The Chemical Composition of Cells

Water is the major component of living cells, but the amount varies greatly. Thus, the pig embryo is 97% water; at birth a new-born pig is only 89% water. A lean 45-kg pig may contain 67% water but a very fat 135-kg animal only 40% water. Similar variations are encountered with other constitutents. The water content of a tissue is often determined by thoroughly drying a weighed sample of tissue at low temperature in vacuum and then weighing it a second time. The solid material can then be extracted with a solvent that will dissolve out the fatty compounds. These are referred to collectively as lipids. After evaporation of the solvent the lipid residue may be weighed. By this procedure a young leafy vegetable might be found to contain 2–5% lipid on a dry weight basis. Even very lean meats contain 10–30% lipid. The residue remaining after removal of the lipid consists predominately of three groups of compounds: proteins, nucleic acids, and carbohydrates. Most of the nitrogen present in tissues is found in the proteins and the protein content is sometimes estimated by determining the percentage of nitrogen and multiplying by 6.25. In a young green plant, 20–30% of the dry matter may be protein, while in very lean meat it may reach 50–70%.
A dried tissue sample may be burned at a high temperature to an ash, which commonly amounts to 3–10% and is higher in specialized tissues such as bone. It is a measure of the inorganic constituents of tissues. The carbohydrate content can be estimated by the difference of the sum of lipid, protein, and ash from 100%. It amounts to 50–60% in young green plants and only 2–10% in typical animal tissues. In exceptional cases the carbohydrate content of animal tissues may be higher; the glycogen content of oysters is 28%. The amount of nucleic acid in tissues varies from 0.1% in yeast and 0.5–1% in muscle and in bacteria to 15–40% in thymus gland and sperm cells. In these latter materials of high nucleic acid content it is clear that multiplication of % N by 6.25 is not a valid measure of protein content. For diploid cells of the body the DNA content per cell is nearly constant. Table 1-4 compares the composition of a bacterium, of a green plant, and of an active animal tissue (rat liver). Although the solid matter of cells consists principally of C, H, O, N, S, and P, many other chemical elements are also present. Among the cations, Na+, K+, Ca2+, and Mg2+ are found in relatively large amounts. Thus, the body of a 70 kg person contains 1050 g Ca (mostly in the bones), 245 g K, 105 g Na, and 35 g Mg. Iron (3 g), zinc (2.3 g), and rubidium (1.2 g) are the next most abundant. Of these iron and zinc are essential to life but rubidium is probably not. It is evidently taken up by the body together with potassium. The other metallic elements in the human body amount to less than 1 g each, but at least seven of them play essential roles. They include copper (100 mg), manganese (20 mg), and cobalt (~5 mg). Others, such as chromium (<6 onblur="try {parent.deselectBloggerImageGracefully();} catch(e) {}" href="https://blogger.googleusercontent.com/img/b/R29vZ2xl/AVvXsEgvALacdidwP3KwCrgrKpGoaFSumV9DBTg2_Ug3l6JiSCyezVRcqQhZKKQuFG5rTAtMVWhMg4F6qVfkM7gY55AXuh6_8piCmXvb-GFGuCMQxOwOw3lnnLySMwWQ9IefASlU-GLg3l_gNrxZ/s1600/20.JPG">

Elements known to be essential to living things (after da Silva and Williams157). Essential elements are enclosed within shaded boxes. The 11 elements–C, H, O, N, S, P, Na, K, Mg, Ca, and Cl–make up 99.9% of the mass of a human being. An additional 13 are known to be essential for higher animals in trace amounts. Boron is essential to higher plants but apparently not to animals, microorganisms, or algae.

Higher Plants and Plant Tissues

Botanists recognize two divisions of higher plants. The Bryophyta or moss plants consist of the Musci (mosses) and Hepaticae (liverworts). These plants grow predominantly on land and are characterized by swimming sperm cells and a dominant gametophyte (haploid) phase. Tracheophyta, or vascular plants, contain conducting tissues. About 2 x 105 species are known. The ferns (class Filicineae, formerly Pteridophyta) are characterized by a dominant diploid plant and alternation with a haploid phase. Seed plants are represented by two classes: Gymnosperms (cone-bearing trees) and Angiosperms, the true flowering plants.
Genetically the simplest of the angiosperms is the little weed Arabidopsis thaliana, whose generation time is as short as five weeks. Its five chromosomes contain only 108 base pairs in all, the smallest known genome among angiosperms153 and one whose complete nucleotide sequence is being determined. Its biochemistry, physiology, and developmental biology are under intensive study. It may become the “fruit fly” of the plant kingdom. There are several kinds of plant tissues. Undifferentiated, embryonic cells found in rapidly growing regions of shoots and roots form the meristematic tissue. By differentiation, the latter yields the simple tissues, the parenchyma, collenchyma, and sclerenchyma. Parenchyma cells are among the most abundant and least specialized in plants. They give rise through further differentiation to the cambium layer, the growing layer of roots and stems. They also make up the pith or pulp in the center of stems and roots, where they serve as food storage cells. The collenchyma, present in herbs, is composed of elongated supporting cells and the sclerenchyma of woody plants is made up of supporting cells with hard lignified cell walls and a low water content. This tissue includes fiber cells, which may be extremely long; e.g., pine stems contain fiber cells of 40 µm diameter and 4 mm long. Two complex tissues, the xylem and phloem, provide the conducting network or “circulatory system” of plants. In the xylem or woody tissue, most of the cells are dead and the thick-walled tubes (tracheids) serve to transport water and dissolved minerals from the roots to the stems and leaves. The phloem cells provide the principal means of downward conduction of foods from the leaves. Phloem cells are joined end to end by sieve plates, so-called because they are perforated by numerous minute pores through which cytoplasm of adjoining sieve cells appears to be connected by strands 5–9 µm in diameter. Mature sieve cells have no nuclei, but each sieve cell is paired with a nucleated“companion” cell. Epidermal tissue of plants consists of flat cells, usually containing no chloroplasts, with a thick outer wall covered by a heavy waxy cuticle about 2 µm thick. Only a few specialized cells are found in the epidermis. Among them are the paired guard cells that surround the small openings known as stomata on the undersurfaces of leaves and control transpiration of water. Specialized cells in the root epidermis form root hairs, long extensions (~1 mm) of diameter 5–17 µm. Each hair is a single cell with the nucleus located near the tip. Figure 1-16 shows a section from a stem of a typical angiosperm. Note the thin cambium layer between the phloem and the xylem. Its cells continuously undergo differentiation to form new layers of xylem increasing the woody part of the stem. New phloem cells are also formed, and as the stem expands all of the tissues external to the cambium are renewed and the older cells are converted into bark.
Plant seeds consist of three distinct portions. The embryo develops from a zygote formed by fusion of a sperm nucleus originating from the pollen and an egg cell. The fertilized egg is surrounded in the gymnosperms by a nutritive layer or endosperm which is haploid and is derived from the same gametophyte tissue that produced the egg. In angiosperms two sperm nuclei form; one of these fertilizes the egg, while the other fuses with two haploid polar nuclei derived from the female gametophyte. (The polar nuclei are formed by the same mitotic divisions that formed the egg.) From this develops a 3n triploid endosperm.

Section of the stem of an angiosperm. Enlarged sections showing tubes of the phloem (left) and xylem (right). From S. Biddulph and O. Biddulph. Drawn by Bunji Tagawa.

Cell Types and Tissues

Isolated animal cells in tissue culture, no matter how highly differentiated, tend to revert quickly to one of three basic types known as epitheliocytes, mechanocytes, and amebocytes. Epitheliocytes are closely adherent cells derived from epithelial tissues and thought to be related in their origins to the two surface layers of the embryonic blastula. Mechanocytes, often called fibroblasts or fibrocytes, are derived from muscle, supporting, or connective tissue. Like the amebocytes, they arise from embryonic mesenchymal tissue cells that have migrated inward from the lower side of the blastula. Neurons, neuroglia, and lymphocytes are additional distinct cell types.
Tissues. Cells aggregate to form four major kinds of tissue. Epithelial tissues line the primary surfaces of the body: the skin, the digestive tract, urogenital tract, and glands. External skin is composed of flat platelike squamous epithelial cells whereas internal surfaces are often formed by colummar epithelial cells. Glands (sweat, oil, mammary, and internal secretory) as well as the sensory organs of the tongue, nose, and ear are all composed of epithelial cells. Epithelial cells are among the most highly polarized of cells. One side of each cell faces the outside, either air or water, while the other side is often directly against a basement membrane.
Supporting and connective tissues include the fatty adipose tissue as well as cartilage and bone. Both of the latter contain large amounts of intercellular material or ground substance consisting largely of complex polymers. Embryonic fibroblasts differentiate into white fibers, which produce collagen, and yellow fibers, which form elastin. The fibrils of both of these proteins are assembled in the intercellular space where they are embedded in the ground substance. Osteoblasts form bone by deposition of calcium phosphate in 3–7 µm thick layers within a ground substance that contains special proteins. A third tissue is muscle, which is classified into three types: striated (voluntary skeletal muscle), cardiac (involuntary striated muscle), and smooth (involuntary) muscle. There are two major groups of cells in nervous tissue, the fourth tissue type. Neurons are the actual conducting cells whose cell membranes
carry nerve impulses. Several kinds of glial cells lie between and around the neurons.
Blood cells. Blood and the linings of blood vessels may be regarded as a fifth tissue type. The human body contains 5 x 109 erythrocytes or red blood cells per ml, a total of 2.5 x 1013 cells in the five liters of blood present in the body. Erythrocytes are rapidly synthesized in the bone marrow. The nucleus is destroyed, leaving a cell almost completely filled with hemoglobin. With an average lifetime of 125 days, human red blood cells are destroyed by leukocytes in the spleen and liver. The white blood cells or leukocytes are nearly a thousandfold less numerous than red cells. About 7 x 106 cells are present per ml of blood. There are three types of leukocytes: lymphocytes (~26% of the total), monocytes (~7% of the total), and polymorphonuclear leukocytes or granulocytes (~70% of the total). Lymphocytes are about the same size as erythrocytes and are made in lymphatic tissue. Individual lymphocytes may survive for as long as ten years. They function in antibody formation and are responsible for maintenance of long-term immunity.
Monocytes, two times larger, are active in ingesting bacteria. These cells stay in the blood only a short time before they migrate into the tissues where they become macrophages, relatively fixed phagocytic cells. Macrophages not only phagocytize and kill invading bacteria, protozoa, and fungi but also destroy cancer cells. They also destroy damaged cells and cellular debris as part of the normal turnover of tissues. They play an essential role in the immune system by “processing” antigens and in releasing stimulatory proteins.
Granulocytes of diameter 9–12 µm are formed in the red bone marrow. Three types are distinguished by staining: neutrophils, eosinophils, and basophils. Neutrophils are the most numerous phagocytic cells of our blood and provide the first line of defense against bacterial infections. The functions of eosinophils and basophils are less well understood. The number of eosinophils rises during attacks of hay fever and asthma and under the influence of some parasites, while the basophil count is increased greatly in leukemia and also by inflammatory diseases. Granules containing histamine, heparin, and leukotrienes are present in the basophils. Blood platelets or thrombocytes are tiny (2–3 µm diameter) cell-like bodies essential for rapid coagulation of blood. They are formed by fragmentation of the cytoplasm of bone marrow megakaryocytes. One mature megakaryocyte may contribute 3000 platelets to the 1–3 x 108 per ml present in whole blood.
Cell culture. Laboratory growth of isolated animal cells has become very important in biochemistry. Sometimes it is necessary to have many cells with as nearly as possible identical genetic makeup. Such bacterial cells are obtained by plating out the bacteria and selecting a small colony that has grown from a single cell to propagate a “pure strain.” Similarly, single eukaryotic cells may be selected for tissue culture and give rise to a clone of cells which remains genetically identical until altered by mutations. The culture of embryonic fibroblasts is used to obtain enough cells to perform prenatal diagnosis of inherited metabolic diseases. Tissue culture is easiest with embryonic or cancer cells, but many other tissues can be propagated. However, the cells that grow best and which can be propogated indefinitely are not entirely normal; the well-known HeLa strain of human cancer cells which was widely grown for many years throughout the world contains 70–80 chromosomes per cell compared with the normal 46.

The Variety of Animal Forms

In this section, we will consider only a few biochemical and other aspects of multicellular animals or Metazoa. The sudden appearance of a large number of Metazoans about 0.5 x 109 years ago may have been an outcome of the appearance of split genes (see Section B, 1). As a result of gene duplication the coding pieces of split genes, the exons, could be moved to new locations in a chromosome where they could have become fused with other pieces of DNA to form entirely new genes.

Major Groups of Multicellular Animals

The simplest metazoa are tiny symbiotic worms of the phylum (or subkingdom) Mesozoa, which live in the kidneys of deep sea-dwelling cephalopods (octopi and squid). Each worm is made up of only 25 cells in a single layer enclosing one or a small number of elongated axial cells. Mesozoa have been regarded as parasitic, but they appear to facilitate excretion of NH3 by the host through acidification of the urine. Porifera or sponges are the most primitive of multicelled animals. They lack distinct tissues but contain several specialized types of cells. The body is formed by stationary cells that pump water through the pores to bring food to the sponge. Within the body amebocytes work in groups to form the spicules of
calcium carbonate, silicon dioxide, or the protein spongin. Sponges appear to lack a nervous
system. Individuals of the next most complex major phylum, Cnidaria (formerly oelenterata), are radially symmetric with two distinct cell layers, the endoderm and ectoderm. Many species exist both as a polyp or hydra form and as a medusa or jellyfish. The jellyfish apparently has no brain but the ways in which its neurons interconnect in a primitive radial net are of interest. The Cnidaria have a very simple body form with remarkable regenerative powers. The freshwater hydra, a creature about 1 cm long, contains a total of ~105 cells. A complete hydra can be regenerated from a small piece of tissue if the latter contains some of both the inner and the outer cell layers. The body of flatworms (phylum Platyhelminthes) consists of two external cell layers (endoderm and ectoderm) with a third layer between. A distinct excretory system is present. In addition to a nerve net resembling that of the Cnidaria, there are a cerebral ganglion and distinct eyes. One large group of flatworms, the planarians (typically about 15 mm in length, inhabit freshwater streams. They are said to be the simplest creatures in which behavior can be studied.

Some lower forms of Metazoa. (A) Mesozoa (25 cells). After C. P. Hickman. (B) A small asconoid sponge. After C. A. Villee, W. F. Walker, Jr., and R. D. Barnes. (C) Ameboid cells of a sponge forming spicules. After Hickman.

Many parasitic flatworms (tapeworms and flukes) attack higher organisms. Among them are the Schistosoma, tiny worms that are transmitted to humans through snails and which attack the blood vessels. The resulting schistosomiasis is one of the most widespread debilitating diseases on earth today, affecting 200 million people or more. The roundworms (Nematoda) have, in addition to the enteron (alimentary tract), a separate body cavity. Free-living nematodes abound in water and soil but many species are parasitic. They do enormous damage to plants and to some animal species. Trichina, hookworms129a, and filaria worms attack humans. However, in the laboratory the 1-mm-long, 810-cell nematode, Caenorhabditis elegans has become an important animal. In 1963 Sydney Brenner launched what has become a worldwide effort to make this tiny worm the equivalent in the animal kingdom of . coli in the bacterial world. The 108 nucleotides in the worm’s six chromosomes contain ~13,600 genes. C. elegans has become an important animal in which to study differentiation. Already the exact lineage of every cell has been traced, as has every connection among the 302 neurons in the animal’s nervous system. The elated rotifers, 130 with whirling“wheels” of cilia on their heads and transparent bodies, are a delight to the microscopist. Like nematodes, they are “cell constant” organisms. The total number of cells in the body is constant as is that in almost every part of every organ. Part of the develop mental plan of such organisms is a “programmed cell death”.

(A) Hydra. After Loomis. (B) The medusa stage of Obelia, a hydroid coelenterate.

The Annelida (segmented worms) are believed to be evolutionary antecedents of the arthropods. Present-day members include earthworms, leeches, and ~105 species of marine polychaetes. Annelids have a true body cavity separate from the alimentary canal and lined by a peritoneum. They have a welldeveloped circulatory system and their blood usually contains a type of hemoglobin. About 106 species of arthropods (80% of all known animals) have been described. Most are very small. These creatures, which have a segmented exoskeleton of chitin and other materials, include the horseshoe crabs, the Arachnida (scorpions, spiders, and mites), the Crustacea, Myriopoda (centipedes and millipedes), and the Insecta. Important biochemical problems are associated with the development and use of insecticides and with our understanding of the metamorphosis that occurs during the growth of arthropods. The fruit fly Drosophila melanogaster has provided much of our basic knowledge of genetics and continues to be the major species in which development is studied. Among the molluscs (phylum Mollusca) the squids and octopuses have generated the most interest among biochemists. The neurons of squid contain giant axons, the study of which has led to much of our knowledge of nerve conduction. Octopuses show signs of intelligence not observed in other invertebrates whose nervous reactions seem to be entirely “preprogrammed.” The brains of some snails contain only neurons, some of which are unusually large. The Echinodermata or spiny-skinned animals (starfish, sea urchins, and sea cucumbers) are regarded as a highly advanced phylum. Their embryological development has been studied intensively. The phylum Chordata, to which we ourselves belong, includes not only the vertebrates but also more primitive marine animals that have a spinal cord. Among these primitive species, which may be related to early ancestral forms, are the tunicates or sea squirts. They have a very high concentration of vanadium in their blood.

(A) A planarian, length 15 mm. After Hickman. Diagram of digestive and nervous sytems;cutaway section shows ventral mouth. Small drawing shows pharynx extended through ventral mouth. (B) The nematode Caenorhabditis elegans. Ascaris is very similar inappearance. From Buchsbaum. (C) A rotifer, Philodina (~103 cells). After C. A. Villee et al.

Algae

Algae are chlorophyll-containing eukaryotic organisms which may be either unicellular or colonial. The colonial forms are usually organized as long filaments, either straight or branched, but in some cases as blades resembling leaves. However, there is little differentiation among cells. The gold-brown, brown, and red algae contain special pigments in addition to the chlorophylls.
The euglenids (Euglenophyta) and dinoflagellates (Pyrrophyta), discussed in the protozoa section, can equally well be regarded as algae. The bright green Chlorophyta, unicellular or filamentous algae, are definitely plants, however. Of biochemical interest is Chlamydomonas, a rather animal-like creature with two flagella and a carotenoid-containing eyespot or stigma. Chlamydomonas contains a single chloroplast. The“pyrenoid”, a center for the synthesis of starch, lies, along with the eyespot, within the chloroplast. The
organism is haploid with “plus” and “minus” strains and motile gametes. Zygotes immediately undergo meiosis to form haploid spores. With a well-established genetic map, Chlamydomonas is another important organism for studies of biochemical genetics. The filamentous Ulothrix shows its relationship to the animals through formation of asexual spores with four flagella and biflagellate gametes. Only the zygote is diploid. On the other hand, the incomparably beautiful Spirogyra has no motile cells. The ameboid male gamete flows through a tube formed between the two mating cells, a behavior suggesting a relationship to higher green plants.

Two frequently studied fungi. Top (including ascus): the yeast Saccharomycescerevisiae. Below: Neurospora crassa showing various stages. After J. Webster.

Some unicellular algae grow to a remarkable size. One of these is Acetabularia, which lives in the warm waters of the Mediterranean and other tropical seas. The cell contains a single nucleus which lies in the base or rhizoid portion. In the mature alga, whose life cycle in the laboratory is 6 months, a cap of characteristic form develops. When cap development is complete, the nucleus divides into about 104 secondary nuclei which migrate up the stalk and out into the rays of the cap where they form cysts. After the cap decays and the cysts are released, meiosis occurs and the flagellated gametes fuse in pairs to form zygotes which again grow into diploid algae. Because of its large size and the location of the nucleus in the base, the cells can be cut and grafted. Nuclei can be removed or transplanted and growth and development can be studied in the presence or absence of a nucleus. The green algae Volvox live in wheel-like colonies of up to several thousand cells and are useful for biochemical studies of differentiation. Look through the microscope at almost any sample of algae from a pond or aquarium and you will see little boatlike diatoms slowly gliding through the water. The most prominent members of the division Chrysophyta, diatoms are characterized by their external “shells” of silicon dioxide. Large and ancient deposits of diatomaceous earth contain these durable silica skeletons which are finely marked, often with beautiful patterns. The slow motion of diatoms is accomplished by streaming of protoplasm through a groove on the surface of the cell. Diatoms are an important part of marine plankton, and it is estimated that three-fourths of the organic material of the world is
produced by diatoms and dinoflagellates. Like the brown algae, Chrysophyta contain the pigment fucoxanthin. Other groups of algae are the brown and red marine algae or seaweed. The former (Phaeophyta) include the giant kelps from which the polysaccharide algin is obtained. The Rhodophyta are delicately branched plants containing the red pigment phycoerythrin. The polysaccharides, agar and carrageenin, a popular additive to chocolate drinks and other foods, come from red algae. Symbiotic associations of fungi with either true
algae or with cyanobacteria are known as lichens. Over 15,000 varieties of lichens grow on rocks and in other dry and often cold places. While the algae appear to benefit little from the association, the fungi penetrate the algae cells and derive nutrients from them. Although either of the two partners in a lichen can be cultured separately, the combination of the two
is capable of producing special pigments and phenolic substances known as depsides which are not formed by either partner alone.

A few species of algae.

Fungi

Lacking photosynthetic ability, living most often in soil but sometimes in water, the fungi are represented by almost half as many species (~105) as are the vascular plants. The distinguishing characteristics of fungi are the lack of chlorophyll and growth as a series of manybranched tubules (usually 6–8 m diameter), the hyphae, which constitute the mycelium. The hyphae are not made up of separate cells but contain a mass of protoplasm with many nuclei. Only occasional septa divide the tubules. Most fungi are saprophytic, living on decaying plants or animal tissues. However, others are parasites that produce serious and difficult-to-treat infections in humans. An important medical problem is the lack of adequate antibiotics for treating fungal infections (mycoses). On the other hand, fungi produce important antibiotics such as penicillin. Still others form some of the most powerful toxins known! The lower fungi or Phycomycetes include simple aquatic molds and mildew organisms. Higher fungi are classified as Ascomycetes or Basidiomycetes according to the manner in which the sexual spores are born. In the Ascomycetes these spores are produced in a small sac called an ascus. Each ascus contains four or eight spores in a row, a set of four representing the results of a single pair of meiotic divisions. A subsequent mitotic division will give eight spores. This is one of the features that has made Neurospora crassa a favorite subject for genetic studies. The ascospores can be dissected out in order from the ascus and cultivated separately to observe the results of crossing-over during meiosis. Neurospora also reproduces via haploid spores called conidia. The haploid mycelia exist as two mating types and conidia or mycelia from one type can fertilize cells in a special body (the protoperithecium) of the other type to form zygotes. The latter immediately undergo meiosis and mitosis to form the eight ascospores. Among other Ascomycetes are the highly prized edible truffles and morels. However, most mushrooms and puffballs are fruiting bodies of Basidiomycetes. Other Basidiomycetes include the rusts, which cause enormous damage to wheat and other grain crops. Yeasts are fungi adapted to life in an environment of high sugar content and which usually remain unicellular and reproduce by budding. Occasionally the haploid cells fuse in pairs to form diploid cells and sexual spores. Some yeasts are related to the Ascomycetes, others to Basidiomycetes. Saccharomyces cerevisiae, the organism of both baker’s and brewer’s yeast, is an Ascomycete. It can grow indefinitely in either the haploid or diploid phase. The genetics and biochemistry of this yeast have been studied extensively. The genome is relatively small with 13.5 x 106 base pairs in 17 chromosomes. The sequence of the 315,000 base pairs of chromosome III was determined in 1992101,102 and the sequence of the entire genome is now known. Fungi often grow in symbiotic association with other organisms. Of special importance are the mycorrhizae (fungus roots) formed by colonization of fine roots by beneficial soil fungi. Almost all plants of economic importance form mycorrihizae.

Protozoa

Among the best known of the animal-like protista is the ameba (subphylum Sarcodina or Rhizopoda). The most striking feature of the ameba is its method of locomotion, which involves the transformation of cytoplasm from a liquid state to a semisolid gel. As the ameba moves, the cytoplasm at the rear liquifies and flows to the front and into the extending pseudopodia where it solidifies along the edges. The ameba poses several important biochemical questions: What chemistry underlies the reversible change from liquid to solid cytoplasm? How can the cell membranes break and reform so quickly when an ameba engulfs food particles? Relatives of the ameba include the Radiolaria, marine organisms of remarkable symmetry with complex internal skeletons containing the carbohydrate polymer chitin together with silica (SiO2) or strontium sulfate. The Foraminifera deposit external shells of calcium carbonate or silicon dioxide. Over 20,000 species are known and now as in the distant past their minute shells fall to the bottom of the ocean and form limestone deposits. Tiny ameboid parasites of the subphylum Sporozoa attack members of all other animal phyla. Several genera of Coccidia parasitize rabbits and poultry causing enormous damage. Humans are often the victims of species of the genus Plasmodium which invade red blood cells and other tissues to cause malaria, one of our most serious ailments on a worldwide basis. Throughout history malaria has probably killed more persons than any other disease. Toxoplasma gondii is another parasite which, in its haploid phase, is found throughout the world in wild animals and in humans. Although its presence usually elicits no symptoms, it sometimes causes blindness and mental retardation in children and can be fatal to persons with AIDS. Its sexual cycle occurs exclusively in cats. Another subphylum of protozoa, the Mastigophora, are propelled by a small number of flagella and are intermediate between animals and the algae. One of these is Euglena viridis, a small freshwater organism with a long flagellum in front, a flexible tapered body, green chloroplasts, and a light-sensitive “eyespot” which it apparently uses to keep itself in the sunshine. Euglena is also able to live as a typical animal if there is no light. Treatment with streptomycin causes Euglena to lose its chloroplasts and to become an animal permanently. The dinoflagellates, some colorless and some green, occur in great numbers among the plankton of the sea. Giardia lamblia is a troublesome intestinal parasite. The hemoflagellates are responsible for some of our most terrible diseases. Trypanosomes (genus Trypanosoma) invade the cells of the nervous system causing African sleeping sickness. Mutating their surface proteins frequently by genescrambling mechanisms, these and other parasites are able to evade the immune response of the host. For the same reason it is difficult to prepare vaccines against them. Other flagellates live in a symbiotic relationship within the alimentary canals of termites and roaches. Termites depend upon bacteria that live within the cells of these symbiotic protozoans to provide the essential enzymes needed to digest the cellulose in wood. Members of the subphylum Ciliophora, structurally the most complex of the protozoa, are covered with a large number of cilia which beat together in an organized pattern. The following question immediately comes to mind: How are the cilia able to communicate with each other to provide this organized pattern? Two ciliates that are often studied by biochemists are Tetrahymena, one of the simplest, and Paramecium, one of the more complex. TheMyxomycetes or “slime molds” are more closely related to protozoa than to fungi. Members of the family Acrasieae, the best studied member of which is Dictyostelium discoideum, start life as small amebas. After a time, when the food supply runs low, some of the amebas begin to secrete pulses of a chemical attractant cyclic AMP. Neighboring amebas respond to the pulses of cyclic AMP by emitting their own pulses about 15 s later, then moving toward the original source. The ultimate effect is to cause the amebas to stream to centers where they aggregate and form fungus-like fruiting bodies. Asexual spores are formed and the life cycle begins again. Other Myxomycetes grow as a multinucleate (diploid) plasmodium containing millions of nuclei but no individual cell membranes. Physarumpolycephalum, a species whose plasmodium may spread to a diameter of 30 cm, has become popular with biochemists. The 800,000 nuclei per square millimeter all divide synchronously.

A few well-known protists.

Survey of the Protists

Unicellular eukaryotes have traditionally been grouped together with multicellular organisms in which all cells have similar functions, with little or no differentiation into tissues, as the kingdom Protista. The fungi may also be included or may be regarded as a separate kingdom.
79 With present-day emphasis on DNA sequence comparisons the traditional classification is changing, however.

Haploid and Diploid Phases

In human beings and other higher animals, meiosis leads directly to formation of the gametes, the egg and sperm cells. These fuse to form a diploid nucleus and the adult develops by repeated mitosis of the diploid cells. While meiosis also occurs in the life cycle of all eukaryotic creatures, it is not always at a point corresponding to that in the human life cycle. Thus, the cells of many protozoa and of fungi are ordinarily haploid. When two haploid nuclei fuse to form a diploid cell, meiosis quickly occurs to produce haploid individuals again. Among lower plants and animals there is often an alternation of haploid and diploid phases of the life cycle. For example, gametes of ferns fall to the ground and germinate to form a low-growing green mosslike haploid or gametophyte form. The latter produces motile haploid gametes which fuse to a diploid zygote that grows into the larger and more obvious sporophyte form of the fern. It is presumably the ability to survive as a heterozygote, even with one or more highly deleterious mutations, that has led to the dominance of the diploid phase in higher plants and animals. However, to the biochemical geneticist organisms with a haploid phase offer experimental advantages because recessive mutants can be detected readily.

Genetic Recombination, Sex, and Chromosomes

Bacteria usually reproduce by simple fission. The single DNA molecule of the chromosome is duplicated and the bacterium divides, each daughter cell receiving an identical chromosome. However, genetic recombination, which is accomplished in several ways by bacteria, provides a deliberate process for mixing of genes. This process has been most fully developed in eukaryotic organisms that undergo sexual reproduction. The growth of a multicelled individual begins with the fusion of two haploid gametes, an egg and a spermatozoon. Each gamete carries a complete set of genetic instructions, and after the nuclei fuse the fertilized egg or zygote is diploid. Each diploid cell contains two complete sets of genetic blueprints of quite different origin. Even if a gene from one parent is defective, the chances are that the gene from the other parent will be good. Sexual reproduction and the associated genetic recombination also provide a means for mixing of genes. When eukaryotic cells prepare to divide in the process called mitosis, the DNA molecules of the nucleus, which become spread out through a large volume, coil and fold. Together with proteins and other molecules they form the compact bodies known as chromosomes. Some organisms, such as Ascaris (a roundworm), have only two chromosomes, a homologous pair, one inheritied from the father and one from the mother. Both chromosomes divide in every mitotic cell division so that every cell of the organism has the homologous pair. Higher organisms usually have a larger number of chromosomes. Thus, humans have 23 homologous pairs. The mouse has 20, the toad 11, onions 8, mosquitos 3, and Drosophila 4. Human chromosomes vary in size but are usually 4–6 m long and ~1 m in diameter. By the successive divisions of mitosis, a single fertilized eukaryotic egg cell can grow to an adult. Less than 50 successive mitotic divisions will produce the ~1014 cells of a human. However, ormation of gametes, which are haploid, requires the special process of meiosis, by which the number of chromosomes is divided in half. During meiosis one chromosome of each of the homologous pairs of the diploid cell is passed to each of the gametes that are formed. In an organism such as Ascaris, which contains only a single pair of chromosomes, a gamete receives either the chromosome of maternal origin or that of paternal origin but not both. In organisms that have several pairs of chromosomes, one chromosome of each pair is passed to the gamete in a random fashion during meiosis. Most gametes receive some chromosomes of maternal and some of paternal origin. An important feature of meiosis is the genetic recombination that occurs during crossing-over. In this process, the strands of DNA are cut and genetic material is exchanged between the chromosomes of maternal and
paternal origin. Thus, crossing-over breaks the linkage between genes and provides for greater variability in the offspring than would otherwise be possible. Each of us receives half of our genes from our mother and half from our father, but some of these genes have been inherited from each grandparent on both sides of the family, some from each great-grandparent, etc. Many genes are passed down through many generations without substantial change, but others are evidently designed to be scrambled readily within somatic cells. Cell surface proteins75 and antibody molecules are among the proteins whose genes undergo alteration during growth and differentiation of the tissues of the body.

A Changing Genome

How is it possible for the genome of an organism to increase in size as it evolved from a lower form to a higher one? Simple mutations that cause alterations in protein sequences could lead to changes in form and behavior of the organisms but could not, by themselves,
account for the increase in genetic material that accompanied evolution. As a result of new techniques of genetic mapping and determining the sequence of nucleotides in DNA we are rapidly acquiring a detailed knowledge of the organization of the genome. It has been found that genes are often present as duplicate but not entirely identical copies. This suggests that there are mechanisms by which cells can acquire extra copies of one or more genes. Indeed it seems probable that at some time in the past the entire genome of bacteria was doubled and that it was later doubled again. Evidence for this is that the masses of bacterial chromosomes group around values of 0.5, 1.4, and 2.7 x 109 Da. Genes can also be duplicated during the process of genetic recombination. In addition, the size of the genome may have increased by incorporation of genetic material from extrachromosomal plasmids.
A possible advantage to a cell possessing an extra copy of a gene is that the cell would survive even when mutations rendered unusable the protein encoded by one of the copies. As long as one of the genes remained “good,” the organism could grow and reproduce. The extra, mutated gene could be carried for many generations. As long as it produced only harmless, nonfunctioning proteins there might be little selection pressure to eliminate it and it might undergo repeated mutations. After many mutations and many generations later, the protein for which it coded could prove useful to the cell in some new way. An example of evolution via gene duplication is provided by the oxygen-carrying proteins of blood. It appears that about a billion years ago, the gene for an ancestral globin, the protein of hemoglobin, was doubled. One gene evolved into that of present-day globins and the other into the gene of the muscle protein myoglobin. Still later, the globin gene again doubled leading to the present-day α and β chains of hemoglobin. These are two distinctly different but related protein subunits whose genes are not even on the same chromosome. To complicate the picture further, most human beings have two or more copies of their α chain gene74 as well as genes for fetal and embryonic forms of hemoglobin. However, some populations have lost one or more α chain genes. Thus, the genome changes in many details, even today.

Inheritance, Metabolic Variation, and Evolution of Eukaryotes

The striking differences between eukaryotic and prokaryotic cells have led to many speculations about the evolutionary relationship of these two great classes of living organisms. A popular theory is that mitochondria, which are characteristic of most eukary-
otes, arose from aerobic bacteria. After cyanobacteria had developed and oxygen had become abundant, a symbiotic relationship could have arisen in which small aerobic bacteria lived within cells of larger bacteria that had previously been obligate anaerobes. Sequence similarities of proteins suggest that these symbionts may have been related to present-day methanogens60 and thermophilic sulfur bacteria. The aerobes presumably used up any oxy-
gen present, protecting the surrounding anaerobic organisms from its toxicity. The elationship became permanent and led eventually to the mitochondriacontaining eukaryotic cell. Further symbiosis with cyanobacteria or prochlorophytes could have led to the chloroplasts of the eukaryotic plants. A fact that supports such ideas is the existence among present-day organisms of many endosymbiotic relationships. For example, the green paramecium (Paramecium bursaria) contains, within its cytoplasm, an alga (Chlorella), a common green plant that is quite capable of living on its own. Perhaps by accident it took up residence within the paramecium. Some dinoflagellates contain endosymbiotic cyano-
bacteria66 and recently a ciliate that contains endosymbiotic purple photosynthetic bacteria has been discovered. These bacteria do not produce O2 but utilize products of the host ciliates’ metabolism such as acetate, lactate, and H2 as electron donors for photosyntheses. They also utilize O2 for respiration and may protect their hosts from the toxicity of O2, just as may have happened in the distant past. According to this theory the symbionts would eventually have lost their photosynthetic ability and have become mitochondria. The elationship of mitochondria to bacteria is also supported by many biochemical similarities. Fossils of bacteria and blue-green algae have been obtained from rocks whose age, as determined by geochemical dating, is more than 3 × 109 years. However the first eukaryotic cells may have appeared about 1 × 109 years ago70 and started to evolve into the more than one million species that now exist.

INHERITED METABOLIC DISEASES

In 1908 Archibald Garroda,b proposed that cystinuria and several other defects in amino acid and sugar metabolism were “inborn errors of metabolism”, i.e. inherited diseases. Since that time the number of recognized genetic defects of human metabolism has increased at an accelerating rate to ~4000. c–e Hundreds of other genetic problems have also been identified. For over 800 of these the defective gene has been mapped to a specific chromosome. An example is sickle cell anemia in which a defective hemoglobin differs from the normal protein at one position in one of its constituent polypeptide chains. Many other defects involve loss of activity of some important enzyme. Most genetic diseases are rare, affecting about one person in 10,000. However, cysticfibrosis affects one in 2500. There are so many metabolic diseases that over 0.5% of all persons born may develop one. Many die at an early age. A much greater number (>5%) develop such conditions as diabetes and mental illness which are, in part, of genetic origin. Since new mutations are always arising, genetic diseases present a problem of continuing significance. At what rate do new mutations appear? From the haploid DNA content (Table 1-2) we can estimate that the total coding capacity of the DNA in a human cell exceeds two million genes (actually two million pairs of genes in diploid cells). However, only a fraction of the DNA codes for proteins. There are perhaps 50,000 pairs of structural genes in human DNA. The easily detectable rate of mutation in bacteria is about 10–6 per gene, or 10–9 per base per replication. g
As a result of sophisticated “proofreading” and repair systems, it may be as low as 10–10 per base in humans. h Thus, in the replication of the 3 x 109 base pairs in diploid human chromosomes we might anticipate about one mistake per cell division. Only about 1/50 of these would be in structural genes and potentially harmful. Thus, if there are 1016 division
cycles in a normal life spanh each parent may pass on to future generations about 2 mutations in protein sequences. The ~1014 body cells (somatic cells) also undergo mutations which may lead to cancer and to other problems of aging. Most mutations may be harmless or nearly so and a few may be beneficial. However, many are damaging and some are lethal. If a mutation is lethal, a homozygote will not survive and will be lost in an early (and usually undetected) spontaneous abortion. Healthy individuals carry as many as ten lethal recessive mutations as well as at least 3–5 autosomal recessive mutations of a seriously harmful type. Harmful dominant mutations are also frequent in the population. These include an elevated lipoprotein content of the blood and an elevated cholesterol level which are linked to early heart disease. Biochemical disorders are also important because
of the light they shed on metabolic processes. No other species is observed as carefully as Homo sapiens. As a consequence frequent reference will be made to genetic diseases throughout the book. A goal is to find ways to prevent or ameliorate the effects of these disorders. For example, in the treatment of phenylketonuria or of galactosemia, a change in the diet can prevent irreversible damage to the brain, the organ most frequently affected by many of these diseases. Injection of a missing enzyme is giving life to victims of Gaucher’s disease. In many other cases no satisfactory therapy is presently available, but the possibilities of finding some way to supply missing enzymes or to carry out “genetic surgery” are among the most exciting developments of contemporary medical biochemistry.

Photomicrograph of human male metaphase chromosomes. © Photo
Researchers

Cell Coats, Walls, and Shells

Like bacteria, most cells of higher plants and animals are surrounded by extracellular materials. Plants have rigid walls rich in cellulose and other carbohydrate polymers. Outside surfaces of plant cells are covered with a cuticle containing layers of a polyester called cutin and of wax. Surfaces of animal cells are usually lined with carbohydrate molecules which are attached to specific surface proteins to form glycoproteins. Spaces between cells are filled with such “cementing substances” as pectins in plants and hyaluronic acid in animals. Insoluble proteins such as collagen and elastin surround connective tissue cells. Cells that lie on a surface (epithelial and endothelial cells) are often lined on one side with a thin, collagen-containing basement membrane. Inorganic deposits such as calcium phosphate (in bone), calcium carbonate (eggshells and spicules of sponges), and silicon dioxide (shells of diatoms) are laid down, often by cooperative action of several or many cells.

Centrioles, Cilia, Flagella, and Microtubules

Many cells contain centrioles, little cylinders about 0.15 µm in diameter and 0.5 µm long, which are not enclosed by membranes. Each centriole contains a series of fine microtubules of 25 nm diameter. A pair of centrioles are present near the nucleus in most animal cells and play an important role in cell division. Together with surrounding materials they form the centrosome. However, centrioles have never been
observed in plant cells. Related in structure to centrioles are the long flagella and shorter cilia (the two words are virtually synonymous) which are commonly present as organ- elles of locomotion in eukaryotic cells. Stationary cells of our own bodies also often have cilia. For example, there are 109 cilia/cm2 in bronchial epithelium. Modified flagella form the receptors of light in our eyes and of taste in our tongues. Flagella and cilia have a diameter of about 0.2 µm and a characteristic internal structure. Eleven hollow microtubules of ~24 nm diameter are usually arranged in a “9 + 2” pattern with nine pairs of fused tubules surrounding a pair of single tubules. Each microtubule resembles a bacterial flagellum in appearance, but there are distinct and significant chemical differences. The basal body of the flagellum, the kinetosome, resembles a centriole in structure, dimensions, and mode of replication. Recently a small 6–9 megabase pair DNA has been found in basal bodies of the protozoan Chlamydomonas. Microtubules similar to those found in flagella are also present in the cytoplasm. Together with thinner microfilaments of several kinds they form an internal
cytoskeleton that provides rigidity to cells. Microtubules also form the “spindle” of dividing cells. In nerve axons (Chapter 30) the microtubules run parallel to the length of the axons and are part of a mechanical transport system for cell constituents.

Structure of cilia and flagella of eukaryotes. After P. Satir

Mitochondria, Plastids, and Peroxisomes

Mitochondria, complex bodies about the size of bacteria and bounded on the outside by a double membrane, are present in all eukaryotic cells that use oxygen for respiration. The numbers per cell appear to vary from the one for certain tiny trypanosomes to as many as 3 x 105 in some oocytes. Liver cells often contain more than 1000 mitochondria
apiece. Study of ultrathin serial sections of a single yeast cell by electron microscopy has shown that under some growth conditions all of the yeast mitochondria are interconnected. More recent evidence from new imaging procedures, e.g. using the green fluorescent protein also supports the idea that mitochondria are interconnected in a reticulum that can become fragmented under some conditions. The inner membrane of a mitochondrion is often highly folded to form the cristae (crests). The outer membrane is porous to small molecules but the passage of substances into and out of the inner space of the mitochondrion, known as the matrix, is tightly controlled by the inner membrane. Although some of the oxidative chemical activites of the cells are located in the ER and in peroxisomes, the major energy-yielding reactions for aerobic organisms are found in the mitochondria, which are also the principal site of utilization of oxygen. Within each mitochondrion is a small circular molecule of DNA whose genes encode only a few of the many proteins needed in this organelle. Also present within mitochondria are ribosomes of a size similar to those of bacteria and smaller than those lining the rough ER. Plastids are organelles of plant cells that serve a variety of purposes. Most important are the chloroplasts, the chlorophyll-containing sites of photosynthesis. Like mitochondria they contain folded internal membranes and several small molecules of DNA. Fragile organelles, the peroxisomes or microbodies, occur in many cells. In green leaves they may occur in numbers up to one-third those of mitochondria. Peroxisomes are often about the size of mitochondria
but have only a single membrane and do not contain DNA. They often contain an apparently crystalline “core.” The single membrane of peroxisomes is porous to small molecules such as sucrose. This permits these organelles to be separated from itochondria by cen trifugation in a sucrose gradient where the microbodies assume a density of about 1.25 g/cm3 compared to 1.19 for the impervious mitochondria. Peroxisomes are rich in enzymes that produce and decompose hydrogen peroxide. They often make a major contribution to the oxidative metabolism of cells. In germinating oilseeds glyoxysomes, a type of peroxisome, contain enzymes that catalyze reactions of
the biosynthetic “glyoxylate pathway” of metabolism. Organelles that resemble eroxisomes in appearance Endoplasmic reticulum Microsomes15 but which are functionally more closely related to mitochondria are the hydrogenosomes of anaerobic protozoa. As the name suggests, these organelles are the site of formation of molecular hydrogen, a ommon product of anaerobic metabolism.

The Endoplasmic Reticulum and Golgi Membranes

Although cytoplasm is fluid and in some organisms can undergo rapid streaming, the electron microscope has revealed that within the liquid portion, the cytosol, there is a complex network of membranes known as the endoplasmic reticulum (ER). The membranes of the ER form tubes, vesicles, and flattened sacs called cisternae. The intracisternal spaces appear to connect with the perinuclear space and to a series of 3–12
flattened, slightly curved disk-shaped membranes known as the Golgi apparatus. This organelle was first reported by Camillo Golgi in 1898. Its existence was long doubted, but it is known now to play a vital role in metabolism. The ER, the Golgi membranes, and secretion granules apparently represent an organized system for synthesis of secreted protein and formation of new membranes. Parts of the ER, the rough endoplasmic reticulum are lined with many ribosomes of 21–25 nm diameter. While resembling those of bacteria, these
eukaryotic ribosomes are about 50% heavier (4 x 106 Da). The smooth endoplasmic reticulum lacks ribosomes but proteins made in the rough ER may be modified in the smooth ER, e.g., by addition of carbohydrate chains. Small membrane vesicles break off rom the smooth ER and pass to the Golgi membranes which lie close to the smooth ER on the side toward the center of the cell. Here additional modification reactions occur (Chapter
20). At the outer edges the membranes of the Golgi apparatus pinch off to form vacuoles which are often densely packed with enzymes or other proteins. These secretion granules move to the surface and are released from the cell. In this process of exocytosis the membranes surrounding the granules fuse with the outer cell membrane. The rough ER appears to contribute membrane material to the smooth ER and Golgi apparatus, while material from Golgi membranes can become incorporated into the outer cell membrane and into lysosomes. Outer mitochondrial membranes and membranes around vacuoles in plant cells may also be derived directly from the ER. Outer membrane materials are probably “recycled” by endocytosis. The term microsome, frequently met in the biochemical literature, refers to small particles of 50–150 nm diameter which are mostly fragments of the ER together with some material from the plasma membrane. Microsomes are formed when cells are ground or homogenized. Upon centrifugation of the disrupted cells, nuclei and other large fragments sediment first, then the mitochondria. At very high speeds (e.g., at 100,000 times the force of gravity) the microsomes, whose masses are 108 –109 Da, settle. With the electron microscope we see that in the microsomes the membrane fragments have closed to give small sacs to the outside of which the ribosomes still cling:

Vacuoles, Endocytosis, and Lysosomes

Cells often contain vacuoles or smaller vesicles that are separated from the cytosol by a single membrane. Their content is often quite acidic. 40 Small vesicles sometimes bud inward from the plasma membrane in a process called endocytosis. In this manner the cell may engulf particles (phagocytosis) or droplets of the external medium (pinocytosis). The resulting endocytotic vesicles or endosomes often fuse with lysosomes, which are small acidified vesicles containing a battery of enzymes powerful enough to digest almost anything in the cell. In cells that engulf bits of food (e.g., ameba) lysosomes provide the digestive enzymes. Lysosomes also take up and digest denatured or damaged proteins and may digest “worn out” or excess cell parts including mitochondria. Lysosomes are vital components of cells, 41 and several serious human diseases result from a lack of specific lysosomal enzymes.

Electron micrograph of a thin section of a young epidermal cell of a sunflower. The tissue was fixed and stained with uranyl acetate and lead citrate. Clearly visible are the nucleus (N), mitochondria (M), chloroplasts (C), a Golgi body dictyosome (G), endoplasmic reticulum, vacuole (V), cell wall, plasmodesmata, and cuticle (upper right, thin dark layer).Micrograph courtesy of H. T. Horner.

The Plasma Membrane

The thin (8 nm) outer cell membrane or“plasma-lemma” controls the flow of materials into and out of cells, conducts impulses in nerve cells and along muscle fibrils, and participates in chemical communication with other cells. Deep infoldings of the outer membrane sometimes run into the cytoplasm. An example, is the “T system” of tubules which functions in excitation of muscle contraction. Surfaces of cells designated to secrete materials or to absorb substances from the surrounding fluid, such as the cells lining kidney tubules and pancreatic secretory cells, are often covered with very fine projections or microvilli which greatly increase the surface area. In other cases projections from one cell interdigitate with those of an adjacent cell to give more intimate contact.

The Nucleus

In a typical animal cell the nucleus has a diameter of ~5 µm and a volume of 65 µm3. Except at the time of cell division, it is densely and almost uniformly packed with DNA. The amount of DNA present is larger than that in bacteria as is indicated in Table 1-3. Yeast contains about three times as much genetic matter as E. coli and a human being or a mouse about 700 times as much. However, genes are sometimes duplicated in higher organisms and large amounts of repetitive DNA of uncertain significance are often present. Some amphibians have 25 times more DNA per cell than do humans. The fruit fly Drosophila
contains about 13,600 functioning genes and a human being perhaps 50,000. 37
Because of its acidic character, DNA is stained by basic dyes. Long before the days of modern biochemistry, the name chromatin was given to the material in the nucleus that was colored by basic dyes. At the time of cell division, the chromatin is consolidated into
distinct chromosomes which contain, in addition to 15% DNA, about 10% RNA and 75% protein. Nearly all of the RNA of the cell is synthesized (transcribed) in the nucleus, according to the instructions encoded in the DNA. Some of the RNA then moves out of the nucleus into the cytoplasm where it functions in protein synthesis and in some other ways.
Many eukaryotic genes consist of several sequences that may be separated in the DNA of a chromosome by intervening sequences of hundreds or thousands of base pairs. The long RNA transcripts made from these split genes must be cut and spliced in the nucleus to form the correct messenger RNA molecules which are then sent out to the ribosomes in the cytoplasm. Each cell nucleus contains one or more dense nucleoli, regions that are rich in RNA and may contain 10–20% of the total RNA of cells. Nucleoli are sites of synthesis and of temporary storage of ribosomal RNA, which is needed for assembly of ribosomes. The nuclear envelope is a pair of membranes, usually a few tens of nanometers apart, that surround the nucleus. The two membranes of the pair separate off a thin perinuclear space. The membranes contain “pores” ~130 nm in diameter with a complex structure. 38,39 There is a central channel ~42 nm in diameter, which provides a route for controlled passage of RNA and other large molecules from the nucleus into the cytoplasm and also from the cytoplasm to the nucleus. Smaller ~10 nm channels allow passive diffusion of ions and small molecules.

Eukaryotic Cells

Cells of the eukaryotes contain true nuclei and are much larger and more complex internally than are those of prokaryotes. The nucleus of a cell contains most of its DNA and is separated from the cytoplasm by membranes. Within the cytoplasm are various organelles with characteristic structures. These include mitochondria, lysosomes, peroxisomes, and centrioles. Eukaryotic cells come in so many sizes and shapes and with so many specialized features that it is impossible to say what is typical. Nevertheless, Fig. 1-6 is an attempt to portray some sort of “average” cell, partly plant and partly animal. As can be seen from Table 1-2, which lists the diameters and volumes of several roughly spherical cells, there is a great variation in size. However, a diameter of 10–20 µm may be regarded as typical for both plants and animals. For growth of a large cell such as the ovum, many adjacent cells assist in synthesis of foodstuffs which are transferred to the developing egg cell. Plant cells are often large but usually 90% or more of the cell is filled with a vacuole or tonoplast, 36
which is drawn unrealistically small in Fig. 1-6. The metabolically active protoplasm of lant cells often lies in a thin layer at their peripheries. Many cells are far from spherical; for example, human red blood cells are discs 8 x 8 x 1 to 2 µm with a volume of 80 µm3. Plant fiber cells may be several millimeters in length. Nerve cells of animals have long extensions, the axons, which in the human sometimes attain a length of a meter. Muscle cells fuse to give very long multinucleate fibers.

Photosynthetic and Nitrogen-Fixing Prokaryotes

It is likely that the earth was once a completely anaerobic place containing water, ammonia, methane, formaldehyde, and more complicated organic compounds. Perhaps the first forms of life, which may have originated about 3.5 x 109 years ago, resembled present-day anaerobic bacteria. The purple and green photosynthetic bacteria may be related to organisms that developed at a second stage of evolution: those able to capture energy from sunlight. Most of these gram-negative photosynthetic bacteria are strict anaerobes. None can make oxygen as do higher plants. Rather, the hydrogen needed to carry out the reduction of carbon dioxide in the photosynthetic process is obtained by the splitting of inorganic compounds, such as H2S, thiosulfate, or H2, or is taken from organic compounds. Today, photosynthetic bacteria are found principally in sulfur springs and in deep lakes, but at one time they were probably far more abundant and the only photosynthetic organisms on earth. Before organisms could produce oxygen a second complete photosynthetic system, which could cleave H2O to O2, had to be developed. The simplest oxygenproducing creatures existing today are the cyanobacteria, 34 also known as blue-green algae. Many
cyanobacteria are unicellular, but others such as Oscilatoria, a slimy “plant” that often coats the inside walls of household aquaria, consist of long filaments about 6 m in diameter (see Fig. 1-11). All cyanobacteria contain two groups of pigments not found in other prokaryotes:
chlorophyll a and -carotene, pigments that are also found in the chloroplasts of true algae and in higher plants. A recently discovered group of bacteria, the prochlorophytes, are even closer to chloroplasts in their pigment composition. In addition to pigmented cells, some cyanobacteria contain paler cells known as heterocysts. They have a specialized function of fixing molecular nitrogen. The development of the ability to convert N2 into organic nitrogen compounds represents another important evolutionary step. Because they can both fix nitrogen and carry out photosynthesis, the blue-green algae have the simplest nutritional requirements of any organisms. They need only N2, CO2, water, light, and minerals for growth. Evolution of the photosynthetic cleavage of water to oxygen was doubtless a major event with farreaching consequences. Biologists generally believe that as oxygen accumulated in the earth’s atmosphere, the obligate anaerobes, which are poisoned by oxygen, became limited to strictly anaerobic environments. Meanwhile, a new group of bacteria, the aerobes, appeared with mechanisms for detoxifying oxygen and for using oxygen to oxidize complex organic compounds to obtain energy.

Abbreviations:

BM, basement membrane, ER, rough endoplasmic reticulum, (with ribosomes attached; smooth, ER is depicted nearer the nucleus and on the right side of the cell.), DI, deep indentation of plasma membrane, GI, glycogen granules Gap, space ~10-20 nm thickbetween adjacent cells, M, mitochondrion, Mb, microbody, L, lysosome, D, desmosome, TJ, tight junction, Mv, microvilli, C, cillium, SG, secretion granule, V, vacuole, Nu, nucleolus, G, Golgi apparatus, CW, cell wall (of a plant), Ct, centrioles, P, plasmodesmata, N, nucleus, Cp, chloroplast, St, starch granule.

The “average” eukaryotic cell. This composite drawing shows the principal organelles of both animal and plant
cells approximately to the correct scale. (Adapted from a drawing by Michael Metzler.)

Nutrition and Growth of Bacteria

Autotrophic (self-nourishing) bacteria can synthesize all of their organic cell constituents from carbon dioxide, water, and inorganic forms of nitrogen and sulfur. The photoautotrophs extract their energy from sunlight, while the chemoautotrophs obtain energy from inorganic chemical reactions. For example, the hydrogen bacteria oxidize H2 to H2O and sulfur bacteria oxidize H2S to H2SO4. Like the fungi and animals, most bacteria are chemoheterotrophic; they obtain energy from the breakdown of organic compounds. Some of these heterotrophic bacteria are anaerobes which live without O2. Many of them
metabolize complex organic substances such as sugars in the absence of oxygen, a process called fermentation. Others oxidize organic compounds with an inorganic oxidant such as nitrite or sulfate. Members of the genus Clostridium are poisoned by oxygen and are known as obligate anaerobes. Others, including E. coli, are facultative anaerobes, able to grow ither in the presence or in the absence of oxygen. Obligate aerobes depend for energy upon ombustion of organic compounds with oxygen. One of the largest groups of strictly aerobic heterotrophic bacteria, the pseudomonads (Pseudomonas and related genera), are of interest to biochemists because of their ability to oxidize organic compounds, such as alkanes, aromatic hydrocarbons, and steroids, which are not attacked by most other bacteria. Often, the number of oxidative reactions used by any one species of bacteria is limited. For example, the acetic acid bacteria that live in wine and beer obtain all of their energy by oxidation of ethanol to acetic acid:
CH3CH2OH + O2 → CH3COOH + H2O
Bacteria can grow incredibly fast. Under some conditions, it takes a bacterial cell only 10–20 min to double its size and to divide to form two cells. 4 An animal cell may take 24 h for the same process. Equally impressive are the rates at which bacteria transform their foods into other materials. One factor contributing to the high rate of bacterial metabolism may be the large surface to volume ratio. For a small spherical bacterium (coccus) of diameter 0.5 m, the ratio of the surface area to the volume is 12 x 106 m–1, while for an ameba of diameter 150 m the ratio is only 4 x 104 m–1 (the ameba can increase this by sticking out some pseudopods). Thimann33 estimated that for a 90-kg human, the ratio is only 30 m–1. When food is limited, some bacteria such as the Bacillus form spores. These are compact little cells that form inside the vegetative cell and are therefore called endospores. They sometimes have only 1/10 the volume of the parent cell. Their water content is very low, their metabolic rate is near zero, and they are extremely resistant to heat and furthe desiccation. Under suitable conditions, the spores can “germinate” and renew their vegetative growth. Spore formation is one of several examples of the development of specialized cells or differentiation among prokaryotes.

Classification and Evolution of Bacteria

Bacteria vary greatly in their chemistry and metabolism, and it is difficult to classify them in a rational way. In higher organisms species are often defined as forms that cannot interbreed and produce fertile offspring, but such a criterion is meaningless for bacteria whose reproduction is largely asexual and which are able readily to accept “visiting genes” from other bacteria. The classification into species and genera is therefore somewhat arbitrary. A currently used scheme 20 classifies the prokaryotes into 35 groups on the basis of many characteristics including shape, staining behavior, and chemical activities. Table
1-1 also includes genus names of most of the bacteria discussed in this book.
Bacteria may have the shape of spheres or straight or curved rods. Some, such as the actinomycetes, grow in a branching filamentous form. Words used to describe bacteria often refer to these shapes: a coccus is a sphere, a bacillus a rod, and a vibrio a curved rod with a flagellum at one end. A spirillum is screwshaped. These same words are frequently used to
name particular genera or families. Other names are derived from some chemical activity of the bacterium being described. The gram stain provides an important criterion of classification that depends upon differences in the structure of the cell wall (see Chapter 20). Bacterial cells are described as gram-positive or gram-negative according to their ability to retain the basic dye crystal violet as an iodine complex. This difference distinguishes two of four large categories of bacteria. 20 Most actinomycetes, the spore-forming bacilli, and most cocci are gram-positive, while E. coli, other enterobacteria, and pseudomonads are gram-negative. A third category consists of eubacteria that lack cell walls, e.g. the mycoplasma. Comparisons of amino acid sequences of proteins and the nucleotide sequences of DNA and RNA have provided a new approach to classification of bacteria. Although the origins of life are obscure, we can easily observe that the genome changes with time through
mutation and through the enzyme-catalyzed process of genetic recombination. The latter gives rise to the deletion of some nucleotides and the insertion of others into a DNA chain. When we examine sequences of closely related species, such as E. coli and Salmonella typhimurium, we find that the sequences are very similar. However, they differ greatly from those of many other bacteria. Consider the 23S ribosomal RNA, a molecule found in the ribosomes of all bacteria. It contains ~3300 nucleotides in a single highly folded chain. The basic structure is highly conserved but between any two species of bacteria there are many nucleotide substitutions caused by mutations as well as deletions and insertions. By asking what is the minimum number of mutations that could have converted one 23S RNA into another and by assuming a more or less constant rate of mutation over millions of years it is possible to construct a phylogenetic tree such as that shown in. One conclusion from these comparisons is that the methane-producing bacteria, the methanogens, 24 are only distantly related to most other bacteria. Methanogens together with the cell wall-less Thermoplasma,
28 some salt-loving halobacteria, and some thermophilic (heat-loving) sulfur bacteria form a fourth major category. They are often regarded as a separate kingdom, the archaeobacteria, 25 which together with the kingdom of the eubacteria form the superkingdom prokaryota. Certain archaeobacteria have biochemical characteristics resembling those of eukaryotes and some biologists therefore classify them as archaea and
rank their kingdom as equal to that of the bacteria and the eukaryotes. 27,29,30,30a,30b Others disagree. 31 In Table 1-1, the archaeobacteria are found in groups 31–35. Most bacteria are very small in size but there are species large enough to be confused with eukaryotic protozoa. The record for bacteria seems to be held by Epulopiscium fishelsoni, a parasite of the surgeonfish intestinal tract. A single cell measured > 600 m by 80 m diameter, over 106 times larger in volume than a cell of E. coli. 32 The organism is a gram-positive bacterium as judged by analysis of its cloned ribosomal RNA genes.

Universal phylogenetic tree. From Wheelis et al.

Flagella and Pili

Many bacteria swim at speeds of 20–60 µm/s, ten or more body lengths per second! Very thin threadlike flagella of diameter 13–20 nm coiled into a helical form are rotated by the world’s smallest “electric motors” to provide the motion. 14 While some bacteria
have a single flagellum, the corkscrew-like Spirillum (Fig. 1-3) synchronously moves tufts of flagella at both ends. Some strains of E. coli have no flagella, but others contain as many as eight flagella per cell distributed over the surface. The flagella stream out behind in a bundle when the bacterium swims. The flagella of the helical spirochetes are located inside the outer membrane. In addition to flagella, extremely thin, long, straight filaments known as pili or fimbriae project from the surfaces of many bacteria. 14 The “sex pili” (F pili and I pili) of E. coli have a specific role in sexual conjugation. The similar but more numerous common pili or fimbriae range in thickness from 3 to 25 nm and in length from 0.2 to 2 m. Pili are involved in adhesion of bacteria to surrounding materials or to other bacteria and facilitate bacterial infections. A typical E. coli cell has 100–300 pili.

Membranes and Cell Walls

Like the mycoplasma, the E. coli cell is bounded by an 8-nm membrane which consists of ~50% protein and 50% lipid. When “stained” (e.g., with permanganate) for electron microscopy, this single membrane appears as two very thin (2.0 nm) dark lines separated by an unstained center band (~3.5 nm) (Fig. 1-4; see also Fig. 8-4). Single membranes of approximately the same thickness and staining behavior occur in all cells, both of bacteria and of eukaryotes. A cell membrane is much more than just a sack. It serves to control the passage of small molecules into and out of the cell. Its outer surface carries receptors for recognition of various materials. The inside surface of bacterial membranes contains enzymes that catalyze most of the oxidative metabolism of the cells. Bacterial cell membranes are sometimes folded inward to form internal structures involved in photosynthesis or other specialized reactions of metabolism such as oxidation
of ammonia to nitrate. 2 In E. coli replication of DNA seems to occur on certain parts of the membrane surface, probably under the control of membrane-bound enzymes. The formation of the new membrane which divides multiplying cells proceeds synchronously with the synthesis of DNA. A characteristic of true bacteria (eubacteria) is a rigid cell wall which surrounds the cell membrane. The 40-nm-thick wall of E. coli is a complex, layered structure five times thicker than the cell membrane. Its chemical makeup is considered in Chapter 8. One of the layers is often referred to as the outer membrane. In some bacteria the wall may be as much as 80 nm thick and may be further surrounded by a thick capsule or glycocalyx (slime layer). 13 The main function of the wall seems to be to prevent osmotic
swelling and bursting of the bacterial cell when the surrounding medium is hypotonic. If the osmotic pressure of the medium is not too low, bacterial cell walls can sometimes be issolved, leaving living cells bounded only by their membranes. Such protoplasts can be produced by action of the enzyme lysozyme on gram-positive bacteria such as Bacillusmegaterium. Treatment of cells of gram-negative bacteria with penicillin (Box 20-G) produces sphero lasts, cells with partially disrupted walls. Spheroplasts and protoplasts are useful in biochemical studies because substances enter cells more readily when the cell wall is absent. Strains of bacteria lacking rigid walls are known as L forms.

Escherichia coli

The biochemist’s best friend is Escherichia coli, an ordinarily harmless inhabitant of our intestinal tract. This bacterium is easy to grow in the laboratory and has become the best understood organism at the molecular level. 4,9 It may be regarded as a typical true bacterium or eubacterium. The cell of E. coli (Figs. 1-1, 1-2) is a rod ~2 µm long and 0.8 µm in diameter with a volume of ~1 µm3 and a density of ~1.1 g/cm3. The mass is ~1 x 10–12 g, i.e., 1 picogram (pg) or ~0.7 x 1012 daltons (Da) (see Box 1-B). 4 It is about 100 times bigger than the smallest mycoplasma but the internal structure, as revealed by the electron microscope, resembles that of a mycoplasma. Each cell of E. coli contains from one to four identical DNA molecules, depending upon how fast the cell is growing, and ~15,000–30,000 ribosomes. Other particles that are sometimes seen within bacteria include food stores such as fat droplets and granules (Fig. 1-3). The granules often consist of poly- - hydroxybutyric acid10 accounting for up to 25% of the weight of Bacillus megaterium. Polymetaphosphate, a highly polymerized phosphoric acid, is sometimes stored in “metachromatic granules.” In addition, there may be droplets of a separate aqueous phase, known as vacuoles.

The Bacterial Genome

The genetic instructions for a cell are found in the DNA molecules. All DNA is derived from four different kinds of monomers, which we call nucleotides. DNA molecules are
double-stranded: two polymer chains are coiled together, their nucleotide units being associated as nucleotide pairs (see Fig. 5-7). The genetic messages in the DNA are in the form of sequences of nucleotides. These sequences usually consist of a series of code “words” or codons. Each codon is composed of three successive nucleotides and specifies which one of the 20 different kinds of amino acids will be used at a particular location in a protein. The sequence of codons in the DNA tells a cell how to order the amino acids for construction of its many
different proteins.

Transmission electron micrograph of a dividing cell of Escherichia coli O157:H7 attached to the intestinal epithelium of a neonatal calf. These bacteria, which are able to efface the intestinal microvilli, form characteristic attachments, and secrete shiga toxins, are responsible for ~73,000 illnesses and 60 deaths per year in the U. S. 10a After embedding, the glutaraldehyde-fixed tissue section was immunostained with goat anti-O157 IgG followed by protein A conjugated to 10-nm gold particles. These are seen around the periphery of the cell bound to the O-antigen (see Fig. 8-28). Notice the two microvilli of the epithelium. Courtesy of Evelyn A. Dean-Nystrom, National Animal Disease Center, USDA, Agricultural Research Service, Ames, IA.

A cell of a Spirillum negatively stained with
phosphotungstic acid. Note the tufts of flagella at the ends,
the rough appearance of the outer surface, the dark granules
of poly- -hydroxybutyric acid and the light-colored gran-
ules of unknown nature. Courtesy of F. D. Williams, Gail E.
VanderMolen, and C. F. Amstein.

Assume that a typical protein molecule consists of a folded chain of 400 amino acids. Its structural gene will therefore be a sequence of 1200 nucleotide pairs. Allowing a few more nucleotides to form spacer regions between genes we can take ~1300 as the number of
nucleotide pairs in a typical bacterial gene. However, some genes may be longer and some may be much shorter. The genome is the quantity of DNA that carries a complete set of genetic instructions for an organism. In bacteria, the genome is a single chromosome con-
sisting of one double-stranded DNA molecule. Mycoplasma genitalium is the smallest organism for which the DNA sequence is known. 11 Its genome is a double-
helical DNA circle of 580,070 nucleotide pairs and appears to contain about 480 genes (an average of ~1200 nucleotides per gene). The average mass of a nucleotide pair (as the
disodium salt) is 664 Da. It follows that the DNA of M. genitalium has a mass of ~385 x 106 Da. The relative molecular mass (Mr) is 0.385 x 109 (See Box 1-B for definitions of dalton and Mr). The DNA of E.coli is about seven times larger with a mass of ~2.7 x 109 Da. It contains ~4.2 x 106 nucleotide pairs and encodes over 4000 different proteins (see Table 1-3). Each nucleotide pair contributes 0.34 nm to the length of the DNA molecule; thus, the total length of DNA of an E. coli chromosome is 1.4 mm. This is about 700 times the length of the cell which contains it. Clearly, the molecules of DNA are highly folded, a fact that accounts for their appearance in the electron microscope as dense aggregates called nucleoids, which occupy about one-fifth of the cell volume. Each bacterial nucleoid contains a complete set of genetic“blueprints” and functions independently. Each nucleoid is haploid, meaning that it contains only a single complete set of genes. In addition to their chromosome, bacteria often contain smaller DNA molecules known as plasmids. These plasmids also carry genetic information that may be useful to bacteria. For example, they often encode proteins that confer resistance to antibiotics. The ability to acquire new genes from plasmids is one mechanism that allows bacteria to adapt readily to new environments.
Plasmids are also used in the laboratory in the cloning of genes and in genetic engineering.

(A) Thin (~60 nm) section of an aquatic gram-
negative bacterium, Aquaspirillum fasciculus. Note the light-
colored DNA, the dark ribosomes, the double membrane
characteristics of gram-negative bacteria (Chapter 8, Section
E), and the cell wall. In addition, an internal “polar mem-
brane” is seen at the end. It may be involved in some way in
the action of the flagella. (B) A thin section of dividing cell
of Streptococcus, a gram-positive organism. Note the DNA
(light-stranded material). A portion of a mesosome is seen
in the center and septum can be seen forming between the
cells. Micrographs courtesy of F. D. Williams, Gail E.
VanderMolen, and C. F. Amstein.

The Simplest Living Things

The simplest organisms are the bacteria. Their cells are called prokaryotic (or procaryotic) because no membrane-enclosed nucleus is present. Cells of all other organisms contain nuclei separated from the cytoplasm by membranes. They are called eukaryotic.
While viruses (Chapter 5) are sometimes regarded as living beings, these amazing parasitic objects are not complete organisms and have little or no metabolism of their own. The smallest bacteria are the mycoplasmas. They do not have the rigid cell wall characteristic of
most bacteria. For this reason they are easily deformedand often pass through filters designed to stop bacteria. They are nutritionally fussy and are usually, if not always, parasitic. Some live harmlessly in mucous membranes of people, but others cause diseases.
For example, Mycoplasma pneumoniae is responsible for primary atypical pneumonia.
Cells of mycoplasmas sometimes grow as filaments but are often spherical and as small as 0.3 micrometer (µm) in diameter. Their outer surface consists of a thin cell membrane about 8 nanometers (nm) thick. This membrane encloses the cytoplasm, a fluid material
containing many dissolved substances as well as submicroscopic particles. At the center of each cell is a single, highly folded molecule of DNA, which constitutes the bacterial chromosome. Besides the DNA there may be, in a small spherical mycoplasma, about
1000 particles ~20 nm in diameter, the ribosomes. These ribosomes are the centers of protein synthesis. Included in the cytoplasm are many different kinds of proteins, but there is room for a total of only about 50,000 protein molecules. Several types of RNA as
well as many smaller molecules are also present. Although we don’t know what minimum quantities of proteins, DNA, and other materials are needed to make a living cell, it is clear that they must all fit into the tiny cell of the mycoplasma.

Escherichia coli and some smaller bacteria.