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FIGURE 1-5 Organisms can be classified according to their source of energy (sunlight or oxidizable chemical compounds) and their source of carbon for the synthesis of cellular material.

domains, sometimes called Archaea and Bacteria. All eu-karyotic organisms, which make up the third domain, Eukarya, evolved from the same branch that gave rise to the Archaea; archaebacteria are therefore more closely related to eukaryotes than to eubacteria.

Within the domains of Archaea and Bacteria are subgroups distinguished by the habitats in which they live. In aerobic habitats with a plentiful supply of oxygen, some resident organisms derive energy from the transfer of electrons from fuel molecules to oxygen. Other environments are anaerobic, virtually devoid of oxygen, and microorganisms adapted to these environments obtain energy by transferring electrons to nitrate (forming N2), sulfate (forming H2S), or CO2 (forming CH4). Many organisms that have evolved in anaerobic environments are obligate anaerobes: they die when exposed to oxygen.

We can classify organisms according to how they obtain the energy and carbon they need for synthesizing cellular material (as summarized in Fig. 1-5). There are two broad categories based on energy sources: pho-totrophs (Greek trophe, "nourishment") trap and use sunlight, and chemotrophs derive their energy from oxidation of a fuel. All chemotrophs require a source of organic nutrients; they cannot fix CO2 into organic compounds. The phototrophs can be further divided into those that can obtain all needed carbon from CO2 (au-totrophs) and those that require organic nutrients (heterotrophs). No chemotroph can get its carbon atoms exclusively from CO2 (that is, no chemotrophs are autotrophs), but the chemotrophs may be further classified according to a different criterion: whether the fuels they oxidize are inorganic (lithotrophs) or organic (organotrophs).

Most known organisms fall within one of these four broad categories—autotrophs or heterotrophs among the photosynthesizers, lithotrophs or organotrophs among the chemical oxidizers. The prokaryotes have several general modes of obtaining carbon and energy. Escherichia coli, for example, is a chemoorganoheterotroph; it requires organic compounds from its environment as fuel and as a source of carbon. Cyanobacteria are photo-lithoautotrophs; they use sunlight as an energy source and convert CO2 into biomolecules. We humans, like E. coli, are chemoorganoheterotrophs.

Escherichia coli Is the Most-Studied Prokaryotic Cell

Bacterial cells share certain common structural features, but also show group-specific specializations (Fig. 1-6). E. coli is a usually harmless inhabitant of the human intestinal tract. The E. coli cell is about 2 ^m long and a little less than 1 ^m in diameter. It has a protective outer membrane and an inner plasma membrane that encloses the cytoplasm and the nucleoid. Between the inner and outer membranes is a thin but strong layer of polymers called peptidoglycans, which gives the cell its shape and rigidity. The plasma membrane and the

Ribosomes Bacterial ribosomes are smaller than eukaryotic ribosomes, but serve the same function— protein synthesis from an RNA message.

Ribosomes Bacterial ribosomes are smaller than eukaryotic ribosomes, but serve the same function— protein synthesis from an RNA message.

Bacterial Ribosome Granul

Cell envelope

Structure varies with type of bacteria.

Cell envelope

Structure varies with type of bacteria.

Gram-negative bacteria Gram-positive bacteria

Outer membrane; No outer membrane;

peptidoglycan layer thicker peptidoglycan layer

Gram-negative bacteria Gram-positive bacteria

Outer membrane; No outer membrane;

peptidoglycan layer thicker peptidoglycan layer

Cyanobacteria

Gram-negative; tougher peptidoglycan layer; extensive internal membrane system with photosynthetic pigments

Archaebacteria

No outer membrane; peptidoglycan layer outside plasma membrane

Cyanobacteria

Gram-negative; tougher peptidoglycan layer; extensive internal membrane system with photosynthetic pigments

Archaebacteria

No outer membrane; peptidoglycan layer outside plasma membrane

FIGURE 1-6 Common structural features of bacterial cells. Because of differences in the cell envelope structure, some eubacteria (grampositive bacteria) retain Gram's stain, and others (gram-negative bacteria) do not. E. coli is gram-negative. Cyanobacteria are also eubacteria but are distinguished by their extensive internal membrane system, in which photosynthetic pigments are localized. Although the cell envelopes of archaebacteria and gram-positive eubacteria look similar under the electron microscope, the structures of the membrane lipids and the polysaccharides of the cell envelope are distinctly different in these organisms.

layers outside it constitute the cell envelope. In the Archaea, rigidity is conferred by a different type of polymer (pseudopeptidoglycan). The plasma membranes of eubacteria consist of a thin bilayer of lipid molecules penetrated by proteins. Archaebacterial membranes have a similar architecture, although their lipids differ strikingly from those of the eubacteria.

The cytoplasm of E. coli contains about 15,000 ribosomes, thousands of copies each of about 1,000 different enzymes, numerous metabolites and cofac-tors, and a variety of inorganic ions. The nucleoid contains a single, circular molecule of DNA, and the cytoplasm (like that of most bacteria) contains one or more smaller, circular segments of DNA called plas-mids. In nature, some plasmids confer resistance to toxins and antibiotics in the environment. In the laboratory, these DNA segments are especially amenable to experimental manipulation and are extremely useful to molecular geneticists.

Most bacteria (including E. coli) lead existences as individual cells, but in some bacterial species cells tend to associate in clusters or filaments, and a few (the myxobacteria, for example) demonstrate simple social behavior.

Eukaryotic Cells Have a Variety of Membranous Organelles, Which Can Be Isolated for Study

Typical eukaryotic cells (Fig. 1-7) are much larger than prokaryotic cells—commonly 5 to 100 ^m in diameter, with cell volumes a thousand to a million times larger than those of bacteria. The distinguishing characteristics of eukaryotes are the nucleus and a variety of membrane-bounded organelles with specific functions: mitochondria, endoplasmic reticulum, Golgi complexes, and lysosomes. Plant cells also contain vacuoles and chloroplasts (Fig. 1-7). Also present in the cytoplasm of many cells are granules or droplets containing stored nutrients such as starch and fat.

In a major advance in biochemistry, Albert Claude, Christian de Duve, and George Palade developed methods for separating organelles from the cytosol and from each other—an essential step in isolating biomolecules and larger cell components and investigating their

(a) Animal cell

Ribosomes are protein-synthesizing machines

Peroxisome destroys peroxides

Cytoskeleton supports cell, aids in movement of organells

Lysosome degrades intracellular debris

Transport vesicle shuttles lipids and proteins between ER, Golgi, and plasma membrane

Golgi complex processes, packages, and targets proteins to other organelles or for export

Smooth endoplasmic reticulum (SER) is site of lipid synthesis and drug metabolism

Ribosomes are protein-synthesizing machines

Peroxisome destroys peroxides

Cytoskeleton supports cell, aids in movement of organells

Cytoskeleton

Nuclear envelope segregates chromatin (DNA + protein) from cytoplasm

Chloroplast harvests sunlight, produces ATP and carbohydrates

Starch granule temporarily stores carbohydrate products of photosynthesis

Smooth endoplasmic reticulum (SER) is site of lipid synthesis and drug metabolism

Golgi complex

Thylakoids are site of light-driven ATP synthesis

Plasmodesma provides path between two plant cells

Cell wall of adjacent cell

Glyoxysome contains enzymes of the glyoxylate cycle

FIGURE 1-7 Eukaryotic cell structure. Schematic illustrations of the two major types of eukaryotic cell: (a) a representative animal cell and (b) a representative plant cell. Plant cells are usually 10 to 100 ^m in diameter—larger than animal cells, which typically range from 5 to 30 ^m. Structures labeled in red are unique to either animal or plant cells.

Nuclear envelope segregates chromatin (DNA + protein) from cytoplasm

Plasma membrane separates cell from environment, regulates movement of materials into and out of cell

Cytoskeleton

Chloroplast harvests sunlight, produces ATP and carbohydrates

Starch granule temporarily stores carbohydrate products of photosynthesis

Thylakoids are site of light-driven ATP synthesis

Cell wall provides shape and rigidity; protects cell from osmotic swelling

Golgi complex

Vacuole degrades and recycles macromolecules, stores metabolites

Plasmodesma provides path between two plant cells

Cell wall of adjacent cell

Glyoxysome contains enzymes of the glyoxylate cycle

FIGURE 1-7 Eukaryotic cell structure. Schematic illustrations of the two major types of eukaryotic cell: (a) a representative animal cell and (b) a representative plant cell. Plant cells are usually 10 to 100 ^m in diameter—larger than animal cells, which typically range from 5 to 30 ^m. Structures labeled in red are unique to either animal or plant cells.

(b) Plant cell structures and functions. In a typical cell fractionation (Fig. 1-8), cells or tissues in solution are disrupted by gentle homogenization. This treatment ruptures the plasma membrane but leaves most of the organelles intact. The homogenate is then centrifuged; organelles such as nuclei, mitochondria, and lysosomes differ in size and therefore sediment at different rates. They also differ in specific gravity, and they "float" at different levels in a density gradient.

(a) Differential centrifugation

Tissue homogenization

Tissue homogenization

Low-speed centrifugation (l,000 g, l0 min)

Tissue homogenate

Low-speed centrifugation (l,000 g, l0 min)

Tissue homogenate

Pellet contains whole cells, nuclei, cytoskeletons plasma membranes

Supernatant subjected to medium-speed centrifugation (20,000 g, 20 min)

Supernatant subjected to high-speed centrifugation (80,000 g, l h)

Pellet contains whole cells, nuclei, cytoskeletons plasma membranes

Pellet contains mitochondria, lysosomes, peroxisomes

Supernatant subjected to very high-speed centrifugation (150,000 g, 3 h)

Supernatant subjected to very high-speed centrifugation (150,000 g, 3 h)

Pellet contains microsomes (fragments of ER), small vesicles

Differential centrifugation results in a rough fraction-ation of the cytoplasmic contents, which may be further purified by isopycnic ("same density") centrifugation. In this procedure, organelles of different buoyant densities (the result of different ratios of lipid and protein in each type of organelle) are separated on a density gradient. By carefully removing material from each region of the gradient and observing it with a microscope, the biochemist can establish the sedimentation position of each organelle

FIGURE 1-8 Subcellular fractionation of tissue. A tissue such as liver is first mechanically homogenized to break cells and disperse their contents in an aqueous buffer. The sucrose medium has an osmotic pressure similar to that in organelles, thus preventing diffusion of water into the organelles, which would swell and burst. (a) The large and small particles in the suspension can be separated by centrifugation at different speeds, or (b) particles of different density can be separated by isopycnic centrifugation. In isopycnic centrifugation, a centrifuge tube is filled with a solution, the density of which increases from top to bottom; a solute such as sucrose is dissolved at different concentrations to produce the density gradient. When a mixture of organelles is layered on top of the density gradient and the tube is centrifuged at high speed, individual organelles sediment until their buoyant density exactly matches that in the gradient. Each layer can be collected separately.

(b) Isopycnic

(sucrose-density) centrifugation

Pellet contains ribosomes, large macromolecules

Supernatant contains soluble proteins

Centrifugation

Centrifugation

Sample

Sucrose gradient

Less dense — component

More dense -component

Fractionation

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