Didinium swims around in the water at high speed by means of the synchronous beating of its cilia. When it encounters a suitable prey, usually another type of protozoan, such as a Paramecium, it releases numerous small paralyzing darts from its snout region. Then the Didinium attaches to and devours the Paramecium, inverting like a hollow ball to engulf the other cell, which is as large as itself. Most of this complex behavior - swimming, and paralyzing and capturing its prey - is generated by the cytoskeletal structures lying just beneath the plasma membrane. Included in this cell cortex, for example, are the parallel bundles of microtubules that form the core of each cilium and enable it to beat.

Predatory behavior of this sort and the set of features on which it depends - large size, the capacity for phagocytosis, and the ability to move in pursuit of prey - are peculiar to eucaryotes. Indeed, it is probable that these features came very early in eucaryotic evolution, making possible the subsequent capture of bacteria and their domestication as mitochondria and chloroplasts.

In Eucaryotic Cells the Genetic Material Is Packaged in Complex Ways

Eucaryotic cells contain a very large quantity of DNA. In human cells, for example, there is about 1000 times more DNA than in typical bacteria. The length of DNA in eucaryotic cells is so great that the risk of entanglement and breakage becomes severe. Probably for this reason, proteins unique to eucaryotes, the histones, have evolved to bind to the DNA and wrap it up into compact and manageable chromosomes (Figure 1-29). Tight packaging of the DNA in chromosomes is an essential part of the preparation for cell division in eucaryotes (Figure 1-30). All eucaryotes (with minor exceptions) have histones bound to their DNA, and the importance of these proteins is reflected in their remarkable conservation in evolution: several of the histones of a pea plant are almost exactly the same, amino acid for amino acid, as those of a cow.

The membranes enclosing the nucleus in eucaryotic cells further protect the structure of the DNA and its associated control machinery, sheltering them from entanglement with the moving cytoskeleton and from many of the chemical changes that take place in the cytoplasm. They also allow the segregation of two crucial steps in the expression of genetic information: (1) the copying of DNA sequences into RNA sequences (DNA transcription) and (2) the use of these RNA sequences, in turn, to direct the synthesis of specific proteins (RNA translation). In procaryotic cells there is no compartmentalization of these processes - the translation of RNA sequences into protein begins as soon as they are transcribed, even before their synthesis is completed. In eucaryotes, however (except in mitochondria and chloroplasts, which in this respect as in others are closer to bacteria), the two steps in the path from gene to protein are kept strictly separate: transcription occurs in the nucleus, translation in the cytoplasm. The RNA has to leave the nucleus before it can be used to guide protein synthesis. While in the nucleus it undergoes elaborate changes in which some parts of the RNA molecule are discarded and other parts are modified (RNA processing).

Because of these complexities, the genetic material of a eucaryotic cell offers many more opportunities for control than are present in bacteria.

Summary

Present-day living cells are classified as procaryotic (bacteria and their close relatives) or eucaryotic. Although they have a relatively simple structure, procaryotic cells are biochemically versatile and diverse: for example, all of the major metabolic pathways can be found in bacteria, including the three principal energy-yielding processes of glycolysis, respiration, and photosynthesis. Eucaryotic cells are larger and more complex than procaryotic cells and contain more DNA, together with components that allow this DNA to be handled in elaborate ways. The DNA of the eucaryotic cell is enclosed in a membrane-bounded nucleus, while the cytoplasm contains many other membrane-bounded organelles, including mitochondria, which carry out the oxidation of food molecules, and, in plant cells, chloroplasts, which carry out photosynthesis. Mitochondria and chloroplasts are almost certainly the descendants of earlier procaryotic cells that established themselves as internal symbionts of a larger anaerobic cell. Eucaryotic cells are also unique in containing a cytoskeleton of protein filaments that helps organize the cytoplasm and provides the machinery for movement.

Introduction

Single-cell organisms, such as bacteria and protozoa, have been so successful in adapting to a variety of different environments that they comprise more than half of the total biomass on earth. Unlike animals, many of these unicellular organisms can synthesize all of the substances they need from a few simple nutrients, and some of them divide more than once every hour. What, then, was the selective advantage that led to the evolution of multicellular organisms?

A short answer is that by collaboration and by division of labor it becomes possible to exploit resources that no single cell could utilize so well. This principle, applying at first to simple associations of cells, has been taken to an extreme in the multicellular organisms we see today. Multicellularity enables a plant, for example, to become physically large; to have roots in the ground, where one set of cells can take up water and nutrients; and to have leaves in the air, where another set of cells can efficiently capture radiant energy from the sun. Specialized cells in the stem of the plant form channels for transporting water and nutrients between the roots and the leaves. Yet another set of specialized cells forms a layer of epidermis to prevent water loss and to provide a protected internal environment (see Panel 1-2, pp. 28-29). The plant as a whole does not compete directly with unicellular organisms for its ecological niche; it has found a radically different way to survive and propagate.

As different animals and plants appeared, they changed the environment in which further evolution occurred. Survival in a jungle calls for different talents than survival in the open sea. Innovations in movement, sensory detection, communication, social organization - all enabled eucaryotic organisms to compete, propagate, and survive in ever more complex ways.

Single Cells Can Associate to Form Colonies

It seems likely that an early step in the evolution of multicellular organisms was the association of unicellular organisms to form colonies. The simplest way of achieving this is for daughter cells to remain together after each cell division. Even some procaryotic cells show such social behavior in a primitive form. Myxobacteria, for example, live in the soil and feed on insoluble organic molecules that they break down by secreting degradative enzymes. They stay together in loose colonies in which the digestive enzymes secreted by individual cells are pooled, thus increasing the efficiency of feeding (the "wolf-pack" effect). These cells indeed represent a peak of social sophistication among procaryotes, for when food supplies are exhausted, the cells aggregate tightly together and form a multicellular fruiting body (Figure 1-31), within which the bacteria differentiate into spores that can survive even in extremely hostile conditions. When conditions are more favorable, the spores in a fruiting body germinate to produce a new swarm of bacteria.

Green algae (not to be confused with the procaryotic "blue-green algae" or cyanobacteria) are eucaryotes that exist as unicellular, colonial, or multicellular forms (Figure 1-32). Different species of green algae can be arranged in order of complexity, illustrating the kind of progression that probably occurred in the evolution of higher plants and animals. Unicellular green algae, such as Chlamydomonas, resemble flagellated protozoa except that they possess chloroplasts, which enable them to carry out photosynthesis. In closely related genera, groups of flagellated cells live in colonies held together by a matrix of extracellular molecules secreted by the cells themselves. The simplest species (those of the genus Gonium) have the form of a concave disc made of 4, 8, 16, or 32 cells. Their flagella beat independently, but since they are all oriented in the same direction, they are able to propel the colony through the water. Each cell is equivalent to every other, and each can divide to give rise to an entirely new colony. Larger colonies are found in other genera, the most spectacular being Volvox, some of whose species have as many as 50,000 or more cells linked together to form a hollow sphere. In Volvox the individual cells forming a colony are connected by fine cytoplasmic bridges so that the beating of their flagella is coordinated to propel the entire colony along like a rolling ball (see Figure 1-32). Within the Volvox colony there is some division of labor among cells, with a small number of cells being specialized for reproduction and serving as precursors of new colonies. The other cells are so dependent on one another that they cannot live in isolation, and the organism dies if the colony is disrupted.

The Cells of a Higher Organism Become Specialized and Cooperate

In some ways Volvox is more like a multicellular organism than a simple colony. All of its flagella beat in synchrony as it spins through the water, and the colony is structurally and functionally polarized and can swim toward a distant source of light. The reproductive cells are usually confined to one end of the colony, where they divide to form new miniature colonies, which are initially sheltered inside the parent sphere. Thus, in a primitive way, Volvox displays the two essential features of all multicellular organisms: its cells become specialized, and they cooperate. By specialization and cooperation the cells combine to form a coordinated single organism with more capabilities than any of its component parts.

Organized patterns of cell differentiation occur even in some procaryotes. For example, many kinds of cyanobacteria remain together after cell division, forming filamentous chains that can be as much as a meter in length. At regular intervals along the filament, individual cells take on a distinctive character and become able to incorporate atmospheric nitrogen into organic molecules. These few specialized cells perform nitrogen fixation for their neighbors and share the products with them. But eucaryotic cells appear to be very much better at this sort of organized division of labor; they, and not procaryotes, are the living units from which all the more complex multicellular organisms are constructed.

Multicellular Organization Depends on Cohesion Between Cells

To form a multicellular organism, the cells must be somehow bound together, and eucaryotes have evolved a number of different ways to satisfy this need. In Volvox, as noted above, the cells do not separate entirely at cell division but remain connected by cytoplasmic bridges. In higher plants the cells not only remain connected by cytoplasmic bridges (called plasmodesmata), they also are imprisoned in a rigid honeycomb of chambers walled with cellulose that the cells themselves have secreted (cell walls).

The cells of most animals do not have rigid walls, and cytoplasmic bridges are unusual. Instead, the cells are bound together by a relatively loose meshwork of large extracellular organic molecules (called the extracellular matrix) and by adhesions between their plasma membranes. Very often, side-to-side attachments between the cells hold them together to form a multicellular sheet, or epithelium.

Epithelial Sheets of Cells Enclose a Sheltered Internal Environment

Of all the ways in which animal cells are woven together into multicellular tissues, the epithelial arrangement is perhaps the most fundamentally important. The epithelial sheet has much the same significance for the evolution of complex multicellular organisms that the cell membrane has for the evolution of complex single cells.

The importance of epithelial sheets is well illustrated in the lowly group of animals known as coelenterates. The group includes sea anemones, jellyfish, and corals, as well as the small freshwater organism Hydra. Coelenterates are constructed from two layers of epithelium, the outer layer being the ectoderm, the inner being the endoderm. The endodermal layer surrounds a cavity, the coelenteron, in which food is digested (Figure 1-33). Among the endodermal cells are some that secrete digestive enzymes into the coelenteron, while other cells absorb and further digest the nutrient molecules that these enzymes release. By forming a tightly coherent epithelial sheet that prevents all these molecules from being lost to the exterior, the endodermal cells create for themselves an environment in the coelenteron that is suited to their own digestive tasks. Meanwhile, the ectodermal cells, facing the exterior, remain specialized for encounters with the outside world. In the ectoderm, for example, are cells that contain a poison capsule with a coiled dart that can be unleashed to kill the small animals on which Hydra feeds. The majority of other ectodermal and endodermal cells have musclelike properties, enabling Hydra to move, as a predator must.

Within the double layer of ectoderm and endoderm is another compartment, separate both from the coelenteron and from the outside world. Here nerve cells lie, occupying narrow enclosed spaces between the epithelial cells, below the external surface where the specialized cell junctions between the epithelial cells form an impermeable barrier. The animal can change its shape and move by contractions of the musclelike cells in the epithelia, and it is the nerve cells that convey electrical signals to control and coordinate these contractions (Figures 1-33, 1-34, and 1-35). As we shall see later, the concentrations of simple inorganic ions in the medium surrounding a nerve cell are crucial for its function. Most nerve cells - our own included - are designed to operate when bathed in a solution with an ionic composition roughly similar to that of seawater. This may well reflect the conditions under which the first nerve cells evolved. Most coelenterates still live in the sea, but not all. Hydra, in particular, lives in fresh water. It has evidently been able to colonize this new habitat only because its nerve cells are contained in a space that is sealed and isolated from the exterior within sheets of epithelial cells that maintain the internal environment necessary for nerve cell function.

Cell-Cell Communication Controls the Spatial Pattern of Multicellular Organisms¹⁹

The cells of Hydra are not only bound together mechanically and connected by junctions that seal off the interior from the exterior environment, they also communicate with one another along the length of the body. If one end of a Hydra is cut off, the remaining cells react to the absence of the amputated part by adjusting their characters and rearranging themselves so as to regenerate a complete animal. Evidently, signals pass from one part of the organism to the other, governing the development of its body pattern - with tentacles and a mouth at one end and a foot at the other. Moreover, these signals are independent of the nervous system. If a developing Hydra is treated with a drug that prevents nerve cells from forming, the animal is unable to move about, catch prey, or feed itself. Its digestive system still functions normally, however, so that it can be kept alive by anyone with the patience to stuff its normal prey into its mouth. In such force-fed animals the body pattern is maintained, and lost parts are regenerated just as well as in an animal that has an intact nervous system.

The vastly more complex higher animals have evolved from simpler ancestors resembling coelenterates, and these higher animals owe their complexity to more sophisticated exploitation of the same basic principles of cell cooperation that underlie the construction of Hydra. Epithelial sheets of cells line all external and internal surfaces in the body, creating sheltered compartments and controlled internal environments in which specialized functions are performed by differentiated cells. Specialized cells interact and communicate with one another, setting up signals to govern the character of each cell according to its place in the structure as a whole. To show how it is possible to generate multicellular organisms of such size, precision, and complexity as a tree, a fly, or a mammal, however, it is necessary to consider more closely the sequence of events in development.

Cell Memory Permits the Development of Complex Patterns

The cells of almost every multicellular organism are generated by repeated division from a single precursor cell; they constitute a clone. As proliferation continues and the clone grows, some of the cells, as we have seen, become differentiated from others, adopting a different structure, a different chemistry, and a different function, usually in response to cues from their neighbors. It is remarkable that eucaryotic cells and their progeny will usually persist in their differently specialized states even after the influences that originally directed their differentiation have disappeared - in other words, these cells have a memory. Consequently, their final character is not determined simply by their final environment, but rather by the entire sequence of influences to which the cells have been exposed in the course of development. Thus as the body grows and matures, progressively finer details of the adult body pattern become specified, creating an organism of gradually increasing complexity whose ultimate form is the expression of a long developmental history.

Basic Developmental Programs Tend to Be Conserved in Evolution²⁰

The final structure of an animal or plant reflects its evolutionary history, which, like development, presents a chronicle of progress from the simple to the complex. What then is the connection between the two perspectives, of evolution on the one hand and development on the other?

During evolution many of the developmental devices that evolved in the simplest multicellular organisms have been conserved as basic principles for the construction of their more complex descendants. We have already mentioned, for example, the organization of cells into epithelia. Some specialized cell types, such as nerve cells, are found throughout nearly the whole of the animal kingdom, from Hydra to humans. Molecular studies, to be discussed later in this book, reveal an astonishing number of developmental resemblances at a fundamental genetic level, even between species as remotely related as mammals and insects. In terms of anatomy, furthermore, early developmental stages of animals whose adult forms appear radically different are often surprisingly similar; it takes an expert eye to distinguish, for example, a young chick embryo from a young human embryo (Figure 1-36).

Such observations are not difficult to understand. Consider the process by which a new anatomical feature - say, an elongated beak - appears in the course of evolution. A random mutation occurs that changes the amino acid sequence of a protein or the timing of its synthesis and hence its biological activity. This alteration may, by chance, affect the cells responsible for the formation of the beak in such a way that they make one that is longer. But the mutation must also be compatible with the development of the rest of the organism; only then will it be propagated by natural selection. There would be little selective advantage in forming a longer beak if, in the process, the tongue was lost or the ears failed to develop. A catastrophe of this type is more likely if the mutation affects events occurring early in development than if it affects those near the end. The early cells of an embryo are like cards at the bottom of a house of cards - a great deal depends on them, and even small changes in their properties are likely to result in disaster. Fundamental steps appear to have been "frozen" into developmental processes, just as the genetic code or protein synthesis mechanisms have become frozen into the basic biochemical organization of the cell. In contrast, cells produced near the end of development (or produced early but forming accessory structures such as the placenta that are not incorporated in the adult body) have more freedom to change. It is presumably for this reason that the embryos of different species so often resemble each other in their early stages and, as they develop, seem sometimes to replay the steps of evolution.

The Cells of the Vertebrate Body Exhibit More Than 200 Different Modes of Specialization

The wealth of diverse specializations to be found among the cells of a higher animal is far greater than any procaryote can show. In a vertebrate more than 200 distinct cell types are plainly distinguishable, and many of these types of cells certainly include, under a single name, a large number of more subtly different varieties. Panel 1-3 (pp. 36-37) shows a small selection. In this profusion of specialized behaviors one can see displayed, in a single organism, the astonishing versatility of the eucaryotic cell. Much of our current knowledge of the general properties of eucaryotic cells has depended on the study of such specialized types of cells, because they demonstrate exceptionally well particular features on which all cells depend in some measure. Each feature and each organelle of the prototype that we have outlined in Panel 1-1 (pp. 18-19) is developed to an unusual degree or revealed with special clarity in one cell type or another. To take one arbitrary example, consider the neuromuscular junction, where just three types of cells are involved: a muscle cell, a nerve cell, and a Schwann cell. Each has a very different role (Figure 1-37):

1. The muscle cell has made contraction its specialty. Its cytoplasm is packed with organized arrays of protein filaments, including vast numbers of actin filaments. There are also many mitochondria interspersed among the protein filaments, supplying ATP as fuel for the contractile apparatus.

2. The nerve cell stimulates the muscle to contract, conveying an excitatory signal to the muscle from the brain or spinal cord. The nerve cell is therefore extraordinarily elongated: its main body, containing the nucleus, may lie a meter or more from the junction with the muscle. The cytoskeleton is consequently well developed so as to maintain the unusual shape of the cell and to transport materials efficiently from one end of the cell to the other. The most crucial specialization of the nerve cell, however, is its plasma membrane, which contains proteins that act as ion pumps and ion channels, causing a movement of ions that is equivalent to a flow of electricity. Whereas all cells contain such pumps and channels in their plasma membranes, the nerve cell has exploited them in such a way that a pulse of electricity can propagate in a fraction of a second from one end of the cell to the other, conveying a signal for action.

3. Lastly, Schwann cells are specialists in the mass production of plasma membrane, which they wrap around the elongated portion of the nerve cell, laying down layer upon layer of membrane like a roll of tape, to form a myelin sheath that serves as insulation.

Genes Can Be Switched On and Off

The various specialized cell types in a single higher plant or animal appear as different from one another as any cells could be. This seems paradoxical, since all of the cells in a multicellular organism are closely related, having recently descended from the same precursor cell - the fertilized egg. Common lineage implies similar genes; how then do the differences arise? In a few cases cell specialization involves the loss of genetic material. An extreme example is the mammalian red blood cell, which loses its entire nucleus in the course of differentiation. But the overwhelming majority of cells in most plant and animal species retain all of the genetic information contained in the fertilized egg. Specialization depends on changes in gene expression, not on the loss or acquisition of genes.

Even bacteria do not make all of their types of protein all of the time but are able to adjust the level of synthesis according to external conditions. Proteins required specifically for the metabolism of lactose, for example, are made by many bacteria only when this sugar is available for use; and when conditions are unfavorable for cell proliferation, some bacteria arrest most of their normal metabolic processes and form spores, which have tough, impermeable outer walls and a cytoplasm of altered composition.

Eucaryotic cells have evolved far more sophisticated mechanisms for controlling gene expression, and these affect entire systems of interacting gene products. Groups of genes are activated or repressed in response to both external and internal signals. Membrane composition, cytoskeleton, secretory products, even metabolism - all these and other features must change in a coordinated manner when cells become differentiated. The radical differences of character between cell types reflect stable changes in gene expression. The controls that bring about these changes have evolved in eucaryotes to a degree unmatched in procaryotes, defining the complex rules of cell behavior that can generate an organized multicellular organism from a single egg.

Sequence Comparisons Reveal Hundreds of Families of Homologous Genes^{12, 21}

To outward appearances, evolution has transformed the universe of living things to such a degree that they are no longer recognizable as relatives. A human being, a fly, a daisy, a yeast, a bacterium - they seem so different that it scarcely makes sense to compare them. Yet all are descendants of one ancestor, and as we probe their inner workings more and more deeply, we find more and more evidence of their common origins. We now know that the basic molecular machinery of life has been conserved to an extent that would surely have astonished the originators of the theory of evolution. As we have seen, all life forms have essentially the same chemistry, based on amino acids, sugars, fatty acids, and nucleotides; all synthesize these chemical constituents in an essentially similar way; all store their genetic information in DNA and express it through RNA and protein. But the degree of evolutionary conservatism becomes even more striking when we examine the detailed sequences of nucleotides in specific genes and of amino acids in specific proteins. The chances are that the bacterial enzyme catalyzing any particular common reaction, such as the splitting of a six-carbon sugar into two three-carbon sugars in glycolysis, will have an amino acid sequence (and a three-dimensional structure) unmistakably similar to the enzyme catalyzing the same reaction in human beings. The two enzymes - and, equivalently, the genes that specify them - not only have a similar function, but also almost certainly a common evolutionary origin. One can exploit these relationships to trace ancient evolutionary pathways; and by comparing gene sequences and recognizing homologies, one discovers hidden parallels and similarities between different organisms.

Family resemblances are also often found among genes coding for proteins that carry out related functions within a single organism. These genes are also evolutionarily related, and their existence reveals a basic strategy by which increasingly complex organisms have arisen: genes and portions of genes become duplicated, and the new copies then diverge from the old by mutation and recombination to serve new, additional purposes. In this way, starting from a relatively small set of genes in primitive cells, the more complex life forms have been able to evolve the more than 50,000 genes thought to be present in a higher animal or plant. From an understanding of one gene or protein, we consequently gain insight into a whole family of others homologous to it. Thus molecular biology both underscores the unity of the living world and gives us tools to discover the general mechanisms that underlie its endless variety of inventions.

In the next chapter we begin our account of these mechanisms with a discussion of the most basic components of the biological construction kit - the small molecules from which all larger components of living cells are made.

Summary

The evolution of large multicellular organisms depended on the ability of eucaryotic cells to express their hereditary information in many different ways and to function cooperatively in a single collective. In animals one of the earliest developments was probably the formation of epithelial cell sheets, which separate the internal space of the body from the exterior. In addition to epithelial cells, primitive differentiated cell types would have included nerve cells, muscle cells, and connective tissue cells, all of which can be found in very simple present-day animals. The evolution of higher animals and plants (Figure 1-38) depended on production of an increasing number of specialized cell types and more sophisticated methods of coordination among them, reflecting an increasingly elaborate system of controls over gene expression in the individual component cells.